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Isolation of a Common Receptor for Coxsackie B Viruses and Adenoviruses 2 and 5
Abstract
A complementary DNA clone has been isolated that encodes a coxsackievirus and adenovirus receptor (CAR). When transfected
with CAR complementary DNA, nonpermissive hamster cells became susceptible to coxsackie B virus attachment and infection.
Furthermore, consistent with previous studies demonstrating that adenovirus infection depends on attachment of a viral fiber
to the target cell, CAR-transfected hamster cells bound adenovirus in a fiber-dependent fashion and showed a 100-fold increase
in susceptibility to virus-mediated gene transfer. Identification of CAR as a receptor for these two unrelated and structurally
distinct viral pathogens is important for understanding viral pathogenesis and has implications for therapeutic gene delivery
with adenovirus vectors.
Supplementary resources (2)
Nucleotide Sequence
August 1996
Nucleotide Sequence
January 1997
... One mechanism by which viruses infect cardiomyocytes is through internalization via receptor complexes interaction, especially the coxsackie/adenoviral receptor (CAR) [50]. Internalization of the virus is facilitated by concurrent binding to decay accelerating factor (DAF; also known as CD55), a ubiquitously expressed host protein that inhibits complement activation [50,51]. ...
... One mechanism by which viruses infect cardiomyocytes is through internalization via receptor complexes interaction, especially the coxsackie/adenoviral receptor (CAR) [50]. Internalization of the virus is facilitated by concurrent binding to decay accelerating factor (DAF; also known as CD55), a ubiquitously expressed host protein that inhibits complement activation [50,51]. ...
The role of the immune system in myocarditis onset and progression involves a range of complex cellular and molecular pathways. Both innate and adaptive immunity contribute to myocarditis pathogenesis, regardless of its infectious or non-infectious nature and across different histological and clinical subtypes. The heterogeneity of myocarditis etiologies and molecular effectors is one of the determinants of its clinical variability, manifesting as a spectrum of disease phenotype and progression. This spectrum ranges from a fulminant presentation with spontaneous recovery to a slowly progressing, refractory heart failure with ventricular dysfunction, to arrhythmic storm and sudden cardiac death. In this review, we first examine the updated definition and classification of myocarditis at clinical, biomolecular and histopathological levels. We then discuss recent insights on the role of specific immune cell populations in myocarditis pathogenesis, with particular emphasis on established or potential therapeutic applications. Besides the well-known immunosuppressive agents, whose efficacy has been already demonstrated in human clinical trials, we discuss the immunomodulatory effects of other drugs commonly used in clinical practice for myocarditis management. The immunological complexity of myocarditis, while presenting a challenge to simplistic understanding, also represents an opportunity for the development of different therapeutic approaches with promising results.
... CAR is the primary receptor for HAdV-C5 and HAdV-C2, as well as most species of C Ads [8,[17][18][19]. As the name suggests, most Ads have some degree of affinity for CAR [18], although, interestingly, not all Ads use it as their primary receptor. ...
Pathogenic adenovirus (Ad) infections are widespread but typically mild and transient, except in the immunocompromised. As vectors for gene therapy, vaccine, and oncology applications, Ad-based platforms offer advantages, including ease of genetic manipulation, scale of production, and well-established safety profiles, making them attractive tools for therapeutic development. However, the immune system often poses a significant challenge that must be overcome for adenovirus-based therapies to be truly efficacious. Both pre-existing anti-Ad immunity in the population as well as the rapid development of an immune response against engineered adenoviral vectors can have detrimental effects on the downstream impact of an adenovirus-based therapeutic. This review focuses on the different challenges posed, including pre-existing natural immunity and anti-vector immunity induced by a therapeutic, in the context of innate and adaptive immune responses. We summarise different approaches developed with the aim of tackling these problems, as well as their outcomes and potential future applications.
... Due to the large number of target receptors on the host cells, the tropism of Adenovirus is very broad. The HAd2 and HAd5 from the specie C can bind the coxsackie adenovirus receptor (CAR), localized on epithelial and endothelial cells [51]. The species HAd3 binds to CD80/CD86 receptors on the APCs [52] and HAd35 recognize CD46 expressed on different cells [53]. ...
... Unlike conventional receptors, αv integrins were responsible for virus uptake into cells rather than initial virus attachment. Interestingly, the primary AdV2 attachment receptor, designated CAR (Coxsackie and Adenovirus Receptor) was not identified until 1997 by Jeff Bergelson and his colleagues at the University of Pennsylvania [17]. ...
Numerous human adenovirus (AdV) types are endowed with arginine–glycine–aspartic acid (RGD) sequences that enable them to recognize vitronectin-binding (αv) integrins. These RGD-binding cell receptors mediate AdV entry into host cells, a crucial early step in virus infection. Integrin interactions with adenoviruses not only initiate receptor-mediated endocytosis but also facilitate AdV capsid disassembly, a prerequisite for membrane penetration by AdV protein VI. This review discusses fundamental aspects of AdV–host interactions mediated by integrins. Recent efforts to re-engineer AdV vectors and non-viral nanoparticles to target αv integrins for bioimaging and the eradication of cancer cells will also be discussed.
... by the vector, which have been reported to play a role on the magnitude of transgene expression after AdV vaccination [21,22,[28][29][30][31][32]. To this extent, Ad5 uses the coxsackie adenovirus receptor (CAR) to transduce cells [33], which is broadly expressed across tissues in mice (including endothelial and epithelial tissues) [34]; whereas Ad26 utilizes CD46 as the main receptor for transduction [35,36], which is mainly restricted to the testis in mice [37], and sialic acids [38] and integrins [39] as alternative receptors. The broader receptor availability at the site of immunization and draining organs could lead to higher transduction rates in Ad5 immunized mice, explaining the higher magnitude of peak transgene expression. ...
Non-replicating adenovirus-based vectors have been broadly used for the development of prophylactic vaccines in humans and are licensed for COVID-19 and Ebola virus disease prevention. Adenovirus-based vectored vaccines encode for one or more disease specific transgenes with the aim to induce protective immunity against the target disease. The magnitude and duration of transgene expression of adenovirus 5- based vectors (human type C) in the host are key factors influencing antigen presentation and adaptive immune responses. Here we characterize the magnitude, duration, and organ biodistribution of transgene expression after single intramuscular administration of adenovirus 26-based vector vaccines in mice and evaluate the differences with adenovirus 5-based vector vaccine to understand if this is universally applicable across serotypes. We demonstrate a correlation between peak transgene expression early after adenovirus 26-based vaccination and transgene-specific cellular and humoral immune responses for a model antigen and SARS-CoV-2 spike protein, independent of innate immune activation. Notably, the memory immune response was similar in mice immunized with adenovirus 26-based vaccine and adenovirus 5-based vaccine, despite the latter inducing a higher peak of transgene expression early after immunization and a longer duration of transgene expression. Together these results provide further insights into the mode of action of adenovirus 26-based vector vaccines.
... The immediate idea is fiber replacement. While HAdV-5 employs the coxsackie virus and adenovirus receptor (CAR) on the cell membrane as an initial receptor [16,17], HAdV-B fibers bind to CD46 or DSG2 [18]. For example, replacement of the fiber knob with that from HAdV-B35 will assign the HAdV-5 vector a capability to transduce cells expressing CD46. ...
The variable domain of a heavy-chain antibody (VHH) has the potential to be used to redirect the cell tropism of adenoviral vectors. Here, we attempted to establish platforms to simplify the screening of VHHs for their specific targeting function when being incorporated into the fiber of adenovirus. Both fowl adenovirus 4 (FAdV-4) and simian adenovirus 1 (SAdV-1) have two types of fiber, one of which is dispensable for virus propagation and is a proper site for VHH display. An intermediate plasmid, pMD-FAV4Fs, was constructed as the start plasmid for FAdV-4 fiber2 modification. Foldon from phage T4 fibritin, a trigger for trimerization, was employed to bridge the tail/shaft domain of fiber2 and VHHs against human CD16A, a key membrane marker of natural killer (NK) cells. Through one step of restriction-assembly, the modified fiber2 was transferred to the adenoviral plasmid, which was linearized and transfected to packaging cells. Five FAdV-4 viruses carrying the GFP gene were finally rescued and amplified, with three VHHs being displayed. One recombinant virus, FAdV4FC21-EG, could hardly transduce human 293 or Jurkat cells. In contrast, when it was used at a multiplicity of infection of 1000 viral particles per cell, the transduction efficiency reached 51% or 34% for 293 or Jurkat cells expressing exogenous CD16A. Such a strategy of fiber modification was transplanted to the SAdV-1 vector to construct SAdV1FC28H-EG, which moderately transduced primary human NK cells while the parental virus transduced none. Collectively, we reformed the strategy of integrating VHH to fiber and established novel platforms for screening VHHs to construct adenoviral vectors with a specific tropism.
... Ono što je najbitnije za ovu fazu bolesti jeste činjenica da se pojedina stanja poput neuhranjenosti organizma, trudnoća, veliki napor, nedostatak sna, deficijencija selena kao i polni hormone češće opisuju kao predisponirajući faktori kod osoba sa miokarditisom iako epidemiološki podaci ukazuju da ne postoji različita sklonost ka nastanku drugih infekcija kod osoba koje su preležale i osoba koje nisu preležale miokarditis (13). Obimnije genetske studije na ljudima koji su preležali miokarditis nedostaju ali se lokus HLA-DQ i CD45 polimorfizam navode kao mogući genetski faktori za nastanak bolesti (21,22). Intrigantan je i podatak dobijen eksperimentom na miševima sa deficitom selena kojim je intraperitonelano dat avirulentni soj coxackiae virusa (CVB3/0), da isti soj virusa mutira i prelazi u virulentni soj (23). ...
Miokarditis tj. inflamacija tkiva miokarda predstavlja veoma kompleksan klinički entititet koji u mnogim svojim segmentima predstavlja enigmu za lekare i istraživače. Pored virusa koji su u najvećem broju slučajeva glavni inicijatori inflamacije srca u dece, miokarditis može biti uzrokovan i toksinima, ishemijom, mehaničkim oštećenjem, lekovima, ali i imunim reakcijama kod transplantirane dece ili u sklopu sistemskih bolesti vezivnog tkiva. Iako je incidenca klinički manifestnih formi miokarditisa relativno mala, evolucija bolesti i često nepovoljan tok: razvoj dilatantne kardiomiopatije, kliničke slike aritmogene displazije desne komore ili infarkta miokarda i ne retko fulminantni tok bolesti, pridaju mu veliki sociomedicinski značaj. Iako je danas nedvosmisleno pokazano da je ključna komponenta koja određuje način i formu ispoljavanja bolesti kao i njen konačan ishod upravo kvalitet imunog odgovora, još uvek postoje velike dileme vezane za način kliničke evaluacije i terapiju kako dece tako i odraslih osoba sa miokarditisom.
... The order parameter S was defined as S = (3cos 2 θ − 1)/2 and showed that the nanometer-scale ordering of ZO-1 molecules at cell junctions was disorganized in claudin/JAM-A/CAR KO cells (n = 5-6 junctions). Although CAR was originally identified as an adenovirus and coxsackievirus receptor (Bergelson et al., 1997), subsequent studies demonstrated that it can also interact with ZO-1 and become localized at TJs (Cohen et al., 2001). It was shown that adenoviruses and coxsackie viruses infect epithelial cells through TJs using CAR as their receptors and that the epithelial barrier function is transiently disrupted during infection (Walters et al., 2002;Coyne and Bergelson, 2006). ...
Epithelia must be able to resist mechanical force to preserve tissue integrity. While intercellular junctions are known to be important for the mechanical resistance of epithelia, the roles of tight junctions (TJs) remain to be established. We previously demonstrated that epithelial cells devoid of the TJ membrane proteins claudins and JAM-A completely lack TJs and exhibit focal breakages of their apical junctions. Here, we demonstrate that apical junctions fracture when claudin/JAM-A–deficient cells undergo spontaneous cell stretching. The junction fracture was accompanied by actin disorganization, and actin polymerization was required for apical junction integrity in the claudin/JAM-A–deficient cells. Further deletion of CAR resulted in the disruption of ZO-1 molecule ordering at cell junctions, accompanied by severe defects in apical junction integrity. These results demonstrate that TJ membrane proteins regulate the mechanical resistance of the apical junctional complex in epithelial cells.
... 32,33 For instance, the binding of wild-type (WT) Ad5 to target cells is mediated by the interaction of the viral fiberknob domain with coxsackievirus and adenovirus receptor (CAR) ubiquitously expressed on many normal cells while absent or low expressed on many cancer cells. [34][35][36] Therefore, fiber-knob modifications allowing for CAR-independent binding of Ad5-based vectors are needed to achieve efficient tumor cell transduction and minimize damage to normal cells. 37 Efficient tumor cell transduction is crucial not only for initial virus internalization but also for subsequent intratumoral spread. ...
Oncolytic adenoviruses (Ads) stand out as a promising strategy for the targeted infection and lysis of tumor cells, with well-established clinical utility across various malignancies. This study delves into the therapeutic potential of oncolytic Ads in the context of neurofibromatosis type 1 (NF1)-associated malignant peripheral nerve sheath tumors (MPNSTs). Specifically, we evaluate conditionally replicative adenoviruses (CRAds) driven by the cyclooxygenase 2 (COX2) promoter, as selective agents against MPNSTs, demonstrating their preferential targeting of MPNST cells compared with non-malignant Schwann cell control. COX2-driven CRAds, particularly those with modified fiber-knobs exhibit superior binding affinity toward MPNST cells and demonstrate efficient and preferential replication and lysis of MPNST cells, with minimal impact on non-malignant control cells. In vivo experiments involving intratumoral CRAd injections in immunocompromised mice with human MPNST xenografts significantly extend survival and reduce tumor growth rate compared with controls. Moreover, in immunocompetent mouse models with MPNST-like allografts, CRAd injections induce a robust infiltration of CD8+ T cells into the tumor microenvironment (TME), indicating the potential to promote a pro-inflammatory response. These findings underscore oncolytic Ads as promising, selective, and minimally toxic agents for MPNST therapy, warranting further exploration.
... To explore potential new cell-surface antigens induced by hypoxia, we focused on proteins with divergent normalized relative abundances between hypoxia and normoxia according to TS-MAP data. We decided to select five candidates for validation experiments: CXADR, known as a receptor for coxsackievirus and adenovirus that has shown potential to promote tumorigenesis and as a target for oncolytic viral therapy [28][29][30]; CD81, known as a receptor of HCV that has been implicated in tumor progression and as a potential immunotherapeutic target in lymphoma and breast cancer [31][32][33][34]; CD47, suggested as a targetable immune checkpoint in GBM and other malignancies, and was shown to be hypoxia/ HIF1α-regulated in breast cancer [35][36][37]; BSG, shown to be overexpressed and hypoxia-induced in GBM [38], and was suggested as an ADC target in hepatocellular carcinoma [39]; and FXYD6, which is a relatively unexplored regulator of the Na + /K + -ATPase, and was suggested as therapeutic target in cancer [40]. In all cases, we could successfully validate TS-MAP data by showing increased surface expression in hypoxia, in both GBM spheroid cores and 2D cultures (Fig. 5A-C, and Additional file 1: Fig. S6A). ...
Immunotherapies with antibody–drug-conjugates (ADC) and CAR-T cells, targeted at tumor surface antigens (surfaceome), currently revolutionize clinical oncology. However, target identification warrants a better understanding of the surfaceome and how it is modulated by the tumor microenvironment. Here, we decode the surfaceome and endocytome and its remodeling by hypoxic stress in glioblastoma (GBM), the most common and aggressive brain tumor in adults. We employed a comprehensive approach for global and dynamic profiling of the surfaceome and endocytosed (endocytome) proteins and their regulation by hypoxia in patient-derived GBM cultures. We found a heterogeneous surface-endocytome profile and a divergent response to hypoxia across GBM cultures. We provide a quantitative ranking of more than 600 surface resident and endocytosed proteins, and their regulation by hypoxia, serving as a resource to the cancer research community. As proof-of-concept, the established target antigen CD44 was identified as a commonly and abundantly expressed surface protein with high endocytic activity. Among hypoxia induced proteins, we reveal CXADR, CD47, CD81, BSG, and FXYD6 as potential targets of the stressed GBM niche. We could validate these findings by immunofluorescence analyses in patient tumors and by increased expression in the hypoxic core of GBM spheroids. Selected candidates were finally confronted by treatment studies, showing their high capacity for internalization and ADC delivery. Importantly, we highlight the limited correlation between transcriptomics and proteomics, emphasizing the critical role of membrane protein enrichment strategies and quantitative mass spectrometry. Our findings provide a comprehensive understanding of the surface-endocytome and its remodeling by hypoxia in GBM as a resource for exploration of targets for immunotherapeutic approaches in GBM.
... Since the best studied adenovirus receptor is the coxsackievirus and adenovirus receptor (CAR) [31], we decided to examine its expression in murine neurons infected with HAdV serotypes. According to literature data, CAR occurs in cells of various organs, including the brain, and its expression allows the entry of adenoviruses belonging to subgroups A (HAdV12), C (HAdV2, HAdV5), E (HAdV4), and F (HAdV41) [32]. ...
Human adenovirus (HAdV) is a common pathogen, which can lead to various clinical symptoms and—in some cases—central nervous system (CNS) dysfunctions, such as encephalitis and meningitis. Although the initial events of virus entry have already been identified in various cell types, the mechanism of neuronal uptake of adenoviruses is relatively little understood. The aim of this study was to investigate early events during adenoviral infection, in particular to determine the connection between cellular coxsackievirus and adenovirus receptor (CAR), clathrin, caveolin, and early endosomal proteins (EEA1 and Rab5) with the entry of HAdVs into primary murine neurons in vitro. An immunofluorescence assay and confocal microscopy analysis were carried out to determine HAdV4, 5, and 7 correlation with CAR, clathrin, caveolin, and early endosomal proteins in neurons. The quantification of Pearson’s coefficient between CAR and HAdVs indicated that the HAdV4 and HAdV5 types correlated with CAR and that the correlation was more substantial for HAdV5. Inhibition of clathrin-mediated endocytosis using chlorpromazine limited the infection with HAdV, whereas inhibition of caveolin-mediated endocytosis did not affect virus entry. Thus, the entry of tested HAdV types into neurons was most likely associated with clathrin but not caveolin. It was also demonstrated that HAdVs correlate with the Rab proteins (EEA1, Rab5) present in early vesicles, and the observed differences in the manner of correlation depended on the serotype of the virus. With our research, we strove to expand knowledge regarding the mechanism of HAdV entry into neurons, which may be beneficial for developing potential therapeutics in the future.
... Species C adenoviruses infect respiratory epithelial cells by interacting with the cellular receptor CAR using the protruding fiber protein, followed by secondary interactions between the penton base and α v β 3 integrins that trigger entry (1,17,18). This fundamentally simple model is likely used in immortalized cell lines but is highly unlikely in the polarized epithelium where CAR is hidden deep within the tight junctions (19)(20)(21). ...
Adenovirus (AdV) infection of the respiratory epithelium is common but poorly understood. Human AdV species C types, such as HAdV-C5, utilize the Coxsackie-adenovirus receptor (CAR) for attachment and subsequently integrins for entry. CAR and integrins are however located deep within the tight junctions in the mucosa where they would not be easily accessible. Recently, a model for CAR-independent AdV entry was proposed. In this model, human lactoferrin (hLF), an innate immune protein, aids the viral uptake into epithelial cells by mediating interactions between the major capsid protein, hexon, and yet unknown host cellular receptor(s). However, a detailed understanding of the molecular interactions driving this mechanism is lacking. Here, we present a new cryo-EM structure of HAdV-5C hexon at high resolution alongside a hybrid structure of HAdV-5C hexon complexed with human lactoferrin (hLF). These structures reveal the molecular determinants of the interaction between hLF and HAdV-C5 hexon. hLF engages hexon primarily via its N-terminal lactoferricin (Lfcin) region, interacting with hexon’s hypervariable region 1 (HVR-1). Mutational analyses pinpoint critical Lfcin contacts and also identify additional regions within hLF that critically contribute to hexon binding. Our study sheds more light on the intricate mechanism by which HAdV-C5 utilizes soluble hLF/Lfcin for cellular entry. These findings hold promise for advancing gene therapy applications and inform vaccine development.
IMPORTANCE
Our study delves into the structural aspects of adenovirus (AdV) infections, specifically HAdV-C5 in the respiratory epithelium. It uncovers the molecular details of a novel pathway where human lactoferrin (hLF) interacts with the major capsid protein, hexon, facilitating viral entry, and bypassing traditional receptors such as CAR and integrins. The study’s cryo-EM structures reveal how hLF engages hexon, primarily through its N-terminal lactoferricin (Lfcin) region and hexon’s hypervariable region 1 (HVR-1). Mutational analyses identify critical Lfcin contacts and other regions within hLF vital for hexon binding. This structural insight sheds light on HAdV-C5’s mechanism of utilizing soluble hLF/Lfcin for cellular entry, holding promise for gene therapy and vaccine development advancements in adenovirus research.
... The original system for obtaining iPSCs is using retroviral vectors, which integrate the transgenes into the host genome [16]. Adenovirus vectors are generally quite poor in cell gene transfer, probably due to the availability of primary receptors and/or co-receptors required for in vitro cell binding and internalization [17,18]. Stem cells gene delivery by these vectors is relatively weak, although there are capsid modification techniques that improve delivery efficiency [19]. ...
Regenerative medicine expects to replace the function of tissue or organs damaged by disease, trauma, or congenital issues. The tools used to realize these outcomes are tissue engineering and cellular therapies. Cellular therapy is considered a regenerative medicine strategy based on the use of stem cells. Pluripotent stem cells are the hotspots of cellular therapy due to their features that have been showed promising results. Embryonic stem cells (ESCs) are pluripotent, self-renewing cells that are derived from the inner cell mass (ICM) of the developing blastocyst. Pluripotency is the main feature that lead single cell to generate all cell lineages of the developing and adult organism. The use of human ESC (hESC) is ethically controversial, to overcome this problem the induced pluripotent stem cells (iPSCs) were developed. The aim of the present mini-review is to report a comprehensive summary of the different cellular reprogramming techniques from its initial conception to the present day.
... Myocarditis is defined by the presence of an inflammatory infiltrate with myocardial necrosis of non-ischemic origin, in three forms: fulminant, acute and chronic [1,2]. The diagnosis is guided by clinical signs, electrocardiogram (ECG), echocardiography and biology (troponins); it is then confirmed by magnetic resonance imaging (MRI) and myocardial biopsy [3][4][5][6][7][8]. ...
Introduction: Myocarditis is defined by an inflammatory myocardial infiltrate with necrosis of non-ischemic origin in three forms: fulminant, acute and chronic. Diagnosis is guided by clinical presentation, ECG, echocardiography and biology, and confirmed by MRI and myocardial biopsy. The prognosis depends on clinical manifestations, echocardiographic features and serum troponin levels. Management is based on the treatment of heart failure (HF). For two years, the world has been experiencing a pandemic related to SARS-CoV2 that can affect the heart with ischemic or non-ischemic lesions (myocarditis, most often fulminant) whose treatment is non-specific. Trials with corticosteroids and immunosuppressant drugs have yielded discordant results. Objective: To describe the evolutionary modalities of COVID-19 associated myocarditis and identify factors of poor ejection fraction recovery under HF treatment. Method: This observational, retrospective, single-center study, in 2021, included patients with non-fulminant COVID-19 associated myocarditis suspected at echocardiography and biology and confirmed on MRI. Patients with previous HF and reduced left ventricular ejection fraction (LVEF) were excluded (n=06). Patients were divided into two groups according to LVEF three months later (LVEF>50% v. LVEF<50%) and compared to identify factors predicting a poor LVEF recovery. Results: 33 patients (19♂/14♀) aged between 30–61 years with acute non-fulminant COVID-19 associated myocarditis were included. All had ECG repolarization abnormalities. The mean LVEF at baseline was 44.3% +/- 6.3 (30–52%) with an average troponin level 480 times normal (20–2,100). Beta-blocker and RASB treatment was initiated in all patients, spironolactone (37.5 mg) in 13 patients with LVEF <40% and furosemide if congestive signs (17 patients/51.5%). Clinical, electrical, biological and echocardiographic monitoring was performed at one and three months. Eight patients developed uncomplicated pericardial effusion. A significant improvement in LVEF>50% was observed in 29 patients. One patient with LVEF of 38% presented with incessant ventricular tachyarrhythmia that necessitated an ICD. Three patients kept LVEF<50%. Sex, congestive signs, ECG and coronary angiogram abnormalities do not seem to influence the LVEF evolution (p at 0.62, 1.00, 1.00, 0.56, 0.50, and 0.23, respectively). Age >60 years, troponins >1,200 times normal, pericardial effusion and a combined criterion of the three seem to be a good predictor of poor LVEF evolution (p at 0.07, 0.02, 0.035 and 0.01, respectively). Discussion: The absence of fulminant forms in our series explains the absence of mortality at three months (>30% in the literature). Acute non-fulminant COVID-19 associated myocarditis has a good prognosis with LVEF recovery in 87.88%. The factors of poor LVEF recovery are the age >60 years, troponins >1,200 times normal, pericardial effusion, and the combined criterion of the three (p respectively at 0.07, 0.02, 0.035, 0.01). The routine prescription of corticosteroids in the COVID-19 protocol made it impossible to analyze its impact on COVID-19 associated myocarditis. Interpretation: Cardiac manifestations are not uncommon during COVID-19; they can be ischemic or non-ischemic. There is no specific therapy for non-fulminant COVID-19 associated myocarditis and the evolution seems favorable. Patients with predictive factors of poor progress should have longer follow-ups. Informed consent: All participants gave their informed consent to participate in this study and share the results
... Each subgroup utilizes specific receptor(s) to enter the host cell. Subgroup A and C-F serotypes mainly use the coxsackievirus-adenovirus receptor (CAR) for virus entry 25,32,33 ; subgroup B utilizes desmoglein-2 (DSG-2), CD46, CD80, and CD86 [26][27][28] ; and subgroup G uses the host protein polysialic acid. 31 Ad displays a high infection rate in humans, and almost all children under the age of 10 years have experienced at least one Ad infection. ...
Owing to the limitations of conventional cancer therapies, including chemotherapy, radiotherapy, and surgery, gene therapy has become a prominent strategy for cancer treatment over the past few decades. Gene therapy is a medical approach for targeting and destroying cancer cells by delivering exogenous genes into the target cancerous cells or surrounding tissues. However, successful delivery of foreign genes into target cells and tissues remains a key issue in such therapy. Efficient gene delivery systems would undoubtedly be important for improving the medical outcomes of gene therapy. With genetic modifications, viral vectors can target specific cells with high gene transduction efficiency, thus, the use of viral vectors is a promising technology for improving foreign gene delivery. Currently, four viral vectors—adenovirus, adeno‐associated virus, herpes simplex virus, and retrovirus—are dominantly being investigated and used in preclinical and clinical trials. In this review, we provide an overview of the mechanisms and latest applications of the four above‐mentioned viral vectors, and summarize the current development of several other viral vectors. In addition, we discuss the challenges and provide insights into future development of viral vectors in cancer treatment.
... The primary receptor for most human adenoviruses (Ad) is CAR, a 46-kDa member of the immunoglobulin (Ig) superfamily (5,39). This receptor is distributed on many cell types in vivo, and its location in tight junctions on polarized epithelial cells (12) plays a role in intercellular dissemination of the virus (45). ...
The adenovirus (Ad) fiber protein mediates Ad binding to the coxsackievirus and Ad receptor (CAR) and is thus a major determinant of viral tropism. The fiber contains three domains: an N-terminal tail that anchors the fiber to the viral capsid, a central shaft region of variable length and flexibility, and a C-terminal knob domain that binds to cell receptors. Ad type 37 (Ad37), a subgroup D virus associated with severe ocular infections, is unable to use CAR efficiently to infect host cells, despite containing a CAR binding site in its fiber knob. We hypothesized that the relatively short, inflexible Ad37 fiber protein restricts interactions with CAR at the cell surface. To test this hypothesis, we analyzed the infectivity and binding of recombinant Ad particles containing modified Ad37 or Ad5 fiber proteins. Ad5 particles equipped with a truncated Ad5 fiber or with a chimeric fiber protein comprised of the Ad5 knob fused to the short, rigid Ad37 shaft domain had significantly reduced infectivity and attachment. In contrast, placing the Ad37 knob onto the long, flexible Ad5 shaft allowed CAR-dependent virus infection and cell attachment, demonstrating the importance of the shaft domain in receptor usage. Increasing fiber rigidity by substituting the predicted flexibility modules in the Ad5 shaft with the corresponding regions of the rigid Ad37 fiber dramatically reduced both virus infection and cell attachment. Cryoelectron microscopy (cryo-EM) single-particle analysis demonstrated the increased rigidity of this chimeric fiber. These studies demonstrate that both length and flexibility of the fiber shaft regulate CAR interaction and provide a molecular explanation for the use of alternative receptors by subgroup D Ad with ocular tropism. We present a molecular model for Ad-CAR interactions at the cell surface that explains the significance of fiber flexibility in cell attachment.
... Despite this enormous number, a conclusive cure has not been discovered (17) . Depending on whether virus targets tumour cells, oncolytic virus therapy is a potential treatment for a variety of cancers (18) . CAR is one of the most widely used viral vectors and was found to be expressed in many malignancies (19) . ...
Background: The Coxsackie virus and Adenovirus Receptor (CAR) is a cellular protein that has a role in cell adhesion, signaling, and viral infection. There is much disagreement over the significance of CAR expression in colorectal carcinoma development, with some research suggesting CAR downregulation and others indicating that CAR enabled complicated effects during colorectal carcinogenesis. Objective: This study aimed to elucidate the difference in CAR expression levels in colorectal cancer (CRC) tissue versus normal colon tissue, and to correlate the expression levels with the disease stage.
Patients and methods: Fifty patients with proven colorectal cancer were enrolled in this study. During surgical excision treatment, 50 pairs of CRC tissue and normal tissue samples were obtained and examined for CAR expression levels using reverse transcriptase Real time PCR.
Results: CRC specimens showed significantly downregulated CAR gene expression when compared to nearby safety margin specimens. No significant differences were found in CAR gene expression levels in CRC tissue based on patients’ gender, tumor site, size, associated LN metastasis and tumor stage (p > 0.05 for each). However, stratifying cases into early (stages I and II) and advanced (stages III and IV) revealed that lower CAR gene expression was significantly associated with advanced CRC stages.
Conclusion: Low CAR gene expression may have a potential role in colorectal carcinogenesis and its level is associated with advanced CRC stages with poorer prognosis.
... OAd infects tumor cells through the interaction of adenoviral fiber knob with receptors on the cell surface (23,24). HAdV-5 was the most com monly used serotype for the design of conventional OAds and binds primarily to the coxsackievirus and adenovirus receptor (CAR) (25,26). Since CAR is absent or poorly expressed in many cancer cells, alternative strategies using non-CAR binding HAdVs were developed. ...
Among human species B adenovirus, HAdV-11, has the particular feature of exploiting two primary receptors, desmoglein 2 (DSG2) and CD46, to mediate cell attachment. This serotype represents a particularly promising class of oncolytic viral vaccines. For instance, Enadenotucirev, a species B HAdV-11/HAdV-3 chimeric adenoviral vector, demonstrated preclinical tumor-selective cytotoxicity. Understanding the mechanism of HAdV-11 entry is a major challenge for the development of powerful vectors. Interactions between HAdV-11 and CD46 are well-characterized and clearly depend on the fiber protein, but the mechanism whereby HAdV-11 engages DSG2 has been still unresolved. In this study, we described a new cryo-EM structure of the HAdV-11 fiber in complex with DSG2. Kinetic interaction studies demonstrated that the binding stability of the HAdV-11 in complex with DSG2 was significantly lower than with CD46. Furthermore, competition assays with pseudotyped viruses suggested that the interaction of the HAdV-11 fiber with DSG2 was not required for infection but that DSG2 could serve as a receptor in the absence of CD46. Unexpectedly, we succeeded to isolate a HAdV-11 fiber/DSG2/CD46 “super complex” showing that these two receptors could simultaneously bind to the same trimeric HAdV-11K. Our work deciphers the use of two independent receptors by HAdV-11 from a biochemical and structural point of view, which can have an impact for future vectors design.
IMPORTANCE
The main limitation of oncolytic vectors is neutralization by blood components, which prevents intratumoral administration to patients. Enadenotucirev, a chimeric HAdV-11p/HAdV-3 adenovirus identified by bio-selection, is a low seroprevalence vector active against a broad range of human carcinoma cell lines. At this stage, there’s still some uncertainty about tropism and primary receptor utilization by HAdV-11. However, this information is very important, as it has a direct influence on the effectiveness of HAdV-11-based vectors. The aim of this work is to determine which of the two receptors, DSG2 and CD46, is involved in the attachment of the virus to the host, and what role they play in the early stages of infection.
... We have not studied the mechanism of antiviral action in vivo and how reduction of virus replication is affected by the extract in heart tissues of the infected mice but there are several hypothesis. Among them, methanolic extract inhibited the virus replication by blocking him Coxsackievirus and Adenovirus Receptor (CAR) which represent the first primary step to virus entry into host cells [22,23]. Several drugs such as WIN compounds have been reported to inhibit the interaction between CVB and CAR [24]. ...
... Virus surface movement is connected to their plasma membrane and receptor interactions (30). HAdV type C and F long fiber knobs bind CAR, while HAdV type F short fiber knobs interact with heparan sulfates (HS) (16,17,31). We investigated the EAdV virus preparations for short vs long fiber ratios and found that they contain more short than long fiber (data not shown) in line with published data (32). ...
Enteric adenovirus types F40 and 41 (EAdVs) are a leading cause of diarrhea and diarrhea-associated death in young children and have recently been proposed to cause acute hepatitis in children. EAdVs have a unique capsid architecture and exhibit — unlike other human adenoviruses — a relatively strict tropism for gastrointestinal tissues with, to date, understudied infection mechanism and unknown target cells. In this study, we turn to potentially limiting host factors by comparing EAdV entry in cell lines with respiratory and intestinal origin by cellular perturbation, virus particle tracking, and transmission electron microscopy. Our analyses highlight kinetic advantages for EAdVs in duodenal HuTu80 cell infection and reveal a larger fraction of mobile particles, faster virus uptake, and infectious particle entry in intestinal cells. Moreover, EAdVs display a dependence on clathrin- and dynamin-dependent pathways in intestinal cells. Detailed knowledge of virus entry routes and host factor requirements is essential to understanding pathogenesis and developing new countermeasures. Hence, this study provides novel insights into the entry mechanisms of a medically important virus with emerging tropism in a cell line originating from a relevant tissue.
IMPORTANCE
Enteric adenoviruses have historically been difficult to grow in cell culture, which has resulted in lack of knowledge of host factors and pathways required for infection of these medically relevant viruses. Previous studies in non-intestinal cell lines showed slow infection kinetics and generated comparatively low virus yields compared to other adenovirus types. We suggest duodenum-derived HuTu80 cells as a superior cell line for studies to complement efforts using complex intestinal tissue models. We show that viral host cell factors required for virus entry differ between cell lines from distinct origins and demonstrate the importance of clathrin-mediated endocytosis.
... Species C adenoviruses infect respiratory epithelial cells by interacting with the cellular receptor CAR using the protruding fiber protein, followed by secondary interactions between the penton base and αvβ3 integrins that triggers entry (1,17,18). This fundamentally simple model is likely used in immortalized cell lines but is highly unlikely in the polarized epithelium where CAR is hidden deep withing the tight junctions (19)(20)(21). ...
Adenovirus (AdV) infection of the respiratory epithelium is common, but poorly understood. Human AdV species C types, such as HAdV-C5, utilize the Coxsackie-adenovirus receptor (CAR) for attachment and subsequently integrins for entry. CAR and integrins are however located deep within the tight junctions in the mucosa where they would not be easily accessible. Recently, a model for CAR-independent AdV entry was proposed. In this model, human lactoferrin (hLF), an innate immune protein, aids the viral uptake into epithelial cells by mediating interactions between the major capsid protein, hexon, and yet unknown host cellular receptor(s). However, a detailed understanding of the molecular interactions driving this mechanism is lacking. Here, we present a new cryo-EM structure of HAdV-5C hexon at high resolution alongside a hybrid structure of HAdV-5C hexon complexed with human lactoferrin (hLF). These structures reveal the molecular determinants of the interaction between hLF and HAdV-C5 hexon. hLF engages hexon primarily via its N-terminal lactoferricin (Lfcin) region, interacting with hexon's hypervariable region 1 (HVR-1). Mutational analyses pinpoint critical Lfcin contacts and also identify additional regions within hLF that critically contribute to hexon binding. Our study sheds more light on the intricate mechanism by which HAdV-C5 utilizes soluble hLF/Lfcin for cellular entry. These findings hold promise for advancing gene therapy applications and inform vaccine development.
... The advantages of SAdVs and HAdVs as gene vectors include the capacity for large heterologous gene insertion, well-defined methods for preparation and purification, versatility for a wide host cell range (able to infect cells in mitotic and non-mitotic stages), and non-integration into the host genome. Adenovirus can recognize a variety of cell surface receptors, such as Coxsackie-adenovirus receptors (CARs) (35), CD46 (36), desmoglein-2 (DSG2) (37,38), integrin (39), and so on. Both HAdVs and SAdVs have been genetically modified and used as delivery vehicles for gene therapy and vaccine applications, including Ebola and SARS-CoV-2, and also as oncolytic agents, e.g., for head and neck cancer and hepatocellular carcinoma (40)(41)(42)(43)(44)(45)(46)(47). ...
Both human and non-human simian adenoviruses (HAdVs and SAdVs, respectively) have been used as gene therapy and vaccine vectors. The high prevalence of HAdVs and the neutralizing antibodies associated with prior infection, may limit HAdV-based vector use in human subjects. To overcome this drawback, a vector derived from a newly isolated and characterized macaque adenovirus was constructed. SAdVs (33.9%) were screened from 115 SAdV fecal samples collected at a zoological park. One novel SAdV was isolated and the whole genome was sequenced and analyzed. The pre-existing neutralizing antibody levels were very low against this isolate (10%). Interestingly, SAdV vector constructs that lack E3 region could not produce infectious progeny in HEK293 cells, suggesting that the E3 region is necessary for SAdV replication. The absence of E3 region could be compensated for by replacement with HAdV-5 E4orf6; the resultant construct could replicate well in HEK293 cells. The enhanced Green Fluorescent Protein (eGFP) was inserted into SAdV E3 region and expressed at high level. One-step growth curve showed that the replication of the SAdVs with HAdV-5 E4orf6 substitution and E1/E3 deletion was similar to that of wild-type SAdVs in HEK293 cells, but the modified SAdVs were replication-deficient in A549 cells which lack HAdV-5 E1A and E1B. Finally, we demonstrated that GZ3-12 could infect cells expressing hCAR or hDSG2 receptors. The successful isolation, characterization, and modification of novel SAdVs provide a potentially important vaccine and gene therapy candidate and a new strategy for the rapid acquisition and development of non-HAdV-based alternative vectors for human health applications.
IMPORTANCE
Adenoviruses are widely used in gene therapy and vaccine delivery. Due to the high prevalence of human adenoviruses (HAdVs), the pre-existing immunity against HAdVs in humans is common, which limits the wide and repetitive use of HAdV vectors. In contrast, the pre-existing immunity against simian adenoviruses (SAdVs) is low in humans. Therefore, we performed epidemiological investigations of SAdVs in simians and found that the SAdV prevalence was as high as 33.9%. The whole-genome sequencing and sequence analysis showed SAdV diversity and possible cross species transmission. One isolate with low level of pre-existing neutralizing antibodies in humans was used to construct replication-deficient SAdV vectors with E4orf6 substitution and E1/E3 deletion. Interestingly, we found that the E3 region plays a critical role in its replication in human cells, but the absence of this region could be compensated for by the E4orf6 from HAdV-5 and the E1 expression intrinsic to HEK293 cells.
... HAdV-B serotypes (3,7,11,15) share a binding mechanism by attaching to the short consensus repeat domains SCR I and SCR II of CD46 [11]. Most adenovirus types predominantly bind to the single-channel transmembrane protein coxsackie virus and adenovirus receptor (CAR), which facilitates their initial attachment and entry into host cells [12]. Integrin, DSG2, liver tropism, and glycoproteins containing sialic acid also serve as characteristic adenovirus receptors for various serotypes [13]. ...
The increasing incidence of severe adenovirus cases underscores the imperative for efficacious anti-adenovirus medications, given the current absence of targeted therapeutic options. This study explores the potential antiviral effects of the natural product Resveratrol (RSV) on various cell lines, marking the first-time discovery of such properties. The results suggest that RSV suppresses the upregulated pathways (NF-κB, JAK-STAT) triggered by viral infections, with its mechanism contingent upon the presence of the SIRT1 protein. Consequently, RSV mitigates the expression of inflammatory factors (IL-6, IL-8) as detected by ELISA. Subsequently, we elucidate the specific amino acid sites on both RSV and SIRT1 using macromolecular docking and protein docking methodologies. This revelation represents the pioneering elucidation of the mechanism and mode of action of resveratrol as an antiviral agent. These promising results establish the substantial potential of RSV as a therapeutic agent against adenovirus-7. The capacity to proficiently hinder adenovirus-7 replication and demonstrate potent antiviral properties in vitro experiments introduces novel avenues for crafting targeted therapies to combat adenovirus infections.
... HAdV species A, C, D, E, and F gain entry to host cells by means of the widely expressed coxsackie-adenovirus receptor (CAR) [48,49]. This is recognised by the knob domain of the virus trimeric fibre capsid protein. ...
Over 100 human adenoviruses (HAdVs) have been isolated and allocated to seven species, A-G. Species F comprises two members-HAdV-F40 and HAdV-F41. As their primary site of infection is the gastrointestinal tract they have been termed, with species A, enteric adenoviruses. HAdV-F40 and HAdV-F41 are a common cause of gastroenteritis and diarrhoea in children. Partly because of difficulties in propagating the viruses in the laboratory, due to their restrictions on growth in many cell lines, our knowledge of the properties of individual viral proteins is limited. However, the structure of HAdV-F41 has recently been determined by cryo-electron microscopy. The overall structure is similar to those of HAdV-C5 and HAdV-D26 although with some differences. The sequence and arrangement of the hexon hypervariable region 1 (HVR1) and the arrangement of the C-terminal region of protein IX differ. Variations in the penton base and hexon HVR1 may play a role in facilitating infection of intestinal cells by HAdV-F41. A unique feature of HAdV-F40 and F41, among human adenoviruses, is the presence and expression of two fibre genes, giving long and short fibre proteins. This may also contribute to the tropism of these viruses. HAdV-F41 has been linked to a recent outbreak of severe acute hepatitis “of unknown origin” in young children. Further investigation has shown a very high prevalence of adeno-associated virus-2 in the liver and/or plasma of some cohorts of patients. These observations have proved controversial as HAdV-F41 had not been reported to infect the liver and AAV-2 has generally been considered harmless.
... RVs are the main cause of common cold in humans, but may also have lower respiratory tract involvement, with a clinical presentation of bronchitis or pneumonia [86,87]. Human Adenovirus (AdV) is a non-enveloped DNA virus that causes a wide range of illnesses, such as conjunctivitis, gastroenteritis, and respiratory infections [88]. Human Parainfluenza (HPIV) are single-stranded, enveloped RNA viruses that cause a broad spectrum of respiratory clinical manifestations, including colds, bronchiolitis, and pneumonia [89]. ...
Pathogenic adenovirus (Ad) infections are widespread but typically mild and transient, except in the immunocompromised. As vectors for gene therapy, vaccine and oncology applications, Ad based platforms offer advantages including ease of genetic manipulation, scale of production and well established safety
profiles, making them attractive tools for therapeutic development. However, the immune system often poses
a significant challenge that may need to be overcome for adenovirus based therapies to be truly efficacious.
Both pre-existing anti-Ad immunity in the population as well as the rapid development of an immune response against engineered adenoviral vectors can have detrimental effects upon the downstream impact of an adenovirus-based therapeutic. This review focusses on the different challenges posed, including pre-existing natural immunity and anti-vector immunity induced by a therapeutic, both in context of the innate and
adaptive immune responses. We summarize different approaches developed with the aim of tackling these problems, as well as their outcomes and potential future applications.
It is necessary to explore new targets for the treatment of colon adenocarcinoma (COAD) according to the tumor microenvironment. The expression levels of JAML and CXADR were analyzed by bioinformatics analysis and validation of clinical samples. JAML over-expression CD8+ T cell line was constructed, and the proliferation activity was detected by MTT. The production of inflammatory factors was detected by ELISA. The expression of immune checkpoint PD-1 and TIM-3 was detected by Western blot. The apoptosis level was detected by flow cytometry and apoptosis markers. The AOM/DSS mouse model of colorectal cancer was constructed. The expression levels of JAML, CXADR and PD-1 were detected by PCR and Western blot, and the proportion of CD8+ T cells and exhausted T cells were detected by flow cytometry. The expression levels of JAML and CXADR were significantly decreased in colon cancer tissues. Overexpression of JAML can promote the proliferation of T cells, secrete a variety of inflammatory factors. Overexpression of CXADR can reduce the proliferation of colorectal cancer cells, promote apoptosis, and down-regulate the migration and invasion ability of tumor cells. Both JAML agonists and PD-L1 inhibitors can effectively treat colorectal cancer, and the combined use of JAML agonists and PD-L1 inhibitors can enhance the effect. JAML can promote the proliferation and toxicity of CD8+ T cells and down-regulate the expression of immune checkpoints in colon cancer. CXADR can inhibit the proliferation of cancer cells and promote the apoptosis. JAML agonist can effectively treat colorectal cancer by regulating CD8+ T cells.
Hepatitis viruses constitute a significant global health threat, resulting in substantial morbidity and mortality on a global scale. The pursuit of effective therapeutic strategies to combat hepatitis virus infections remains a paramount objective within the realm of medical research. Among the promising candidates, neutralizing antibodies (NAbs) have emerged as noteworthy therapeutic agents due to their capacity to impede viral entry and replication. This chapter provides an overview of recent advancements in the investigation of NAbs targeting various hepatitis viruses, encompassing hepatitis A, B, C, D, and E viruses. Our discourse delves into elucidating the mechanisms underlying neutralization, as well as their practical applications in the therapeutic domain. Additionally, we shed light on the challenges inherent in this research area and delineate the prospective trajectories for future exploration.
Adenoviruses (Ad) have the potential to induce severe infections in vulnerable patient groups. Therefore, understanding Ad biology and antiviral processes is important to comprehend the signaling cascades during an infection and to initiate appropriate diagnostic and therapeutic interventions. In addition, Ad vector-based vaccines have revealed significant potential in generating robust immune protection and recombinant Ad vectors facilitate efficient gene transfer to treat genetic diseases and are used as oncolytic viruses to treat cancer. Continuous improvements in gene delivery capacity, coupled with advancements in production methods, have enabled widespread application in cancer therapy, vaccine development, and gene therapy on a large scale. This review provides a comprehensive overview of the virus biology, and several aspects of recombinant Ad vectors, as well as the development of Ad vector, are discussed. Moreover, we focus on those Ads that were used in preclinical and clinical applications including regenerative medicine, vaccine development, genome engineering, treatment of genetic diseases, and virotherapy in tumor treatment.
BACKGROUND
Viral cardiac infection represents a significant clinical challenge encompassing several etiological agents, disease stages, complex presentation, and a resulting lack of mechanistic understanding. Myocarditis is a major cause of sudden cardiac death in young adults, where current knowledge in the field is dominated by later disease phases and pathological immune responses. However, little is known regarding how infection can acutely induce an arrhythmogenic substrate before significant immune responses. Adenovirus is a leading cause of myocarditis, but due to species specificity, models of infection are lacking, and it is not understood how adenoviral infection may underlie sudden cardiac arrest. Mouse adenovirus type-3 was previously reported as cardiotropic, yet it has not been utilized to understand the mechanisms of cardiac infection and pathology.
METHODS
We have developed mouse adenovirus type-3 infection as a model to investigate acute cardiac infection and molecular alterations to the infected heart before an appreciable immune response or gross cardiomyopathy.
RESULTS
Optical mapping of infected hearts exposes decreases in conduction velocity concomitant with increased Cx43Ser368 phosphorylation, a residue known to regulate gap junction function. Hearts from animals harboring a phospho-null mutation at Cx43Ser368 are protected against mouse adenovirus type-3–induced conduction velocity slowing. Additional to gap junction alterations, patch clamping of mouse adenovirus type-3–infected adult mouse ventricular cardiomyocytes reveals prolonged action potential duration as a result of decreased I K1 and I Ks current density. Turning to human systems, we find human adenovirus type-5 increases phosphorylation of Cx43Ser368 and disrupts synchrony in human induced pluripotent stem cell-derived cardiomyocytes, indicating common mechanisms with our mouse whole heart and adult cardiomyocyte data.
CONCLUSIONS
Together, these findings demonstrate that adenoviral infection creates an arrhythmogenic substrate through direct targeting of gap junction and ion channel function in the heart. Such alterations are known to precipitate arrhythmias and likely contribute to sudden cardiac death in acutely infected patients.
Delivering vectorized information into cells with the help of viruses has been of high interest to fundamental and applied science, and bears significant therapeutic promise. Human adenoviruses (HAdVs) have been at the forefront of gene delivery for many years, and the subject of intensive development resulting in several generations of agents, including replication-competent, -defective or retargeted vectors, and recently also helper-dependent (HD), so-called gutless vectors lacking any viral protein coding information. While it is possible to produce HD-AdVs in significant amounts, physical properties of these virus-like particles and their efficiency of transduction have not been addressed. Here, we used single-cell and single virus particle assays to probe the effect of genome length on HAdV-C5 vector transduction. Our results demonstrate that first-generation C5 vectors lacking the E1/E3 regions of the viral genome as well as HD-AdV-C5 particles with a wild type (wt) ∼36 kbp or an undersized double-strand DNA genome are similar to human adenovirus C5 (HAdV-C5) wt regarding attachment to human lung epithelial cells, endocytic uptake, endosome penetration and dependency on the E3 RING ubiquitin ligase Mind Bomb 1 for DNA uncoating at the nuclear pore complex. Atomic force microscopy measurements of single virus particles indicated that small changes in the genome length from 94% to 103% of HAdV-C5 have no major impact on physical and mechanical features of AdV vectors. In contrast, an HD-AdV-C5 with ∼30 kbp genome was slightly stiffer and less heat-resistant than the other particles, despite comparable entry and transduction efficiencies in tissue culture cell lines, including murine alveolar macrophage-like Max-Planck-Institute (MPI)-2 cells. Together, our in vitro studies reinforce the use of HD-AdV vectors for effective single round gene delivery. The results illustrate how physical properties and cell entry behavior of single virus particles can provide functional information for anticipated therapeutic vector applications.
Purpose
To sequence, identify, and perform phylogenetic and recombination analysis on three clinical adenovirus samples taken from the vitreous humor at the Bascom Palmer Eye Institute.
Methods
The PacBio Sequel II was used to sequence the genomes of the three clinical adenovirus isolates. To identify the isolates, a full genome-based multiple sequence alignment (MSA) of 722 mastadenoviruses was generated using multiple alignment using fast Fourier transform (MAFFT). MAFFT was also used to generate genome-based human adenovirus B (HAdV-B) MSAs, as well as HAdV-B fiber, hexon, and penton protein-based MSAs. To examine recombination within HAdV-B, RF-Net 2 and Bootscan software programs were used.
Results
In the course of classifying three new atypical ocular adenovirus samples, taken from the vitreous humor, we found that all three isolates were HAdV-B species. The three Bascom Palmer HAdV-B genomes were then combined with over 300 HAdV-B genome sequences, including nine ocular HAdV-B genome sequences. Attempts to categorize the penton, hexon, and fiber serotypes using phylogeny of the three Bascom Palmer samples were inconclusive due to incongruence between serotype and phylogeny in the dataset. Recombination analysis using a subset of HAdV-B strains to generate a hybridization network detected recombination between nonhuman primate and human-derived strains, recombination between one HAdV-B strain and the HAdV-E outgroup, and limited recombination between the B1 and B2 clades.
Conclusions
The discordance between serotype and phylogeny detected in this study suggests that the current classification system does not accurately describe the natural history and phylogenetic relationships among adenoviruses.
The notion that genetic material could be transferred to a host patient for therapeutic benefit has been around for over half a century. However, it wasn’t until 2017 that the concept became a clinical reality, following the approval of the first CAR-T cell therapy in the US by the FDA and subsequent approval by the E.U. Since the approval of this first product, many more gene therapies have come to market, and the number of products in late-stage clinical trials indicate that gene therapy could become one of the fastest growing sectors in the biopharmaceutical industry. A key aspect for the commercial success of any biopharmaceutical is the ability to economically manufacture the therapeutic product at a large enough scale to meet market demand. To date, manufacturing processes have been able to produce gene therapy viral vectors at the necessary scale to satisfy the demands of clinical studies. However, current biomanufacturing processes will need to be scaled-up and optimized to meet commercial demand, especially for therapies that treat diseases with large patient populations.
Efficient and targeted delivery of a DNA payload is vital for developing safe gene therapy. Owing to the recent success of commercial oncolytic vector and multiple COVID-19 vaccines, adenovirus vectors are back in the spotlight. Adenovirus vectors can be used in gene therapy by altering the wild-type virus and making it replication-defective; specific viral genes can be removed and replaced with a segment that holds a therapeutic gene, and this vector can be used as delivery vehicle for tissue specific gene delivery. Modified conditionally replicative–oncolytic adenoviruses target tumors exclusively and have been studied in clinical trials extensively. This comprehensive review seeks to offer a summary of adenovirus vectors, exploring their characteristics, genetic enhancements, and diverse applications in clinical and preclinical settings. A significant emphasis is placed on their crucial role in advancing cancer therapy and the latest breakthroughs in vaccine clinical trials for various diseases. Additionally, we tackle current challenges and future avenues for optimizing adenovirus vectors, promising to open new frontiers in the fields of cell and gene therapies.
In the past, histological diagnosis of (post-)viral myocarditis was based on the so-called Dallas criteria, which have been criticized because of high interobserver variability and sampling error. Immunohistochemical qualification and quantification of interstitial intramyocardial leucocytes was established and standard values concerning adults were published. Fatal casualties due to a viral myocarditis are rare as far as babies and children are concerned (sudden unexpected death in infancy; SUDI). Cases of sudden unexpected death in the first year of life are frequently regarded as sudden infant death syndrome (SIDS). To diagnose myocarditis when there are only single focal lymphocytic infiltrates in the myocardium, the number of samples taken by autopsy is relevant. But even in babies, immunohistochemical qualification and quantification of interstitial lymphocytes and macrophages can lead to standard values allowing diagnosis of myocarditis. Depending on the course of a viral infection, molecular pathological detection of viral genome in the myocardium is possible to support the diagnosis. Using the mentioned methods gradually, there are more cases of suspected SIDS, which are in fact cases of virus-induced myocarditis as cause of death. Primary enteroviruses (coxsackie viruses) and adenoviruses were found but also Epstein-Barr virus and PVB-19.
Human adenoviruses (HAdV) are widespread pathogens causing usually mild infections. The Species D (HAdV-D) cause gastrointestinal tract infections and epidemic keratoconjunctivitis (EKC). Despite being significant pathogens, knowledge around HAdV-D mechanism of cell infection is lacking. Sialic acid (SA) usage has been proposed as a cell infection mechanism for EKC causing HAdV-D. Here we highlight an important role for SA engagement by many HAdV-D. We provide apo state crystal structures of 7 previously undetermined HAdV-D fiber-knob proteins, and structures of HAdV-D25, D29, D30 and D53 fiber-knob proteins in complex with SA. Biologically, we demonstrate that removal of cell surface SA reduced infectivity of HAdV-C5 vectors pseudotyped with HAdV-D fiber-knob proteins, whilst engagement of the classical HAdV receptor CAR was variable. Our data indicates variable usage of SA and CAR across HAdV-D. Better defining these interactions will enable improved development of antivirals and engineering of the viruses into refined therapeutic vectors.
Adenoviruses (AdVs) are being developed for oncolytic or vaccination therapy against existing and emerging conditions. Well-characterized replication-competent human and human primate AdVs expressing multiple payloads are desirable, but their replication in rodent models is limited. To score the timing of adenoviral gene expression in cell cultures, we developed fully replication-competent transcriptional reporter viruses for HAdV-C5, -B3 and -B35. The picornavirus-derived 2A sequence, which induces cotranslational peptide splitting and reinitiation (skipping), was linked to GFP and the fused sequence was inserted C-terminal of the early gene E1A, the intermediate early gene protein IX and the late fiber gene. The 2A peptide induced ribosomal skipping during translation of the mRNA and gave rise to GFP from the corresponding viral promoters, as shown by immunoblotting and flow cytometry analyses of human and rodent cells. In human cells, both species B and C AdV exhibited highest reporter expression for fiber, followed by protein IX and lowest for E1A. Inoculation with either HAdV-C5 or -B3/35 viruses encoding protein IX- or fiber-GFP gave rise to higher GFP levels in hamster than mouse cells. Remarkably, despite rather low 2A ribosomal skipping efficiency of ∼50% for E1A-2A-GFP, protein IX-2A-GFP and fiber-2A-GFP, unprocessed protein IX-2A-GFP and fiber-2A-GFP fusion proteins were efficiently incorporated into HAdV-B3 virions, respectively. These data indicate that the B3 C-termini of protein IX and fiber can be considered for retargeting engineered oncolytic or vaccination vectors, or for antigen display. The variable expression levels of transgenes from different subviral promoters may be used to improve oncolytic AdV vectors expressing therapeutic genes.
Fibroblast activation protein (FAP) is a cell surface serine protease that is highly expressed on reactive stromal fibroblasts, such as cancer-associated fibroblasts (CAFs), and generally absent in healthy adult tissues. FAP expression in the tumor stroma has been detected in more than 90% of all carcinomas, rendering CAFs excellent target cells for a tumor site-specific adenoviral delivery of cancer therapeutics. Here, we present a tropism-modified human adenovirus 5 (Ad5) vector that targets FAP through trivalent, designed ankyrin repeat protein (DARPin)-based retargeting adapters. We describe the development and validation of these adapters via cell-based screening assays and demonstrate adapter-mediated Ad5 retargeting to FAP+ fibroblasts in vitro and in vivo. We further show efficient in vivo delivery and in-situ production of a therapeutic payload by CAFs in the tumor microenvironment (TME), resulting in attenuated tumor growth. We thus propose using our FAP-Ad5 vector to convert CAFs into a 'biofactory', secreting encoded cancer therapeutics into the TME to enable a safe and effective cancer treatment.
Vaccines are instrumental tools to fight against novel and re-emerging pathogens and curb pandemics. Vaccination has been an integral part of the multifaceted public health response to the COVID-19 pandemic. Diverse vaccine platforms have been designed and are currently at different stages of development. Some vaccines are still in early biological testing, while others have been launched after being approved by regulatory agencies worldwide. Genomic vaccines that deliver parts of the viral DNA or RNA to host cells have gained popularity recently due to their high efficiency and fast manufacture. Furthermore, recent clinical studies encouraged the use of different vaccine platforms within the primary vaccination course to enhance the efficacy of vaccination. Herein, we discuss COVID-19 genomic vaccines, which deliver viral genetic material to host cells through diverse biotechnology platforms, including viral vector vaccines, messenger RNA nucleic acid vaccines, and DNA nucleic acid vaccines. We compare and contrast vaccine characteristics, composition, and pros and cons among different genomic vaccine platforms as well as non-genomic vaccines. This review summarizes all current knowledge about COVID-19 genomic vaccines, which could be highly valuable to researchers interested in public health and vaccine development.
Interferon-γ (IFN-γ) is well known to reduce the infectivity of viral pathogens by altering their tissue tropism. This effect is induced by upregulation of cholesterol 25-hydroxylase (CH25H). Given the similarity of viral pathogens and ligand-functionalized nanoparticles in the underlying strategy of receptor-mediated cell recognition, it appears conceivable that IFN-γ exceeds similar effects on nanoparticles. Concretely, IFN-γ-induced activation of CH25H could decrease nanoparticle avidity for target cells via depletion of clathrin-coated pits. We hypothesized that this effect would cause deterioration of target-cell specific accumulation of nanoparticles. To prove our hypothesis, we investigated the cell tropism of angiotensin II functionalized nanoparticles (NPLys-Ang II) in a co-culture system of angiotensin II subtype 1 receptor (AT1R) positive rat mesangial target cells (rMCs) and AT1R-negative HeLa off-target cells. In the presence of IFN-γ we observed an up to 5-fold loss of target cell preference for NPLys-Ang II. Thus, our in vitro results suggest a strong influence of IFN-γ on nanoparticle distribution, which is relevant in the context of nanotherapeutic approaches to cancer treatment, as IFN-γ is strongly expressed in tumors. For the target cell tropism of viruses, our results provide a conclusive hypothesis for the underlying mechanism behind non-directed viral distribution in the presence of IFN-γ.
A prospective study of 116 patients with acute pancreatitis included routine screening for evidence of viral infection. Five patients (all female) exhibited significant rising antibody titres to Coxsackie B or mumpsvirus, while none of the remaining 111 patients did. Diarrhoea was a prodromal feature of the pancreatitis in those patients with evidence of viral disease. Screening patients with acute pancreatitis for Coxsackie B and mumpsvirus infections is worthwhile in the identification of aetiological factors and may minimise protracted biliary investigations. The incidence of "idiopathic" acute pancreatitis in this study was 5-2% (six patients).
We examined the different steps necessary for the enzymatic digestion of proteins in the polyacrylamide matrix after gel electrophoresis. As a result, we developed an improved method for obtaining peptides for internal sequence analysis from 1-2 micrograms of in-gel-digested proteins. The long washing-lyophilization-equilibration steps necessary to eliminate the dye, sodium dodecyl sulfate, and other gel-associated contaminants that perturb protein digestion in Coomassie blue-stained gels have been replaced by washing for 40 min with 50% acetonitrile, drying for 10 min at room temperature, and then rehydrating with a protease solution. The washing and drying steps result in a substantial reduction of the gel slice volume that, when next swollen in the protease solution, readily absorbs the enzyme, facilitating digestion. The Coomassie blue staining procedure has also been modified by reducing acetic acid and methanol concentrations in the staining solution and by eliminating acetic acid in the destaining solution. The peptides resulting from the in-gel digestion are easily recovered by passive elution, in excellent yields for structural characterization. This simple and rapid method has been successfully applied for the internal sequence analysis of membrane proteins from the rat mitochondria resolved in preparative two-dimensional gel electrophoresis.
5'-Noncoding sequences have been compiled from 699 vertebrate mRNAs. (GCC)GCCAGCCATGG emerges as the consensus sequence for initiation of translation in vertebrates. The most highly conserved position in that
motif is the purine in position -3 (three nucleotides upstream from the ATG codon); 97% of vertebrate mRNAs have a purine,
most often A, in that position- The periodical occurrence of G (in positions −3, −6, −9) is discussed. Upstream ATG codons
occur in fewer than 10% of vertebrate mRNAs-at-large; a notable exception are oncogene transcripts, two-thirds of which have
ATG codons preceding the start of the major open reading frame. The leader sequences of most vertebrate mRNAs fall in the
size range of 20 to 100 nucleotides. The significance of shorter and longer 5' -noncoding sequences is discussed.
HeLa or KB cells each contain around 10(4) receptor sites for adenovirus type 2. These are inactivated by treatment of live cells with subtilisin. The receptor activity of the enzyme-treated cells is regained after 4 to 8 hr of incubation in complete medium. A technique that utilizes the difference in buoyant density between free virus and virus-receptor complex was developed to demonstrate receptor activity. Cellular fractionation revealed that receptors were confined mainly to the plasma membrane fraction and that negligible receptor activity could be demonstrated in enzyme-treated cells. Subtilisin probably did not penetrate the cell membrane; thus, the receptors are limited to the cell surface. Purified fiber of the virion completely prevents attachment of adenovirus types 2 and 5 to receptor sites at a ratio of 10(5) protein molecules per cell. Adsorption studies indicate that 10(5) to 10(6) receptor sites are available for the structural protein. The fiber does not affect attachment of poliovirus type 1.
A coxsackievirus B3 (CB3) isolate adapted to growth in RD cells shows an alteration in cell tropism as a result of its capacity to bind a 70-kDa cell surface molecule expressed on these cells. We now show that this molecule is the complement regulatory protein, decay-accelerating factor (DAF) (CD55). Anti-DAF antibodies prevented CB3 attachment to the cell surface. Radiolabeled CB3 adapted to growth in RD cells bound to CHO cells transfected with human DAF, whereas CB3 (strain Nancy), the parental strain, did not bind to DAF transfectants. These results indicate that growth of CB3 in RD cells selected for a virus strain that uses DAF for cell surface attachment.
The adenovirus fiber protein is used for attachment of the virus to a specific receptor on the cell surface. Structurally, the protein consists of a long, thin shaft that protrudes from the vertex of the virus capsid and terminates in a globular domain termed the knob. To verify that the knob is the domain which interacts with the cellular receptor, we have cloned and expressed the knob from adenovirus type 5 together with a single repeat of the shaft in Escherichia coli. The protein was purified by conventional chromatography and functionally characterized for its interaction with the adenovirus receptor. The recombinant knob domain bound about 4,700 sites per HeLa cell with an affinity of 3 x 10(9) M-1 and blocked adenovirus infection of human cells. Antibodies raised against the knob also blocked virus infection. By gel filtration and X-ray diffraction analysis of protein crystals, the knob was shown to consist of a homotrimer of 21-kDa subunits. The results confirm that the trimeric knob is the ligand for attachment to the adenovirus receptor.
Anti-VLA-2 antibodies protected HeLa cells from infection by echoviruses 1 and 8 but not from infection by other echovirus serotypes. Echoviruses 1 and 8 bound to and infected nonpermissive hamster cells transfected with the alpha 2 subunit of human VLA-2. These results indicate that the human alpha 2 subunit is critical for infection by echoviruses 1 and 8 but that other echovirus serotypes must bind receptors other than VLA-2.
In this review we describe current strategies for adenoviral mediated gene transfer (AMGT) and adeno-associated viral mediated gene transfer (AAVMGT). We consider the structure and molecular biology of adenoviruses and adeno-associated viruses and detail the current advantages and disadvantages of AMGT and AAVMGT. Potential solutions to some of the specific drawbacks to AMGT, including the development of new vectors, addition of gp19k, organoides, and the use of non-human adenoviral vectors, are discussed.
A healthy 10-year-old boy was admitted to the hospital in diabetic ketoacidosis within three days of onset of symptoms of a flu-like illness. He died seven days later and post-mortem examination showed lymphocytic infiltration of the islets of Langerhans and necrosis of beta cells. Inoculation of mouse, monkey and human cell cultures with homogenates from the patient's pancreas led to isolation of a virus. Serologic studies revealed a rise in the titer of neutralizing antibody to this virus from less than 4 on the second hospital day to 32 on the day of death. Neutralization data showed that the virus was related to a diabetogenic variant derived from Coxsackievirus B4. Inoculation of mice with the human isolate produced hyperglycemia, inflammatory cells in the islets of Langerhans and beta-cell necrosis. Staining of mouse pancreatic sections with fluorescein-labeled antiviral antibody revealed viral antigens in beta cells. Both the clinical picture and animal studies suggested that the patient's...
The amino acid sequence of the fiber from adenovirus type 5 has been deduced from the nucleotide sequence of the fiber gene. This sequence is compared with that of adenovirus type 2, a closely related serotype. We find 69% homology for the fiber protein whereas the known nonstructural proteins of these two serotypes have 99% sequence homology. A detailed sequence analysis was performed in the context of the model proposed by Green et al., [(1983), EMBO J., 8, 1357–1365] for the folding of the polypeptide chain of the adenovirus type 2 fiber. The N-terminal region, which in the virion is associated with the capsid, is identical for these two serotypes. In the shaft of the fiber the features, such as periodicity of the prolines and of the hydrophobic residues in the amino acid sequence, on which the model for the adenovirus type 2 is based are very well preserved in adenovirus type 5. On the other hand, there are large differences all along the sequence of the shaft of the fiber showing that there is a very limited homology between the amino acids in the two serotypes when they do not have a key role in establishing the structure. In the knob the homology between serotypes is 64%. These results are consistent with the differences between serotypes being confined to the exposed proteins.
THE specific receptors for several non-enveloped viruses are present on cells in only limited numbers (1 × 104-10 × 104), and can therefore be saturated with excess virus1-5. An excess of inactivated poliovirus type 1 inhibits the attachment of infectious poliovirus of all 3 serotypes, but not the attachment of other viruses, including B Coxsackie viruses6. Most of the B Coxsackie viruses probably share the same receptor7, but Coxsackie viruses B1 and B3 do not compete for the poliovirus receptor1. Adenovirus 2 and 5 also appear to share the same receptor, since their attachment to host cells is blocked by an excess of the adenovirus type 2 fibre protein, which seems to be the receptor-recognising portion of the virus particle2. A number of other adenoviruses may also belong to the same receptor family since soluble antigens block their attachment to erythrocytes8. We here report results which permit the construction of a `receptor map' for a number of non-enveloped viruses.
A healthy 10-year-old boy was admitted to the hospital in diabetic ketoacidosis within three days of onset of symptoms of a flu-like illness. He died seven days later and post-mortem examination showed lymphocytic infiltration of the islets of Langerhans and necrosis of beta cells. Inoculation of mouse, monkey and human cell cultures with homogenates from the patient's pancreas led to isolation of a virus. Serologic studies revealed a rise in the titer of neutralizing antibody to this virus from less than 4 on the second hospital day to 32 on the day of death. Neutralization data showed that the virus was related to a diabetogenic variant derived from Coxsackievirus B4. Inoculation of mice with the human isolate produced hyperglycemia, inflammatory cells in the islets of Langerhans and beta-cell necrosis. Staining of mouse pancreatic sections with fluorescein-labeled antiviral antibody revealed viral antigens in beta cells. Both the clinical picture and animal studies suggested that the patient's diabetes was virus induced.
Cell surface receptors for echovirus, a common human pathogen, were identified with monoclonal antibodies that protected susceptible
cells from infection with echovirus 1. These monoclonal antibodies, which prevented virus attachment to specific receptor
sites, recognized the alpha and beta subunits of the integrin VLA-2 (alpha 2 beta 1), a receptor for collagen and laminin.
RD rhabdomyosarcoma cells expressed little VLA-2, did not bind to 35S-labeled virus, and resisted infection until transfected
with complementary DNA encoding the alpha 2 subunit of VLA-2. Thus, integrins, adhesion receptors important in interactions
between cells and with the extracellular matrix, can mediate virus attachment and infection.
A 50-kilodalton receptor protein (Rp-a) for the group B coxsackieviruses (CB) was isolated in a virus-receptor complex from detergent-solubilized HeLa cells (J. E. Mapoles, D. L. Krah, and R. L. Crowell, J. Virol. 55:560-566, 1985). It was used as an immunogen for preparation of a mouse monoclonal antibody (RmcB) which protected HeLa cells and Buffalo green monkey kidney cells from infection by all six serotypes of CB. RmcB did not protect HeLa cells from infection by poliovirus, echovirus 6, or coxsackievirus A18. This monoclonal antibody differed in receptor epitope specificity from a previously isolated antibody (RmcA) (R. L. Crowell, A. K. Field, W. A. Schleif, W. L. Long, R. J. Colonno, J. E. Mapoles, and E. A. Emini, J. Virol. 57:438-445, 1986) which blocked receptors only for type 1 CB (CB1), CB3, CB5, and echovirus 6. RmcA and RmcB recognized two distinct saturable receptors on HeLa cells, designated HR2 and HR1, respectively. Human rhabdomyosarcoma (RD) cells have the HR2 receptor for CB3-RD (a variant of CB3), but lack the HR1 receptor for CB3. Therefore, RD cells were resistant to infection by CB3. Although binding of CB3-RD to the HR2 receptor on RD cells can lead to infection, binding of CB3-RD to the HR2 receptor on HeLa cells did not lead to infection. Apparently, both CB3 and CB3-RD use only the HR1 receptor for infection of HeLa cells. Thus, a given virus may use two distinct receptors to bind to cells when only one virus-receptor interaction leads to infection.
Coxsackievirus B3 (CB3) firmly attaches to HeLa cells, forming a specific complex between the virus and its receptor on the cell surface. We extracted this virus-receptor complex (VRC) with the detergents sodium deoxycholate and Triton X-100. The VRC was identified by its sedimentation coefficient (140S), which was less than that of virions (155S). Formation of the VRC from cell lysates and 3H-CB3 occurred with the same specificity as did attachment of virions to cells, in that its formation was blocked by unlabeled CB3 but not by poliovirus. The VRC was purified 30,000-fold by differential and sucrose gradient centrifugation. Iodination with Na125I revealed that the purified VRC consisted of the normal CB3 proteins and one additional protein (RP-a) with an approximate molecular weight of 49,500. RP-a was eluted from the VRC and was shown to rebind with CB3 and CB1 virions but not with poliovirus type 1. We propose that Rp-a is a protein in the plasma membrane receptor complex which is responsible for the specific recognition and binding of the group B coxsackieviruses.
We have found that the replication of human adenovirus (Ad2) is restricted in multiple Chinese hamster cell lines including CHO and V79. The major site of restriction involves differential accumulation of late viral proteins as demonstrated by immunofluorescence assay and polyacrylamide gel electrophoresis with and without prior immunoprecipitation. Synthesis of fiber and penton base are markedly reduced, whereas others, such as the 100K polypeptide, are synthesized efficiently. This pattern of restriction is similar to that previously reported for Ad2 infection of several monkey cell lines; however, the restriction is more marked in the Chinese hamster cell lines. The restriction is most likely due to a deficient cellular function since stable cell hybrids between V79 or CHO and human cells are permissive for virus replication. By analysis of a series of hybrids with reduced numbers of human chromosomes, fiber synthesis was correlated with the presence of the short arm of human chromosome 3. More hybrids showed restoration of fiber synthesis than production of progeny virus, suggesting that more than one unlinked function is required for the latter.
The adenovirion has been shown to contain at least nine different polypeptides demonstrable by dissociation and acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS). Each polypeptide of adenovirus type 2 is chemically distinct by isotopic ratio analysis, and they all contain lysine, arginine, tryptophan, valine, and threonine. The molecular weights of these polypeptides determined by gel electrophoresis range from 120,000 for the largest and most prominent component to 7500 for the smallest. Comparison of nontumorigenic type 2 with tumorigenic types 7A and 12 by double-isotope labeling revealed a generally similar peptide pattern for all types. However, there were distinct differences between the corresponding peptides of all three types. These results imply extensive differences in the genes for most of the capsid proteins.
Chinese hamster ovary (CHO) cells infected with adenovirus type 2 (Ad2) produced amounts of viral deoxyribonucleic acid (DNA)
equal to that synthesized in permissively infected HeLa cells. However, there was 6,000-fold less virion produced in CHO cells.
Since the structural viral polypeptides were not detected by pulse-labeling CHO cells at various times postinfection, the
block in virion formation is located between the synthesis of viral DNA and late proteins. Extracts of CHO cells could also
function in a recently reported in vitro Ad2 DNA synthesis system which is dependent upon the addition of exogenous Ad2 DNA
covalently linked to a 5'-terminal protein (Ikeda et al., Proc. Natl. Acad. Sci. U.S.A. 77:5827-5831, 1980). Extracts of infected
CHO cytoplasm were able to complement uninfected CHO nuclear extracts to synthesize viral DNA on Ad2 templates. This in vitro
replication system has the potential to probe host DNA synthesis requirements as well as viral factors.
The penton base gene from adenovirus type 12 (Ad12) was sequenced and encodes a 497-residue polypeptide, 74 residues shorter than the penton base from Ad2. The Ad2 and Ad12 proteins are highly conserved at the amino- and carboxy-terminal ends but diverge radically in the central region, where 63 residues are missing from the Ad12 sequence. Conserved within this variable region is the sequence Arg-Gly-Asp (RGD), which, in the Ad2 penton base, binds to integrins in the target cell membrane, enhancing the rate or the efficiency of infection. The Ad12 penton base was expressed in Escherichia coli, and the purified refolded protein assembled in vitro with Ad2 fibers. In contrast to the Ad2 penton base, the Ad12 protein failed to cause the rounding of adherent cells or to promote attachment of HeLa S3 suspension cells; however, A549 cells did attach to surfaces coated with either protein and pretreatment of the cells with an integrin alpha v beta 5 monoclonal antibody reduced attachment to background levels. Treatment of HeLa and A549 cells with integrin alpha v beta 3 or alpha v beta 5 monoclonal antibodies or with an RGD-containing fragment of the Ad2 penton base protein inhibited infection by Ad12 but had no effect on and in some cases enhanced infection by Ad2. Purified Ad2 fiber protein reduced the binding of radiolabeled Ad2 and Ad12 virions to HeLa and A549 cells nearly to background levels, but the concentrations of fiber that strongly inhibited infection by Ad2 only weakly inhibited Ad12 infection. These data suggest that alpha v-containing integrins alone may be sufficient to support infection by Ad12 and that this pathway is not efficiently used by Ad2.
Infection with Coxsackie B viruses has been linked to insulin-dependent diabetes mellitus. Nine of 14 serum samples (64%) taken from children at the onset of diabetes were positive for enterovirus RNA by PCR. All of the children were under age six, and five were under age three. By contrast, enterovirus sequences were detected in only two of 45 serum samples from appropriate comparison children (4%). Sequences from six of the positive patients showed strong homology with Coxsackie B3 and B4 viruses, and there were some common patterns among the sequences from infected diabetic children. This is evidence for a role for enteroviruses in childhood diabetes.
The diagnosis of viral myocarditis remains difficult and generally depends on clinical and histological criteria. Viral cultures and serology are often unrewarding, with low yields. The purpose of this study was to analyze the usefulness of polymerase chain reaction (PCR) in the rapid diagnosis of acute myocarditis in children.
PCR was used to analyze 38 myocardial tissue samples from 34 patients with suspected acute viral myocarditis and 17 control patients with congenital heart disease (14) or hypertrophic cardiomyopathy (3). Myocardial samples were obtained at the time of right ventricular biopsy (13 samples), from explanted hearts (18 samples) at transplantation, and from cardiac autopsy specimens (24 samples) and were evaluated for the presence of enterovirus, cytomegalovirus (CMV), adenovirus, and herpes simplex virus (HSV) using PCR primers designed to consensus and unique sequences of these viral genomes. Blood also was obtained at the time of biopsy (11) or transplant (18). In 26 of 38 myocardial samples (68%), viral genome was detected by PCR (15 adenoviral, 8 enteroviral, 2 HSV, 1 CMV), whereas all control myocardial samples and blood samples were negative. Four patients had positive viral cultures, and these matched the PCR findings. Disagreement with histopathology occurred in 13 of 26 PCR-positive specimens, usually associated with adenovirus.
PCR offers a rapid, sensitive diagnostic method for myocardial viral infection. While enterovirus is an important etiological agent, adenovirus was more prevalent in this series and should be evaluated when etiology is sought. PCR used in conjunction with standard endomyocardial biopsy appears to enhance the likelihood of detecting viral genome in the myocardium of patients with clinical evidence of myocarditis.
Group B coxsackieviruses (CVBs) are etiologic agents of a number of human diseases that range in severity from asymptomatic to lethal infections. They are small, single-stranded RNA icosahedral viruses that belong to the enterovirus genus of the picornavirus family. Structural studies were initiated in light of the information available on the cellular receptors for this virus and to assist in the design of antiviral capsid-binding compounds for the CVBs.
The structure of coxsackievirus B3 (CVB3) has been solved to a resolution of 3.5 A. The beta-sandwich structure of the viral capsid proteins VP1, VP2 and VP3 is conserved between CVB3 and other picornaviruses. Structural differences between CVB3 and other enteroviruses and rhinoviruses are located primarily on the viral surface. The hydrophobic pocket of the VP1 beta-sandwich is occupied by a pocket factor, modeled as a C16 fatty acid. An additional study has shown that the pocket factor can be displaced by an antiviral compound. Myristate was observed covalently linked to the N terminus of VP4. Density consistent with the presence of ions was observed on the icosahedral threefold and fivefold axes.
The canyon and twofold depression, major surface depressions, are predicted to be the primary and secondary receptor-binding sites on CVB3, respectively. Neutralizing immunogenic sites are predicted to lie on the extreme surfaces of the capsid at sites that lack amino acid sequence conservation among the CVBs. The ions located on the icosahedral threefold and fivefold axes together with the pocket factor may contribute to the pH stability of the coxsackieviruses.
Cytosine arabinoside (ara-C) is a cytidine analog that incorporates into replicating DNA and induces lethal DNA strand breaks. Although ara-C is a potent antitumor agent for hematologic malignancies, it has only minimal activity against most solid tumors. The rate-limiting step in intracellular ara-C activation is phosphorylation of the prodrug by deoxycytidine kinase (dCK). The present results demonstrate that both retroviral and adenoviral vector-mediated transduction of the dCK cDNA results in marked sensitization of glioma cells lines to the cytotoxic effects of ara-C in vitro. We also demonstrate that ara-C treatment of established intradermal and intracerebral gliomas transduced with dCK results in significant antitumor effects in vivo. These data suggest that viral vector transduction of the dCK gene followed by treatment with ara-C represents a new chemosensitization strategy for cancer gene therapy.
Viral envelope (Env)-receptor interactions have been implicated in the cell death associated with infection by subgroups B and D avian leukosis-sarcoma viruses (ALVs). A chicken protein, CAR1, was identified that permitted infection of mammalian cells by these viral subgroups. CAR1 bound to a viral Env fusion protein, comprising an ALV-B surface Env protein and the Fc region of an immunoglobulin, indicating that it is a specific viral receptor. CAR1 contains two extracellular cysteine-rich domains characteristic of the TNFR family and a cytoplasmic region strikingly similar to the death domain of TNFR1 and Fas, implicating this receptor in cell killing. Chicken embryo fibroblasts susceptible to ALV-B infection and transfected quail QT6 cells expressing CAR1 underwent apoptosis in response to the Env-Ig fusion protein, demonstrating that this cytopathic ALV receptor can mediate cell death.
The receptors on human cells which mediate adsorption of adenoviruses have not been identified. We found that murine A9 cells and Chinese hamster ovary (CHO) cells failed to bind significant levels of radiolabeled adenovirus type 2 (Ad2) virions but that derivatives of these cells carrying human chromosome 21 exhibited high levels of virus binding that was specific for the viral fiber protein. G418-resistant A9 cell transformants expressing Ad2 receptors were detected at a frequency of about 10(-4) following cotransfection with high-molecular-weight DNAs from mouse cells containing human chromosome 21 and plasmid DNA containing a neomycin resistance gene. The Ad2 receptors on the transformed A9 cells were similar to those on human cells with respect to their concentration on the cell membrane, their affinity for the viral fiber protein, and their ability to direct virus into cells along a pathway leading to delivery of the viral DNA genome into the cell nucleus. Furthermore, identical human DNA fragments were present in three independent mouse cell transformants expressing Ad2 receptors, supporting the conclusion that these human DNA fragments correspond to a gene or locus on chromosome 21 that directs the expression of Ad2 receptors in these cells.
Adenovirus contains a heterodimeric protein complex consisting of 186 kd fiber protein that mediates high affinity virus attachment to cells and a 400 kd pentavalent subunit (penton base) that contains five Arg-Gly-Asp sequences, implying a role for integrins in adenovirus infection. We demonstrate that the vitro-nectin-binding integrins alpha v beta 3 and alpha v beta 5 promote viral infection in a novel way since antibodies against these receptors or soluble penton base block virus internalization without affecting attachment. Moreover, adenovirus binds to cultured cells lacking alpha v integrins but fail to become internalized, thus restricting infection of these cells. Transfection of alpha v(-) cells with a cDNA encoding alpha v results in the expression of integrins alpha v beta 3 and alpha v beta 5 and allows virus internalization and infection. These data indicate that adenovirus attachment and uptake into cells are separate but cooperative events that result from the interaction of distinct viral coat proteins with a receptor for attachment and alpha v integrin receptors for internalization.
A corresponding expressed sequence tag (EST) cDNA (emb F05145; IMAGE Consortium Clone ID 25001) isolated from an infant brain library
- G G Lennon
- C Auffray
- M Polymeropoulos
The protein bands were excised. After in-gel digestion with trypsin the sequences of four peptides were determined at the Harvard Microchemistry Facility by collisionally activated dissociation on a Finnigan
EMBL) accession number Y10320] was obtained from a mouse liver cDNA library (Gibco- BRL)
- Car Murine
- Cdna
Adenovirus fibers were purified from the top 10 ml of the CsCl gradients by chromatography on Mono Q and Mono S ion exchange columns (Pharmacia)
- J V D O Maizel
- M D White