RESUMEN
Genetic screens have transformed our ability to interrogate cellular factor requirements for viral infections1,2, but most current approaches are limited in their sensitivity, biased towards early stages of infection and provide only simplistic phenotypic information that is often based on survival of infected cells2-4. Here, by engineering human cytomegalovirus to express single guide RNA libraries directly from the viral genome, we developed virus-encoded CRISPR-based direct readout screening (VECOS), a sensitive, versatile, viral-centric approach that enables profiling of different stages of viral infection in a pooled format. Using this approach, we identified hundreds of host dependency and restriction factors and quantified their direct effects on viral genome replication, viral particle secretion and infectiousness of secreted particles, providing a multi-dimensional perspective on virus-host interactions. These high-resolution measurements reveal that perturbations altering late stages in the life cycle of human cytomegalovirus (HCMV) mostly regulate viral particle quality rather than quantity, establishing correct virion assembly as a critical stage that is heavily reliant on virus-host interactions. Overall, VECOS facilitates systematic high-resolution dissection of the role of human proteins during the infection cycle, providing a roadmap for in-depth study of host-herpesvirus interactions.
Asunto(s)
Sistemas CRISPR-Cas , Infecciones por Citomegalovirus , Citomegalovirus , Interacciones Huésped-Patógeno , ARN Guía de Sistemas CRISPR-Cas , Replicación Viral , Humanos , Línea Celular , Sistemas CRISPR-Cas/genética , Citomegalovirus/genética , Citomegalovirus/fisiología , Infecciones por Citomegalovirus/genética , Infecciones por Citomegalovirus/virología , Genoma Viral/genética , Interacciones Huésped-Patógeno/genética , ARN Guía de Sistemas CRISPR-Cas/genética , ARN Guía de Sistemas CRISPR-Cas/metabolismo , Virión/genética , Virión/metabolismo , Ensamble de Virus/genética , Liberación del Virus/genética , Replicación Viral/genéticaRESUMEN
Enveloped viruses undergo a complex multistep process of assembly, maturation, and release into the extracellular space utilizing host secretory machinery. Several studies of the herpesvirus subfamily have shown that secretory vesicles derived from the trans-Golgi network (TGN) or endosomes transport virions into the extracellular space. However, the regulatory mechanism underlying the release of Epstein-Barr virus, a human oncovirus, remains unclear. We demonstrate that disruption of BBLF1, a tegument component, suppressed viral release and resulted in the accumulation of viral particles on the inner side of the vesicular membrane. Organelle separation revealed the accumulation of infectious viruses in fractions containing vesicles derived from the TGN and late endosomes. Deficiency of an acidic amino acid cluster in BBLF1 reduced viral secretion. Moreover, truncational deletion of the C-terminal region of BBLF1 increased infectious virus production. These findings suggest that BBLF1 regulates the viral release pathway and reveal a new aspect of tegument protein function. IMPORTANCE Several viruses have been linked to the development of cancer in humans. Epstein-Barr virus (EBV), the first identified human oncovirus, causes a wide range of cancers. Accumulating literature has demonstrated the role of viral reactivation in tumorigenesis. Elucidating the functions of viral lytic genes induced by reactivation, and the mechanisms of lytic infection, is essential to understanding pathogenesis. Progeny viral particles synthesized during lytic infection are released outside the cell after the assembly, maturation, and release steps, leading to further infection. Through functional analysis using BBLF1-knockout viruses, we demonstrated that BBLF1 promotes viral release. The acidic amino acid cluster in BBLF1 was also important for viral release. Conversely, mutants lacking the C terminus exhibited more efficient virus production, suggesting that BBLF1 is involved in the fine-tuning of progeny release during the EBV life cycle.
Asunto(s)
Herpesvirus Humano 4 , Vesículas Secretoras , Proteínas Virales , Liberación del Virus , Replicación Viral , Humanos , Infecciones por Virus de Epstein-Barr/virología , Herpesvirus Humano 4/fisiología , Vesículas Secretoras/metabolismo , Vesículas Secretoras/virología , Virión/fisiología , Replicación Viral/fisiología , Células HEK293 , Proteínas Virales/metabolismo , Liberación del Virus/genéticaRESUMEN
Tetherin prevents viral cross-species transmission by inhibiting the release of multiple enveloped viruses from infected cells. With the evolution of simian immunodeficiency virus of chimpanzees (SIVcpz), a pandemic human immunodeficiency virus type 1 (HIV-1) precursor, its Vpu protein can antagonize human tetherin (hTetherin). Macaca leonina (northern pig-tailed macaque [NPM]) is susceptible to HIV-1, but host-specific restriction factors limit virus replication in vivo. In this study, we isolated the virus from NPMs infected with strain stHIV-1sv (with a macaque-adapted HIV-1 env gene from simian-human immunodeficiency virus SHIV-KB9, a vif gene replaced by SIVmac239, and other genes originating from HIV-1NL4.3) and found that a single acidic amino acid substitution (G53D) in Vpu could increase its ability to degrade the tetherin of macaques (mTetherin) mainly through the proteasome pathway, resulting in an enhanced release and resistance to interferon inhibition of the mutant stHIV-1sv strain, with no influence on the other functions of Vpu. IMPORTANCE HIV-1 has obvious host specificity, which has greatly hindered the construction of animal models and severely restricted the development of HIV-1 vaccines and drugs. To overcome this barrier, we attempted to isolate the virus from NPMs infected with stHIV-1sv, search for a strain with an adaptive mutation in NPMs, and develop a more appropriate nonhuman primate model of HIV-1. This is the first report identifying HIV-1 adaptations in NPMs. It suggests that while tetherin may limit HIV-1 cross-species transmission, the Vpu protein in HIV-1 can overcome this species barrier through adaptive mutation, increasing viral replication in the new host. This finding will be beneficial to building an appropriate animal model for HIV-1 infection and promoting the development of HIV-1 vaccines and drugs.
Asunto(s)
Antígeno 2 del Estroma de la Médula Ósea , VIH-1 , Macaca , Proteínas Virales , Liberación del Virus , VIH-1/genética , VIH-1/patogenicidad , Proteínas Virales/genética , Proteínas Virales/metabolismo , Mutación , Antígeno 2 del Estroma de la Médula Ósea/metabolismo , Ubiquitina/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Liberación del Virus/genética , Sustitución de Aminoácidos/genética , Infecciones por VIH/virología , Modelos Animales de Enfermedad , Replicación Viral/genéticaRESUMEN
The current COVID-19 pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The positive-sense single-stranded RNA virus contains a single linear RNA segment that serves as a template for transcription and replication, leading to the synthesis of positive and negative-stranded viral RNA (vRNA) in infected cells. Tools to visualize vRNA directly in infected cells are critical to analyze the viral replication cycle, screen for therapeutic molecules, or study infections in human tissue. Here, we report the design, validation, and initial application of FISH probes to visualize positive or negative RNA of SARS-CoV-2 (CoronaFISH). We demonstrate sensitive visualization of vRNA in African green monkey and several human cell lines, in patient samples and human tissue. We further demonstrate the adaptation of CoronaFISH probes to electron microscopy. We provide all required oligonucleotide sequences, source code to design the probes, and a detailed protocol. We hope that CoronaFISH will complement existing techniques for research on SARS-CoV-2 biology and COVID-19 pathophysiology, drug screening, and diagnostics.
Asunto(s)
COVID-19/diagnóstico , Hibridación Fluorescente in Situ/métodos , ARN Viral/genética , SARS-CoV-2/genética , Replicación Viral/genética , Adenosina Monofosfato/análogos & derivados , Adenosina Monofosfato/farmacología , Alanina/análogos & derivados , Alanina/farmacología , Animales , Antivirales/farmacología , COVID-19/virología , Células CACO-2 , Línea Celular Tumoral , Chlorocebus aethiops , Humanos , Hibridación in Situ/métodos , Microscopía Electrónica/métodos , ARN Viral/ultraestructura , Reproducibilidad de los Resultados , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/fisiología , Sensibilidad y Especificidad , Células Vero , Liberación del Virus/efectos de los fármacos , Liberación del Virus/genética , Liberación del Virus/fisiología , Replicación Viral/efectos de los fármacos , Replicación Viral/fisiología , Tratamiento Farmacológico de COVID-19RESUMEN
Molecular details of field rabies virus (RABV) adaptation to cell culture replication are insufficiently understood. A better understanding of adaptation may not only reveal requirements for efficient RABV replication in cell lines, but may also provide novel insights into RABV biology and adaptation-related loss of virulence and pathogenicity. Using two recombinant field rabies virus clones (rRABV Dog and rRABV Fox), we performed virus passages in three different cell lines to identify cell culture adaptive mutations. Ten passages were sufficient for the acquisition of adaptive mutations in the glycoprotein G and in the C-terminus of phosphoprotein P. Apart from the insertion of a glycosylation sequon via the mutation D247N in either virus, both acquired additional and cell line-specific mutations after passages on BHK (K425N) and MDCK-II (R346S or R350G) cells. As determined by virus replication kinetics, complementation, and immunofluorescence analysis, the major bottleneck in cell culture replication was the intracellular accumulation of field virus G protein, which was overcome after the acquisition of the adaptive mutations. Our data indicate that limited release of extracellular infectious virus at the plasma membrane is a defined characteristic of highly virulent field rabies viruses and we hypothesize that the observed suboptimal release of infectious virions is due to the inverse correlation of virus release and virulence in vivo.
Asunto(s)
Antígenos Virales/genética , Virus de la Rabia/genética , Proteínas del Envoltorio Viral/genética , Liberación del Virus/genética , Animales , Anticuerpos Antivirales/sangre , Antígenos Virales/metabolismo , Técnicas de Cultivo de Célula , Línea Celular , Perros , Glicoproteínas/genética , Glicosilación , Mutación Puntual/genética , Rabia/virología , Proteínas del Envoltorio Viral/metabolismo , Proteínas Virales/genética , Virión/metabolismo , Virulencia/genética , Replicación Viral/genéticaRESUMEN
Ebolavirus (EBOV) is a negative-sense RNA virus that causes severe hemorrhagic fever in humans. The matrix protein VP40 facilitates viral budding by binding to lipids in the host cell plasma membrane and driving the formation of filamentous, pleomorphic virus particles. The C-terminal domain of VP40 contains two highly-conserved cysteine residues at positions 311 and 314, but their role in the viral life cycle is unknown. We therefore investigated the properties of VP40 mutants in which the conserved cysteine residues were replaced with alanine. The C311A mutation significantly increased the affinity of VP40 for membranes containing phosphatidylserine (PS), resulting in the assembly of longer virus-like particles (VLPs) compared to wild-type VP40. The C314A mutation also increased the affinity of VP40 for membranes containing PS, albeit to a lesser degree than C311A. The double mutant behaved in a similar manner to the individual mutants. Computer modeling revealed that both cysteine residues restrain a loop segment containing lysine residues that interact with the plasma membrane, but Cys311 has the dominant role. Accordingly, the C311A mutation increases the flexibility of this membrane-binding loop, changes the profile of hydrogen bonding within VP40 and therefore binds to PS with greater affinity. This is the first evidence that mutations in VP40 can increase its affinity for biological membranes and modify the length of Ebola VLPs. The Cys311 and Cys314 residues therefore play an important role in dynamic interactions at the plasma membrane by modulating the ability of VP40 to bind PS.
Asunto(s)
Ebolavirus/genética , Proteínas de la Matriz Viral/genética , Animales , Células COS , Membrana Celular/metabolismo , Chlorocebus aethiops , Cisteína/genética , Ebolavirus/metabolismo , Humanos , Lípidos/fisiología , Simulación de Dinámica Molecular , Fosfatidilserinas/metabolismo , Polimorfismo de Nucleótido Simple/genética , Unión Proteica , Multimerización de Proteína , Proteínas de la Matriz Viral/metabolismo , Proteínas de la Matriz Viral/ultraestructura , Virión/metabolismo , Ensamble de Virus/genética , Liberación del Virus/genéticaRESUMEN
Ebola virus (EBOV) attaches to target cells using two categories of cell surface receptors: C-type lectins and phosphatidylserine (PS) receptors. PS receptors typically bind to apoptotic cell membrane PS and orchestrate the uptake and clearance of apoptotic debris. Many enveloped viruses also contain exposed PS and can therefore exploit these receptors for cell entry. Viral infection can induce PS externalization in host cells, resulting in increased outer PS levels on budding virions. Scramblase enzymes carry out cellular PS externalization; thus, we targeted these proteins in order to manipulate viral envelope PS levels. We investigated two scramblases previously identified to be involved in EBOV PS levels, transmembrane protein 16F and Xk-related protein 8 (XKR8), as possible mediators of cellular and viral envelope surface PS levels during the replication of recombinant vesicular stomatitis virus containing its native glycoprotein (rVSV/G) or the EBOV glycoprotein (rVSV/EBOV-GP). We found that rVSV/G and rVSV/EBOV-GP virions produced in XKR8 knockout cells contain decreased levels of PS on their surfaces, and the PS-deficient rVSV/EBOV-GP virions are 70% less efficient at infecting cells through PS receptors. We also observed reduced rVSV and EBOV virus-like particle (VLP) budding in ΔXKR8 cells. Deletion of XKR8 in HAP1 cells reduced rVSV/G and rVSV/EBOV-GP budding by 60 and 65%, respectively, and reduced Ebola VLP budding more than 60%. We further demonstrated that caspase cleavage of XKR8 is required to promote budding. This suggests that XKR8, in addition to mediating virion PS levels, may also be critical for enveloped virus budding at the plasma membrane. IMPORTANCE Within the last decade, countries in western and central Africa have experienced the most widespread and deadly Ebola outbreaks since Ebola virus was identified in 1976. While outbreaks are primarily attributed to zoonotic transfer events, new evidence is emerging outbreaks may be caused by a combination of spillover events and viral latency or persistence in survivors. The possibility that Ebola virus can remain dormant and then reemerge in survivors highlights the critical need to prevent the virus from entering and establishing infection in human cells. Thus far, host cell scramblases TMEM16F and XKR8 have been implicated in Ebola envelope surface phosphatidylserine (PS) and cell entry using PS receptors. We assessed the contributions of these proteins using CRISPR knockout cells and two EBOV models: rVSV/EBOV-GP and EBOV VLPs. We observed that XKR8 is required for optimal EBOV envelope PS levels and infectivity and particle budding across all viral models.
Asunto(s)
Ebolavirus/metabolismo , Fosfatidilserinas/metabolismo , Liberación del Virus/fisiología , Línea Celular , Ebolavirus/patogenicidad , Glicoproteínas/metabolismo , Fiebre Hemorrágica Ebola/virología , Humanos , Fosfatidilserinas/fisiología , Proteínas de Transferencia de Fosfolípidos/metabolismo , Proteínas de Transferencia de Fosfolípidos/fisiología , Proteínas del Envoltorio Viral/metabolismo , Virión/metabolismo , Ensamble de Virus/genética , Ensamble de Virus/fisiología , Liberación del Virus/genéticaRESUMEN
A deficient interferon (IFN) response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has been implicated as a determinant of severe coronavirus disease 2019 (COVID-19). To identify the molecular effectors that govern IFN control of SARS-CoV-2 infection, we conducted a large-scale gain-of-function analysis that evaluated the impact of human IFN-stimulated genes (ISGs) on viral replication. A limited subset of ISGs were found to control viral infection, including endosomal factors inhibiting viral entry, RNA binding proteins suppressing viral RNA synthesis, and a highly enriched cluster of endoplasmic reticulum (ER)/Golgi-resident ISGs inhibiting viral assembly/egress. These included broad-acting antiviral ISGs and eight ISGs that specifically inhibited SARS-CoV-2 and SARS-CoV-1 replication. Among the broad-acting ISGs was BST2/tetherin, which impeded viral release and is antagonized by SARS-CoV-2 Orf7a protein. Overall, these data illuminate a set of ISGs that underlie innate immune control of SARS-CoV-2/SARS-CoV-1 infection, which will facilitate the understanding of host determinants that impact disease severity and offer potential therapeutic strategies for COVID-19.
Asunto(s)
Antígenos CD/genética , Interacciones Huésped-Patógeno/genética , Factores Reguladores del Interferón/genética , Interferón Tipo I/genética , SARS-CoV-2/genética , Proteínas Virales/genética , Animales , Antígenos CD/química , Antígenos CD/inmunología , Sitios de Unión , Línea Celular Tumoral , Chlorocebus aethiops , Retículo Endoplásmico/genética , Retículo Endoplásmico/inmunología , Retículo Endoplásmico/virología , Proteínas Ligadas a GPI/química , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/inmunología , Regulación de la Expresión Génica , Aparato de Golgi/genética , Aparato de Golgi/inmunología , Aparato de Golgi/virología , Células HEK293 , Interacciones Huésped-Patógeno/inmunología , Humanos , Inmunidad Innata , Factores Reguladores del Interferón/clasificación , Factores Reguladores del Interferón/inmunología , Interferón Tipo I/inmunología , Simulación del Acoplamiento Molecular , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , SARS-CoV-2/inmunología , Transducción de Señal , Células Vero , Proteínas Virales/química , Proteínas Virales/inmunología , Internalización del Virus , Liberación del Virus/genética , Liberación del Virus/inmunología , Replicación Viral/genética , Replicación Viral/inmunologíaRESUMEN
The Ebola virus matrix protein VP40 forms distinct structures linked to distinct functions in the virus life cycle. Dimeric VP40 is a structural protein associated with virus assembly, while octameric, ring-shaped VP40 is associated with transcriptional control. In this study, we show that suitable nucleic acid is sufficient to trigger a dynamic transformation of VP40 dimer into the octameric ring. Deep sequencing reveals a binding preference of the VP40 ring for the 3' untranslated region of cellular mRNA and a guanine- and adenine-rich binding motif. Complementary analyses of the nucleic-acid-induced VP40 ring by native mass spectrometry, electron microscopy, and X-ray crystal structures at 1.8 and 1.4 Å resolution reveal the stoichiometry of RNA binding, as well as an interface involving a key guanine nucleotide. The host factor-induced structural transformation of protein structure in response to specific RNA triggers in the Ebola virus life cycle presents unique opportunities for therapeutic inhibition.
Asunto(s)
Regiones no Traducidas 3' , Ebolavirus/genética , Guanina/química , Interacciones Huésped-Patógeno/genética , Nucleoproteínas/química , Proteínas del Núcleo Viral/química , Sitios de Unión , Cristalografía por Rayos X , Ebolavirus/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Guanina/metabolismo , Células HEK293 , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Modelos Moleculares , Nucleoproteínas/genética , Nucleoproteínas/metabolismo , Motivos de Nucleótidos , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas del Núcleo Viral/genética , Proteínas del Núcleo Viral/metabolismo , Ensamble de Virus/genética , Liberación del Virus/genéticaRESUMEN
Hepatitis C virus (HCV) causes acute hepatitis C and can lead to life-threatening complications if it becomes chronic. The HCV genome is a single plus strand of RNA. Its intracellular replication is a spatiotemporally coordinated process of RNA translation upon cell infection, RNA synthesis within a replication compartment, and virus particle production. While HCV is mainly transmitted via mature infectious virus particles, it has also been suggested that HCV-infected cells can secrete HCV RNA carrying exosomes that can infect cells in a receptor independent manner. In order to gain insight into these two routes of transmission, we developed a series of intracellular HCV replication models that include HCV RNA secretion and/or virus assembly and release. Fitting our models to in vitro data, in which cells were infected with HCV, suggests that initially most secreted HCV RNA derives from intracellular cytosolic plus-strand RNA, but subsequently secreted HCV RNA derives equally from the cytoplasm and the replication compartments. Furthermore, our model fits to the data suggest that the rate of virus assembly and release is limited by host cell resources. Including the effects of direct acting antivirals in our models, we found that in spite of decreasing intracellular HCV RNA and extracellular virus concentration, low level HCV RNA secretion may continue as long as intracellular RNA is available. This may possibly explain the presence of detectable levels of plasma HCV RNA at the end of treatment even in patients that ultimately attain a sustained virologic response.
Asunto(s)
Hepacivirus/genética , Hepacivirus/fisiología , Modelos Biológicos , Antivirales/farmacología , Biología Computacional , Simulación por Computador , Exosomas/virología , Hepacivirus/patogenicidad , Hepatitis C Crónica/tratamiento farmacológico , Hepatitis C Crónica/virología , Interacciones Microbiota-Huesped/genética , Interacciones Microbiota-Huesped/fisiología , Humanos , Conceptos Matemáticos , ARN Viral/biosíntesis , ARN Viral/genética , Compartimentos de Replicación Viral/fisiología , Virión/genética , Virión/fisiología , Ensamble de Virus/efectos de los fármacos , Ensamble de Virus/genética , Ensamble de Virus/fisiología , Liberación del Virus/genética , Liberación del Virus/fisiología , Replicación Viral/efectos de los fármacos , Replicación Viral/genética , Replicación Viral/fisiologíaRESUMEN
Herpesviruses uniquely express two essential nuclear egress-regulating proteins forming a heterodimeric nuclear egress complex (core NEC). These core NECs serve as hexameric lattice-structured platforms for capsid docking and recruit viral and cellular NEC-associated factors that jointly exert nuclear lamina as well as membrane-rearranging functions (multicomponent NEC). The regulation of nuclear egress has been profoundly analyzed for murine and human cytomegaloviruses (CMVs) on a mechanistic basis, followed by the description of core NEC crystal structures, first for HCMV, then HSV-1, PRV and EBV. Interestingly, the highly conserved structural domains of these proteins stand in contrast to a very limited sequence conservation of the key amino acids within core NEC-binding interfaces. Even more surprising, although a high functional consistency was found when regarding the basic role of NECs in nuclear egress, a clear specification was identified regarding the limited, subfamily-spanning binding properties of core NEC pairs and NEC multicomponent proteins. This review summarizes the evolving picture of the relationship between sequence coevolution, structural conservation and properties of NEC interaction, comparing HCMV to α-, ß- and γ-herpesviruses. Since NECs represent substantially important elements of herpesviral replication that are considered as drug-accessible targets, their putative translational use for antiviral strategies is discussed.
Asunto(s)
Transporte Activo de Núcleo Celular/genética , Alphaherpesvirinae/genética , Citomegalovirus/genética , Gammaherpesvirinae/genética , Liberación del Virus/genética , Transporte Activo de Núcleo Celular/fisiología , Alphaherpesvirinae/metabolismo , Secuencia de Aminoácidos/genética , Cápside/metabolismo , Proteínas de la Cápside/genética , Citomegalovirus/metabolismo , Gammaherpesvirinae/metabolismo , Humanos , Membrana Nuclear/metabolismo , Lámina Nuclear/fisiología , Liberación del Virus/fisiologíaRESUMEN
Kaposi sarcoma is the most common cancer in human immunodeficiency virus-positive individuals and is caused by Kaposi sarcoma-associated herpesvirus (KSHV). It is believed that a small number of latently infected Kaposi sarcoma tumor cells undergo spontaneous lytic reactivation to produce viral progeny for infection of new cells. Here, we use matched donor-derived human dermal blood and lymphatic endothelial cells (BEC and LEC, respectively) to show that KSHV-infected BECs progressively lose viral genome as they proliferate. In sharp contrast, KSHV-infected LECs predominantly entered lytic replication, underwent cell lysis, and released new virus. Continuous lytic cell lysis and de novo infection allowed LEC culture to remain infected for a prolonged time. Because of the strong propensity of LECs toward lytic replication, LECs maintained virus as a population, despite the death of individual host cells from lytic lysis. The master regulator of lymphatic development, Prox1, bound the promoter of the RTA gene to upregulate its expression and physically interacted with RTA protein to coregulate lytic genes. Thus, LECs may serve as a proficient viral reservoir that provides viral progeny for continuous de novo infection of tumor origin cells, and potentially BECs and mesenchymal stem cells, which give rise to Kaposi sarcoma tumors. Our study reveals drastically different host cell behaviors between BEC and LEC and defines the underlying mechanisms of the lymphatic cell environment supporting persistent infection in Kaposi sarcoma tumors. SIGNIFICANCE: This study defines the mechanism by which Kaposi's sarcoma could be maintained by virus constantly produced by lymphatic cells in HIV-positive individuals.
Asunto(s)
Herpesvirus Humano 8/fisiología , Proteínas de Homeodominio/fisiología , Vasos Linfáticos/virología , Sarcoma de Kaposi , Microambiente Tumoral/fisiología , Proteínas Supresoras de Tumor/fisiología , Liberación del Virus/genética , Replicación Viral/genética , Transformación Celular Viral/genética , Células Cultivadas , Células Endoteliales/metabolismo , Células Endoteliales/patología , Células Endoteliales/virología , Regulación Viral de la Expresión Génica , Células HEK293 , VIH/fisiología , Humanos , Vasos Linfáticos/metabolismo , Vasos Linfáticos/patología , Sarcoma de Kaposi/genética , Sarcoma de Kaposi/patología , Sarcoma de Kaposi/virología , Latencia del Virus/genéticaRESUMEN
Persistent viruses cause chronic disease, and threaten the lives of immunosuppressed individuals. Here, we elucidate a mechanism supporting the persistence of human adenovirus (AdV), a virus that can kill immunosuppressed patients. Cell biological analyses, genetics and chemical interference demonstrate that one of five AdV membrane proteins, the E3-19K glycoprotein specifically triggers the unfolded protein response (UPR) sensor IRE1α in the endoplasmic reticulum (ER), but not other UPR sensors, such as protein kinase R-like ER kinase (PERK) and activating transcription factor 6 (ATF6). The E3-19K lumenal domain activates the IRE1α nuclease, which initiates mRNA splicing of X-box binding protein-1 (XBP1). XBP1s binds to the viral E1A-enhancer/promoter sequence, and boosts E1A transcription, E3-19K levels and lytic infection. Inhibition of IRE1α nuclease interrupts the five components feedforward loop, E1A, E3-19K, IRE1α, XBP1s, E1A enhancer/promoter. This loop sustains persistent infection in the presence of the immune activator interferon, and lytic infection in the absence of interferon.
Asunto(s)
Infecciones por Adenoviridae/inmunología , Adenoviridae/patogenicidad , Proteínas E3 de Adenovirus/metabolismo , Endorribonucleasas/metabolismo , Regulación Viral de la Expresión Génica/inmunología , Proteínas Serina-Treonina Quinasas/metabolismo , Células A549 , Adenoviridae/genética , Adenoviridae/inmunología , Infecciones por Adenoviridae/genética , Infecciones por Adenoviridae/virología , Proteínas E1A de Adenovirus/genética , Enfermedad Crónica , Retículo Endoplásmico/metabolismo , Endorribonucleasas/genética , Técnicas de Silenciamiento del Gen , Técnicas de Inactivación de Genes , Células HeLa , Interacciones Huésped-Patógeno/genética , Humanos , Huésped Inmunocomprometido , Interferón gamma/genética , Interferón gamma/inmunología , Interferón gamma/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Empalme del ARN , Latencia del Virus , Liberación del Virus/genética , Proteína 1 de Unión a la X-Box/genéticaRESUMEN
Newly assembled herpesvirus nucleocapsids traverse the intact nuclear envelope by a vesicle-mediated nucleo-cytoplasmic transport for final virion maturation in the cytoplasm. For this, they bud at the inner nuclear membrane resulting in primary enveloped particles in the perinuclear space (PNS) followed by fusion of the primary envelope with the outer nuclear membrane (ONM). While the conserved viral nuclear egress complex orchestrates the first steps, effectors of fusion of the primary virion envelope with the ONM are still mostly enigmatic but might include cellular proteins like SUN2 or ESCRT-III components. Here, we analyzed the influence of the only known AAA+ ATPases located in the endoplasmic reticulum and the PNS, the Torsins (Tor), on nuclear egress of the alphaherpesvirus pseudorabies virus. For this overexpression of wild type and mutant proteins as well as CRISPR/Cas9 genome editing was applied. Neither single overexpression nor gene knockout (KO) of TorA or TorB had a significant impact. However, TorA/B double KO cells showed decreased viral titers at early time points of infection and an accumulation of primary virions in the PNS pointing to a delay in capsid release during nuclear egress.
Asunto(s)
ATPasas Asociadas con Actividades Celulares Diversas/metabolismo , Transporte Activo de Núcleo Celular/fisiología , Núcleo Celular/virología , Herpesvirus Suido 1/metabolismo , ATPasas Asociadas con Actividades Celulares Diversas/genética , Animales , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Citoplasma/virología , Herpesvirus Suido 1/genética , Chaperonas Moleculares/metabolismo , Membrana Nuclear/metabolismo , Conejos , Proteínas Virales/metabolismo , Liberación del Virus/genética , Liberación del Virus/fisiologíaRESUMEN
Epstein-Barr virus (EBV) genomic DNA is replicated and packaged into procapsids in the nucleus to form nucleocapsids, which are then transported into the cytoplasm for tegumentation and final maturation. The process is facilitated by the coordination of the viral nuclear egress complex (NEC), which consists of BFLF2 and BFRF1. By expression alone, BFLF2 is distributed mainly in the nucleus. However, it colocalizes with BFRF1 at the nuclear rim and in cytoplasmic nuclear envelope-derived vesicles in coexpressing cells, suggesting temporal control of the interaction between BFLF2 and BFRF1 is critical for their proper function. The N-terminal sequence of BFLF2 is less conserved than that of alpha- and betaherpesvirus homologs. Here, we found that BFLF2 amino acids (aa) 2 to 102 are required for both nuclear targeting and its interaction with BFRF1. Coimmunoprecipitation and confocal analysis indicated that aa 82 to 106 of BFLF2 are important for its interaction with BFRF1. Three crucial amino acids (R47, K50, and R52) and several noncontinuous arginine and histidine residues within aa 59 to 80 function together as a noncanonical nuclear localization signal (NLS), which can be transferred onto yellow fluorescent protein (YFP)-LacZ for nuclear targeting in an importin ß-dependent manner. Virion secretion is defective in 293 cells harboring a BFLF2 knockout EBV bacmid upon lytic induction and is restored by trans-complementation of wild-type BFLF2, but not NLS or BFRF1-interacting defective mutants. In addition, multiple domains of BFRF1 were found to bind BFLF2, suggesting multiple contact regions within BFRF1 and BFLF2 are required for proper nuclear egress of EBV nucleocapsids.IMPORTANCE Although Epstein-Barr virus (EBV) BFRF1 and BFLF2 are homologs of conserved viral nuclear egress complex (NEC) in all human herpesviruses, unique amino acid sequences and functions were identified in both proteins. In this study, the nuclear targeting and BFRF1-interacting domains were found within the N terminus of BFLF2. We showed that amino acids (aa) 82 to 106 are the major region required for BFLF2 to interact with BFRF1. However, the coimmunoprecipitation (Co-IP) data and glutathione transferase (GST) pulldown experiments revealed that multiple regions of both proteins contribute to reciprocal interactions. Different from the canonical nuclear localization signal (NLS) in other herpes viral homologs, BFLF2 contains a novel importin-dependent nuclear localization signal, including R47, K50, and R52 and several neighboring discontinuous arginine and histidine residues. Using a bacmid complementation system, we show that both the nuclear targeting and the novel nuclear localization signal within aa 82 to 106 of BFLF2 are required for virion secretion.
Asunto(s)
Núcleo Celular/virología , Herpesvirus Humano 4/genética , Proteínas Virales/metabolismo , Liberación del Virus/fisiología , Secuencia de Aminoácidos , Línea Celular , Citoplasma/metabolismo , Glutatión Transferasa/metabolismo , Células HEK293 , Células HeLa , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Membrana Nuclear , Señales de Localización Nuclear/metabolismo , Conformación Proteica , Análisis de Secuencia de Proteína , Proteínas Virales/química , Proteínas Virales/genética , Virión/metabolismo , Liberación del Virus/genética , beta CarioferinasRESUMEN
The oncogenic gammaherpesviruses, including human Epstein-Barr virus (EBV), human Kaposi's sarcoma-associated herpesvirus (KSHV), and murine gammaherpesvirus 68 (MHV68, γHV68, MuHV-4) establish life-long latency in circulating B cells. The precise determinants that mediate in vivo gammaherpesvirus latency and tumorigenesis remain unclear. The EBV-encoded RNAs (EBERs) are among the first noncoding RNAs ever identified and have been the subject of decades of studies; however, their biological roles during in vivo infection remain unknown. Herein, we use a series of refined virus mutants to define the active isoform of MHV68 noncoding RNA TMER4 and demonstrate that EBV EBER1 functionally conserves this activity in vivo to promote egress of infected B cells from lymph nodes into peripheral circulation.
Asunto(s)
Gammaherpesvirinae/genética , ARN no Traducido , ARN Viral , Liberación del Virus/genética , Animales , Células Cultivadas , Infecciones por Herpesviridae/virología , Ratones , Conformación de Ácido Nucleico , ARN no Traducido/química , ARN no Traducido/genética , ARN no Traducido/fisiología , ARN Viral/química , ARN Viral/genética , ARN Viral/fisiología , Bazo/citología , Bazo/virología , Latencia del Virus/genéticaRESUMEN
Human astroviruses (HAstVs), non-enveloped RNA viruses with positive-sense RNA genomes, are an important cause of acute gastroenteritis in young children, although the processes that produce infectious virions are not clearly defined. To track the viral replication complex (RC) upon HAstV1 infection, the subcellular distribution of double-stranded (ds) RNA and of ORF1b, a viral RNA polymerase, was examined by immunocytochemistry. Foci that were positive for dsRNA and for ORF1b were co-localized, and both foci were also co-localized with resident proteins of the endoplasmic reticulum (ER). Focusing on the association between the HAstV RC and ER, we examined the expression of unfolded protein response (UPR) markers and found that targets of eukaryotic translation initiation factor 2α (eIF2α)-activating transcription factor 4 (ATF4), including CCAAT/enhancer-binding protein homologous protein (CHOP), a proapoptotic transcription factor, were upregulated at the late phase in HAstV-infected cells. Consistently, eIF2α was phosphorylated at the late phase of HAstV infection. The formation of foci resembling stress granules, another known downstream response to eIF2α phosphorylation, was also observed at the same period. Phosphorylation of eIF2α was attenuated in protein kinase R (PKR)-knockdown cells, suggesting that, unlike the canonical ER stress response, PKR was involved in eIF2α phosphorylation in response to HAstV infection. Studies have indicated that immature HAstV capsid protein is processed by caspases, and caspase cleavage is integral to particle release. Inhibition of CHOP upregulation reduced caspase activation and the release of HAstV RNA from cells during HAstV infection. Our results suggest that the eIF2α-ATF4-CHOP pathway participates in HAstV propagation.
Asunto(s)
Infecciones por Astroviridae/genética , Infecciones por Astroviridae/virología , Caspasas/genética , Mamastrovirus/patogenicidad , Factor de Transcripción CHOP/genética , Regulación hacia Arriba/genética , Liberación del Virus/genética , Factor de Transcripción Activador 4/genética , Células CACO-2 , Línea Celular Tumoral , Estrés del Retículo Endoplásmico/genética , Factor 2 Eucariótico de Iniciación/genética , Humanos , Fosforilación/genética , Transducción de Señal/genética , Respuesta de Proteína Desplegada/genéticaRESUMEN
The influenza A virus (IAV) M2 protein is a multifunctional protein with critical roles in virion entry, assembly, and budding. M2 is targeted to the apical plasma membrane of polarized epithelial cells, and the interaction of the viral proteins M2, M1, HA, and NA near glycolipid rafts in the apical plasma membrane is hypothesized to coordinate the assembly of infectious virus particles. To determine the role of M2 protein apical targeting in IAV replication, a panel of M2 proteins with basolateral plasma membrane (M2-Baso) or endoplasmic reticulum (M2-ER) targeting sequences was generated. MDCK II cells stably expressing M2-Baso, but not M2-ER, complemented the replication of M2-stop viruses. However, in primary human nasal epithelial cell (hNEC) cultures, viruses encoding M2-Baso and M2-ER replicated to negligible titers compared to those of wild-type virus. M2-Baso replication was negatively correlated with cell polarization. These results demonstrate that M2 apical targeting is essential for IAV replication: targeting M2 to the ER results in a strong, cell type-independent inhibition of virus replication, and targeting M2 to the basolateral membrane has greater effects in hNECs than in MDCK cells.IMPORTANCE Influenza A virus assembly and particle release occur at the apical membrane of polarized epithelial cells. The integral membrane proteins encoded by the virus, HA, NA, and M2, are all targeted to the apical membrane and believed to recruit the other structural proteins to sites of virus assembly. By targeting M2 to the basolateral or endoplasmic reticulum membranes, influenza A virus replication was significantly reduced. Basolateral targeting of M2 reduced the infectious virus titers with minimal effects on virus particle release, while targeting to the endoplasmic reticulum resulted in reduced infectious and total virus particle release. Therefore, altering the expression and the intracellular targeting of M2 has major effects on virus replication.
Asunto(s)
Subtipo H1N1 del Virus de la Influenza A/crecimiento & desarrollo , Subtipo H3N2 del Virus de la Influenza A/crecimiento & desarrollo , Proteínas de la Matriz Viral/genética , Proteínas de la Matriz Viral/metabolismo , Ensamble de Virus/genética , Liberación del Virus/genética , Animales , Línea Celular , Membrana Celular/virología , Perros , Retículo Endoplásmico/virología , Células HEK293 , Humanos , Subtipo H1N1 del Virus de la Influenza A/genética , Subtipo H3N2 del Virus de la Influenza A/genética , Células de Riñón Canino Madin Darby , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismoRESUMEN
The Ebola virus (EBOV) matrix protein VP40 drives budding of virions and encodes 2 overlapping late domain motifs at amino acid positions 7-13 (PTAPPEY). However, these motifs are not absolutely essential for replication in cell culture, and recently a potential third late domain motif (YPx(6)I) was proposed at amino acid positions 18-26 of VP40. To analyze the importance of this motif in viral budding, we used a transcription and replication-competent virus-like particle system. Using this system, we show that this motif does not contribute to EBOV budding or particle propagation.
Asunto(s)
Ebolavirus/genética , Fiebre Hemorrágica Ebola/virología , Transcripción Genética/genética , Replicación Viral/genética , Secuencias de Aminoácidos/genética , Secuencia de Aminoácidos , Línea Celular , Células HEK293 , Interacciones Huésped-Patógeno/genética , Humanos , Nucleoproteínas , Proteínas del Núcleo Viral , Virión/genética , Liberación del Virus/genéticaRESUMEN
gp41, one of the baculovirus core genes, encodes the only recognized tegument (O-glycosylated) protein of the occlusion-derived virion (ODV) phenotype so far. A previous study using a temperature-sensitive Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) mutant showed that GP41 plays a crucial role in budded virion (BV) formation. However, the precise function of GP41 in the baculovirus replication cycle remains unclear. In this study, AcMNPV GP41 was found to accumulate around the ring zone (RZ) region within the infected nucleus and finally assembled into both BVs and ODVs. Deletion of gp41 from the AcMNPV genome showed that BVs were no longer formed and ODVs were no longer assembled, suggesting the essential role of this gene in baculovirus virion morphogenesis. In infected cells, besides the 42-kDa monomers, dimers and trimers were detected under nonreducing conditions, whereas only trimeric GP41 forms were selectively incorporated into BVs or ODVs. Mutations of all five cysteines in GP41 individually had minor effects on GP41 oligomer formation, albeit certain mutations impaired infectious BV production, suggesting flexibility in the intermolecular disulfide bonding. Single mutations of key leucines within two predicted leucine zipper-like motifs did not interfere with GP41 oligomerization or BV and ODV formation, but double leucine mutations completely blocked oligomerization of GP41 and progeny BV production. In the latter case, the usual subcellular localization, especially RZ accumulation, of GP41 was abolished. The above findings clearly point out a close correlation between GP41 oligomerization and function and therefore highlight the oligomeric state as the functional form of GP41 in the baculovirus replication cycle.IMPORTANCE The tegument, which is sandwiched between the nucleocapsid and the virion envelope, is an important substructure of many enveloped viruses. It is composed of one or more proteins that have important functions during virus entry, replication, assembly, and egress. Unlike another large DNA virus (herpesvirus) that encodes an extensive set of tegument components, baculoviruses very likely exploit the major tegument protein, GP41, to execute functions in baculovirus virion morphogenesis and assembly. However, the function of this O-glycosylated baculovirus tegument protein remains largely unknown. In this study, we identified trimers as the functional structure of GP41 in baculovirus virion morphogenesis and showed that both disulfide bridging and protein-protein interactions via the two leucine zipper-like domains are involved in the formation of different oligomeric states. This study advances our understanding of the unique viral tegument protein GP41 participating in the life cycle of baculoviruses.