RESUMEN
Rift Valley fever virus (RVFV) is a zoonotic pathogen with pandemic potential. RVFV entry is mediated by the viral glycoprotein (Gn), but host entry factors remain poorly defined. Our genome-wide CRISPR screen identified low-density lipoprotein receptor-related protein 1 (mouse Lrp1/human LRP1), heat shock protein (Grp94), and receptor-associated protein (RAP) as critical host factors for RVFV infection. RVFV Gn directly binds to specific Lrp1 clusters and is glycosylation independent. Exogenous addition of murine RAP domain 3 (mRAPD3) and anti-Lrp1 antibodies neutralizes RVFV infection in taxonomically diverse cell lines. Mice treated with mRAPD3 and infected with pathogenic RVFV are protected from disease and death. A mutant mRAPD3 that binds Lrp1 weakly failed to protect from RVFV infection. Together, these data support Lrp1 as a host entry factor for RVFV infection and define a new target to limit RVFV infections.
Asunto(s)
Interacciones Huésped-Patógeno , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Virus de la Fiebre del Valle del Rift/fisiología , Internalización del Virus , Animales , Especificidad de Anticuerpos/inmunología , Secuencia de Bases , Encéfalo/patología , Encéfalo/virología , Sistemas CRISPR-Cas/genética , Membrana Celular/metabolismo , Células Cultivadas , Glicoproteínas/metabolismo , Glicosaminoglicanos/metabolismo , Glicosilación , Humanos , Proteína Asociada a Proteínas Relacionadas con Receptor de LDL/metabolismo , Ligandos , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/deficiencia , Glicoproteínas de Membrana/metabolismo , Ratones , Unión Proteica , Desnaturalización Proteica , Fiebre del Valle del Rift/patología , Fiebre del Valle del Rift/prevención & control , Fiebre del Valle del Rift/virología , Virus de la Fiebre del Valle del Rift/inmunologíaRESUMEN
Oropouche orthobunyavirus (OROV; Peribunyaviridae) is a mosquito-transmitted virus that causes widespread human febrile illness in South America, with occasional progression to neurologic effects. Host factors mediating the cellular entry of OROV are undefined. Here, we show that OROV uses the host protein low-density lipoprotein-related protein 1 (Lrp1) for efficient cellular infection. Cells from evolutionarily distinct species lacking Lrp1 were less permissive to OROV infection than cells with Lrp1. Treatment of cells with either the high-affinity Lrp1 ligand receptor-associated protein (RAP) or recombinant ectodomain truncations of Lrp1 significantly reduced OROV infection. In addition, chimeric vesicular stomatitis virus (VSV) expressing OROV glycoproteins (VSV-OROV) bound to the Lrp1 ectodomain in vitro. Furthermore, we demonstrate the biological relevance of the OROV-Lrp1 interaction in a proof-of-concept mouse study in which treatment of mice with RAP at the time of infection reduced tissue viral load and promoted survival from an otherwise lethal infection. These results with OROV, along with the recent finding of Lrp1 as an entry factor for Rift Valley fever virus, highlight the broader significance of Lrp1 in cellular infection by diverse bunyaviruses. Shared strategies for entry, such as the critical function of Lrp1 defined here, provide a foundation for the development of pan-bunyaviral therapeutics.
Asunto(s)
Infecciones por Bunyaviridae , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad , Orthobunyavirus , Internalización del Virus , Animales , Infecciones por Bunyaviridae/metabolismo , Infecciones por Bunyaviridae/virología , Técnicas de Inactivación de Genes , Humanos , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/genética , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Ratones , Orthobunyavirus/fisiología , América del SurRESUMEN
Endogenous retroviruses (ERVs), remnants of ancient germline infections, comprise 8% of the human genome. The most recently integrated includes human ERV-K (HERV-K) where several envelope (env) sequences remain intact. Viral pseudotypes decorated with one of those Envs are infectious. Using a recombinant vesicular stomatitis virus encoding HERV-K Env as its sole attachment and fusion protein (VSV-HERVK) we conducted a genome-wide haploid genetic screen to interrogate the host requirements for infection. This screen identified 11 genes involved in heparan sulfate biosynthesis. Genetic inhibition or chemical removal of heparan sulfate and addition of excess soluble heparan sulfate inhibit infection. Direct binding of heparin to soluble HERV-K Env and purified VSV-HERVK defines it as critical for viral attachment. Cell surface bound VSV-HERVK particles are triggered to infect on exposure to acidic pH, whereas acid pH pretreatment of virions blocks infection. Testing of additional endogenous HERV-K env sequences reveals they bind heparin and mediate acid pH triggered fusion. This work reconstructs and defines key steps in the infectious entry pathway of an extinct virus.
Asunto(s)
Retrovirus Endógenos/fisiología , Heparitina Sulfato/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Tropismo Viral/fisiología , Internalización del Virus , HumanosRESUMEN
UNLABELLED: Virus entry into cells is a multistep process that often requires the subversion of subcellular machineries. A more complete understanding of these steps is necessary to develop new antiviral strategies. While studying the potential role of the actin network and one of its master regulators, the small GTPase Cdc42, during Junin virus (JUNV) entry, we serendipitously uncovered the small molecule ZCL278, reported to inhibit Cdc42 function as an entry inhibitor for JUNV and for vesicular stomatitis virus, lymphocytic choriomeningitis virus, and dengue virus but not for the nonenveloped poliovirus. Although ZCL278 did not interfere with JUNV attachment to the cell surface or virus particle internalization into host cells, it prevented the release of JUNV ribonucleoprotein cores into the cytosol and decreased pH-mediated viral fusion with host membranes. We also identified SVG-A astroglial cell-derived cells to be highly permissive for JUNV infection and generated new cell lines expressing fluorescently tagged Rab5c or Rab7a or lacking Cdc42 using clustered regularly interspaced short palindromic repeat (CRISPR)-caspase 9 (Cas9) gene-editing strategies. Aided by these tools, we uncovered that perturbations in the actin cytoskeleton or Cdc42 activity minimally affect JUNV entry, suggesting that the inhibitory effect of ZCL278 is not mediated by ZCL278 interfering with the activity of Cdc42. Instead, ZCL278 appears to redistribute viral particles from endosomal to lysosomal compartments. ZCL278 also inhibited JUNV replication in a mouse model, and no toxicity was detected. Together, our data suggest the unexpected antiviral activity of ZCL278 and highlight its potential for use in the development of valuable new tools to study the intracellular trafficking of pathogens. IMPORTANCE: The Junin virus is responsible for outbreaks of Argentine hemorrhagic fever in South America, where 5 million people are at risk. Limited options are currently available to treat infections by Junin virus or other viruses of the Arenaviridae, making the identification of additional tools, including small-molecule inhibitors, of great importance. How Junin virus enters cells is not yet fully understood. Here we describe new cell culture models in which the cells are susceptible to Junin virus infection and to which we applied CRISPR-Cas9 genome engineering strategies to help characterize early steps during virus entry. We also uncovered ZCL278 to be a new antiviral small molecule that potently inhibits the cellular entry of the Junin virus and other enveloped viruses. Moreover, we show that ZCL278 also functions in vivo, thereby preventing Junin virus replication in a mouse model, opening the possibility for the discovery of ZCL278 derivatives of therapeutic potential.
Asunto(s)
Antivirales/farmacología , Benzamidas/farmacología , Descubrimiento de Drogas , Tiourea/análogos & derivados , Internalización del Virus/efectos de los fármacos , Actinas/metabolismo , Animales , Línea Celular , Células Cultivadas , Clatrina/metabolismo , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Endocitosis/efectos de los fármacos , Endosomas/efectos de los fármacos , Endosomas/virología , Técnicas de Inactivación de Genes , Fiebre Hemorrágica Americana/genética , Fiebre Hemorrágica Americana/metabolismo , Fiebre Hemorrágica Americana/virología , Humanos , Virus Junin/efectos de los fármacos , Virus Junin/fisiología , Ratones , Unión Proteica , Transporte de Proteínas , Proteolisis , Ribonucleoproteínas/metabolismo , Tiourea/farmacología , Carga Viral , Proteínas Virales/metabolismo , Acoplamiento Viral/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Proteína de Unión al GTP cdc42/genética , Proteína de Unión al GTP cdc42/metabolismoRESUMEN
Pre-mRNA processing is mechanistically linked to transcription with RNA pol II serving as a platform to recruit RNA processing factors to nascent transcripts. The TREX complex member, REF/Aly, has been suggested to play roles in transcription and nuclear RNA stability in addition to its more broadly characterized role in mRNA export. We employed RNA-seq to identify a subset of transcripts with decreased expression in both nuclear and cytoplasmic fractions upon REF/Aly knockdown, which implies that REF/Aly affects their expression upstream of its role in mRNA export. Transcription inhibition experiments and metabolic labeling assays argue that REF/Aly does not affect stability of selected candidate transcripts. Instead, ChIP assays and nuclear run-on analysis reveal that REF/Aly depletion diminishes the transcription of these candidate genes. Furthermore, we determined that REF/Aly binds directly to candidate transcripts, supporting a direct effect of REF/Aly on candidate gene transcription. Taken together, our data suggest that the importance of REF/Aly is not limited to RNA export, but that REF/Aly is also critical for gene expression at the level of transcription. Our data are consistent with the model that REF/Aly is involved in linking splicing with transcription efficiency.
Asunto(s)
Proteínas Nucleares/metabolismo , ARN Polimerasa II/metabolismo , Proteínas de Unión al ARN/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética , Animales , Línea Celular , Regulación hacia Abajo , Técnicas de Silenciamiento del Gen , Humanos , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/genética , Estabilidad del ARN , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/antagonistas & inhibidores , Proteínas de Unión al ARN/genética , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/genéticaRESUMEN
Viral inclusion bodies (IBs) are potential sites of viral replication and assembly. How viral IBs form remains poorly defined. Here we describe a combined biophysical and cellular approach to identify the components necessary for IB formation during Ebola virus (EBOV) infection. We find that the eNP0VP35 complex containing Ebola nucleoprotein (eNP) and viral protein 35 (eVP35), the functional equivalents of nucleoprotein (N) and phosphoprotein (P) in non-segmented negative strand viruses (NNSVs), phase separates to form inclusion bodies. Phase separation of eNP0VP35 is reversible and modulated by ionic strength. The multivalency of eVP35, and not eNP, is also critical for phase separation. Furthermore, overexpression of an eVP35 peptide disrupts eNP0VP35 complex formation, leading to reduced frequency of IB formation and limited viral infection. Together, our results show that upon EBOV infection, the eNP0VP35 complex forms the minimum unit to drive IB formation and viral replication.
Asunto(s)
Ebolavirus , Fiebre Hemorrágica Ebola , Cuerpos de Inclusión , Nucleoproteínas , Replicación Viral , Humanos , Ebolavirus/metabolismo , Ebolavirus/fisiología , Fiebre Hemorrágica Ebola/virología , Cuerpos de Inclusión/virología , Nucleoproteínas/metabolismo , Proteínas Reguladoras y Accesorias Virales/metabolismoRESUMEN
Pharmacologically active compounds with known biological targets were evaluated for inhibition of SARS-CoV-2 infection in cell and tissue models to help identify potent classes of active small molecules and to better understand host-virus interactions. We evaluated 6,710 clinical and preclinical compounds targeting 2,183 host proteins by immunocytofluorescence-based screening to identify SARS-CoV-2 infection inhibitors. Computationally integrating relationships between small molecule structure, dose-response antiviral activity, host target, and cell interactome produced cellular networks important for infection. This analysis revealed 389 small molecules with micromolar to low nanomolar activities, representing >12 scaffold classes and 813 host targets. Representatives were evaluated for mechanism of action in stable and primary human cell models with SARS-CoV-2 variants and MERS-CoV. One promising candidate, obatoclax, significantly reduced SARS-CoV-2 viral lung load in mice. Ultimately, this work establishes a rigorous approach for future pharmacological and computational identification of host factor dependencies and treatments for viral diseases.
RESUMEN
Arenaviruses cause fatal hemorrhagic disease in humans. Old World arenavirus glycoproteins (GPs) mainly engage α-dystroglycan as a cell-surface receptor, while New World arenaviruses hijack transferrin receptor. However, the Lujo virus (LUJV) GP does not cluster with New or Old World arenaviruses. Using a recombinant vesicular stomatitis virus containing LUJV GP as its sole attachment and fusion protein (VSV-LUJV), we demonstrate that infection is independent of known arenavirus receptor genes. A genome-wide haploid genetic screen identified the transmembrane protein neuropilin 2 (NRP2) and tetraspanin CD63 as factors for LUJV GP-mediated infection. LUJV GP binds the N-terminal domain of NRP2, while CD63 stimulates pH-activated LUJV GP-mediated membrane fusion. Overexpression of NRP2 or its N-terminal domain enhances VSV-LUJV infection, and cells lacking NRP2 are deficient in wild-type LUJV infection. These findings uncover this distinct set of host cell entry factors in LUJV infection and are attractive focus points for therapeutic intervention.
Asunto(s)
Lujo virus/fisiología , Neuropilina-2/metabolismo , Tetraspanina 30/metabolismo , Proteínas Virales de Fusión/metabolismo , Proteínas Virales/metabolismo , Internalización del Virus , Proteínas Portadoras , Línea Celular , Interacciones Huésped-Patógeno/fisiología , Células Endoteliales de la Vena Umbilical Humana , Humanos , Lujo virus/genética , Lujo virus/patogenicidad , Dominios y Motivos de Interacción de Proteínas , Receptores de Superficie Celular/metabolismo , Receptores de Transferrina , Proteínas Virales de Fusión/genética , Proteínas Virales/genéticaRESUMEN
RNA-protein complexes are critical for almost all aspects of gene expression. Analysis of RNA-protein interactions can be complicated by the disruption of native complexes and the formation of new, reassorted complexes upon cell lysis. Before concluding that a specific RNA and protein interact in vivo, cell-mixing experiments can be performed to ensure that observed RNA-protein complexes are not formed after lysis of cells.
Asunto(s)
ARN/metabolismo , Ribonucleoproteínas Nucleares Pequeñas/metabolismo , Animales , Northern Blotting , Western Blotting , Extractos Celulares/química , Fraccionamiento Celular , Sistema Libre de Células , Células Cultivadas , Humanos , Inmunoprecipitación , ARN/química , Ribonucleoproteínas Nucleares Pequeñas/químicaRESUMEN
Lassa virus spreads from a rodent to humans and can lead to lethal hemorrhagic fever. Despite its broad tropism, chicken cells were reported 30 years ago to resist infection. We found that Lassa virus readily engaged its cell-surface receptor α-dystroglycan in avian cells, but virus entry in susceptible species involved a pH-dependent switch to an intracellular receptor, the lysosome-resident protein LAMP1. Iterative haploid screens revealed that the sialyltransferase ST3GAL4 was required for the interaction of the virus glycoprotein with LAMP1. A single glycosylated residue in LAMP1, present in susceptible species but absent in birds, was essential for interaction with the Lassa virus envelope protein and subsequent infection. The resistance of Lamp1-deficient mice to Lassa virus highlights the relevance of this receptor switch in vivo.
Asunto(s)
Virus Lassa/fisiología , Proteína 1 de la Membrana Asociada a los Lisosomas/metabolismo , Receptores Virales/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Internalización del Virus , Secuencia de Aminoácidos , Animales , Línea Celular , Membrana Celular/metabolismo , Membrana Celular/virología , Células Cultivadas , Pollos , Distroglicanos/genética , Distroglicanos/metabolismo , Glicosilación , Humanos , Concentración de Iones de Hidrógeno , Fiebre de Lassa/virología , Proteína 1 de la Membrana Asociada a los Lisosomas/química , Lisosomas/metabolismo , Lisosomas/virología , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Unión Proteica , Sialiltransferasas/metabolismo , beta-Galactosida alfa-2,3-SialiltransferasaRESUMEN
TREX is a conserved multiprotein complex that is necessary for efficient mRNA export to the cytoplasm. In Saccharomyces cerevisiae, the TREX complex is additionally implicated in RNA quality control pathways, but it is unclear whether this function is conserved in mammalian cells. The Kaposi's sarcoma-associated herpesvirus (KSHV) ORF57 protein binds and recruits the TREX component REF/Aly to viral mRNAs. Here, we demonstrate that REF/Aly is recruited to the KSHV noncoding polyadenylated nuclear (PAN) RNA by ORF57. This recruitment correlates with ORF57-mediated stabilization of PAN RNA, suggesting that REF/Aly promotes nuclear RNA stability. Further supporting this idea, tethering REF/Aly to PAN RNA is sufficient to increase the nuclear abundance and half-life of PAN RNA but is not sufficient to promote its export. Interestingly, REF/Aly appears to protect the poly(A) tail from deadenylation, and REF/Aly-stabilized transcripts are further adenylated over time, consistent with previous reports linking poly(A) tail length with nuclear RNA surveillance. These studies show that REF/Aly can stabilize nuclear RNAs independently of their export and support a broader conservation of RNA quality control mechanisms from yeast to humans.