Human Cytomegalovirus Entry Research Articles

Human Cytomegalovirus-Regulated Paxillin in Monocytes Links Cellular Pathogenic Motility to the Process of Viral Entry

We have established that human cytomegalovirus (HCMV) infection modulates the biology of target primary peripheral blood monocytes, allowing HCMV to use monocytes as "vehicles" for its systemic spread. HCMV infection of monocytes results in rapid induction of phosphatidylinositol-3-kinase [PI(3)K] and NF-κB activities. Integrins, which are upstream of the PI(3)K and NF-κB pathways, were shown to be involved in HCMV binding to and entry into fibroblasts, suggesting that receptor ligand-mediated signaling following viral binding to integrins on monocytes could trigger the functional changes seen in infected monocytes. We now show that integrin engagement and the activation of the integrin/Src signaling pathway are essential for the induction of HCMV-infected monocyte motility. To investigate how integrin engagement by HCMV triggers monocyte motility, we examined the infected-monocyte transcriptome and found that the integrin/Src signaling pathway regulates the expression of paxillin, which is an important signal transducer in the regulation of actin rearrangement during cell adhesion and movement. Functionally, we observed that paxillin is activated via the integrin/Src signaling pathway and is required for monocyte motility. Because motility is intimately connected to cellular cytoskeletal organization, a process that is also important in viral entry, we investigated the role paxillin regulation plays in the process of viral entry into monocytes. New results confirmed that HCMV entry into target monocytes was significantly reduced in cells deficient in paxillin expression or the integrin/Src/paxillin signaling pathway. From our data, HCMV-cell interactions emerge as an essential trigger for the cellular changes that allow for HCMV entry and hematogenous dissemination.

The Glycoprotein B Disintegrin-Like Domain Binds Beta 1 Integrin To Mediate Cytomegalovirus Entry

Cellular integrins were identified as human cytomegalovirus (HCMV) entry receptors and signaling mediators in both fibroblasts and endothelial cells. The goal of these studies was to determine the mechanism by which HCMV binds to cellular integrins to mediate virus entry. HCMV envelope glycoprotein B (gB) has sequence similarity to the integrin-binding disintegrin-like domain found in the ADAM (a disintegrin and metalloprotease) family of proteins. To test the ability of this region to bind to cellular integrins, we generated a recombinant soluble version of the gB disintegrin-like domain (gB-DLD). The gB-DLD protein bound to human fibroblasts in a specific, dose-dependent and saturable manner that required the expression of an intact beta1 integrin ectodomain. Furthermore, a physical association between gB-DLD and beta1 integrin was demonstrated through in vitro pull-down assays. The function of this interaction was shown by the ability of cell-bound gB-DLD to efficiently block HCMV entry and the infectivity of multiple in vivo target cells. Additionally, rabbit polyclonal antibodies raised against gB-DLD neutralized HCMV infection. Mimicry of the ADAM family disintegrin-like domain by HCMV gB represents a novel mechanism for integrin engagement by a virus and reveals a unique therapeutic target for HCMV neutralization. The strong conservation of the DLD across beta- and gammaherpesviruses suggests that integrin recognition and utilization may be a more broadly conserved feature throughout the Herpesviridae.

Human cytomegalovirus tropism for endothelial/epithelial cells: scientific background and clinical implications

Human cytomegalovirus (HCMV) has been routinely isolated from and propagated in vitro in human embryonic lung fibroblast (HELF) cell cultures, while in vivo it is known to infect predominantly endothelial and epithelial cells. In recent years, genetic determinants of the HCMV tropism for endothelial/epithelial cells were identified in the UL131A/UL130/UL128 locus of HCMV genome of wild-type strains. UL131A-UL128 gene products form a complex with glycoprotein H (gH) and L (gL) resulting in a gH/gL/UL131A-UL128 complex that is required for HCMV entry into endothelial/epithelial cells. In contrast, virus entry into fibroblasts has its genetic determinants in the complex gH/gL/gO (or gH/gL). During primary HCMV infection, the neutralising antibody response measured in endothelial cells (EC) is potent, occurs very early and is directed mostly against combinations of two or three gene products of the UL131A-128 locus. On the contrary, neutralising antibodies measured in fibroblasts appear late, are relatively weak in potency and are directed against gH and gB. The T-cell immune response to UL131A-UL128 gene products remains to be investigated. Recently, a role has been proposed for neutralising antibody in conferring prevention/protection against HCMV infection/disease in pregnant women with primary HCMV infection. However, the level of cooperation between humoral immunity and the well-established T-cell protection remains to be defined.

Activation of EGFR on monocytes is required for human cytomegalovirus entry and mediates cellular motility

Human cytomegalovirus (HCMV) rapidly induces a mobile and functionally unique proinflammatory monocyte following infection that is proposed to mediate viral spread. The cellular pathways used by HCMV to initiate these biological changes remain unknown. Here, we document the expression of the epidermal growth factor receptor (EGFR) on the surface of human peripheral blood monocytes but not on other blood leukocyte populations. Inhibition of EGFR signaling abrogated viral entry into monocytes, indicating that EGFR can serve as a cellular tropism receptor. Moreover, HCMV-activated EGFR was required for the induction of monocyte motility and transendothelial migration, two biological events required for monocyte extravasation into peripheral tissue, and thus viral spread. Transcriptome analysis revealed that HCMV-mediated EGFR signaling up-regulated neural Wiskott-Aldrich syndrome protein (N-WASP), an actin nucleator whose expression and function are normally limited in leukocytes. Knockdown of N-WASP expression blocked HCMV-induced but not phorbol 12-myristate 13-acetate (PMA)-induced monocyte motility, suggesting that a switch to and/or the distinct use of a new actin nucleator controlling motility occurs during HCMV infection of monocytes. Together, these data provide evidence that EGFR plays an essential role in the immunopathobiology of HCMV by mediating viral entry into monocytes and stimulating the aberrant biological activity that promotes hematogenous dissemination.

Endothelial cells in human cytomegalovirus infection: One host cell out of many or a crucial target for virus spread?

Endothelial cells (EC) are assumed to play a central role in the spread of human cytomegalovirus (HCMV) throughout the body. Results from in-situ analyses of infected tissues and data from cell culture systems together strongly suggest that vascular EC can support productive replication of HCMV and thus contribute to its haematogeneous dissemination. By inducing an angiogenic response, HCMV may even promote growth of its own habitat. The particular role of EC is further supported by the fact that entry of HCMV into EC is dependent on a complex of the envelope glycoproteins gH and gL with a set of proteins (UL128-131A) which is dispensable for HCMV entry into most other cell types. These molecular requirements may also be reflected by cell type-dependent differences in entry routes, i.e. endocytosis versus fusion at the plasma membrane. An animal model with trackable murine CMV is now available to clarify the pathogenetic role of EC during haematogeneous dissemination of this virus.

HCMV gH/gL/UL128–131 interferes with virus entry into epithelial cells: Evidence for cell type-specific receptors

Human cytomegalovirus (HCMV) forms two different membrane protein complexes, gH/gL/gO and gH/gL/UL128/UL130/UL131, that function in different cell types. gH/gL/gO appears to be important for HCMV entry into or spread between fibroblasts, processes that occur at neutral pH. We demonstrated that HCMV entry into epithelial and endothelial cells requires gH/gL/UL128-131 and involves endocytosis and low pH. A complex of all five HCMV proteins, gH, gL, UL128, UL130, and UL131, is the functionally important mediator of this entry pathway into epithelial/endothelial cells. Here, we report that expression of gH/gL/UL128-131 in ARPE-19 epithelial cells causes the cells to be resistant to HCMV infection. Another HCMV glycoprotein, gB, did not interfere, and expression of all five gH/gL/UL128-131 proteins was required for this interference. gH/gL/UL128-131 interference was at the stage of virus entry into cells rather than the initial adsorption onto cell surfaces or after-entry defects. By contrast, expression of gH/gL/UL128-131 in primary human fibroblasts did not block HCMV infection. Previously, interference by retrovirus and herpes-simplex-virus entry mediators resulted from sequestration or obstruction of receptors. We concluded that epithelial cells express gH/gL/UL128-131 receptors that mediate HCMV entry. Fibroblasts either lack the gH/gL/UL128-131 receptors, the receptors are more numerous, or fibroblasts express other functional receptors.

Platelet-derived growth factor-α receptor activation is required for human cytomegalovirus infection

Human cytomegalovirus (HCMV) is a ubiquitous human herpesvirus that can cause life-threatening disease in the fetus and the immunocompromised host. Upon attachment to the cell, the virus induces robust inflammatory, interferon- and growth-factor-like signalling. The mechanisms facilitating viral entry and gene expression are not clearly understood. Here we show that platelet-derived growth factor-alpha receptor (PDGFR-alpha) is specifically phosphorylated by both laboratory and clinical isolates of HCMV in various human cell types, resulting in activation of the phosphoinositide-3-kinase (PI(3)K) signalling pathway. Upon stimulation by HCMV, tyrosine-phosphorylated PDGFR-alpha associated with the p85 regulatory subunit of PI(3)K and induced protein kinase B (also known as Akt) phosphorylation, similar to the genuine ligand, PDGF-AA. Cells in which PDGFR-alpha was genetically deleted or functionally blocked were non-permissive to HCMV entry, viral gene expression or infectious virus production. Re-introducing human PDGFRA gene into knockout cells restored susceptibility to viral entry and essential viral gene expression. Blockade of receptor function with a humanized PDGFR-alpha blocking antibody (IMC-3G3) or targeted inhibition of its kinase activity with a small molecule (Gleevec) completely inhibited HCMV viral internalization and gene expression in human epithelial, endothelial and fibroblast cells. Viral entry in cells harbouring endogenous PDGFR-alpha was competitively inhibited by pretreatment with PDGF-AA. We further demonstrate that HCMV glycoprotein B directly interacts with PDGFR-alpha, resulting in receptor tyrosine phosphorylation, and that glycoprotein B neutralizing antibodies inhibit HCMV-induced PDGFR-alpha phosphorylation. Taken together, these data indicate that PDGFR-alpha is a critical receptor required for HCMV infection, and thus a target for novel anti-viral therapies.

Differential Initiation of Innate Immune Responses Induced by Human Cytomegalovirus Entry into Fibroblast Cells

Infection of permissive fibroblasts with human CMV (HCMV, AD169) is accompanied by a robust activation of innate immune defense. In this study, we show that inflammatory cytokine (IC) secretion and activation of the type I IFN pathway (alphabeta IFN) are initiated through distinct mechanisms. HCMV is recognized by TLR2 leading to the NF-kappaB activation and IC secretion. However, the IFN response to HCMV is not a TLR2-dependent process, as a dominant negative TLR2 does not affect the antiviral response to infection. Additionally, bafilomycin, an endosomal acidification inhibitor, has no effect on HCMV-induced IFN responses suggesting that IFN signaling is independent of endosomal resident TLRs. By contrast, disruption of lipid rafts by depletion of cellular cholesterol inhibits both HCMV entry as well as IFN responses. Cholesterol depletion had no effect on the induction of ICs by HCMV, illustrating a biological distinction at the cellular level with the initiation of innate immune pathways. Furthermore, HCMV entry inhibitors block IFN responses but not IC signaling. In particular, blocking the interaction of HCMV with beta(1) integrin diminished IFN signaling, suggesting that this virus-cell interaction or subsequent downstream steps in the entry pathway are critical for downstream signal transduction events. These data show that HCMV entry and IFN signaling are coordinated processes that require cholesterol-rich microdomains, whereas IC signaling is activated through outright sensing via TLR2. These findings further highlight the complexity and sophistication of innate immune responses at the earliest points in HCMV infection.

Phosphorothioate-Modified Oligodeoxynucleotides Inhibit Human Cytomegalovirus Replication by Blocking Virus Entry

Studies in animal models have provided evidence that Toll-like receptor 9 (TLR9) agonists, such as synthetic oligodeoxynucleotides (ODNs) that contain immunostimulatory deoxycytidyl-deoxyguanosine (CpG) motifs (CpG ODNs), protect against a wide range of viral pathogens. This antiviral activity has been suggested to be indirect and secondary to CpG-induced cytokines and inflammatory responses triggered through TLR9 activation. However, few studies have addressed the potential of CpG ODNs as direct antiviral agents. Here, we report on the ability of some CpG ODNs to directly suppress, almost completely, human cytomegalovirus (HCMV) replication in both primary fibroblasts and endothelial cells. Murine CMV replication was inhibited as well, whereas no inhibition was observed for herpes simplex virus type 1, adenovirus, or vesicular stomatitis virus. The antiviral activity of these ODNs was significantly reduced when they were added after virus adsorption, indicating that their action may be primarily targeted to the very early phases of the HCMV cycle. In fact, the B-class prototype CpG ODN 2006 effectively prevented the nuclear localization of pp65 and input viral DNA, which suggests that it inhibits HCMV entry. Moreover, a CpG 2006 control, ODN 2137 without CpG motifs, also showed a potent inhibitory activity on the HCMV entry phase, indicating that the anticytomegaloviral activity is independent of the CpG motif. In contrast, a phosphodiester version of CpG 2006 showed reduced antiviral activity, indicating that the inhibitory activity is dependent on the phosphorothioate backbone of the ODN. These results suggest that this yet-unrecognized activity of CpG ODNs may be of interest in the development of novel anticytomegaloviral molecules.

Characterization of the Human Cytomegalovirus gH/gL/UL128-131 Complex That Mediates Entry into Epithelial and Endothelial Cells

The entry of human cytomegalovirus (HCMV) into biologically relevant epithelial and endothelial cells involves endocytosis followed by low-pH-dependent fusion. This entry pathway is facilitated by the HCMV UL128, UL130, and UL131 proteins, which form one or more complexes with the virion envelope glycoprotein gH/gL. gH/gL/UL128-131 complexes appear to be distinct from the gH/gL/gO complex, which likely facilitates entry into fibroblasts. In order to better understand the assembly and protein-protein interactions of gH/gL/UL128-131 complexes, we generated HCMV mutants lacking UL128-131 proteins and nonreplicating adenovirus vectors expressing gH, gL, UL128, UL130, and UL131. Our results demonstrate that UL128, UL130, and UL131 can each independently assemble onto gH/gL scaffolds. However, the binding of individual UL128-131 proteins onto gH/gL can significantly affect the binding of other proteins; for example, UL128 increased the binding of both UL130 and UL131 to gH/gL. Direct interactions between gH/UL130, UL130/UL131, gL/UL128, and UL128/UL130 were also observed. The export of gH/gL complexes from the endoplasmic reticulum (ER) to the Golgi apparatus and cell surface was dramatically increased when all of UL128, UL130, and UL131 were coexpressed with gH/gL (with or without gO expression). Incorporation of gH/gL complexes into the virion envelope requires transport beyond the ER. Thus, we concluded that UL128, UL130, and UL131 must all bind simultaneously onto gH/gL for the production of complexes that can function in entry into epithelial and endothelial cells.

Cytomegalovirus UL131-128 products promote gB conformational transition and gB-gH interaction during entry into endothelial cells.

Herpesviruses use gB and gH-gL glycoproteins to execute fusion. Other virus-specific glycoproteins are required for receptor binding and fusion activation. The human cytomegalovirus (HCMV) UL131-128 proteins are essential for the infection of leukocytes, endothelial cells (ECs), and many epithelial cell lines. Here we show that UL131-128 play a role in a chain of events involving gB and gH during HCMV entry into ECs. An HCMV strain bearing the wild-type (wt) UL131-128 locus exhibited a gB transition from a protease-resistant to protease-sensitive form, a conformational change that was suppressed by a thiourea inhibitor of fusion (WY1768); in contrast, gH was susceptible to proteolysis throughout entry. Moreover, gB and gH transiently interacted, and a lipid mixing assay showed that the wt strain had carried out fusion by 60 min postinfection. However, these events were greatly altered when UL131-128-defective strains were used for infection or when there was an excess of soluble pUL128 during wt infection: the gB conformational change became WY1768 resistant, the gB-gH complex was no longer observed, and fusion was prevented. Both gB and gH in this case showed late protease resistance, related to their endocytic uptake. Our data point to the involvement of UL131-128 proteins in driving gB through a WY1768-sensitive fold transition, thus promoting a short-lived gB-gH complex and fusion; they also suggest that HCMV fuses with the EC plasma membrane and that endocytosis ensues only when the virus cannot trigger UL131-128-dependent steps.

Rational Development of β-Peptide Inhibitors of Human Cytomegalovirus Entry

Human cytomegalovirus (HCMV) is a pervasive and significant pathogen. At present, there is no HCMV vaccine, and the available drugs target only replication events. Thus, new therapeutic strategies are needed. HCMV fusion appears to require interactions of alpha-helical regions in viral surface glycoproteins gB and gH. Oligomers of beta-amino acids ("beta-peptides") are attractive unnatural scaffolds for mimicry of specific protein surfaces, because beta-peptides adopt predictable helical conformations and resist proteolysis. Here, we report the development of beta-peptides designed to mimic the gB heptad repeat and block HCMV entry. The most potent beta-peptide inhibits HCMV infection in a cell based-assay with an IC50 of approximately 30 microm. Consistent with our structure-based design strategy, inhibition is highly specific for HCMV relative to other related viruses. Mechanistic studies indicate that inhibitory beta-peptides act by disrupting membrane fusion. Our findings raise the possibility that beta-peptides may provide a general platform for development of a new class of antiviral agents and that inhibitory beta-peptides will constitute new tools for elucidating viral entry mechanisms.

Human cytomegalovirus entry into epithelial and endothelial cells depends on genes UL128 to UL150 and occurs by endocytosis and low-pH fusion.

Human cytomegalovirus (HCMV) replication in epithelial and endothelial cells appears to be important in virus spread, disease, and persistence. It has been difficult to study infection of these cell types because HCMV laboratory strains (e.g., AD169 and Towne) have lost their ability to infect cultured epithelial and endothelial cells during extensive propagation in fibroblasts. Clinical strains of HCMV (e.g., TR and FIX) possess a cluster of genes (UL128 to UL150) that are largely mutated in laboratory strains, and recent studies have indicated that these genes facilitate replication in epithelial and endothelial cells. The mechanisms by which these genes promote infection of these two cell types are unclear. We derived an HCMV UL128-to-UL150 deletion mutant from strain TR, TRdelta4, and studied early events in HCMV infection of epithelial and endothelial cells, and the role of genes UL128 to UL150. Analysis of wild-type TR indicated that HCMV enters epithelial and endothelial cells by endocytosis followed by low-pH-dependent fusion, which is different from the pH-independent fusion with the plasma membrane observed with human fibroblasts. TRdelta4 displayed a number of defects in early infection processes. Adsorption and entry of TRdelta4 on epithelial cells were poor compared with those of TR, but these defects could be overcome with higher doses of virus and the use of polyethylene glycol (PEG) to promote fusion between virion and cellular membranes. High multiplicity and PEG treatment did not promote infection of endothelial cells by TRdelta4, yet virus particles were internalized. Together, these data indicate that genes UL128 to UL150 are required for HCMV adsorption and penetration of epithelial cells and to promote some early stage of virus replication, subsequent to virus entry, in endothelial cells.

Integrin αvβ3 is a coreceptor for human cytomegalovirus

Human cytomegalovirus (HCMV) is a widespread opportunistic pathogen that causes birth defects in newborns and severe disease in immunocompromised individuals. The broad tropism of HCMV infection suggests that it uses multiple receptors. We recently showed that the epidermal growth factor receptor (EGFR) serves as a receptor for HCMV. Here we show that HCMV also uses integrin alphavbeta3 as a coreceptor. Upon infection, HCMV glycoproteins gB and gH independently bind to EGFR and alphavbeta3, respectively, to initiate viral entry and signaling. Alphavbeta3 then translocates to lipid rafts where it interacts with EGFR to induce coordinated signaling. The coordination between EGFR and alphavbeta3 is essential for the early events of HCMV infection, including viral entry, RhoA downregulation, stress-fiber disassembly and viral nuclear trafficking. Our findings support a model in which EGFR and alphavbeta3 work together as coreceptors for HCMV entry and signaling. This discovery is fundamental to understanding HCMV pathogenesis and developing treatment strategies targeted to viral receptors.

Cellular integrins function as entry receptors for human cytomegalovirus via a highly conserved disintegrin-like domain

Human cytomegalovirus (HCMV) is capable of manifesting disease in nearly every organ system in immunocompromised patients. This broad pathogenic tropism correlates with the ability of the virus to infect all tested vertebrate cell types in vitro, a characteristic that has made receptor identification extremely difficult. During virus entry, HCMV induces cellular morphological changes and signaling cascades consistent with engagement of cellular integrins; however, HCMV structural proteins do not possess the widely used RGD integrin-binding motif. We identified an integrin-binding disintegrin-like domain within HCMV envelope glycoprotein B, a protein required for virus entry and fusion throughout the Herpesviridae. Accepted receptor criteria are met through the use of function-blocking integrin Abs, beta1 integrin knockout mouse fibroblasts, and glycoprotein B disintegrin-like peptides, all of which support a critical role for alpha2beta1, alpha6beta1, and alphaVbeta3 integrins as HCMV entry receptors and signaling mediators acting during the penetration stage of the entry pathway. Strikingly, the glycoprotein B disintegrin-like domain is conserved in many human and animal herpesviruses, suggesting that integrins may support entry across this medically important virus family.

Human cytomegalovirus carries a cell-derived phospholipase A2 required for infectivity.

Human cytomegalovirus (HCMV) is known to carry host cell-derived proteins and mRNAs whose role in cell infection is not understood. We have identified a phospholipase A2 (PLA2) activity borne by HCMV by using an assay based on the hydrolysis of fluorescent phosphatidylcholine. This activity was found in all virus strains analyzed and in purified strains. It was calcium dependent and was sensitive to inhibitors of cytosolic PLA2 (cPLA2) but not to inhibitors of soluble PLA2 or calcium-independent PLA2. No other phospholipase activity was detected in the virus. Purified virus was found to contain human cellular cPLA2alpha, as detected by monoclonal antibody. No homology with PLA2 was found in the genome of HCMV, indicating that HCMV does not code for a PLA2. Decreased de novo expression of immediate-early proteins 1 and 2 (IE1 and IE2), tegument phosphoprotein pp65, and virus production was observed when HCMV was treated with inhibitors of cPLA2. Cell entry of HCMV was not altered by those inhibitors, suggesting the action of cPLA2 was postentry. Together, our results indicate a selective sorting of a cell-derived cPLA2 during HCMV maturation, which is further required for infectivity.

Receptors and immune sensors: the complex entry path of human cytomegalovirus

The ability of human cytomegalovirus (HCMV) to infect an extensive range of cell types has complicated efforts to identify cellular receptors for this significant pathogen. Recent findings demonstrate that epidermal growth factor receptor (EGFR) serves also as a receptor for HCMV. Additional evidence has shown that HCMV entry occurs in concert with immune detection through toll-like receptors. Here, the implications of EGFR activation, the existence of other receptors and the coordination of entry with the innate sensing are discussed.

Microtubule network facilitates nuclear targeting of human cytomegalovirus capsid.

We assessed the requirement of the host cytoskeleton for the intracytosolic transport of incoming human cytomegalovirus (HCMV) capsids. Treatments with microtubule (MT)-depolymerizing drugs nocodazole and colchicine led to a drastic decrease in levels of IE1 antigen, whereas cytochalasin B had no effect on the level of IE1 as determined by Western blot analyses. Sequential treatment including nocodazole washout and removal of cell surface virion revealed that HCMV entry into the cells occurred normally in the absence of the MT network. This finding was also supported by data obtained by monitoring pUL83 signals with an immunofluorescent assay (IFA). Furthermore, we demonstrated a close association of incoming HCMV capsids with MTs by IFA and ultrastructural analyses. In the absence of the MT network, the capsids which had entered the cytoplasm did not move to close proximity of the nucleus. These data suggest that HCMV capsids associate with the MT network to facilitate their own movement to the nucleus before the onset of immediate-early (IE) gene expression and that this association is required to start efficient IE gene expression.

Impaired Killing of HCMV-Infected Retinal Pigment Epithelial Cells by Anti-pp65 CD8+Cytotoxic T Cells

Host defense against infection by human cytomegalovirus (HCMV) is ensured in great part by cytotoxic CD8(+) T lymphocytes (CTLs) directed against the tegument protein pp65. The hyperimmediate release of incoming pp65 into the major histocompatibility complex (MHC) class I pathway after fusion of the virus with the cell membrane provides a very early mechanism of defense. In retinal pigment epithelial (RPE) cells HCMV is known to enter through endocytosis. This study was conducted to determine whether this means of penetration into the cells would allow the virus to elude immune surveillance. Infection of RPE cells with HCMV AD169 was performed for 6 hours, 48 hours, and 8 days. Expression of intracellular pp65 in RPE cells and in the astrocytoma reference cell line U373MG was evaluated by flow cytometry, fluorescence microscopy, and Western blot analysis. Killing of both HCMV-infected cell lines by HLA-A2-restricted CD8(+) CTLs directed against pp65 was monitored by (51)Cr-release assays. RPE cells were not lysed by CTLs directed against incoming pp65, contrary to U373MG. Moreover, both cell lines were not killed by anti-pp65 CTLs later after infection, because of the MHC class-I-downregulating effect of HCMV unique short (US2-11) proteins. In RPE cells, both HCMV entry through endocytosis and the immunosuppressive effect of US proteins could allow the virus to evade immune surveillance at any stage of infection, which could promote viral spreading within the retina.

Altered cellular mRNA levels in human cytomegalovirus-infected fibroblasts: viral block to the accumulation of antiviral mRNAs.

The effect of human cytomegalovirus (HCMV) infection on cellular mRNA accumulation was analyzed by gene chip technology. During a 48-h time course after infection of human diploid fibroblasts, 1,425 cellular mRNAs were found to be up-regulated or down-regulated by threefold or greater in at least two consecutive time points. Several classes of genes were prominently affected, including interferon response genes, cell cycle regulators, apoptosis regulators, inflammatory pathway genes, and immune regulators. The number of mRNAs that were up-regulated or down-regulated were roughly equal over the complete time course. However, for the first 8 h after infection, the number of up-regulated mRNAs was significantly less than the number of down-regulated mRNAs. By analyzing the mRNA expression profile of cells infected in the presence of cycloheximide, it was found that a minimum of 25 mRNAs were modulated by HCMV in the absence of protein synthesis. These included mRNAs encoded by a small number of interferon-responsive genes, as well as beta interferon itself. Cellular mRNA levels in cytomegalovirus-infected cells were compared to the levels in cells infected with UV-inactivated virus. The inactivated virus caused the up-regulation of a much greater number of mRNAs, many of which encoded proteins with antiviral roles, such as interferon-responsive genes and proinflammatory cytokines. These data argue that one or more newly synthesized viral gene products block the induction of antiviral pathways that are triggered by HCMV binding and entry.