RESUMEN
Human cytomegalovirus (HCMV) forms two gH/gL glycoprotein complexes, gH/gL/gO and gH/gL/pUL(128,130,131A), which determine the tropism, the entry pathways and the mode of spread of the virus. For murine cytomegalovirus (MCMV), which serves as a model for HCMV, a gH/gL/gO complex functionally homologous to the HCMV gH/gL/gO complex has been described. Knock-out of MCMV gO does impair, but not abolish, virus spread indicating that also MCMV might form an alternative gH/gL complex. Here, we show that the MCMV CC chemokine MCK-2 forms a complex with the glycoprotein gH, a complex which is incorporated into the virion. We could additionally show that mutants lacking both, gO and MCK-2 are not able to produce infectious virus. Trans-complementation of these double mutants with either gO or MCK-2 showed that both proteins can promote infection of host cells, although through different entry pathways. MCK-2 has been extensively studied in vivo by others. It has been shown to be involved in attracting cells for virus dissemination and in regulating antiviral host responses. We now show that MCK-2, by forming a complex with gH, strongly promotes infection of macrophages in vitro and in vivo. Thus, MCK-2 may play a dual role in MCMV infection, as a chemokine regulating the host response and attracting specific target cells and as part of a glycoprotein complex promoting entry into cells crucial for virus dissemination.
Asunto(s)
Quimiocinas CC/metabolismo , Infecciones por Herpesviridae/inmunología , Inmunidad Innata , Macrófagos/inmunología , Muromegalovirus/fisiología , Proteínas del Envoltorio Viral/metabolismo , Proteínas Virales/metabolismo , Internalización del Virus , Animales , Línea Celular , Células Cultivadas , Quimiocinas CC/química , Quimiocinas CC/genética , Femenino , Infecciones por Herpesviridae/metabolismo , Infecciones por Herpesviridae/patología , Infecciones por Herpesviridae/virología , Hígado/inmunología , Hígado/patología , Hígado/virología , Macrófagos/patología , Macrófagos/virología , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/patología , Macrófagos Peritoneales/virología , Ratones , Ratones Endogámicos BALB C , Muromegalovirus/inmunología , Mutación , Multimerización de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Organismos Libres de Patógenos Específicos , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/genética , Proteínas Virales/química , Proteínas Virales/genética , Virión/inmunología , Virión/fisiologíaRESUMEN
The herpesvirus replication cycle comprises maturation processes in the nucleus and cytoplasm of the infected cells. After their nuclear assembly viral capsids translocate via primary envelopment towards the cytoplasm. This event is mediated by the nuclear envelopment complex, which is composed by two conserved viral proteins belonging to the UL34 and UL31 protein families. Here, we generated recombinant viruses, which express affinity-tagged pM50 and/or pM53, the pUL34 and pUL31 homologues of the murine cytomegalovirus. We extracted pM50- and pM53-associated protein complexes from infected cells and analysed their composition after affinity purification by mass spectrometry. We observed reported interaction partners and identified new putative protein-protein interactions for both proteins. Endophilin-A2 was observed as the most prominent cellular partner of pM50. We found that endophilin-A2 binds to pM50 directly, and this interaction seems to be conserved in the pUL34 family.
Asunto(s)
Aciltransferasas/metabolismo , Muromegalovirus/fisiología , Proteínas Mutantes Quiméricas/metabolismo , Proteínas Virales/metabolismo , Aciltransferasas/antagonistas & inhibidores , Aciltransferasas/genética , Animales , Citosol/metabolismo , Citosol/virología , Expresión Génica , Interacciones Huésped-Patógeno , Espectrometría de Masas , Ratones , Proteínas Mutantes Quiméricas/genética , Membrana Nuclear/metabolismo , Membrana Nuclear/virología , Unión Proteica , Mapeo de Interacción de Proteínas , ARN Interferente Pequeño/genética , Técnicas del Sistema de Dos Híbridos , Proteínas Virales/genética , Liberación del VirusRESUMEN
The gene M94 of murine cytomegalovirus (MCMV) as well as its homologues UL16 in alphaherpesviruses is involved in viral morphogenesis. For a better understanding of its role in the viral life cycle, a library of random M94 mutants was generated by modified transposon-based linker scanning mutagenesis. A comprehensive set of M94 mutants was reinserted into the MCMV genome and tested for their capacity to complement the M94 null mutant. Thereby, 34 loss-of-function mutants of M94 were identified, which were tested in a second screen for their capacity to inhibit virus replication. This analysis identified two N-terminal insertion mutants of M94 with a dominant negative effect. We compared phenotypes induced by the conditional expression of these dominant negative M94 alleles with the null phenotype of the M94 deletion. The viral gene expression cascade and the nuclear morphogenesis steps were not affected in either setting. In both cases, however, secondary envelopment did not proceed in the absence of functional M94, and capsids subsequently accumulated in the center of the cytoplasmic assembly complex. In addition, deletion of M94 resulted in a block of cell-to-cell spread. Moreover, the dominant negative mutant of M94 demonstrated a defect in interacting with M99, the UL11 homologue of MCMV.
Asunto(s)
Muromegalovirus/fisiología , Proteínas Virales/metabolismo , Ensamble de Virus , ADN Viral/química , ADN Viral/genética , Eliminación de Gen , Prueba de Complementación Genética , Datos de Secuencia Molecular , Muromegalovirus/genética , Mutagénesis , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Análisis de Secuencia de ADN , Proteínas Virales/genéticaRESUMEN
Bacterial artificial chromosomes (BACs) are recombinant DNA molecules designed for propagation of large and instable foreign DNA fragment in Escherichia coli. BACs are used in genetics of large DNA viruses such as herpes and baculoviruses for propagation and manipulation of complex genomic regions or even entire viral genomes in one piece. Viral genomes in BACs are ready for the advanced tools of E. coli genetics. These techniques based on homologous or site-specific recombination allow engineering of virtually any kind of genetic changes. In the recent years, BAC technology was also adapted to manipulation of adenovirus genomes and became an effective alternative to traditional genetic engineering of recombinant adenoviruses.