RESUMEN
We investigated the localization of host mRNA during Rift Valley fever virus (RVFV) infection. Fluorescence in situ hybridization revealed that infection with RVFV altered the localization of host mRNA. mRNA accumulated in the nuclei of RVFV-infected but not mock-infected cells. Further, overexpression of the NSS gene, but not the N, GN or NSM genes correlated with mRNA nuclear accumulation. Nuclear accumulation of host mRNA was not observed in cells infected with a strain of RVFV lacking the gene encoding NSS, confirming that expression of NSS is likely responsible for this phenomenon.
Asunto(s)
Núcleo Celular/metabolismo , ARN Mensajero/metabolismo , Fiebre del Valle del Rift/metabolismo , Virus de la Fiebre del Valle del Rift/metabolismo , Proteínas no Estructurales Virales/metabolismo , Transporte Activo de Núcleo Celular , Núcleo Celular/genética , Núcleo Celular/virología , Células HeLa , Interacciones Huésped-Patógeno , Humanos , ARN Mensajero/genética , Fiebre del Valle del Rift/genética , Fiebre del Valle del Rift/virología , Virus de la Fiebre del Valle del Rift/genética , Proteínas no Estructurales Virales/genéticaRESUMEN
Cynomolgus macaques were vaccinated by intramuscular electroporation with DNA plasmids expressing codon-optimized glycoprotein (GP) genes of Ebola virus (EBOV) or Marburg virus (MARV) or a combination of codon-optimized GP DNA vaccines for EBOV, MARV, Sudan virus and Ravn virus. When measured by ELISA, the individual vaccines elicited slightly higher IgG responses to EBOV or MARV than did the combination vaccines. No significant differences in immune responses of macaques given the individual or combination vaccines were measured by pseudovirion neutralization or IFN-γ ELISpot assays. Both the MARV and mixed vaccines were able to protect macaques from lethal MARV challenge (5/6 vs. 6/6). In contrast, a greater proportion of macaques vaccinated with the EBOV vaccine survived lethal EBOV challenge in comparison to those that received the mixed vaccine (5/6 vs. 1/6). EBOV challenge survivors had significantly higher pre-challenge neutralizing antibody titers than those that succumbed.
Asunto(s)
Electroporación , Filoviridae/inmunología , Fiebre Hemorrágica Ebola/prevención & control , Enfermedad del Virus de Marburg/prevención & control , Vacunas de ADN/inmunología , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Codón , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Ensayo de Inmunoadsorción Enzimática , Ensayo de Immunospot Ligado a Enzimas , Femenino , Filoviridae/genética , Glicoproteínas/genética , Glicoproteínas/inmunología , Inmunoglobulina G/sangre , Inyecciones Intramusculares , Interferón gamma/metabolismo , Leucocitos Mononucleares/inmunología , Macaca fascicularis , Masculino , Pruebas de Neutralización , Plásmidos , Análisis de Supervivencia , Resultado del Tratamiento , Vacunas de ADN/administración & dosificación , Vacunas de ADN/genéticaRESUMEN
Rift Valley fever virus (RVFV), an ambisense member of the family Bunyaviridae, genus Phlebovirus, is the causative agent of Rift Valley fever, an important zoonotic infection in Africa and the Middle East. Phlebovirus proteins are translated from virally transcribed mRNAs that, like host mRNA, are capped but, unlike host mRNAs, are not polyadenylated. Here, we investigated the role of PABP1 during RVFV infection of HeLa cells. Immunofluorescence studies of infected cells demonstrated a gross relocalization of PABP1 to the nucleus late in infection. Immunofluorescence microscopy studies of nuclear proteins revealed costaining between PABP1 and markers of nuclear speckles. PABP1 relocalization was sharply decreased in cells infected with a strain of RVFV lacking the gene encoding the RVFV nonstructural protein S (NSs). To determine whether PABP1 was required for RVFV infection, we measured the production of nucleocapsid protein (N) in cells transfected with small interfering RNAs (siRNAs) targeting PABP1. We found that the overall percentage of RVFV N-positive cells was not changed by siRNA treatment, indicating that PABP1 was not required for RVFV infection. However, when we analyzed populations of cells producing high versus low levels of PABP1, we found that the percentage of RVFV N-positive cells was decreased in cell populations producing physiologic levels of PABP1 and increased in cells with reduced levels of PABP1. Together, these results suggest that production of the NSs protein during RVFV infection leads to sequestration of PABP1 in the nuclear speckles, creating a state within the cell that favors viral protein production.
Asunto(s)
Núcleo Celular/metabolismo , Interacciones Huésped-Patógeno , Proteína I de Unión a Poli(A)/metabolismo , Virus de la Fiebre del Valle del Rift/fisiología , Proteínas no Estructurales Virales/metabolismo , Replicación Viral , Eliminación de Gen , Células HeLa , Humanos , Microscopía Confocal , Microscopía Fluorescente , Virus de la Fiebre del Valle del Rift/genética , Proteínas no Estructurales Virales/genéticaRESUMEN
We evaluated the immunogenicity and protective efficacy of DNA vaccines expressing the codon-optimized envelope glycoprotein genes of Zaire ebolavirus, Sudan ebolavirus, and Marburg marburgvirus (Musoke and Ravn). Intramuscular or intradermal delivery of the vaccines in BALB/c mice was performed using the TriGrid™ electroporation device. Mice that received DNA vaccines against the individual viruses developed robust glycoprotein-specific antibody titers as determined by ELISA and survived lethal viral challenge with no display of clinical signs of infection. Survival curve analysis revealed there was a statistically significant increase in survival compared to the control groups for both the Ebola and Ravn virus challenges. These data suggest that further analysis of the immune responses generated in the mice and additional protection studies in nonhuman primates are warranted.
