Identification of Human Cytomegalovirus UL84 Virus- and Cell-Encoded Binding Partners by Using Proteomics Analysis

Gao, Yang; Colletti, Kelly; Pari, Gregory S.

doi:10.1128/jvi.01559-07

Cited by 53 publications

(78 citation statements)

References 46 publications

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“…Finally, the viral proteins pUL25, pUL84, and pUL38 were observed. HCMV pUL84 has been previously demonstrated to associate with p53, the NuRD component RBBP4, and importin ␣ (Table 1) (42,63,64). Our results suggest that pUL29/28 interacts with multiple viral and cellular proteins at 24 hpi, including the tumor suppressor protein p53.…”

Section: Hcmv Pul29/28 Interacts With Cellular P53 During Infectionsupporting

confidence: 53%

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

Savaryn

Reitsma

Bigley

et al. 2013

J Virol

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c During infection by human cytomegalovirus (HCMV), the tumor suppressor protein p53, which promotes efficient viral gene expression, is stabilized. However, the expression of numerous p53-responsive cellular genes is not upregulated. The molecular mechanism used to manipulate the transcriptional activity of p53 during infection remains unclear. The HCMV proteins IE1, IE2, pUL44, and pUL84 likely contribute to the regulation of p53. In this study, we used a discovery-based approach to identify the protein targets of the HCMV protein pUL29/28 during infection. Previous studies have demonstrated that pUL29/28 regulates viral gene expression by interacting with the chromatin remodeling complex NuRD. Here, we observed that pUL29/28 also associates with p53, an additional deacetylase complex, and several HCMV proteins, including pUL38. We confirmed the interaction between p53 and pUL29/28 in both the presence and absence of infection. HCMV pUL29/28 with pUL38 altered the activity of the 53-regulatable p21CIP1 promoter. During infection, pUL29/28 and pUL38 contributed to the inhibition of p21CIP1 as well as caspase 1 expression. The expression of several other p53-regulating genes was not altered. Infection using a UL29-deficient virus resulted in increased p53 binding and histone H3 acetylation at the responsive promoters. Furthermore, expression of pUL29/28 and its interacting partner pUL38 contributed to an increase in the steady-state protein levels of p53. This study identified two additional HCMV proteins, pUL29/28 and pUL38, which participate in the complex regulation of p53 transcriptional activity during infection. Human cytomegalovirus (HCMV) is a member of the betaherpesvirus family, which also includes human herpesviruses 6 and 7. Infection by HCMV is a leading cause of birth defects and can cause severe disease upon immunosuppression (reviewed in reference 1). HCMV disease in immunosuppressed individuals is often successfully managed using the antiviral compound ganciclovir, valganciclovir, cidofovir, or foscarnet. Congenital HCMV infection, however, remains a significant problem because of limited diagnostics and treatment options as well as the lack of community awareness (2). The initial infection leads to systemic viral spread and a balance between latent and lytic replication cycles among diverse cell types within the body. These complex replication cycles result in a persistent lifelong infection.Successful HCMV infection involves viral proteins interacting with and disconnecting cellular stress response pathways. Many of these pathways and the associated proteins are also altered in cancers and are conserved targets among diverse herpesviruses. Examples include DAXX (death domain-associated protein) (3-6), PML (promyelocytic leukemia protein) (7-11), IFI16 (interferoninducible protein 16) (12, 13), Tip60 (Tat-interactive protein, 60 kDa) (14, 15), and p53 (16-24). Upon infection, delivery of the HCMV tegument protein pp71 (UL82) results in the degradation of cellular DAXX and disruption of an intri...

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Section: Hcmv Pul29/28 Interacts With Cellular P53 During Infectionsupporting

confidence: 53%

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

Savaryn

Reitsma

Bigley

et al. 2013

J Virol

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“…The UL44 protein is structurally homologous to the eukaryotic proliferating cell nuclear antigen (PCNA), which interacts with multiple proteins (31). Recent proteomic analyses demonstrated that the UL44 protein interacts with a variety of proteins, including transcription factors other than viral replication-related proteins (13,(45)(46)(47). Therefore, it is conceivable that pre-RCs including UL44 protein act to optimize the timing of late gene transcription.…”

Section: Discussionmentioning

confidence: 99%

The Human Cytomegalovirus Gene Products Essential for Late Viral Gene Expression Assemble into Prereplication Complexes before Viral DNA Replication

Isomura

Stinski

Murata

et al. 2011

J Virol

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The regulation of human cytomegalovirus (HCMV) late gene expression by viral proteins is poorly understood, and these viral proteins could be targets for novel antivirals. HCMV open reading frames (ORFs) UL79, -87, and -95 encode proteins with homology to late gene transcription factors of murine gammaherpesvirus 68 ORFs 18, 24, and 34, respectively. To determine whether these HCMV proteins are also essential for late gene transcription of a betaherpesvirus, we mutated HCMV ORFs UL79, -87, and -95. Cells were infected with the recombinant viruses at high and low multiplicities of infection (MOIs). While viral DNA was detected with the recombinant viruses, infectious virus was not detected unless the wild-type viral proteins were expressed in trans. At a high MOI, mutation of ORF UL79, -87, or -95 had no effect on the level of major immediate-early ( Human cytomegalovirus (HCMV) is a member of the betaherpesvirus family. The viral genome is 240,000 bp long and has at least 150 predicted open reading frames (ORFs) (9, 57). Although infection by HCMV occurs in most individuals, it is usually asymptomatic. The virus is reactivated under immunosuppressive conditions and causes pneumonitis, hepatitis, retinitis, and gastrointestinal diseases. The virus replicates productively in terminally differentiated cells such as fibroblasts, epithelial and endothelial cells, and monocyte-derived macrophages (11,12,18,28,41,42,48).During productive infection, HCMV genes are expressed in a temporal cascade, with temporal phases designated immediate-early (IE), early, and late. The major IE genes (MIE) UL123 and UL122 (IE1/IE2) play a critical role in subsequent viral gene expression and the efficiency of viral replication (19,20,24,(33)(34)(35). The early viral genes encode proteins necessary for viral DNA replication (36). Following viral DNA replication, delayed early and late viral genes are expressed which encode structural proteins for the virion. Expression of true late (gamma 2 class) viral genes is prevented by inhibition of viral DNA synthesis. UL75 (gH), UL99 (pp28), and the middle transcription start site of UL44 are examples of HCMV genes that are true late transcripts (26,29,32,53). While the regulation of HCMV immediate-early and early gene expression has been studied extensively, little is known about the specific mechanisms of regulating late gene expression.With HCMV, the IE2 protein can be found in microfoci early after infection and is associated with parental viral genomes by direct DNA contact. Some of the microfoci enlarge to form replication compartments (RCs) at late times after infection (43). However, the HCMV IE genes alone are not sufficient for activation of the true late viral genes. Which HCMV early genes are required for late gene expression is not known.ORFs 18, 24, 30, and 34 of murine gammaherpesvirus 68 (MHV-68) are essential for late gene transcription (3,54,55) and have homology to HCMV ORFs UL79, -87, and -95. The MHV-68 gene products function specifically to stimulate the promoters of late g...

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“…To date, no quantitative or nonquantitative proteomic analysis of the MAM fraction has yet been reported. Although proteomic analyses have identified HCMV virion components (42) and cellular proteins that interact with HCMV products (43,44) as well as defined the HCMV-induced secretome (40), this is the first quantitative proteomic analyses of HCMV-infected HFFs and the effects of virus on ER-mitochondrial contacts. Using this quantitative proteomic approach, we observed dramatic changes in the MAM proteome by late times of HCMV infection, consistent with its global reprogramming of cellular metabolism.…”

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confidence: 99%

Quantitative Proteomic Analyses of Human Cytomegalovirus-Induced Restructuring of Endoplasmic Reticulum-Mitochondrial Contacts at Late Times of Infection

Zhang

Williamson

Wong

et al. 2011

Molecular & Cellular Proteomics

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Endoplasmic reticulum-mitochondrial contacts, known as mitochondria-associated membranes, regulate important cellular functions including calcium signaling, bioenergetics, and apoptosis. Human cytomegalovirus is a medically important herpesvirus whose growth increases energy demand and depends upon continued cell survival. To gain insight into how human cytomegalovirus infection affects endoplasmic reticulum-mitochondrial contacts, we undertook quantitative proteomics of mitochondriaassociated membranes using differential stable isotope labeling by amino acids in cell culture strategy and liquid chromatography-tandem MS analysis. This is the first reported quantitative proteomic analyses of a suborganelle during permissive human cytomegalovirus infection. Human fibroblasts were uninfected or human cytomegalovirus-infected for 72 h. Heavy mitochondria-associated membranes were isolated from paired unlabeled, uninfected cells and stable isotope labeling by amino acids in cell culture-labeled, infected cells and analyzed by liquid chromatography-tandem MS analysis. The results were verified by a reverse labeling experiment. Human cytomegalovirus infection dramatically altered endoplasmic reticulum-mitochondrial contacts by late times. Notable is the increased abundance of several fundamental networks in the mitochondria-associated membrane fraction of human cytomegalovirus-infected fibroblasts. Chaperones, including HSP60 and BiP, which is required for human cytomegalovirus assembly, were prominently increased at endoplasmic reticulum-mitochondrial contacts after infection. Minimal translational and translocation machineries were also associated with endoplasmic reticulum-mitochondrial contacts and increased after human cytomegalovirus infection as were glucose regulated protein 75 and the voltage dependent anion channel, which can form an endoplasmic reticulum-mitochondrial calcium signaling complex. Surprisingly, mitochondrial metabolic enzymes and cytosolic glycolytic enzymes were confidently detected in the mitochondria-associated membrane fraction and increased therein after infection. Finally, proapoptotic regulatory proteins, including Bax, cytochrome c, and Opa1, were augmented in endoplasmic reticulum-mitochondrial contacts after infection, suggesting attenuation of proapoptotic signaling by their increased presence therein. Together, these results suggest that human cytomegalovirus infection restructures

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Identification of Human Cytomegalovirus UL84 Virus- and Cell-Encoded Binding Partners by Using Proteomics Analysis

Cited by 53 publications

References 46 publications

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

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

The Human Cytomegalovirus Gene Products Essential for Late Viral Gene Expression Assemble into Prereplication Complexes before Viral DNA Replication

Quantitative Proteomic Analyses of Human Cytomegalovirus-Induced Restructuring of Endoplasmic Reticulum-Mitochondrial Contacts at Late Times of Infection

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