[Show abstract][Hide abstract] ABSTRACT: The genomes of HCMV clinical strains (e.g. FIX, TR, PH, etc) contain a 15 kb region that encodes 20 putative ORFs. The region, termed ULb', is lost after serial passage of virus in human foreskin fibroblast (HFF) cell culture. Compared to clinical strains, laboratory strains replicate faster and to higher titers of infectious virus. We made recombinant viruses with 22, 14, or 7 ORFs deleted from the ULb' region using FIX and TR as model clinical strains. We also introduced a stop codon into single ORFs between UL133 and UL138 to prevent protein expression. All deletions within ULb' and all stop codon mutants within the UL133 to UL138 region increased to varying degrees, viral major immediate early RNA and protein, DNA, and cell-free infectious virus compared to the wild type viruses. The wild type viral proteins slowed down the viral replication process along with cell-free infectious virus release from human fibroblast cells.
[Show abstract][Hide abstract] ABSTRACT: In the human host, infection by cytomegaloviruses (CMVs) induces a variety of syndromes ranging from the classic cytomegalic inclusion disease to intrauterine death, prematurity, congenital defects, infectious mononucleosis, postperfusion syndrome, and interstitial pneumonia in transplantation patients (for reviews, see Weller, 1971; Rapp, 1980; Ho, 1982). These diseases caused by human CMV (HCMV) frequently represent infections after reactivation of latent virus and are usually associated with immuno-suppression due to a variety of situations such as malignant disease and chemotherapy (Weller, 1971; Plummer, 1973). Therefore, the HCMV genome has evolved to remain in human cells in a quiescent state from the time of primary infection. Although the majority of humans carry this virus, overt disease is a rare event. Unfortunately, little is known about the molecular biology of the latent CMV genome; consequently, this chapter will emphasize the events after reactivation of the viral genome. Reactivation is the event that allows for replication of the viral DNA genome and eventual release of infectious virus to cause overt disease. Latency and reactivation of HCMV replication relate directly to the regulation of the viral genome by host- and/or virus-specified genes in the eukaryotic cell.
No preview · Article · Mar 2014 · Methods in molecular biology (Clifton, N.J.)
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Expression of the human cytomegalovirus (HCMV) major immediate-early (MIE) genes is regulated by a strong enhancer-containing promoter with multiple binding sites for various transcription factors, including cyclic AMP response element binding protein 1 (CREB1). Here we show that overexpression of CREB1 potently blocked MIE transcription and HCMV replication. Surprisingly, CREB1 still exhibited strong inhibition of the MIE promoter when all five CREB binding sites within the enhancer were mutated, suggesting that CREB1 regulated the MIE gene expression indirectly. Promoter deletion analysis and site-directed mutagenesis identified the region between -130 and -50 upstream of the transcription start site of the MIE gene as the "CREB1 responsive region". Mutations of SP1/3 and NF-κB binding sites within this region interrupted the inhibitory effect induced by CREB1 overexpression. Our findings suggest that overexpression of CREB1 can cause repression of HCMV replication and may contribute to the development of new anti-HCMV strategies.
Full-text · Article · Dec 2013 · Antiviral research
[Show abstract][Hide abstract] ABSTRACT: Introduction:
During productive infection, human cytomegalovirus (HCMV) genes are expressed in a temporal cascade, with temporal phases designated as immediate-early (IE), early, and late. The major IE (MIE) genes, UL123 and UL122 (IE1/IE2), play a critical role in subsequent viral gene expression and the efficiency of viral replication. The early viral genes encode proteins necessary for viral DNA replication. Following viral DNA replication, delayed-early and late viral genes are expressed which encode structural proteins for the virion. The late genes can be divided into two broad classes. At early times the gamma-1 or leaky-late class are expressed at low levels after infection and are dramatically upregulated at late times. In contrast, the gamma-2 or 'true' late genes are expressed exclusively after viral DNA replication. Expression of true late (gamma-2 class) viral genes is completely prevented by inhibition of viral DNA synthesis.
This review addresses the viral genes required for HCMV late gene transcription. Recombinant viruses that are defective for late gene transcription allow for early viral gene expression and viral DNA synthesis, but not infectious virus production. Since current HCMV prophylaxis is limited by several shortcomings, the use of defective recombinant viruses to induce HCMV cell-mediated and humoral immunity is discussed.
HCMV DNA replication and late gene transcription are not completely linked. Viral-encoded trans-acting factors are required. Recombinant viruses proficient in MIE and early viral gene expression and defective in late gene expression may be an alternative therapeutic vaccine candidates for the induction of cell-mediated and humoral immunity.
No preview · Article · Dec 2012 · Expert Opinion on Therapeutic Targets
[Show abstract][Hide abstract] ABSTRACT: The regulation of human cytomegalovirus (HCMV) late gene expression by viral proteins is poorly understood, and these viral proteins could be targets for novel antivirals. HCMV open reading frames (ORFs) UL79, -87, and -95 encode proteins with homology to late gene transcription factors of murine gammaherpesvirus 68 ORFs 18, 24, and 34, respectively. To determine whether these HCMV proteins are also essential for late gene transcription of a betaherpesvirus, we mutated HCMV ORFs UL79, -87, and -95. Cells were infected with the recombinant viruses at high and low multiplicities of infection (MOIs). While viral DNA was detected with the recombinant viruses, infectious virus was not detected unless the wild-type viral proteins were expressed in trans. At a high MOI, mutation of ORF UL79, -87, or -95 had no effect on the level of major immediate-early (MIE) gene expression or viral DNA replication, but late viral gene expression from the UL44, -75, and -99 ORFs was not detected. At a low MOI, preexpression of UL79 or -87, but not UL95, in human fibroblast cells negatively affected the level of MIE viral gene expression and viral DNA replication. The products of ORFs UL79, -87, and -95 were expressed as early viral proteins and recruited to prereplication complexes (pre-RCs), along with UL44, before the initiation of viral DNA replication. All three HCMV ORFs are indispensable for late viral gene expression and viral growth. The roles of UL79, -87, and -95 in pre-RCs for late viral gene expression are discussed.
Full-text · Article · Jul 2011 · Journal of Virology
[Show abstract][Hide abstract] ABSTRACT: The major immediate-early (MIE) gene locus of human cytomegalovirus (HCMV) is the master switch that determines the outcomes of both lytic and latent infections. Here, we provide evidence that alteration in the splicing of HCMV (Towne strain) MIE genes affects infectious-virus replication, movement through the cell cycle, and cyclin-dependent kinase activity. Mutation of a conserved 24-nucleotide region in MIE exon 4 increased the abundance of IE1-p38 mRNA and decreased the abundance of IE1-p72 and IE2-p86 mRNAs. An increase in IE1-p38 protein was accompanied by a slight decrease in IE1-p72 protein and a significant decrease in IE2-p86 protein. The mutant virus had growth defects, which could not be complemented by wild-type IE1-p72 protein in trans. The phenotype of the mutant virus could not be explained by an increase in IE1-p38 protein, but prevention of the alternate splice returned the recombinant virus to the wild-type phenotype. The lower levels of IE1-p72 and IE2-p86 proteins correlated with a delay in early and late viral gene expression and movement into the S phase of the cell cycle. Mutant virus-infected cells had significantly higher levels of cdk-1 expression and enzymatic activity than cells infected with wild-type virus. The mutant virus induced a round-cell phenotype that accumulated in the G(2)/M compartment of the cell cycle with condensation and fragmentation of the chromatin. An inhibitor of viral DNA synthesis increased the round-cell phenotype. The round cells were characteristic of an abortive viral infection.
Preview · Article · Nov 2010 · Journal of Virology
[Show abstract][Hide abstract] ABSTRACT: Human cytomegalovirus (HCMV) can be reactivated under immunosuppressive conditions causing several fatal pneumonitis, hepatitis, retinitis, and gastrointestinal diseases. HCMV also causes deafness and mental retardation in neonates when primary infection has occurred during pregnancy. In the genome of HCMV at least 194 known open reading frames (ORFs) have been predicted, and approximately one-quarter, or 41 ORFs, are required for viral replication in cell culture. In contrast, the majority of the predicted ORFs are nonessential for viral replication in cell culture. However, it is also possible that these ORFs are required for the efficient viral replication in the host. The UL77 gene of HCMV is essential for viral replication and has a role in viral DNA packaging. The function of the upstream UL76 gene in the HCMV-infected cells is not understood. UL76 and UL77 are cistons on the same viral mRNA and a conventional 5' mRNA for UL77 has not been detected. The vast majority of eukaryotic mRNAs are monocistronic, i.e., they encode only a single protein.
To determine whether the UL76 ORF affects UL77 gene expression, we mutated UL76 by ORF frame-shifts, stop codons or deletion of the viral gene. The effect on UL77 protein expression was determined by either transfection of expression plasmids or infection with recombinant viruses. Mutation of UL76 ORF significantly increased the level of UL77 protein expression. However, deletion of UL76 upstream of the UL77 ORF had only marginal effects on viral growth.
While UL76 is not essential for viral replication, the UL76 ORF is involved in regulation of the level of UL77 protein expression in a manner dependent on the translation re-initiation. UL76 may fine-tune the UL77 expression for the efficient viral replication in the HCMV- infected cells.
[Show abstract][Hide abstract] ABSTRACT: Human cytomegalovirus (HCMV) or its immediate-early IE86 protein alone induces cell cycle in quiescent primary human foreskin fibroblasts (HFFs), but blocks its progression at the G1/S interphase and inhibits cellular DNA synthesis by a mechanism that is not clearly understood. It is assumed that, in this phenomenon, the binding of minichromosome maintenance (Mcm) proteins to replication origins is blocked. In this work, we analyzed the initiation of DNA replication in HCMV-permissive U373MG cells and used oriP of Epstein-Barr virus (EBV) as a simplified model of a cellular replication origin. Using U373MG cells we found that HCMV IE86 protein was bound to Mcm3, but did not inhibit the cellular DNA synthesis. Using U373MG-p220.2 cells carrying EBV oriP and expressing Epstein-Barr nuclear antigen 1 (EBNA1), we found that EBNA1 as well as Mcm3 were bound to oriP and that neither HCMV nor IE86 protein inhibited the binding of Mcm3 to oriP. Differences between the effects of HCMV on the cell cycle progression in HFFs and U373MG cells are discussed. Keywords: cell cycle; Human cytomegalovirus; DNA replication.
[Show abstract][Hide abstract] ABSTRACT: One of the two SP1 sites in the proximal enhancer of the human cytomegalovirus (HCMV) major immediate-early (MIE) promoter is essential for transcription in human fibroblast cells (H. Isomura, M. F. Stinski, A. Kudoh, T. Daikoku, N. Shirata, and T. Tsurumi, J. Virol. 79:9597-9607, 2005). Upstream of the two SP1 sites to -223 relative to the +1 transcription start site, there are an additional five DNA binding sites for eukaryotic transcription factors. We determined the effects of the various transcription factor DNA binding sites on viral MIE RNA transcription, viral gene expression, viral DNA synthesis, or infectious virus production. We prepared recombinant HCMV bacterial artificial chromosome (BAC) DNAs with either one site missing or one site present upstream of the two SP1 sites. Infectious recombinant HCMV BAC DNAs were transfected into various cell types to avoid the effect of the virion-associated transactivators. Regardless of the cell type, which included human fibroblast, endothelial, and epithelial cells, the CREB site had the most significant and independent effect on the MIE promoter. The other sites had a minor independent effect. However, the combination of the different transcription factor DNA binding sites was significantly stronger than multiple duplications of the CREB site. These findings indicate that the CREB site in the presence of the other sites has a major role for the replication of HCMV.
Preview · Article · Jul 2009 · Journal of Virology
[Show abstract][Hide abstract] ABSTRACT: The cytomegalovirus (CMV) major immediate early (MIE) enhancer-containing promoter regulates the expression of the downstream MIE genes, which have critical roles in reactivation from latency and acute infection. The enhancer consists of binding sites for cellular transcription factors that are repeated multiple times. The primate and nonprimate CMV enhancers can substitute for one another. The enhancers are not functionally equivalent, but they do have overlapping activities. The CMV MIE enhancers are located between divergent promoters where the leftward genes are critical and essential for reactivation from latency and acute infection and the rightward gene is nonessential. The rightward transcription unit is controlled by an enhancer for murine CMV. In contrast, human CMV has a set of repressor elements that prevents enhancer effects on the rightward viral promoter. The human CMV enhancer that controls the leftward transcription unit has a distal component that is nonessential at high multiplicity of infection (MOI), but has a significant impact on the MIE gene expression at low MOI. The proximal enhancer influences directly the level of transcription of the MIE genes and contains an essential Sp-1 site. The MIE promoter has a site adjacent to the transcription start site that is essential at the earliest stage of infection. The MIE enhancer-containing promoter responds to signal transduction events and to cellular differentiation. The role of the CMV MIE enhancer-containing promoter in acute infection and reactivation from latency are reviewed.
Preview · Article · Jul 2008 · Medical Microbiology and Immunology
[Show abstract][Hide abstract] ABSTRACT: During productive infection, human cytomegalovirus (HCMV) UL44 transcription initiates at three distinct start sites that
are differentially regulated. Two of the start sites, the distal and the proximal, are active at early times, whereas the
middle start site is active only at late times after infection. The UL44 early viral gene product is essential for viral DNA
synthesis. The UL44 gene product from the late viral promoter affects primarily viral gene expression at late times after
infection rather than viral DNA synthesis (H. Isomura, M. F. Stinski, A. Kudoh, S. Nakayama, S. Iwahori, Y. Sato, and T. Tsurumi,
J. Virol. 81:6197, 2007). The UL44 early viral promoters have a canonical TATA sequence, “TATAA.” In contrast, the UL44 late viral promoter
has a noncanonical TATA sequence. Using recombinant viruses, we found that the noncanonical TATA sequence is required for
the accumulation of late viral transcripts. The GC boxes that surround the middle TATA element did not affect the kinetics
or the start site of UL44 late transcription. Replacement of the distal TATA element with a noncanonical TATA sequence did
not affect the kinetics of transcription or the transcription start site, but it did induce an alternative transcript at late
times after infection. The data indicate that a noncanonical TATA box is used at late times after HCMV infection.
Full-text · Article · Mar 2008 · Journal of Virology
[Show abstract][Hide abstract] ABSTRACT: The promoter of the major immediate-early (MIE) genes of human cytomegalovirus (HCMV), also referred to as the CMV promoter,
possesses a cis-acting element positioned downstream of the TATA box between positions −14 and −1 relative to the transcription start site
(+1). We determined the role of the cis-acting element in viral replication by comparing recombinant viruses with the cis-acting element replaced with other sequences. Recombinant virus with the simian CMV counterpart replicated efficiently in
human foreskin fibroblasts, as well as wild-type virus. In contrast, replacement with the murine CMV counterpart caused inefficient
MIE gene transcription, RNA splicing, MIE and early viral gene expression, and viral DNA replication. To determine which nucleotides
in the cis-acting element are required for efficient MIE gene transcription and splicing, we constructed mutations within the cis-acting element in the context of a recombinant virus. While mutations in the cis-acting element have only a minor effect on in vitro transcription, the effects on viral replication are major. The nucleotides
at −10 and −9 in the cis-acting element relative to the transcription start site (+1) affect efficient MIE gene transcription and splicing at early
times after infection. The cis-acting element also acts as a cis-repression sequence when the viral IE86 protein accumulates in the infected cell. We demonstrate that the cis-acting element has an essential role in viral replication.
Full-text · Article · Feb 2008 · Journal of Virology
[Show abstract][Hide abstract] ABSTRACT: The IE86 protein of human cytomegalovirus (HCMV) is unique among viral and cellular proteins because it negatively autoregulates its own expression, activates the viral early and late promoters, and both activates and inhibits cellular promoters. It promotes cell cycle progression from Go/G1 to G1/S and arrests cell cycle progression at the G1/S interface or at G2/M. The IE86 protein is essential because it creates a cellular environment favorable for viral replication. The multiple functions of the IE86 protein during the replication of HCMV are reviewed.
No preview · Article · Jan 2008 · Current topics in microbiology and immunology
[Show abstract][Hide abstract] ABSTRACT: The IE2p86 protein of human cytomegalovirus is an essential activator of early- and late-phase viral gene expression. Whilst IE2p86 activates expression of a number of cellular genes, it also represses certain cellular genes, particularly those activated by nuclear factor kappaB (NF-kappaB). As the interleukin-6 (IL-6) promoter can be activated by both NF-kappaB and IE2p86, it was examined whether there is competition between these two factors. Here, it is reported that both wild-type and mutant IE2p86 can block activation of the IL-6 promoter in response to interleukin-1beta. By using an artificial activator in which the activation domain of NF-kappaB is directed to the promoter by the GAL4 DNA-binding domain, it is shown that the mutant form of IE2p86 can inhibit NF-kappaB-mediated activation at a step subsequent to promoter recruitment. These data therefore suggest a novel mechanism for inhibition of NF-kappaB by IE2p86.
Preview · Article · Oct 2007 · Journal of General Virology
[Show abstract][Hide abstract] ABSTRACT: An AT-rich region of the human cytomegalovirus (CMV) genome between the UL127 open reading frame and the major immediate-early (MIE) enhancer is referred to as the unique region (UR). It has been shown that the UR represses activation of transcription from the UL127 promoter and functions as a boundary between the divergent UL127 and MIE genes during human CMV infection [Angulo, A., Kerry, D., Huang, H., Borst, E.M., Razinsky, A., Wu, J., Hobom, U., Messerle, M., Ghazal, P., 2000. Identification of a boundary domain adjacent to the potent human cytomegalovirus enhancer that represses transcription of the divergent UL127 promoter. J. Virol. 74 (6), 2826-2839; Lundquist, C.A., Meier, J.L., Stinski, M.F., 1999. A strong negative transcriptional regulatory region between the human cytomegalovirus UL127 gene and the major immediate-early enhancer. J. Virol. 73 (11), 9039-9052]. A putative forkhead box-like (FOX-like) site, AAATCAATATT, was identified in the UR and found to play a key role in repression of the UL127 promoter in recombinant virus-infected cells [Lashmit, P.E., Lundquist, C.A., Meier, J.L., Stinski, M.F., 2004. Cellular repressor inhibits human cytomegalovirus transcription from the UL127 promoter. J. Virol. 78 (10), 5113-5123]. However, the cellular factors which associate with the UR and FOX-like region remain to be determined. We reported previously that pancreatic-duodenal homeobox factor-1 (PDX1) bound to a 45-bp element located within the UR [Chao, S.H., Harada, J.N., Hyndman, F., Gao, X., Nelson, C.G., Chanda, S.K., Caldwell, J.S., 2004. PDX1, a Cellular Homeoprotein, Binds to and Regulates the Activity of Human Cytomegalovirus Immediate Early Promoter. J. Biol. Chem. 279 (16), 16111-16120]. Here we demonstrate that two additional cellular homeoproteins, special AT-rich sequence binding protein 1 (SATB1) and CCAAT displacement protein (CDP), bind to the human CMV UR in vitro and in vivo. Furthermore, CDP is identified as a FOX-like binding protein and a repressor of the UL127 promoter, while SATB1 has no effect on UL127 expression. Since CDP is known as a transcription repressor and a nuclear matrix-associated region binding protein, CDP may have a role in the regulation of human CMV transcription.
[Show abstract][Hide abstract] ABSTRACT: Transcription of the DNA polymerase processivity factor gene (UL44) of human cytomegalovirus initiates at three distinct start
sites, which are differentially regulated during productive infection. Two of these start sites, the distal and proximal sites,
are active at early times, and the middle start site is active at only late times after infection (F. Leach and E. S. Mocarski,
J. Virol. 63:1783-1791, 1989). Compared to the wild type, UL44 gene expression was lower for recombinant viruses with the
distal or the middle TATA element mutated. The transcripts initiating from the distal or middle start site facilitated late
viral gene expression. The level of viral DNA synthesis was affected by mutation of the distal TATA element. In contrast,
mutation of the middle TATA element did not affect the level of viral DNA synthesis, but it did affect significantly the level
of late viral gene expression. Recombinant viruses with the distal or middle TATA element mutated grew more slowly than the
wild type at both low and high multiplicities of infection. Reduced expression of the UL44 gene from the late middle viral
promoter correlated with decreased late viral protein expression and decreased viral growth.
Preview · Article · Jul 2007 · Journal of Virology
[Show abstract][Hide abstract] ABSTRACT: The functions of the human cytomegalovirus (HCMV) IE86 protein are paradoxical, as it can both activate and repress viral
gene expression through interaction with the promoter region. Although the mechanism for these functions is not clearly defined,
it appears that a combination of direct DNA binding and protein-protein interactions is involved. Multiple sequence alignment
of several HCMV IE86 homologs reveals that the amino acids 534LPIYE538 are conserved between all primate and nonprimate CMVs. In the context of a bacterial artificial chromosome (BAC), mutation
of both P535 and Y537 to alanines (P535A/Y537A) results in a nonviable BAC. The defective HCMV BAC does not undergo DNA replication,
although the P535A/Y537A mutant IE86 protein appears to be stably expressed. The P535A/Y537A mutant IE86 protein is able to
negatively autoregulate transcription from the major immediate-early (MIE) promoter and was recruited to the MIE promoter
in a chromatin immunoprecipitation (ChIP) assay. However, the P535A/Y537A mutant IE86 protein was unable to transactivate
early viral genes and was not recruited to the early viral UL4 and UL112 promoters in a ChIP assay. From these data, we conclude
that the transactivation and repressive functions of the HCMV IE86 protein can be separated and must occur through independent
Preview · Article · Jul 2007 · Journal of Virology
[Show abstract][Hide abstract] ABSTRACT: Human cytomegalovirus (HCMV) reactivation from latency causes disease in individuals who are immunocompromised or immunosuppressed. Activation of the major immediate-early (MIE) promoter is thought to be an initial step for reactivation. We determined whether expression of the MIE gene products in trans was sufficient to circumvent an HCMV latent-like state in an undifferentiated transformed human promonocytic (THP)-1 cell model system. Expression of the functional MIE proteins was achieved with a replication-defective adenovirus vector, Ad-IE1/2, which contains the MIE gene locus. Expression of the MIE proteins by Ad-IE1/2 prior to HCMV infection induced viral early gene expression accompanied by an increase in active chromatin signals. Expression of the anti-apoptotic protein encoded by UL37x1 increased viral early gene expression. However, viral DNA replication and production of infectious virus was not detected. As expected, cellular differentiation with phorbol 12-myristate 13-acetate and hydrocortisone induced virus production. Cellular differentiation is required for efficient viral reactivation.
[Show abstract][Hide abstract] ABSTRACT: Betaherpesviruses such as human cytomegalovirus (HCMV), human herpesvirus-6A and 6B (HHV-6), and human herpesvirus-7 (HHV-7) replicate more slowly than alphaherpesviruses, are highly species-specific for infection, and establish latency in progenitor cells of the bone marrow and monocytes of the blood. HCMV has been the prototype of the betaherpesviruses for studies of gene expression and regulation. In cell culture, HCMV strains have been adapted to preferentially infect and replicate in fibroblasts. However, low passage isolates replicate well in other cell types, such as endothelial cells, macrophages and dendritic cells. In the host, HCMV replicates in macrophages, dendritic cells, colonic and retinal pigmented epithelial cells, endothelial cells, fibroblasts, smooth muscle cells, neuronal cells, glial cells, hepatocytes, and trophoblasts (Fish et al., 1995, 1996; Hertel et al., 2003; Ibanez et al., 1991; Lathey and Spector, 1991; Maidji et al., 2002; Schmidbauer et al., 1989; Sinzger et al., 1993, 1995, 1996). In contrast, HHV-6 and HHV-7 infect CD4+ lymphocytes (Takahashi et al., 1989) as well as monocyte/macrophages. Although HCMV can be transferred into and out of polymorphonuclear leukocytes via cell-to-cell contact, these cells do not permit viral replication (Grundy et al., 1998; Sinclair and Sissons, 1996; Sinzger and Jahn, 1996).