RESUMEN
Little is known about the repertoire of cellular factors involved in the replication of pathogenic alphaviruses. To uncover molecular regulators of alphavirus infection, and to identify candidate drug targets, we performed a high-content imaging-based siRNA screen. We revealed an actin-remodeling pathway involving Rac1, PIP5K1- α, and Arp3, as essential for infection by pathogenic alphaviruses. Infection causes cellular actin rearrangements into large bundles of actin filaments termed actin foci. Actin foci are generated late in infection concomitantly with alphavirus envelope (E2) expression and are dependent on the activities of Rac1 and Arp3. E2 associates with actin in alphavirus-infected cells and co-localizes with Rac1-PIP5K1-α along actin filaments in the context of actin foci. Finally, Rac1, Arp3, and actin polymerization inhibitors interfere with E2 trafficking from the trans-Golgi network to the cell surface, suggesting a plausible model in which transport of E2 to the cell surface is mediated via Rac1- and Arp3-dependent actin remodeling.
Asunto(s)
Infecciones por Alphavirus/genética , Alphavirus/genética , Movimiento Celular/genética , ARN Interferente Pequeño/genética , Actinas/metabolismo , Alphavirus/metabolismo , Infecciones por Alphavirus/metabolismo , Movimiento Celular/fisiología , Replicación del ADN/genética , Humanos , Transporte de Proteínas/genética , Red trans-Golgi/genética , Red trans-Golgi/metabolismoRESUMEN
Bone marrow stromal antigen 2 (BST-2/tetherin) is a cellular membrane protein that inhibits the release of HIV-1. We show for the first time, using infectious viruses, that BST-2 also inhibits egress of arenaviruses but has no effect on filovirus replication and spread. Specifically, infectious Lassa virus (LASV) release significantly decreased or increased in human cells in which BST-2 was either stably expressed or knocked down, respectively. In contrast, replication and spread of infectious Zaire ebolavirus (ZEBOV) and Lake Victoria marburgvirus (MARV) were not affected by these conditions. Replication of infectious Rift Valley fever virus (RVFV) and cowpox virus (CPXV) was also not affected by BST-2 expression. Elevated cellular levels of human or murine BST-2 inhibited the release of virus-like particles (VLPs) consisting of the matrix proteins of multiple highly virulent NIAID Priority Pathogens, including arenaviruses (LASV and Machupo virus [MACV]), filoviruses (ZEBOV and MARV), and paramyxoviruses (Nipah virus). Although the glycoproteins of filoviruses counteracted the antiviral activity of BST-2 in the context of VLPs, they could not rescue arenaviral (LASV and MACV) VLP release upon BST-2 overexpression. Furthermore, we did not observe colocalization of filoviral glycoproteins with BST-2 during infection with authentic viruses. None of the arenavirus-encoded proteins rescued budding of VLPs in the presence of BST-2. Our results demonstrate that BST-2 might be a broad antiviral factor with the ability to restrict release of a wide variety of human pathogens. However, at least filoviruses, RVFV, and CPXV are immune to its inhibitory effect.
Asunto(s)
Antígenos CD/fisiología , Filoviridae/patogenicidad , Virus Lassa/patogenicidad , Glicoproteínas de Membrana/fisiología , Animales , Antígenos CD/genética , Arenavirus del Nuevo Mundo/genética , Arenavirus del Nuevo Mundo/patogenicidad , Arenavirus del Nuevo Mundo/fisiología , Secuencia de Bases , Línea Celular , ADN Viral/genética , Ebolavirus/genética , Ebolavirus/patogenicidad , Ebolavirus/fisiología , Filoviridae/genética , Filoviridae/fisiología , Proteínas Ligadas a GPI/antagonistas & inhibidores , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/fisiología , Técnicas de Silenciamiento del Gen , Células HeLa , Interacciones Huésped-Patógeno/fisiología , Humanos , Virus Lassa/genética , Virus Lassa/fisiología , Marburgvirus/genética , Marburgvirus/patogenicidad , Marburgvirus/fisiología , Glicoproteínas de Membrana/genética , Ratones , Virus Nipah/genética , Virus Nipah/patogenicidad , Virus Nipah/fisiología , ARN Interferente Pequeño/genética , Transfección , Liberación del Virus/fisiologíaRESUMEN
Machupo virus (MACV) is a highly pathogenic New World arenavirus that causes hemorrhagic fever in humans. MACV, as well as other pathogenic New World arenaviruses, enter cells after their GP1 attachment glycoprotein binds to their cellular receptor, transferrin receptor 1 (TfR1). TfR1 residues essential for this interaction have been described, and a co-crystal of MACV GP1 bound to TfR1 suggests GP1 residues important for this association. We created MACV GP1 variants and tested their effect on TfR1 binding and virus entry to evaluate the functional significance of some of these and additional residues in human and simian cells. We found residues R111, D123, Y122, and F226 to be essential, D155, and P160 important, and D114, S116, D140, and K169 expendable for the GP1-TfR1 interaction and MACV entry. Several MACV GP1 residues that are critical for the interaction with TfR1 are conserved among other New World arenaviruses, indicating a common basis of receptor interaction. Our findings also open avenues for the rational development of viral entry inhibitors.