Functional profiling of a human cytomegalovirus genome

Dunn, Walter; Chou, Cassie; Li, Hong; Hai, Rong; Patterson, David A.; Štolc, Viktor; Zhu, Hua; Liu, Fenyong

doi:10.1073/pnas.2334032100

Cited by 603 publications

(846 citation statements)

References 32 publications

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“…Additionally, TRS1 has been shown to play a role in viral capsid assembly (1), and deletion of the majority of the TRS1 open-reading frame from HCMV results in a growth defect that is most pronounced at a low MOI (8). IRS1, the TRS1 homologue in HCMV, also stimulates reporter gene expression but is not required for efficient replication of HCMV (8,31,42,61,63). In addition to these roles, both TRS1 and IRS1 prevent the phosphorylation of eIF2␣, the activation of RNase L, and the shutoff of translation that occur upon infection with VV⌬E3L and restore the full host cell range of VV⌬E3L (17).…”

Section: Discussionmentioning

confidence: 99%

Binding and Nuclear Relocalization of Protein Kinase R by Human Cytomegalovirus TRS1

Hakki

Marshall²,

Niro³

et al. 2006

J Virol

104

100

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The human cytomegalovirus (HCMV) TRS1 and IRS1 genes block the phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2␣) and the consequent shutoff of cellular protein synthesis that occur during infection with vaccinia virus (VV) deleted of the double-stranded RNA binding protein gene E3L (VV⌬E3L). To further define the underlying mechanism, we first evaluated the effect of pTRS1 on protein kinase R (PKR), the double-stranded RNA (dsRNA)-dependent eIF2␣ kinase. Immunoblot analyses revealed that pTRS1 expression in the context of a VV⌬E3L recombinant decreased levels of PKR in the cytoplasm and increased its levels in the nucleus of infected cells, an effect not seen with wild-type VV or a VV⌬E3L recombinant virus expressing E3L. This effect of pTRS1 was confirmed by visualizing the nuclear relocalization of PKR-EGFP expressed by transient transfection. PKR present in both the nuclear and cytoplasmic fractions was nonphosphorylated, indicating that it was unactivated when TRS1 was present. PKR also accumulated in the nucleus during HCMV infection as determined by indirect immunofluorescence and immunoblot analysis. Binding assays revealed that pTRS1 interacted with PKR in mammalian cells and in vitro. This interaction required the same carboxy-terminal region of pTRS1 that is necessary to rescue VV⌬E3L replication in HeLa cells. The carboxy terminus of pIRS1 was also required for rescue of VV⌬E3L and for mediating an interaction of pIRS1 with PKR. These results suggest that these HCMV genes directly interact with PKR and inhibit its activation by sequestering it in the nucleus, away from both its activator, cytoplasmic dsRNA, and its substrate, eIF2␣.Double-stranded RNA (dsRNA) activates the host cell response to viral infection in numerous ways, one of which involves the interferon-induced, dsRNA-dependent protein kinase R (PKR) (reviewed in reference 43). After binding to dsRNA, PKR dimerizes, autophosphorylates, and then phosphorylates the eukaryotic initiation factor 2 (eIF2) on its ␣ subunit. Phosphorylated eIF2␣ sequesters the guanine nucleotide exchange factor eIF2B, resulting in the inhibition of protein synthesis at the level of translation initiation. Since viruses depend on the cellular translational machinery, the shutoff of host cell protein synthesis inhibits viral replication and spread.Many viruses have mechanisms for inhibiting the PKR-mediated phosphorylation of eIF2␣ (56). The vaccinia virus (VV) E3L protein prevents the activation of PKR by binding to and sequestering dsRNA via a carboxy-terminal double-stranded RNA binding domain (dsRBD) (39). VV from which the E3L gene has been deleted (VV⌬E3L) exhibits a dsRNA-dependent restriction of host cell range, and this phenotype can be reversed by expression of the dsRBD from pE3L or dsRNAbinding proteins from other viruses (4, 47, 62). We previously found that the products of the human cytomegalovirus (HCMV) genes TRS1 and IRS1 restore the host cell range of VV⌬E3L, inhibit the phosphorylation of eIF2␣, and prevent the shutoff...

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Section: Discussionmentioning

confidence: 99%

Binding and Nuclear Relocalization of Protein Kinase R by Human Cytomegalovirus TRS1

Hakki

Marshall²,

Niro³

et al. 2006

J Virol

104

100

View full text Add to dashboard Cite

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“…UL97-null HCMV fails to induce pRb phosphorylation and shows defects in the production of infectious virus progeny (7,8,(14)(15)(16)(17)20). We wanted to determine whether UL97 functionally inactivates pRb and if heterologous pRb inactivation in cells infected with UL97-null virus would rescue deficiencies of UL97-null HCMV.…”

Section: Resultsmentioning

confidence: 99%

“…There is considerable variability reported in the literature regarding the degree and nature of replication defects caused by genetic ablation or pharmacological inhibition of UL97, and some of this variability appears to depend on cell type and culture conditions (10,(14)(15)(16)(17)(18)36). Nonetheless, we are unaware of any previously presented data directly comparing the replication of viruses lacking UL97 activity in dividing versus resting cells.…”

Section: Discussionmentioning

confidence: 97%

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Human papillomavirus 16 E7 inactivator of retinoblastoma family proteins complements human cytomegalovirus lacking UL97 protein kinase

Kamil¹,

Hume

Jurak³

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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Several different families of DNA viruses encode proteins that inactivate the cellular retinoblastoma tumor suppressor protein (pRb), which normally functions to bind E2F transcription factors and restrict expression of genes necessary for cellular processes including DNA replication. Human cytomegalovirus (HCMV) UL97, a protein kinase functionally orthologous to cellular cyclin-dependent kinases, phosphorylates pRb on inactivating residues during HCMV infection. To assess if such phosphorylation is biologically relevant, we tested whether the human papillomavirus type 16 E7 protein, which inactivates pRb family proteins by direct binding and destabilization, could substitute for UL97 during HCMV infection. In the absence of UL97, expression of wild-type E7 protein, but not a mutant E7 unable to bind pRb family proteins, restored E2F-responsive cellular gene expression, late viral gene expression, and viral DNA synthesis to levels normally observed during wildtype virus infection of quiescent cells. UL97-null mutants exhibited more pronounced defects in virus production and DNA synthesis in quiescent cells as compared to serum-fed, cycling cells. E7 expression substantially enhanced infectious virus production in quiescent cells, but did not complement the defects observed during UL97-null virus infection of cycling cells. Thus, a primary role of UL97 is to inactivate pRb family proteins during infection of quiescent cells, and this inactivation likely abets virus replication by induction of cellular E2F-responsive genes. Our findings have implications for human cytomegalovirus disease and for drugs that target UL97.E2F ͉ cyclin-dependent kinase ͉ cell cycle ͉ antiviral drugs ͉ retinoblastoma protein

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“…The mAP coding sequence completely overlaps with and is within the 3Ј coding sequence of another viral capsid protein, M80 (21). Both mAP and M80 are essential for viral capsid formation and CMV replication (22,23). We showed that the constructed ribozymes efficiently cleaved the target mRNA sequence in vitro and effectively inhibited mAP expression and viral growth in cultured cells.…”

mentioning

confidence: 96%

Effective inhibition in animals of viral pathogenesis by a ribozyme derived from RNase P catalytic RNA

Bai¹,

Trang

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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A functional RNase P ribozyme (M1GS RNA) was constructed to target the overlapping mRNA region of two murine cytomegalovirus (MCMV) capsid proteins essential for viral replication: the assembly protein (mAP) and M80. The customized ribozyme efficiently cleaved the target mRNA sequence in vitro. Moreover, 80% reduction in the expression of mAP and M80 and a 2,000-fold reduction in viral growth were observed in cells expressing the ribozyme. In contrast, there was no significant reduction in viral gene expression and growth in cells that either did not express the ribozyme or produced a ''disabled'' ribozyme carrying mutations that abolished its catalytic activity. When the ribozyme-expressing constructs were delivered into MCMV-infected SCID mice via a modified ''hydrodynamic transfection'' procedure, expression of ribozymes was observed in the livers and spleens. Compared with the control animals that did not receive any M1GS constructs or received the disabled ribozyme construct, animals receiving the functional ribozyme construct exhibited a significant reduction of viral gene expression and infection. Viral titers in the spleens, livers, lungs, and salivary glands of the functional ribozyme-treated SCID mice at 21 days after infection were 200-to 2,000-fold lower than those in the control animals. Moreover, survival of the infected animals significantly improved upon receiving the functional ribozyme construct. Our study examines the use of M1GS ribozymes for inhibition of gene expression in animals and demonstrates the utility of RNase P ribozymes for gene targeting applications in vivo.cytomegalovirus ͉ gene targeting ͉ herpesvirus ͉ antisense H uman cytomegalovirus (CMV), a common opportunistic pathogen, causes significant morbidity and mortality in immunocompromised or immunologically immature individuals, including neonates, AIDS patients, and transplant recipients (1). The emergence of drug-resistant strains of CMV has posed a need for the development of new drugs and treatment strategies. Murine cytomegalovirus (MCMV) infection of mice resembles its human counterpart with respect to pathogenesis, thus providing an animal model for studying CMV infection in vivo and for screening novel agents and developing new antiviral approaches (1-4). For example, the CB17 SCID mice, which lack functional T and B lymphocytes, are highly susceptible to MCMV infection (5, 6). Analysis of viral replication in these mice can be used for studying whether new therapeutic approaches block CMV opportunistic infection and prevent viral-associated diseases in immunocompromised hosts.Nucleic acid-based gene interference technologies represent promising gene-targeting strategies for specific inhibition of mRNA sequences of choice (7-9). For example, ribozymes have been shown to cleave viral mRNA sequences and inhibit viral replication in human cells (10). More recently, siRNAs were proven to be effective in inducing endogenous RNase of the RNA-induced silencing complex (RISC) in the RNAi pathway to inhibit gene expression and g...

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Functional profiling of a human cytomegalovirus genome

Cited by 603 publications

References 32 publications

Binding and Nuclear Relocalization of Protein Kinase R by Human Cytomegalovirus TRS1

Binding and Nuclear Relocalization of Protein Kinase R by Human Cytomegalovirus TRS1

Human papillomavirus 16 E7 inactivator of retinoblastoma family proteins complements human cytomegalovirus lacking UL97 protein kinase

Effective inhibition in animals of viral pathogenesis by a ribozyme derived from RNase P catalytic RNA

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