Host protein Snapin interacts with human cytomegalovirus pUL130 and affects viral DNA replication

The interplay between the host and Human cytomegalovirus (HCMV) plays a pivotal role in the outcome of an infection. HCMV growth in endothelial and epithelial cells requires expression of viral proteins UL128, UL130, and UL131 proteins (UL128-131), of which UL130 is the largest gene and the only one that is not interrupted by introns. Mutation of the C terminus of the UL130 protein causes reduced tropism of endothelial cells (EC). However, very few host factors have been identified that interact with the UL130 protein. In this study, HCMV UL130 protein was shown to directly interact with the human protein Snapin in human embryonic kidney HEK293 cells by Yeast two-hybrid screening, in vitro glutathione S-transferase (GST) pull-down, and co-immunoprecipitation. Additionally, heterologous expression of protein UL130 revealed co-localization with Snapin in the cell membrane and cytoplasm of HEK293 cells using fluorescence confocal microscopy. Furthermore, decreasing the level of Snapin via specific small interfering RNAs decreased the number of viral DNA copies and titer in HCMV-infected U373-S cells. Taken together, these results suggest that Snapin, the pUL130 interacting protein, has a role in modulating HCMV DNA synthesis.     

The Cellular Protein MCM3AP Is Required for Inhibition of Cellular DNA Synthesis by the IE86 Protein of Human Cytomegalovirus

Like all DNA viruses, human cytomegalovirus (HCMV) infection is known to result in profound effects on host cell cycle. Infection of fibroblasts with HCMV is known to induce an advance in cell cycle through the G₀-G₁ phase and then a subsequent arrest of cell cycle in early S-phase, presumably resulting in a cellular environment optimum for high levels of viral DNA replication whilst precluding replication of cellular DNA. Although the exact mechanisms used to arrest cell cycle by HCMV are unclear, they likely involve a number of viral gene products and evidence points to the ability of the virus to prevent licensing of cellular DNA synthesis. One viral protein known to profoundly alter cell cycle is the viral immediate early 86 (IE86) protein - an established function of which is to initially drive cells into early S phase but then inhibit cellular DNA synthesis. Here we show that, although IE86 interacts with the cellular licensing factor Cdt1, it does not inhibit licensing of cellular origins. Instead, IE86-mediated inhibition of cellular DNA synthesis requires mini-chromosome-maintenance 3 (MCM3) associated protein (MCM3AP), which can cause subsequent inhibition of initiation of cellular DNA synthesis in a licensing-independent manner.     

Induction of alpha type DNA polymerases in human cytomegalovirus-infected WI-38 cells.

Productive infection of WI-38 cells with human cytomegalovirus (HCMV) induced the increase in the activity of DNA polymerases as well as the synthesis of viral and cellular DNA. Sedimentation analyses in sucrose gradients of high ionic strength showed that the HCMV infection caused marked increase in the activity of alpha-type polymerases (resolved into alpha1, 8 S, and alpha 2, 6 S, in the present experiments), while the infection little affected the level of beta-type polymerase (about 3.5 S) activity in both the nuclei and cytoplasm. Such increase in alpha-type polymerases was also observed when DNA synthesis in WI-38 cells was enhanced by SV40 infection or by an increased concentration of serum in medium. Phosphonacetate, which selectively blocked the synthesis of HCMV DNA, did not significantly affect the HCMV-mediated induction of DNA polymerases. However, phosphonoacetate added in the reaction mixture for DNA polymerase assay inhibited the activity of the HCMV-induced polyperase alpha, but not of the polymerases alpha2 and beta. These results support the idea that alpha-type polymerases are involved in the replicative synthesis of cellular and viral DNA.     

The essential human cytomegalovirus gene UL52 is required for cleavage-packaging of the viral genome

Replication of human cytomegalovirus (HCMV) produces large DNA concatemers of head-to-tail-linked viral genomes that upon packaging into capsids are cut into unit-length genomes. The mechanisms underlying cleavage-packaging and the subsequent steps prior to nuclear egress of DNA-filled capsids are incompletely understood. The hitherto uncharacterized product of the essential HCMV UL52 gene was proposed to participate in these processes. To investigate the function of pUL52, we constructed a ΔUL52 mutant as well as a complementing cell line. We found that replication of viral DNA was not impaired in noncomplementing cells infected with the ΔUL52 virus, but viral concatemers remained uncleaved. Since the subnuclear localization of the known cleavage-packaging proteins pUL56, pUL89, and pUL104 was unchanged in ΔUL52-infected fibroblasts, pUL52 does not seem to act via these proteins. Electron microscopy studies revealed only B capsids in the nuclei of ΔUL52-infected cells, indicating that the mutant virus has a defect in encapsidation of viral DNA. Generation of recombinant HCMV genomes encoding epitope-tagged pUL52 versions showed that only the N-terminally tagged pUL52 supported viral growth, suggesting that the C terminus is crucial for its function. pUL52 was expressed as a 75-kDa protein with true late kinetics. It localized preferentially to the nuclei of infected cells and was found to enclose the replication compartments. Taken together, our results demonstrate an essential role for pUL52 in cleavage-packaging of HCMV DNA. Given its unique subnuclear localization, the function of pUL52 might be distinct from that of other cleavage-packaging proteins.     

Human cytomegalovirus UL84 oligomerization and heterodimerization domains act as transdominant inhibitors of oriLyt-dependent DNA replication: evidence that IE2-UL84 and UL84-UL84 interactions are required for lytic DNA replication

Human cytomegalovirus (HCMV) UL84 encodes a 75-kDa protein required for oriLyt-dependent DNA replication and interacts with IE2 in infected and transfected cells. UL84 localizes to the nucleus of transfected and infected cells and is found in viral replication compartments. In transient assays it was shown that UL84 can interfere with the IE2-mediated transactivation of the UL112/113 promoter of HCMV. To determine whether UL84 protein-protein interactions are necessary for lytic DNA synthesis, we purified UL84 and used this protein to generate a monoclonal antibody. Using this antibody, we now show that UL84 forms a stable interaction with itself in vivo. The point of self-interaction maps to a region of the protein between amino acids 151 and 200, a domain that contains a series of highly charged amino acid residues. Coimmunoprecipitation assays determined that UL84 interacts with a protein domain present within the first 215 amino acids of IE2. We also show that an intact leucine zipper domain of UL84 is required for a stable interaction with IE2 and UL84 leucine zipper mutants fail to complement oriLyt-dependent DNA replication. UL84 leucine zipper mutants no longer interfere with IE2-mediated transactivation of the UL112/113 promoter, confirming that the leucine zipper is essential for a functional interaction with IE2. In addition, we demonstrate that both the leucine zipper and oligomerization domains of UL84 can act as transdominant-negative inhibitors of lytic replication in the transient assay, strongly suggesting that both an IE2-UL84 and a UL84-UL84 interaction are required for DNA synthesis.     

Infection-dependent nuclear localization of US17, a member of the US12 family of human cytomegalovirus-encoded seven-transmembrane proteins

The human cytomegalovirus (HCMV) US12 gene family is a group of predicted seven-transmembrane, G-protein-coupled receptor-related proteins, about which little is known. Specific rabbit polyclonal antibodies detected US17 and US18 beginning 54 and 36 h after infection, respectively, with expression of both proteins dependent on viral DNA synthesis. While US14 and US18 are expressed exclusively in the cytoplasm, we unexpectedly found abundant expression of US17 in both the cytoplasm and nucleoplasm. N- and C-terminally tagged versions of US17 were readily detected in the cytoplasm of transfected mammalian cells, but not in nuclei, suggesting that nuclear localization involves other viral proteins or an infection-triggered cellular process. There was no specific colocalization between US17 and other nuclear expressed HCMV-encoded proteins (IE-2, DNA polymerase processivity factor, and pp28/UL99). To determine whether the observed nuclear localization might be the product of a process by which a soluble C-terminal segment of the full-length protein is expressed, we constructed a recombinant virus that incorporates a synthetic epitope at its N terminus, which in conjunction with the antipeptide antibody that targets its predicted cytoplasmic C-terminal segment, enables simultaneous independent detection of both termini. In cells infected with the recombinant, the US17 N and C termini had limited colocalization, with the N-terminal segment not detected in nuclei, supporting the segmentation hypothesis. Consistent with this, a fragment with an apparent molecular size of 10 kDa was detected by immunoblotting. We have identified the first viral example of a seven-transmembrane protein that is either segmented or expressed in nuclei. Further study will be required to learn the mechanism by which this occurs and the function of the nuclear localizing segment. This likely represents yet another mechanism by which a virus has hijacked or modified cellular regulatory pathways for its benefit.     

A Novel DDB2-ATM Feedback Loop Regulates Human Cytomegalovirus Replication

Human cytomegalovirus (HCMV) genome replication requires host DNA damage responses (DDRs) and raises the possibility that DNA repair pathways may influence viral replication. We report here that a nucleotide excision repair (NER)-associated-factor is required for efficient HCMV DNA replication. Mutations in genes encoding NER factors are associated with xeroderma pigmentosum (XP). One of the XP complementation groups, XPE, involves mutation in ddb2, which encodes DNA damage binding protein 2 (DDB2). Infectious progeny virus production was reduced by >2 logs in XPE fibroblasts compared to levels in normal fibroblasts. The levels of immediate early (IE) (IE2), early (E) (pp65), and early/late (E/L) (gB55) proteins were decreased in XPE cells. These replication defects were rescued by infection with a retrovirus expressing DDB2 cDNA. Similar patterns of reduced viral gene expression and progeny virus production were also observed in normal fibroblasts that were depleted for DDB2 by RNA interference (RNAi). Mature replication compartments (RCs) were nearly absent in XPE cells, and there were 1.5- to 2.0-log reductions in viral DNA loads in infected XPE cells relative to those in normal fibroblasts. The expression of viral genes (UL122, UL44, UL54, UL55, and UL84) affected by DDB2 status was also sensitive to a viral DNA replication inhibitor, phosphonoacetic acid (PAA), suggesting that DDB2 affects gene expression upstream of or events associated with the initiation of DNA replication. Finally, a novel, infection-associated feedback loop between DDB2 and ataxia telangiectasia mutated (ATM) was observed in infected cells. Together, these results demonstrate that DDB2 and a DDB2-ATM feedback loop influence HCMV replication.     

The Human Cytomegalovirus DNA Polymerase Processivity Factor UL44 Is Modified by SUMO in a DNA-Dependent Manner

During the replication of human cytomegalovirus (HCMV) genome, the viral DNA polymerase subunit UL44 plays a key role, as by binding both DNA and the polymerase catalytic subunit it confers processivity to the holoenzyme. However, several lines of evidence suggest that UL44 might have additional roles during virus life cycle. To shed light on this, we searched for cellular partners of UL44 by yeast two-hybrid screenings. Intriguingly, we discovered the interaction of UL44 with Ubc9, an enzyme involved in the covalent conjugation of SUMO (Small Ubiquitin-related MOdifier) to cellular and viral proteins. We found that UL44 can be extensively sumoylated not only in a cell-free system and in transfected cells, but also in HCMV-infected cells, in which about 50% of the protein resulted to be modified at late times post-infection, when viral genome replication is accomplished. Mass spectrometry studies revealed that UL44 possesses multiple SUMO target sites, located throughout the protein. Remarkably, we observed that binding of UL44 to DNA greatly stimulates its sumoylation both in vitro and in vivo. In addition, we showed that overexpression of SUMO alters the intranuclear distribution of UL44 in HCMV-infected cells, and enhances both virus production and DNA replication, arguing for an important role for sumoylation in HCMV life cycle and UL44 function(s). These data report for the first time the sumoylation of a viral processivity factor and show that there is a functional interplay between the HCMV UL44 protein and the cellular sumoylation system.     

Human cytomegalovirus UL84 insertion mutant defective for viral DNA synthesis and growth

Human cytomegalovirus (HCMV) UL84 is required for oriLyt-dependent DNA replication, and evidence from transient transfection assays suggests that UL84 directly participates in DNA synthesis. In addition, because of its apparent interaction with IE2, UL84 is implicated as a possible regulatory protein. To address the role of UL84 in the context of the viral genome, we generated a recombinant HCMV bacterial artificial chromosome (BAC) construct that did not express the UL84 gene product. This construct, BAC-IN84/Ep, displayed a null phenotype in that it failed to produce infectious virus after transfection into human fibroblast cells, whereas a revertant virus readily produced viral plaques and, subsequently, infectious virus. Real-time quantitative PCR showed that BAC-IN84/Ep was defective for DNA synthesis in that no increase in the accumulation of viral DNA was observed in transfected cells. We were unable to complement BAC-IN84/Ep in trans; however, oriLyt-dependent DNA replication was observed by the cotransfection of UL84 and BAC-IN84/Ep. An analysis of viral mRNA by real-time PCR indicated that, even in the absence of DNA synthesis, all representative kinetic classes of genes were expressed in cells transfected with BAC-IN84/Ep. The detection of UL44 and IE2 by immunofluorescence in BAC-IN84/Ep-transfected cells showed that these proteins failed to partition into replication compartments, indicating that UL84 expression is essential for the formation of these proteins into replication centers within the context of the viral genome. These results show that UL84 provides an essential DNA replication function and influences the subcellular localization of other viral proteins.     

Down-regulation of human cytomegalovirus UL138, a novel latency-associated determinant, by hcmv-miR-UL36

MicroRNAs (miRNAs) are small RNAs, 19–23 nucleotides in length, which regulate a variety of cellular processes. Human cytomegalovirus (HCMV) encodes only one intronic miRNA: human cytomegalovirus microRNA UL36 (hcmv-miR-UL36). In this study, we found that over-expression of hcmv-miR-UL36 resulted in a more than threefold increase in HCMV DNA synthesis at 24 h post infection. Fifteen putative targets of hcmv-miR-UL36 were identified using hybrid PCR, one being the HCMV UL138 gene that has previously been identified as a novel latency-associated determinant of HCMV infection. Down-regulation of UL138 expression by hcmv-miR-UL36 was validated using luciferase reporter assays and Western blot analysis in HEK293 cells. In the presence of hcmv-miR-UL36, we observed a 74.6% decrease in luciferase activity and a 46.2% decrease in HCMV UL138 protein expression. Our results indicate that hcmv-miR-UL36 may be a viral miRNA contributing to HCMV replication.     

Human cytomegalovirus labeled with green fluorescent protein for live analysis of intracellular particle movements

Human cytomegalovirus (HCMV) replicates in the nuclei of infected cells. Successful replication therefore depends on particle movements between the cell cortex and nucleus during entry and egress. To visualize HCMV particles in living cells, we have generated a recombinant HCMV expressing enhanced green fluorescent protein (EGFP) fused to the C terminus of the capsid-associated tegument protein pUL32 (pp150). The resulting UL32-EGFP-HCMV was analyzed by immunofluorescence, electron microscopy, immunoblotting, confocal microscopy, and time-lapse microscopy to evaluate the growth properties of this virus and the dynamics of particle movements. UL32-EGFP-HCMV replicated similarly to wild-type virus in fibroblast cultures. Green fluorescent virus particles were released from infected cells. The fluorescence stayed associated with particles during viral entry, and fluorescent progeny particles appeared in the nucleus at 44 h after infection. Surprisingly, strict colocalization of pUL32 and the major capsid protein pUL86 within nuclear inclusions indicated that incorporation of pUL32 into nascent HCMV particles occurred simultaneously with or immediately after assembly of the capsid. A slow transport of nuclear particles towards the nuclear margin was demonstrated. Within the cytoplasm, most particles performed irregular short-distance movements, while a smaller fraction of particles performed centripetal and centrifugal long-distance movements. Although numerous particles accumulated in the cytoplasm, release of particles from infected cells was a rare event, consistent with a release rate of about 1 infectious unit per h per cell in HCMV-infected fibroblasts as calculated from single-step growth curves. UL32-EGFP-HCMV will be useful for further investigations into the entry, maturation, and release of this virus.     

Human cytomegalovirus UL84 is a phosphoprotein that exhibits UTPase activity and is a putative member of the DExD/H box family of proteins

Human cytomegalovirus (HCMV) UL84 is required for lytic DNA replication and is proposed to be the key factor in initiation of viral DNA synthesis. We now show that UL84 has a high degree of homology to the DExD/H (where x can be any amino acid) box family of helicases, displays UTPase activity, and is phosphorylated at serine residues. Affinity column-purified UL84-FLAG fusion protein was used in an in vitro nucleoside triphosphatase (NTPase) assay to show that UL84 has NTPase activity, preferring UTP. This UTPase activity was linear with respect to enzyme concentration and slightly enhanced by the addition of nucleic acid substrates. UL84 UTPase was the highest at low salt concentrations, a pH of 7.5, and a temperature of 45 degrees C. The enzyme preferred Mg2+ as the divalent cation but was also able to catalyze the UTPase reaction in the presence of Mn2+, Ca2+, and Zn2+ albeit at lower levels. The evidence presented here suggests that the UL84 UTPase activity may be part of an energy-generating system for helicase activity associated with the initiation of HCMV DNA replication.