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Prolonged activation of NF-kappaB by human cytomegalovirus promotes efficient viral replication and late gene expression.

ABSTRACT: Infection of fibroblasts by human cytomegalovirus (HCMV) rapidly activates the NF-kappaB signaling pathway, which we documented promotes efficient transactivation of the major immediate-early promoter (DeMeritt, I.B., Milford, L.E., Yurochko, A.D. (2004). Activation of the NF-kappaB pathway in human cytomegalovirus-infected cells is necessary for efficient transactivation of the major immediate-early promoter. J. Virol. 78, 4498-4507). Because a second, sustained increase in NF-kappaB activity following the initial phase of NF-kappaB activation was also observed, we investigated the role that this prolonged NF-kappaB activation played in viral replication and late gene expression. We first investigated HCMV replication in cells in which NF-kappaB activation was blocked by pretreatment with NF-kappaB inhibitors: HCMV replication was significantly decreased in these cultures. A decrease in replication was also observed when NF-kappaB was inhibited up to 48 h post-infection, suggesting a previously unidentified role for NF-kappaB in the regulation of the later class of viral genes.

SUBMITTER: DeMeritt IB

PROVIDER: S-EPMC2600890 | biostudies-literature | 2006 Mar

REPOSITORIES: biostudies-literature

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Prolonged activation of NF-kappaB by human cytomegalovirus promotes efficient viral replication and late gene expression.

DeMeritt Ian B IB Podduturi Jagat P JP Tilley A Michael AM Nogalski Maciej T MT Yurochko Andrew D AD

Virology 20051121 1

Infection of fibroblasts by human cytomegalovirus (HCMV) rapidly activates the NF-kappaB signaling pathway, which we documented promotes efficient transactivation of the major immediate-early promoter (DeMeritt, I.B., Milford, L.E., Yurochko, A.D. (2004). Activation of the NF-kappaB pathway in human cytomegalovirus-infected cells is necessary for efficient transactivation of the major immediate-early promoter. J. Virol. 78, 4498-4507). Because a second, sustained increase in NF-kappaB activity f ...[more]

PMID: 16303162

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Project description:Alphaviruses are enveloped, positive-sense RNA viruses that are important causes of viral encephalomyelitis. Sindbis virus (SINV) infects the neurons of rodents and is a model for studying factors that regulate infection of neuronal cells. The outcome of alphavirus infection of the central nervous system is dependent on neuronal maturation status. Differentiated mature neurons survive and control viral replication better than undifferentiated immature neurons. The cellular factors involved in age-dependent susceptibility include higher levels of antiapoptotic and innate immune factors in mature neurons. Because NF-?B pathway activation is required for the initiation of both apoptosis and the host antiviral response, we analyzed the role of NF-?B during SINV infection of differentiated and undifferentiated rat neuronal cells. SINV infection induced canonical NF-?B activation, as evidenced by the degradation of I?B? and the phosphorylation and nuclear translocation of p65. Inhibition or deletion of the upstream I?B kinase substantially reduced SINV replication in differentiated but not in undifferentiated neuronal cells or mouse embryo fibroblasts. NF-?B inhibition did not affect the establishment of infection, replication complex formation, the synthesis of nonstructural proteins, or viral RNA synthesis in differentiated neurons. However, the translation of structural proteins was impaired, phosphorylation of the ? subunit of eukaryotic translation initiation factor 2 (eIF2?) was decreased, and host protein synthesis was maintained, suggesting that NF-?B activation was involved in the regulation of translation during infection of mature neurons. Inhibition or deletion of double-stranded RNA-activated protein kinase (PKR) also decreased eIF2? phosphorylation, the translation of viral structural proteins, and virus production. Therefore, canonical NF-?B activation synergizes with PKR to promote SINV replication in differentiated neurons by facilitating viral structural protein translation.IMPORTANCE Mosquito-borne alphaviruses are a significant and growing cause of viral encephalomyelitis worldwide. The outcome of alphaviral neuronal infections is host age dependent and greatly affected by neuronal maturation status, with differentiated, mature neurons being more resistant to infection than undifferentiated, immature neurons. The biological factors that change during neuronal maturation and that influence the outcome of viral infection are currently only partially defined. These studies investigated the role of NF-?B in determining the outcome of alphaviral infection in mature and immature neurons. Inhibition of canonical NF-?B activation decreased alphavirus replication in mature neurons by regulating protein synthesis and limiting the production of the viral structural proteins but had little effect on viral replication in immature neurons or fibroblasts. Therefore, NF-?B is a signaling pathway that influences the maturation-dependent outcome of alphaviral infection in neurons and that highlights the importance of cellular context in determining the effects of signal pathway activation.

Project description:Human cytomegalovirus (HCMV) is a prototypic betaherpesvirus which causes severe manifestations in individuals with impaired or immature immunity. To investigate cytomegalovirus-induced pathogenesis and virus-specific immune responses, mouse cytomegalovirus (MCMV) infections in mice are employed as accepted small animal model. MCMV and HCMV share co-linear genomes and encode several homologous proteins. Due to the size and complexity of CMV genomes, the molecular functions of numerous cytomegaloviral gene products remain to be elucidated. While the essential nature of viral genes highlights their biological relevance, it renders functional studies particularly cumbersome by precluding experiments in the infection context. The HCMV-encoded protein pUL34 binds the HCMV genome and regulates viral gene expression (e.g., of US3). Several groups provided compelling evidence that UL34 is essential for HCMV replication. MCMV encodes the homologous protein pM34 (34% identical and 55% similar). Based on unsuccessful attempts to reconstitute M34-deficient virus from a bacterial artificial chromosome (BAC), M34 was previously classified as essential for MCMV replication. To characterize pM34 during viral infection, we engineered and analyzed an MCMV mutant expressing an HA-epitope-tagged pM34 which was expressed with early-late kinetics and localized in the nucleus. Additionally, we generated an M34-deficient ("ΔM34") MCMV-BAC by replacing the entire M34 coding sequence by a kanamycin resistance cassette. The deletion of M34 was confirmed by Southern blot and PCR. Unexpectedly, we could reconstitute replicating ΔM34-MCMV upon transfection of the BAC DNA into mouse embryonic fibroblasts. The absence of M34 from the genome of the replicating ΔM34-MCMV was also confirmed. Accordingly, a ΔM34-MCMV, in which the kanamycin cassette was excised by frt/Flp-mediated recombination, was also replication competent. In order to corroborate the absence of pM34 protein, the M34 deletion was recapitulated on the background of M34HA, which yielded replicating virus devoid of detectable pM34HA protein. The replication of MCMVs lacking M34 was found to be 10- to 100-fold reduced as compared to wt-MCMV which might explain previous unsuccessful reconstitution attempts conducted by others. Taken together, our findings reveal that MCMV remains replication competent despite the absence of M34, enabling functional studies in the infection context.

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Prolonged activation of NF-kappaB by human cytomegalovirus promotes efficient viral replication and late gene expression.

Publications

Prolonged activation of NF-kappaB by human cytomegalovirus promotes efficient viral replication and late gene expression.

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