RESUMEN
During entry, human papillomavirus (HPV) traffics from the endosome to the trans Golgi network (TGN) and Golgi and then the nucleus to cause infection. Although dynein is thought to play a role in HPV infection, how this host motor recruits the virus to support infection and which entry step(s) requires dynein are unclear. Here we show that the dynein cargo adaptor BICD2 binds to the HPV L2 capsid protein during entry, recruiting HPV to dynein for transport of the virus along the endosome-TGN/Golgi axis to promote infection. In the absence of BICD2 function, HPV accumulates in the endosome and TGN and infection is inhibited. Cell-based and in vitro binding studies identified a short segment near the C-terminus of L2 that can directly interact with BICD2. Our results reveal the molecular basis by which the dynein motor captures HPV to promote infection and identify this virus as a novel cargo of the BICD2 dynein adaptor.
Asunto(s)
Proteínas de la Cápside , Papillomavirus Humano 16 , Proteínas Oncogénicas Virales , Infecciones por Papillomavirus , Humanos , Proteínas de la Cápside/metabolismo , Papillomavirus Humano 16/metabolismo , Proteínas Oncogénicas Virales/metabolismo , Infecciones por Papillomavirus/metabolismo , Infecciones por Papillomavirus/virología , Dineínas/metabolismo , Endosomas/metabolismo , Endosomas/virología , Red trans-Golgi/metabolismo , Red trans-Golgi/virología , Internalización del Virus , Unión Proteica , Células HeLa , Proteínas Asociadas a Microtúbulos/metabolismo , Dineínas Citoplasmáticas/metabolismoRESUMEN
The membrane-associated RING-CH 8 protein (MARCH8), a member of the E3 ubiquitin ligase family, has broad-spectrum antiviral activity. However, some viruses hijack MARCH8 to promote virus replication, highlighting its dual role in the viral lifecycle. Most studies on MARCH8 have focused on RNA viruses, leaving its role in DNA viruses largely unexplored. Pseudorabies virus (PRV) is a large DNA virus that poses a potential threat to humans. In this study, we found that MARCH8 inhibited PRV replication at the cell-to-cell fusion stage. Interestingly, our findings proved that MARCH8 blocks gB cleavage by recruiting furin but this activity does not inhibit viral infection in vitro. Furthermore, we confirmed that MARCH8 inhibits cell-to-cell fusion independent of its E3 ubiquitin ligase activity but dependent on the interaction with the cell-to-cell fusion complex (gB, gD, gH, and gL). Finally, we discovered that the distribution of the cell-to-cell fusion complex is significantly altered and trapped within the trans-Golgi network. Overall, our results indicate that human MARCH8 acts as a potent antiviral host factor against PRV via trapping the cell-to-cell fusion complex in the trans-Golgi network.
Asunto(s)
Fusión Celular , Herpesvirus Suido 1 , Ubiquitina-Proteína Ligasas , Replicación Viral , Red trans-Golgi , Animales , Humanos , Línea Celular , Herpesvirus Suido 1/fisiología , Red trans-Golgi/metabolismo , Red trans-Golgi/virología , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
The membrane-associated RING-CH (MARCH) family of proteins are members of the E3 ubiquitin ligase family and are essential for a variety of biological functions. Currently, MARCH proteins are discovered to execute antiviral functions by directly triggering viral protein degradation or blocking the furin cleavage of viral class I fusion proteins. Here, we report a novel antiviral mechanism of MARCH1 and MARCH2 (MARCH1/2) in the replication of Pseudorabies virus (PRV), a member of the Herpesviridae family. We discovered MARCH1/2 restrict PRV replication at the cell-to-cell fusion step. Furthermore, MARCH1/2 block gB cleavage, and this is dependent on their E3 ligase activity. Interestingly, the blocking of gB cleavage by MARCH1/2 does not contribute to their antiviral activity in vitro. We discovered that MARCH1/2 are associated with the cell-to-cell fusion complex of gB, gD, gH, and gL and trap these viral proteins in the trans-Golgi network (TGN) rather than degrading them. Overall, we conclude that MARCH1/2 inhibit PRV by trapping the viral cell-to-cell fusion complex in TGN.
Asunto(s)
Herpesvirus Suido 1 , Ubiquitina-Proteína Ligasas , Replicación Viral , Red trans-Golgi , Herpesvirus Suido 1/fisiología , Animales , Red trans-Golgi/virología , Red trans-Golgi/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Fusión Celular , Porcinos , Línea Celular , Humanos , Proteínas Virales/metabolismo , Proteínas Virales/genética , Células HEK293 , Seudorrabia/virologíaRESUMEN
Mechanisms of infection are deciphered at the host-pathogen interface.
Asunto(s)
Antivirales , Tratamiento Farmacológico de COVID-19 , Endosomas , Neuropilina-1 , Receptores Virales , SARS-CoV-2 , Antivirales/farmacología , Antivirales/uso terapéutico , Endosomas/virología , Interacciones Huésped-Patógeno/efectos de los fármacos , Neuropilina-1/antagonistas & inhibidores , Receptores Virales/antagonistas & inhibidores , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/fisiología , Red trans-Golgi/virología , Internalización del Virus/efectos de los fármacos , HumanosRESUMEN
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
During initial infection, human papillomaviruses (HPV) take an unusual trafficking pathway through their host cell. It begins with a long period on the cell surface, during which the capsid is primed and a virus entry platform is formed. A specific type of clathrin-independent endocytosis and subsequent retrograde trafficking to the trans-Golgi network follow this. Cellular reorganization processes, which take place during mitosis, enable further virus transport and the establishment of infection while evading intrinsic cellular immune defenses. First, the fragmentation of the Golgi allows the release of membrane-encased virions, which are partially protected from cytoplasmic restriction factors. Second, the nuclear envelope breakdown opens the gate for these virus-vesicles to the cell nucleus. Third, the dis- and re-assembly of the PML nuclear bodies leads to the formation of modified virus-associated PML subnuclear structures, enabling viral transcription and replication. While remnants of the major capsid protein L1 and the viral DNA remain in a transport vesicle, the viral capsid protein L2 plays a crucial role during virus entry, as it adopts a membrane-spanning conformation for interaction with various cellular proteins to establish a successful infection. In this review, we follow the oncogenic HPV type 16 during its long journey into the nucleus, and contrast pro- and antiviral processes.
Asunto(s)
Células Epiteliales/virología , Papillomavirus Humano 16/fisiología , Infecciones por Papillomavirus/virología , Internalización del Virus , Animales , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Endocitosis , Papillomavirus Humano 16/genética , Humanos , Infecciones por Papillomavirus/fisiopatología , Red trans-Golgi/virologíaRESUMEN
Bovine herpesvirus envelope glycoprotein E (gE) and, in particular, the gE cytoplasmic tail (CT) is a virulence determinant in cattle. Also, the gE CT contributes to virus cell-to-cell spread and anterograde neuronal transport. In this study, our goal was to map the gE CT sub-domains that contribute to virus cell-to-cell spread property. A panel of gE-CT specific mutant viruses was constructed and characterized, in vitro, with respect to their plaque phenotypes, gE recycling and gE basolateral membrane targeting. The results revealed that disruption of the tyrosine-based motifs, 467YTSL470 and 563YTVV566, individually produced smaller plaque phenotypes than the wild type. However, they were slightly larger than the gE CT-null virus plaques. The Y467A mutation affected the gE endocytosis, gE trans-Golgi network (TGN) recycling, and gE virion incorporation properties. However, the Y563A mutation affected only the gE basolateral cell-surface redistribution function. Notably, the simultaneous Y467A/Y563A mutations produced gE CT-null virus-like plaque phenotypes.
Asunto(s)
Enfermedades de los Bovinos/virología , Citoplasma/virología , Infecciones por Herpesviridae/veterinaria , Herpesvirus Bovino 1/metabolismo , Proteínas Virales/química , Proteínas Virales/metabolismo , Secuencias de Aminoácidos , Animales , Bovinos , Endocitosis , Infecciones por Herpesviridae/virología , Herpesvirus Bovino 1/genética , Proteínas Virales/genética , Red trans-Golgi/virologíaRESUMEN
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
Previous analysis of postentry events revealed that human cytomegalovirus (HCMV) displays a unique, extended nuclear translocation pattern in monocytes. We determined that c-Src signaling through pentamer engagement of integrins is required upon HCMV entry to avoid sorting of the virus into late endosomes and subsequent degradation. To follow up on this previous study, we designed experiments to investigate how HCMV-induced signaling through the other major axis-the epidermal growth factor receptor (EGFR) kinase-regulates viral postentry events. Here we show that HCMV induces chronic and functional EGFR signaling that is distinct to the virus as compared to the natural EGFR ligand: EGF. This chronic EGFR kinase activity in infected monocytes is required for the proper subcellular localization of the viral particle during trafficking events, as well as for promoting translocation of viral DNA into the host nucleus. Our data indicate that HCMV glycoprotein B (gB) binds to EGFR at the monocyte surface, the virus and EGFR are internalized together, and gB remains bound to EGFR throughout viral postentry events until de-envelopment to promote the chronic EGFR kinase activity required for viral trafficking and nuclear translocation. These data highlight how initial EGFR signaling via viral binding is necessary for entry, but not sufficient to promote each viral trafficking event. HCMV appears to manipulate the EGFR kinase postentry, via gB-EGFR interaction, to be active at the critical points throughout the trafficking process that leads to nuclear translocation and productive infection of peripheral blood monocytes.
Asunto(s)
Núcleo Celular/metabolismo , Citomegalovirus/fisiología , Monocitos/virología , Proteínas del Envoltorio Viral/metabolismo , Núcleo Celular/virología , Células Cultivadas , ADN Viral/metabolismo , Endosomas/metabolismo , Endosomas/virología , Factor de Crecimiento Epidérmico/metabolismo , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/metabolismo , Interacciones Huésped-Patógeno , Humanos , Monocitos/metabolismo , Unión Proteica , Transducción de Señal , Red trans-Golgi/metabolismo , Red trans-Golgi/virologíaRESUMEN
Axonal sorting, the controlled passage of specific cargoes from the cell soma into the axon compartment, is critical for establishing and maintaining the polarity of mature neurons. To delineate axonal sorting events, we took advantage of two neuroinvasive alpha-herpesviruses. Human herpes simplex virus 1 (HSV-1) and pseudorabies virus of swine (PRV; suid herpesvirus 1) have evolved as robust cargo of axonal sorting and transport mechanisms. For efficient axonal sorting and subsequent egress from axons and presynaptic termini, progeny capsids depend on three viral membrane proteins (Us7 (gI), Us8 (gE), and Us9), which engage axon-directed kinesin motors. We present evidence that Us7-9 of the veterinary pathogen pseudorabies virus (PRV) form a tripartite complex to recruit Kif1a, a kinesin-3 motor. Based on multi-channel super-resolution and live TIRF microscopy, complex formation and motor recruitment occurs at the trans-Golgi network. Subsequently, progeny virus particles enter axons as enveloped capsids in a transport vesicle. Artificial recruitment of Kif1a using a drug-inducible heterodimerization system was sufficient to rescue axonal sorting and anterograde spread of PRV mutants devoid of Us7-9. Importantly, biophysical evidence suggests that Us9 is able to increase the velocity of Kif1a, a previously undescribed phenomenon. In addition to elucidating mechanisms governing axonal sorting, our results provide further insight into the composition of neuronal transport systems used by alpha-herpesviruses, which will be critical for both inhibiting the spread of infection and the safety of herpesvirus-based oncolytic therapies.
Asunto(s)
Axones/virología , Cápside/metabolismo , Herpes Simple/metabolismo , Herpesvirus Humano 1/metabolismo , Herpesvirus Suido 1/metabolismo , Cinesinas/metabolismo , Seudorrabia/metabolismo , Animales , Transporte Axonal , Axones/metabolismo , Herpes Simple/genética , Herpes Simple/virología , Herpesvirus Humano 1/genética , Herpesvirus Suido 1/genética , Interacciones Huésped-Patógeno , Humanos , Cinesinas/genética , Unión Proteica , Seudorrabia/genética , Seudorrabia/virología , Porcinos , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismo , Red trans-Golgi/metabolismo , Red trans-Golgi/virologíaRESUMEN
Previous studies have shown that the endoplasmic reticulum (ER)-anchored protein VAP is strictly required by human papillomavirus type 16 (HPV-16) for successful infectious entry. Entry appeared to be mediated in part through the induction of endosomal tubulation and subsequent transport of the virion to the trans-Golgi network (TGN). In this study, we were interested in investigating whether this mechanism of infectious entry is conserved across multiple Papillomavirus types. To do this, we analyzed the role of VAP and endosomal tubulation following infection with Pseudovirions (PsVs) derived from the alpha, beta, delta, kappa, and pi papillomavirus genera, reflecting viruses that are important human and animal pathogens. We demonstrate that VAP is essential for infection with all PV types analyzed. Furthermore, we find that VAP and EGFR-dependent endosomal tubulation is also induced by all these different Papillomaviruses. These results indicate an evolutionarily conserved requirement for VAP-induced endocytic tubulation during Papillomavirus infectious entry.
Asunto(s)
Endosomas/metabolismo , Endosomas/virología , Infecciones por Papillomavirus/virología , Internalización del Virus , Alphapapillomavirus/patogenicidad , Animales , Transporte Biológico Activo , Proteínas de la Cápside/metabolismo , Endocitosis , Retículo Endoplásmico/virología , Endosomas/genética , Técnicas de Inactivación de Genes , Células HEK293 , Células HeLa , Humanos , Proteínas de Microfilamentos/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Virión/metabolismo , Red trans-Golgi/genética , Red trans-Golgi/metabolismo , Red trans-Golgi/virologíaRESUMEN
The Ebola virus-encoded major matrix protein VP40 traffics to the plasma membrane, which leads to the formation of filamentous viral particles and subsequent viral egress. However, the cellular machineries underlying this process are not fully understood. In the present study, we have assessed the role of host endocytic recycling in Ebola virus particle formation. We found that a small GTPase Rab11, which regulates recycling of molecules among the trans-Golgi network, recycling endosomes, and the plasma membrane, was incorporated in Ebola virus-like particles. Although Rab11 predominantly localized in the perinuclear region, it distributed diffusely in the cytoplasm and partly localized in the periphery of the cells transiently expressing VP40. In contrast, Rab11 exhibited a perinuclear distribution when 2 VP40 derivatives that lack ability to traffic to the plasma membrane were expressed. Finally, expression of a dominant-negative form of Rab11 or knockdown of Rab11 inhibited both VP40-induced clusters at the plasma membrane and release of viral-like particles. Taken together, our findings demonstrate that Ebola virus exploits host endocytic recycling machinery to facilitate the trafficking of VP40 to the cell surface and the subsequent release of viral-like particles for its establishment of efficient viral egress.
Asunto(s)
Ebolavirus/metabolismo , Fiebre Hemorrágica Ebola/metabolismo , Fiebre Hemorrágica Ebola/virología , Vesículas Transportadoras/metabolismo , Virión/metabolismo , Liberación del Virus/fisiología , Proteínas de Unión al GTP rab/metabolismo , Animales , Membrana Celular/metabolismo , Membrana Celular/fisiología , Membrana Celular/virología , Chlorocebus aethiops , Endosomas/metabolismo , Endosomas/fisiología , Endosomas/virología , GTP Fosfohidrolasas/metabolismo , Células HEK293 , Humanos , Nucleoproteínas/metabolismo , Transporte de Proteínas/fisiología , Células Vero , Proteínas del Núcleo Viral/metabolismo , Red trans-Golgi/metabolismo , Red trans-Golgi/virologíaRESUMEN
Endocytic trafficking plays a major role in transport of incoming human papillomavirus (HPVs) from plasma membrane to the trans Golgi network (TGN) and ultimately into the nucleus. During this infectious entry, several cellular sorting factors are recruited by the viral capsid protein L2, which plays a critical role in ensuring successful transport of the L2/viral DNA complex to the nucleus. Later in the infection cycle, two viral oncoproteins, E5 and E6, have also been shown to modulate different aspects of endocytic transport pathways. In this review, we highlight how HPV makes use of and perturbs normal endocytic transport pathways, firstly to achieve infectious virus entry, secondly to produce productive infection and the completion of the viral life cycle and, finally, on rare occasions, to bring about the development of malignancy.
Asunto(s)
Endocitosis , Papillomaviridae/fisiología , Proteínas de la Cápside/metabolismo , Membrana Celular/virología , Núcleo Celular/virología , Humanos , Proteínas Oncogénicas Virales/metabolismo , Internalización del Virus , Red trans-Golgi/virologíaRESUMEN
The microtubule cytoskeleton is a primary organizer of viral infections for delivering virus particles to their sites of replication, establishing and maintaining subcellular compartments where distinct steps of viral morphogenesis take place, and ultimately dispersing viral progeny. One of the best characterized examples of virus motility is the anterograde transport of the wrapped virus form of vaccinia virus (VACV) from the trans-Golgi network (TGN) to the cell periphery by kinesin-1. Yet many aspects of this transport event are elusive due to the speed of motility and the challenges of imaging this stage at high resolution over extended time periods. We have established a novel imaging technology to track virus transport that uses photoconvertible fluorescent recombinant viruses to track subsets of virus particles from their site of origin and determine their destination. Here we image virus exit from the TGN and their rate of egress to the cell periphery. We demonstrate a role for kinesin-1 engagement in regulating virus exit from the TGN by removing A36 and F12 function, critical viral mediators of kinesin-1 recruitment to virus particles. Phototracking viral particles and components during infection is a powerful new imaging approach to elucidate mechanisms of virus replication.
Asunto(s)
Citoplasma/metabolismo , Virus Vaccinia/fisiología , Virión/fisiología , Liberación del Virus , Red trans-Golgi/fisiología , Transporte Biológico , Citoplasma/virología , Células HeLa , Humanos , Cinesinas/metabolismo , Imagen Óptica , Virus Vaccinia/genética , Proteínas Virales/genética , Proteínas Estructurales Virales/genética , Replicación Viral , Red trans-Golgi/virologíaRESUMEN
ORF9p (homologous to herpes simplex virus 1 [HSV-1] VP22) is a varicella-zoster virus (VZV) tegument protein essential for viral replication. Even though its precise functions are far from being fully described, a role in the secondary envelopment of the virus has long been suggested. We performed a yeast two-hybrid screen to identify cellular proteins interacting with ORF9p that might be important for this function. We found 31 ORF9p interaction partners, among which was AP1M1, the µ subunit of the adaptor protein complex 1 (AP-1). AP-1 is a heterotetramer involved in intracellular vesicle-mediated transport and regulates the shuttling of cargo proteins between endosomes and the trans-Golgi network via clathrin-coated vesicles. We confirmed that AP-1 interacts with ORF9p in infected cells and mapped potential interaction motifs within ORF9p. We generated VZV mutants in which each of these motifs was individually impaired and identified leucine 231 in ORF9p to be critical for the interaction with AP-1. Disrupting ORF9p binding to AP-1 by mutating leucine 231 to alanine in ORF9p strongly impaired viral growth, most likely by preventing efficient secondary envelopment of the virus. Leucine 231 is part of a dileucine motif conserved among alphaherpesviruses, and we showed that VP22 of Marek's disease virus and HSV-2 also interacts with AP-1. This indicates that the function of this interaction in secondary envelopment might be conserved as well.IMPORTANCE Herpesviruses are responsible for infections that, especially in immunocompromised patients, can lead to severe complications, including neurological symptoms and strokes. The constant emergence of viral strains resistant to classical antivirals (mainly acyclovir and its derivatives) pleads for the identification of new targets for future antiviral treatments. Cellular adaptor protein (AP) complexes have been implicated in the correct addressing of herpesvirus glycoproteins in infected cells, and the discovery that a major constituent of the varicella-zoster virus tegument interacts with AP-1 reveals a previously unsuspected role of this tegument protein. Unraveling the complex mechanisms leading to virion production will certainly be an important step in the discovery of future therapeutic targets.
Asunto(s)
Complejo 1 de Proteína Adaptadora/metabolismo , Subunidades mu de Complejo de Proteína Adaptadora/metabolismo , Vesículas Cubiertas por Clatrina/metabolismo , Herpesvirus Humano 3/metabolismo , Proteínas Virales/metabolismo , Red trans-Golgi/metabolismo , Complejo 1 de Proteína Adaptadora/genética , Subunidades mu de Complejo de Proteína Adaptadora/genética , Secuencias de Aminoácidos , Sustitución de Aminoácidos , Línea Celular Tumoral , Vesículas Cubiertas por Clatrina/genética , Vesículas Cubiertas por Clatrina/virología , Herpesvirus Humano 3/genética , Humanos , Mutación Missense , Proteínas Virales/genética , Red trans-Golgi/genética , Red trans-Golgi/virologíaRESUMEN
The HIV type 1 pathogenicity factor Nef enhances viral replication by modulating multiple host cell pathways, including tuning the activation state of infected CD4 T lymphocytes to optimize virus spread. For this, Nef inhibits anterograde transport of the Src family kinase (SFK) Lck toward the plasma membrane (PM). This leads to retargeting of the kinase to the trans-Golgi network, whereas the intracellular transport of a related SFK, Fyn, is unaffected by Nef. The 18-amino acid Src homology 4 (SH4) domain membrane anchor of Lck is necessary and sufficient for Nef-mediated retargeting, but other details of this process are not known. The goal of this study was therefore to identify characteristics of SH4 domains responsive to Nef and the transport machinery used. Screening a panel of SFK SH4 domains revealed two groups that were sensitive or insensitive for trans-Golgi network retargeting by Nef as well as the importance of the amino acid at position 8 for determining Nef sensitivity. Anterograde transport of Nef-sensitive domains was characterized by slower delivery to the PM and initial targeting to Golgi membranes, where transport was arrested in the presence of Nef. For Nef-sensitive SH4 domains, ectopic expression of the lipoprotein binding chaperone Unc119a or the GTPase Arl3 or reduction of their endogenous expression phenocopied the effect of Nef. Together, these results suggest that, analogous to K-Ras, Nef-sensitive SH4 domains are transported to the PM by a cycle of solubilization and membrane insertion and that intrinsic properties define SH4 domains as cargo of this Nef-sensitive lipoprotein binding chaperone-GTPase transport cycle.
Asunto(s)
Membrana Celular/metabolismo , VIH-1/metabolismo , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/metabolismo , Linfocitos T/virología , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/metabolismo , Red trans-Golgi/metabolismo , Factores de Ribosilacion-ADP/genética , Factores de Ribosilacion-ADP/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Membrana Celular/virología , Regulación de la Expresión Génica , VIH-1/genética , Interacciones Huésped-Patógeno , Humanos , Células Jurkat , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/química , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/genética , Cultivo Primario de Células , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Transporte de Proteínas , Proteínas Proto-Oncogénicas c-fyn/genética , Proteínas Proto-Oncogénicas c-fyn/metabolismo , Transducción de Señal , Linfocitos T/metabolismo , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/genética , Red trans-Golgi/virologíaRESUMEN
Human papillomavirus (HPV) infection involves complex interactions with the endocytic transport machinery, which ultimately facilitates the entry of the incoming viral genomes into the trans-Golgi network (TGN) and their subsequent nuclear entry during mitosis. The endosomal pathway is a highly dynamic intracellular transport system, which consists of vesicular compartments and tubular extensions, although it is currently unclear whether incoming viruses specifically alter the endocytic machinery. In this study, using MICAL-L1 as a marker for tubulating endosomes, we show that incoming HPV-16 virions induce a profound alteration in global levels of endocytic tubulation. In addition, we also show a critical requirement for the endoplasmic reticulum (ER)-anchored protein VAP in this process. VAP plays an essential role in actin nucleation and endosome-to-Golgi transport. Indeed, the loss of VAP results in a dramatic decrease in the level of endosomal tubulation induced by incoming HPV-16 virions. This is also accompanied by a marked reduction in virus infectivity. In VAP knockdown cells, we see that the defect in virus trafficking occurs after capsid disassembly but prior to localization at the trans-Golgi network, with the incoming virion-transduced DNA accumulating in Vps29/TGN46-positive hybrid vesicles. Taken together, these studies demonstrate that infection with HPV-16 virions induces marked alterations of endocytic transport pathways, some of which are VAP dependent and required for the endosome-to-Golgi transport of the incoming viral L2/DNA complex.IMPORTANCE Human papillomavirus infectious entry involves multiple interactions with the endocytic transport machinery. In this study, we show that incoming HPV-16 virions induce a dramatic increase in endocytic tubulation. This tubulation requires ER-associated VAP, which plays a critical role in ensuring the delivery of cargoes from the endocytic compartments to the trans-Golgi network. Indeed, the loss of VAP blocks HPV infectious entry at a step after capsid uncoating but prior to localization at the trans-Golgi network. These results define a critical role for ER-associated VAP in endocytic tubulation and in HPV-16 infectious entry.
Asunto(s)
Endocitosis , Endosomas/metabolismo , Papillomavirus Humano 16/metabolismo , Infecciones por Papillomavirus/metabolismo , Virión/metabolismo , Internalización del Virus , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Transporte Biológico Activo/genética , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Endosomas/genética , Endosomas/virología , Células HeLa , Papillomavirus Humano 16/genética , Humanos , Proteínas con Dominio LIM/genética , Proteínas con Dominio LIM/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de Microfilamentos , Oxigenasas de Función Mixta , Infecciones por Papillomavirus/genética , Infecciones por Papillomavirus/patología , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Virión/genética , Red trans-Golgi/genética , Red trans-Golgi/metabolismo , Red trans-Golgi/virologíaRESUMEN
Cathepsins, endosomal acid proteases, are transported from the trans-Golgi network to late endosomes by the mannose-6-phosphate receptor (M6PR). We have previously demonstrated that some rotavirus strains, like UK, Wa, WI61, DS-1, and YM, require the cation-dependent (CD-) M6PR and cathepsins to enter from late endosomes to the cytoplasm in MA104 cells, while other strains, like the simian strain RRV, which enter cells from maturing endosomes, do not. However, the role of other trans-Golgi network-late endosome transporters, such as the cation-independent (CI-) M6PR and sortillin-1, has not been evaluated. In this work, we found that several rotavirus strains that require the CD-M6PR for cell entry are also dependent on CI-M6PR and sortilin-1. Furthermore, we showed that the infectivity of all these rotavirus strains also requires cathepsins to enter not only MA104 cells, but also human intestinal Caco-2 cells. This study identifies sortilin-1 as a novel cell factor necessary for the infectivity of a virus; in addition, our results strongly suggest that cathepsins could be common cell factors needed for the infectivity of most rotavirus strains.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/genética , Catepsinas/genética , Interacciones Huésped-Patógeno , Receptor IGF Tipo 2/genética , Rotavirus/metabolismo , Internalización del Virus , Proteínas Adaptadoras del Transporte Vesicular/antagonistas & inhibidores , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Animales , Transporte Biológico , Células CACO-2 , Catepsinas/antagonistas & inhibidores , Catepsinas/metabolismo , Línea Celular , Endosomas/metabolismo , Endosomas/virología , Fibroblastos/metabolismo , Fibroblastos/virología , Regulación de la Expresión Génica , Genotipo , Humanos , Macaca mulatta , Ratones , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Receptor IGF Tipo 2/antagonistas & inhibidores , Receptor IGF Tipo 2/metabolismo , Rotavirus/clasificación , Rotavirus/genética , Rotavirus/crecimiento & desarrollo , Especificidad de la Especie , Red trans-Golgi/metabolismo , Red trans-Golgi/virologíaRESUMEN
The K15P membrane protein of Kaposi's sarcoma-associated herpesvirus (KSHV) interacts with multiple cellular signaling pathways and is thought to play key roles in KSHV-associated endothelial cell angiogenesis, regulation of B-cell receptor (BCR) signaling, and the survival, activation, and proliferation of BCR-negative primary effusion lymphoma (PEL) cells. Although full-length K15P is â¼45 kDa, numerous lower-molecular-weight forms of the protein exist as a result of differential splicing and poorly characterized posttranslational processing. K15P has been reported to localize to numerous subcellular organelles in heterologous expression studies, but there are limited data concerning the sorting of K15P in KSHV-infected cells. The relationships between the various molecular weight forms of K15P, their subcellular distribution, and how these may differ in latent and lytic KSHV infections are poorly understood. Here we report that a cDNA encoding a full-length, â¼45-kDa K15P reporter protein is expressed as an â¼23- to 24-kDa species that colocalizes with the trans-Golgi network (TGN) marker TGN46 in KSHV-infected PEL cells. Following lytic reactivation by sodium butyrate, the levels of the â¼23- to 24-kDa protein diminish, and the full-length, â¼45-kDa K15P protein accumulates. This is accompanied by apparent fragmentation of the TGN and redistribution of K15P to a dispersed peripheral location. Similar results were seen when lytic reactivation was stimulated by the KSHV protein replication and transcription activator (RTA) and during spontaneous reactivation. We speculate that expression of different molecular weight forms of K15P in distinct cellular locations reflects the alternative demands placed upon the protein in the latent and lytic phases.IMPORTANCE The K15P protein of Kaposi's sarcoma-associated herpesvirus (KSHV) is thought to play key roles in disease, including KSHV-associated angiogenesis and the survival and growth of primary effusion lymphoma (PEL) cells. The protein exists in multiple molecular weight forms, and its intracellular trafficking is poorly understood. Here we demonstrate that the molecular weight form of a reporter K15P molecule and its intracellular distribution change when KSHV switches from its latent (quiescent) phase to the lytic, infectious state. We speculate that expression of different molecular weight forms of K15P in distinct cellular locations reflects the alternative demands placed upon the protein in the viral latent and lytic stages.
Asunto(s)
Regulación Viral de la Expresión Génica , Linfoma de Efusión Primaria/metabolismo , Sarcoma de Kaposi/metabolismo , Fracciones Subcelulares/metabolismo , Proteínas Virales/metabolismo , Activación Viral/fisiología , Latencia del Virus/fisiología , Membrana Celular/metabolismo , Membrana Celular/virología , Herpesvirus Humano 8/fisiología , Humanos , Linfoma de Efusión Primaria/virología , Mitocondrias/metabolismo , Mitocondrias/virología , Sarcoma de Kaposi/virología , Fracciones Subcelulares/virología , Red trans-Golgi/metabolismo , Red trans-Golgi/virologíaRESUMEN
The human papillomavirus type 16 (HPV16) L2 protein acts as a chaperone to ensure that the viral genome (vDNA) traffics from endosomes to the trans-Golgi network (TGN) and eventually the nucleus, where HPV replication occurs. En route to the nucleus, the L2/vDNA complex must translocate across limiting intracellular membranes. The details of this critical process remain poorly characterized. We have developed a system based on subcellular compartmentalization of the enzyme BirA and its cognate substrate to detect membrane translocation of L2-BirA from incoming virions. We find that L2 translocation requires transport to the TGN and is strictly dependent on entry into mitosis, coinciding with mitotic entry in synchronized cells. Cell cycle arrest causes retention of L2/vDNA at the TGN; only release and progression past G2/M enables translocation across the limiting membrane and subsequent infection. Microscopy of EdU-labeled vDNA reveals a rapid and dramatic shift in vDNA localization during early mitosis. At late G2/early prophase vDNA egresses from the TGN to a pericentriolar location, accumulating there through prometaphase where it begins to associate with condensed chromosomes. By metaphase and throughout anaphase the vDNA is seen bound to the mitotic chromosomes, ensuring distribution into both daughter nuclei. Mutations in a newly defined chromatin binding region of L2 potently blocked translocation, suggesting that translocation is dependent on chromatin binding during prometaphase. This represents the first time a virus has been shown to functionally couple the penetration of limiting membranes to cellular mitosis, explaining in part the tropism of HPV for mitotic basal keratinocytes.