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1.
Cell Rep ; 43(8): 114571, 2024 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-39093698

RESUMO

Rice stripe virus (RSV) establishes infection in the ovaries of its vector insect, Laodelphax striatellus. We demonstrate that RSV infection delays ovarian maturation by inhibiting membrane localization of the vitellogenin receptor (VgR), thereby reducing the vitellogenin (Vg) accumulation essential for egg development. We identify the host protein L. striatellus Rab1 protein (LsRab1), which directly interacts with RSV nucleocapsid protein (NP) within nurse cells. LsRab1 is required for VgR surface localization and ovarian Vg accumulation. RSV inhibits LsRab1 function through two mechanisms: NP binding LsRab1 prevents GTP binding, and NP binding LsRab1-GTP complexes stimulates GTP hydrolysis, forming an inactive LsRab1 form. Through this dual inhibition, RSV infection prevents LsRab1 from facilitating VgR trafficking to the cell membrane, leading to inefficient Vg uptake. The Vg-VgR pathway is present in most oviparous animals, and the mechanisms detailed here provide insights into the vertical transmission of other insect-transmitted viruses of medical and agricultural importance.


Assuntos
Receptores de Superfície Celular , Tenuivirus , Proteínas rab1 de Ligação ao GTP , Animais , Feminino , Proteínas rab1 de Ligação ao GTP/metabolismo , Tenuivirus/fisiologia , Tenuivirus/metabolismo , Receptores de Superfície Celular/metabolismo , Proteínas do Ovo/metabolismo , Proteínas de Insetos/metabolismo , Proteínas de Insetos/genética , Vitelogeninas/metabolismo , Proteínas do Nucleocapsídeo/metabolismo , Hemípteros/virologia , Hemípteros/metabolismo , Ovário/virologia , Ovário/metabolismo , Ligação Proteica , Transporte Proteico , Membrana Celular/metabolismo , Membrana Celular/virologia , Doenças das Plantas/virologia
2.
Viruses ; 16(8)2024 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-39205315

RESUMO

The efficiency of virus internalization into target cells is a major determinant of infectivity. SARS-CoV-2 internalization occurs via S-protein-mediated cell binding followed either by direct fusion with the plasma membrane or endocytosis and subsequent fusion with the endosomal membrane. Despite the crucial role of virus internalization, the precise kinetics of the processes involved remains elusive. We developed a pipeline, which combines live-cell microscopy and advanced image analysis, for measuring the rates of multiple internalization-associated molecular events of single SARS-CoV-2-virus-like particles (VLPs), including endosome ingression and pH change. Our live-cell imaging experiments demonstrate that only a few minutes after binding to the plasma membrane, VLPs ingress into RAP5-negative endosomes via dynamin-dependent scission. Less than two minutes later, VLP speed increases in parallel with a pH drop below 5, yet these two events are not interrelated. By co-imaging fluorescently labeled nucleocapsid proteins, we show that nucleocapsid release occurs with similar kinetics to VLP acidification. Neither Omicron mutations nor abrogation of the S protein polybasic cleavage site affected the rate of VLP internalization, indicating that they do not confer any significant advantages or disadvantages during this process. Finally, we observe that VLP internalization occurs two to three times faster in VeroE6 than in A549 cells, which may contribute to the greater susceptibility of the former cell line to SARS-CoV-2 infection. Taken together, our precise measurements of the kinetics of VLP internalization-associated processes shed light on their contribution to the effectiveness of SARS-CoV-2 propagation in cells.


Assuntos
COVID-19 , Endossomos , SARS-CoV-2 , Internalização do Vírus , SARS-CoV-2/fisiologia , SARS-CoV-2/metabolismo , Humanos , Cinética , COVID-19/virologia , COVID-19/metabolismo , Endossomos/metabolismo , Endossomos/virologia , Endocitose , Animais , Concentração de Íons de Hidrogênio , Chlorocebus aethiops , Glicoproteína da Espícula de Coronavírus/metabolismo , Células Vero , Membrana Celular/metabolismo , Membrana Celular/virologia , Vírion/metabolismo
3.
J Virol ; 98(9): e0064924, 2024 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-39136462

RESUMO

Human immunodeficiency virus (HIV)-1 assembly is initiated by Gag binding to the inner leaflet of the plasma membrane (PM). Gag targeting is mediated by its N-terminally myristoylated matrix (MA) domain and PM phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Upon Gag assembly, envelope (Env) glycoproteins are recruited to assembly sites; this process depends on the MA domain of Gag and the Env cytoplasmic tail. To investigate the dynamics of Env recruitment, we applied a chemical dimerizer system to manipulate HIV-1 assembly by reversible PI(4,5)P2 depletion in combination with super resolution and live-cell microscopy. This approach enabled us to control and synchronize HIV-1 assembly and track Env recruitment to individual nascent assembly sites in real time. Single virion tracking revealed that Gag and Env are accumulating at HIV-1 assembly sites with similar kinetics. PI(4,5)P2 depletion prevented Gag PM targeting and Env cluster formation, confirming Gag dependence of Env recruitment. In cells displaying pre-assembled Gag lattices, PI(4,5)P2 depletion resulted in the disintegration of the complete assembly domain, as not only Gag but also Env clusters were rapidly lost from the PM. These results argue for the existence of a Gag-induced and -maintained membrane micro-environment, which attracts Env. Gag cluster dissociation by PI(4,5)P2 depletion apparently disrupts this micro-environment, resulting in the loss of Env from the former assembly domain.IMPORTANCEHuman immunodeficiency virus (HIV)-1 assembles at the plasma membrane of infected cells, resulting in the budding of membrane-enveloped virions. HIV-1 assembly is a complex process initiated by the main structural protein of HIV-1, Gag. Interestingly, HIV-1 incorporates only a few envelope (Env) glycoproteins into budding virions, although large Env accumulations surrounding nascent Gag assemblies are detected at the plasma membrane of HIV-expressing cells. The matrix domain of Gag and the Env cytoplasmatic tail play a role in Env recruitment to HIV-1 assembly sites and its incorporation into nascent virions. However, the regulation of these processes is incompletely understood. By combining a chemical dimerizer system to manipulate HIV-1 assembly with super resolution and live-cell microscopy, our study provides new insights into the interplay between Gag, Env, and host cell membranes during viral assembly and into Env incorporation into HIV-1 virions.


Assuntos
Membrana Celular , HIV-1 , Fosfatidilinositol 4,5-Difosfato , Montagem de Vírus , Produtos do Gene env do Vírus da Imunodeficiência Humana , Produtos do Gene gag do Vírus da Imunodeficiência Humana , HIV-1/fisiologia , HIV-1/metabolismo , Humanos , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética , Membrana Celular/metabolismo , Membrana Celular/virologia , Fosfatidilinositol 4,5-Difosfato/metabolismo , Produtos do Gene env do Vírus da Imunodeficiência Humana/metabolismo , Produtos do Gene env do Vírus da Imunodeficiência Humana/genética , Vírion/metabolismo , Células HeLa , Microscopia/métodos
4.
J Virol ; 98(9): e0059924, 2024 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-39136459

RESUMO

Herpes simplex virus 1 (HSV-1) is an alpha herpesvirus that infects a majority of the world population. The mechanisms and cellular host factors involved in the intracellular transport and exocytosis of HSV-1 particles are not fully understood. To elucidate these late steps in the replication cycle, we developed a live-cell fluorescence microscopy assay of HSV-1 virion intracellular trafficking and exocytosis. This method allows us to track individual virus particles and identify the precise moment and location of particle exocytosis using a pH-sensitive reporter. We show that HSV-1 uses the host cell's post-Golgi secretory pathway during egress. The small GTPase, Rab6, binds to nascent secretory vesicles at the trans-Golgi network and plays important, but non-essential, roles in vesicle traffic and exocytosis at the plasma membrane, therefore making it a useful marker of the Golgi and post-Golgi secretory pathway. We show that HSV-1 particles colocalize with Rab6a in the region of the Golgi, cotraffic with Rab6a to the cell periphery, and undergo exocytosis from Rab6a vesicles. Consistent with previous reports, we find that HSV-1 particles accumulate at preferential egress sites in infected cells. The secretory pathway mediates this preferential/polarized egress, since Rab6a vesicles accumulate near the plasma membrane similarly in uninfected cells. These data suggest that, following particle envelopment, HSV-1 egress follows a pre-existing cellular secretory pathway to exit infected cells rather than novel, virus-induced mechanisms. IMPORTANCE: Herpes simplex virus 1 (HSV-1) infects a majority of people. It establishes a life-long latent infection and occasionally reactivates, typically causing characteristic oral or genital lesions. Rarely in healthy natural hosts, but more commonly in zoonotic infections and in elderly, newborn, or immunocompromised patients, HSV-1 can cause severe herpes encephalitis. The precise cellular mechanisms used by HSV-1 remain an important area of research. In particular, the egress pathways that newly assembled virus particles use to exit from infected cells are unclear. In this study, we used fluorescence microscopy to visualize individual virus particles exiting from cells and found that HSV-1 particles use the pre-existing cellular secretory pathway.


Assuntos
Exocitose , Complexo de Golgi , Herpesvirus Humano 1 , Via Secretória , Liberação de Vírus , Proteínas rab de Ligação ao GTP , Herpesvirus Humano 1/fisiologia , Herpesvirus Humano 1/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Humanos , Animais , Complexo de Golgi/metabolismo , Complexo de Golgi/virologia , Células Vero , Rede trans-Golgi/metabolismo , Rede trans-Golgi/virologia , Chlorocebus aethiops , Herpes Simples/virologia , Herpes Simples/metabolismo , Vírion/metabolismo , Células HeLa , Membrana Celular/metabolismo , Membrana Celular/virologia
5.
J Virol ; 98(8): e0088024, 2024 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-39078176

RESUMO

Porcine deltacoronavirus (PDCoV) is an emerging swine enteric coronavirus with zoonotic potential. The coronavirus spike (S) glycoprotein, especially the S1 subunit, mediates viral entry by binding to cellular receptors. However, the functional receptor of PDCoV remains poorly understood. In this study, we used the soluble PDCoV S1 protein as bait to capture the S1-binding cellular transmembrane proteins in combined immunoprecipitation and mass spectrometry analyses. A single guide RNA screen identified d-glucuronyl C5-epimerase (GLCE), a heparan sulfate-modifying enzyme, as a proviral host factor for PDCoV infection. GLCE knockout significantly inhibited the attachment and internalization stages of PDCoV infection. We also demonstrated the interaction between GLCE and PDCoV S with coimmunoprecipitation in both an overexpression system and PDCoV-infected cells. GLCE could be localized to the cell membrane, and an anti-GLCE antibody suppressed PDCoV infection. Although GLCE expression alone did not render nonpermissive cells susceptible to PDCoV infection, GLCE promoted the binding of PDCoV S to porcine amino peptidase N (pAPN), acting synergistically with pAPN to enhance PDCoV infection. In conclusion, our results demonstrate that GLCE is a novel cell-surface factor facilitating PDCoV entry and provide new insights into PDCoV infection. IMPORTANCE: The identification of viral receptors is of great significance, potentially extending our understanding of viral infection and pathogenesis. Porcine deltacoronavirus (PDCoV) is an emerging enteropathogenic coronavirus with the potential for cross-species transmission. However, the receptors or coreceptors of PDCoV are still poorly understood. The present study confirms that d-glucuronyl C5-epimerase (GLCE) is a positive regulator of PDCoV infection, promoting viral attachment and internalization. The anti-GLCE antibody suppressed PDCoV infection. Mechanically, GLCE interacts with PDCoV S and promotes the binding of PDCoV S to porcine amino peptidase N (pAPN), acting synergistically with pAPN to enhance PDCoV infection. This work identifies GLCE as a novel cell-surface factor facilitating PDCoV entry and paves the way for further insights into the mechanisms of PDCoV infection.


Assuntos
Deltacoronavirus , Glicoproteína da Espícula de Coronavírus , Internalização do Vírus , Animais , Glicoproteína da Espícula de Coronavírus/metabolismo , Glicoproteína da Espícula de Coronavírus/genética , Suínos , Deltacoronavirus/metabolismo , Humanos , Carboidratos Epimerases/metabolismo , Carboidratos Epimerases/genética , Ligação Proteica , Infecções por Coronavirus/virologia , Infecções por Coronavirus/metabolismo , Infecções por Coronavirus/veterinária , Doenças dos Suínos/virologia , Doenças dos Suínos/metabolismo , Linhagem Celular , Receptores Virais/metabolismo , Ligação Viral , Células HEK293 , Membrana Celular/metabolismo , Membrana Celular/virologia
6.
J Virol ; 98(7): e0071324, 2024 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-38899931

RESUMO

Herpesvirus assembly requires the cytoplasmic association of large macromolecular and membrane structures that derive from both the nucleus and cytoplasmic membrane systems. Results from the study of human cytomegalovirus (HCMV) in cells where it organizes a perinuclear cytoplasmic virus assembly compartment (cVAC) show a clear requirement for the minus-end-directed microtubule motor, dynein, for virus assembly. In contrast, the assembly of herpes simplex virus -1 (HSV-1) in epithelial cells where it forms multiple dispersed, peripheral assembly sites is only mildly inhibited by the microtubule-depolymerizing agent, nocodazole. Here, we make use of a neuronal cell line system in which HSV-1 forms a single cVAC and show that dynein and its co-factor dynactin localize to the cVAC, and dynactin is associated with membranes that contain the virion tegument protein pUL11. We also show that the virus membrane-associated structural proteins pUL51 and the viral envelope glycoprotein gE arrive at the cVAC by different routes. Specifically, gE arrives at the cVAC after retrieval from the plasma membrane, suggesting the need for an intact retrograde transport system. Finally, we demonstrate that inhibition of dynactin function profoundly inhibits cVAC formation and virus production during the cytoplasmic assembly phase of infection.IMPORTANCEMany viruses reorganize cytoplasmic membrane systems and macromolecular transport systems to promote the production of progeny virions. Clarifying the mechanisms by which they accomplish this may reveal novel therapeutic strategies and illustrate mechanisms that are critical for normal cellular organization. Here, we explore the mechanism by which HSV-1 moves macromolecular and membrane cargo to generate a virus assembly compartment in the infected cell. We find that the virus makes use of a well-characterized, microtubule-based transport system that is stabilized against drugs that disrupt microtubules.


Assuntos
Membrana Celular , Complexo Dinactina , Dineínas , Herpesvirus Humano 1 , Proteínas Associadas aos Microtúbulos , Neurônios , Proteínas do Envelope Viral , Montagem de Vírus , Herpesvirus Humano 1/fisiologia , Herpesvirus Humano 1/metabolismo , Dineínas/metabolismo , Membrana Celular/metabolismo , Membrana Celular/virologia , Humanos , Neurônios/virologia , Neurônios/metabolismo , Complexo Dinactina/metabolismo , Proteínas do Envelope Viral/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Linhagem Celular , Animais , Compartimentos de Replicação Viral/metabolismo , Microtúbulos/metabolismo
7.
J Virol ; 98(7): e0036824, 2024 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-38940586

RESUMO

Chikungunya virus (CHIKV) is a mosquito-borne pathogen responsible for an acute musculoskeletal disease in humans. Replication of the viral RNA genome occurs in specialized membranous replication organelles (ROs) or spherules, which contain the viral replication complex. Initially generated by RNA synthesis-associated plasma membrane deformation, alphavirus ROs are generally rapidly endocytosed to produce type I cytopathic vacuoles (CPV-I), from which nascent RNAs are extruded for cytoplasmic translation. By contrast, CHIKV ROs are poorly internalized, raising the question of their fate and functionality at the late stage of infection. Here, using in situ cryogenic-electron microscopy approaches, we investigate the outcome of CHIKV ROs and associated replication machinery in infected human cells. We evidence the late persistence of CHIKV ROs at the plasma membrane with a crowned protein complex at the spherule neck similar to the recently resolved replication complex. The unexpectedly heterogeneous and large diameter of these compartments suggests a continuous, dynamic growth of these organelles beyond the replication of a single RNA genome. Ultrastructural analysis of surrounding cytoplasmic regions supports that outgrown CHIKV ROs remain dynamically active in viral RNA synthesis and export to the cell cytosol for protein translation. Interestingly, rare ROs with a homogeneous diameter are also marginally internalized in CPV-I near honeycomb-like arrangements of unknown function, which are absent in uninfected controls, thereby suggesting a temporal regulation of this internalization. Altogether, this study sheds new light on the dynamic pattern of CHIKV ROs and associated viral replication at the interface with cell membranes in infected cells.IMPORTANCEThe Chikungunya virus (CHIKV) is a positive-stranded RNA virus that requires specialized membranous replication organelles (ROs) for its genome replication. Our knowledge of this viral cycle stage is still incomplete, notably regarding the fate and functional dynamics of CHIKV ROs in infected cells. Here, we show that CHIKV ROs are maintained at the plasma membrane beyond the first viral cycle, continuing to grow and be dynamically active both in viral RNA replication and in its export to the cell cytosol, where translation occurs in proximity to ROs. This contrasts with the homogeneous diameter of ROs during internalization in cytoplasmic vacuoles, which are often associated with honeycomb-like arrangements of unknown function, suggesting a regulated mechanism. This study sheds new light on the dynamics and fate of CHIKV ROs in human cells and, consequently, on our understanding of the Chikungunya viral cycle.


Assuntos
Vírus Chikungunya , RNA Viral , Replicação Viral , Vírus Chikungunya/fisiologia , Humanos , RNA Viral/metabolismo , RNA Viral/genética , Febre de Chikungunya/virologia , Compartimentos de Replicação Viral/metabolismo , Organelas/virologia , Organelas/ultraestrutura , Organelas/metabolismo , Membrana Celular/virologia , Membrana Celular/metabolismo , Linhagem Celular , Microscopia Crioeletrônica , Animais , Genoma Viral
8.
J Mol Biol ; 436(16): 168639, 2024 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-38838849

RESUMO

HIV-1 Gag polyprotein plays a pivotal role in assembly and budding of new particles, by specifically packaging two copies of viral gRNA in the host cell cytoplasm and selecting the cell plasma membrane for budding. Both gRNA and membrane selections are thought to be mediated by the compact form of Gag. This compact form binds to gRNA through both its matrix (MA) and nucleocapsid (NC) domains in the cytoplasm. At the plasma membrane, the membrane competes with gRNA for Gag binding, resulting in a transition to the extended form of Gag found in immature particles with MA bound to membrane lipids and NC to gRNA. The Gag compact form was previously evidenced in vitro. Here, we demonstrated the compact form of Gag in cells by confocal microscopy, using a bimolecular fluorescence complementation approach with a split-GFP bipartite system. Using wild-type Gag and Gag mutants, we showed that the compact form is highly dependent on the binding of MA and NC domains to RNA, as well as on interactions between MA and CA domains. In contrast, Gag multimerization appears to be less critical for the accumulation of the compact form. Finally, mutations altering the formation of Gag compact form led to a strong reduction in viral particle production and infectivity, revealing its key role in the production of infectious viral particles.


Assuntos
HIV-1 , Montagem de Vírus , Produtos do Gene gag do Vírus da Imunodeficiência Humana , HIV-1/metabolismo , HIV-1/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/química , Montagem de Vírus/genética , Humanos , Vírion/metabolismo , Vírion/genética , Ligação Proteica , RNA Viral/metabolismo , RNA Viral/genética , Membrana Celular/metabolismo , Membrana Celular/virologia
9.
ACS Infect Dis ; 10(8): 2728-2740, 2024 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-38873897

RESUMO

Nonenveloped viruses employ unique entry mechanisms to breach and infect host cells. Understanding these mechanisms is crucial for developing antiviral strategies. Prevailing perspective suggests that nonenveloped viruses release membrane pore-forming peptides to breach host membranes. However, the precise involvement of the viral capsid in this entry remains elusive. Our study presents direct observations elucidating the dynamically distinctive steps through which metastable reovirus capsids disrupt host lipid membranes as they uncoat into partially hydrophobic intermediate particles. Using both live cells and model membrane systems, our key finding is that reovirus capsids actively deform and permeabilize lipid membranes in a cholesterol-dependent process. Unlike membrane pore-forming peptides, these metastable viral capsids induce more extensive membrane perturbations, including budding, bridging between adjacent membranes, and complete rupture. Notably, cholesterol enhances subviral particle adsorption, resulting in the formation of pores equivalent to the capsid size. This cholesterol dependence is attributed to the lipid condensing effect, particularly prominent at an intermediate cholesterol level. Furthermore, our results reveal a positive correlation between membrane disruption extent and efficiency of viral variants in establishing infection. This study unveils the crucial role of capsid-lipid interaction in nonenveloped virus entry, providing new insights into how cholesterol homeostasis influences virus infection dynamics.


Assuntos
Capsídeo , Membrana Celular , Colesterol , Reoviridae , Internalização do Vírus , Colesterol/metabolismo , Capsídeo/metabolismo , Membrana Celular/virologia , Membrana Celular/metabolismo , Reoviridae/fisiologia , Humanos , Animais , Proteínas do Capsídeo/metabolismo , Proteínas do Capsídeo/química
10.
Front Immunol ; 15: 1340332, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38919631

RESUMO

During infection, positive-stranded RNA causes a rearrangement of the host cell membrane, resulting in specialized membrane structure formation aiding viral genome replication. Double-membrane vesicles (DMVs), typical structures produced by virus-induced membrane rearrangements, are platforms for viral replication. Nidoviruses, one of the most complex positive-strand RNA viruses, have the ability to infect not only mammals and a few birds but also invertebrates. Nidoviruses possess a distinctive replication mechanism, wherein their nonstructural proteins (nsps) play a crucial role in DMV biogenesis. With the participation of host factors related to autophagy and lipid synthesis pathways, several viral nsps hijack the membrane rearrangement process of host endoplasmic reticulum (ER), Golgi apparatus, and other organelles to induce DMV formation. An understanding of the mechanisms of DMV formation and its structure and function in the infectious cycle of nidovirus may be essential for the development of new and effective antiviral strategies in the future.


Assuntos
Nidovirales , Replicação Viral , Nidovirales/fisiologia , Animais , Humanos , Infecções por Nidovirales , Proteínas não Estruturais Virais/metabolismo , Proteínas não Estruturais Virais/genética , Retículo Endoplasmático/virologia , Retículo Endoplasmático/metabolismo , Membrana Celular/metabolismo , Membrana Celular/virologia , Interações Hospedeiro-Patógeno
11.
Biochem Biophys Res Commun ; 716: 149954, 2024 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-38704887

RESUMO

Membrane lipids and proteins form dynamic domains crucial for physiological and pathophysiological processes, including viral infection. Many plasma membrane proteins, residing within membrane domains enriched with cholesterol (CHOL) and sphingomyelin (SM), serve as receptors for attachment and entry of viruses into the host cell. Among these, human coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), use proteins associated with membrane domains for initial binding and internalization. We hypothesized that the interaction of lipid-binding proteins with CHOL in plasma membrane could sequestrate lipids and thus affect the efficiency of virus entry into host cells, preventing the initial steps of viral infection. We have prepared CHOL-binding proteins with high affinities for lipids in the plasma membrane of mammalian cells. Binding of the perfringolysin O domain four (D4) and its variant D4E458L to membrane CHOL impaired the internalization of the receptor-binding domain of the SARS-CoV-2 spike protein and the pseudovirus complemented with the SARS-CoV-2 spike protein. SARS-CoV-2 replication in Vero E6 cells was also decreased. Overall, our results demonstrate that the integrity of CHOL-rich membrane domains and the accessibility of CHOL in the membrane play an essential role in SARS-CoV-2 cell entry.


Assuntos
Membrana Celular , Colesterol , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Internalização do Vírus , Células Vero , Chlorocebus aethiops , Colesterol/metabolismo , Animais , SARS-CoV-2/metabolismo , SARS-CoV-2/fisiologia , Membrana Celular/metabolismo , Membrana Celular/virologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Humanos , Proteínas de Transporte/metabolismo , COVID-19/virologia , COVID-19/metabolismo , Ligação Proteica
12.
Methods Mol Biol ; 2807: 61-76, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38743221

RESUMO

The 20-year revolution in optical fluorescence microscopy, supported by the optimization of both spatial resolution and timely acquisition, allows the visualization of nanoscaled objects in cell biology. Currently, the use of a recent generation of super-resolution fluorescence microscope coupled with improved fluorescent probes gives the possibility to study the replicative cycle of viruses in living cells, at the single-virus particle or protein level. Here, we highlight the protocol for visualizing HIV-1 Gag assembly at the host T-cell plasma membrane using super-resolution light microscopy. Total internal reflection fluorescence microscopy (TIRF-M) coupled with single-molecule localization microscopy (SMLM) enables the detection and characterization of the assembly of viral proteins at the plasma membrane of infected host cells at the single protein level. Here, we describe the TIRF equipment, the T-cell culture for HIV-1, the sample preparation for single-molecule localization microscopies such as PALM and STORM, acquisition protocols, and Gag assembling cluster analysis.


Assuntos
Membrana Celular , HIV-1 , Microscopia de Fluorescência , Imagem Individual de Molécula , Linfócitos T , Montagem de Vírus , Produtos do Gene gag do Vírus da Imunodeficiência Humana , HIV-1/fisiologia , Humanos , Membrana Celular/metabolismo , Membrana Celular/virologia , Imagem Individual de Molécula/métodos , Linfócitos T/virologia , Linfócitos T/metabolismo , Microscopia de Fluorescência/métodos , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo
13.
J Gen Virol ; 105(4)2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38687323

RESUMO

The human cytomegalovirus (HCMV) pUS2 glycoprotein exploits the host's endoplasmic reticulum (ER)-associated degradation (ERAD) pathway to degrade major histocompatibility complex class I (MHC-I) and prevent antigen presentation. Beyond MHC-I, pUS2 has been shown to target a range of cellular proteins for degradation, preventing their cell surface expression. Here we have identified a novel pUS2 target, ER-resident protein lectin mannose binding 2 like (LMAN2L). pUS2 expression was both necessary and sufficient for the downregulation of LMAN2L, which was dependent on the cellular E3 ligase TRC8. Given the hypothesized role of LMAN2L in the trafficking of glycoproteins, we employed proteomic plasma membrane profiling to measure LMAN2L-dependent changes at the cell surface. A known pUS2 target, integrin alpha-6 (ITGA6), was downregulated from the surface of LMAN2L-deficient cells, but not other integrins. Overall, these results suggest a novel strategy of pUS2-mediated protein degradation whereby pUS2 targets LMAN2L to impair trafficking of ITGA6. Given that pUS2 can directly target other integrins, we propose that this single viral protein may exhibit both direct and indirect mechanisms to downregulate key cell surface molecules.


Assuntos
Citomegalovirus , Retículo Endoplasmático , Proteínas do Envelope Viral , Proteínas Virais , Humanos , Citomegalovirus/genética , Citomegalovirus/metabolismo , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/virologia , Proteínas Virais/metabolismo , Proteínas Virais/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Proteólise , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Lectinas de Ligação a Manose/metabolismo , Lectinas de Ligação a Manose/genética , Degradação Associada com o Retículo Endoplasmático , Interações Hospedeiro-Patógeno , Membrana Celular/metabolismo , Membrana Celular/virologia
14.
ACS Infect Dis ; 10(5): 1590-1601, 2024 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-38684073

RESUMO

Ebola virus (EBOV) is an enveloped virus that must fuse with the host cell membrane in order to release its genome and initiate infection. This process requires the action of the EBOV envelope glycoprotein (GP), encoded by the virus, which resides in the viral envelope and consists of a receptor binding subunit, GP1, and a membrane fusion subunit, GP2. Despite extensive research, a mechanistic understanding of the viral fusion process is incomplete. To investigate GP-membrane association, a key step in the fusion process, we used two approaches: high-throughput measurements of single-particle diffusion and single-molecule measurements with optical tweezers. Using these methods, we show that the presence of the endosomal Niemann-Pick C1 (NPC1) receptor is not required for primed GP-membrane binding. In addition, we demonstrate this binding is very strong, likely attributed to the interaction between the GP fusion loop and the membrane's hydrophobic core. Our results also align with previously reported findings, emphasizing the significance of acidic pH in the protein-membrane interaction. Beyond Ebola virus research, our approach provides a powerful toolkit for studying other protein-membrane interactions, opening new avenues for a better understanding of protein-mediated membrane fusion events.


Assuntos
Ebolavirus , Proteínas do Envelope Viral , Ebolavirus/metabolismo , Ebolavirus/fisiologia , Ebolavirus/genética , Ebolavirus/química , Proteínas do Envelope Viral/metabolismo , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética , Humanos , Ligação Proteica , Internalização do Vírus , Proteína C1 de Niemann-Pick/metabolismo , Membrana Celular/metabolismo , Membrana Celular/virologia , Doença pelo Vírus Ebola/virologia , Concentração de Íons de Hidrogênio
15.
J Virol ; 98(2): e0194823, 2024 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-38299843

RESUMO

The eukaryotic translation initiation factor eIF4E can regulate cellular translation via phosphorylation on serine 209. In a recent study, by two rounds of TMT relative quantitative proteomics, we found that phosphorylated eIF4E (p-eIF4E) favors the translation of selected mRNAs, and the encoded proteins are mainly involved in ECM-receptor, focal adhesion, and PI3K-Akt signaling. The current paper is focused on the relationship between p-eIF4E and the downstream host cell proteins, and their presumed effect on efficient entry of PEDV. We found that the depletion of membrane-residential factor TSPAN3, CD63, and ITGB2 significantly inhibited viral invasion of PEDV, and reduced the entry of pseudotyped particles PEDV-pp, SARS-CoV-pp, and SARS-CoV-2-pp. The specific antibodies of TSPAN3, CD63, and ITGB2 blocked the adsorption of PEDV into host cells. Moreover, we detected that eIF4E phosphorylation was increased at 1 h after PEDV infection, in accordance with the expression of TSPAN3, CD63, and ITGB2. Similar trends appeared in the intestines of piglets in the early stage of PEDV challenge. Compared with Vero cells, S209A-Vero cells in which eIF4E cannot be phosphorylated showed a decrease of invading PEDV virions. MNK kinase inhibitor blocked PEDV invasion, as well as reduced the accumulation of TSPAN3, CD63, and ITGB2. Further study showed that the ERK-MNK pathway was responsible for the regulation of PEDV-induced early phosphorylation of eIF4E. This paper demonstrates for the first time the connections among p-eIF4E stimulation and membrane-residential host factors. Our findings also enrich the understanding of the biological function of phosphorylated eIF4E during the viral life cycle.IMPORTANCEThe eukaryotic translation initiation factor eIF4E can regulate cellular translation via phosphorylation. In our previous study, several host factors susceptible to a high level of p-eIF4E were found to be conducive to viral infection by coronavirus PEDV. The current paper is focused on cell membrane-residential factors, which are involved in signal pathways that are sensitive to phosphorylated eIF4E. We found that the ERK-MNK pathway was activated, which resulted in the stimulation of phosphorylation of eIF4E in early PEDV infection. Phospho-eIF4E promoted the viral invasion of PEDV by upregulating the expression of host factors TSPAN3, CD63, and ITGB2 at the translation level rather than at the transcription level. Moreover, TSPAN3, CD63, or ITGB2 facilitates the efficient entry of coronavirus SARS-CoV, SARS-CoV-2, and HCoV-OC43. Our findings broaden our insights into the dynamic phosphorylation of eIF4E during the viral life cycle, and provide further evidence that phosphorylated eIF4E regulates selective translation of host mRNA.


Assuntos
Membrana Celular , Fator de Iniciação 4E em Eucariotos , Vírus da Diarreia Epidêmica Suína , Biossíntese de Proteínas , Internalização do Vírus , Animais , Membrana Celular/química , Membrana Celular/genética , Membrana Celular/metabolismo , Membrana Celular/virologia , Chlorocebus aethiops , Fator de Iniciação 4E em Eucariotos/química , Fator de Iniciação 4E em Eucariotos/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Cadeias beta de Integrinas/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Vírus da Diarreia Epidêmica Suína/fisiologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteômica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Suínos , Tetraspaninas/metabolismo , Células Vero
16.
Nat Commun ; 14(1): 1148, 2023 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-36854760

RESUMO

Globally, most cases of gastroenteritis are caused by pandemic GII.4 human norovirus (HuNoV) strains with no approved therapies or vaccines available. The cellular pathways that these strains exploit for cell entry and internalization are unknown. Here, using nontransformed human jejunal enteroids (HIEs) that recapitulate the physiology of the gastrointestinal tract, we show that infectious GII.4 virions and virus-like particles are endocytosed using a unique combination of endosomal acidification-dependent clathrin-independent carriers (CLIC), acid sphingomyelinase (ASM)-mediated lysosomal exocytosis, and membrane wound repair pathways. We found that besides the known interaction of the viral capsid Protruding (P) domain with host glycans, the Shell (S) domain interacts with both galectin-3 (gal-3) and apoptosis-linked gene 2-interacting protein X (ALIX), to orchestrate GII.4 cell entry. Recognition of the viral and cellular determinants regulating HuNoV entry provides insight into the infection process of a non-enveloped virus highlighting unique pathways and targets for developing effective therapeutics.


Assuntos
Membrana Celular , Norovirus , Internalização do Vírus , Humanos , Clatrina , Norovirus/fisiologia , Transdução de Sinais , Membrana Celular/virologia
17.
J Microbiol Immunol Infect ; 56(2): 257-266, 2023 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-36127231

RESUMO

BACKGROUND: The exploration of virology knowledge was limited by the optical technology for the observation of virus. Previously, a three-dimensional multi-resolution real-time microscope system (3D-MRM) was developed to observe the uptake of HIV-1-tat peptide-modified nanoparticles in cell membrane. In this study, we labeled HIV-1 virus-like particles (VLPs) with passivated giant quantum dots (gQDs) and recorded their interactive trajectories with human Jurkat CD4 cells through 3D-MRM. METHODS: The labeled of gQDs of the HIV-1 VLPs in sucrose-gradient purified viral lysates was first confirmed by Cryo-electronic microscopy and Western blot assay. After the infection with CD4 cells, the gQD-labeled VLPs were visualized and their extracellular and intracellular trajectories were recorded by 3D-MRM. RESULTS: A total of 208 prime trajectories was identified and classified into three distinct patterns: cell-free random diffusion pattern, directional movement pattern and cell-associated movement pattern, with distributions and mean durations were 72.6%/87.6 s, 9.1%/402.7 s and 18.3%/68.7 s, respectively. Further analysis of the spatial-temporal relationship between VLP trajectories and CD4 cells revealed the three stages of interactions: (1) cell-associated (extracellular) diffusion stage, (2) cell membrane surfing stage and (3) intracellular directional movement stage. CONCLUSION: A complete trajectory of HIV-1 VLP interacting with CD4 cells was presented in animation. This encapsulating method could increase the accuracy for the observation of HIV-1-CD4 cell interaction in real time and three dimensions.


Assuntos
Linfócitos T CD4-Positivos , Membrana Celular , HIV-1 , Microscopia Eletrônica , Pontos Quânticos , Produtos do Gene tat do Vírus da Imunodeficiência Humana , Humanos , Linfócitos T CD4-Positivos/fisiologia , Linfócitos T CD4-Positivos/ultraestrutura , Linfócitos T CD4-Positivos/virologia , HIV-1/fisiologia , HIV-1/ultraestrutura , Imageamento Tridimensional/métodos , Produtos do Gene tat do Vírus da Imunodeficiência Humana/fisiologia , Peptídeos Penetradores de Células/fisiologia , Membrana Celular/fisiologia , Membrana Celular/ultraestrutura , Membrana Celular/virologia , Nanopartículas/ultraestrutura , Nanopartículas/virologia , Partículas Artificiais Semelhantes a Vírus/fisiologia , Microscopia Eletrônica/métodos
18.
J Phys Chem Lett ; 13(21): 4642-4649, 2022 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-35593652

RESUMO

Like all viral infections, SARS-CoV-2 acts at multiple levels, hijacking fundamental cellular functions and assuring its replication and immune system evasion. In particular, the viral 3' Open Reading Frame (ORF3a) codes for a hydrophobic protein, which embeds in the cellular membrane, where it acts as an ion viroporin and is related to strong inflammatory response. Here we report equilibrium and enhanced sampling molecular dynamic simulation of the SARS-CoV-2 ORF3a in a model lipid bilayer, showing how the protein permeabilizes the lipid membrane, via the formation of a water channel, which in turn assures ion transport. We report the free energy profile for both K+ and Cl- transfer from the cytosol to the extracellular domain. The important role of ORF3a in the viral cycle and its high conservation among coronaviruses may also make it a target of choice for future antiviral development, further justifying the elucidation of its mechanism at the atomistic level.


Assuntos
COVID-19 , Membrana Celular , Proteínas Viroporinas , Membrana Celular/virologia , Humanos , Lipídeos , SARS-CoV-2
19.
J Virol ; 96(12): e0021522, 2022 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-35608346

RESUMO

The baculovirus envelope protein GP64 is an essential component of the budded virus and is necessary for efficient virion assembly. Little is known regarding intracellular trafficking of GP64 to the plasma membrane, where it is incorporated into budding virions during egress. To identify host proteins and potential cellular trafficking pathways that are involved in delivery of GP64 to the plasma membrane, we developed and characterized a stable Drosophila cell line that inducibly expresses the AcMNPV GP64 protein and used that cell line in combination with a targeted RNA interference (RNAi) screen of vesicular protein trafficking pathway genes. Of the 37 initial hits from the screen, we validated and examined six host genes that were important for trafficking of GP64 to the cell surface. Validated hits included Rab GTPases Rab1 and Rab4, Clathrin heavy chain, clathrin adaptor protein genes AP-1-2ß and AP-2µ, and Snap29. Two gene knockdowns (Rab5 and Exo84) caused substantial increases (up to 2.5-fold) of GP64 on the plasma membrane. We found that a small amount of GP64 is released from cells in exosomes and that some portion of cell surface GP64 is endocytosed, suggesting that recycling helps to maintain GP64 at the cell surface. IMPORTANCE While much is known regarding trafficking of viral envelope proteins in mammalian cells, little is known about this process in insect cells. To begin to understand which factors and pathways are needed for trafficking of insect virus envelope proteins, we engineered a Drosophila melanogaster cell line and implemented an RNAi screen to identify cellular proteins that aid transport of the model baculovirus envelope protein (GP64) to the cell surface. For this we developed an experimental system that leverages the large array of tools available for Drosophila and performed a targeted RNAi screen to identify cellular proteins involved in GP64 trafficking to the cell surface. Since viral envelope proteins are often critical for production of infectious progeny virions, these studies lay the foundation for understanding how either pathogenic insect viruses (baculoviruses) or insect-vectored viruses (e.g., flaviviruses, alphaviruses) egress from cells in tissues such as the midgut to enable systemic virus infection.


Assuntos
Baculoviridae , Membrana Celular , Proteínas de Insetos , Proteínas do Envelope Viral , Animais , Baculoviridae/metabolismo , Linhagem Celular , Membrana Celular/virologia , Drosophila melanogaster/virologia , Proteínas de Insetos/genética , Interferência de RNA , Proteínas do Envelope Viral/metabolismo
20.
Nature ; 603(7902): 706-714, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35104837

RESUMO

The SARS-CoV-2 Omicron BA.1 variant emerged in 20211 and has multiple mutations in its spike protein2. Here we show that the spike protein of Omicron has a higher affinity for ACE2 compared with Delta, and a marked change in its antigenicity increases Omicron's evasion of therapeutic monoclonal and vaccine-elicited polyclonal neutralizing antibodies after two doses. mRNA vaccination as a third vaccine dose rescues and broadens neutralization. Importantly, the antiviral drugs remdesivir and molnupiravir retain efficacy against Omicron BA.1. Replication was similar for Omicron and Delta virus isolates in human nasal epithelial cultures. However, in lung cells and gut cells, Omicron demonstrated lower replication. Omicron spike protein was less efficiently cleaved compared with Delta. The differences in replication were mapped to the entry efficiency of the virus on the basis of spike-pseudotyped virus assays. The defect in entry of Omicron pseudotyped virus to specific cell types effectively correlated with higher cellular RNA expression of TMPRSS2, and deletion of TMPRSS2 affected Delta entry to a greater extent than Omicron. Furthermore, drug inhibitors targeting specific entry pathways3 demonstrated that the Omicron spike inefficiently uses the cellular protease TMPRSS2, which promotes cell entry through plasma membrane fusion, with greater dependency on cell entry through the endocytic pathway. Consistent with suboptimal S1/S2 cleavage and inability to use TMPRSS2, syncytium formation by the Omicron spike was substantially impaired compared with the Delta spike. The less efficient spike cleavage of Omicron at S1/S2 is associated with a shift in cellular tropism away from TMPRSS2-expressing cells, with implications for altered pathogenesis.


Assuntos
COVID-19/patologia , COVID-19/virologia , Fusão de Membrana , SARS-CoV-2/metabolismo , SARS-CoV-2/patogenicidade , Serina Endopeptidases/metabolismo , Internalização do Vírus , Adulto , Idoso , Idoso de 80 Anos ou mais , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19/imunologia , Vacinas contra COVID-19/imunologia , Linhagem Celular , Membrana Celular/metabolismo , Membrana Celular/virologia , Chlorocebus aethiops , Convalescença , Feminino , Humanos , Soros Imunes/imunologia , Intestinos/patologia , Intestinos/virologia , Pulmão/patologia , Pulmão/virologia , Masculino , Pessoa de Meia-Idade , Mutação , Mucosa Nasal/patologia , Mucosa Nasal/virologia , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Técnicas de Cultura de Tecidos , Virulência , Replicação Viral
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