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(A to C) HIV infection induces nuclear herniations. HIV virions were harvested from 293T cell cultures 48 hours after cotransfection with pNL4-3 env Vpr proviral DNA and VSV-G-and Vpr-encoding expression vectors. These pseudotyped virions were used to infect GHOST cells that contained an integrated HIV-1 LTR-GFP reporter plasmid. The images presented were acquired 17 hours after infection. NE herniation is highlighted by arrows in (B). One of these cells is productively infected by HIV, as evidenced by Tat-mediated activation of the integrated HIV-1 LTR-GFP reporter (green epifluorescence), whereas the second cell displays NE herniation at an earlier stage of infection. (C) Images acquired 20 min later, illustrating the dynamic nature of these NE abnormalities.
Source publication
Human immunodeficiency virus–1 (HIV-1) Vpr expression halts the proliferation of human cells at or near the G2cell-cycle checkpoint. The transition from G2 to mitosis is normally controlled by changes in the state of phosphorylation and subcellular compartmentalization of key
cell-cycle regulatory proteins. In studies of the intracellular trafficki...
Citations
... Conversely, several viruses have been shown to rely on lamins or their interactors for efficient nuclear entry [18,19]. For example, during nuclear entry, HIV viral Vpr protein triggers local disassembly of the nuclear lamina, resulting in localized herniations in the NE that burst occasionally [20], while parvoviruses (hyper)phosphorylate nuclear lamins to trigger local lamin depolymerization [19]. Herpesvirus HCMV viral proteins pUL50 and pUL53 can induce structural modifications of the nuclear lamina in order to complete nuclear egress [21]. ...
... Viral titers were expressed in terms of viral genome equivalents (VGE) and were determined based on the protocol of Biryukov [26]. siRNA gene silencing siRNA mediated knockdown (kd) of LMNA (LMNAkd) (Dharmacon®, D-001050- [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20], LMNB1 (LMNB1kd) (Dharmacon®, M-005270-01-0005), LMNB2 (LMNB2kd) (Dharmacon®, M-005290-00-0005) and PML (PMLkd) (Dharmacon®, M-006547-01-0005) was performed using Lipofectamine® RNAiMAX (Thermo fisher scientific, 13,778,075) according to manufacturer's instructions. Cells transfected with non-targeting siRNA (Thermo fisher scientific, 12935300) were used as control cells (CTRLkd). ...
Human papillomavirus (HPV) infection is a primary cause of cervical and head-and-neck cancers. The HPV genome enters the nucleus during mitosis when the nuclear envelope disassembles. Given that lamins maintain nuclear integrity during interphase, we asked to what extent their loss would affect early HPV infection. To address this question, we infected human cervical cancer cells and keratinocytes lacking the major lamins with a HPV16 pseudovirus (HP-PsV) encoding an EGFP reporter. We found that a sustained reduction or complete loss of lamin B1 significantly increased HP-PsV infection rate. A corresponding greater nuclear HP-PsV load in LMNB1 knockout cells was directly related to their prolonged mitotic window and extensive nuclear rupture propensity. Despite the increased HP-PsV presence, EGFP transcript levels remained virtually unchanged, indicating an additional defect in protein turnover. Further investigation revealed that LMNB1 knockout led to a substantial decrease in autophagic capacity, possibly linked to the persistent activation of cGAS by cytoplasmic chromatin exposure. Thus, the attrition of lamin B1 increases nuclear perviousness and attenuates autophagic capacity, creating an environment conducive to unrestrained accumulation of HPV capsids. Our identification of lower lamin B1 levels and nuclear BAF foci in the basal epithelial layer of several human cervix samples suggests that this pathway may contribute to an increased individual susceptibility to HPV infection.
Graphical abstract
... This latter biomarker can result particularly challenging considering that some pathogens, namely some types of viruses, alter or disrupt nuclear integrity upon infection as a mechanism to extend their genomes from this compartment onto the cytoplasm on their way out of the cell. This is particularly relevant for pathogens such as retroviruses (De Noronha et al., 2001;Takeshima et al., 2022;Wen et al., 2022). ...
Cellular senescence is a key biological process characterized by irreversible cell cycle arrest. The accumulation of senescent cells creates a pro-inflammatory environment that can negatively affect tissue functions and may promote the development of aging-related diseases. Typical biomarkers related to senescence include senescence-associated β-galactosidase activity, histone H2A.X phosphorylation at serine139 (γH2A.X), and senescence-associated heterochromatin foci (SAHF) with heterochromatin protein 1γ (HP-1γ protein) Moreover, immune cells undergoing senescence, which is known as immunosenescence, can affect innate and adaptative immune functions and may elicit detrimental effects over the host’s susceptibility to infectious diseases. Although associations between senescence and pathogens have been reported, clear links between both, and the related molecular mechanisms involved remain to be determined. Furthermore, it remains to be determined whether infections effectively induce senescence, the impact of senescence and immunosenescence over infections, or if both events coincidently share common molecular markers, such as γH2A.X and p53. Here, we review and discuss the most recent reports that describe cellular hallmarks and biomarkers related to senescence in immune and non-immune cells in the context of infections, seeking to better understand their relationships. Related literature was searched in Pubmed and Google Scholar databases with search terms related to the sections and subsections of this review.
... Whether preformed NEIs, present in HeLa cells, but not in CD4 + T cells, or only those induced by HIV-1, are involved in the nuclear transfer of HIV-1 components has yet to be explored. In addition to, or in synergy with, the VOR complex, a possible contributory role for HIV-1 Vpr protein in NEI induction cannot be excluded, based on the finding that Vpr induced transient, localized herniations (probably NEI) in the nuclear envelope, associated with defects in the nuclear lamina 84 . Interestingly, a recent study attributed to Vpr in HIV-1 virions a reprogramming role of resting T cells into tissue-resident memory T cells 85 . ...
The mechanism of human immunodeficiency virus 1 (HIV-1) nuclear entry, required for productive infection, is not fully understood. Here, we report that in HeLa cells and activated CD4⁺ T cells infected with HIV-1 pseudotyped with VSV-G and native Env protein, respectively, Rab7⁺ late endosomes containing endocytosed HIV-1 promote the formation of nuclear envelope invaginations (NEIs) by a molecular mechanism involving the VOR complex, composed of the outer nuclear membrane protein VAP-A, hyperphosphorylated ORP3 and Rab7. Silencing VAP-A or ORP3 and drug-mediated impairment of Rab7 binding to ORP3-VAP-A inhibited the nuclear transfer of the HIV-1 components and productive infection. In HIV-1-resistant quiescent CD4⁺ T cells, ORP3 was not hyperphosphorylated and neither VOR complex nor NEIs were formed. This new cellular pathway and its molecular players are potential therapeutic targets, perhaps shared by other viruses that require nuclear entry to complete their life cycle.
... The nuclear envelope (NE) encloses genomic DNA to protect it from a variety of insults and plays pivotal roles in regulating genomic function [1,2]. Previous studies demonstrated that the NE is damaged upon various types of stresses, leading to its dysfunction [3][4][5][6][7]. Such stresses are referred to as "NE stress" [8][9][10][11]. ...
The nuclear envelope (NE) is often challenged by various stresses (known as “NE stress”), leading to its dysfunction. Accumulating evidence has proven the pathological relevance of NE stress in numerous diseases ranging from cancer to neurodegenerative diseases. Although several proteins involved in the reassembly of the NE after mitosis have been identified as the NE repair factors, the regulatory mechanisms modulating the efficiency of NE repair remain unclear. Here, we showed that response to NE stress varied among different types of cancer cell lines. U251MG derived from glioblastoma exhibited severe nuclear deformation and massive DNA damage at the deformed nuclear region upon mechanical NE stress. In contrast, another cell line derived from glioblastoma, U87MG, only presented mild nuclear deformation without DNA damage. Time-lapse imaging demonstrated that repairing of ruptured NE often failed in U251MG, but not in U87MG. These differences were unlikely to have been due to weakened NE in U251MG because the expression levels of lamin A/C, determinants of the physical property of the NE, were comparable and loss of compartmentalization across the NE was observed just after laser ablation of the NE in both cell lines. U251MG proliferated more rapidly than U87MG concomitant with reduced expression of p21, a major inhibitor of cyclin-dependent kinases, suggesting a correlation between NE stress response and cell cycle progression. Indeed, visualization of cell cycle stages using fluorescent ubiquitination-based cell cycle indicator reporters revealed greater resistance of U251MG to NE stress at G 1 phase than at S and G 2 phases. Furthermore, attenuation of cell cycle progression by inducing p21 in U251MG counteracted the nuclear deformation and DNA damage upon NE stress. These findings imply that dysregulation of cell cycle progression in cancer cells causes loss of the NE integrity and its consequences such as DNA damage and cell death upon mechanical NE stress.
... Nuclear membrane rupture is defined as the rapid loss of nucleus compartmentalization in interphase resulting in the mixing of nuclear and cytoplasmic proteins and organelles (Vos et al., 2011;Noronha et al., 2001;Vargas et al., 2012). In the nucleus, membrane rupture is typically repaired within minutes with limited effects on cell proliferation or viability (Raab et al., 2016;Denais et al., 2016). ...
Nuclear membrane rupture is a physiological response to in vivo situations, such as cell migration. When disregulated, it can have deleterious consequences for the cell including increased genome instability and inflammatory signaling. However, many of the molecular mechanisms underlying this process are unclear and only a few regulators have been uncovered. In this study, we developed a nuclear rupture reporter that is size excluded from re-compartmentalization following nuclear rupture events. This allows for robust detection of factors influencing nuclear integrity in fixed cells. Additionally, we created and validated an automated pipeline for the analysis of fixed cell images that we applied in a high-content siRNA screen. Our screen identified conditions that led to both increased and decreased nuclear rupture frequency. Analysis of the hits demonstrated an enrichment in factors in the nuclear membrane and ER, and demonstrated that nuclear size is not strongly correlated with rupture frequency. Based on these results, we validated a new function for the protein phosphatase CTDNEP1 in maintaining nucleus stability. These findings provide new insights into the regulation of nuclear stability and define a novel method of rupture analysis that removes a substantial barrier to understanding the molecular mechanisms underlying this process.
... For example, EBV lytic gene BGLF4 encodes a viral kinase that phosphorylates lamina A causing its degradation [116]. Alternatively, the nuclear lamina can also be involved in the formation of viral replication compartments [117][118][119][120][121]. EBV latent infection of B cells induces Lamin A/C expression, and Type III but not Type I EBV+ B cells express Lamin A/C [122]. ...
Epstein–Barr Virus (EBV) is a human gamma-herpesvirus that is widespread worldwide. To this day, about 200,000 cancer cases per year are attributed to EBV infection. EBV is capable of infecting both B cells and epithelial cells. Upon entry, viral DNA reaches the nucleus and undergoes a process of circularization and chromatinization and establishes a latent lifelong infection in host cells. There are different types of latency all characterized by different expressions of latent viral genes correlated with a different three-dimensional architecture of the viral genome. There are multiple factors involved in the regulation and maintenance of this three-dimensional organization, such as CTCF, PARP1, MYC and Nuclear Lamina, emphasizing its central role in latency maintenance.
... One of the surprising facts from the nuclear membrane rupture analysis is that cells have a strong nuclear membrane repair mechanism during the interphase. The length of an individual rupture of the nuclear membrane is only a few minutes (de Noronha et al., 2001) but the membrane repair occurs within a few minutes to a few hours, and has been observed in many in vitro settings (Hatch and Hetzer, 2014;Denais et al., 2016;Raab et al., 2016;Yang et al., 2017). Remarkably, most of the cells are capable of restoring the NE integrity even after repeated NE rupture events (De vos et al., 2011;Vargas et al., 2012;Denais et al., 2016;Hatch and Hetzer, 2016;Raab et al., 2016;Robijns et al., 2016). ...
The eukaryotic genome is enclosed in a nuclear envelope that protects it from potentially damaging cellular activities and physically segregates transcription and translation.Transport across the NE is highly regulated and occurs primarily via the macromolecular nuclear pore complexes.Loss of nuclear compartmentalization due to defects in NPC function and NE integrity are tied to neurological and ageing disorders like Alzheimer’s, viral pathogenesis, immune disorders, and cancer progression.Recent work implicates inner-nuclear membrane proteins of the conserved LEM domain family and the ESCRT machinery in NE reformation during cell division and NE repair upon rupture in migrating cancer cells, and generating seals over defective NPCs. In this review, we discuss the recent in-roads made into defining the molecular mechanisms and biochemical networks engaged by LEM and many other integral inner nuclear membrane proteins to preserve the nuclear barrier.
... One of the consequences of NE destabilization is the appearance of NE ruptures that cause loss of nuclear compartmentalization. This has been observed in laminopathy patient cells and animal models (13)(14)(15), in cells undergoing a viral infection (16,17) or lacking specific components of the lamina (13,18). Additionally, mechanical stress can cause NE rupture, for example in vivo when cells are migrating through dense tissues (19)(20)(21), or in vitro when cells are cultured in 2D on stiff substrates (22). ...
Nestor–Guillermo progeria syndrome (NGPS) is caused by a homozygous alanine-to-threonine mutation at position 12 (A12T) in barrier-to-autointegration factor (BAF). It is characterized by accelerated aging with severe skeletal abnormalities. BAF is an essential protein binding to DNA and nuclear envelope (NE) proteins, involved in NE rupture repair. Here, we assessed the impact of BAF A12T on NE integrity using NGPS-derived patient fibroblasts. We observed a strong defect in lamin A/C accumulation to NE ruptures in NGPS cells, restored upon homozygous reversion of the pathogenic BAF A12T mutation with CRISPR/Cas9. By combining in vitro and cellular assays, we demonstrated that while the A12T mutation does not affect BAF 3D structure and phosphorylation by VRK1, it specifically decreases the interaction between BAF and lamin A/C. Finally, we revealed that the disrupted interaction does not prevent repair of NE ruptures but instead generates weak points in the NE that lead to a higher frequency of NE re-rupturing in NGPS cells. We propose that this NE fragility could directly contribute to the premature aging phenotype in patients.
... Conversely, several viruses have been shown to rely on lamins or their interactors for e cient nuclear entry (18,19). For example, during nuclear entry, HIV viral Vpr protein triggers local disassembly of the nuclear lamina, resulting in localized herniations in the NE that burst occasionally (20), while parvoviruses (hyper)phosphorylate nuclear lamins to trigger local lamin depolymerization (19). Herpesvirus HCMV viral proteins pUL50 and pUL53 can induce structural modi cations of the nuclear lamina in order to complete nuclear egress (21). ...
Human papillomavirus (HPV) infection is the prime elicitor of cervical and head-and-neck cancers. The HPV genome enters the nucleus during mitosis when the nuclear envelope dismantles. Since lamins safeguard nuclear integrity during interphase, we asked to what extent their loss would affect early HPV infection. We challenged human cervical cancer cells knocked out for the major lamin genes with a HPV16 pseudovirus (PsV) encoding an EGFP reporter and found that loss of lamin B1 amplified infection rate. A prolonged mitotic window and extensive nuclear rupture propensity during interphase led to a higher nuclear PsV load in LMNB1 knockout cells, but unchanged EGFP transcript levels pointed to an additional defect in protein turnover. We found a strong decrease in autophagic capacity in LMNB1 knockout cells, which we connect to the persistent activation of cGAS. Thus, loss of lamin B1 increases nuclear perviousness and blunts the autophagic capacity, which primes cells for unrestrained buildup of HPV capsids.
... Recent studies have described that a variety of cellular stresses, such as cell migration through constricted environments and virus infection, disrupt the structure and function of the NE [14][15][16][17]. Di Micco et. ...
The nuclear envelope (NE) separates genomic DNA from the cytoplasm and provides the molecular platforms for nucleocytoplasmic transport, higher-order chromatin organization, and physical links between the nucleus and cytoskeleton. Recent studies have shown that the NE is often damaged by various stresses termed “NE stress”, leading to critical cellular dysfunction. Accumulating evidence has revealed the crucial roles of NE stress in the pathology of a broad spectrum of diseases. In the central nervous system (CNS), NE dysfunction impairs neural development and is associated with several neurological disorders, such as Alzheimer’s disease and autosomal dominant leukodystrophy. In this review, the structure and functions of the NE are summarized, and the concepts of NE stress and NE stress responses are introduced. Additionally, the significant roles of the NE in the development of CNS and the mechanistic connections between NE stress and neurological disorders are described.
