Glycoprotein H (gH, UL75) of human cytomegalovirus (HCMV) is an essential envelope glycoprotein that functions in viral entry and the activation of gene expression. To understand the regulation of this important viral gene, the promoter of the UL75 late gene was characterized in HCMV-infected cells at the late stages of viral infection. Primer extension analysis revealed a single major start site located 26 bp downstream of a putative TATA element. Deletion analysis showed the presence of a dominant activation domain from +14 to +35 that masked regulatory sequences upstream of the TATA element. Mutational analysis demonstrated that a PEA3-like element in this downstream domain was important for promoter activation. In addition, gel shift analysis revealed direct protein binding to the PEA3-like element. Together, these studies reveal that the gH promoter is regulated in a complex manner with sequences both upstream and downstream of the cap site influencing promoter activation.

Cytomegaloviruses likely encode numerous gene products involved in regulating virus–host cell interactions and pathogenesis. We previously identified a region of murine cytomegalovirus (MCMV) within HindIII-J and -I that regulates pathogenesis of the virus [open reading frames (ORFs) M139–M141] or is likely required for MCMV replication (ORFs m142 and m143). As a prerequisite for further studies on the structure and function of this gene region, we mapped the transcripts encoded within MCMV HindIII-I. Probes for ORFs M140 and M141 hybridized to 5.4- and 7.0-kb RNA, respectively, which were transcribed with early kinetics and were 3′ coterminal with HindIII-J ORF M139. Probes representing ORFs m142, m143, or m144 hybridized to 3′ coterminal transcripts of 1.8, 3.8, and 5.1 kb, respectively. ORFs m142 and m143 were transcribed with immediate-early kinetics but were most abundantly expressed at early times. Probes for the rightmost end of HindIII-I hybridized to a 5.1-kb early/late RNA corresponding to m144 and to a 1.8-kb early RNA transcribed from m145. All of the major transcripts were polyadenylated and therefore are likely coding. Additional minor transcripts of intermediate sizes were also detected. ORFs M139–m143 showed homology to the betaherpesvirus-specific HCMV US22 gene family. Because deletion of these viral genes results in attenuated or helper-dependent phenotypes, this conserved region of US22 family genes may have a role in virus replication as well as in the pathogenesis of betaherpesviruses in their natural hosts.

ABSTRACT Blood monocytes or tissue macrophages play a pivotal role in the pathogenesis of murine cytomegalovirus (MCMV) infection, providing functions beneficial to both the virus and the host. In vitro and in vivo studies have indicated that differentiated macrophages support MCMV replication, are target cells for MCMV infection within tissues, and harbor latent MCMV DNA. However, this cell type presumably initiates early, antiviral immune responses as well. In addressing this paradoxical role of macrophages, we provide evidence that the proficiency of MCMV replication in macrophages positively correlates with virulence in vivo. An MCMV mutant from which the open reading frames M139, M140, and M141 had been deleted (RV10) was defective in its ability to replicate in macrophages in vitro and was highly attenuated for growth in vivo. However, depletion of splenic macrophages significantly enhanced, rather than deterred, replication of both wild-type (WT) virus and RV10 in the spleen. The ability of RV10 to replicate in intact or macrophage-depleted spleens was independent of cytokine production, as this mutant virus was a poor inducer of cytokines compared to WT virus in both intact organs and macrophage-depleted organs. Macrophages were, however, a major contributor to the production of tumor necrosis factor alpha and gamma interferon in response to WT virus infection. Thus, the data indicate that tissue macrophages serve a net protective role and may function as “filters” in protecting other highly permissive cell types from MCMV infection. The magnitude of virus replication in tissue macrophages may dictate the amount of virus accessible to the other cells. Concomitantly, infection of this cell type initiates the production of antiviral immune responses to guarantee efficient clearance of acute MCMV infection.

ABSTRACT We used a live attenuated murine cytomegalovirus (MCMV) mutant to analyze mechanisms of vaccination against acute and latent CMV infection. We selected MCMV mutant RV7 as a vaccine candidate since this virus grows well in tissue culture but is profoundly attenuated for growth in normal and severe combined immunodeficient (SCID) mice (V. J. Cavanaugh et al., J. Virol. 70:1365–1374, 1996). BALB/c mice were immunized twice (0 and 14 days) subcutaneously (s.c.) with tissue culture-passaged RV7 and then challenged with salivary gland-passaged wild-type MCMV (sgMCMV) intraperitoneally (i.p.) on day 28. RV7 vaccination protected mice against challenge with 105 PFU of sgMCMV, a dose that killed 100% of mock-vaccinated mice. RV7 vaccination reduced MCMV replication 100- to 500-fold in the spleen between 1 and 8 days after challenge. We used the capacity to control replication of MCMV in the spleen 4 days after challenge as a surrogate for protection. Protection was antigen specific and required both live RV7 and antigen-specific lymphocytes. Interestingly, RV7 was effective when administered s.c., i.p., perorally, intranasally, and intragastrically, demonstrating that attenuated CMV applied to mucosal surfaces can elicit protection against parenteral virus challenge. B cells and immunoglobulin G were not essential for RV7-induced immunity since B-cell-deficient mice were effectively vaccinated by RV7. CD8 T cells, but not CD4 T cells, were critical for RV7-induced protection. Depletion of CD8 T cells by passive transfer of monoclonal anti-CD8 (but not anti-CD4) antibody abrogated RV7-mediated protection, and RV7 vaccination was less efficient in CD8 T-cell-deficient mice with a targeted mutation in the β2-microglobulin gene. Although gamma interferon is important for innate resistance to MCMV, it was not essential for RV7 vaccination since gamma interferon receptor-deficient mice were protected by RV7 vaccination. Establishment of and/or reactivation from latency by sgMCMV was decreased by RV7 vaccination, as measured by diminished reactivation of MCMV from splenic explants. We found no evidence for establishment of splenic latency by RV7 after s.c. vaccination. We conclude that RV7 administered through both systemic and mucosal routes is an effective vaccine against MCMV infection. It may be possible to design human CMV vaccines with similar properties.

Previous analysis of the human cytomegalovirus (HCMV) DNA polymerase (UL54) early gene promoter demonstrated that transcriptional activation of this gene is dependent upon the interaction of cellular transcription factors with viral transactivators (J. A. Kerry, M. A. Priddy, T. Y. Jervey, C. P. Kohler, T. L. Staley, C. D. Vanson, T. R. Jones, A. C. Iskenderian, D. G. Anders, and R. M. Stenberg, J. Virol. 70:373-382, 1996). A sequence element, IR1, was shown to be the primary regulatory element of this promoter in transient assays. However, assessment of this element in the context of the viral genome revealed IR1-independent activation at late times after infection. To extend these studies, we aim to identify additional sequence elements involved in the activation of the UL54 promoter. Our present studies demonstrate that the level of binding of proteins to the ATF site in the UL54 promoter is enhanced by viral infection. Furthermore this increase is sensitive to treatment with phosphonoacetic acid (PAA), a DNA synthesis inhibitor. These data suggest that the increase in the level of ATF binding activity is regulated, either directly or indirectly, by HCMV late gene expression. By using specific antibodies, we determined that ATF-1 was a major component of the proteins binding to the UL54 ATF site at late times. In addition, we have demonstrated direct binding of recombinant ATF-1 to the UL54 ATF site. To assess the biological significance of these events, a recombinant virus construct was generated that contained the UL54 promoter with a mutation in the ATF site regulating expression of the chloramphenicol acetyltransferase (CAT) reporter gene inserted between open reading frames US9 and US10. Analysis of this virus (RVATFmCAT) revealed that mutation of the ATF site does not alter the kinetics of UL54 promoter activation. However, levels of CAT mRNA and activity were reduced by 5- to 10-fold compared to those of the wild-type promoter at all stages of infection. These findings indicate that ATF-1 can regulate the levels of UL54 promoter activity at both early and late times. Furthermore, these results imply that HCMV can regulate the activity of cellular factors involved in early gene regulation.

The pp28 (UL99) gene of human cytomegalovirus is expressed as a true late gene, in that DNA synthesis is absolutely required for mRNA expression. Our previous studies demonstrated that pp28 promoter sequences from position -40 to +106 are sufficient for late gene expression in the context of the viral genome (C. P. Kohler, J. A. Kerry, M. Carter, V. P. Muzithras, T. R. Jones, and R. M. Stenberg, J. Virol. 68:6589-6597, 1994). To extend these studies, we have examined the sequences in the downstream leader region of the pp28 gene for their role in late gene expression. Deletion of sequences from position -6 to +46 (deltaSS) results in a threefold increase in gene expression in transient assays. In contrast, deletion of sequences from position +46 to +88 (deltaA) has little effect on gene expression. These results indicate that the sequences from position -6 to +46 may repress gene expression. To further analyze this region, site-directed mutagenesis was performed. Mutation of residues from either position +1 to +6 (SS1) or position +12 to +17 (SS2) duplicated the effect of the deltaSS deletion mutant, indicating that sequences from position +1 to +17 were important for the inhibitory effect. To assess the biological significance of these events, a recombinant virus construct containing the deltaSS mutant promoter regulating expression of the chloramphenicol acetyltransferase (CAT) reporter gene was generated. Analysis of this virus (RV delta SSCAT) revealed that deletion of sequences from position -6 to +46 does not alter the kinetic class of this promoter. However, the ratio of CAT protein to CAT mRNA levels in RV delta SSCAT-infected cells was 8- to 12-fold higher than that observed in the parental RV24/26CAT-infected cells. These results imply that the leader sequences within the pp28 gene can regulate the translation of this late gene.

The development of an induced transcript environment was investigated at the supramolecular level through comparative localization of the human cytomegalovirus immediate early (IE) transcripts and specific nuclear domains shortly after infection. Compact aggregates of IE transcripts form only adjacent to nuclear domain 10 (ND10), and the viral protein IE86 accumulates exclusively juxtaposed to the subpopulation of ND 10 with transcripts. The stream of transcripts is funneled from ND10 into the spliceosome assembly factor SC35 domain through the accumulation of IE86 protein, which recruits some components of the basal transcription machinery. Concomitantly the IE72 protein binds to ND10 and later disperses them. The domain containing the zinc finger region of IE72 is essential for this dispersal. Positional analysis of proteins IE86 and IE72, IE transcripts, ND10, the spliceosome assembly factor SC35, and basal transcription factors defines spatially and temporally an immediate transcript environment, the basic components of which exist in the cell before viral infection, providing the structural environment for the virus to usurp.

Immediate-early genes function to regulate viral and cellular gene expression during the course of virus replication. The major immediate-early gene region of human cytomegalovirus plays a key role in affecting activation and repression of viral and cellular genes. These proteins intimately associate and/or interact with viral and cellular proteins during this process. Herein, we will discuss the current understanding of this complex gene region.

As previously shown, 11 loci are required to complement human cytomegalovirus (HCMV) DNA replication in a transient-transfection assay (G. S. Pari and D. G. Anders, J. Virol. 67:6979-6988, 1993). Six of these loci encode known or candidate replication fork proteins, as judged by sequence and biochemical similarities to herpes simplex virus homologs of known function; three encode known immediate early regulatory proteins (UL36-38, IRS1/TRS1, and the major immediate early region spanning UL122-123); and two encode early, nucleus-localized proteins of unknown functions (UL84 and UL112-113). We speculated that proteins of the latter five loci might cooperate to promote and regulate expression of the six replication fork proteins. To test this hypothesis we made luciferase reporter plasmids for each of the replication fork gene promoters and measured their activation by the candidate effectors, expressed under the control of their respective native promoters, using a transient-cooperativity assay in which the candidate effectors were subtracted individually from a transfection mixture containing all five loci. The combination of UL36-38, UL112-113, IRS1, or TRS1 and the major immediate early region produced as much as 100-fold-higher expression than the major immediate early region alone; omitting any one of these four loci from complementing mixtures produced a significant reduction in expression. In contrast, omitting UL84 had insignificant (less than twofold), promoter-dependent effects on reporter activity, and these data do not implicate UL84 in regulating HCMV early-gene expression. Most of the effector interactions showed significant positive cooperativity, producing synergistic enhancement of expression. Similar responses to these effectors were observed for the each of the promoters controlling expression of replication fork proteins. However, subtracting UL112-113 had little if any effect on expression by the UL112-113 promoter or by the simian virus 40 promoter-enhancer under the same conditions. Several lines of evidence argue that the cooperative interactions observed in our transient-transfection assays are important to viral replication in permissive cells. Therefore, the data suggest a model in which coordinate expression of multiple essential replication proteins during permissive infection is vitally dependent upon the cooperative regulatory interactions of proteins encoded by multiple loci and thus have broad implications for our understanding of HCMV biology.

Murine cytomegalovirus (MCMV) gene products dispensable for growth in cell culture are likely to have important functions within the infected host, influencing tissue tropism, dissemination, or immunological responses against the virus. To identify such genes, our strategy was to delete large regions of the MCMV genome likely to contain genes nonessential for virus replication in NIH 3T3 cells. Mutant virus RV7 contained a deletion of 7.7 kb spanning portions of MCMV HindIII-J and -I. This virus grew comparably to wild-type (WT) virus in NIH 3T3 fibroblasts, primary embryo fibroblasts, and bone marrow macrophages. However, RV7 failed to replicate in target organs of immunocompetent BALB/c mice and severe combined immunodeficient mice, which are exquisitely sensitive to MCMV infection. This defect in vivo growth may be related to the observation that RV7 grew poorly in the peritoneal macrophage cell line IC-21, which is highly permissive for growth of WT MCMV. Two other mutant viruses with an insertion or smaller deletion in the region common to the RV7 deletion grew comparably to WT virus in the macrophage cell line and replicated in salivary gland tissue. The poor growth of RV7 in IC-21 cells was due to a block in immediate-early gene expression, as levels of RNA from immediate-early gene IE1 were reduced eightfold compared with levels for WT virus in macrophages infected with RV7. Consequently, levels of RNA from early and late genes were also reduced. The lower expression of IE1 in RV7-infected IC-21 macrophages was not due to defective entry of virus into the cells, as equal amounts of viral DNA were present in cells 3 h after infection with RV7 or WT MCMV. These studies demonstrate that deletion of sequences in HindIII-J and -I confer altered cell and tissue tropism.

The human cytomegalovirus (HCMV) DNA polymerase gene (UL54; also called pol) is a prototypical early gene in that expression is mandatory for viral DNA replication. Recently, we have identified the major regulatory element in the UL54 promoter responsive to the major immediate early (MIE) proteins (UL122 and UL123) (J. A. Kerry, M. A. Priddy, and R. M. Stenberg, J. Virol, 68:4167-4176, 1994), Mutation of this element, inverted repeat sequence 1 (IR1), abrogates binding of cellular proteins to the UL54 promoter and reduces promoter activity in response to viral proteins in transient-transfection assays. To extend our studies on the UL54 promoter, we aimed to examine the role of IR1 in UL54 regulation throughout the course of infection, These studies show that viral proteins in addition to the MIE proteins can activate the UL54 promoter, Proteins from UL112-113 and IRS1/TRS1, recently identified as essential loci for transient complementation of HCMV oriLyt-dependent DNA replication? were found to function as transactivators of the UL54 promoter in association with MIE proteins, UL112-113 enhanced UL54 promoter activation by MIE proteins three- to fourfold, Constitutive expression of UL112-113 demonstrated that the MIE protein dependence of UL112-113 transactivational activity was not related to activation of cognate promoter sequences, suggesting that UL112-113 proteins function in cooperation with the MIE proteins. Mutation of IR1 was found to abrogate stimulation of the UL54 promoter by UL112-113, suggesting that this element is also involved in UL112-113 stimulatory activity, These results demonstrate that additional viral proteins influence UL54 promoter expression in transient-transfection assays via the IR1 element, To confirm the biological relevance of IR1 in regulating UL54 promoter activity during viral infection, a recombinant virus construct containing the UL54 promoter with a mutated IR1 element regulating expression of the chloramphenicol acetyltransferase (CAT) reporter gene (RVIRmCAT) was generated, Analysis of RVIRmCAT revealed that mutation of IR1 dramatically reduces UL54 promoter activity at early times after infection, However, at late times after infection CAT expression by RVIRmCAT, as assessed by RNA and protein levels, was approximately equivalent to expression by wild-type RVpolCAT. These data demonstrate IR1-independent regulation of the UL54 promoter at late times after infection. Together these results show that multiple regulatory events affect UL54 promoter expression during the course of infection.

The purpose of this study was to identified nonessential murine cytomegalovirus (MCMV) genes involved in pathogenesis in vivo. Our approach to identifyjng these genes consisted of constructing MCMV mutants, and then analyzing these mutants in vitro and in vivo. Recombinant viruses (RV) expressing the β-glucuronidase marker gene were constructed by site-directed insertion and deletion mutagenesis of the MCMV Hind III-J and -I regions of the viral genome. Mutations were targeted to this region of the MCMV genome because the corresponding region of the human CMV genome is nonessential and is involved in down-regulating major histocompatibility complex (MHC) class I expression in infected human cells. Four mutant viruses were created: RV5, an insertion mutant in Hind III-J; RV6, a deletion mutant missing 2.8 kilobase (kb) pairs from Hind III-J; RV7, a deletion mutant lacking 7.7 kb from Hind III-J and -I; and RV9, a deletion mutant missing 10.7 kb from Hind III-J and -I. RV9 is a helper-dependent virus, and therefore, was not included in subsequent experiments. In vitro, RV5, RV6, and RV7 grew similarly to wild-type (WT) MCMV in NIH3T3 fibroblasts and in primary embryo fibroblasts, confirming that the mutations introduced into these viruses are nonessential for MCMV replication in cultured fibroblasts. In IC-21 macrophages, RV5 and RV6 great similarly to WT virus, however, RV7 grew 2 to 3 logs lower. A one-step growth curve of RV7 in these macrophages indicated that the reduced growth of this mutant virus was primarily due to a defect in virus replication. Northern blot analyses of immediate-early (IE), early, and late RNAs demonstrated that the block in RV7 replication occured at the IE phase of virus replication. A simultaneous Southern blot analysis of RV7-infected IC-21 macrophages indicated that RV7 entered these cells as efficiently as WT MCMV. Therefore, poor replication of RV7 in these cells is not the result of a defect in virus entry, but instead is due to inadequate IE viral gene expression. These data are highly significant because interactions between CMV and macrophages play a critical role in pathogenesis in vivo, particularly in virus dissemination and latency. The MCMV mutants were analyzed bv Western blot with an antibody to the H-2Kb class I heavy chain to determine if sequences within Hind III-J or -I were involved in the down-regulation of MHC class I expression observed in WT-infected cells. RV5, RV6, and RV7 significantly reduced the level of the class I heavy chain in infected fibroblasts, indicating that this nonessential region of the MCMV genome is not involved in class I down-regulation. In infected mice, all of the MCMV mutants displayed significantly reduced growth in the salivary glands, an organ central to the biology of CMV. In fact, the growth of RV7 in the salivary glands was barely detectable, indicating that the genes deleted from these viruses are required for the efficient replication of MCMV in the salivary gland.

During the past several years, studies have indicated that cytomegalovirus (CMV) genes can be grouped into two broad categories; those essential for replication in cell culture and those dispensible for virus replication. The latter group of genes are likely to be important for pathogenesis and host-virus interactions. As the field progresses, the need to utilize and establish biological systems capable of addressing gene function during a natural infection of cells in culture, or in the infected animal, is becoming more apparent. Herein, we describe the current status of some of those systems, what has been learned and where these studies may lead. Specifically, we address studies that assess mechanisms of gene activation and function in biologically relevant systems. These include (i) the identification of genes dispensible for replication in cell culture, (ii) the use of dispensible regions of the CMV genome to manipulate genetic information for assessing gene function and activation, and (iii) the identification of a related group of essential loci important for replication of human CMV (HCMV) DNA and what is presently known of the function of those genes during HCMV infection.

The major immediate-early (IE) gene region of human cytomegalovirus (HCMV) encodes several proteins as a result of differential RNA splicing events. By expression vector cloning of HCMV IE mRNA, we isolated and characterized a cDNA for a novel splice variant from the major IE gene region. The RNA product is a derivative of the IE55 mRNA and contains an additional splice from nucleotides 170,635 to 170,307 in the IE2 gene region (UL122), resulting in a 1.4-kb mRNA. The predicted open reading frame codes for a 164-amino-acid protein with a calculated molecular mass of 18 kDa (IE18). Mung bean nuclease analysis and PCR were used to characterize expression of IE18 mRNA in HCMV-infected cells. While the 1.4-kb mRNA was detected in infected human fibroblasts in the presence of a protein synthesis inhibitor, it was not detectable during a normal infection. However, the 1.4-kb mRNA was readily detected in infected human monocyte-derived macrophages at IE times. These results suggest that the novel IE18 mRNA exhibits cell type-specific expression indicating differential regulation of the major IE gene region in different permissive cell types.

Reduction of major histocompatibility complex class I cell surface expression occurs in adenovirus-, herpes simplex virus-, human cytomegalovirus (HCMV)-, and murine cytomegalovirus-infected cell systems. Recently, it was demonstrated that the down-regulation mediated by HCMV infection is posttranslational, as a result of increased turnover of class I heavy chains in the endoplasmic reticulum (M. F. C. Beersma, M. J. E. Bijlmakers, and H. L. Ploegh, J. Immunol. 151:4455-4464, 1993; Y. Yamashita, K. Shimokata, S. Saga, S. Mizuno, T. Tsurumi, and Y. Nishiyama, J. Virol. 68:7933-7943, 1994. To identify HCMV genes involved in class I regulation, we screened our bank of HCMV deletion mutants for this phenotype. A mutant with a 9-kb deletion in the S component of the HCMV genome (including open reading frames IRS1 to US9 and US11) failed to down-regulate class I heavy chains. By examining the effects of smaller deletions within this portion of the HCMV genome, a 7-kb region containing at least nine open reading frames was shown to contain the genes required for reduction in heavy-chain expression. Furthermore, it was determined that at least two independent loci within the 7-kb region were able to cause class I heavy-chain down-regulation. One of these, US11, encodes a 32-kDa glycoprotein which causes down-regulation of class I heavy chains in the absence of other viral gene products. Hence, a specific function associated with a phenotype of the HCMV replicative cycle has been mapped to a dispensable gene region. These loci may be important for evasion of the host's immune response and viral persistence.