Human Cytomegalovirus UL97 Kinase Is Required for the Normal Intranuclear Distribution of pp65 and Virion Morphogenesis

Prichard, Mark N.; Britt, William J.; Daily, Shannon; Hartline, Caroll B.; Kern, Earl R.

doi:10.1128/jvi.79.24.15494-15502.2005

Cited by 106 publications

(146 citation statements)

References 38 publications

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“…As has been previously reported by others as data not shown (20), UL97-null viruses produced higher titers in serum-fed cycling cells than in quiescent cells, consistently showing only a Ϸ10-fold replication defect relative to WT in cycling cells (Fig. 4B).…”

Section: Hpv16 E7 Expression Increases Infectious Virion Production Asupporting

confidence: 67%

“…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%

“…Specific defects of these mutants during viral replication include reduced viral DNA synthesis, impaired exit of capsids from the nucleus (nuclear egress), and altered intracellular localization of virion components; pharmacological inhibition of UL97 results in similar phenotypes (8,10,15,17,19,20). Our discovery that UL97 directly phosphorylates pRb prompted us to examine the roles that both UL97 and pRb play during HCMV infection.…”

mentioning

confidence: 99%

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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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Section: Hpv16 E7 Expression Increases Infectious Virion Production Asupporting

confidence: 67%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

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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“…The failure of viral proteins to localize at the AC after inhibition of viral DNA synthesis by GCV, is in concert with previous studies using anti-HCMV molecules. 13,64 Interestingly, RhoB was still able to be recruited at the HCMV AC, even when the Late phase of the viral life-cycle was inhibited by GCV and proper AC was not formed (Fig. 5, h-i and q-r).…”

Section: Discussionmentioning

confidence: 99%

RhoB is a component of the human cytomegalovirus assembly complex and is required for efficient viral production

et al. 2015

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Human Cytomegalovirus (HCMV), an ubiquitous b-herpesvirus, is a significant pathogen that causes medically severe diseases in immunocompromised individuals and in congenitally infected neonates. RhoB belongs to the family of Rho GTPases, which regulates diverse cellular processes. Rho proteins are implicated in the entry and egress from the host cell of mainly a-and g-herpesviruses, whereas b-herpesviruses are the least studied in this regard. Here, we studied the role of RhoB GTPase during HCMV lytic infection. Microscopy analysis, both in fixed and live infected cells showed that RhoB was translocated to the assembly complex/compartment (AC) of HCMV, a cytoplasmic zone in infected cells where many viral structural proteins are known to accumulate and assembly of new virions takes place. Furthermore, RhoB was localized at the AC even when the expression of the late HCMV AC proteins was inhibited. At the very late stages of infection, cellular projections were formed containing RhoB and HCMV virions, potentially contributing to the successful viral spread. Interestingly, the knockdown of RhoB in HCMV-infected cells resulted in a significant reduction of the virus titer and could also affect the accumulation of AC viral proteins at this subcellular compartment. RhoB knockdown also affected actin fibers' structure. Actin reorganization was observed at late stages of infection originating from the viral AC and surrounding the cellular projections, implying a potential interplay between RhoB and actin during HCMV assembly and egress. In conclusion, our results demonstrate for the first time that RhoB is a constituent of the viral AC and is required for HCMV productive infection.

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“…The pp65 nuclear localization signals (NLS) have been mapped and are contained in the carboxy-terminal one-third of the protein (6,24); however, the underlying cause of the relocalization of pp65 to the cytoplasm late in infection has not yet been elucidated. A recent report has described the aggregation of pp65 in cells infected with an HCMV viral kinase UL97 mutant (15). The aggregation of pp65 in large nuclear and cytoplasmic structures was also detected upon treatment of cells with maribavir, a UL97 inhibitor.…”

mentioning

confidence: 95%

Nuclear Export of the Human Cytomegalovirus Tegument Protein pp65 Requires Cyclin-Dependent Kinase Activity and the Crm1 Exporter

Sánchez

Mahr²,

Orazio

et al. 2007

J Virol

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In this report, we add to these findings by demonstrating alterations in the phosphorylation and localization of pp65 (UL83) in cells treated with roscovitine. We observed that inhibition of cdk activity causes the retention of pp65 within the nucleus at late times postinfection. At the same time, we observed a change in the phosphorylation pattern of the protein. Interestingly, mutation of potential cdk phosphorylation sites did not affect the ability of pp65 to localize to the nucleus or to relocalize to the cytoplasm late in infection. However, we found that the cytoplasmic accumulation of pp65 late in infection was sensitive to the Crm1 inhibitor leptomycin B.Human cytomegalovirus (HCMV), the largest member of the herpesvirus family, is a ubiquitous pathogen that remains the leading viral cause of birth defects (13). Like that of other herpesviruses, HCMV gene expression is temporally regulated (10). The immediate-early (IE) class of genes is expressed shortly after infection, and their expression requires no de novo protein synthesis. The IE genes encode proteins important for the modulation of the apoptotic response to infection (for a review, see reference 4) and for the expression of the early (E) genes, which are necessary for viral DNA replication. The early proteins have also been implicated in the altered expression of key cell cycle proteins during infection (8,19). Viral DNA synthesis is a prerequisite for the synthesis of the late RNAs, which encode structural components of the virion and proteins that function in virion maturation (10).Numerous studies have examined the assembly pathways for the herpesviruses (for a review, see reference 9). The current model describes the encapsidation of the viral DNA in the nucleus, followed by egress of subviral particles through the nuclear envelope. This process is thought to occur through an initial envelopment at the inner nuclear membrane and a subsequent de-envelopment step at the outer nuclear membrane. The immature virions are then transported to the final site of envelopment in the cytoplasm. The acquisition of the tegument proteins likely occurs in both the nucleus and the cytoplasm, since the steady-state localization of some HCMV tegument proteins is restricted during infection (1,7,22,26). The distribution of other tegument proteins is temporally regulated (5,11,18,27). The best-studied example of this temporal regulation of virion proteins is the biphasic localization of the abundant tegument protein pp65. As part of the incoming virion, pp65 is targeted to the nucleus immediately after infection (24). Expression of UL83 is an early-late event, and the newly synthesized pp65 is observed in the nucleus until some time after 48 h postinfection (p.i.). Thereafter, pp65 accumulates in the cytoplasm, and the nucleus becomes devoid of the protein (18). The pp65 nuclear localization signals (NLS) have been mapped and are contained in the carboxy-terminal one-third of the protein (6, 24); however, the underlying cause of the relocalization of pp65 to th...

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Human Cytomegalovirus UL97 Kinase Is Required for the Normal Intranuclear Distribution of pp65 and Virion Morphogenesis

Cited by 106 publications

References 38 publications

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

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

RhoB is a component of the human cytomegalovirus assembly complex and is required for efficient viral production

Nuclear Export of the Human Cytomegalovirus Tegument Protein pp65 Requires Cyclin-Dependent Kinase Activity and the Crm1 Exporter

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