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.     

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.     

A Mutation Deleting Sequences Encoding the Amino Terminus of Human Cytomegalovirus UL84 Impairs Interaction with UL44 and Capsid Localization

Protein-protein interactions are required for many biological functions. Previous work has demonstrated an interaction between the human cytomegalovirus DNA polymerase subunit UL44 and the viral replication factor UL84. In this study, glutathione S-transferase pulldown assays indicated that residues 1 to 68 of UL84 are both necessary and sufficient for efficient interaction of UL84 with UL44 in vitro. We created a mutant virus in which sequences encoding these residues were deleted. This mutant displayed decreased virus replication compared to wild-type virus. Immunoprecipitation assays showed that the mutation decreased but did not abrogate association of UL84 with UL44 in infected cell lysate, suggesting that the association in the infected cell can involve other protein-protein interactions. Further immunoprecipitation assays indicated that IRS1, TRS1, and nucleolin are candidates for such interactions in infected cells. Quantitative real-time PCR analysis of viral DNA indicated that the absence of the UL84 amino terminus does not notably affect viral DNA synthesis. Western blotting experiments and pulse labeling of infected cells with [(35)S]methionine demonstrated a rather modest downregulation of levels of multiple proteins and particularly decreased levels of the minor capsid protein UL85. Electron microscopy demonstrated that viral capsids assemble but are mislocalized in nuclei of cells infected with the mutant virus, with fewer cytoplasmic capsids detected. In sum, deletion of the sequences encoding the amino terminus of UL84 affects interaction with UL44 and virus replication unexpectedly, not viral DNA synthesis. Mislocalization of viral capsids in infected cell nuclei likely contributes to the observed decrease in virus replication.     

Identification of an essential domain in the herpes simplex virus 1 UL34 protein that is necessary and sufficient to interact with UL31 protein

Previous results have indicated that the herpes simplex virus 1 UL31 and UL34 proteins interact and form a complex at the inner nuclear membranes of infected cells, where both play important roles in the envelopment of nucleocapsids at the inner nuclear membrane. In the work described here, mapping studies using glutathione S-transferase pull-down assays indicated that amino acids 137 to 181 of the UL34 protein are sufficient to mediate an interaction with the UL31 protein. A recombinant virus (v3480) lacking UL34 codons 138 to 181 was constructed. Similar to a UL34 null virus, v3480 failed to replicate on Vero cells and grew to a limited extent on rabbit skin cells. A UL34-expressing cell line restored v3480 growth and plaque formation. Similar to the localization of UL31 protein in cells infected with a UL34 null virus, the UL31 protein was present in the nuclei of Hep2 cells infected with v3480. Hep2 cells infected with v3480 contained the UL34 protein in the cytoplasm, the nucleus, and the nuclear membrane, and this was noted to be similar to the appearance of cells infected with a UL31 null virus. In transient expression assays, the interaction between UL34 amino acids 137 to 181 and the UL31 protein was sufficiently robust to target green fluorescent protein and emerin to intranuclear sites that contained the UL31 protein. These data indicate that amino acids 137 to 181 of the UL34 protein are (i) sufficient to mediate interactions with the UL31 protein in vitro and in vivo, (ii) necessary for the colocalization of UL31 and UL34 in infected cells, and (iii) essential for normal viral replication.     

Interactions among four proteins encoded by the human cytomegalovirus UL112-113 region regulate their intranuclear targeting and the recruitment of UL44 to prereplication foci

Four phosphoproteins, of 34, 43, 50, and 84 kDa, with common amino termini are synthesized via alternative splicing from the UL112-113 region of the human cytomegalovirus genome. Although genetic studies provided evidence that both the UL112 and UL113 loci in the viral genome are required for efficient viral replication, whether the four proteins play specific roles or cooperate in replication is not understood. Here we present evidence, using in vitro and in vivo coimmunoprecipitation assays, that the four UL112-113 proteins both self-interact and interact with each other. A mapping study of the 84-kDa protein showed that the N-terminal region encompassing amino acids 1 to 125, which is shared in all UL112-113 proteins and highly conserved among betaherpesviruses, is required for both self-interaction and nuclear localization as foci. Further localization studies revealed that, unlike the 43-, 50-, and 84-kDa proteins, which were distributed as nuclear punctate forms, the 34-kDa form was located predominantly in the cytoplasm. However, when all four proteins were coexpressed simultaneously, all of the UL112-113 proteins were efficiently localized to the promyelocytic leukemia oncogenic domains. We also found that the ability of the UL112-113 proteins to relocate UL44 (the viral polymerase processivity factor) to prereplication foci relied on self-interaction and reached maximal levels when the four proteins were coexpressed. Therefore, our data suggest that interactions occurring among UL112-113 proteins via their shared N-terminal regions are important to both their intranuclear targeting and the recruitment of UL44 to subnuclear sites for viral replication.     

Human cytomegalovirus DNA polymerase catalytic subunit pUL54 possesses independently acting nuclear localization and ppUL44 binding motifs

The catalytic subunit of human cytomegalovirus (HCMV) DNA polymerase pUL54 is a 1242-amino-acid protein, whose function, stimulated by the processivity factor, phosphoprotein UL44 (ppUL44), is essential for viral replication. The C-terminal residues (amino acids 1220-1242) of pUL54 have been reported to be sufficient for ppUL44 binding in vitro. Although believed to be important for functioning in the nuclei of infected cells, no data are available on either the interaction of pUL54 with ppUL44 in living mammalian cells or the mechanism of pUL54 nuclear transport and its relationship with that of ppUL44. The present study examines for the first time the nuclear import pathway of pUL54 and its interaction with ppUL44 using dual color, quantitative confocal laser scanning microscopy on live transfected cells and quantitative gel mobility shift assays. We showed that of two nuclear localization signals (NLSs) located at amino acids 1153-1159 (NLSA) and 1222-1227 (NLSB), NLSA is sufficient to confer nuclear localization on green fluorescent protein (GFP) by mediating interaction with importin alpha/beta. We also showed that pUL54 residues 1213-1242 are sufficient to confer ppUL44 binding abilities on GFP and that pUL54 and ppUL44 can be transported to the nucleus as a complex. Our work thus identified distinct sites within the HCMV DNA polymerase, which represent potential therapeutic targets and establishes the molecular basis of UL54 nuclear import.     

A nonconventional nuclear localization signal within the UL84 protein of human cytomegalovirus mediates nuclear import via the importin alpha/beta pathway

The open reading frame UL84 of human cytomegalovirus encodes a multifunctional regulatory protein which is required for viral DNA replication and binds with high affinity to the immediate-early transactivator IE2-p86. Although the exact role of pUL84 in DNA replication is unknown, the nuclear localization of this protein is a prerequisite for this function. To investigate whether the activities of pUL84 are modulated by cellular proteins we used the Saccharomyces cerevisiae two-hybrid system to screen a cDNA-library for interacting proteins. Strong interactions were found between pUL84 and four members of the importin alpha protein family. These interactions could be confirmed in vitro by pull down experiments and in vivo by coimmunoprecipitation analysis from transfected cells. Using in vitro transport assays we showed that the pUL84 nuclear import required importin alpha, importin beta, and Ran, thus following the classical importin-mediated import pathway. Deletion mutagenesis of pUL84 revealed a domain of 282 amino acids which is required for binding to the importin alpha proteins. Its function as a nuclear localization signal (NLS) was confirmed by fusion to heterologous proteins. Although containing a cluster of basic amino acids similar to classical NLSs, this cluster did not contain the NLS activity. Thus, a complex structure appears to be essential for importin alpha binding and import activity.     

Herpes simplex virus 1 protein kinase Us3 and major tegument protein UL47 reciprocally regulate their subcellular localization in infected cells

Us3 is a serine-threonine protein kinase encoded by herpes simplex virus 1 (HSV-1). We have identified UL47, a major virion protein, as a novel physiological substrate of Us3. In vitro kinase assays and systematic analysis of mutations at putative Us3 phosphorylation sites near the nuclear localization signal of UL47 showed that serine at residue 77 (Ser-77) was required for Us3 phosphorylation of UL47. Replacement of UL47 Ser-77 by alanine produced aberrant accumulation of UL47 at the nuclear rim and impaired the nuclear localization of UL47 in a significant fraction of infected cells. The same defect in UL47 localization was produced by an amino acid substitution in Us3 that inactivated its protein kinase activity. In contrast, a phosphomimetic mutation at UL47 Ser-77 restored wild-type nuclear localization. The UL47 S77A mutation also reduced viral replication in the mouse cornea and the development of herpes stromal keratitis in mice. In addition, UL47 formed a stable complex with Us3 in infected cells, and nuclear localization of Us3 was significantly impaired in the absence of UL47. These results suggested that Us3 phosphorylation of UL47 Ser-77 promoted the nuclear localization of UL47 in cell cultures and played a critical role in viral replication and pathogenesis in vivo. Furthermore, UL47 appeared to be required for efficient nuclear localization of Us3 in infected cells. Therefore, Us3 protein kinase and its substrate UL47 demonstrated a unique regulatory feature in that they reciprocally regulated their subcellular localization in infected cells.     

Human cytomegalovirus UL44 concentrates at the periphery of replication compartments, the site of viral DNA synthesis

The formation of replication compartments, the subnuclear structures in which the viral DNA genome is replicated, is a hallmark of herpesvirus infections. The localization of proteins and viral DNA within human cytomegalovirus replication compartments is not well characterized. Immunofluorescence analysis demonstrated the accumulation of the viral DNA polymerase subunit UL44 at the periphery of replication compartments and the presence of different populations of UL44 in infected cells. In contrast, the viral single-stranded-DNA binding protein UL57 was distributed throughout replication compartments. Using "click chemistry" to detect 5-ethynyl-2'-deoxyuridine (EdU) incorporation into replicating viral DNA and pulse-chase protocols, we found that viral DNA synthesis occurs at the periphery of replication compartments and that replicated viral DNA subsequently localizes to the interior of replication compartments. The interiors of replication compartments also contain regions in which UL44 and EdU-labeled DNA are absent. The treatment of cells with a viral DNA polymerase inhibitor reversibly caused the dispersal of both UL44 and EdU-labeled viral DNA from replication compartments, indicating that ongoing viral DNA synthesis is necessary to maintain the organization of replication compartments. Our results reveal a previously unappreciated complexity of the organization of human cytomegalovirus replication compartments.     

Identification of nuclear export signal in UL37 protein of herpes simplex virus type 2

The UL37 gene of herpes simplex virus (HSV) encodes a 120-kDa phosphoprotein associated with the virion. In this study, we have generated a rabbit polyclonal antiserum against HSV-2 UL37 protein, and examined its intracellular localization by immunofluorescence study. In infected cells, specific fluorescence was detectable in the perinuclear region. In transfected cells, UL37 protein was observed mainly in the cytoplasm. Transfection assays of deletion mutants of UL37 protein suggested that the leucine rich region (LRR) containing amino acids 263-273 may be important for cytoplasmic localization. Deletion of the LRR or substitution of the leucine residues resulted in nuclear remaining of UL37 protein. Moreover, the LRR could export green fluorescent protein (GFP) to the cytoplasm as a fusion protein and this export was blocked by leptomycin B treatment, indicating that the LRR acted as a nuclear export signal. These results suggest that UL37 protein fulfills a role as a shuttle between the nucleus and the cytoplasm through the LRR.     

Correct intranuclear localization of herpes simplex virus DNA polymerase requires the viral ICP8 DNA-binding protein.

We used indirect immunofluorescence to examine the factors determining the intranuclear location of herpes simplex virus (HSV) DNA polymerase (Pol) in infected cells. In the absence of viral DNA replication, HSV Pol colocalized with the HSV DNA-binding protein ICP8 in nuclear framework-associated structures called prereplicative sites. In the presence of viral DNA replication, HSV Pol colocalized with ICP8 in globular intranuclear structures called replication compartments. In cells infected with mutant viruses encoding defective ICP8 molecules, Pol localized within the cell nucleus but showed a general diffuse intranuclear distribution. In uninfected cells transfected with a plasmid expressing Pol, Pol similarly showed a diffuse intranuclear distribution. Therefore, Pol can localize to the cell nucleus without other viral proteins, but functional ICP8 is required for Pol to localize to prereplicative sites. In cells infected with mutant viruses encoding defective Pol molecules, ICP8 localized to prereplicative sites. Thus, Pol or the portions of Pol not expressed by the mutant viruses are not essential for the formation of prereplicative sites or the localization of ICP8 to these structures. These results demonstrate that a specific nuclear protein can influence the intranuclear location of another nuclear protein.     

Characterization of human cytomegalovirus uracil DNA glycosylase (UL114) and its interaction with polymerase processivity factor (UL44)

Here, we report the molecular characterization of the human cytomegalovirus uracil DNA glycosylase (UNG) UL114. Purified UL114 was shown to be a DNA glycosylase, which removes uracil from double-stranded and single-stranded DNA. However, kinetic analysis has shown that viral UNG removed uracil more slowly compared with the core form of human UNG (Δ84hUNG), which has a catalytic efficiency (k_cat/K_M) 350- to 650-fold higher than that of UL114. Furthermore, UL114 showed a maximum level of DNA glycosylase activity at equimolar concentrations of the viral polymerase processivity factor UL44. Next, UL114 was coprecipitated with DNA immobilized to magnetic beads only in the presence of UL44, suggesting that UL44 facilitated the loading of UL114 on DNA. Moreover, mutant analysis demonstrated that the C-terminal part of UL44 (residues 291-433) is important for the interplay with UL114. Immunofluorescence microscopy revealed that UL44 and UL114 colocalized in numerous small punctuate foci at the immediate-early (5 and 8 hpi) phases of infection and that these foci grew in size throughout the infection. Furthermore, coimmunoprecipitation assays with cellular extracts of infected cells confirmed that UL44 associated with UL114. Finally, the nuclear concentration of UL114 was estimated to be 5- to 10-fold higher than that of UL44 in infected cells, which indicated a UL44-independent role of UL114. In summary, our data have demonstrated a catalytically inefficient viral UNG that was highly enriched in viral replication foci, thus supporting an important role of UL114 in replication rather than repair of the viral genome.     

Interaction Network of Proteins Associated with Human Cytomegalovirus IE2-p86 Protein during Infection: A Proteomic Analysis

Human cytomegalovirus protein IE2-p86 exerts its functions through interaction with other viral and cellular proteins. To further delineate its protein interaction network, we generated a recombinant virus expressing SG-tagged IE2-p86 and used tandem affinity purification coupled with mass spectrometry. A total of 9 viral proteins and 75 cellular proteins were found to associate with IE2-p86 protein during the first 48 hours of infection. The protein profile at 8, 24, and 48 h post infection revealed that UL84 tightly associated with IE2-p86, and more viral and cellular proteins came into association with IE2-p86 with the progression of virus infection. A computational analysis of the protein-protein interaction network indicated that all of the 9 viral proteins and most of the cellular proteins identified in the study are interconnected to varying degrees. Of the cellular proteins that were confirmed to associate with IE2-p86 by immunoprecipitation, C1QBP was further shown to be upregulated by HCMV infection and colocalized with IE2-p86, UL84 and UL44 in the virus replication compartment of the nucleus. The IE2-p86 interactome network demonstrated the temporal development of stable and abundant protein complexes that associate with IE2-p86 and provided a framework to benefit future studies of various protein complexes during HCMV infection.     

Characterization of nuclear structures in cells infected with herpes simplex virus type 1 in the absence of viral DNA replication.

Herpes simplex virus type 1 DNA replication occurs in nuclear domains termed replication compartments, which are areas of viral single-stranded DNA-binding protein (UL29) localization (M.P. Quinlan, L. B. Chen, and D. M. Knipe, Cell 36:857-868). In the presence of herpesvirus-specific polymerase inhibitors, UL29 localizes to punctate nuclear foci called prereplicative sites. Using versions of the helicase-primase complex proteins containing short peptide epitopes which can be detected in an immunofluorescence assay, we have found that the helicase-primase complex localizes to prereplicative sites and replication compartments. To determine if prereplicative site formation is dependent upon these and other essential viral replication proteins, we have studied UL29 localization in cells infected with replication-defective viruses. Cells infected with viruses that fail to express one of the three helicase-primase subunits or the origin-binding protein show a diffuse nuclear staining for UL29. However, in the presence of polymerase inhibitors, mutant-infected cells contain UL29 in prereplicative sites. Replication-defective viruses containing subtle mutations in the helicase or origin-binding proteins behaved identically to their null mutant counterparts. In contrast, cells infected with viral mutants which fail to express the polymerase protein contain prereplicative sites in the absence and presence of polymerase inhibitors. We propose that active viral polymerase prevents the formation of prereplicative sites. Models of the requirement of essential viral replication proteins in the assembly of prereplicative sites are presented.     

A protein kinase CK2 site flanking the nuclear targeting signal enhances nuclear transport of human cytomegalovirus ppUL44

The processivity factor of the human cytomegalovirus (HCMV) DNA polymerase phosphoprotein ppUL44 plays an essential role in viral replication, showing nuclear localization in infected cells. The present study examines ppUL44's nuclear import pathway for the first time, ectopic expression of ppUL44 revealing a strong nuclear localization in transfected COS-7 and other cell types, implying that no other HCMV proteins are required for nuclear transportation and retention. We show that of the two potential nuclear localization signals (NLSs) located at amino acids 162-168 (NLS1) and 425-431 (NLS2), NLS2 is necessary and sufficient to confer nuclear localization. Moreover, using enzyme-linked immunosorbent assays and gel mobility shift assays, we show that NLS2 is recognized with high affinity by the importin (IMP) alpha/beta heterodimer. Using gel mobility shift and transient transfection assays, we find that flanking sequences containing a cluster of potential phosphorylation sites, including a consensus site for protein kinase CK2 (CK2) at Ser413 upstream of the NLS, increase NLS2-dependent IMP binding and nuclear localization, suggesting a role for these sites in enhancing UL44 nuclear transport. Results from site-directed mutagenic analysis and live-cell imaging of green fluorescent protein (GFP)-UL44 fusion protein-expressing cells treated with the CK2-specific inhibitor 4,5,6,7-tetrabromobenzotriazole are consistent with phosphorylation of Ser413 enhancing ppUL44 nuclear transport.     

Functional order of assembly of herpes simplex virus DNA replication proteins into prereplicative site structures.

Herpes simplex virus replicates its DNA within nuclear structures called replication compartments. In contrast, in cells in which viral DNA replication is inhibited, viral replication proteins localize to punctate structures called prereplicative sites. We have utilized viruses individually mutated in each of the seven essential replication genes to assess the function of each replication protein in the assembly of these proteins into prereplicative sites. We observed that four replication proteins, UL5, UL8 UL52, and UL9, are necessary for the localization of ICP8 (UL29) to prereplicative sites natural infection conditions. Likewise, four of the seven viral DNA replication proteins, UL5, UL52, UL9, and ICP8, are necessary for the localization of the viral DNA polymerase to prereplicative sites. On the basis of these results, we present a model for prereplicative site formation in infected cells in which the helicase-primase components (UL5, UL8, and UL52), the origin-binding protein (UL9), and the viral single-stranded DNA-binding protein (ICP8) assemble together to initiate the process. This is followed by the recruitment of the viral polymerase into the structures, a step facilitated by the polymerase accessory protein, UL42. Host cell factors can apparently substitute for some of these viral proteins under certain conditions, because the viral protein requirements for prereplicative site formation are reduced in transfected cells and in infected cells treated with drugs that inhibit DNA synthesis.