Defective Growth Correlates with Reduced Accumulation of a Viral DNA Replication Protein after Low-Multiplicity Infection by a Human Cytomegalovirus ie1 Mutant

To investigate the importance of the IE1 p72 regulatory protein during human cytomegalovirus replication, a recombinant virus unable to synthesize IE1 p72 was constructed. The Towne strain mutant CR208 lacked exon 4 of the major immediate-early gene and was isolated and complemented in an IE1-expressing immortalized human fibroblast line (ihfie1.3). Replication of CR208 in primary human fibroblasts was completed after an input multiplicity of 10 PFU/cell but was severely impaired at 0.1 PFU/cell. CR208 formed plaques with lower efficiency on primary fibroblasts than on ihfie1.3 cells, and the relationship between the CR208 inoculum size and the resulting number of undersized plaques was nonlinear, indicating that multiple particles of CR208 were required to initiate lytic replication in a single primary fibroblast. After infection of primary fibroblasts with CR208 at 5 PFU/cell, a normal pattern of viral antigens was detected, although IE1 p72 was absent. During lower-multiplicity infections, IE2 protein was consistently detected at similar levels in a similar proportion of CR208-infected cells relative to the case for a Towne infection, but many fewer CR208-infected cells contained the ppUL44 polymerase accessory protein when evaluated at 24 or 48 h after infection. Furthermore, fibroblasts infected with CR208 at a low multiplicity failed to form viral DNA replication compartments, despite having expressed IE2 p86. These low-multiplicity growth and expression defects were corrected in two rescued derivatives of CR208 able to synthesize IE1 p72. One rescued virus (CR249) carried a deletion removing the large intron between exons 1 and 2 of the ie1-ie2 locus, revealing that this intron was dispensable for growth in cell culture.     

Human cytomegalovirus tegument protein pp71 (ppUL82) enhances the infectivity of viral DNA and accelerates the infectious cycle.

Three tegument proteins of human cytomegalovirus (HCMV), ppUL82 (pp71), pUL69, and ppUL83 (pp65), were examined for the ability to stimulate the production of infectious virus from human diploid fibroblasts transfected with viral DNA. Although viral DNA alone had a low intrinsic infectivity of 3 to 8 plaques/microg of viral DNA, cotransfection of a plasmid expressing pp71 increased the infectivity of HCMV DNA 30- to 80-fold. The increase in infectivity produced by pp71 was reflected in an increased number of nuclei observed to express high levels of the major immediate-early proteins IE1 and IE2. Cotransfection of viral DNA with plasmids directing expression of IE1 and IE2 also resulted in extensive IE1 and IE2 expression in the transfected cells; however, the infectivity of viral DNA was only marginally increased. pp71 also facilitated late gene expression, virus transmission to adjacent cells, and plaque formation. In contrast, expression of pUL69 reduced the pp71- and IE1/IE2-mediated enhancement of HCMV DNA infectivity and also failed to produce any increase in the number of cells expressing IE1 and IE2 over that seen with viral DNA alone. Expression of pp65 did not alter the infectivity of HCMV DNA, nor did it modify the effects of pp71 or pUL69. These results imply that pp71 plays a critical role in the initiation of infection apart from its function as a transactivator of IE1 and IE2.     

Cytomegalovirus-mediated induction of antisense mRNA expression to UL44 inhibits virus replication in an astrocytoma cell line: identification of an essential gene.

We have used an antisense RNA approach in the analysis of gene function in human cytomegalovirus (HCMV). An astrocytoma cell line (U373-MG) that is permissive for virus replication was permanently transfected with a construct bearing sequence from HCMV UL44 (coding for the major late DNA-binding protein, ppUL44, also known as pp52 or ICP36) in an antisense orientation and under the control of the immediate-early enhancer-promoter element. Upon HCMV infection at a high multiplicity, we found a marked reduction in UL44 protein products (the ICP36 family of proteins) in established cell transfectants and a strong inhibition of virus yield in infected-cell supernatants at two weeks postinfection, while herpes simplex virus replication was not affected. In infected cells, viral DNA replication was strongly inhibited. While gene products such as pUS22 and pUL32 were also inhibited, pUL123 and pUL82 accumulated in the infected cells over time. Our data suggest an essential role for the UL44 family of proteins in HCMV replication and represent a model of virus inhibition by virus-induced antisense RNA synthesis in genetically modified cells.     

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.     

Human Cytomegalovirus Protein pUL117 Targets the Mini-Chromosome Maintenance Complex and Suppresses Cellular DNA Synthesis

Modulation of host DNA synthesis is essential for many viruses to establish productive infections and contributes to viral diseases. Human cytomegalovirus (HCMV), a large DNA virus, blocks host DNA synthesis and deregulates cell cycle progression. We report that pUL117, a viral protein that we recently identified, is required for HCMV to block host DNA synthesis. Mutant viruses in which pUL117 was disrupted, either by frame-shift mutation or by a protein destabilization-based approach, failed to block host DNA synthesis at times after 24 hours post infection in human foreskin fibroblasts. Furthermore, pUL117-deficient virus stimulated quiescent fibroblasts to enter S-phase, demonstrating the intrinsic ability of HCMV to promote host DNA synthesis, which was suppressed by pUL117. We examined key proteins known to be involved in inhibition of host DNA synthesis in HCMV infection, and found that many were unlikely involved in the inhibitory activity of pUL117, including geminin, cyclin A, and viral protein IE2, based on their expression patterns. However, the ability of HCMV to delay the accumulation of the mini-chromosome maintenance (MCM) complex proteins, represented by MCM2 and MCM4, and prevent their loading onto chromatin, was compromised in the absence of pUL117. When expressed alone, pUL117 slowed cell proliferation, delayed DNA synthesis, and inhibited MCM accumulation. Knockdown of MCM proteins by siRNA restored the ability of pUL117-deficient virus to block cellular DNA synthesis. Thus, targeting MCM complex is one mechanism pUL117 employs to help block cellular DNA synthesis during HCMV infection. Our finding substantiates an emerging picture that deregulation of MCM is a conserved strategy for many viruses to prevent host DNA synthesis and helps to elucidate the complex strategy used by a large DNA virus to modulate cellular processes to promote infection and pathogenesis.     

Human cytomegalovirus (HCMV) DNA polymerase processivity factor ppUL44 dimerizes in the cytosol before translocation to the nucleus

Replication of the human cytomegalovirus genome takes place in the nuclei of infected cells and is mediated by a viral-encoded DNA polymerase complex formed by the catalytic subunit pUL54 and the processivity factor ppUL44. Although it has recently been shown that the dimerization ability of recombinant pUL44 appears to be crucial for effective DNA binding in vitro, whether ppUL44 can dimerize or not in a cellular context is unknown. Here, we show, by using co-immunoprecipitation and dual-color live imaging approaches on cells expressing fluorescent and differently tagged ppUL44 fusion proteins, that ppUL44 dimerizes in the cytoplasm via its N-terminal domain, before translocating to the nucleus. Furthermore, we show that nuclear translocation of differently tagged ppUL44 heterodimers can occur even when one subunit carries a nonfunctional nuclear localization signal. Importantly, the latter cotransfection assay represents a system to test small-molecule inhibitors for their ability to impair ppUL44 dimerization.     

Localization of Human Cytomegalovirus Structural Proteins to the Nuclear Matrix of Infected Human Fibroblasts

The intranuclear assembly of herpesvirus subviral particles remains an incompletely understood process. Previous studies have described the nuclear localization of capsid and tegument proteins as well as intranuclear tegumentation of capsid-like particles. The temporally and spatially regulated replication of viral DNA suggests that assembly may also be regulated by compartmentalization of structural proteins. We have investigated the intranuclear location of several structural and nonstructural proteins of human cytomegalovirus (HCMV). Tegument components including pp65 (ppUL83) and ppUL69 and capsid components including the major capsid protein (pUL86) and the small capsid protein (pUL48/49) were retained within the nuclear matrix (NM), whereas the immediate-early regulatory proteins IE-1 and IE-2 were present in the soluble nuclear fraction. The association of pp65 with the NM resisted washes with 1 M guanidine hydrochloride, and direct binding to the NM could be demonstrated by far-Western blotting. Furthermore, pp65 exhibited accumulation along the nuclear periphery and in far-Western analysis bound to proteins which comigrated with proteins of the size of nuclear lamins. A direct interaction between pp65 and lamins was demonstrated by coprecipitation of lamins in immune complexes containing pp65. Together, our findings provide evidence that major virion structural proteins localized to a nuclear compartment, the NM, during permissive infection of human fibroblasts.     

Human Cytomegalovirus UL29/28 Protein Interacts with Components of the NuRD Complex Which Promote Accumulation of Immediate-Early RNA

Histone deacetylation plays a pivotal role in regulating human cytomegalovirus gene expression. In this report, we have identified candidate HDAC1-interacting proteins in the context of infection by using a method for rapid immunoisolation of an epitope-tagged protein coupled with mass spectrometry. Putative interactors included multiple human cytomegalovirus-coded proteins. In particular, the interaction of pUL38 and pUL29/28 with HDAC1 was confirmed by reciprocal immunoprecipitations. HDAC1 is present in numerous protein complexes, including the HDAC1-containing nucleosome remodeling and deacetylase protein complex, NuRD. pUL38 and pUL29/28 associated with the MTA2 component of NuRD, and shRNA-mediated knockdown of the RBBP4 and CHD4 constituents of NuRD inhibited HCMV immediate-early RNA and viral DNA accumulation; together this argues that multiple components of the NuRD complex are needed for efficient HCMV replication. Consistent with a positive acting role for the NuRD elements during viral replication, the growth of pUL29/28- or pUL38-deficient viruses could not be rescued by treating infected cells with the deacetylase inhibitor, trichostatin A. Transient expression of pUL29/28 enhanced activity of the HCMV major immediate-early promoter in a reporter assay, regardless of pUL38 expression. Importantly, induction of the major immediate-early reporter activity by pUL29/28 required functional NuRD components, consistent with the inhibition of immediate-early RNA accumulation within infected cells after knockdown of RBBP4 and CHD4. We propose that pUL29/28 modifies the NuRD complex to stimulate the accumulation of immediate-early RNAs.     

The Flexible Loop of the Human Cytomegalovirus DNA Polymerase Processivity Factor ppUL44 Is Required for Efficient DNA Binding and Replication in Cells

Phosphoprotein ppUL44 of the human cytomegalovirus (HCMV) DNA polymerase plays an essential role in viral replication, conferring processivity to the DNA polymerase catalytic subunit pUL54 by tethering it to the DNA. Here, for the first time, we examine in living cells the function of the highly flexible loop of ppUL44 (UL44-FL; residues 162 to 174 [PHTRVKRNVKKAP¹⁷⁴]), which has been proposed to be directly involved in ppUL44''s interaction with DNA. In particular, we use a variety of approaches in transfected cells to characterize in detail the behavior of ppUL44Δloop, a mutant derivative in which three of the five basic residues within UL44-FL are replaced by nonbasic amino acids. Our results indicate that ppUL44Δloop is functional in dimerization and binding to pUL54 but strongly impaired in binding nuclear structures within the nucleus, as shown by its inability to form nuclear speckles, reduced nuclear accumulation, and increased intranuclear mobility compared to wild-type ppUL44. Moreover, analysis of cellular fractions after detergent and DNase treatment indicates that ppUL44Δloop is strongly reduced in DNA-binding ability, in similar fashion to ppUL44-L86A/L87A, a point mutant derivative impaired in dimerization. Finally, ppUL44Δloop fails to transcomplement HCMV oriLyt-dependent DNA replication in cells and also inhibits replication in the presence of wild-type ppUL44, possibly via formation of heterodimers defective for double-stranded DNA binding. UL44-FL thus emerges for the first time as an important determinant for HCMV replication in cells, with potential implications for the development of novel antiviral approaches by targeting HCMV replication.The Betaherpesviridae subfamily member human cytomegalovirus (HCMV) is a major human pathogen, causing serious disease in newborns following congenital infection and in immunocompromised individuals (28, 42). Replication of its double-stranded DNA (dsDNA) genome occurs in the nuclei of infected cells via a rolling-circle process mediated by 11 virally encoded proteins (32, 33), including a viral DNA polymerase holoenzyme, comprising a catalytic subunit, pUL54, and a proposed processivity factor, ppUL44 (14). ppUL44 is readily detectable in virus-infected cells as a 52-kDa phosphoprotein of 433 amino acids with strong dsDNA-binding ability (30, 45). Defined as a “polymerase accessory protein” (PAP) whose function is highly conserved among herpesviruses, ppUL44 is an essential factor for viral replication in cultured cells and hence represents a potential therapeutic target to combat HCMV infection (39). It is a multifunctional protein capable of self-associating (5, 10), as well as interacting with a plethora of viral and host cell proteins, including the viral kinase pUL97 (29), the viral transactivating protein pUL84 (15), the viral uracil DNA glycosylase ppUL114 (37), and the host cell importin α/β (IMPα/β) heterodimer, which is responsible for its transport into the nucleus (4). The activities of ppUL44 as a processivity factor, including the ability to dimerize, as well as bind to, pUL54 and DNA, reside in the N-terminal portion (26, 45), whereas the C terminus is essential for phosphorylation-regulated, IMPα/β-dependent nuclear targeting of ppUL44 monomers and dimers (4-6). Once within the nucleus, ppUL44 is thought to tether the DNA polymerase holoenzyme to the DNA, thus increasing its processivity (14).Recent studies have identified specific residues responsible for ppUL44 interaction with pUL54, as well as for the interaction with IMPα/β and homodimerization (4, 10, 27, 41). The crystal structure of ppUL44''s N-terminal domain (Fig. ​(Fig.1A)1A) reveals striking similarity to that of other processivity factors, such as proliferating cell nuclear antigen (PCNA) and its herpes simplex virus type 1 (HSV-1) homologue UL42 (10, 46). Unlike the PCNA trimeric ring, however, both ppUL44 and UL42, which bind to dsDNA as dimers and monomers, respectively, have an open structure, which is believed to be the basis for their ability to bind to dsDNA in the absence of clamp loaders and ATP (9, 10, 46). Both ppUL44 and UL42 share a very basic “back” face, which appears to be directly involved in DNA binding via electrostatic interactions (19, 22, 23, 38, 46). One striking difference between ppUL44 and UL42 is the presence on the former of an extremely basic flexible loop (UL44-FL, PHTRVKRNVKKAP¹⁷⁴) protruding from the basic back face of the protein (Fig. ​(Fig.1A).1A). Comparison of ppUL44 homologues from different betaherpesviruses, including human herpesvirus 6 (HHV-6) and 7 (HHV-7), showed that all possess similar sequences in the same position (44) (Fig. ​(Fig.1B),1B), implying functional significance.Open in a separate windowFIG. 1.The highly conserved flexible loop (residues 162 to 174) within ppUL44 protrudes from ppUL44 basic face and is important for efficient nuclear accumulation and localization in nuclear speckles. (A) Schematic representation of ppUL44 N-terminal domain (residues 9 to 270, protein data bank accession no. 1T6L) generated using the Chimera software based on the published crystal structure (10, 35). Color: yellow, β-sheets; red, α-helices. Residues involved in ppUL44 dimerization (P85, L86, L87, L93, F121, and M123), as well as basic residues potentially involved in DNA binding (K21, R28, K32, K35, K128, K158, K224, and K237), are represented as spacefill in orange and green, respectively. Residues P162 and C175, in black, are indicated by arrowheads, while residues 163 to 174 are not visible in the electron density maps and could potentially extend in the cavity formed by ppUL44''s basic face to directly contact DNA. Residues forming ppUL44 connector loop (128-142) are in blue. (B) Sequence alignment between HCMVUL44-FL and the corresponding region of several betaherpesvirus ppUL44 homologues. The single-letter amino acid code is used, with basic residues in boldface. (C) COS-7 cells were transfected to express the indicated GFP fusion proteins and imaged live 16 h after transfection using CLSM and a 40× water immersion objective lens. (D) Quantitative results for the Fn/c and speckle formation for GFP-UL44 fusion proteins. The data for the Fn/c ratios represent the mean Fn/c relative to each protein indicated as a percentage of the mean Fn/c relative to GFP-UL44wt ± the standard error of the mean, with the number of analyzed cells in parentheses. (E) HEK 293 cells expressing the indicated GFP-UL44 fusion proteins were lysed, separated by PAGE, and analyzed by Western blotting as described in Materials and Methods, using either the anti-GFP or the anti-α-tubulin MAbs.A recent study revealed that substitution of UL44-FL basic residues with alanine residues strongly impairs the ability of a bacterially expressed N-terminal fragment of UL44 to bind 30-bp dsDNA oligonucleotides in vitro, suggesting that UL44-FL could be involved in dsDNA-binding during viral replication (22). However, the role of UL44-FL in mediating the binding of full-length UL44 to dsDNA in cells and its role in DNA replication have not been investigated. We use here a variety of approaches to delineate the role of UL44-FL in living cells, our data revealing that UL44-FL is not required for ppUL44 dimerization or binding to the catalytic subunit pUL54 but is crucial for HCMV oriLyt-dependent DNA replication, being required for the formation of nuclear aggregates, nuclear accumulation/retention, and DNA binding of ppUL44. Importantly, ppUL44Δloop exhibits a transdominant-negative phenotype, inhibiting HCMV oriLyt-dependent DNA replication in the presence of wild-type ppUL44, possibly via formation of heterodimers defective for dsDNA binding. This underlines ppUL44-FL as an important determinant for HCMV replication in a cellular context for the first time, with potential implications for the development of novel antiviral approaches.     

PUL21a-Cyclin A2 Interaction is Required to Protect Human Cytomegalovirus-Infected Cells from the Deleterious Consequences of Mitotic Entry

Entry into mitosis is accompanied by dramatic changes in cellular architecture, metabolism and gene expression. Many viruses have evolved cell cycle arrest strategies to prevent mitotic entry, presumably to ensure sustained, uninterrupted viral replication. Here we show for human cytomegalovirus (HCMV) what happens if the viral cell cycle arrest mechanism is disabled and cells engaged in viral replication enter into unscheduled mitosis. We made use of an HCMV mutant that, due to a defective Cyclin A2 binding motif in its UL21a gene product (pUL21a), has lost its ability to down-regulate Cyclin A2 and, therefore, to arrest cells at the G1/S transition. Cyclin A2 up-regulation in infected cells not only triggered the onset of cellular DNA synthesis, but also promoted the accumulation and nuclear translocation of Cyclin B1-CDK1, premature chromatin condensation and mitotic entry. The infected cells were able to enter metaphase as shown by nuclear lamina disassembly and, often irregular, metaphase spindle formation. However, anaphase onset was blocked by the still intact anaphase promoting complex/cyclosome (APC/C) inhibitory function of pUL21a. Remarkably, the essential viral IE2, but not the related chromosome-associated IE1 protein, disappeared upon mitotic entry, suggesting an inherent instability of IE2 under mitotic conditions. Viral DNA synthesis was impaired in mitosis, as demonstrated by the abnormal morphology and strongly reduced BrdU incorporation rates of viral replication compartments. The prolonged metaphase arrest in infected cells coincided with precocious sister chromatid separation and progressive fragmentation of the chromosomal material. We conclude that the Cyclin A2-binding function of pUL21a contributes to the maintenance of a cell cycle state conducive for the completion of the HCMV replication cycle. Unscheduled mitotic entry during the course of the HCMV replication has fatal consequences, leading to abortive infection and cell death.     

Cyclin-dependent kinase activity is required for efficient expression and posttranslational modification of human cytomegalovirus proteins and for production of extracellular particles

We have previously shown that the addition of the cyclin-dependent kinase (cdk) inhibitor Roscovitine at the beginning of infection of cells with human cytomegalovirus (HCMV) significantly disrupts immediate-early gene expression and the progression of the infection. In the present study, we have examined the effects of cdk inhibition on late viral events by delaying addition of Roscovitine until 24 h postinfection. Although viral DNA replication was inhibited two- to threefold by treatment of infected cells with Roscovitine, the drop did not correspond to the 1- to 2-log-unit decrease in virus titer. Quantification of viral DNA in the supernatant from cells revealed that there was a significant reduction in the production or release of extracellular particles. We observed a lag in the expression of several viral proteins but there was a significant decrease in the steady-state levels of IE2-86. Likewise, the steady-state level of the essential tegument protein UL32 (pp150) was reduced. The levels of pp150 and IE2-86 mRNA were not greatly affected by treatment with Roscovitine and thus did not correlate with the reduced levels of protein. In contrast, the expression of the tegument protein ppUL69 was higher in drug-treated samples, and the protein accumulated in a hyperphosphorylated form. ppUL69 localized to intranuclear aggregates that did not overlap with viral replication centers in cells treated with Roscovitine. Taken together, these data indicate that cdk activity is required at multiple steps during HCMV infection, including the expression, modification, and localization of virus-encoded proteins.     

Exon 3 of the human cytomegalovirus major immediate-early region is required for efficient viral gene expression and for cellular cyclin modulation

The human cytomegalovirus (HCMV) major immediate-early (IE) proteins share an 85-amino-acid N-terminal domain specified by exons 2 and 3 of the major IE region, UL122-123. We have constructed IE Delta30-77, a recombinant virus that lacks the majority of IE exon 3 and consequently expresses smaller forms of both IE1 72- and IE2 86-kDa proteins. The mutant virus is viable but growth impaired at both high and low multiplicities of infection and exhibits a kinetic defect that is not rescued by growth in fibroblasts expressing IE1 72-kDa protein. The kinetics of mutant IE2 protein accumulation in IE Delta30-77 virus-infected cells are approximately normal compared to wild-type virus-infected cells, but the IE Delta30-77 virus is delayed in expression of early viral genes, including UL112-113 and UL44, and does not sustain expression of mutant IE1 protein as the infection progresses. Additionally, cells infected with IE Delta30-77 exhibit altered expression of cellular proteins compared to wild-type HCMV-infected cells. PML is not dispersed but is retained at ND10 sites following infection with IE Delta30-77 mutant virus. While the deletion mutant retains the ability to mediate the stabilization of cyclin B1, cdc6, and geminin in infected cells, its capacity to upregulate the expression of cyclin E has been reduced. These data indicate that the activity of one or both of the HCMV major IE proteins is required in vivo for the modulation of cell cycle proteins observed in cells infected with wild-type HCMV.     

Interactions between proteins encoded within the human cytomegalovirus UL133-UL138 locus

We previously described a novel genetic locus within the ULb' region of the human cytomegalovirus (HCMV) genome that, while dispensable for replication in fibroblasts, suppresses replication in hematopoietic progenitors and augments replication in endothelial cells. This locus, referred to as the UL133-UL138 locus, encodes four proteins, pUL133, pUL135, pUL136, and pUL138. In this work, we have mapped the interactions among these proteins. An analysis of all pairwise interactions during transient expression revealed a robust interaction between pUL133 and pUL138. Potential interactions between pUL136 and both pUL133 and pUL138 were also revealed. In addition, each of the UL133-UL138 locus proteins self-associated, suggesting a potential to form higher-order homomeric complexes. As both pUL133 and pUL138 function in promoting viral latency in CD34(+) hematopoietic progenitor cells (HPCs) infected in vitro, we further focused on this interaction. pUL133 and pUL138 are the predominant complex detected when all proteins are expressed together and require no other proteins in the locus for their association. During infection, the interaction between pUL133 and pUL138 or pUL136 can be detected. A recombinant virus that fails to express both pUL133 and pUL138 exhibited a latency phenotype similar to that of viruses that fail to express either pUL133 or pUL138, indicating that these proteins function cooperatively in latency and do not have independent functions that additively contribute to HCMV latency. These studies identify protein interactions among proteins encoded by the UL133-UL138 locus and demonstrate an important interaction impacting the outcome of HCMV infection.