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Address correspondence to M. Kent Froberg, DVM, MD,Department of Pathology and Laboratory Medicine, Universityof Minnesota–Duluth School of Medicine, 1035 UniversityDrive, Duluth, MN 55812, USA; tel 218 726 7223; fax 218726 7559; e-mail kfroberg@d.umn.edu.

Review: CMV Escapes!

M. Kent FrobergDepartment of Pathology and Laboratory Medicine, School of Medicine, University of Minnesota–Duluth,Duluth, Minnesota

Abstract. Cytomegalovirus (CMV) is an opportunistic pathogen that establishes life-long latent infectionwithout clinical disease in immunocompetent individuals, but can cause severe illness in newborns, transplantrecipients, and patients with HIV. CMV has evolved complex molecular mechanisms to avoid host immunedetection and destruction. Collectively these mechanisms have been termed “immunoevasion” or“escapology.” Perhaps the most essential mechanism of virus survival within the host is latency, a form ofreversible nonproductive infection of host cells by replication-competent virus. During periods of activevirus replication, however, there are multiple strategies by which CMV evades host defenses. These includemethods referred to as camouflage, which aid the virus in hiding from immune defenses, and those referredto as sabotage, whereby the virus disrupts or manipulates host inflammatory or immune responses. Theultimate pathogen survival strategy, host cell transformation, has been demonstrated in vitro for CMV, butto date has not been demonstrated in vivo. This review surveys the current literature on CMV immunoevasionand suggests a paradigm whereby CMV survives host defenses and contributes to atherogenesis. (received 20

November 2003; accepted 28 November 2003)

Keywords: cytomegalovirus, escapology, immunoevasion, cytokines, atherosclerosis

Introduction

Cytomegalovirus is a ubiquitous beta-herpesviruswhich causes most adult persons to become sero-positive by the age of 35 yr [1]. Most CMV infect-ons are mild or asymptomatic. However, like otherherpes viruses, CMV establishes life-long latentinfection with the potential of causing clinical diseasefollowing reactivation. Acute clinical illness is mostoften seen in neonates, solid organ or bone marrowtransplant patients, or patients with AIDS. Whilethe extremes of infection, latent virus withoutclinical disease vs life threatening disease in immuno-compromised patients, are well recognized, the invivo battle where neither virus or host appears towin, is less clearly defined.

There is substantial evidence that CMVinfection plays a role in atherosclerosis, restenosisafter angioplasty, and the de novo atherosclerosis thatmay arise following heart transplantation [2-5].Since atherosclerosis is a chronic inflammatorydisease of blood vessel walls, and latent CMVinfection occurs in tissues at these sites, one mayreason that reactivation of the virus could contributeto the chronic inflammatory process and exacerbateor accelerate vascular lesions. Understanding howCMV survives in these tissues and the mechanismsof reactivation and immunoevasion could establishthe extent to which CMV contributes toatherogenesis and could lead to new methods tosuppress the reactivation of CMV and its accom-panying inflammatory cascade.

CMV has a large genome compared to manyviruses and appears to have acquired several hostgenes over a long period of co-evolution with itshost [6-9]. Many of these genes allow CMV tomodulate both the host immune response and virus

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replication in the face of host cell mitogenic orproinflammatory activation. Collectively these genesand proteins have been termed “immunoevasins”and the overall process termed viral “escapology” [7,8]. CMV avoids immune detection throughcamouflage, the art of hiding from host defenses, aswell as subverting the immune system by sabotage,the synthesis of molecules that disrupt or manipulatehost immune/inflammatory responses. Virallyproduced molecules involved in these processesinclude cytokine homologs (virokines), solublecytokine scavengers that sequester free chemokines,and cell–surface receptor homologs (viroceptors) [7].Most studies of CMV escapology have been cond-ucted on human CMV (hCMV) or murine CMV(mCMV) using in vitro techniques. In combination,these immunoevasive activities may allow CMV tocontribute to atherosclerotic disease by surviving theonslaught of host defenses and contributing tochronic inflammation during periods of reactivation.Herein, we review the numerous and complex waysin which CMV interacts with host defenses and weattempt to formulate a hypothesis to define CMV’srole in atherogenesis.

Primary infection/latency

Like other herpes viruses, CMV establishes life-longlatent infection once the host is exposed [10,11].Replication is controlled by three groups of genestermed immediate early (IE), early (E), and late (L)phase genes. IE genes are under the control of themajor IE promoter (MIEP), which controls theexpression of two genetic elements IE-1 and IE-2.IE genes, in turn, regulate expression of E and Lgenes. MIEP is a potent transactivator of severalviral and host genes and along with IE genes appearsto play an important role in viral pathogenesis [12].IE gene products appear to be particularly importantin the proinflammatory response seen in CMVinfection and may (a) increase levels of adhesionmolecules such as ICAM, (b) increase productionof cytokines such as IL-1 and TNF-α; (c) upregulateseveral chemokines like MCP-1, MIP-1β, RANTES,and IL-8; and (d) stimulate expression of growthfactors and mitogens including IL-6, TGF-β andGM-CSF [13-16].

Control of latency is defined as the reversiblenon-productive infection of cells by replicative-competent virus and probably presents the best wayfor a virus to camouflage itself from immunedetection. It implies that either no or relatively fewviral genes are expressed and that no virions areproduced. CMV may infect and be transferredbetween monocytes, endothelial cells, and smoothmuscle cells, which are all central to atherogenesis.These cells may be activated by CMV infection andproduce proinflammatory cytokines and chemo-kines that attract inflammatory cells to sites ofvascular injury, adhesion molecules that enhancebinding of inflammatory cells to the vessel wall,mitogens that may stimulate replication of smoothmuscle cells or fibroblasts, and agents that stimulatelipid oxidation and free radical accumulation indamaged vascular walls [17-19]. The exact mechan-isms of latency and reactivation of CMV are notwell understood, but recent studies suggestreactivation may be more common than previouslybelieved and may occur with minor stress-relatedevents such as work overload, the occurrence of oralherpes, or alcohol ingestion [20]. These events,which are relatively common in many patients, couldprovide the CMV virus with multiple opportunitiesto contribute to the chronic inflammatory processwe know as atherogenesis.

Apoptosis/mitogenesis

A potential way by which CMV may prevent virionsfrom being exposed to host defenses would be toreduce apoptosis of CMV infected cells, thuslimiting the amount of extra-cellular virus releasedfrom these cells. Virally infected or damaged cellsmay undergo apoptosis as a host defense mechanismto maintain stable cell populations and perhaps limittissue injury or oncogenesis. Apoptotic cells arepresent in atherosclerotic lesions and may play asignificant role in restenosis following coronaryangioplasty [3,4,21]. Angioplasty induces excessivesmooth muscle cell proliferation, with rapidrestenosis occurring in 25–50% of patients under-going coronary angioplasty [4]. CMV appears toblock TNF-α induced apoptosis through the actionof IE1 and IE2 genes [22-25]. p53 is a nucleoprotein

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that directs cells to programmed cell death, and thehCMV protein IE2-84 binds to and inhibits p53transcriptional activity. p53 is sequestered withinthe cytoplasm of CMV-infected human endothelialcells in vitro [26]. By inhibiting apoptosis, CMVmay contribute to the excessive proliferation of SMCseen in restenosis.

CMV genes also induce proliferation of hostcells and cause transformation of several cell lines invitro. The MIEP of CMV transactivates NFκB,which in turn activates several host and viral genesenhancing viral replication and infectivity [27,28].Indeed, inflammation and activation of macrophagescan lead to upregulation of PGE2, TNF-α, and IL-1β, which can alone or synergistically upregulateMIEP in vitro [29,30]. Thus, it is possible thatactivation of macrophages can lead to reactivationof CMV from the latent state facilitating viralpathogenesis within atherosclerotic lesions. Otherfactors that appear to activate CMV include reactiveoxygen species (ROS) and nitric oxide (NO) [18,19,31]. ROS are generated during atherogenesis, andNO is produced by activated macrophages andendothelium, suggesting that renewal of inflam-mation within an atheromatous plaque couldreactivate latent virus within the lesion and escalatethe degree of tissue injury. These in vitro obser-vations are supported by data indicating cyclo-oxygenase inhibitors may reduce the thrombogenictendency of atheromatous lesions and may reduceCMV-induced production of ROS as well as CMVreplication in smooth muscle cells [32].

Blocking antigen presentation

While latency may be the ultimate method by whicha virus may survive within the host without exposureof viral antigens to host defenses, it has the distinctdisadvantage of preventing spread of the virus toother tissues or transmission to new hosts. CMV,in part, overcomes this by establishing latentinfection in most host tissues, but maintainingchronic persistent infection of salivary glands tofacilitate transmission to susceptible hosts. If hCMVis reactivated readily from relatively commonstresses, there may be frequent opportunities for thehost immune system to recognize and destroy the

virus. The CMV genome contains several openingreading frames (ORFs) that produce proteins thatblock or subvert viral antigen expression.

hCMV uses several strategies to inhibitpresentation of viral antigens in conjunction withMHC class I and MHC class II complexes. MHCclass I proteins are ubiquitously expressed andpresent antigen to CD8+ T lymphocytes, whileMHC II expression is limited to antigen presentingcells, B cells, and monocytes/macrophages, whichpresent antigen to CD4+ T lymphocytes. hCMVencodes several unique short (US) segments thatinhibit multiple steps of the antigen presentationpathway [33,34] (Fig. 1). US2 causes degradationof the MHC class I complex [34]. US6 inhibitspeptide translocation to the endoplasmic reticulumby the MHC-encoded TAP peptide transporter [35].US3 binds to MHC class I complexes and leads toretention of peptide-loaded complexes within theendoplasmic reticulum (ER) [36]. US11 dislocatesclass I complexes to the cytosol, where they arerapidly degraded [37]. Taken together, theseglycoproteins produce significant reduction of viralantigen presentation at the cell surface with MHCclass I complexes. This could potentially lead to anincrease in cell killing of CMV-infected cells bynatural killer (NK) cells, since these cells areinhibited from activation by the expression of MHCclass I antigens on the surface of host cells. hCMVmay overcome this problem by production of anMHC class I homolog, a product of the UL18 geneof hCMV, which can bind endogenous peptide, beexpressed on cell surfaces, and may protect virus-infected cells against NK cell cytotoxicity byengaging inhibitory receptors on the surface of NKcells [38-40]. Similar sequential disruption of MHCclass I processing has been demonstrated for mCMV[33,41,42].

CMV also interferes with antigen presentationby MHC class II complexes to CD 4+ lymphocytes.In vitro studies indicate that CMV inhibits MHCclass II expression through the Jak/Stat pathway bydecreasing levels of Janus kinase 1 [43]. The US2 ofhCMV destroys two essential proteins in the MHCclass II antigen presentation pathway [44]. MCMVinduces early host IL-10 production, which down-regulates MHC class II expression on macrophages

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Fig. 1. Human cytomegalo-virus disrupts presentationof viral antigens by theMHC Class I complex.Intracellular viral proteinsare normally cleaved by theproteasome, transportedinto the endoplasmic retic-ulum (ER) through the TAPtransporter, and bind to theMHC I complex for present-ation to CD8+ lymphocyteson the cell surface. US6blocks viral peptidetransport through TAP; US3retains the MHC Class Icomplex within the ER; andUS2 and US11 divert MHCheavy chains to the cytosolwhere they are degraded.(US = unique short, α =MHC heavy chains, β = β2-microglobulin)

Fig. 2. An atheromatouslesion depicting multiplecells, cytokines, chemokines,growth factors, lipids, andcomponents of free radicalformation that are commonto atherosclerosis and CMVinfection. (CMV = cytomeg-alovirus, ROS = reactiveoxygen species, ox-LDL =oxidized low density lipo-protein, VCAM = vascularcell adhesion molecule,ICAM = intercellular adhes-ion molecule, MCP-1 =monocyte chemoattractantprotein-1, M-CSF = mono-cyte-colony stimulatingfactor, PDGF = plateletderived growth factor, MΦ =monocytes/macrophages[purple], SMC = smoothmuscle cells [red], vascularendothelium [brown]).

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[45,46]. In addition, CMV produces its own IL-10homolog, which may inhibit host production ofcytokines including TNF-α, IFN-γ, IL-2, andgranulocyte-monocyte colony stimulating factor[47]. This could lead to a TH1/TH2 switch, sinceIL-10 is known to be inhibitory to the TH1 pathway.This may be important for virus survival, becauseprotection against CMV, which, like many intra-cellular pathogens, depends more on cell-mediatedimmune mechanisms. Extracellular viral antigensmay be neutralized by specific antibody recognition;however, both hCMV and mCMV produce an Fcreceptor that binds IgG [48,49]. Other viruses andsome bacteria also produce Fc receptors that mayreduce the efficacy of neutralizing antibodies,complement fixation, or interfere with antibody-mediated immune cytotoxicity.

G Protein-coupled receptor homologs

Both murine and human CMV contain ORFs thatencode G protein-coupled receptor homologs,which bind host chemokines and may sequesterchemokines from the extracellular environment [50].HCMV contains UL33, US27, and US28, whichare all transcribed during CMV infection [51].Unlike wild type virus, US28-deleted virus failed todownregulate either of the chemokines RANTESor MCP-1 in the medium of cultured fibroblasts[50]. Moreover, US28 mediates vascular smoothmuscle cell migration in vitro [52], which is animportant facet of atherogenesis. An mCMV ORFtermed M33 encodes a G protein-coupled receptorwith homology to UL33 [53]. An M33-deletedMCMV grows normally in vitro, but shows atten-uated growth in vivo. MCP-1 is a C-C chemokineproduced in vascular lesions; it causes chemotaxisof monocytes and T lymphocytes, which are majorcells found in atheromatous lesions. mCMVinfection accelerates inflammation in vascular tissuethat overexpress MCP-1 [54]. The present authoris investigating whether wild type mCMV is moreeffective than an M33-depleted strain of mCMV inreducing serum chemokine levels in MCP-1transgenic mice. If so, it would indicate that M33may sequester chemokines in vivo and modulate thehost inflammatory response.

Lipid metabolism and CMV

Hyperlipidemia, especially of low density lipoprotein(LDL), is a risk factor for atherosclerosis. The earliesthistologically recognizable lesion of atherogenesisconsists of a few lipid laden macrophages (foam cells)beneath the vascular endothelium. Oxidative modif-ication of LDL activates endothelial cells, macro-phages, and smooth muscle cells, and hasproinflammatory, proatherogenic and prothrombo-genic effects. A possible connection between CMVand lipid metabolism was first recognized whenMarek’s Disease Virus (MDV), a herpesvirus, wasshown to induce atherosclerotic lesions in the aortasof chickens as well as to alter lipid metabolism andlead to accumulation of cholesterol and cholesterolester within these lesions [55]. hCMV also increaseslevels of neutral lipids in cultured human saphenousvein smooth muscle cells [56]. In CMV-infectedmonocytes, the presence of oxidized LDL andendothelial cells within the culture mediumincreased MIEP activity over seven-fold [57].hCMV infection of human aortic smooth musclecells in vitro stimulated the expression of scavengerreceptor mRNA and the uptake of oxidized andacetylated LDL [18]. We reported that CMV-seropositvity is associated with higher serumcholesterol levels in young females, although theclinical significance of this finding is unclear [58].

Conclusions

Cytomegalovirus infection has been linked toatherosclerosis through epidemiologic studies, thefinding of viral DNA within atheromatous lesions,and studies demonstrating increased developmentof atherosclerosis in animal models [13,59].Together, these studies provide strong circumstantialevidence that CMV may be a risk factor foratherosclerosis. However, a causal relationship hasnot been proven and it is possible that themicroenvironment of atheromatous lesions is simplymore suitable to CMV survival. Even if CMV doesnot initiate vascular lesions, it may be an importantcontributing factor to atherogenesis by acceleratingor exacerbating existing lesions. hCMV infectionhas been shown to produce in vitro many of the

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inflammatory responses that are hallmarks ofatherosclerosis (Fig. 2). These responses are commonto many chronic inflammatory conditions, and arenot unique to either CMV infection or athero-sclerosis. Although CMV antigens and DNA canbe found in atheromatous plaques, other organisms,such as Chlamydia pneumoniae, may also be present[60]. However, these infections may be largely latentwith little or no inflammatory response attributableto the presence of the microorganism.

There is broad agreement that atherosclerosis isa chronic inflammatory response of the vascular wallto injury and may progress through multiple periodsof acute inflammation followed by relativequiescence [2]. Several important factors may allowCMV infection to contribute to formation of theatherogenic lesion: (a) reactivation from latencyappears to be a common event giving the virus ampleopportunity to contribute to atherogenesis; (b)CMV is often present in atheromatous plaques andvirus replication is induced by several factorsactivated during inflammatory episodes known tooccur during atherogenesis, again amplifying theopportunity for CMV to contribute to theinflammatory damage; (c) CMV may be capable ofcontributing to the lipid perturbations seen withinaffected vascular walls, and in particular may increasethe oxidation of LDL and affect cholesterolaccumulation (or conversely, CMV may simplythrive in the particular lipid microenvironmentfound in atheromatous lesions); (d) once activated,the IE genes of CMV in turn activate many pro-inflammatory host genes creating the probability ofa cyclic succession of inflammatory damage tovascular walls, no matter what the initiating factormay be; and (e) CMV dampens the inflammatoryassault by immunoevasive techniques that allowthe virus to survive in inflamed tissues untilreactivated by factors directly involving the arterialwall, or more distant factors that may elicit virusreactivation.

CMV infection or reactivation elicits and thendampens the inflammatory response. For example,IFN-γ and TNF-α are both upregulated inmonocytes following infection. However, viralinfection also induces differentiation of monocytesinto hCMV-permissive macrophages, which are

resistant to the antiviral effects of these cytokines[61], thus protecting viruses from the detrimentalaction of these cytokines. Despite recurrent orpersistent inflammation of the artery wall, CMVmay be able to survive within these vascular lesionsand persist through multiple episodes of reactivationand viral replication. In this manner CMV maycontribute to the chronic fibro-inflammatoryprogression of atheroma formation and vesselstenosis. Finding reactivated hCMV within earlyactive atheromatous lesions in young patients wouldsupport the concept that CMV could contribute toatherogenesis during lesion development. It wouldalso be useful to demonstrate virus reactivation inhumans or animal models in concert withexacerbations by other known risk factors, such asperiods of hypertension or hyperlipidemia.

It is likely that CMV infection or reactivationis simply one of many environmental factors thatmay contribute to atherogenesis. However, it maybe possible to control or prevent that contributionby controlling or eliminating the infection. If thatis the case, clinical improvement and prevention ofthe sequelae of atherosclerosis should be possiblewith adequate antiviral therapy.

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