RESUMO
During internalization and trafficking, human papillomavirus (HPV) moves from the cell surface to the endosome where the transmembrane protease γ-secretase promotes insertion of the viral L2 capsid protein into the endosome membrane. Protrusion of L2 through the endosome membrane into the cytosol allows the recruitment of cytosolic host factors that target the virus to the Golgi en route for productive infection. How endosome-localized HPV is delivered to γ-secretase, a decisive infection step, is unclear. Here we demonstrate that cytosolic p120 catenin, likely via an unidentified transmembrane protein, interacts with HPV at early time-points during viral internalization and trafficking. In the endosome, p120 is not required for low pH-dependent disassembly of the HPV L1 capsid protein from the incoming virion. Rather, p120 is required for HPV to interact with γ-secretase-an interaction that ensures the virus is transported along a productive route. Our findings clarify an enigmatic HPV infection step and provide critical insights into HPV infection that may lead to new therapeutic strategies against HPV-induced diseases.
Assuntos
Alphapapillomavirus/patogenicidade , Cateninas/metabolismo , Infecções por Papillomavirus/virologia , Internalização do Vírus , Alphapapillomavirus/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Proteínas do Capsídeo/metabolismo , Endossomos/metabolismo , Células HeLa/virologia , Humanos , Membranas Intracelulares/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Infecções por Papillomavirus/metabolismo , Transporte Proteico/fisiologia , Vírion/metabolismoRESUMO
Despite their importance as human pathogens, entry of human papillomaviruses (HPVs) into cells is poorly understood. The transmembrane protease γ-secretase executes a crucial function during the early stages of HPV infection, but the role of γ-secretase in infection and the identity of its critical substrate are unknown. Here we demonstrate that γ-secretase harbors a previously uncharacterized chaperone function, promoting low pH-dependent insertion of the HPV L2 capsid protein into endosomal membranes. Upon membrane insertion, L2 recruits the cytosolic retromer, which enables the L2 viral genome complex to enter the retrograde transport pathway and traffic to the Golgi en route for infection. Although a small fraction of membrane-inserted L2 is also cleaved by γ-secretase, this proteolytic event appears dispensable for HPV infection. Our findings demonstrate that γ-secretase is endowed with an activity that can promote membrane insertion of L2, thereby targeting the virus to the productive infectious pathway.
Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Proteínas do Capsídeo/metabolismo , Papillomavirus Humano 16/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Infecções por Papillomavirus/metabolismo , Secretases da Proteína Precursora do Amiloide/genética , Proteínas do Capsídeo/genética , Endossomos/genética , Endossomos/metabolismo , Endossomos/patologia , Endossomos/virologia , Complexo de Golgi/genética , Complexo de Golgi/metabolismo , Complexo de Golgi/patologia , Complexo de Golgi/virologia , Células HEK293 , Células HeLa , Papillomavirus Humano 16/genética , Humanos , Concentração de Íons de Hidrogênio , Membranas Intracelulares/metabolismo , Membranas Intracelulares/patologia , Membranas Intracelulares/virologia , Chaperonas Moleculares/genética , Proteínas Oncogênicas Virais/genética , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/patologia , ProteóliseRESUMO
The molecular mechanism by which non-enveloped viruses penetrate biological membranes remains enigmatic. The non-enveloped polyomavirus SV40 penetrates the endoplasmic reticulum (ER) membrane to reach the cytosol and cause infection. We previously demonstrated that SV40 creates its own membrane penetration structure by mobilizing select transmembrane proteins to distinct puncta in the ER membrane called foci that likely function as the cytosol entry sites. How these ER membrane proteins reorganize into the foci is unknown. B12 is a transmembrane J-protein that mobilizes into the foci to promote cytosol entry of SV40. Here we identify two closely related ER membrane proteins Erlin1 and Erlin2 (Erlin1/2) as B12-interaction partners. Strikingly, SV40 recruits B12 to the foci by inducing release of this J-protein from Erlin1/2. Our data thus reveal how a non-enveloped virus promotes its own membrane translocation by triggering the release and recruitment of a critical transport factor to the membrane penetration site.
Assuntos
Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Vírus 40 dos Símios/fisiologia , Internalização do Vírus , Linhagem Celular , Retículo Endoplasmático/virologia , Técnicas de Silenciamento de Genes , Humanos , Membranas Intracelulares/metabolismo , Membranas Intracelulares/virologia , Infecções por Polyomavirus/metabolismoRESUMO
Membrane penetration by nonenveloped viruses remains enigmatic. In the case of the nonenveloped polyomavirus simian virus 40 (SV40), the virus penetrates the endoplasmic reticulum (ER) membrane to reach the cytosol and then traffics to the nucleus to cause infection. We previously demonstrated that the cytosolic Hsc70-SGTA-Hsp105 complex is tethered to the ER membrane, where Hsp105 and SGTA facilitate the extraction of SV40 from the ER and transport of the virus into the cytosol. We now find that Hsc70 also ejects SV40 from the ER into the cytosol in a step regulated by SGTA. Although SGTA's N-terminal domain, which mediates homodimerization and recruits cellular adaptors, is dispensable during ER-to-cytosol transport of SV40, this domain appears to exert an unexpected post-ER membrane translocation function during SV40 entry. Our study thus establishes a critical function of Hsc70 within the Hsc70-SGTA-Hsp105 complex in promoting SV40 ER-to-cytosol membrane penetration and unveils a role of SGTA in controlling this step.IMPORTANCE How a nonenveloped virus transports across a biological membrane to cause infection remains mysterious. One enigmatic step is whether host cytosolic components are co-opted to transport the viral particle into the cytosol. During ER-to-cytosol membrane transport of the nonenveloped polyomavirus SV40, a decisive infection step, a cytosolic complex composed of Hsc70-SGTA-Hsp105 was previously shown to associate with the ER membrane. SGTA and Hsp105 have been shown to extract SV40 from the ER and transport the virus into the cytosol. We demonstrate here a critical role of Hsc70 in SV40 ER-to-cytosol penetration and reveal how SGTA controls Hsc70 to impact this process.
Assuntos
Proteínas de Transporte/metabolismo , Citosol/virologia , Retículo Endoplasmático/virologia , Proteínas de Choque Térmico HSC70/metabolismo , Vírus 40 dos Símios/fisiologia , Internalização do Vírus , Animais , Transporte Biológico/fisiologia , Células COS , Proteínas de Transporte/genética , Linhagem Celular , Chlorocebus aethiops , Citosol/metabolismo , Retículo Endoplasmático/fisiologia , Regulação da Expressão Gênica , Células HEK293 , Proteínas de Choque Térmico HSC70/genética , Interações Hospedeiro-Patógeno/genética , Humanos , Membranas Intracelulares/virologia , Chaperonas Moleculares/metabolismo , RNA Interferente PequenoRESUMO
Polyomavirus (Py) is a non-enveloped, double stranded DNA virus that causes a myriad of devastating human diseases for immunocompromised individuals. To cause infection, Py binds to its receptors on the plasma membrane, is endocytosed, and sorts to the endoplasmic reticulum (ER). From here, Py penetrates the ER membrane to reach the cytosol. Ensuing nuclear entry enables the virus to cause infection. How Py penetrates the ER membrane to access the cytosol is a decisive infection step that is enigmatic. In this review, I highlight the mechanisms by which host cell functions facilitate Py translocation across the ER membrane into the cytosol.
RESUMO
Destabilization of a non-enveloped virus generates a membrane transport-competent viral particle. Here we probe polyomavirus SV40 endoplasmic reticulum (ER)-to-cytosol membrane transport, a decisive infection step where destabilization initiates this non-enveloped virus for membrane penetration. We find that a member of the ER membrane protein complex (EMC) called EMC1 promotes SV40 ER membrane transport and infection. Surprisingly, EMC1 does so by using its predicted transmembrane residue D961 to bind to and stabilize the membrane-embedded partially destabilized SV40, thereby preventing premature viral disassembly. EMC1-dependent stabilization enables SV40 to engage a cytosolic extraction complex that ejects the virus into the cytosol. Thus EMC1 acts as a molecular chaperone, bracing the destabilized SV40 in a transport-competent state. Our findings reveal the novel principle that coordinated destabilization-stabilization drives membrane transport of a non-enveloped virus.
Assuntos
Retículo Endoplasmático/metabolismo , Proteínas/metabolismo , Vírus 40 dos Símios/fisiologia , Internalização do Vírus , Animais , Transporte Biológico , Células COS , Células HEK293 , Humanos , Proteínas de MembranaRESUMO
Endoplasmic reticulum (ER)-associated degradation (ERAD) represents a principle quality control mechanism to clear misfolded proteins in the ER; however, its physiological significance and the nature of endogenous ERAD substrates remain largely unexplored. Here we discover that IRE1α, the sensor of the unfolded protein response (UPR), is a bona fide substrate of the Sel1L-Hrd1 ERAD complex. ERAD-mediated IRE1α degradation occurs under basal conditions in a BiP-dependent manner, requires both the intramembrane hydrophilic residues of IRE1α and the lectin protein OS9, and is attenuated by ER stress. ERAD deficiency causes IRE1α protein stabilization, accumulation and mild activation both in vitro and in vivo. Although enterocyte-specific Sel1L-knockout mice (Sel1L(ΔIEC)) are viable and seem normal, they are highly susceptible to experimental colitis and inflammation-associated dysbiosis, in an IRE1α-dependent but CHOP-independent manner. Hence, Sel1L-Hrd1 ERAD serves a distinct, essential function in restraint of IRE1α signalling in vivo by managing its protein turnover.
Assuntos
Degradação Associada com o Retículo Endoplasmático/genética , Endorribonucleases/genética , Proteínas Serina-Treonina Quinases/genética , Resposta a Proteínas não Dobradas/genética , Animais , Sequência de Bases , Western Blotting , Células Cultivadas , Retículo Endoplasmático/metabolismo , Chaperona BiP do Retículo Endoplasmático , Endorribonucleases/metabolismo , Enterócitos/metabolismo , Feminino , Perfilação da Expressão Gênica , Células HEK293 , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Lectinas/genética , Lectinas/metabolismo , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas/genética , Proteínas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Mammalian cytosolic Hsp110 family, in concert with the Hsc70:J-protein complex, functions as a disaggregation machinery to rectify protein misfolding problems. Here we uncover a novel role of this machinery in driving membrane translocation during viral entry. The non-enveloped virus SV40 penetrates the endoplasmic reticulum (ER) membrane to reach the cytosol, a critical infection step. Combining biochemical, cell-based, and imaging approaches, we find that the Hsp110 family member Hsp105 associates with the ER membrane J-protein B14. Here Hsp105 cooperates with Hsc70 and extracts the membrane-penetrating SV40 into the cytosol, potentially by disassembling the membrane-embedded virus. Hence the energy provided by the Hsc70-dependent Hsp105 disaggregation machinery can be harnessed to catalyze a membrane translocation event.
Assuntos
Retículo Endoplasmático/virologia , Interações Hospedeiro-Parasita/fisiologia , Infecções por Polyomavirus/metabolismo , Vírus 40 dos Símios/patogenicidade , Infecções Tumorais por Vírus/metabolismo , Transporte Biológico/fisiologia , Linhagem Celular , Retículo Endoplasmático/metabolismo , Proteínas de Choque Térmico HSC70/metabolismo , Proteínas de Choque Térmico HSP110/metabolismo , Humanos , Immunoblotting , Imunoprecipitação , Microscopia de Fluorescência , Transdução de Sinais/fisiologia , TransfecçãoRESUMO
UNLABELLED: The nonenveloped polyomavirus (PyV) simian virus 40 (SV40) traffics from the cell surface to the endoplasmic reticulum (ER), where it penetrates the ER membrane to reach the cytosol before mobilizing into the nucleus to cause infection. Prior to ER membrane penetration, ER lumenal factors impart structural rearrangements to the virus, generating a translocation-competent virion capable of crossing the ER membrane. Here we identify ERdj5 as an ER enzyme that reduces SV40's disulfide bonds, a reaction important for its ER membrane transport and infection. ERdj5 also mediates human BK PyV infection. This enzyme cooperates with protein disulfide isomerase (PDI), a redox chaperone previously implicated in the unfolding of SV40, to fully stimulate membrane penetration. Negative-stain electron microscopy of ER-localized SV40 suggests that ERdj5 and PDI impart structural rearrangements to the virus. These conformational changes enable SV40 to engage BAP31, an ER membrane protein essential for supporting membrane penetration of the virus. Uncoupling of SV40 from BAP31 traps the virus in ER subdomains called foci, which likely serve as depots from where SV40 gains access to the cytosol. Our study thus pinpoints two ER lumenal factors that coordinately prime SV40 for ER membrane translocation and establishes a functional connection between lumenal and membrane events driving this process. IMPORTANCE: PyVs are established etiologic agents of many debilitating human diseases, especially in immunocompromised individuals. To infect cells at the cellular level, this virus family must penetrate the host ER membrane to reach the cytosol, a critical entry step. In this report, we identify two ER lumenal factors that prepare the virus for ER membrane translocation and connect these lumenal events with events on the ER membrane. Pinpointing cellular components necessary for supporting PyV infection should lead to rational therapeutic strategies for preventing and treating PyV-related diseases.
Assuntos
Retículo Endoplasmático/enzimologia , Proteínas de Choque Térmico HSP40/metabolismo , Chaperonas Moleculares/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Vírus 40 dos Símios/patogenicidade , Animais , Vírus BK/patogenicidade , Transporte Biológico , Linhagem Celular , Chlorocebus aethiops , Dissulfetos/metabolismo , Proteínas de Choque Térmico HSP40/genética , Humanos , Proteínas de Membrana/metabolismo , Chaperonas Moleculares/genética , Oxirredução , Infecções por Polyomavirus/genética , Infecções por Polyomavirus/patologia , Infecções por Polyomavirus/virologia , Isomerases de Dissulfetos de Proteínas/genética , Interferência de RNA , RNA Interferente Pequeno , Infecções Tumorais por Vírus/genética , Infecções Tumorais por Vírus/patologia , Infecções Tumorais por Vírus/virologia , Internalização do VírusRESUMO
UNLABELLED: The nonenveloped simian polyomavirus (PyV) simian virus 40 (SV40) hijacks the endoplasmic reticulum (ER) quality control machinery to penetrate the ER membrane and reach the cytosol, a critical infection step. During entry, SV40 traffics to the ER, where host-induced conformational changes render the virus hydrophobic. The hydrophobic virus binds and integrates into the ER lipid bilayer to initiate membrane penetration. However, prior to membrane transport, the hydrophobic SV40 recruits the ER-resident Hsp70 BiP, which holds the virus in a transport-competent state until it is ready to cross the ER membrane. Here we probed how BiP disengages from SV40 to enable the virus to penetrate the ER membrane. We found that nucleotide exchange factor (NEF) Grp170 induces nucleotide exchange of BiP and releases SV40 from BiP. Importantly, this reaction promotes SV40 ER-to-cytosol transport and infection. The human BK PyV also relies on Grp170 for successful infection. Interestingly, SV40 mobilizes a pool of Grp170 into discrete puncta in the ER called foci. These foci, postulated to represent the ER membrane penetration site, harbor ER components, including BiP, known to facilitate viral ER-to-cytosol transport. Our results thus identify a nucleotide exchange activity essential for catalyzing the most proximal event before ER membrane penetration of PyVs. IMPORTANCE: PyVs are known to cause debilitating human diseases. During entry, this virus family, including monkey SV40 and human BK PyV, hijacks ER protein quality control machinery to breach the ER membrane and access the cytosol, a decisive infection step. In this study, we pinpointed an ER-resident factor that executes a crucial role in promoting ER-to-cytosol membrane penetration of PyVs. Identifying a host factor that facilitates entry of the PyV family thus provides additional therapeutic targets to combat PyV-induced diseases.
Assuntos
Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico/metabolismo , Membranas Intracelulares/metabolismo , Infecções por Polyomavirus/fisiopatologia , Vírus 40 dos Símios/fisiologia , Transporte Biológico/fisiologia , Citosol/virologia , Retículo Endoplasmático/virologia , Chaperona BiP do Retículo Endoplasmático , Imunofluorescência , Técnicas de Silenciamento de Genes , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Células HEK293 , Proteínas de Choque Térmico HSP70/genética , Humanos , Luciferases , Reação em Cadeia da PolimeraseRESUMO
The icosahedral capsid structure of simian virus 40 (diameter, 45 nm) consists of 72 pentameric subunits, with each subunit formed by five VP1 molecules. Electron microscopy, immuno-gold labeling, and ζ-potential analysis showed that purified recombinant VP1 pentamers covered polystyrene beads measuring 100, 200, and 500 nm in diameter, as well as silica beads. In addition to covering spherical beads, VP1 pentamers covered cubic magnetite beads, as well as the distorted surface structures of liposomes. These findings indicate that VP1 pentamers could coat artificial beads of various shapes and sizes larger than the natural capsid. Technology based on VP1 pentamers may be useful in providing a capsid-like surface for enclosed materials, enhancing their stability and cellular uptake for drug delivery systems.
RESUMO
Nonenveloped viruses undergo conformational changes that enable them to bind to, disrupt, and penetrate a biological membrane leading to successful infection. We assessed whether cytosolic factors play any role in the endoplasmic reticulum (ER) membrane penetration of the nonenveloped SV40. We find the cytosolic SGTA-Hsc70 complex interacts with the ER transmembrane J-proteins DnaJB14 (B14) and DnaJB12 (B12), two cellular factors previously implicated in SV40 infection. SGTA binds directly to SV40 and completes ER membrane penetration. During ER-to-cytosol transport of SV40, SGTA disengages from B14 and B12. Concomitant with this, SV40 triggers B14 and B12 to reorganize into discrete foci within the ER membrane. B14 must retain its ability to form foci and interact with SGTA-Hsc70 to promote SV40 infection. Our results identify a novel role for a cytosolic chaperone in the membrane penetration of a nonenveloped virus and raise the possibility that the SV40-induced foci represent cytosol entry sites.
Assuntos
Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Infecções por Polyomavirus/metabolismo , Vírus 40 dos Símios/fisiologia , Animais , Linhagem Celular , Cromatografia em Gel , Humanos , Imunoprecipitação , Membranas Intracelulares/metabolismo , Microscopia de Fluorescência , RNA Interferente Pequeno , TransfecçãoRESUMO
Endoplasmic reticulum (ER) membrane-bound E3 ubiquitin ligases promote ER-associated degradation (ERAD) by ubiquitinating a retro-translocated substrate that reaches the cytosol from the ER, targeting it to the proteasome for destruction. Recent findings implicate ERAD-associated deubiquitinases (DUBs) as positive and negative regulators during ERAD, reflecting the different consequences of deubiquitinating a substrate prior to proteasomal degradation. These observations raise the question of whether a DUB can control the fate of a nonubiquitinated ERAD substrate. In this study, we probed the role of the ERAD-associated DUB, YOD1, during retro-translocation of the nonubiquitinated cholera toxin A1 (CTA1) peptide, a critical intoxication step. Through combining knockdown, overexpression, and binding studies, we demonstrated that YOD1 negatively controls CTA1 retro-translocation, likely by deubiquitinating and inactivating ubiquitinated ERAD components that normally promote toxin retro-translocation. YOD1 also antagonizes the proteasomal degradation of nonglycosylated pro-α factor, a postulated nonubiquitinated yeast ERAD substrate, in mammalian cells. Our findings reveal that a cytosolic DUB exerts a negative function during retro-translocation of nonubiquitinated substrates, potentially by acting on elements of the ERAD machinery.
Assuntos
Endopeptidases/metabolismo , Degradação Associada com o Retículo Endoplasmático/genética , Retículo Endoplasmático/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Tioléster Hidrolases/metabolismo , Ubiquitina/metabolismo , Toxina da Cólera/metabolismo , Citosol/metabolismo , Endopeptidases/genética , Retículo Endoplasmático/enzimologia , Regulação da Expressão Gênica , Células HEK293 , Humanos , Peptídeos/metabolismo , Precursores de Proteínas/metabolismo , Transporte Proteico , Proteólise , Transdução de Sinais , Tioléster Hidrolases/genética , Ubiquitina/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
Artificial beads including magnetite and fluorescence particles are useful to visualize pathologic tissue, such as cancers, from harmless types by magnetic resonance imaging (MRI) or fluorescence imaging. Desirable properties of diagnostic materials include high dispersion in body fluids, and the ability to target specific tissues. Here we report on the development of novel magnetic nanoparticles (MNPs) intended for use as diagnosis and therapy that are coated with viral capsid protein VP1-pentamers of simian virus 40, which are monodispersive in body fluid by conjugating epidermal growth factor (EGF) to VP1. Critically, the coating of MNPs with VP1 facilitated stable dispersion of the MNPs in body fluids. In addition, EGF was conjugated to VP1 coating on MNPs (VP1-MNPs). EGF-conjugated VP1-MNPs were successfully used to target EGF receptor-expressing tumor cells in vitro. Thus, using viral capsid protein VP1 as a coating material would be useful for medical diagnosis and therapy.
Assuntos
Proteínas do Capsídeo/química , Fator de Crescimento Epidérmico/química , Nanopartículas/química , Animais , Proteínas do Capsídeo/administração & dosagem , Linhagem Celular Tumoral , Ácido Cítrico/química , Fator de Crescimento Epidérmico/administração & dosagem , Receptores ErbB/metabolismo , Humanos , Ferro/sangue , Fenômenos Magnéticos , Camundongos , Camundongos Endogâmicos BALB C , Nanopartículas/administração & dosagem , Soro/químicaRESUMO
Cholera toxin (CT) traffics from the host cell surface to the endoplasmic reticulum (ER), where the toxin's catalytic CTA1 subunit retrotranslocates to the cytosol to induce toxicity. In the ER, CT is captured by the E3 ubiquitin ligase Hrd1 via an undefined mechanism to prepare for retrotranslocation. Using loss-of-function and gain-of-function approaches, we demonstrate that the ER-resident factor ERdj5 promotes CTA1 retrotranslocation, in part, via its J domain. This Hsp70 cochaperone regulates binding between CTA and the ER Hsp70 BiP, a chaperone previously implicated in toxin retrotranslocation. Importantly, ERdj5 interacts with the Hrd1 adaptor Sel1L directly through Sel1L's N-terminal lumenal domain, thereby linking ERdj5 to the Hrd1 complex. Sel1L itself also binds CTA and facilitates toxin retrotranslocation. By contrast, EDEM1 and OS-9, two established Sel1L binding partners, do not play significant roles in CTA1 retrotranslocation. Our results thus identify two ER factors that promote ER-to-cytosol transport of CTA1. They also indicate that ERdj5, by binding to Sel1L, triggers BiP-toxin interaction proximal to the Hrd1 complex. We postulate this scenario enables the Hrd1-associated retrotranslocation machinery to capture the toxin efficiently once the toxin is released from BiP.
Assuntos
Toxina da Cólera/metabolismo , Proteínas de Choque Térmico HSP40/metabolismo , Chaperonas Moleculares/metabolismo , Oligopeptídeos/metabolismo , Proteínas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Anticorpos/imunologia , Linhagem Celular , Toxina da Cólera/química , Toxina da Cólera/toxicidade , Retículo Endoplasmático/metabolismo , Células HEK293 , Proteínas de Choque Térmico HSP40/genética , Proteínas de Choque Térmico HSP40/imunologia , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Lectinas/genética , Lectinas/imunologia , Lectinas/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/imunologia , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/imunologia , Proteínas de Neoplasias/metabolismo , Transporte Proteico , Proteínas/genética , Proteínas/imunologia , Interferência de RNA , RNA Interferente Pequeno , Vibrio cholerae/patogenicidadeRESUMO
To cause infection, a virus enters a host cell, replicates, and assembles, with the resulting new viral progeny typically released into the extracellular environment to initiate a new infection round. Virus entry, replication, and assembly are dynamic and coordinated processes that require precise interactions with host components, often within and surrounding a defined subcellular compartment. Accumulating evidence pinpoints the endoplasmic reticulum (ER) as a crucial organelle supporting viral entry, replication, and assembly. This review focuses on the molecular mechanism by which different viruses co-opt the ER to accomplish these crucial infection steps. Certain bacterial toxins also hijack the ER for entry. An interdisciplinary approach, using rigorous biochemical and cell biological assays coupled with advanced microscopy strategies, will push to the next level our understanding of the virus-ER interaction during infection.
Assuntos
Retículo Endoplasmático/fisiologia , Polyomavirus/fisiologia , Montagem de Vírus/fisiologia , Internalização do Vírus , Replicação Viral/fisiologia , Toxina da Cólera/metabolismo , Retículo Endoplasmático/metabolismo , Interações Hospedeiro-Patógeno , Evasão da Resposta Imune , Modelos BiológicosRESUMO
Simian virus 40 (SV40) is a nonenveloped DNA virus that traffics through the endoplasmic reticulum (ER) en route to the nucleus, but the mechanisms of capsid disassembly and ER exit are poorly understood. We conducted an unbiased RNA interference screen to identify cellular genes required for SV40 infection. SV40 infection was specifically inhibited by up to 50-fold by knockdown of four different DNAJ molecular cochaperones or by inhibition of BiP, the Hsp70 partner of DNAJB11. These proteins were not required for the initiation of capsid disassembly, but knockdown markedly inhibited SV40 exit from the ER. In addition, BiP formed a complex with SV40 capsids in the ER in a DNAJB11-dependent fashion. These experiments identify five new cellular proteins required for SV40 infection and suggest that the binding of BiP to the capsid is required for ER exit. Further studies of these proteins will provide insight into the molecular mechanisms of polyomavirus infection and ER function.
Assuntos
Retículo Endoplasmático/enzimologia , Retículo Endoplasmático/virologia , Proteínas de Choque Térmico HSP40/metabolismo , Proteínas de Choque Térmico/metabolismo , Interações Hospedeiro-Patógeno , Chaperonas Moleculares/metabolismo , Vírus 40 dos Símios/patogenicidade , Animais , Linhagem Celular , Chlorocebus aethiops , Retículo Endoplasmático/metabolismo , Chaperona BiP do Retículo Endoplasmático , HumanosRESUMO
Non-enveloped viruses penetrate host membranes to infect cells. A cell-based assay was used to probe the endoplasmic reticulum (ER)-to-cytosol membrane transport of the non-enveloped SV40. We found that, upon ER arrival, SV40 is released into the lumen and undergoes sequential disulfide bond disruptions to reach the cytosol. However, despite these ER-dependent conformational changes, SV40 crosses the ER membrane as a large and intact particle consisting of the VP1 coat, the internal components VP2, VP3, and the genome. This large particle subsequently disassembles in the cytosol. Mutant virus and inhibitor studies demonstrate VP3 and likely the viral genome, as well as cellular proteasome, control ER-to-cytosol transport. Our results identify the sequence of events, as well as virus and host components, that regulate ER membrane penetration. They also suggest that the ER membrane supports passage of a large particle, potentially through either a sizeable protein-conducting channel or the lipid bilayer.
Assuntos
Citosol/virologia , Retículo Endoplasmático/virologia , Vírus 40 dos Símios/fisiologia , Vírion/fisiologia , Animais , Transporte Biológico , Proteínas do Capsídeo/metabolismo , Linhagem Celular , Chlorocebus aethiops , Citosol/metabolismo , Citosol/ultraestrutura , Dissulfetos/metabolismo , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Genoma Viral/genética , Imunoprecipitação , Membranas Intracelulares/metabolismo , Membranas Intracelulares/virologia , Bicamadas Lipídicas/metabolismo , Microscopia Eletrônica , Modelos Biológicos , Complexo de Endopeptidases do Proteassoma/metabolismo , RNA Interferente Pequeno , Vírus 40 dos Símios/química , Vírus 40 dos Símios/genética , Vírus 40 dos Símios/ultraestrutura , Vírion/química , Vírion/genética , Vírion/ultraestruturaRESUMO
Virus-receptor interaction represents a crucial step during virus entry. In this issue of Cell Host & Microbe, Neu et al. (2010) identify a receptor motif that engages JC virus, a human polyomavirus known to cause progressive multifocal leukoencephalopathy in immunocompromised individuals.
RESUMO
Nitrogen-containing bisphosphonates are pyrophosphate analogs that have long been the preferred prescription for treating osteoporosis. Although these drugs are considered inhibitors of prenylation and are believed to exert their effects on bone resorption by disrupting the signaling pathways downstream of prenylated small GTPases, this explanation seems to be insufficient. Because other classes of prenylation inhibitors have recently emerged as potential antiviral therapeutic agents, we first investigated here the effects of bisphosphonates on simian virus 40 and adenovirus infections and, to our surprise, found that viral infections are suppressed by bisphosphonates through a prenylation-independent pathway. By in-house affinity-capture techniques, dynamin-2 was identified as a new molecular target of bisphosphonates. We present evidence that certain bisphosphonates block endocytosis of adenovirus and a model substrate by inhibiting GTPase activity of dynamin-2. Hence, this study has uncovered a previously unknown mechanism of action of bisphosphonates and offers potential novel use for these drugs.