HCMV INFECTION: MODULATING THE CELL CYCLE AND CELL DEATH

HCMV INFECTION: MODULATING THE CELL CYCLEAND CELL DEATH

JONATHAN P. CASTILLO and TIMOTHY F. KOWALIKProgram in Immunology and Virology,Department of Molecular Genetics and Microbiology,University of Massachusetts Medical School, Worcester, Massachusetts, USA

Human cytomegalovirus (HCMV) is a member of the Herpesviridae family and is recog-nized as a significant pathogen to certain subgroups of the human population. It has be-come apparent that HCMV manipulation of the host cell cycle as well as the immuneresponse promotes the replication and propagation of the virus. The ability of HCMVto modulate components of the host immune system and the response to infection mostlikely contributes to the pathology associated with this virus. This review will addressthe mechanisms HCMV has adapted to modulate the cell cycle to promote viral repli-cation as well as the different ways it can prevent the ‘‘death’’ of an infected cell.

Keywords: herpesvirus, cytomegalovirus, cell cycle, apoptosis, immune evasion

HUMAN CYTOMEGALOVIRUS

Background

HCMV, also referred to as human herpesvirus 5 (HHV5) is the proto-typic member of the subfamily Betaherpesvirinae. Like other betaher-pesviruses, HCMV exhibits a restricted host range such that it onlyproductively infects human cells. The virus contains a 230,000 basepair linear, double-stranded DNA genome, which is encased withinan icosahedral capsid and surrounded by a tegument and a lipidbilayer envelope containing numerous HCMV-encoded glycoproteins[1]. The HCMV genome is divided into two covalently linked segments,designated UL (unique long) and US (unique short), that are flanked by

Address correspondence to Timothy F. Kowalik, Program in Immunology andVirology, Department of Molecular genetics and Microbiology, University of Massachu-setts Medical School, Worcester, MA 01655, USA. E-mail: [email protected]

International Reviews of Immunology, 23: 113–139, 2004

Copyright # Taylor & Francis Inc.

ISSN: 0883-0185 print/1563-5244 online

DOI: 10.1080=08830180490265565

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inverted repeat sequences. Because of its large genome size, HCMVpossesses the highest potential coding capacity among the herpes-viruses. In fact, the HCMV genome encodes for over 200 open readingframes (ORFs), with only a subset of the gene products identified andfunctionally characterized.

Analogous to the other herpesviruses, HCMV replication is tightlyregulated and is dependent on a multistep process involving theordered expression of its viral gene products. Specifically, HCMV geneexpression occurs in sequential phases designated immediate early(IE), early, and late. Expression of the IE gene products occurs im-mediately after infection and takes place in the absence of de novo pro-tein synthesis. IE gene expression is required for the transcription ofearly genes, which encode factors needed for viral DNA replication.The expression of the late viral genes, which encode many HCMVstructural proteins, is further dependent upon the initiation of viralDNA replication [1].

Because of its high specificity for human cells, human fibroblastsare the cells most often used to analyze HCMV replication in vitro.There is considerable evidence that in vivo the virus targets a widevariety of cells including epithelial and endothelial cells, fibroblasts,smooth muscle cells, and peripheral blood leukocytes including mono-cytes and granulocytes [2]. Unlike the other herpesviruses, HCMV hasa slow replicative cycle (> 24h) and requires a longer period of time toinduce its cytopathic effects [1]. HCMV-infected cells typically becomeenlarged (cytomegalia) and eventually develop nuclear and cytoplas-mic inclusions that are characteristic of HCMV infection [2].

HCMV Pathogenesis

HCMV, which is also referred to as human herpesvirus 5 (HHV5),infects a large proportion of the population. Although the virus is en-demic within the population, HCMV infection rarely causes sympto-matic disease in healthy, immunocompetent individuals. Rather, likeother herpesviruses, HCMV can remain latent or persistant within ahealthy host indefinitely. The host immune system plays a crucialrole in regulating HCMV infection. Reactivation of latent virus inimmunocompromised and immunosuppressed individuals is associa-ted with many diseases, including HCMV-associated pneumonitisand retinitis. Additionally, the virus poses a serious threat to HIV-positive individuals because HCMV may accelerate the developmentof AIDS as well as contribute to the morbidity associated withincreased immunodeficiency [2]. Moreover, reactivation of latentHCMV or transmission of the virus to organ transplant recipients

114 J. P. Castillo and T. F. Kowalik

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can result in severe complications such as disseminated viremia and,in some instances, organ dysfunction.

HCMV infection is also problematic for pregnant women and chil-dren, especially infants. HCMV is recognized as the most common con-genital viral infection and is the leading cause of various neurologicalabnormalities associated with an infectious agent during early child-hood [3]. Infants congenitally infected with HCMV are prone to hear-ing loss, chorioretinitis, and other disorders involving the perceptualorgans (e.g., inner ears and eyes) and the central nervous system.

HCMV and Proliferative Disorders

There is a plethora of evidence suggesting a link between HCMV and anumber of proliferative disorders. Although HCMV does not appear tobe oncogenic, HCMV exhibits the capacity to transform rodent embryofibroblasts [4,5] and, in some instances, human cells [6]. Additionally,the detection of HCMV DNA and antigen in tumor tissues isolatedfrom patient biopsies, in addition to elevated HCMV antibody titersin these patients, imply a relationship between HCMV and severalcancers including cervical carcinoma, prostate cancer, and adenocarci-noma of the colon [7,8]. However, it is unlikely that HCMV directlycauses cancer since the low incidence observed for each of the cancerslinked to the virus do not reflect the ubiquitous nature of HCMVwithin the population. It has been suggested that HCMV may act asa coetiologic agent in the development of tumors through a ‘‘hit andrun’’ mechanism [9,10] in which HCMV promotes cellular transform-ation by causing genetic instability or by preventing cells from under-going apoptosis. Thus it appears that HCMV may contribute to tumorformation, but its specific role in the transformation process remainsundefined.

Recent evidence suggests that HCMV may also play a role in the de-velopment of restenosis, a disorder that is characterized by the over-proliferation, migration, and accumulation of arterial smooth musclecells (SMCs) along the vessel walls of coronary arteries followingballoon angioplasty [11,12]. The accumulation of SMCs often leadsto the reocclusion of the vessel, which can be worse than the initialblockage. The presence of HCMV DNA and protein in SMCs from rest-enotic lesions implies an association between the virus and restenosis.Additionally, epidemiological evidence suggest a correlation existsbetween HCMV seropositivity and restenosis occurrence becauseHCMV-seropositive patients exhibit a higher propensity of developingrestenosis following coronary angioplasty as compared to HCMV-seronegative patients [13].

Modulation of Cell Cycle and Apoptosis by HCMV 115

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Although there is no definitive proof that HCMV causes restenosis,it has been hypothesized that the virus promotes certain eventsthat contribute to restenosis. HCMV infection enhances SMCmigration in vitro [14], and this effect may be attributed to the in-creased expression of various chemokines such as RANTES, mono-cyte chemoattractant protein-1 (MCP-1), and interleukin-8 (IL-8)in HCMV-infected cells [15–17]. HCMV infection also induces theexpression of surface adhesion molecules that augment the migrationand adhesion of SMCs and inflammatory cells to the endothelium[16]. In addition, HCMV encodes a chemokine receptor (US28) that,when expressed on the surface of SMCs, promotes their cellularmigration in vitro [17].

In contrast to our understanding of HCMV and SMC migration, therelationship between HCMV and the overproliferation of SMCs re-mains vague. Studies examining the effect of HCMV on cellular pro-liferation have yielded conflicting results. Early studies on HCMVand the cell cycle suggested that HCMV-infected cells exhibitincreased rates of DNA synthesis [18–20]. Notably, HCMV infectioninduces proliferation in SMCs in vitro that may be attributed in partto the HCMV-mediated increase in NF-jB transcriptional activityand platelet-derived growth factor (PDGF) receptor in these cells[14,21]. Additionally, HCMV inhibits apoptosis in response to variousstimuli [9,22]. In this manner, HCMV may contribute to the develop-ment of restenosis by promoting the overproliferation of SMCs in vivoand abrogating their ability to undergo apoptosis. But contrary to theproliferative effects seen in SMCs, HCMV infection causes humanfibroblasts to undergo growth arrest [23–26]. Given the divergenteffects that HCMV mediates on the cell cycle, it remains unclear ifHCMV contributes to the overproliferation of SMCs in vivo. Theseobservations bring into question the issue of how HCMV alters the cellcycle signaling pathway(s) in general.

MODULATING THE HOST CELL CYCLE

Viruses and the Cell Cycle: Small DNA Viruses

There is a precedent for DNA viruses altering cell cycle control to theiradvantage. The small DNA tumor viruses, such as adenovirus, simianvirus 40 (SV-40), and human papillomavirus (HPV), can each perturbthe replication machinery of the host cell to facilitate the replication oftheir viral DNA [27]. Their ability to overcome the normal regulationof cell proliferation control is largely dependent upon their oncogeneproducts, which target and inactivate members of the Retinoblastoma


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(RB) family of proteins. The RB protein family consists of threemembers, p107, p130, and pRb, that function in the maintenance ofa quiescent cellular state as well as the regulation of the transitionfrom G0=G1 to S phase by modulating the activity of the E2F familyof transcription factors [28]. The E2F proteins play an essentialrole in regulating the expression of genes required for DNA replication[29]. Active, hypophosphorylated RB proteins repress the expression ofE2F target genes by binding to E2F proteins at their C-terminaltransactivation domain and recruiting transcriptional repressorsto promoters of target genes [28]. When the RB proteins becomehyperphosphorylated by cyclin=cyclin-dependent kinase complexesactivated during mid-late G1, the RB proteins no longer bind E2Fproteins, resulting in the derepression of E2F target genes [30].

Each of the small DNA tumor viruses encodes proteins that canbind to the RB proteins, displace their interaction with E2F, and al-leviate the repression of E2F proteins. The E1A protein of adenovirus,large T antigen of SV-40, and the E7 protein of HPV each contain anLxCxE motif that facilitates their binding to the RB ‘‘pocket domain’’,thereby displacing E2F proteins from their interaction with the RBproteins at this region [27]. The release of E2F proteins from the RBproteins enables E2Fs to transactivate the promoters of their targetgenes, including genes required for S phase [31,32].

A consequence of the aberrant growth signal that is induced whenRB proteins are inactivated by viral proteins is the activation of p53[33]. The p53 tumor suppressor protein is a sequence-specific DNA-binding transcription factor that transactivates the promoters of manyp53-responsive target genes including the cyclin-dependent kinase in-hibitor, p21, which mediates cellular growth inhibition [34,35]. Ad-ditionally, p53 induction can also trigger signals leading to celldeath or apoptosis. Each of the small DNA tumor viruses expressesa protein that binds and inactivates p53. The adenovirus E1B55kDa protein and the large T antigen of SV-40 bind p53 and inhibitits function [36–40]. In contrast, the HPV E6 protein promotes thedegradation of p53 through the ubiquitin independent proteolytic sys-tem [41]. In all of these instances, expression of the viral oncoproteinsalters p53 function to prevent the infected cell from undergoingp53-mediated growth arrest and=or apoptosis.

Because each of the small DNA tumor viruses infects quiescent cellsand lacks certain components needed to replicate their viral DNA, it isthought that manipulation of both the RB proteins and p53 facilitatesthe DNA replication of each of the small DNA tumor viruses bybypassing arrest and death signals and thereby forcing cells into theS phase.


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HCMVHCMV normally infects quiescent cells in vivo—cells that have lim-

ited quantities of deoxyribonucleotides and cofactors available for thereplication of viral or cellular DNA. This lack of ‘‘S phase constituents’’likely presents an unfavorable environment for viral DNA replication.Based on the precedence established with the small DNA tumorviruses, it would be advantageous for HCMV to modulate the cell cycleto maximize optimal viral DNA replication within the infected cell. In-deed, HCMV has been shown to have varying effects on the cell cycle.Some early studies show that HCMV infection stimulates cellularDNA synthesis [18–20]. However, these experiments were done incells that are not permissive to virus replication. In contrast to theseobservations, more recent studies suggest that HCMV induces a G1

and, occasionally, a G2=M growth arrest in productively infected hu-man fibroblasts [23–26]. Although these latter recent studies describea ‘‘G1 arrest’’ in infected cells, biochemically these cells exhibit hall-marks of early S phase entry including pRb hyperphosphorylation,increased E2F transcriptional activity, elevated cyclin E and cyclinA kinase activity, and expression of many S phase genes such as dihy-drofolic reductase (DHFR), DNA polymerase alpha, proliferating cellnuclear antigen (PCNA), and topoisomerase II [25,26,42]. In addition,HCMV infection of human endothelial cells or a differentiated monocy-tic cell line suggest that HCMV can drive some permissive cells into Sphase [14,43]. Together, these observations suggest that HCMV med-iates differing effects on the cell cycle.

Like the small DNA tumor viruses, HCMV infects noncycling cellsin vivo, and these cells are not likely to be conducive to viral DNA rep-lication. To bypass this situation, HCMV expresses several proteinsthat alter the cell cycle towards a more favorable, S phase-like en-vironment. As discussed below, HCMV also expresses several geneproducts that inhibit cell cycle progression, probably at the early Sphase-like state seen in infected fibroblasts. The ability of HCMV pro-teins to differentially modulate the cell cycle as summarized in Figure 1may represent a strategy that promotes viral DNA replication overcellular DNA replication. This section will review the biology of theseHCMV proteins, in particular their putative effects on the cell cycle.

pp71pp71 is an early gene product that is expressed from the UL82 ORF

and is packaged as a component of the viral tegument [44–46]. Follow-ing virus entry, pp71 localizes to the nucleus where it functions as atranscription factor that transactivates the major immediate early


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promoter (MIEP) [47]. Besides enhancing the transactivation of theMIEP, pp71 can bind to all three RB family members in vitro andcan accelerate the transition from G1 to S phase [48,49]. As a result,cells expressing pp71 are able to enter S phase and commence cellularDNA replication faster than cells that do not express pp71. Addition-ally, pp71 expression induces quiescent cells to enter S phase [48]. Stu-dies examining the relationship between pp71 and the cell cycle revealthat pp71 contains a sequence (LACSD) that is similar to the RB-bind-ing motif (LxCxE) that is present in adenovirus E1A, SV-40 large Tantigen, and HPV E7 [50]. Mutating residues within the pp71 LACSDmotif abrogates its ability to induce DNA synthesis in quiescent cellsbut does not affect the ability of pp71 to accelerate cells through G1

into S phase [50]. Taken together, the function of pp71 may be to drivequiescent, G0 cells into S phase by targeting and inactivating each ofthe RB protein family members.

IE1-72IE1-72 is expressed as a 491 aa nuclear protein from the UL123

ORF during the IE phase of infection. Transcription from this ORFgives rise to an alternatively spliced 1.95 kb mRNA that is the initialand most abundant viral transcript generated during HCMV infection[51]. Although it is not absolutely required for HCMV infectivity and

FIGURE 1 Modulation of the host cell cycle by HCMV. The RB family of pro-teins governs cell cycle progression from G1 to S phase. HCMV encodes severalgene products (shown in green) that target the RB family members to stimu-late cell cycle progression towards early S phase. However, progressionthrough S phase is prevented by the expression of gene products (shown inred) that causes cells to arrest in early S phase.


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viral DNA replication, IE1-72 transactivates the promoters of numer-ous HCMV early genes including gene products that facilitate the rep-lication process [52,53]. IE1-72 also interacts with the p107 proteinthrough a domain contained within the first 85 aa of the N-terminusof IE1-72 [54,55]. Binding of IE1-72 to p107 alleviates the p107-mediated repression of E2F-responsive promoters and abrogatesp107-mediated growth suppression [55]. IE1-72 expression stimu-lates transcription from the promoters of several E2F-responsivegenes such as DHFR and DNA polymerase a [56,57]. Therefore, itappears that IE1-72 can induce E2F activity; however, it is unclearwhether IE1-72 can activate the full repertoire of E2F-responsivegenes. Additionally, it has been suggested through in vitro kinaseassays that IE1-72 can exhibit kinase activity and phosphorylatep107 and p130 to disrupt their interaction with one member of theE2F family, E2F4 [58]. Besides p107 and p130, IE1-72 appears tophosphorylate the E2F proteins, E2F1, E2F2, and E2F3 in vitro [58]and can interact with E2F1 [56]; however, the significance of theseevents in virally infected cells is unknown.

Since p107-E2F complexes accumulate at or near the G1=S bound-ary and govern the transition from G1 to S phase [59], one mayhypothesize that the disruption of these complexes by IE1-72 shouldpromote S phase entry. The expression of IE1-72 can promote S phaseentry and delay exit from the cell cycle into G0, but only in cellslacking p53 or p21 [60,61]. In contrast, IE1-72 expression causeswild-type cells to arrest, most likely in G1. This effect is due toincreased levels of p53 protein resulting from IE1-72-mediatedinduction of p19ARF=Mdm2=p53 protein stabilization pathway andphosphorylation of p53 at Ser15, which enhances p53 transcriptionalactivity. Together, these changes to p53 levels and activity result ina p53-dependent induction of p21 expression and subsequent growtharrest [60].

IE2-86IE2-86 is a 579 aa nuclear protein that is expressed from the UL122

ORF during the IE phase of infection. Unlike IE1-72, IE2-86 is essen-tial for HCMV replication, as demonstrated by the inability to gener-ate infectious HCMV mutants lacking IE2-86 [62]. The failure ofHCMV to replicate in the absence of IE2-86 is consistent with its roleas a potent transactivator of numerous viral early genes required forvirus replication [53].

IE2-86 specifically interacts with pRb [55,63,64]. IE2-86 lacks theconsensus pocket domain-binding motif, LxCxE, and therefore doesnot bind to pRb at its pocket domain. Attempts to map the pRb-binding


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domain on IE2-86 show that it interacts with pRb through more thanone domain and that binding of IE2-86 to pRb relieves the pRb-mediated repression of E2F responsive promoters [63,64]. However,the precise mechanism by which IE2-86 blocks pRb function isunclear. IE2-86 induces the expression of numerous E2F-target genes,including factors associated with the bioenzymatic machinery neces-sary for DNA replication. For example, IE2-86 induces an increasein the mRNA levels of c-myc, cyclin E, cdk2, E2F1, ribonucleotide re-ductase 1 and 2, thymidine synthetase, MCM3, and MCM7 [65],implying that IE2-86 disruption of pRb-E2F complexes may freeE2Fs to transactivate their target genes.

Initial studies addressing how IE2-86 affects the cell cycle suggestthat IE2-86 may block cell cycle progression in G1 [23–26]; however,subsequent analyses indicate that IE2-86 induces cells to enter Sphase and can delay cells from exiting the cell cycle following serumwithdrawal [61,66]. The ability of IE2-86 to mediate these growth-pro-moting effects may be attributed in part to IE2-86 transactivation ofthe cyclin E promoter and induction of E2F activity [65,67]. Anotherpossibility is that IE2-86 binding to p53 [68] and p21 [43] inhibits theirrespective growth arrest functions, thereby contributing to the gener-ation of an ‘‘S phase-like’’ environment within HCMV-infected cells.

pUL69pUL69 is the product of the UL69 ORF and functions as a tegument

associated-transactivator that has sequence similarity to the herpessimplex virus ICP27 gene product [69,70]. The ability of pUL69 tofunction as a viral transactivator may be attributed to its interactionwith hSPT6, a protein involved in the regulation of chromatin struc-ture [71]. Additionally, transient expression of pUL69 in human fibro-blasts results in the accumulation of cells in the G1-phase of the cellcycle indicating that pUL69 inhibits cell cycle progression [72].Results of a subsequent study utilizing an HCMV mutant lackingtheUL69 coding region also demonstrates that pUL69 promotes an ac-cumulation of cells in G1-phase [73]. Although the mechanism bywhich pUL69 mediates the accumulation at G1-phase is not known,these findings demonstrate that a component of the HCMV tegumentcan mediate an immediate effect on the cell cycle. Furthermore, UV-inactivated HCMV can also mediate an inhibitory effect on the hostcell cycle, indicating that the presence of UL69 in virions may contri-bute to the growth arrest phenotype observed in cells following HCMVinfection [23–26].

Upon virus entry, the nuclear translocation of the pp71 tegumentprotein stimulates the cellular replication machinery and produces


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an S phase-like environment. In addition to pp71, the expression ofIE1-72 and IE2-86 continues to promote this S phase-like state duringthe later stages of infection. However, pUL69, and possibly IE1-72,negatively affect further cell cycle progression by limiting the cell cycleto an early S phase-like state. Through this approach, which may wellinvolve other viral proteins, HCMV activates host cell factors that fa-cilitate the replication of the viral genome and at the same timeensures that replication of viral DNA takes precedence over cellularDNA replication within the infected cell.

BLOCKING CELL DEATH

Inhibiting Extrinsic Killing of the Infected Host Cell

From the standpoint of the host, it is imperative to limit the spread ofthe invading virus to the surrounding cells. One way that this is ac-complished is through the action of cytotoxic T-lymphocytes (CTLs),which regulate viral dissemination by targeting the infected host cellfor destruction. During the course of a viral infection, the T cell recep-tor complexes that are expressed on CTLs recognize MHC class I mole-cules on infected and antigen-presenting cells. The MHC class Imolecules present peptides derived from processed viral antigens tothe CTLs, thus activating them. The activated CTLs eventually pro-mote lysis of the infected cell and the secretion of factors that furtherenhance the antiviral response.

In addition to CTLs, natural killer (NK) cells can also lyse targetcells, albeit in a less specific manner as compared to the CTLs. Theability of a NK cell to kill a target cell is governed by the engagementof MHC class I molecules on the target cell with receptors expressed onthe surface of the NK cell. At present, two types of NK cell receptorsthat vary by the sequence motifs contained in their cytoplasmicdomains have been identified [74]. One type of receptor containsimmunoreceptor tyrosine-based activation motifs (ITAMs) in its cyto-plasmic tail that, upon receptor engagement, triggers a signaling cas-cade that activates the NK cell. Once activated, the NK cell lyses thetarget cell. The other type of NK cell receptor contains immunorecep-tor tyrosine-based inhibitory motifs (ITIMs) that block the activationsignals and in effect neutralize NK cell activity.

HCMV has evolved numerous strategies to impede the cytolytic ac-tivity of CTLs and NK cells to subvert this aspect of the host immuneresponse. HCMV encodes several gene products (the US2, US3, US6,and US11 proteins) that can interfere with the presentation of viralantigen by the MHC class I molecules on the surface of infected cells.


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As shown in Figure 2A, each protein disrupts a particular step used inthe pathway to generate MHC class I-peptide complexes. BecauseHCMV can interfere with the surface expression of MHC class I mole-cules, one would expect HCMV-infected cells to be more susceptible toNK-cell mediated lysis since NK cells kill target cells that do not ex-press MHC class I molecules on their surface. Therefore, it is advan-tageous for the virus to also block this form of killing. HCMV mayinhibit NK-cell–mediated lysis though the expression of viral proteinsthat engage NK cell receptors (UL18, gpUL40, and gpUL16) or bymodulating the levels of certain surface proteins on the infected cell,as shown in Figure 2B. The mechanisms employed by HCMV proteinsthat enable the infected cell to elude CTL-mediated and NK-cell–mediated killing will be addressed in this section.

US3US3 is a 23 kDa glycoprotein that is expressed from the US3 ORF

during the IE phase of infection [75,76]. The US3 protein localizes tothe endoplasmic reticulum (ER), where it binds to peptide-loadedMHC class I molecules and retains them in the ER. US3 expressionis effective in blocking the transition of specific MHC class I alleles(HLA-A, HLA-B, HLA-C, and HLA-G) to the Golgi apparatus [77].

US2 and US11The US2 and US11 ORFs encode type I glycoproteins that are

expressed during the early phase of infection [78]. Both the US2 andUS11 proteins enhance the degradation of nascent MHC class I mole-cules by promoting the relocalization of the MHC class I heavy chainsfrom the ER to the cytosol. In both instances, the heavy chains areredirected into the cytosol through the Sec61 core complex on theER membrane in an ATP-dependent and redox-sensitive manner[79,80]. Once in the cytosol, the heavy chains are degraded by the pro-teosome. Recent evidence has shown that the US2 and US11 proteinscan target the heavy chains from distinct MHC class I molecules(HLA-A and HLA-B) for destruction [81,82]. However, the heavychains derived from the HLA-E or HLA-G loci appear to be resistantto US2- and US11-directed degradation [83]. The mechanism ofMHC class I heavy chain degradation mediated by US2 or US11 isanalogous to an endogenous cellular process that eliminates misfoldedor improperly assembled protein complexes formed in the ER [79,84].Therefore, it is feasible that US2 or US11 may function to mimic thisprocess on the heavy chains of select MHC class I molecules.


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US6US6 is a 21 kDa type I glycoprotein that is expressed from the US6

ORF during both early and late phases of HCMV infection [85,86]. Inaddition to its membrane-bound form, US6 is expressed as a soluble

FIGURE 2 HCMV blocks CTL-mediated and NK-cell–mediated lysis of theinfected host cell through several mechanisms. (A) HCMV-infected cells escapeimmune detection by CTLs. HCMV encodes several gene products (shown inred) that alter various steps in the MHC class I processing and presentationpathway. These proteins act in concert to hinder the processing and presen-tation of virus-derived peptides by MHC class I molecules, thereby allowingthe infected host cell to elude virus-specific CTL and killing. (B) HCMVimpedes NK-cell–mediated killing of the infected host cell. NK cells expressreceptors, which upon binding to their respective ligands either activate or in-hibit NK cell activity. HCMV expresses a number of gene products that func-tion alone or in conjunction with host cell proteins to block NK cell activation.Green lines depict pathways that block NK cell activity to promote the sur-vival of the infected host cell. Orange lines represent pathways that triggerNK cell activity and result in NK-cell–mediated lysis of the infected cell. Dot-ted lines represent pathways and their putative effects on NK-cell–mediatedlysis.


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protein lacking both the transmembrane region and cytoplasmic tail.Both forms of US6 protein transiently interact with the transporterassociated with antigen processing (TAP) in the ER lumen [87]. Thisinteraction occurs when the TAP forms a complex with MHC class Iheavy chain and b2 microglobulin and does not hinder peptide bindingto the TAP. It is hypothesized that US6 inhibits peptide translocationacross the ER membrane by blocking the exit pore of the TAP complex[88,89]. Through this process, US6 may prevent the loading of pep-tides onto the MHC class I molecules in the ER.

pp65pp65 is a tegument phosphoprotein that exhibits kinase activity. In

contrast to the immunomodulatory proteins encoded within the US re-gion of the viral genome that target MHC class I complexes, the pp65protein appears to influence the activity of a specific subset of CTLs bymodulating the processing of a particular HCMV antigen, IE1-72.During the IE and early phases of infection, a strong CTL response

FIGURE 2. (continued )


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is directed against peptides derived from IE1-72 [90]. However, whenIE1-72 and pp65 are coexpressed, the CTL response to IE1-72 isdiminished [91]. Although it is unclear how pp65 may contribute tothis reduction in CTL activity, it seems that the modification of IE1-72, possibly through phosphorylation, may interfere with its proces-sing and=or degradation.

UL18UL18 is an MHC class I homolog that is expressed from the UL18

ORF and is capable of binding to b2 microglobulin and peptide [92].The UL18 protein is a type I transmembrane protein that interactswith leukocyte immunoglobulin-like receptor 1 (LIR-1), a member ofthe immunoglobulin-like killer inhibitory receptors that is expressedon the surface of a small subset of NK cells [93,94] (Figure 2B). Itwas originally speculated that binding of UL18 to LIR-1 on NK cellsinhibits NK cell cytolytic activity. Indeed, transient expression ofUL18 in an MHC class I-deficient cell line results in increased resist-ance to NK-cell–mediated lysis [95,96]. Subsequent analyses revealedthat UL18 expression in other cell types enhanced NK-cell–mediatedkilling of target cells [97,98]. Furthermore, recent evidence demon-strating that LIR-1 has been detected on the surface of monocytes,dendritic cells, and B-cells suggests that UL18 may affect the activityof these cells [99]. However, the function of UL18 in vivo has yet to bedetermined.

gpUL40gpUL40 provides a peptide that is utilized in the maturation of

the nonclassical MHC molecule, HLA-E [100]. In contrast to theother HLA alleles, HLA-E specifically binds to and presents peptidesthat are derived from the leader sequence of classical MHC class Imolecules [101]. HLA-E surface expression is dependent on bindingto the MHC class-I–derived peptide. The leader sequence of gpUL40contains an HLA-E–binding peptide that is homologous to thepeptide produced from the MHC class I signal sequences. Binding ofthe gpUL40-derived peptide to HLA-E enhances HLA-E surfaceexpression and confers onto the target cell resistance to NK-cell–mediated lysis [100,102]. Contrary to this, cells infected witha gpUL40-deletion mutant virus demonstrate a higher susceptibilityto NK-cell–mediated killing [103], implying that gpUL40 appears toplay an important role in protecting HCMV-infected cells from beinglysed by NK cells. Additionally, HLA-E interacts with the NKcell CD94=NKG2 inhibitory receptor to suppress NK-cell–mediatedkilling [104].


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gpUL16gpUL16 is a nonessential gene product that is expressed as a type I

membrane glycoprotein and as a soluble protein [105]. In its solubleform, gpUL16 can bind to the MHC class I homolog (MICB) and tothe UL16-binding proteins (ULBPs) [106], both of which are ligandsfor the NKG2D=DAP10-activating receptor that is expressed on NKcells and on a small subset of T cells [107]. Binding of MICB and theULBPs to NK2D=DAP10 activates NK cells in vitro, and this interac-tion can be blocked by soluble gpUL16 [106]. Thus, HCMV-infectedcells may be able to repress NK cell activity by sequestering the acti-vating ligands recognized by receptors.

Altering Expression of Surface MoleculesAltering the expression of certain surface molecules may be another

method employed by HCMV to deter killing of the infected host cellby NK cells. One molecule whose surface expression may be alteredby HCMV is leukocyte functional antigen-3 (LFA-3). Under normalconditions, LFA-3 binds to CD2, which is expressed on the surfaceof NK cells, to strengthen the interaction between NK cells and theirtarget cells. Certain strains of HCMV can cause the surface levels ofLFA-3 to decrease on infected cells, and this correlates with theincreased resistance of HCMV-infected cells to NK cytotoxicity [108].One possible explanation for this observation is that decreased levelsof LFA-3 on the cell surface may dampen the binding of an infected cellwith NK cells and may enhance the resistance of infected cells toNK cell-mediated lysis. however, this possibility is complicated bythe observation that HCMV clinical isolates confer resistance to NKcell-mediated killing without modulating LFA-3 surface expressionon infected cells. Therefore, the relationship between LFA-3 surfacelevels and NK cell activity remains unclear.

Inhibiting Intrinsic Killing (Apoptosis) of the Infected Host Cell

In response to negative stimuli such as viral infection, cells activatethe apoptotic program to cause their self-destruction. But maintainingcell viability until the end of the replication cycle is essential for maxi-mal virion production. Therefore, preventing the apoptotic processwithin an infected cell is a necessary step for sustaining an appropri-ate environment for virus replication. The small DNA tumor virusesencode proteins such as the adenovirus E1B protein, SV-40 large Tantigen, and HPV E6 protein that block p53 activity and inhibitp53-mediated apoptosis. As shown in Figure 3, HCMV interrupts


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several signaling pathways to override attempts by an infected cell toundergo apoptosis. HCMV proteins expressed from two particular loci,UL36-38 and UL122=123, have been shown to exhibit much of thisantiapoptotic activity. This section will cover the different ways thatHCMV can block apoptosis in an infected cell.

UL36-38The UL36-38 loci of HCMV gives rise to several distinct IE tran-

scripts, pUL36, pUL37, and pUL37� 1, through the activation of

FIGURE 3 HCMV inhibits apoptosis through multiple pathways. Pro-grammed cell death can be triggered within a cell through several means.HCMV can disrupt key events in the death receptor-signaling (TNF=Fas)pathway to prolong the survival of the infected host cell. Likewise, HCMVexpresses other proteins that can prevent programmed cell death by inducingfactors that promote cell survival. These effects most likely counteract theapoptotic signals generated by HCMV infection and the effects of certain viralgene products. Green lines represent viral-mediated events that are antiapop-totic. Orange lines are indicative of viral-mediated events that are proapopto-tic. Dotted lines represent putative effects on apoptosis.


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different promoters and alternative splicing events [109]. These pro-teins appear to obstruct the apoptotic signals that stem from the en-gagement of the tumor necrosis factor (TNF) family of receptorsincluding Fas. When Fas is bound by Fas-ligand (FasL) Fas-associateddeath domain (FADD) is activated, and this promotes the cleavage andactivation of procaspase-8 (FLICE). The active caspase-8 causes thesubsequent cleavage and activation of downstream effector caspaseswhich lead to the induction of apoptosis within the cell [110].

The product of the UL36 ORF, also referred to as viral inhibitor ofcaspase-8–induced apoptosis (vICA) can inhibit the signaling cascadeinitiated by the interaction of Fas and FasL. This signaling disruptionis accomplished by pUL36 binding to procaspase-8 and preventing itfrom being cleaved into its active form [111]. By suppressing recep-tor-induced cell death in this manner, pUL36 is functionally simi-lar to the FLICE-ligand inhibitor proteins (FLIPs), which inhibitFas- and TNF-mediated apoptosis [112].

Like pUL36, expression of the UL37 ORF gene product, pUL37,occurs during the IE phase of infection. The pUL37 protein is a heavilyglycosylated type I transmembrane protein that localizes to the mito-chondria following infection [113]. Similarly, the antiapoptotic ca-pacity of pUL37, which is also known as the viral mitochondrialinhibitor of apoptosis (vMIA), stems from its ability to inhibit down-stream events in the Fas-mediated apoptotic pathway.Although theprecise function of pUL37 is not known, pUL37 appears to act down-stream of caspase-8 activation and Bid cleavage but upstream of cyto-chrome C release [113]. Because pUL37 localizes to mitochondrialmembranes, it was originally hypothesized to be an HCMV-encodedform of the Bcl-2 protein. However, this does not appear to be the casesince pUL37 does not share amino acid sequence homology to the Bcl-2proteins and lacks the Bcl2-homology (BH) domains that are con-served in all Bcl-2 family members [113].

Expression through the UL37 ORF gives rise to an additional IEgene product, pUL37 exon 1 (pUL37� 1), which is identical in se-quence to pUL37 with the exception of one residue [114]. Recent evi-dence has demonstrated the importance of the pUL37� 1 protein tocell survival and virus growth. The inability to generate infectious vi-rus from BAC-derived mutants lacking pUL37� 1 hints at the impor-tance of this protein to HCMV replication [115]. In fact, infection withthe pUL37� 1 mutants results in extensive cell death. Analogous topUL37, pUL37� 1 localizes to the membrane of mitochondria [116].However, unlike pUL37, pUL37� 1 interacts with the adenosinenucleotide transporter, a pore complex expressed on the inner mem-brane of mitochondria that regulates cytochrome C release [115].


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pUL37x1 can block the release of cytochrome C to prevent the acti-vation of the Fas-mediated apoptotic pathway [113,116].

The combined activities of the three IE proteins expressed from theUL36-38 loci function to prevent the infected cell from undergoingapoptosis following the engagement of Fas and possibly other typesof death receptors.

UL122=UL123The gene products expressed from these loci may also exert an anti-

apoptotic effect on the host cell following HCMV infection. The pro-teins encoded by these ORFs are IE1-72, IE2-86, and IE2-55. TheIE2-55 gene product differs from IE2-86 because of an additional spli-cing event in exon 5 that results in the deletion of 155 amino acidsbetween residues 365–519 in the C-terminus [117].

Both IE1-72 and IE2-86 induce p53 protein [61,118]; howeveronly IE2-86 can interact with p53 and block its transactivation func-tion [12,68]. Therefore, one may assume that in addition to inhibitingp53-mediated growth arrest, IE2-86 may also block p53-mediatedapoptosis. Indeed, IE2-86 inhibits apoptosis of cells following treat-ment with doxorubicin, a DNA damage-inducing agent that inducesp53, suggesting that IE2-86 can suppress p53-mediated apoptosis fol-lowing DNA damage [119]. However, it should be noted that IE2-86fails to protect cells from apoptosis following UV irradiation [9], imply-ing that IE2-86 may be ineffective in blocking apoptosis mediated byother stimuli.

There is a growing line of evidence suggesting that IE1-72 and IE2-86 can interfere with the mechanisms involved with p53-independentapoptosis. IE2-86 can block apoptosis mediated by the TNF-mediateddeath receptor-signaling pathway [22]. Additionally, the expression ofIE1-72 and IE2-86 in a TAFII250-temperature-sensitive mutant cellline (ts13) is sufficient to block the apoptosis mediated by growth ofthe cells at the nonpermissive temperature [9]. Moreover, the resultsfrom a more recent study suggest that IE1-72 and IE2-86 activateAkt, a component of the phosphatidylinositide 30OH (PI3) kinase path-way, to mediate their antiapoptotic effects in ts13 cells [120]. The Aktkinase can phosphorylate a variety of substrates including Bad andcaspase-9, and the IjB kinase [121]. Although the precise mechanismby which expression of IE1-72 and IE2-86 activates Akt is not known,the phosphorylation of these particular substrates by Akt may deterthe cell from undergoing apoptosis.

Another way that the IE proteins can inhibit apoptosis is byactivating NF-jB. The NF-jB transcription factor family inducesthe expression of a family of genes that encode cellular inhibitors of


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apoptosis (cIAPs), which protect cells from apoptosis. All three of theseIE proteins are capable of inducing NF-jB activity or at least a compo-nent of NF-jB. While IE2-86 can induce NF-jB transcriptional ac-tivity, IE1-72 contributes to the induction of NF-jB transcription bytransactivating the promoter of one its constituent proteins, p65[122,123]. The transient expression of IE2-55 also increases NF-jBlevels by enhancing the transactivation of the promoter regulatingthe expression of the p65 and p105=p50 subunits of NF-jB[122,123]. Based on these observations, one prediction is that the in-duction of NF-jB by these IE proteins increases protection from apop-tosis and, as a consequence, should contribute to a more favorableenvironment for virus replication. However, this has not yet beendetermined.

CONCLUSION

From the standpoint of HCMV infecting a cell, it is imperative for thevirus to generate and sustain an environment that is conducive toviral replication. Although HCMV encodes numerous gene productsthat participate in viral DNA replication, it is apparent that HCMVutilizes components of the host cell cycle machinery to facilitate thereplication of its genome. By targeting p53 and the RB family of pro-teins, HCMV can stimulate the cell cycle to progress towards a morefavorable setting, namely an S phase-like state. While modulatingthe cell cycle, the virus also utilizes several tactics to delay thedestruction of the infected host cell from either internal (apoptosis)or external (cytolytic killing by CTLs and NK cells) assaults. Throughthese processes, the conditions needed for HCMV optimal replicationto occur are achieved and maintained within the infected cell.

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HCMV INFECTION: MODULATING THE CELL CYCLE AND CELL DEATH