Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 32
Filtrar
1.
Cell ; 163(7): 1692-701, 2015 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-26687357

RESUMO

Vesicular nucleo-cytoplasmic transport is becoming recognized as a general cellular mechanism for translocation of large cargoes across the nuclear envelope. Cargo is recruited, enveloped at the inner nuclear membrane (INM), and delivered by membrane fusion at the outer nuclear membrane. To understand the structural underpinning for this trafficking, we investigated nuclear egress of progeny herpesvirus capsids where capsid envelopment is mediated by two viral proteins, forming the nuclear egress complex (NEC). Using a multi-modal imaging approach, we visualized the NEC in situ forming coated vesicles of defined size. Cellular electron cryo-tomography revealed a protein layer showing two distinct hexagonal lattices at its membrane-proximal and membrane-distant faces, respectively. NEC coat architecture was determined by combining this information with integrative modeling using small-angle X-ray scattering data. The molecular arrangement of the NEC establishes the basic mechanism for budding and scission of tailored vesicles at the INM.


Assuntos
Transporte Ativo do Núcleo Celular , Capsídeo/metabolismo , Membrana Nuclear/metabolismo , Membrana Nuclear/ultraestrutura , Vesículas Transportadoras/ultraestrutura , Animais , Capsídeo/ultraestrutura , Chlorocebus aethiops , Microscopia Crioeletrônica , Tomografia com Microscopia Eletrônica , Herpesvirus Humano 1/metabolismo , Herpesvirus Suídeo 1/metabolismo , Membrana Nuclear/química , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Dímeros de Pirimidina , Espalhamento a Baixo Ângulo , Vesículas Transportadoras/metabolismo , Células Vero , Proteínas Virais/química , Proteínas Virais/metabolismo
2.
PLoS Pathog ; 18(8): e1010575, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35925870

RESUMO

Human Cytomegalovirus (HCMV) can infect a variety of cell types by using virions of varying glycoprotein compositions. It is still unclear how this diversity is generated, but spatio-temporally separated envelopment and egress pathways might play a role. So far, one egress pathway has been described in which HCMV particles are individually enveloped into small vesicles and are subsequently exocytosed continuously. However, some studies have also found enveloped virus particles inside multivesicular structures but could not link them to productive egress or degradation pathways. We used a novel 3D-CLEM workflow allowing us to investigate these structures in HCMV morphogenesis and egress at high spatio-temporal resolution. We found that multiple envelopment events occurred at individual vesicles leading to multiviral bodies (MViBs), which subsequently traversed the cytoplasm to release virions as intermittent bulk pulses at the plasma membrane to form extracellular virus accumulations (EVAs). Our data support the existence of a novel bona fide HCMV egress pathway, which opens the gate to evaluate divergent egress pathways in generating virion diversity.


Assuntos
Citomegalovirus , Montagem de Vírus , Citoplasma/metabolismo , Humanos , Vírion
3.
Mol Microbiol ; 117(6): 1317-1323, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35607767

RESUMO

Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus and the leading cause of congenital disabilities as well as a significant cause of disease in immunocompromised patients. The envelopment and egress of HCMV particles is an essential step of the viral life cycle as it determines viral spread and potentially tropism. Here we review the current literature on HCMV envelopment and egress with a particular focus on the role of virus-containing multivesicular body-like vesicles for virus egress and spread. We discuss the difficulties of determining the cellular provenance of these structures in light of viral redistribution of cellular marker proteins and provide potential paths to illuminate their genesis. Finally, we discuss how divergent egress pathways could result in virions of different tropisms.


Assuntos
Citomegalovirus , Montagem de Vírus , Citomegalovirus/metabolismo , Humanos , Proteínas/metabolismo , Vírion
4.
PLoS Pathog ; 17(12): e1010132, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34910768

RESUMO

Herpes simplex virus capsids are assembled and packaged in the nucleus and move by diffusion through the nucleoplasm to the nuclear envelope for egress. Analyzing their motion provides conclusions not only on capsid transport but also on the properties of the nuclear environment during infection. We utilized live-cell imaging and single-particle tracking to characterize capsid motion relative to the host chromatin. The data indicate that as the chromatin was marginalized toward the nuclear envelope it presented a restrictive barrier to the capsids. However, later in infection this barrier became more permissive and the probability of capsids to enter the chromatin increased. Thus, although chromatin marginalization initially restricted capsid transport to the nuclear envelope, a structural reorganization of the chromatin counteracted that to promote capsid transport later. Analyses of capsid motion revealed that it was subdiffusive, and that the diffusion coefficients were lower in the chromatin than in regions lacking chromatin. In addition, the diffusion coefficient in both regions increased during infection. Throughout the infection, the capsids were never enriched at the nuclear envelope, which suggests that instead of nuclear export the transport through the chromatin is the rate-limiting step for the nuclear egress of capsids. This provides motivation for further studies by validating the importance of intranuclear transport to the life cycle of HSV-1.


Assuntos
Transporte Biológico Ativo/fisiologia , Capsídeo/metabolismo , Cromatina/metabolismo , Membrana Nuclear/metabolismo , Simplexvirus/metabolismo , Animais , Chlorocebus aethiops , Herpes Simples , Células Vero , Replicação Viral/fisiologia
5.
Angew Chem Int Ed Engl ; 62(38): e202308271, 2023 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-37435767

RESUMO

The metabolic labeling of nucleic acids in living cells is highly desirable to track the dynamics of nucleic acid metabolism in real-time and has the potential to provide novel insights into cellular biology as well as pathogen-host interactions. Catalyst-free inverse electron demand Diels-Alder reactions (iEDDA) with nucleosides carrying highly reactive moieties such as axial 2-trans-cyclooctene (2TCOa) would be an ideal tool to allow intracellular labeling of DNA. However, cellular kinase phosphorylation of the modified nucleosides is needed after cellular uptake as triphosphates are not membrane permeable. Unfortunately, the narrow substrate window of most endogenous kinases limits the use of highly reactive moieties. Here, we apply our TriPPPro (triphosphate pronucleotide) approach to directly deliver a highly reactive 2TCOa-modified 2'-deoxycytidine triphosphate reporter into living cells. We show that this nucleoside triphosphate is metabolically incorporated into de novo synthesized cellular and viral DNA and can be labeled with highly reactive and cell-permeable fluorescent dye-tetrazine conjugates via iEDDA to visualize DNA in living cells directly. Thus, we present the first comprehensive method for live-cell imaging of cellular and viral nucleic acids using a two-step labeling approach.


Assuntos
DNA Viral , Nucleotídeos , Nucleosídeos , Corantes Fluorescentes , Reação de Cicloadição
6.
PLoS Pathog ; 16(6): e1008588, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32584886

RESUMO

The human adenovirus type 5 (HAdV5) causes disease of the upper and lower respiratory tract. The early steps of HAdV5 entry up to genome replication in the host nucleus have been extensively studied. However, late stages of infection remain poorly understood. Here, we set out to elucidate the spatiotemporal orchestration of late adenovirus nuclear remodeling in living cells. We generated virus mutants expressing fluorescently tagged protein IX (pIX) and protein V (pV), a capsid and viral genome associated protein, respectively. We found that during progeny virion production both proteins localize to a membrane-less, nuclear compartment, which is highly impermeable such that in immunofluorescence microscopy antibodies can hardly penetrate it. We termed this compartment 'late virion accumulation compartment' (LVAC). Correlation between light- and electron microscopy revealed that the LVAC contains paracrystalline arrays of viral capsids that arrange tightly packed within a honeycomb-like organization of viral DNA. Live-cell microscopy as well as FRAP measurements showed that the LVAC is rigid and restricts diffusion of larger molecules, indicating that capsids are trapped inside.


Assuntos
Infecções por Adenovirus Humanos/metabolismo , Adenovírus Humanos/fisiologia , Proteínas do Capsídeo/metabolismo , DNA Viral/metabolismo , Vírion/metabolismo , Replicação Viral , Células A549 , Infecções por Adenovirus Humanos/genética , Infecções por Adenovirus Humanos/patologia , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/ultraestrutura , DNA Viral/genética , Humanos , Vírion/genética
7.
Int J Mol Sci ; 22(23)2021 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-34884837

RESUMO

The human adenovirus type 5 (HAdV5) infects epithelial cells of the upper and lower respiratory tract. The virus causes lysis of infected cells and thus enables spread of progeny virions to neighboring cells for the next round of infection. The mechanism of adenovirus virion egress across the nuclear barrier is not known. The human adenovirus death protein (ADP) facilitates the release of virions from infected cells and has been hypothesized to cause membrane damage. Here, we set out to answer whether ADP does indeed increase nuclear membrane damage. We analyzed the nuclear envelope morphology using a combination of fluorescence and state-of-the-art electron microscopy techniques, including serial block-face scanning electron microscopy and electron cryo-tomography of focused ion beam-milled cells. We report multiple destabilization phenotypes of the nuclear envelope in HAdV5 infection. These include reduction of lamin A/C at the nuclear envelope, large-scale membrane invaginations, alterations in double membrane separation distance and small-scale membrane protrusions. Additionally, we measured increased nuclear membrane permeability and detected nuclear envelope lesions under cryoconditions. Unexpectedly, and in contrast to previous hypotheses, ADP did not have an effect on lamin A/C reduction or nuclear permeability.


Assuntos
Proteínas E3 de Adenovirus/metabolismo , Adenovírus Humanos/metabolismo , Membrana Nuclear/metabolismo , Proteínas E3 de Adenovirus/genética , Linhagem Celular Tumoral , Humanos , Lamina Tipo A/metabolismo , Microscopia Eletrônica de Varredura , Permeabilidade
8.
Eur J Immunol ; 49(5): 758-769, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30785638

RESUMO

The pathogenesis of primary sclerosing cholangitis (PSC), an autoimmune liver disease, remains unknown. The aim of this study was to characterize peripheral blood and intrahepatic NK cells from patients with PSC. Peripheral blood samples from patients with PSC, other autoimmune liver diseases, and from healthy control individuals were used, as well as liver tissues from PSC patients undergoing liver transplantation. Multiparameter flow cytometry showed that peripheral blood NK cells from PSC patients were significantly enriched for CCR7+ and CXCR3+ cells, and CCR7+ but not CXCR3+ cells were also significantly increased within intrahepatic NK cells. PSC patients undergoing liver transplantation furthermore had significantly higher plasma levels of the CCR7-ligand CCL21, and the CXCR3-ligands CXCL10 and CXCL11, and significantly higher levels of CCL21, but not CXCL10, were detected in liver tissues. CCR7+ and CXCR3+ NK cells from PSC patients exhibited significantly higher functional capacity in peripheral blood, but not liver tissues, consistent with chronic activation of these NK cells in the inflamed liver. These data show that PSC is characterized by intrahepatic CCL21 expression and accumulation of CCR7+ NK cells in the inflamed liver tissue.


Assuntos
Quimiocina CCL21/genética , Colangite Esclerosante/etiologia , Colangite Esclerosante/metabolismo , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Receptores CCR7/metabolismo , Biomarcadores , Quimiocina CCL21/metabolismo , Colangite Esclerosante/patologia , Suscetibilidade a Doenças , Expressão Gênica , Humanos , Fígado/imunologia , Fígado/metabolismo , Fígado/patologia , Contagem de Linfócitos , Especificidade de Órgãos/genética , Receptores CXCR3/metabolismo
10.
PLoS Pathog ; 14(12): e1007527, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30586431

RESUMO

Type III secretion systems (T3SSs) are essential virulence factors of numerous bacterial pathogens. Upon host cell contact the T3SS machinery-also named injectisome-assembles a pore complex/translocon within host cell membranes that serves as an entry gate for the bacterial effectors. Whether and how translocons are physically connected to injectisome needles, whether their phenotype is related to the level of effector translocation and which target cell factors trigger their formation have remained unclear. We employed the superresolution fluorescence microscopy techniques Stimulated Emission Depletion (STED) and Structured Illumination Microscopy (SIM) as well as immunogold electron microscopy to visualize Y. enterocolitica translocons during infection of different target cell types. Thereby we were able to resolve translocon and needle complex proteins within the same injectisomes and demonstrate that these fully assembled injectisomes are generated in a prevacuole, a PI(4,5)P2 enriched host cell compartment inaccessible to large extracellular proteins like antibodies. Furthermore, the operable translocons were produced by the yersiniae to a much larger degree in macrophages (up to 25% of bacteria) than in HeLa cells (2% of bacteria). However, when the Rho GTPase Rac1 was activated in the HeLa cells, uptake of the yersiniae into the prevacuole, translocon formation and effector translocation were strongly enhanced reaching the same levels as in macrophages. Our findings indicate that operable T3SS translocons can be visualized as part of fully assembled injectisomes with superresolution fluorescence microscopy techniques. By using this technology, we provide novel information about the spatiotemporal organization of T3SS translocons and their regulation by host cell factors.


Assuntos
Sistemas de Secreção Tipo III , Yersiniose/transmissão , Yersinia enterocolitica/patogenicidade , Humanos , Microscopia de Fluorescência
11.
PLoS Pathog ; 13(12): e1006813, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-29284065

RESUMO

Upon reactivation from latency and during lytic infections in neurons, alphaherpesviruses assemble cytosolic capsids, capsids associated with enveloping membranes, and transport vesicles harboring fully enveloped capsids. It is debated whether capsid envelopment of herpes simplex virus (HSV) is completed in the soma prior to axonal targeting or later, and whether the mechanisms are the same in neurons derived from embryos or from adult hosts. We used HSV mutants impaired in capsid envelopment to test whether the inner tegument proteins pUL36 or pUL37 necessary for microtubule-mediated capsid transport were sufficient for axonal capsid targeting in neurons derived from the dorsal root ganglia of adult mice. Such neurons were infected with HSV1-ΔUL20 whose capsids recruited pUL36 and pUL37, with HSV1-ΔUL37 whose capsids associate only with pUL36, or with HSV1-ΔUL36 that assembles capsids lacking both proteins. While capsids of HSV1-ΔUL20 were actively transported along microtubules in epithelial cells and in the somata of neurons, those of HSV1-ΔUL36 and -ΔUL37 could only diffuse in the cytoplasm. Employing a novel image analysis algorithm to quantify capsid targeting to axons, we show that only a few capsids of HSV1-ΔUL20 entered axons, while vesicles transporting gD utilized axonal transport efficiently and independently of pUL36, pUL37, or pUL20. Our data indicate that capsid motility in the somata of neurons mediated by pUL36 and pUL37 does not suffice for targeting capsids to axons, and suggest that capsid envelopment needs to be completed in the soma prior to targeting of herpes simplex virus to the axons, and to spreading from neurons to neighboring cells.


Assuntos
Herpesvirus Humano 1/fisiologia , Herpesvirus Humano 1/patogenicidade , Neurônios/virologia , Animais , Transporte Axonal , Axônios/ultraestrutura , Axônios/virologia , Capsídeo/fisiologia , Capsídeo/ultraestrutura , Células Cultivadas , Chlorocebus aethiops , Gânglios Espinais/virologia , Herpes Simples/virologia , Herpesvirus Humano 1/genética , Interações Hospedeiro-Patógeno , Humanos , Camundongos , Microscopia Eletrônica de Transmissão , Movimento/fisiologia , Mutação , Neurônios/ultraestrutura , Células Vero , Proteínas Virais/genética , Proteínas Virais/fisiologia , Proteínas Estruturais Virais/genética , Proteínas Estruturais Virais/fisiologia
12.
Proc Natl Acad Sci U S A ; 112(42): E5725-33, 2015 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-26438852

RESUMO

The nuclear chromatin structure confines the movement of large macromolecular complexes to interchromatin corrals. Herpesvirus capsids of approximately 125 nm assemble in the nucleoplasm and must reach the nuclear membranes for egress. Previous studies concluded that nuclear herpesvirus capsid motility is active, directed, and based on nuclear filamentous actin, suggesting that large nuclear complexes need metabolic energy to escape nuclear entrapment. However, this hypothesis has recently been challenged. Commonly used microscopy techniques do not allow the imaging of rapid nuclear particle motility with sufficient spatiotemporal resolution. Here, we use a rotating, oblique light sheet, which we dubbed a ring-sheet, to image and track viral capsids with high temporal and spatial resolution. We do not find any evidence for directed transport. Instead, infection with different herpesviruses induced an enlargement of interchromatin domains and allowed particles to diffuse unrestricted over longer distances, thereby facilitating nuclear egress for a larger fraction of capsids.


Assuntos
Capsídeo/metabolismo , Núcleo Celular/metabolismo , Herpesviridae/metabolismo , Linhagem Celular , Difusão , Herpesviridae/fisiologia , Microscopia de Fluorescência , Transporte Proteico , Replicação Viral
13.
PLoS Pathog ; 10(12): e1004535, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25474634

RESUMO

Egress of newly assembled herpesvirus particles from infected cells is a highly dynamic process involving the host secretory pathway working in concert with viral components. To elucidate the location, dynamics, and molecular mechanisms of alpha herpesvirus egress, we developed a live-cell fluorescence microscopy method to visualize the final transport and exocytosis of pseudorabies virus (PRV) particles in non-polarized epithelial cells. This method is based on total internal reflection fluorescence (TIRF) microscopy to selectively image fluorescent virus particles near the plasma membrane, and takes advantage of a virus-encoded pH-sensitive probe to visualize the precise moment and location of particle exocytosis. We performed single-particle tracking and mean squared displacement analysis to characterize particle motion, and imaged a panel of cellular proteins to identify those spatially and dynamically associated with viral exocytosis. Based on our data, individual virus particles travel to the plasma membrane inside small, acidified secretory vesicles. Rab GTPases, Rab6a, Rab8a, and Rab11a, key regulators of the plasma membrane-directed secretory pathway, are present on the virus secretory vesicle. These vesicles undergo fast, directional transport directly to the site of exocytosis, which is most frequently near patches of LL5ß, part of a complex that anchors microtubules to the plasma membrane. Vesicles are tightly docked at the site of exocytosis for several seconds, and membrane fusion occurs, displacing the virion a small distance across the plasma membrane. After exocytosis, particles remain tightly confined on the outer cell surface. Based on recent reports in the cell biological and alpha herpesvirus literature, combined with our spatial and dynamic data on viral egress, we propose an integrated model that links together the intracellular transport pathways and exocytosis mechanisms that mediate alpha herpesvirus egress.


Assuntos
Células Epiteliais/metabolismo , Herpesvirus Suídeo 1/fisiologia , Liberação de Vírus/fisiologia , Proteínas de Transporte/metabolismo , Linhagem Celular , Células Epiteliais/virologia , Humanos , Microscopia de Fluorescência , Proteínas rab de Ligação ao GTP/metabolismo
14.
Proc Natl Acad Sci U S A ; 110(37): E3516-25, 2013 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-23980169

RESUMO

A clinical hallmark of human alphaherpesvirus infections is peripheral pain or itching. Pseudorabies virus (PRV), a broad host range alphaherpesvirus, causes violent pruritus in many different animals, but the mechanism is unknown. Previous in vitro studies have shown that infected, cultured peripheral nervous system (PNS) neurons exhibited aberrant electrical activity after PRV infection due to the action of viral membrane fusion proteins, yet it is unclear if such activity occurs in infected PNS ganglia in living animals and if it correlates with disease symptoms. Using two-photon microscopy, we imaged autonomic ganglia in living mice infected with PRV strains expressing GCaMP3, a genetically encoded calcium indicator, and used the changes in calcium flux to monitor the activity of many neurons simultaneously with single-cell resolution. Infection with virulent PRV caused these PNS neurons to fire synchronously and cyclically in highly correlated patterns among infected neurons. This activity persisted even when we severed the presynaptic axons, showing that infection-induced firing is independent of input from presynaptic brainstem neurons. This activity was not observed after infections with an attenuated PRV recombinant used for circuit tracing or with PRV mutants lacking either viral glycoprotein B, required for membrane fusion, or viral membrane protein Us9, required for sorting virions and viral glycoproteins into axons. We propose that the viral fusion proteins produced by virulent PRV infection induce electrical coupling in unmyelinated axons in vivo. This action would then give rise to the synchronous and cyclical activity in the ganglia and contribute to the characteristic peripheral neuropathy.


Assuntos
Herpesvirus Suídeo 1/metabolismo , Neurônios/metabolismo , Neurônios/virologia , Pseudorraiva/metabolismo , Pseudorraiva/virologia , Proteínas Virais/metabolismo , Potenciais de Ação , Animais , Axônios/metabolismo , Axônios/virologia , Sinalização do Cálcio , Herpesvirus Suídeo 1/genética , Herpesvirus Suídeo 1/patogenicidade , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Lipoproteínas/metabolismo , Proteínas Luminescentes/metabolismo , Masculino , Camundongos , Nervos Periféricos/metabolismo , Nervos Periféricos/virologia , Doenças do Sistema Nervoso Periférico/etiologia , Doenças do Sistema Nervoso Periférico/metabolismo , Doenças do Sistema Nervoso Periférico/virologia , Fosfoproteínas/metabolismo , Prurido/etiologia , Prurido/metabolismo , Prurido/virologia , Proteínas Recombinantes/metabolismo , Glândula Submandibular/inervação , Glândula Submandibular/virologia , Proteínas do Envelope Viral/metabolismo , Virulência
15.
PLoS Pathog ; 8(9): e1002908, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22969428

RESUMO

During viral infections cellular gene expression is subject to rapid alterations induced by both viral and antiviral mechanisms. In this study, we applied metabolic labeling of newly transcribed RNA with 4-thiouridine (4sU-tagging) to dissect the real-time kinetics of cellular and viral transcriptional activity during lytic murine cytomegalovirus (MCMV) infection. Microarray profiling on newly transcribed RNA obtained at different times during the first six hours of MCMV infection revealed discrete functional clusters of cellular genes regulated with distinct kinetics at surprising temporal resolution. Immediately upon virus entry, a cluster of NF-κB- and interferon-regulated genes was induced. Rapid viral counter-regulation of this coincided with a very transient DNA-damage response, followed by a delayed ER-stress response. Rapid counter-regulation of all three clusters indicated the involvement of novel viral regulators targeting these pathways. In addition, down-regulation of two clusters involved in cell-differentiation (rapid repression) and cell-cycle (delayed repression) was observed. Promoter analysis revealed all five clusters to be associated with distinct transcription factors, of which NF-κB and c-Myc were validated to precisely match the respective transcriptional changes observed in newly transcribed RNA. 4sU-tagging also allowed us to study the real-time kinetics of viral gene expression in the absence of any interfering virion-associated-RNA. Both qRT-PCR and next-generation sequencing demonstrated a sharp peak of viral gene expression during the first two hours of infection including transcription of immediate-early, early and even well characterized late genes. Interestingly, this was subject to rapid gene silencing by 5-6 hours post infection. Despite the rapid increase in viral DNA load during viral DNA replication, transcriptional activity of some viral genes remained remarkably constant until late-stage infection, or was subject to further continuous decline. In summary, this study pioneers real-time transcriptional analysis during a lytic herpesvirus infection and highlights numerous novel regulatory aspects of virus-host-cell interaction.


Assuntos
Regulação Viral da Expressão Gênica , Infecções por Herpesviridae/genética , Interações Hospedeiro-Patógeno/genética , Muromegalovirus/genética , Animais , Perfilação da Expressão Gênica/métodos , Genes Virais/genética , Infecções por Herpesviridae/virologia , Camundongos , Análise em Microsséries , Família Multigênica/genética , Muromegalovirus/patogenicidade , Células NIH 3T3 , Regiões Promotoras Genéticas/genética , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Transcrição/genética , Transcrição Gênica/genética
16.
Nat Microbiol ; 9(7): 1842-1855, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38918469

RESUMO

The viral nuclear egress complex (NEC) allows herpesvirus capsids to escape from the nucleus without compromising the nuclear envelope integrity. The NEC lattice assembles on the inner nuclear membrane and mediates the budding of nascent nucleocapsids into the perinuclear space and their subsequent release into the cytosol. Its essential role makes it a potent antiviral target, necessitating structural information in the context of a cellular infection. Here we determined structures of NEC-capsid interfaces in situ using electron cryo-tomography, showing a substantial structural heterogeneity. In addition, while the capsid is associated with budding initiation, it is not required for curvature formation. By determining the NEC structure in several conformations, we show that curvature arises from an asymmetric assembly of disordered and hexagonally ordered lattice domains independent of pUL25 or other viral capsid vertex components. Our results advance our understanding of the mechanism of nuclear egress in the context of a living cell.


Assuntos
Capsídeo , Núcleo Celular , Microscopia Crioeletrônica , Membrana Nuclear , Liberação de Vírus , Núcleo Celular/metabolismo , Núcleo Celular/virologia , Humanos , Membrana Nuclear/metabolismo , Capsídeo/metabolismo , Proteínas do Capsídeo/metabolismo , Proteínas do Capsídeo/genética , Nucleocapsídeo/metabolismo , Tomografia com Microscopia Eletrônica , Proteínas Virais/metabolismo , Proteínas Virais/genética , Herpesviridae/fisiologia , Herpesviridae/genética
17.
J Virol ; 86(23): 12512-24, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22993161

RESUMO

Dominant-negative (DN) mutants are powerful tools for studying essential protein-protein interactions. A systematic genetic screen of the essential murine cytomegalovirus (MCMV) protein pM53 identified the accumulation of inhibitory mutations within conserved region 2 (CR2) and CR4. The strong inhibitory potential of these CR4 mutants is characterized by a particular phenotype. The DN effect of the small insertion mutations in CR2 was too weak to analyze (M. Popa, Z. Ruzsics, M. Lötzerich, L. Dölken, C. Buser, P. Walther, and U. H. Koszinowski, J. Virol. 84:9035-9046, 2010); therefore, the present study describes the construction of M53 alleles lacking CR2 (either completely or partially) and subsequent examination of the DN effect on MCMV replication upon conditional expression. Overexpression of CR2-deficient pM53 inhibited virus production by about 10,000-fold. This was due to interference with capsid export from the nucleus and viral genome cleavage/packaging. In addition, the fate of the nuclear envelopment complex in the presence of DN pM53 overexpression was analyzed. The CR2 mutants were able to bind to pM50, albeit to a lesser extent than the wild-type protein, and relocalized the wild-type nuclear envelope complex in infected cells. Unlike the CR4 DN, the CR2 DN mutants did not affect the stability of pM50.


Assuntos
Proteínas do Capsídeo/genética , Muromegalovirus/genética , Membrana Nuclear/virologia , Proteínas Nucleares/genética , Replicação Viral/genética , Alelos , Animais , Southern Blotting , Western Blotting , Proteínas do Capsídeo/metabolismo , Linhagem Celular , Ensaio de Imunoadsorção Enzimática , Teste de Complementação Genética , Imunoprecipitação , Camundongos , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Muromegalovirus/crescimento & desenvolvimento , Mutação/genética , Proteínas Nucleares/metabolismo , Plasmídeos/genética , Reação em Cadeia da Polimerase
18.
Adv Virus Res ; 116: 45-88, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37524482

RESUMO

Individual functional viral morphogenesis events are often dynamic, short, and infrequent and might be obscured by other pathways and dead-end products. Volumetric live cell imaging has become an essential tool for studying viral morphogenesis events. It allows following entire dynamic processes while providing functional evidence that the imaged process is involved in viral production. Moreover, it allows to capture many individual events and allows quantitative analysis. Finally, the correlation of volumetric live-cell data with volumetric electron microscopy (EM) can provide crucial insights into the ultrastructure and mechanisms of viral morphogenesis events. Here, we provide an overview and discussion of suitable imaging methods for volumetric correlative imaging of viral morphogenesis and frame them in a historical summary of their development.


Assuntos
Vírus , Microscopia Eletrônica , Morfogênese , Vírus/ultraestrutura
19.
Nat Microbiol ; 8(9): 1732-1747, 2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37550507

RESUMO

Herpesviruses assemble large enveloped particles that are difficult to characterize structurally due to their size, fragility and complex multilayered proteome with partially amorphous nature. Here we used crosslinking mass spectrometry and quantitative proteomics to derive a spatially resolved interactome map of intact human cytomegalovirus virions. This enabled the de novo allocation of 32 viral proteins into four spatially resolved virion layers, each organized by a dominant viral scaffold protein. The viral protein UL32 engages with all layers in an N-to-C-terminal radial orientation, bridging nucleocapsid to viral envelope. We observed the layer-specific incorporation of 82 host proteins, of which 39 are selectively recruited. We uncovered how UL32, by recruitment of PP-1 phosphatase, antagonizes binding to 14-3-3 proteins. This mechanism assures effective viral biogenesis, suggesting a perturbing role of UL32-14-3-3 interaction. Finally, we integrated these data into a coarse-grained model to provide global insights into the native configuration of virus and host protein interactions inside herpesvirions.


Assuntos
Citomegalovirus , Vírion , Humanos , Citomegalovirus/metabolismo , Vírion/metabolismo , Proteínas Virais/metabolismo , Nucleocapsídeo/metabolismo , Proteoma
20.
PLoS One ; 18(5): e0274065, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37163509

RESUMO

Downstream analysis of virus-infected cell samples, such as reverse transcription polymerase chain reaction (RT PCR) or mass spectrometry, often needs to be performed at lower biosafety levels than their actual cultivation, and thus the samples require inactivation before they can be transferred. Common inactivation methods involve chemical crosslinking with formaldehyde or denaturing samples with strong detergents, such as sodium dodecyl sulfate. However, these protocols destroy the protein quaternary structure and prevent the analysis of protein complexes, albeit through different chemical mechanisms. This often leads to studies being performed in over-expression or surrogate model systems. To address this problem, we generated a protocol that achieves the inactivation of infected cells through ultraviolet (UV) irradiation. UV irradiation damages viral genomes and crosslinks nucleic acids to proteins but leaves the overall structure of protein complexes mostly intact. Protein analysis can then be performed from intact cells without biosafety containment. While UV treatment protocols have been established to inactivate viral solutions, a protocol was missing to inactivate crude infected cell lysates, which heavily absorb light. In this work, we develop and validate a UV inactivation protocol for SARS-CoV-2, HSV-1, and HCMV-infected cells. A fluence of 10,000 mJ/cm2 with intermittent mixing was sufficient to completely inactivate infected cells, as demonstrated by the absence of viral replication even after three sequential passages of cells inoculated with the treated material. The herein described protocol should serve as a reference for inactivating cells infected with these or similar viruses and allow for the analysis of protein quaternary structure from bona fide infected cells.


Assuntos
COVID-19 , Herpesviridae , Humanos , SARS-CoV-2 , Replicação Viral , Inativação de Vírus/efeitos da radiação , Raios Ultravioleta
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA