Human Cytomegalovirus pUL29/28 and pUL38 Repression of p53-Regulated p21CIP1 and Caspase 1 Promoters during Infection

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Human cytomegalovirus (HCMV) is a member of the betaherpesvirus family. During infection, an array of viral proteins manipulates the host cell cycle. We have previously shown that expression of HCMV pUL27 results in increased levels of the cyclindependent kinase (CDK) inhibitor p21Cip1 . In addition, pUL27 is necessary for the full antiviral activity of the pUL97 kinase inhibitor maribavir (MBV). The purpose of this study was to define the relationship between pUL27 and pUL97 and its role in MBV antiviral activity. We observed that expression of wild-type but not kinase-inactive pUL97 disrupted pUL27-dependent induction of p21Cip1 . Furthermore, pUL97 associated with and promoted the phosphorylation of pUL27. During infection, inhibition of the kinase resulted in elevated levels of p21 Cip1 in wild-type virus but not a pUL27-deficient virus. We manipulated the p21 Cip1 levels to evaluate the functional consequence to MBV. Overexpression of p21 Cip1 restored MBV activity against a pUL27-deficient virus, while disruption reduced activity against wild-type virus. We provide evidence that the functional target of p21Cip1 in the context of MBV activity is CDK1. One CDK-like activity of pUL97 is to phosphorylate nuclear lamin A/C, resulting in altered nuclear morphology and increased viral egress. In the presence of MBV, we observed that infection using a pUL27-deficient virus still altered the nuclear morphology. This was prevented by the addition of a CDK inhibitor. Overall, our results demonstrate an antagonistic relationship between pUL27 and pUL97 activities centering on p21Cip1 and support the idea that CDKs can complement some activities of pUL97. IMPORTANCEHCMV infection results in severe disease upon immunosuppression and is a leading cause of congenital birth defects. Effective antiviral compounds exist, yet they exhibit high levels of toxicity, are not approved for use during pregnancy, and can result in antiviral resistance. Our studies have uncovered new information regarding the antiviral efficacy of the HCMV pUL97 kinase inhibitor MBV as it relates to the complex interplay between pUL97 and a second HCMV protein, pUL27. We demonstrate that pUL97 functions antagonistically against pUL27 by phosphorylation-dependent inactivation of pUL27-mediated induction of p21Cip1 . In contrast, we provide evidence that p21 Cip1 functions to antagonize overlapping activities between pUL97 and cellular CDKs. In addition, these studies further support the notion that CDK inhibitors or p21Cip1 activators might be useful in combination with MBV to effectively inhibit HCMV infections. H uman cytomegalovirus (HCMV) infects the majority of the world's population (1). Infection of immunocompetent children and adults is usually asymptomatic or associated with minor disease. In contrast, HCMV infection in immunocompromised patients results in serious disease, especially in organ transplant recipients receiving immunosuppressants (2). HCMV is also the leading congenital infection in the developed world (3). Currently, the...

Section: Cellular P21mentioning

confidence: 99%

“…Cip1 is mediated in part by proteasome-and calpain-dependent protein degradation (47) as well as decreased RNA expression (47)(48)(49).…”

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confidence: 99%

Antagonistic Relationship between Human Cytomegalovirus pUL27 and pUL97 Activities during Infection

Bigley

Reitsma

Terhune

2015

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“…It has equivalents in human herpesvirus 6 and human herpesvirus 7, and UL38-homologous genes are carried by cytomegaloviruses of other species (24)(25)(26)(27). The UL38 gene product pUL38 is a multifunctional protein that regulates viral and host gene expression and, in collaboration with other viral proteins pUL29/28 (28,29), acts as an antiapoptotic molecule during HCMV infection (23,30), interacts with TSC2, and activates mTORC1 (14). However, the mechanisms whereby pUL38 interacts with TSC2 to activate mTORC1 are unclear.…”

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confidence: 99%

Tuberous Sclerosis Complex Protein 2-Independent Activation of mTORC1 by Human Cytomegalovirus pUL38

Bai

Xuan

Liu

et al. 2015

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The mammalian target of rapamycin complex 1 (mTORC1) controls cell growth and anabolic metabolism and is a critical host factor activated by human cytomegalovirus (HCMV) for successful infection. The multifunctional HCMV protein pUL38 previously has been reported to activate mTORC1 by binding to and antagonizing tuberous sclerosis complex protein 2 (TSC2) . pUL38 also plays a role in blocking endoplasmic reticulum stress-induced cell death during HCMV infection. In this study, we showed that a mutant pUL38 lacking the N-terminal 24 amino acids (pHA-UL38 25-331 ) was fully functional in suppressing cell death during infection. Interestingly, pHA-UL38 25-331 lost the ability to interact with TSC2 but retained the ability to activate mTORC1, although to a lesser extent than full-length pHA-UL38. Recombinant virus expressing pHA-UL38 25-331 replicated with ϳ10-fold less efficiency than the wild-type virus at a low multiplicity of infection (MOI), but it grew similarly well at a high MOI, suggesting an MOIdependent importance of pUL38-TSC2 interaction in supporting virus propagation. Site-directed mutational analysis identified a TQ motif at amino acid residues 23 and 24 as critical for pUL38 interaction with TSC2. Importantly, when expressed in isolation, the TQ/AA substitution mutant pHA-UL38 TQ/AA was capable of activating mTORC1 just like pHA-UL38 25-331 . We also created TSC2-null U373-MG cell lines by CRISPR genome editing and showed that pUL38 was capable of further increasing mTORC1 activity in TSC2-null cells. Therefore, this study identified the residues important for pUL38-TSC2 interaction and demonstrated that pUL38 can activate mTORC1 in both TSC2-dependent and -independent manners. IMPORTANCE HCMV, like other viruses, depends exclusively on its host cell to propagate. Therefore, it has developed methods to protect against host stress responses and to usurp cellular processes to complete its life cycle. mTORC1 is believed to be important for virus replication, and HCMV maintains high mTORC1 activity despite the stressful cellular environment associated with infection. mTORC1 inhibitors suppressed HCMV replication in vitro and reduced the incidence of HCMV reactivation in transplant recipients. We demonstrated that mTORC1 was activated by HCMV protein pUL38 in both TSC2-dependent and TSC2-independent manners. The pUL38-independent mode of mTORC1 activation also has been reported. These novel findings suggest the evolution of sophisticated approaches whereby HCMV activates mTORC1, indicating its importance in the biology and pathogenesis of HCMV.( Human cytomegalovirus (HCMV) is a member of the betaherpesvirus family with broad cell tropism. It is capable of escaping the immune surveillance and persists as a lifelong latent and recurrent infection in the host (1, 2). HCMV infection in adults and healthy people normally is asymptomatic or causes mild illness, but it can cause severe and life-threatening diseases in immunocompromised individuals, and importantly, HCMV congenital infection is a leadin...

“…Also, the HCMV pUL123 (IE1) protein inhibits histone deacetylation (34) and, along with pUL122 (IE2), has been shown to affect the repressive actions of HDAC1, 2, and 3 (34)(35)(36)(37). Other HCMV proteins interact with HDACs or HDACcontaining complexes, including pUL29/28, which associates with the nucleosome remodeling and deacetylase complex, NuRD, influencing both viral (25) and cellular gene expression (38). In general, regulation of deacetylation is a central event during infection by other herpesviruses.…”

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confidence: 99%

Human Cytomegalovirus pUL97 Regulates the Viral Major Immediate Early Promoter by Phosphorylation-Mediated Disruption of Histone Deacetylase 1 Binding

Bigley

Reitsma

Mirza

et al. 2013

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Human cytomegalovirus (HCMV) is a common agent of congenital infection and causes severe disease in immunocompromised patients. Current approved therapies focus on inhibiting viral DNA replication. The HCMV kinase pUL97 contributes to multiple stages of viral infection including DNA replication, controlling the cell cycle, and virion maturation. Our studies demonstrate that pUL97 also functions by influencing immediate early (IE) gene expression during the initial stages of infection. Inhibition of kinase activity using the antiviral compound maribavir or deletion of the UL97 gene resulted in decreased expression of viral immediate early genes during infection. Expression of pUL97 was sufficient to transactivate IE1 gene expression from the viral genome, which was dependent on viral kinase activity. We observed that pUL97 associates with histone deacetylase 1 (HDAC1). HDAC1 is a transcriptional corepressor that acts to silence expression of viral genes. We observed that inhibition or deletion of pUL97 kinase resulted in increased HDAC1 and decreased histone H3 lysine 9 acetylation associating with the viral major immediate early (MIE) promoter. IE expression during pUL97 inhibition or deletion was rescued following inhibition of deacetylase activity. HDAC1 associates with chromatin by protein-protein interactions. Expression of active but not inactive pUL97 kinase decreased HDAC1 interaction with the transcriptional repressor protein DAXX. Finally, using mass spectrometry, we found that HDAC1 is uniquely phosphorylated upon expression of pUL97. Our results support the conclusion that HCMV pUL97 kinase regulates viral immediate early gene expression by phosphorylation-mediated disruption of HDAC1 binding to the MIE promoter.