Eric Poeschla

University of Colorado | UCD · Section of Infectious Diseases

The COVID-19 pandemic demonstrated the poor ability of body temperature to reliably identify SARS-CoV-2-infected individuals, an observation that has been made before in the context of other infectious diseases. While acute infection does not always cause fever, it does reliably drive host transcriptional responses as the body responds at the site...

The study of species- and virus-specific differences in restriction factors and their antagonists has been central to deciphering the nature of these key host defenses. FIV is an AIDS-causing lentivirus that has achieved pandemic spread in the domestic cat.

Allosteric integrase (IN) inhibitors (ALLINIs), which are promising preclinical compounds that engage the lens epithelium-derived growth factor (LEDGF)/p75 binding site on IN, can inhibit different aspects of human immunodeficiency virus 1 (HIV-1) replication. During the late phase of replication, ALLINIs induce aberrant IN hyper-multimerization, t...

The emergence of SARS-CoV-2 variants with enhanced transmissibility, pathogenesis, and resistance to vaccines presents urgent challenges for curbing the COVID-19 pandemic. While Spike mutations that enhance virus infectivity or neutralizing antibody evasion may drive the emergence of these novel variants, studies documenting a critical role for int...

A multimer of retroviral integrase (IN) synapses viral DNA ends within a stable intasome nucleoprotein complex for integration into a host cell genome. Reconstitution of the intasome from the maedi-visna virus (MVV), an ovine lentivirus, revealed a large assembly containing sixteen IN subunits. Herein, we report cryo-EM structures of the lentiviral...

Antiviral strategies that target host systems needed for SARS‐CoV‐2 replication and pathogenesis may have therapeutic potential and help mitigate resistance development. Here, we evaluate nafamostat mesylate, a potent broad‐spectrum serine protease inhibitor that blocks host protease activation of the viral spike protein. SARS‐CoV‐2 is used to infe...

Early events of the human immunodeficiency virus 1 (HIV-1) lifecycle, such as post-entry virus trafficking, uncoating and nuclear import, are poorly characterized because of limited understanding of virus–host interactions. Here, we used mass spectrometry-based proteomics to delineate cellular binding partners of curved HIV-1 capsid lattices and id...

Attacking HIV by stabilizing its capsid Current HIV treatments require drugs that must be taken daily, and care would be improved with an effective drug that is long-acting. GS-6207 (Lenacapavir) is a drug developed by Gilead Sciences that shows potential for a 6-month dosing interval and is in phase 2/3 clinical trials. Bester et al. describe stru...

Jails and prisons are exceptionally susceptible to viral outbreaks, such as severe acute respiratory syndrome coronavirus 2. The USA has extremely high rates of incarceration and COVID-19 is causing an urgent health crisis in correctional facilities and detention centres. Epidemics happening in prisons are compounding the elevated risks that COVID-...

Integration is the defining step of the retroviral life cycle and underlies the inability to cure HIV/AIDS through the use of intensified antiviral therapy. The reservoir of latent, replication-competent proviruses that forms early during HIV infection reseeds viremia when patients discontinue medication. HIV cure research is accordingly focused on...

The COVID-19 pandemic suggests that bat innate immune systems are insufficiently characterized relative to the medical importance of these animals. Retroviruses, e.g., HIV-1, can be severe pathogens when they cross species barriers, and bat restrictions corresponding to retroviruses are comparatively unstudied. Here, we compared the abilities of re...

Pathogen-associated molecular patterns, e.g. dsRNA, activate expression of interferon-stimulated genes (ISGs), which protects hosts from infection. While transient ISG upregulation is essential for effective innate immunity, constitutive activation typically causes harmful autoimmunity in mice and humans, often including severe developmental abnorm...

Immune control of retroviruses is notoriously difficult, a fundamental problem that has been most clinically consequential with the HIV-1 pandemic. As humans expand further into previously uninhabited areas, the likelihood of new zoonotic retroviral exposures increases. The role of the innate immune system, including ISGs, in controlling retroviral...

Pathogen-associated molecular patterns (e.g., dsRNA) activate expression of IFN-stimulated genes (ISGs), which protect hosts from infection. Although transient ISG upregulation is essential for effective innate immunity, constitutive activation typically causes harmful autoimmunity in mice and humans, often including severe developmental abnormalit...

In North America, hantaviruses commonly cause hantavirus pulmonary syndrome (HPS). Clinical descriptions of hantavirus-associated renal disease in the Americas are scarce. Herein, we discuss the case of a 61-year-old man whose predominant manifestations were acute kidney injury and proteinuria. Clinical recognition of renal signs in hantavirus infe...

Overexpression and long terminal repeat (LTR) polymorphism of the HRES-1/Rab4 human endogenous retrovirus locus have been associated with T-cell activation and disease manifestations in systemic lupus erythematosus (SLE). Although genomic DNA methylation is overall diminished in SLE, its role in HRES-1/Rab4 expression is unknown. Therefore, we dete...

RNA viruses are a major source of emerging and re-emerging infectious diseases around the world. We developed a method to identify RNA viruses that is based on the fact that RNA viruses produce double-stranded RNA (dsRNA) while replicating. Purifying and sequencing dsRNA from the total RNA isolated from infected tissue allowed us to recover dsRNA v...

RNA viruses are a major source of emerging and re-emerging infectious diseases around the world. We developed a method to identify RNA viruses that is based on the fact that all RNA viruses produce dsRNA while replicating. Purifying and sequencing dsRNA from total RNA isolated from infected tissue allowed us to recover replicating viral sequences....

A 23-year-old recent emigrant from Afghanistan presented in August 2017 with severe wheezing and dyspnea that required hospital admission. Her illness was associated with marked peripheral blood eosinophilia (9,900-15,600/µL; 45.2-68%), as well as mild nausea, epigastric pain, and decreased appetite. She had lived until 3 months earlier in close pr...

HIV-1 integration into the host genome favors actively transcribed genes. Prior work indicated that the nuclear periphery provides the architectural basis for integration site selection, with viral capsid-binding host cofactor CPSF6 and viral integrase-binding cofactor LEDGF/p75 contributing to selection of individual sites. Here, by investigating...

Much remains unknown about how TREX1 influences HIV-1 replication: whether it targets full-length viral DNA versus partial intermediates, how intracellular TREX1 protein levels correlate with ISG induction, and whether TREX1 digestion of cytoplasmic DNA and subsequent cGAS pathway activation affects both initial and subsequent cycles of infection....

Significance HIV-1 requires integration for efficient gene expression, and the local chromatin environment significantly influences the level of HIV-1 transcription. Silent, integrated proviruses constitute the latent HIV reservoir. As HIV-1 commandeers cellular factors to dictate its preferred integration sites, these interactions consequentially...

For many emerging and re-emerging infectious diseases, definitive solutions via sterilizing adaptive immunity may require years or decades to develop, if they are even possible. The innate immune system offers alternative mechanisms that do not require antigen-specific recognition or a priori knowledge of the causative agent. However, it is unclear...

For many emerging and re-emerging infectious diseases, definitive solutions via sterilizing adaptive immunity may require years or decades to develop, if they are even possible. The innate immune system offers alternative mechanisms that do not require antigen-specific recognition or a priori knowledge of the causative agent. However, it is unclear...

RdRP mouse tissues express endogenous dsRNA. Expression of endogenous dsRNA (biotinylated K1 antibody, peroxidase stain) in brains of uninfected WT and RdRP mice. Slides were photographed using an Olympus DP73 camera attached to an Olympus AX70 microscope (left panels 50 μm, right 20 μm). (TIF)

List of genes upregulated in virally-infected wildtype mice. Differential gene expression in spinal cords from wildtype (WT) mice infected with EMCV (n = 2) compared to uninfected WT mice (n = 2). Gene chip was analyzed as described in methods. Only genes with a fold change in expression >4.0 and a p-value of <0.05 are shown. There were no genes si...

List of genes differentially expressed in RdRP THP-1 cells. Differential gene expression in THP-1 cells expressing the RdRP transgene (n = 2) compared to THP-1 empty vector control cells (n = 2). Gene chip was analyzed as described in methods. Only genes with a fold change in expression >4.0 or <-4.0 and a p-value of <0.05 are shown. References to...

RdRP mice inherently express antiviral effectors. (A) Venn diagram summary of murine microarray analyses. Each treatment group (WT+EMCV, uninfected RdRP, or RdRP+EMCV) was compared to the respective uninfected WT control group included in that experiment. Orange: gene upregulated >4-fold in the WT+EMCV treatment group only. Red: genes upregulated >...

RdRP-induced ISG inductions are mediated in a MDA5-MAVS, IFNαβR-dependent manner. (A-E) Knockout mice were bred to RdRP mice and the antiviral ISGs, Oasl2, Isg15, Ifit1, and Rig-I, were analyzed by RT-PCR in CNS tissues of uninfected mice. Mice used were directly compared with sibling controls (n = 5 per genotype, relative to Gapdh mRNA, mean ± SEM...

IFNαβR-dependent antiviral signaling despite an attenuated type I IFN response in RdRP mice. (A) Results from microarray analysis. Gene expression profile of type I IFNs (Ifna1, Refseq ID NM_010502; Ifna2, NM_010503; Ifna4, NM_010504; Ifna5, NM_010505; Ifna9 NM_010507; Ifna11, NM_008333; Ifnab NM_008336; Ifnb1, NM_010510) in spinal cords of uninfec...

List of genes upregulated in uninfected RdRP mice. Differential gene expression in spinal cords from uninfected RdRP mice (n = 3) compared to uninfected WT mice (n = 3). Gene chip was analyzed as described in methods. Only genes with a fold change in expression >4.0 and a p-value of <0.05 are shown. There were no genes significantly downregulated....

List of genes differentially expressed in codon-optimized RdRP THP-1 cells. Differential gene expression in THP-1 cells expressing a codon-optimized RdRP, RdRPΔrna, transgene (n = 2) compared to THP-1 empty vector control cells (n = 2). Gene chip was analyzed as described in methods. Only genes with a fold change in expression >4.0 or <-4.0 and a p...

RdRP expression triggers an innate antiviral response in uninfected hosts. Murine genes upregulated (>4-fold) in tissues of uninfected RdRP mice and human genes upregulated (>4-fold) in RdRP-expressing THP-1 monocytes encode proteins with prominent roles in broad-spectrum viral attenuation. (PDF)

References cited in Supporting Information files. (DOCX)

THP-1 monocytes expressing the RdRP from mRNA with pervasively altered nucleotide sequence display highly augmented antiviral defenses. (A) RdRP cDNA was synthesized with coding-neutral point mutations that maximally disrupt primary and secondary RNA structure (RdRPΔrna). Human codon usage was employed. The WT viral RdRP sequence is shown in the to...

List of genes differentially expressed in virally-infected RdRP mice. Differential gene expression in spinal cords from RdRP mice infected with EMCV (n = 2 females) compared to uninfected WT mice (n = 2 males). Genes listed in italics represent sex-linked genes. Gene chip was analyzed as described in methods. Only genes with a fold change in expres...

List of genes differentially expressed in RdRPΔcat THP-1 cells. Differential gene expression in THP-1 cells expressing the catalytic mutant, RdRPΔcat, transgene (n = 2) compared to THP-1 empty vector control cells (n = 2). Gene chip was analyzed as described in methods. Only genes with a fold change in expression >4.0 or <-4.0 and a p-value of <0.0...

Genomic integration, an obligate step in the HIV-1 replication cycle, is blocked by the integrase inhibitor raltegravir. A consequence is an excess of unintegrated viral DNA genomes, which undergo intramolecular ligation and accumulate as 2-LTR circles. These circularized genomes are also reliably observed in vivo in the absence of antiviral therap...

The host chromatin-binding factor LEDGF/p75 interacts with HIV-1 integrase and directs integration to active transcription units. To understand how LEDGF/p75 recognizes transcription units, we sequenced 1 million HIV-1 integration sites isolated from cultured HEK293T cells. Analysis of integration sites showed that cancer genes were preferentially...

The host chromatin-binding factor LEDGF/P75 interacts with HIV-1 integrase and directs integration to actively transcribed genes. However, it is not clear how LEDGF/P75 recognizes active genes. To better understand HIV integration, we sequenced one million HIV-1 integration sites in cultured HEK293 cells. Approximately 72% of the integration sites...

Ocular hypertension arising from increased resistance to aqueous humor (AH) outflow through the trabecular meshwork is a primary risk factor for open-angle glaucoma, a leading cause of blindness. Ongoing efforts have found little about the molecular and cellular bases of increased resistance to AH outflow through the trabecular meshwork in ocular h...

Background Allosteric HIV-1 integrase (IN) inhibitors (ALLINIs) are an important new class of anti-HIV-1 agents. ALLINIs bind at the IN catalytic core domain (CCD) dimer interface occupying the principal binding pocket of its cellular cofactor LEDGF/p75. Consequently, ALLINIs inhibit HIV-1 IN interaction with LEDGF/p75 as well as promote aberrant I...

Unlabelled: HIV-1 utilizes the cellular protein LEDGF/p75 as a chromosome docking and integration cofactor. The LEDGF/p75 gene, PSIP1, is a potential therapeutic target because, like CCR5, depletion of LEDGF/p75 is tolerated well by human CD4+ T cells, and knockout mice have normal immune systems. RNA interference (RNAi) has been useful for studyi...

As invasive mucormycosis (IM) numbers rise, clinicians suspect prior voriconazole worsens IM incidence and severity, and believe combination anti-fungal therapy improves IM survival. To compare the cumulative incidence (CI), severity and mortality of IM in eras immediately before and after the commercial availability of voriconazole all IM cases fr...

The quinoline-based allosteric HIV-1 integrase (IN) inhibitors (ALLINIs) are promising candidates for clinically useful antiviral agents. Studies using these compounds have highlighted the role of IN in both early and late stages of virus replication. However, dissecting the exact mechanism of action of the quinoline-based ALLINIs has been complica...

Genetic variation and candidate genes associated with breast cancer susceptibility have been identified. Identifying molecular interactions between associated genetic variation and cellular proteins may help to better understand environmental risk. Human MCS5A1 breast cancer susceptibility associated SNP rs7042509 is located in F-box protein 10 (FB...

Unlabelled: BST2/tetherin inhibits the release of enveloped viruses from cells. Primate lentiviruses have evolved specific antagonists (Vpu, Nef, and Env). Here we characterized tetherin proteins of species representing both branches of the order Carnivora. Comparison of tiger and cat (Feliformia) to dog and ferret (Caniformia) genes demonstrated...

The large nucleoporin Nup358/RanBP2 forms eight filaments that project from the nuclear pore into the cytoplasm where they function as docking platforms for nucleocytoplasmic transport receptors. RNAi screens have implicated Nup358 in the HIV-1 life cycle. The 164 C-terminal amino acids of this 3,224 amino acid protein are a cyclophilin homology do...

Aggressive regrowth of recurrent tumors following treatment-induced dormancy represents a major clinical challenge for treatment of malignant disease. We reported previously that recurrent prostate tumors, which underwent complete macroscopic regression followed by aggressive regrowth, could be cured with a vesicular stomatitis virus (VSV)-expresse...

Central initiation of plus strand synthesis is a conserved feature of lentiviruses and certain other retroelements. This complication of the standard reverse transcription mechanism produces a transient "central DNA flap" in the viral cDNA, which has been proposed to mediate its subsequent nuclear import. This model has assumed that the important f...

Classical tumor suppressor genes block neoplasia by regulating cell growth and death. A remarkable puzzle is therefore presented by familial paraganglioma (PGL), a neuroendocrine cancer where the tumor suppressor genes encode subunits of succinate dehydrogenase (SDH), an enzyme of the tricarboxylic acid (TCA) cycle of central metabolism. Loss of SD...

Schematic illustration of fitting parameters used to extract growth curve characteristics for objective scoring of screened compound effects. A. Illustration of lag parameter for multiphasic growth curves. B. Illustration of saturation (sat) and lag parameters in the absence (−) and presence (+) of screened compound. (EPS)

Concentration-dependent growth inhibition curves for example compounds 6035147, 7172827, 7312219, 7619814. Maximal yeast growth is shown as a function of compound concentration to allow estimation of the compound concentration necessary for 50% growth inhibition (IC50) as shown in Table 3. (EPS)

Anaerobic regeneration of NAD+ in yeast vs. human cells. Fungal alcohol dehydrogenase regenerates NAD+ by NADH-dependent reduction of acetaldehyde, whereas NAD+ regeneration in mammals is by NADH-dependent reduction of pyruvate by lactate dehydrogenase. Differential toxicity of alcohol dehydrogenase inhibitors for sdh2Δ mutant yeast suggests that l...

Test for proper integration of NatR gene into NHP6A gene locus. A) PCR products for WT, sdh2Δ, and nhp6aΔ strains resulting from amplification with primers LJM-4338 in the NHP6A gene region and LJM-4339 internal to the NatR gene insert. The expected ∼600 bp PCR product is observed in the nhp6aΔ strain indicating disruption of the NHP6A gene with th...

Verification of yeast strains. A. Serial dilutions across agar plate containing the fermentable carbon source, dextrose (glucose). B. Serial dilutions across agar plate containing non-fermentable carbon source, glycerol. C. Serial dilutions across agar plate containing glucose and clonNat. D–F. Growth curves of yeast strains in liquid media contain...

Compound classes in LOPAC 1280 library. (DOC)

Yeast growth assay in 384 well plate. WT (solid) or sdh2Δ (open) yeast strains were grown in 50 µL cultures in 384 well plates. The plot represents the distribution of the absorbance at 600 nm for each yeast strain grown in YPGal (red) or a 50∶50 mixture of YPGal and YPGly media (black). (EPS)

LOPAC 1280 compounds that significantly inhibit growth of sdh2Δ mutant yeast. (DOC)

Histograms of yeast growth for control plates used to calculate z-factors for HTS. A) Histogram plots of 16 and 48 h OD600 readings for a single 384 well plate ordered by row along the x-axis. Each row alternates between sdh2Δ (red), sdh2Δ plus calmidazolium (green), and media alone (blue). The 48 h readings maximize the differences between sdh2Δ a...

Single cycle reporter viruses that preserve the majority of the HIV-1 genome, long terminal repeat-promoted transcription and Rev-dependent structural protein expression are useful for investigating the viral life cycle. Reporter viruses that encode the viral proteins in cis in this way have been lacking for feline immunodeficiency virus (FIV), whe...

Productive replication of human immunodeficiency virus type 1 (HIV-1) occurs efficiently only in humans. The posttranscriptional stages of the HIV-1 life cycle proceed poorly in mouse cells, with a resulting defect in viral assembly and release. Previous work has shown that the presence of human chromosome 2 increases HIV-1 production in mouse cell...

Lentiviral genomic RNAs are encapsidated by the viral Gag protein during virion assembly. The intracellular location of the initial Gag-RNA interaction is unknown. We previously observed feline immunodeficiency virus (FIV) Gag accumulating at the nuclear envelope during live-cell imaging, which suggested that trafficking of human immunodeficiency v...

Opportunistic infections (OIs) accounted for most of the morbidity and mortality associated with HIV infection in the early era of the pandemic. Introduction of effective primary and secondary antimicrobial prophylaxis regimens markedly decreased their incidence. Later in the HIV/AIDS pandemic, the introduction of effective highly active antiretrov...

A rodent or other small animal model for HIV-1 has not been forthcoming, with the principal obstacles being species-specific restriction mechanisms and deficits in HIV-1 dependency factors. Some Carnivorans may harbor comparatively fewer impediments. For example, in contrast to mice, the domestic cat genome encodes essential nonreceptor HIV-1 depen...

While infections have always played an important role in the history of mankind, advances in science and technology as well as rapid globalization have resulted in an unprecedented wave of new and old infections thrust into the limelight. The recent pandemic of H1N1 influenza virus infection demonstrates the recurrent theme of emerging and reemergi...

Feline immunodeficiency virus (FIV)-based lentiviral vectors are useful for introducing integrated transgenes into nondividing human cells. This protocol describes the production of FIV-based lentiviral vectors using cells grown in CF10 or CF2 devices. It also details the harvesting and concentration of these vectors. The methods are for production...

Feline immunodeficiency virus (FIV)-based lentiviral vectors are useful for introducing integrated transgenes into nondividing human cells. This protocol describes the production and harvesting of vector from cells grown in T75 tissue-culture flasks. The methods are for production for basic science laboratory use and in vivo experimentation. They d...

Feline immunodeficiency virus (FIV)-based lentiviral vectors are useful for introducing integrated transgenes into nondividing human cells. This protocol describes methods for measuring and calculating vector titers in transducing units (TU)/mL. Alternate methods are provided for green fluorescent protein (GFP) vectors and for β-galactosidase vecto...

Feline immunodeficiency virus (FIV)-based lentiviral vectors are useful for introducing integrated transgenes into nondividing human cells. This article describes the production and use of advanced generation FIV vectors. Key properties are discussed in comparison to other lentiviral vectors. Additional topics include the practical implications of...

Studies of the domestic cat have contributed to many scientific advances, including the present understanding of the mammalian cerebral cortex. A practical capability for cat transgenesis is needed to realize the distinctive potential of research on this neurobehaviorally complex, accessible species for advancing human and feline health. For exampl...

Casp8p41, a novel protein generated when HIV-1 protease cleaves caspase 8, independently causes NF-κB activation, proinflammatory cytokine production, and cell death. Here we investigate the mechanism by which Casp8p41 induces cell death. Immunogold staining and electron microscopy demonstrate that Casp8p41 localizes to mitochondria of activated pr...

Recent studies have extended the rapidly developing retroviral restriction factor field to cells of carnivore species. Carnivoran genomes, and the domestic cat genome in particular, are revealing intriguing properties vis-à-vis the primate and feline lentiviruses, not only with respect to their repertoires of virus-blocking restriction factors but...

Retroviral restriction factor research is explaining long-standing lentiviral mysteries. Asking why a particular retrovirus cannot complete a critical part of its life cycle in cells of a particular species has been the starting point for numerous discoveries, including heretofore elusive functions of HIV-1 accessory genes. The potential for therap...

Target cell overexpression of the integrase binding domain (IBD) of LEDGF/p75 (LEDGF) inhibits HIV-1 replication. The mechanism and protein structure requirements for this dominant interference are unclear. More generally, how and when HIV-1 uncoating occurs postentry is poorly defined, and it is unknown whether integrase within the evolving viral...

Nonprimate animal models of HIV-1 infection are prevented by missing cellular cofactors and by antiviral actions of species-specific host defense factors. These blocks are profound in rodents but may be less abundant in certain Carnivora. Here, we enabled productive, spreading replication and passage of HIV-1 in feline cells. Feline fibroblasts, T-...

HIV-1 and certain other retroviruses initiate plus-strand synthesis in the center of the genome as well as at the standard retroviral 3' polypurine tract. This peculiarity of reverse transcription results in a central DNA "flap" structure that has been of controversial functional significance. We mutated both HIV-1 flap-generating elements, the cen...

The chromatin-associated cellular proteins LEDGF/p75 and LEDGF/p52 have been implicated in transcriptional regulation, cell survival and autoimmunity. LEDGF/p75 also appears to act as a chromatin-docking factor or receptor for HIV-1 and other lentiviruses and to play a role in leukemogenesis. For both the viral and cellular roles of this protein, a...

Human immunodeficiency virus type 1 (HIV-1) Gag and genomic RNA determinants required for encapsidation are well established, but where and when encapsidation occurs in the cell is unknown. We constructed MS2 phage coat protein labeling systems to track spatial dynamics of primate and nonprimate lentiviral genomic RNAs (HIV-1 and feline immunodefic...

Cyclooxygenase-2 (COX-2) is a rate-limiting enzyme in prostaglandin (PG) biosynthesis. In the eye, loss of COX-2 expression in aqueous humor-secreting cells has been associated with primary open-angle glaucoma (POAG). Reduction of intraocular pressure (IOP) is the main treatment goal in this disease. We used lentiviral vectors to stably express COX...

Between November, 2006, and May, 2008, a subacute neurological syndrome affected workers from two swine abattoirs in Minnesota and Indiana who had occupational exposure to aerosolised porcine brain. We aimed to describe the pathogenic and immunological characteristics of this illness. All patients from two abattoirs who presented or were referred t...

Permanent integration of the viral genome into a host chromosome is an essential step in the life cycles of lentiviruses and other retroviruses. By archiving the viral genetic information in the genome of the host target cell and its progeny, integrated proviruses prevent curative therapy of HIV-1 and make the development of antiretroviral drug res...

Author Summary Like other retroviruses, HIV-1 integrates a DNA copy of its genome into a host cell chromosome in each replication cycle. The resulting integrated proviruses are the basis for two important clinical problems: the inability to eradicate HIV-1 from the body and the permanent archiving of drug-resistant viruses. The DNA recombining step...

Confocal microscopy of linker histone and LANA31 fusion proteins. (A) H1 fusions and control proteins. See Figure 1 for protein architecture. Proteins were expressed in 293T cells and GFP and DNA (DAPI) were co-imaged. Mitotic cells are highlighted by circling. GFP-199-530 is cytoplasmic and not chromatin bound. GFP-H1.1 and GFP-H1.5 are exclusivel...

Chimera function analysis at different time points. (A) Luciferase expression in TL3 cells stably expressing GFP-H1.5-199-530 (construct F) analyzed 24 hours after challenge with HIVluc. (B) Luciferase expression in TL3 cell lines stably expressing GFP-H1.1-199-530 (construct E) or GFP-H1.5-199-530 (construct F) analyzed two months after challenge...

GFP-IBD interacts with IN and blocks HIV-1 infection. (A) GFP-IBD and IN were expressed by plasmid co-transfection in L cells and imaged by confocal microscopy. Circling highlights a metaphase cell. (B) HIVluc infection of the indicated cell lines. Luciferase activity was measured at 5 days. (0.46 MB TIF)

Cellular localization of GFP-IBD spectral variants. LANA31-GFP-IBD and mCherry-IBD (or CFP-IBD, data not shown) do not colocalize. (0.10 MB TIF)

Immunoblotting of sub-cellular fractions of TL4 cells. Western blotting of TL4 cells expressing LANA31-GFP-IBD or LANA31-GFP-IBDD366N confirms that there is no detectable endogenous LEDGF/p75 in the S2 fractions. (0.08 MB TIF)

Baseline characteristics of stable human T cell lines. (0.03 MB DOC)

Immunoblotting of sub-cellular fractions from stable cell lines. (A) GFP-H1.5-199-530 was stably expressed in cells previously engineered to express GFP-IBD [6]. (B) LANA31-p75P-/ΔAT2R was stably expressed in TL4 cells. The results confirm that the H1 and LANA31 chimeras are confined to the chromatin-bound S2 fraction, while GFP-IBD is found in the...

We injected lentiviral vectors into the eyes of live nonhuman primates to assess potential for glaucoma gene therapy. Anterior chambers of five cynomolgus monkeys were injected with green fluorescent protein (GFP)-encoding feline immunodeficiency viral vectors. The monkeys were monitored for in vivo transgene expression and clinical parameters. The...

RNA interference (RNAi) is a powerful technology for studying the functional significance of genes. The technique is more accessible than gene knockout methods, and is directly applicable to diverse human cells. However, inadequate reductions in target mRNAs can reduce the utility of RNAi and insufficiently rigorous controls can lead to spurious co...

We injected lentiviral vectors into the eyes of live non-human primates to assess potential for glaucoma gene therapy. Anterior chambers of five cynomolgus monkeys were injected with GFP-encoding FIV vectors. The monkeys were followed for in vivo transgene expression and clinical parameters. Their eyes were harvested 2 - 15 months post-injection fo...