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Antagonism of the protein kinase R pathway by the guinea pig cytomegalovirus US22-family gene gp145.

ABSTRACT: Viral double-stranded RNA (dsRNA) activates protein kinase R (PKR), which phosphorylates eIF2? and inhibits translation. In response, viruses have evolved various strategies to evade the antiviral impact of PKR. We investigated whether guinea pig cytomegalovirus (GPCMV), a useful model of congenital CMV infection, encodes a gene that interferes with the PKR pathway. Using a proteomic screen, we identified several GPCMV dsRNA-binding proteins, among which only gp145 rescued replication of a vaccinia virus mutant that lacks E3L. gp145 also reversed the inhibitory effects of PKR on expression of a cotransfected reporter gene. Mapping studies demonstrated that the gp145 dsRNA-binding domain has homology to the PKR antagonists of other CMVs. However, dsRNA-binding by gp145 is not sufficient for it to block PKR. gp145 differs from the PKR antagonists of murine CMV in that it functions alone and from those encoded by human CMV in functioning in cells from both primates and rodents.

SUBMITTER: Bierle CJ

PROVIDER: S-EPMC3444634 | biostudies-literature | 2012 Nov

REPOSITORIES: biostudies-literature

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Antagonism of the protein kinase R pathway by the guinea pig cytomegalovirus US22-family gene gp145.

Bierle Craig J CJ Schleiss Mark R MR Geballe Adam P AP

Virology 20120820 1

Viral double-stranded RNA (dsRNA) activates protein kinase R (PKR), which phosphorylates eIF2α and inhibits translation. In response, viruses have evolved various strategies to evade the antiviral impact of PKR. We investigated whether guinea pig cytomegalovirus (GPCMV), a useful model of congenital CMV infection, encodes a gene that interferes with the PKR pathway. Using a proteomic screen, we identified several GPCMV dsRNA-binding proteins, among which only gp145 rescued replication of a vacci ...[more]

PMID: 22917493

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Project description:The cytomegaloviruses (CMVs) are among the most genetically complex mammalian viruses, with viral genomes that often exceed 230 kbp. Manipulation of cytomegalovirus genomes is largely performed using infectious bacterial artificial chromosomes (BACs), which necessitates the maintenance of the viral genome in Escherichia coli and successful reconstitution of virus from permissive cells after transfection of the BAC. Here we describe an alternative strategy for the mutagenesis of guinea pig cytomegalovirus that utilizes clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated genome editing to introduce targeted mutations to the viral genome. Transient transfection and drug selection were used to restrict lytic replication of guinea pig cytomegalovirus to cells that express Cas9 and virus-specific guide RNA. The result was highly efficient editing of the viral genome that introduced targeted insertion or deletion mutations to nonessential viral genes. Cotransfection of multiple virus-specific guide RNAs or a homology repair template was used for targeted, markerless deletions of viral sequence or to introduce exogenous sequence by homology-driven repair. As CRISPR/Cas9 mutagenesis occurs directly in infected cells, this methodology avoids selective pressures that may occur during propagation of the viral genome in bacteria and may facilitate genetic manipulation of low-passage or clinical CMV isolates.The cytomegalovirus genome is complex, and viral adaptations to cell culture have complicated the study of infection in vivo Recombineering of viral bacterial artificial chromosomes enabled the study of recombinant cytomegaloviruses. Here we report the development of an alternative approach using CRISPR/Cas9-based mutagenesis in guinea pig cytomegalovirus, a small-animal model of congenital cytomegalovirus disease. CRISPR/Cas9 mutagenesis can introduce the same types of mutations to the viral genome as bacterial artificial chromosome recombineering but does so directly in virus-infected cells. CRISPR/Cas9 mutagenesis is not dependent on a bacterial intermediate, and defined viral mutants can be recovered after a limited number of viral genome replications, minimizing the risk of spontaneous mutation.

Project description:BackgroundCytomegalovirus (CMV) is a leading infectious cause of neurologic deficits, both in the settings of congenital and perinatal infection, but few animal models exist to study neurodevelopmental outcomes. This study examined the impact of neonatal guinea pig CMV (GPCMV) infection on spatial learning and memory in a Morris water maze (MWM) model.MethodsNewborn pups were challenged intraperitoneally (i.p.) with a pathogenic red fluorescent protein-tagged GPCMV, or sham inoculated. On days 15-19 post infection (p.i.), pups were tested in the MWM. Viral loads were measured in blood and tissue by quantitative PCR (qPCR), and brain samples collected at necropsy were examined by histology and immunohistochemistry.ResultsViremia (DNAemia) was detected at day 3 p.i. in 7/8 challenged animals. End-organ dissemination was observed, by qPCR, in the lung, liver, and spleen. CD4-positive (CD4+) and CD8-positive (CD8+) T cell infiltrates were present in brains of challenged animals, particularly in periventricular and hippocampal regions. Reactive gliosis and microglial nodules were observed. Statistically significant spatial learning and memory deficits were observed by MWM, particularly for total maze distance traveled (p < 0.0001).ConclusionNeonatal GPCMV infection in guinea pigs results in cognitive defects demonstrable by the MWM. This neonatal guinea pig challenge model can be exploited for studying antiviral interventions.ImpactCMV impairs neonatal neurocognition and memory in the setting of postnatal infection. The MWM can be used to examine memory and learning in a guinea pig model of neonatal CMV infection. CD4+ and CD8+ T cells infiltrate the brain following neonatal CMV challenge. This article demonstrates that the MWM can be used to evaluate memory and learning after neonatal GPCMV challenge. The guinea pig can be used to examine central nervous system pathology caused by neonatal CMV infection and this attribute may facilitate the study of vaccines and antivirals.

Project description:UNLABELLED:Development of a vaccine to prevent congenital cytomegalovirus infection is a major public health priority. Live vaccines attenuated through mutations targeting viral mechanisms responsible for evasion of host defense may be both safe and efficacious. Safety and vaccine efficacy were evaluated using a guinea pig cytomegalovirus (GPCMV) model. Recombinant GPCMV with a targeted deletion of gp145 (designated ?145), a viral protein kinase R (PKR) inhibitor, was generated. Attenuation was evaluated following inoculation of 10(7) PFU of ?145 or parental virus into guinea pigs immunosuppressed with cyclophosphamide. Efficacy was evaluated by immunizing GPCMV-naive guinea pigs twice with either 10(5) or 10(6) PFU of ?145, establishing pregnancy, and challenging the guinea pigs with salivary gland-adapted GPCMV. The immune response, maternal viral load, pup mortality, and congenital infection rates in the vaccine and control groups were compared. ?145 was substantially attenuated for replication in immunocompromised guinea pigs. Vaccination with ?145 induced enzyme-linked immunosorbent assay (ELISA) and neutralizing antibody levels comparable to those achieved in natural infection. In the higher- and lower-dose vaccine groups, pup mortality was reduced to 1/24 (4%) and 4/29 (14%) pups, respectively, whereas it was 26/31 (81%) in unvaccinated control pups (P < 0.0001 for both groups versus the control group). Congenital infection occurred in 20/31 (65%) control pups but only 8/24 (33%) pups in the group vaccinated with 10(6) PFU (P < 0.05). Significant reductions in the magnitude of maternal DNAemia and pup viral load were noted in the vaccine groups compared to those in the controls. Deletion of a GPCMV genome-encoded PKR inhibitor results in a highly attenuated virus that is immunogenic and protective as a vaccine against transplacental infection. IMPORTANCE:Previous attempts to develop successful immunization against cytomegalovirus have largely centered on subunit vaccination against virion proteins but have yielded disappointing results. The advent of bacterial artificial chromosome technologies has enabled engineering of recombinant cytomegaloviruses (CMVs) from which virus genome-encoded immune modulation genes have been deleted, toward the goal of developing a safe and potentially more efficacious live attenuated vaccine. Here we report the findings of studies of such a vaccine against congenital CMV infection based on a virus with a targeted deletion in gp145, a virus genome-encoded inhibitor of protein kinase R, using the guinea pig model of vertical CMV transmission. The deletion virus was attenuated for dissemination in immunocompromised guinea pigs but elicited ELISA and neutralizing responses. The vaccine conferred protection against maternal DNAemia and congenital transmission and resulted in reduced viral loads in newborn guinea pigs. These results provide support for future studies of attenuated CMV vaccines.

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Antagonism of the protein kinase R pathway by the guinea pig cytomegalovirus US22-family gene gp145.

Publications

Antagonism of the protein kinase R pathway by the guinea pig cytomegalovirus US22-family gene gp145.

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