RESUMEN
Nuclear pore complexes (NPC) regulate molecular traffics on nuclear envelope, which plays crucial roles during cell fate specification and diseases. The viral accessory protein NSP9 of SARS-CoV-2 is reported to interact with nucleoporin 62 (NUP62), a structural component of the NPC, but its biological impact on the host cell remain obscure. Here, we established new cell line models with ectopic NSP9 expression and determined the subcellular destination and biological functions of NSP9. Confocal imaging identified NSP9 to be largely localized in close proximity to the endoplasmic reticulum. In agreement with the subcellular distribution of NSP9, association of NSP9 with NUP62 was observed in cytoplasm. Furthermore, the overexpression of NSP9 correlated with a reduction of NUP62 expression on the nuclear envelope, suggesting that attenuating NUP62 expression might have contributed to defective NPC formation. Importantly, the loss of NUP62 impaired translocation of p65, a subunit of NF-κB, upon TNF-α stimulation. Concordantly, NSP9 over-expression blocked p65 nuclear transport. Taken together, these data shed light on the molecular mechanisms underlying the modulation of host cells during SARS-CoV-2 infection.
Asunto(s)
COVID-19/metabolismo , COVID-19/virología , Interacciones Microbiota-Huesped/fisiología , Glicoproteínas de Membrana/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Proteínas de Unión al ARN/metabolismo , SARS-CoV-2/metabolismo , Proteínas no Estructurales Virales/metabolismo , Transporte Activo de Núcleo Celular , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/virología , Técnicas de Silenciamiento del Gen , Células HeLa , Humanos , Glicoproteínas de Membrana/antagonistas & inhibidores , Glicoproteínas de Membrana/genética , Modelos Biológicos , Membrana Nuclear/metabolismo , Membrana Nuclear/virología , Proteínas de Complejo Poro Nuclear/antagonistas & inhibidores , Proteínas de Complejo Poro Nuclear/genética , Proteínas de Unión al ARN/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Factor de Transcripción ReIA/metabolismo , Proteínas no Estructurales Virales/genéticaRESUMEN
Herpesvirus capsids are assembled in the nucleus and undergo a two-step process to cross the nuclear envelope. Capsids bud into the inner nuclear membrane (INM) aided by the nuclear egress complex (NEC) proteins UL31/34. At that stage of egress, enveloped virions are found for a short time in the perinuclear space. In the second step of nuclear egress, perinuclear enveloped virions (PEVs) fuse with the outer nuclear membrane (ONM) delivering capsids into the cytoplasm. Once in the cytoplasm, capsids undergo re-envelopment in the Golgi/trans-Golgi apparatus producing mature virions. This second step of nuclear egress is known as de-envelopment and is the focus of this review. Compared with herpesvirus envelopment at the INM, much less is known about de-envelopment. We propose a model in which de-envelopment involves two phases: (i) fusion of the PEV membrane with the ONM and (ii) expansion of the fusion pore leading to release of the viral capsid into the cytoplasm. The first phase of de-envelopment, membrane fusion, involves four herpes simplex virus (HSV) proteins: gB, gH/gL, gK and UL20. gB is the viral fusion protein and appears to act to perturb membranes and promote fusion. gH/gL may also have similar properties and appears to be able to act in de-envelopment without gB. gK and UL20 negatively regulate these fusion proteins. In the second phase of de-envelopment (pore expansion and capsid release), an alpha-herpesvirus protein kinase, US3, acts to phosphorylate NEC proteins, which normally produce membrane curvature during envelopment. Phosphorylation of NEC proteins reverses tight membrane curvature, causing expansion of the membrane fusion pore and promoting release of capsids into the cytoplasm.
Asunto(s)
Cápside/metabolismo , Infecciones por Herpesviridae/virología , Herpesviridae/fisiología , Proteínas Virales de Fusión/metabolismo , Núcleo Celular/virología , Citoplasma/virología , Herpesviridae/genética , Herpesviridae/ultraestructura , Humanos , Fusión de Membrana , Membrana Nuclear/virología , Fosforilación , Simplexvirus/genética , Simplexvirus/fisiología , Envoltura Viral , Proteínas Virales de Fusión/genética , Virión , Red trans-Golgi/virologíaRESUMEN
The human immunodeficiency virus (HIV) enters the nucleus to establish infection, but the role of nuclear envelope proteins in this process is incompletely understood. Inner nuclear transmembrane proteins SUN1 and SUN2 connect nuclear lamins to the cytoskeleton and participate in the DNA damage response (DDR). Increased levels of SUN1 or SUN2 potently restrict HIV infection through an unresolved mechanism. Here, we find that the antiviral activities of SUN1 and SUN2 are distinct. HIV-1 and HIV-2 are preferentially inhibited by SUN1 and SUN2, respectively. We identify DNA damage inducers that stimulate HIV-1 infection and show that SUN1, but not SUN2, neutralizes this effect. Finally, we show that chromatin movements and nuclear rotations are associated with the effects of SUN proteins and Lamin A/C on infection. These results reveal an emerging role of chromatin dynamics and the DDR in the control of HIV infection by structural components of the nuclear envelope.
Asunto(s)
Cromatina/metabolismo , Infecciones por VIH/virología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Membrana Nuclear/metabolismo , Núcleo Celular/metabolismo , Humanos , Proteínas de la Membrana/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Membrana Nuclear/virología , Proteínas Nucleares/metabolismoRESUMEN
It is well established that the herpesvirus nuclear egress complex (NEC) has an intrinsic ability to deform membranes. During viral infection, the membrane-deformation activity of the NEC must be precisely regulated to ensure efficient nuclear egress of capsids. One viral protein known to regulate herpes simplex virus type 2 (HSV-2) NEC activity is the tegument protein pUL21. Cells infected with an HSV-2 mutant lacking pUL21 (ΔUL21) produced a slower migrating species of the viral serine/threonine kinase pUs3 that was shown to be a hyperphosphorylated form of the enzyme. Investigation of the pUs3 substrate profile in ΔUL21-infected cells revealed a prominent band with a molecular weight consistent with that of the NEC components pUL31 and pUL34. Phosphatase sensitivity and retarded mobility in phos-tag SDS-PAGE confirmed that both pUL31 and pUL34 were hyperphosphorylated by pUs3 in the absence of pUL21. To gain insight into the consequences of increased phosphorylation of NEC components, the architecture of the nuclear envelope in cells producing the HSV-2 NEC in the presence or absence of pUs3 was examined. In cells with robust NEC production, invaginations of the inner nuclear membrane were observed that contained budded vesicles of uniform size. By contrast, nuclear envelope deformations protruding outwards from the nucleus, were observed when pUs3 was included in transfections with the HSV-2 NEC. Finally, when pUL21 was included in transfections with the HSV-2 NEC and pUs3, decreased phosphorylation of NEC components was observed in comparison to transfections lacking pUL21. These results demonstrate that pUL21 influences the phosphorylation status of pUs3 and the HSV-2 NEC and that this has consequences for the architecture of the nuclear envelope.
Asunto(s)
Herpes Simple/patología , Herpesvirus Humano 2/fisiología , Membrana Nuclear/patología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Virales/metabolismo , Liberación del Virus , Animales , Cápside/fisiología , Núcleo Celular/genética , Núcleo Celular/metabolismo , Chlorocebus aethiops , Células HeLa , Herpes Simple/metabolismo , Herpes Simple/virología , Humanos , Membrana Nuclear/metabolismo , Membrana Nuclear/virología , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Células Vero , Proteínas Virales/genética , Ensamble de VirusRESUMEN
During replication of herpesviruses, capsids escape from the nucleus into the cytoplasm by budding at the inner nuclear membrane. This unusual process is mediated by the viral nuclear egress complex (NEC) that deforms the membrane around the capsid by oligomerizing into a hexagonal, membrane-bound scaffold. Here, we found that highly basic membrane-proximal regions (MPRs) of the NEC alter lipid order by inserting into the lipid headgroups and promote negative Gaussian curvature. We also find that the electrostatic interactions between the MPRs and the membranes are essential for membrane deformation. One of the MPRs is phosphorylated by a viral kinase during infection, and the corresponding phosphomimicking mutations block capsid nuclear egress. We show that the same phosphomimicking mutations disrupt the NEC-membrane interactions and inhibit NEC-mediated budding in vitro, providing a biophysical explanation for the in vivo phenomenon. Our data suggest that the NEC generates negative membrane curvature by both lipid ordering and protein scaffolding and that phosphorylation acts as an off switch that inhibits the membrane-budding activity of the NEC to prevent capsid-less budding. IMPORTANCE Herpesviruses are large viruses that infect nearly all vertebrates and some invertebrates and cause lifelong infections in most of the world's population. During replication, herpesviruses export their capsids from the nucleus into the cytoplasm by an unusual mechanism in which the viral nuclear egress complex (NEC) deforms the nuclear membrane around the capsid. However, how membrane deformation is achieved is unclear. Here, we show that the NEC from herpes simplex virus 1, a prototypical herpesvirus, uses clusters of positive charges to bind membranes and order membrane lipids. Reducing the positive charge or introducing negative charges weakens the membrane deforming ability of the NEC. We propose that the virus employs electrostatics to deform nuclear membrane around the capsid and can control this process by changing the NEC charge through phosphorylation. Blocking NEC-membrane interactions could be exploited as a therapeutic strategy.
Asunto(s)
Cápside/metabolismo , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/fisiología , Metabolismo de los Lípidos , Membrana Nuclear/metabolismo , Liberación del Virus , Animales , Núcleo Celular/metabolismo , Chlorocebus aethiops , Humanos , Membrana Nuclear/virología , Fosforilación , Electricidad Estática , Células Vero , Ensamble de Virus , Replicación ViralRESUMEN
HIV-1 can infect non-dividing cells. The nuclear envelope therefore represents a barrier that HIV-1 must traverse in order to gain access to the host cell chromatin for integration. Hence, nuclear entry is a critical step in the early stages of HIV-1 replication. Following membrane fusion, the viral capsid (CA) lattice, which forms the outer face of the retroviral core, makes numerous interactions with cellular proteins that orchestrate the progress of HIV-1 through the replication cycle. The ability of CA to interact with nuclear pore proteins and other host factors around the nuclear pore determines whether nuclear entry occurs. Uncoating, the process by which the CA lattice opens and/or disassembles, is another critical step that must occur prior to integration. Both early and delayed uncoating have detrimental effects on viral infectivity. How uncoating relates to nuclear entry is currently hotly debated. Recent technological advances have led to intense discussions about the timing, location, and requirements for uncoating and have prompted the field to consider alternative uncoating scenarios that presently focus on uncoating at the nuclear pore and within the nuclear compartment. This review describes recent advances in the study of HIV-1 nuclear entry, outlines the interactions of the retroviral CA protein, and discusses the challenges of investigating HIV-1 uncoating.
Asunto(s)
Proteínas de la Cápside/metabolismo , Cápside/metabolismo , Núcleo Celular/virología , Infecciones por VIH/virología , VIH-1/fisiología , Desencapsidación Viral , Animales , Núcleo Celular/metabolismo , VIH-1/genética , Interacciones Huésped-Patógeno , Humanos , Membrana Nuclear/fisiología , Membrana Nuclear/virología , Poro Nuclear/fisiología , Poro Nuclear/virología , Retroviridae/fisiología , Transcripción ReversaRESUMEN
Ebola virus (EBOV) VP24 protein is a nucleocapsid-associated protein that inhibits interferon (IFN) gene expression and counteracts the IFN-mediated antiviral response, preventing nuclear import of signal transducer and activator of transcription 1 (STAT1). Proteomic studies to identify additional EBOV VP24 partners have pointed to the nuclear membrane component emerin as a potential element of the VP24 cellular interactome. Here, we have further studied this interaction and its impact on cell biology. We demonstrate that VP24 interacts with emerin but also with other components of the inner nuclear membrane, such as lamin A/C and lamin B. We also show that VP24 diminishes the interaction between emerin and lamin A/C and compromises the integrity of the nuclear membrane. This disruption is associated with nuclear morphological abnormalities, activation of a DNA damage response, the phosphorylation of extracellular signal-regulated kinase (ERK), and the induction of interferon-stimulated gene 15 (ISG15). Interestingly, expression of VP24 also promoted the cytoplasmic translocation and downmodulation of barrier-to-autointegration factor (BAF), a common interactor of lamin A/C and emerin, leading to repression of the BAF-regulated CSF1 gene. Importantly, we found that EBOV infection results in the activation of pathways associated with nuclear envelope damage, consistent with our observations in cells expressing VP24. In summary, here we demonstrate that VP24 acts at the nuclear membrane, causing morphological and functional changes in cells that recapitulate several of the hallmarks of laminopathy diseases. IMPORTANCE The Ebola virus (EBOV) VP24 protein is a nucleocapsid-associated protein with multiple functions. Proteomic studies have identified the cellular nuclear membrane component emerin as a potential VP24 interactor. Here, we demonstrate that VP24 not only interacts with emerin but also with lamin A/C and lamin B, prompting nuclear membrane disruption. This disruption is associated with nuclear morphological abnormalities, activation of a DNA damage response, the phosphorylation of extracellular signal-regulated kinase (ERK), and the induction of interferon-stimulated gene 15 (ISG15). Interestingly, VP24 also promotes the cytoplasmic translocation and downmodulation of barrier-to-autointegration factor (BAF), leading to repression of the BAF-regulated CSF1 gene. Finally, we show that EBOV infection also results in the activation of pathways associated with nuclear envelope damage, consistent with our observations in cells expressing VP24. These results reveal novel activities of EBOV VP24 protein, resulting in a cell phenotype similar to that of most laminopathies, with potential impact on EBOV replication.
Asunto(s)
Ebolavirus/patogenicidad , Laminopatías/virología , Laminas/metabolismo , Membrana Nuclear/patología , Proteínas Virales/genética , Células A549 , Transporte Activo de Núcleo Celular , Núcleo Celular/patología , Núcleo Celular/virología , Ebolavirus/química , Ebolavirus/genética , Células HEK293 , Células HeLa , Fiebre Hemorrágica Ebola/virología , Humanos , Laminas/clasificación , Proteínas de la Membrana/metabolismo , Membrana Nuclear/virología , Proteínas Nucleares/metabolismo , Fenotipo , Proteínas Virales/metabolismo , Replicación ViralRESUMEN
Nuclear envelope budding in herpesvirus nuclear egress may be negatively regulated, since the pUL31/pUL34 nuclear egress complex heterodimer can induce membrane budding without capsids when expressed ectopically or on artificial membranes in vitro, but not in the infected cell. We have previously described a pUL34 mutant that contained alanine substitutions at R158 and R161 and that showed impaired growth, impaired pUL31/pUL34 interaction, and unregulated budding. Here, we determine the phenotypic contributions of the individual substitutions to these phenotypes. Neither substitution alone was able to reproduce the impaired growth or nuclear egress complex (NEC) interaction phenotypes. Either substitution, however, could fully reproduce the unregulated budding phenotype, suggesting that misregulated budding may not substantially impair virus replication. In addition, the R158A substitution caused relocalization of the NEC to intranuclear punctate structures and recruited lamin A/C to these structures, suggesting that this residue might be important for recruitment of kinases for dispersal of nuclear lamins. IMPORTANCE Herpesvirus nuclear egress is a complex, regulated process coordinated by two virus proteins that are conserved among the herpesviruses that form a heterodimeric nuclear egress complex (NEC). The NEC drives budding of capsids at the inner nuclear membrane and recruits other viral and host cell proteins for disruption of the nuclear lamina, membrane scission, and fusion. The structural basis of individual activities of the NEC, apart from membrane budding, are not clear, nor is the basis of the regulation of membrane budding. Here, we explore the properties of NEC mutants that have an unregulated budding phenotype, determine the significance of that regulation for virus replication, and also characterize a structural requirement for nuclear lamina disruption.
Asunto(s)
Herpes Simple/virología , Herpesvirus Humano 1/fisiología , Mutación , Lámina Nuclear/metabolismo , Proteínas Virales/metabolismo , Replicación Viral , Transporte Activo de Núcleo Celular , Animales , Chlorocebus aethiops , Herpes Simple/genética , Herpes Simple/metabolismo , Humanos , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Membrana Nuclear/metabolismo , Membrana Nuclear/patología , Membrana Nuclear/virología , Lámina Nuclear/patología , Lámina Nuclear/virología , Células Vero , Proteínas Virales/genética , Liberación del VirusRESUMEN
Herpesviral nuclear egress is a regulated process shared by all family members, ensuring the efficient cytoplasmic release of viral capsids. In the case of human cytomegalovirus (HCMV), the core of the nuclear egress complex (NEC) consists of the pUL50-pUL53 heterodimer that builds hexameric lattices for capsid binding and multicomponent interaction, including NEC-associated host factors. A characteristic feature of NEC interaction is the N-terminal hook structure of pUL53 that binds to an alpha-helical groove of pUL50, thus termed as hook-into-groove interaction. This central regulatory element is essential for viral replication and shows structural-functional conservation, which has been postulated as a next-generation target of antiviral strategies. However, a solid validation of this concept has been missing. In the present study, we focused on the properties of oligomeric HCMV core NEC interaction and the antiviral activity of specifically targeted prototype inhibitors. Our data suggest the following: (i) transiently expressed, variably tagged versions of HCMV NEC proteins exert hook-into-groove complexes, putatively in oligomeric assemblies that are distinguishable from heterodimers, as shown by in vitro assembly and coimmunoprecipitation approaches; (ii) this postulated oligomeric binding pattern was further supported by the use of a pUL50::pUL53 fusion construct also showing a pronounced multi-interaction potency; (iii) using confocal imaging cellular NEC-associated proteins were found partly colocalized with the tagged core NECs; (iv) a small inhibitory molecule, recently identified by an in vitro binding inhibition assay, was likewise active in blocking pUL50-pUL53 oligomeric assembly and in exerting antiviral activity in HCMV-infected fibroblasts. In summary, the findings refine the previous concept of HCMV core NEC formation and nominate this drug-accessible complex as a validated antiviral drug target.
Asunto(s)
Antivirales/farmacología , Infecciones por Citomegalovirus/tratamiento farmacológico , Citomegalovirus/crecimiento & desarrollo , Proteínas Virales/metabolismo , Liberación del Virus/efectos de los fármacos , Cápside/metabolismo , Proteínas de la Cápside/metabolismo , Línea Celular , Núcleo Celular/virología , Citomegalovirus/efectos de los fármacos , Infecciones por Citomegalovirus/patología , Células HEK293 , Células HeLa , Humanos , Proteínas de la Membrana/metabolismo , Simulación de Dinámica Molecular , Membrana Nuclear/virología , Unión ProteicaRESUMEN
BACKGROUND INFORMATION: Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection induces an alteration in the endomembrane system of the mammalian cells. In this study, we used transmission electron microscopy and electron tomography to investigate the main structural alterations in the cytoplasm of Vero cells infected with a SARS-CoV-2 isolate from São Paulo state (Brazil). RESULTS: Different membranous structures derived from the zippered endoplasmic reticulum were observed along with virus assembly through membrane budding. Also, we demonstrated the occurrence of annulate lamellae in the cytoplasm of infected cells and the presence of virus particles in the perinuclear space. CONCLUSIONS AND SIGNIFICANCE: This study contributes to a better understanding of the cell biology of SARS-CoV-2 and the mechanisms of the interaction of the virus with the host cell that promote morphological changes, recruitment of organelles and cell components, in a context of a virus-induced membrane remodelling.
Asunto(s)
Retículo Endoplásmico/virología , Membranas Intracelulares/virología , Membrana Nuclear/virología , SARS-CoV-2 , Animales , COVID-19 , Chlorocebus aethiops , Tomografía con Microscopio Electrónico , Retículo Endoplásmico/ultraestructura , Humanos , Membranas Intracelulares/ultraestructura , Microscopía Electrónica de Transmisión , Membrana Nuclear/ultraestructura , SARS-CoV-2/crecimiento & desarrollo , SARS-CoV-2/ultraestructura , Células Vero , Ensamble de Virus , Replicación ViralRESUMEN
It is recognized that HIV-1 capsid cores are disassembled in the cytoplasm, releasing their genomes into the nucleus through nuclear pores, but there is also evidence showing the capsid (CA) exists in the nucleus. Whether HIV-1 enters the nucleus and how it enters the nucleus through the undersized nuclear pore remains mysterious. Based on multicolor labeling and real-time imaging of the viral and cellular components, our observations via light and electron microscopy suggest that HIV-1 selectively gathered at the microtubule organization center (MTOC), leading the nearby nuclear envelope (NE) to undergo deformation, invagination and restoration to form a nuclear vesicle in which the viral particles were wrapped; then, the inner membrane of the nuclear vesicle ruptured to release HIV-1 into the nucleus. This unexpected discovery expands our understanding of the complexity of HIV-1 nuclear entry, which may provide new insights to HIV-1 virology.
Asunto(s)
Proteínas de la Cápside/metabolismo , Núcleo Celular/metabolismo , Endocitosis , VIH-1/metabolismo , Poro Nuclear/metabolismo , Virión/metabolismo , Transporte Activo de Núcleo Celular , Línea Celular Tumoral , Núcleo Celular/ultraestructura , Núcleo Celular/virología , Células HEK293 , Infecciones por VIH/virología , VIH-1/genética , VIH-1/fisiología , Humanos , Microscopía Confocal , Microscopía Electrónica de Transmisión , Centro Organizador de los Microtúbulos/metabolismo , Centro Organizador de los Microtúbulos/virología , Membrana Nuclear/metabolismo , Membrana Nuclear/ultraestructura , Membrana Nuclear/virología , Poro Nuclear/ultraestructura , Poro Nuclear/virología , Imagen de Lapso de Tiempo/métodos , Virión/ultraestructuraRESUMEN
The assembly of human cytomegalovirus (HCMV) and other herpesviruses includes both nuclear and cytoplasmic phases. During the prolonged replication cycle of HCMV, the cell undergoes remarkable changes in cellular architecture that include marked increases in nuclear size and structure as well as the reorganization of membranes in cytoplasm. Similarly, significant changes occur in cellular metabolism, protein trafficking, and cellular homeostatic functions. These cellular modifications are considered integral in the efficient assembly of infectious progeny in productively infected cells. Nuclear egress of HCMV nucleocapsids is thought to follow a pathway similar to that proposed for other members of the herpesvirus family. During this process, viral nucleocapsids must overcome structural barriers in the nucleus that limit transit and, ultimately, their delivery to the cytoplasm for final assembly of progeny virions. HCMV, similar to other herpesviruses, encodes viral functions that co-opt cellular functions to overcome these barriers and to bridge the bilaminar nuclear membrane. In this brief review, we will highlight some of the mechanisms that define our current understanding of HCMV egress, relying heavily on the current understanding of egress of the more well-studied α-herpesviruses, HSV-1 and PRV.
Asunto(s)
Núcleo Celular/virología , Citomegalovirus/fisiología , Liberación del Virus , Cápside/fisiología , Núcleo Celular/ultraestructura , Citoplasma/virología , Replicación del ADN , ADN Viral/metabolismo , Humanos , Membrana Nuclear/virología , Nucleocápside/fisiología , Empaquetamiento del Genoma Viral , Replicación ViralRESUMEN
The glycoprotein M of herpes simplex virus 1 (HSV-1) is dynamically relocated from nuclear membranes to the trans-Golgi network (TGN) during infection, but molecular partners that promote this relocalization are unknown. Furthermore, while the presence of the virus is essential for this phenomenon, it is not clear if this is facilitated by viral or host proteins. Past attempts to characterize glycoprotein M (gM) interacting partners identified the viral protein gN by coimmunoprecipitation and the host protein E-Syt1 through a proteomics approach. Interestingly, both proteins modulate the activity of gM on the viral fusion machinery. However, neither protein is targeted to the nuclear membrane and consequently unlikely explains the dynamic regulation of gM nuclear localization. We thus reasoned that gM may transiently interact with other molecules. To resolve this issue, we opted for a proximity-dependent biotin identification (BioID) proteomics approach by tagging gM with a BirA* biotinylation enzyme and purifying BirA substrates on a streptavidin column followed by mass spectrometry analysis. The data identified gM and 170 other proteins that specifically and reproducibly were labeled by tagged gM at 4 or 12 h postinfection. Surprisingly, 35% of these cellular proteins are implicated in protein transport. Upon testing select candidate proteins, we discovered that XPO6, an exportin, is required for gM to be released from the nucleus toward the TGN. This is the first indication of a host or viral protein that modulates the presence of HSV-1 gM on nuclear membranes.IMPORTANCE The mechanisms that enable integral proteins to be targeted to the inner nuclear membrane are poorly understood. Herpes simplex virus 1 (HSV-1) glycoprotein M (gM) is an interesting candidate, as it is dynamically relocalized from nuclear envelopes to the trans-Golgi network (TGN) in a virus- and time-dependent fashion. However, it was, until now, unclear how gM was directed to the nucleus or evaded that compartment later on. Through a proteomic study relying on a proximity-ligation assay, we identified several novel gM interacting partners, many of which are involved in vesicular transport. Analysis of select proteins revealed that XPO6 is required for gM to leave the nuclear membranes late in the infection. This was unexpected, as XPO6 is an exportin specifically associated with actin/profilin nuclear export. This raises some very interesting questions about the interaction of HSV-1 with the exportin machinery and the cargo specificity of XPO6.
Asunto(s)
Herpesvirus Humano 1/metabolismo , Carioferinas/metabolismo , Glicoproteínas de Membrana/metabolismo , Membrana Nuclear/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Proteínas Virales/metabolismo , Proteína de Unión al GTP ran/metabolismo , Red trans-Golgi/metabolismo , Biotinilación , Ligasas de Carbono-Nitrógeno/química , Ligasas de Carbono-Nitrógeno/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Expresión Génica , Herpesvirus Humano 1/genética , Interacciones Huésped-Patógeno/genética , Humanos , Carioferinas/genética , Glicoproteínas de Membrana/genética , Membrana Nuclear/virología , Unión Proteica , Transporte de Proteínas , Proteómica/métodos , Proteínas Represoras/química , Proteínas Represoras/metabolismo , Coloración y Etiquetado/métodos , Estreptavidina/química , Sinaptotagminas/genética , Sinaptotagminas/metabolismo , Proteínas del Envoltorio Viral/genética , Proteínas Virales/genética , Proteína de Unión al GTP ran/genética , Red trans-Golgi/virologíaRESUMEN
Herpesvirus infection usually relies on the interaction between viral protein and host protein to enhance replication of the enveloped virus. Fish Carassius auratus herpesvirus (CaHV) is highly pathogenic pathogen causing gill acute hemorrhages of crucian carp (Carassius auratus) and high moritality rates among those infected fish. The protein of CaHV (CaHV-138 L) containing two transmembrane (TM) domains and an immunoglobulin C-2 Type (IGc2) domain was predicted as a viral membrane protein. In this investigation, fluorescence observation showed that full-length CaHV-138 L mainly localized on the plasma membrane or around nuclear membrane of fish fathead minnow (FHM) cells in a punctate pattern. The TM domain deletion mutants of CaHV-138 L (ΔTM1, ΔTM2, and ΔTM1&ΔTM2) diffusely distributed in both the cytoplasm and the nucleus, mainly presented patchy fashion in the cytoplasm, and mainly presented both in the nucleus and in the cytoplasm, respectively. Obviously, the TM domain deletion mutants significantly affected CaHV-138 L subcellular localization. Meanwhile, colocalization assay showed that the full-length viral protein colocalized with mitochondria. Furthermore, the interaction between CaHV-138 L and host protein was identified by yeast two-hybrid (Y2H) and co-immunoprecipitation (co-IP) assays. The host mitochondrial protein FoF1 ATP synthase (FoF1-ATPase) that interacts with this viral protein was screened. The data indicated that CaHV-138 L can target to mitochondrial protein FoF1-ATPase, which might provide energy for virus replication through mediating mitochondrial ATP synthesis. This study has provided valuable information for better understanding of the links of herpesvirus proteins with aquaculture animal proteins.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Carpas/virología , Proteínas de Peces/metabolismo , Infecciones por Herpesviridae/veterinaria , Herpesviridae/genética , Proteínas Virales/metabolismo , Animales , Núcleo Celular/virología , Citoplasma/virología , Herpesviridae/patogenicidad , Infecciones por Herpesviridae/virología , Interacciones Microbiota-Huesped , Microscopía Fluorescente , Proteínas Mitocondriales/metabolismo , Membrana Nuclear/virologíaRESUMEN
Although it has been well-accepted that baculoviruses produce a virus envelop within the nucleus, the redistribution of membrane lipids in infected cells has not been demonstrated. Here, we characterize a baculovirus protein (Bm5/Ac13: renamed BION; baculovirus protein associated with both the inner- and outer nuclear membranes) that localizes to both the inner- and outer nuclear membranes and show that the nuclear membrane (NE) protein promotes formation of a virus-induced intranuclear structure, the peristromal region (PR). Consistent with its role in virus envelopment, the PR was found to contain viral membrane proteins and lipids, suggesting PR formation proceeds through intranuclear lipid accumulation. About 50% of the cells infected with a bion-deficient virus exhibited no polyhedra production due to lack of the PR. Association of BION with the NE rather than the PR may contribute to the formation of the PR and polyhedra via NE-to-PR lipid transport.
Asunto(s)
Baculoviridae/fisiología , Lípidos/química , Membrana Nuclear/virología , Replicación Viral/fisiología , Animales , Baculoviridae/ultraestructura , Transporte Biológico , Bombyx , Línea Celular , Células Epiteliales/ultraestructura , Células Epiteliales/virología , Células HeLa , Humanos , Metabolismo de los Lípidos , Membrana Nuclear/ultraestructuraRESUMEN
Morphology of the nucleus is an important regulator of gene expression. Nuclear morphology is in turn a function of the forces acting on it and the mechanical properties of the nuclear envelope. Here, we present a two-parameter, nondimensional mechanical model of the nucleus that reveals a relationship among nuclear shape parameters, such as projected area, surface area, and volume. Our model fits the morphology of individual nuclei and predicts the ratio between forces and modulus in each nucleus. We analyzed the changes in nuclear morphology of liver cells due to hepatitis C virus (HCV) infection using this model. The model predicted a decrease in the elastic modulus of the nuclear envelope and an increase in the pre-tension in cortical actin as the causes for the change in nuclear morphology. These predictions were validated biomechanically by showing that liver cells expressing HCV proteins possessed enhanced cellular stiffness and reduced nuclear stiffness. Concomitantly, cells expressing HCV proteins showed downregulation of lamin-A,C and upregulation of ß-actin, corroborating the predictions of the model. Our modeling assumptions are broadly applicable to adherent, monolayer cell cultures, making the model amenable to investigate changes in nuclear mechanics due to other stimuli by merely measuring nuclear morphology. Toward this, we present two techniques, graphical and numerical, to use our model for predicting physical changes in the nucleus.
Asunto(s)
Módulo de Elasticidad , Hepacivirus/fisiología , Modelos Teóricos , Membrana Nuclear/química , Replicación Viral , Actinas/química , Actinas/metabolismo , Línea Celular Tumoral , Hepatocitos/metabolismo , Hepatocitos/virología , Humanos , Lamina Tipo A/química , Lamina Tipo A/metabolismo , Membrana Nuclear/virologíaRESUMEN
Vpx, a virion-associated protein of Human Immunodeficiency Virus 2 (HIV-2) and Simian Immunodeficiency Virus (SIV) counteracts host restriction factor SAMDH1 for efficient viral DNA synthesis in the cytoplasm and mediates subsequent nuclear translocation of the viral genome. Vpx was found to be indispensable in the viral infection of terminally differentiated target cells and macaques infected with virions carrying truncated Vpx showed delayed pathogenesis, suggesting multiple roles of Vpx at different steps in the virus life cycle. The current study demonstrates a novel function of SIVsmPBj1.9 Vpx on the integrity of the nuclear envelope in HeLa cells. Results from the Super-Resolution Structured Illumination Microscopy (SR-SIM) analysis showed that Vpx puncta alter HeLa cell nuclear envelope assembly. Furthermore, three-dimensional (3D) SIM analysis of such regions suggests that Vpx is primed in a specific way to disrupt the nuclear envelope integrity. The nuclear incursion of cytoplasmic proteins through Vpx mediated ruptured nuclear envelope regions suggest that these events might play a critical role in the nuclear entry of otherwise cytoplasmically sequestered molecules and theirby may be assisting Vpx functions including the transport of viral genome into the nucleus, which is critical for the establishment of virus infection and pathogenesis.
Asunto(s)
Membrana Nuclear/virología , Síndrome de Inmunodeficiencia Adquirida del Simio/metabolismo , Virus de la Inmunodeficiencia de los Simios/fisiología , Proteínas Reguladoras y Accesorias Virales/metabolismo , Animales , Células HEK293 , Células HeLa , Interacciones Huésped-Patógeno , Humanos , Membrana Nuclear/metabolismo , Membrana Nuclear/patología , Síndrome de Inmunodeficiencia Adquirida del Simio/patologíaRESUMEN
Equine herpesvirus-1 (EHV-1) is an important pathogen, which infects horses worldwide with high morbidity but low mortality rates. The respiratory disorders and abortions are the most common indicators. Ab4p (an abortigenic and paralytic virus) is one of the most important and virulent strains. The development and functional characterization of the open reading frame-68 (ORF68) negative EHV-1 Ab4p mutants and an assessment of their roles in the infection at the cellular level were the main targets of the current study. Escherichia coli DH10ß containing the Ab4p bacterial artificial chromosome (pAb4pBAC) and Red/ET expression vector were used to develop different ORF68 mutants. Multi-step growth kinetic experiments were conducted in order to evaluate the growth properties of the constructed mutant viruses. Growth of the Ab4pΔORF68 showed the lowest titer, compared to the Ab4pΔORF68R, Ab4pΔORF68R non-sense, and the parent Ab4p viruses without any significant difference (P > 0.05). The growth of the mutant viruses was almost similar across the cell types, but viruses growth was more efficient in FHK cells as judged by the number of the obtained virus particles. The plaque size of Ab4pΔORF68 was significantly (40%) smaller than those of Ab4p (P < 0.01), Ab4pΔORF68R, and Ab4pΔORF68R non-sense viruses which confirmed the importance of ORF68 protein in the cell-to-cell transmission of EHV-1. Subcellular localization of the green fluorescent protein (GFP) ORF68 gene fusion product showed late expression with intranuclear localization of the transfected cells while immunofluorescent antibody technique (IFAT) localized it at the nucleus and nuclear membranes of the infected cells. Hence, it could be concluded that ORF68 protein may not be essential for EHV-1 Ab4p growth but plays a crucial role in virus penetration and transmission at the cellular level. Therefore, the generated EHV-1 ORF68 negative mutant could be a prospective candidate for the development of a vaccine marker.
Asunto(s)
Herpesvirus Équido 1/crecimiento & desarrollo , Herpesvirus Équido 1/genética , Proteínas Virales/genética , Animales , Línea Celular , Núcleo Celular/virología , Cromosomas Artificiales Bacterianos , Escherichia coli/genética , Genes Reporteros , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/genética , Caballos , Microscopía Fluorescente , Membrana Nuclear/virología , Proteínas Recombinantes de Fusión/análisis , Proteínas Recombinantes de Fusión/genética , Eliminación de Secuencia , Carga Viral , Ensayo de Placa Viral , Proteínas Virales/análisisRESUMEN
The ability of the matrix (M) protein of potato yellow dwarf virus (PYDV) to remodel nuclear membranes is controlled by a di-leucine motif located at residues 223 and 224 of its primary structure. This function can be uncoupled from that of its nuclear localization signal (NLS), which is controlled primarily by lysine and arginine residues immediately downstream of the LL motif. In planta localization of green fluorescent protein fusions, bimolecular fluorescence complementation assays with nuclear import receptor importin-α1 and yeast-based nuclear import assays provided three independent experimental approaches to validate the authenticity of the M-NLS. The carboxy terminus of M is predicted to contain a nuclear export signal, which is belived to be functional, given the ability of M to bind the Arabidopsis nuclear export receptor 1 (XPO1). The nuclear shuttle activity of M has implications for the cell-to-cell movement of PYDV nucleocapsids, based upon its interaction with the N and Y proteins.
Asunto(s)
Señales de Localización Nuclear/genética , Rhabdoviridae/genética , Proteínas de la Matriz Viral/genética , Transporte Activo de Núcleo Celular , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Núcleo Celular/metabolismo , Núcleo Celular/virología , Carioferinas/genética , Carioferinas/metabolismo , Membrana Nuclear/metabolismo , Membrana Nuclear/virología , Unión Proteica , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Rhabdoviridae/metabolismo , alfa Carioferinas/genéticaRESUMEN
The nuclear phase of herpesvirus replication is regulated through the formation of regulatory multi-component protein complexes. Viral genomic replication is followed by nuclear capsid assembly, DNA encapsidation and nuclear egress. The latter has been studied intensely pointing to the formation of a viral core nuclear egress complex (NEC) that recruits a multimeric assembly of viral and cellular factors for the reorganization of the nuclear envelope. To date, the mechanism of the association of human cytomegalovirus (HCMV) capsids with the NEC, which in turn initiates the specific steps of nuclear capsid budding, remains undefined. Here, we provide electron microscopy-based data demonstrating the association of both nuclear capsids and NEC proteins at nuclear lamina budding sites. Specifically, immunogold labelling of the core NEC constituent pUL53 and NEC-associated viral kinase pUL97 suggested an intranuclear NEC-capsid interaction. Staining patterns with phospho-specific lamin A/C antibodies are compatible with earlier postulates of targeted capsid egress at lamina-depleted areas. Important data were provided by co-immunoprecipitation and in vitro kinase analyses using lysates from HCMV-infected cells, nuclear fractions, or infectious virions. Data strongly suggest that nuclear capsids interact with pUL53 and pUL97. Combined, the findings support a refined concept of HCMV nuclear trafficking and NEC-capsid interaction.