Immunology Today, vol. 7, No. 2, 1986
reviewsCytomegalovirus-its cellular immunologyand biology
The biology of persistent virus infections presents many intriguing questions, which extend well beyond classical virology into molecular and cell biology and immunology. Here, Patrick Sissonsand his colleagues review the current state of knowledge of the biology and immunology of one particular medically important persistent human virus - human cytomegalovirus (HCMV) and they discuss insights into the normal and pathological virus-host relationship provided by recent work both on HCMV itself, and on its murine counterpart mouse cytomegalovirus (MCMV). HCMV is one of five human herpes viruses (the others being herpes simplex (HSV) 1 and 2, varicella zoster (VZV) and Epstein-Barr (EBV) viruses). Like the others HCMV persists following primary infection and may subsequently reactivate, and is widely prevalent, infecting 50-100% of adults depending on the particular population studied. EBV has, of course, long been of interest to immunologists because of its tropism for, and ability to transform, B cells, and because it appears to illustrate the importance of T-cell surveillance in containing ~iraiiy induced tumours. Of the other four HCMV as a particular propensity to produce severe morbidity and death in immunosuppressed subjects - principally allograft recipients (especially of bone marrow transplants) and more recently those with acquired immunodeficiency syndrome (AIDS). Current evidence suggests that HCMV displays a twosided relationship with the immune response, being controlled by it and yet capable of itself producing immunosuppression. Although clearly not unique among viruses in this respect (this may be something of a general strategy for persistent viruses with human T-cell leukaemia virus (HTLV)-III perhaps the ultimate example), the widespread distribution, and complex biology of HCMV, and the frequency with which it produces clinical problems, make it a challenging example to study. Molecular virology
HCMV is the largest herpes virus with a linear double stranded DNA genome of about 235 kilobase pairs (kbp), divided into long and short unique regions each flanked by inverted terminal repeat sequences (Fig. 1). The genome has the capacity to code for about 150 proteins, but their characterization is as yet incomplete. Although the molecular biology of HCMV has previously received less attention than that of HSV, the situation is now changing rapidly: the complete nucleotide sequence of FICMV will probably be known soon - this should accelerate characterization of the virus proteins by recognition of open reading frames, and prediction
MRCClinicalImmunologyResearchGroup, RoyalPostgraduateMedical School,London W12 OHS,UK ~) 1986, Elsevier Science Pubtishers B.V., Amsterdam
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49191861502.00
J.G.P. Sissons, L.K. Borysiewicz, Brian Rodgersand Diane Scott of the proteins from the nucleotide sequence. The virus genome is transcribed in a regulated sequence, broadly analogous to that of HSV, with serial transcription of three different classes of viral mRNAs. These are the immediate-early, early and late RNAs - the proteins they code for go by the same designation. The majority of the HCMV immediate-early (IE) genes, including the gene coding for the major 72kDa IE polypeptide, are transcribed from a restricted region of the long unique sequence of the virat genome, contained within the Hind IIIE fragment (Fig. 1). In the case of HSV it is known that the major immediate early proteins can act as trans acting factors - that is, they are capable of binding to and activating transcription from transcription units on independent duplex DNA molecules-their prime function being to activate transcription of the early virus proteins. It is now clear that the IE proteins of HCMV can also activate genes in trans~ : for instance, in short term transfection assays, the Hind IIIE fragment of HCMV DNA produced trans activation of transcription from both viral and cellular promoters. There is considerable current interest in the trans activating properties of other viral proteins, including the adenovirus Ela and HTLV tat proteins, and of oncogenes 2, and it is tempting to speculate that alteration in transcription of host cell genes might be responsible for some of the pathogenic effects of HCMV. The early viral mRNAs are then transcribed, virus DNA replication occurs, and following this the virus late RNAs are transcribed the proteins they code for are mainly the structural proteins of the virus particle. Expression of these individual sets of CMV proteins can be manipulated experimentally by the use of metabolic inhibitors, as shown in Fig. 2. This ability to manipulate expression of the stagespecific proteins, and the fact that the immediateearly and early proteins are only present in the infected cell and not in the virus particle itself, are both pertinent to the experiments discussed below. The replication strategy and sequence of protein expression of MCMV appear to be essentially similar to HCMV and its detailed molecular biology is also under study. However there is very little DNA homology between the two viruses. Cellular tropisms
The cytomegaloviruses (unlike HSV) show a high degree of species specificity, and will only productively infect cells of their particular species in vitro.
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Immunology Today, vol. 7, No. 2, 1986
IRL IRS TRS Thus HCMV cannot be studied in T,, UL /l~ US W r.tJ I~ ~ 235kb the mouse, and whilst there are. r.~ II Hind III E ~ J undoubtedly major similarities in m ECoRI J the biology of MCMV and HCMV, / / it may not necessarily always be Hind III e - region of major ~ ~i~ correct to extrapolate direct from IE transcription of HC R1 the mouse to the human virus. The fibroblast is the only cell that i i is permissive for a full cycle of 1.9skb HCMV replication in vitro, 5kb 2,2kb 1~75 / although one recent report 3 sug- Ilkb 2.15 / gests endothelial cells may also support virus replication (which major 72K IE protein might be relevant to patho~ - ~ Cellular homologies genesis). Other cell types may be Fig.1.6enomestructureand immediateearlytranscriptsof HCMV(Ad 169strain). infected but then presumably lack some host cell factor(s) required for further gene expression. For example MCMV and HCMV can latently bone marrow cells, following their infection with ~infect teratoma cell lines but only then enter a full HCMV in vitro 8'9 - about 10% of PBM expressed replicative cycle when the cell is induced to the major 72kDa HCMV immediate-early protein differentiate by chemical agents4's - t h e block here (assessed with a specific monoclonal antibody), and is at the level of transcription of the IE genes6. It is one study suggested this was mainly in monocytes8. not known whether this occurs in vivo but it is an It can be envisaged that even expression of the IE interesting possibility, perhaps particularly in the proteins alone might produce pathogenic effects context of haemopoietic cell differentiation. (by virtue of their trans activating properties discusThe important question of the sites at which sed above). Interestingly this expression was only CMV persists in vivo still awaits a definitive answer. readily apparent when the infecting virus was a MCMV persists in the salivary gland, and can also recent clinical isolate, as opposed to the usually latently infect mouse macrophages and possibly B used laboratory strains which have been passaged cells. HCMV can be identified in many different cells in fibrobtasts for years, such as AD169. This raises during disseminated clinical infection; however, it is the problem for other workers that although they much less certain in which cell(s) HCMV normally may be working on a molecularly better defined persists in the silently infected host - possible virus, its biology may be of uncertain clinical relecandidates are epithelial cells in salivary gland and vance! renal tubule. Particular uncertainty has surrounded Hybridization studies for virus DNA and for the question of the extent to which HCMV infects, mRNA transcripts would help resolve these quesand can replicate in, monocytes and lymphocytes tions. It is now known that HCMV DNA has regions (discussed in this journal in a recent Compass showing extensive sequence homology with normal article7). Although it is quite widely assumed that cellular DNA 10 , which makes selection of appropriHCMV does infect these cells, this is partly based on ate probes particularly important and interpretation the fact that the virus can be isolated from buffy of most previous studies difficult. In a very recent coat in patients, is transmitted by blood transfusion, report the EcoR1J fragment (from the region of IE and on a measure of extrapolation from the mouse. transcription see Fig. 1.) was used as a probe in a However it is difficult or impossible to establish sensitive in situ hybridization assay which detected productive infection in any peripheral blood mono- virus mRNA: Schrier et aL found that 0.5-2% of nuclear cell (PBM) in vitro. Two recent reports have PBM from normal subjects seropositive for HCMV suggested that limited virus gene expression may showed hybridization (this was without infection in occur in a small proportion of PBM, and also in vitro), and their preliminary evidence suggests most of the positive cells were T cells and monocytes 11. This is probably the hardest evidence yet for PBM being a normal site of persistence for HCMV, and further results using this approach will be i/ of great interest. Immediate early proteins rly proteins late proteins / Like the other herpes viruses, HCMV has been proposed as a Inhibitors Cycloheximide phosphono formate candidate oncogenic virus - one and actinomycin D tumour with which an associaFig.2. Sequentialsynthesisof HCMVproteins and thor r~etabolicinhibition. Inhibitionof protein syntion has particularly been sugthesis with cydofleximideallowsaccumulationof IEgene transcripts, and its removalin the presenceof gested is Kaposrs sarcoma (both the transcriptioninhibitoractinomycinD allowstheir expressionwithout latergene transcription.Expression of the late proteins can be prevented by the use of the virus DNA polymerase inhibithe African endemic and more
tor, phosphonoformate.
immunology Today, vol. 7, No. 2, 1986
reviews-
recent AIDS associated forms), which is thought to antigen, and that two independent populations of be of endothelial cell origin. This area cannot be MCMV specific Tc can then be detected. One is specific for structural (late) viral proteins, and the reviewed here, but it is worth noting that recent attempts to demonstrate HCMV DNA in the cells of other detects an antigen present on target cells AIDS associated Kaposi's sarcoma have been un- treated with cycloheximide and actinomycin D successful. Any causal association thus remains very which express only the MCMV IE proteins. Tc, showing similar specificity for cells expressing late much unproven. antigens and IE proteins, could be generated in Immunoloqical control vitro from adult mice which had been neonatally It seems increasingly likely that of the other four herpes viruses only HSV and VZV manifest classical infected with MCMV, although this required restimulation with antigen as well as IL-219: thus latency, and perhaps persist without replicating because they are latent in neuronal cells, which are memory Tc are maintained during latent or persislong-lived and do not divide. In contrast EBV, which tent infection. The specificity for cells expressing IE probably persists in oropharyngeal epithelial cells proteins has also been shown with Tc clones. The (which have a rapid turnover), seems to replicate at MCMV antigen recognized by the Tc which correlow level for much of the time in the normal lates with IE antigen expression awaits structural carrier 12. It is in this latter situation that the host definition - especially as the IE proteins have hitherto been thought of primarily as nuclear proteins. response may be crucial in keeping this replication Reddehase and colleagues term the antiqen LYDIEA in check at local sites and preventing dissemination. (lymphocyte detected IE antigen) and point to its How then does the persistently infected normal potential similarity with the lymphocyte determined host manage to live with CMV, (a lytic rather than a transforming herpes virus), and what happens membrane antigen (LYDMA) recognized by EBV when the normal relationship breaks down? As yet . specific To. This correlation of specificity with exthere is no complete answer to the question, but pression of a viral nuclear protein is particularly interesting in the context of the recent reported recent work provides some intriguing clues. recognition of the influenza nucleoprotein by cross MCMV and NK cells reactive 'flu specific Tc 20 - and its implication that Natural killer (NK) cells have a postulated role in viral determinants recognised by Tc do not necesvivo against tumours and virus infection; probably sarily have to be the conventional surface glycoprothe most convincing evidence for their playing a teins. role in controlling any virus infection comes from Studies of the relative protection against MCMV studies on MCMV. Genetically determined resist- conferred by transfer of these two different populaance to MCMV correlates with NK cell activity 13, tions of Tc would obviously be of interest. The role and homozygous beige mice (which have defective of delayed type hypersensitivity in MCMV infection NK cell function) are very susceptible to MCMV 14. still remains to be determined despite its demonEspecially persuasive evidence comes from the strated protective role in primary HSV infection in recent studies of Bukowski, Welsh and col- the mouse 21. leagues ls'16. They showed that mice depleted of NK cells with anti-asialo GM1 developed more serious MCMV and immunosuppression There is fairly clear evidence that MCMV can disease from MCMV is. Then using adoptiye transfer experiments they found that spleen cells con- establish persistent infection in immunocompetent ferred protection from lethal MCMV infection in cells: this definitely includes macrophages from suckling mice, and the phenotype of the protective which the virus can be recovered by co-cultivation, cell was that of an NK cell rather than a T cell; and probably a proportion of mouse B cells22, finally they conferred protection by transfer of a MCMV infection also impairs macrophage depencloned NK cell line. This protective effect of NK cells dent T-cell proliferation; this immunosuppressive did not apply to LCM virus infection and so cannot effect is apparently mediated through infection of be assumed to apply to all virus infections ~6, but the macrophage 23, and seems to have its counterthese experiments do suggest NK cells play an part in the human situation. There is also evidence important role at least in limiting the initial infection that MCMV can increase alloreactive respones to major histocompatibility (MHC) antigens and enwith MCMV. hance graft versus host reactions in transplanted MCMV specific cytotoxic T cells m i c e 24. It was shown some time ago that, just as for many other viruses, virus specific cytotoxic T cells Immunology of human CMV infection (To) are generated following MCMV infection, and There are obvious constraints on the approaches their presence correlates with recovery from which can be used in investigating human CMV infection ~7. However, recently Reddehase, Koszin- infection, but there have been recent studies of the owski and colleagues have analysed the T~ response NK and Tc killing of HCMV infected cells both in to MCMV in more detail, with particular reference vitro and in clinical studies. to its specificity for the staged expression of virus proteins ~8'19. They report that during primary HCMV and NK cells MCMV infection, T cells from lymph nodes can be As with other viruses, HCMV infected cells are expanded in vitro in interleukin 2 (IL-2) without more susceptible to NK lysis. A component of this
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enhanced susceptibility is independent of interferon recipients) 3°. This was without any restimulation in acting on the effector population, and related to a vitro, making it difficult to examine specificity in any detail (see below). virus induced change in the cell. HCMV infected cells show increased susceptibility to NK killing at the stage when the virus early antigens are expressed: the effector cells are Leu HCMV and immunosuppression 7 +, Leu 1 1+ large granular lymphocytes - the same Immunosuppressive properties have been attricells which mediate NK lysis of tumour cells25. In all buted to HCMV at a rather anecdotal clinical level systems in which NK cells have been studied there is patients with HCMV disease often have other continuing uncertainty about the target structures opportunistic infections - but there is accumulatwhich the NK cells actually recognize. Proposed ing experimental evidence to give weight to this candidates have included glycolipids, sugars and impression. It was shown some years ago that various membrane glycoproteins, including recently monocytes from patients with primary HCMV the transferrin receptor 26. In the case of virus mononucleosis were less able to support mitogen infected cells there is little general evidence to induced T-cell responses31, as were normal monosuggest that viral proteins themselves are NK cell cytes infected with HCMV in vitro 32. We have target structures, and it seems more likely that any recently shown that the ability of monocytes to such structure is also present on normal cells (a produce interleukin-I (IL-1) activity is abrogated by possible exception is HSV where one group 27 have HCMV infection in vitro; this is associated with the evidence for its glycoproteins being recognized by release of an inhibitor of IL-1 from the monocyte NK cells). We recently found a direct correlation cultures 33. This inhibitor is almost certainly a host between the susceptibility of virus infected cells to cell protein acting at the level of the IL-1 responding NK lysis, and their expression of transferrin cell, and shows similarities to inhibitors of IL-1 and receptor 28. In the case of HCMV the increase in thymocyte proliferation described by others 34"3s. Its transferrin receptor temporally associated with EA release is presumably induced by HCMV - other expression, but we were unable to inhibit NK lysis viruses we studied did not produce this effect. The with purified transferfin receptor. Thus despite character of this inhibitor, and whether it plays any these close correlations it seemed unlikely to be the normal regulatory role in vivo, remain to be detertarget structure for NK cells. The role of human NK mined, as does the extent to which HCMV is cells in vivo remains unknown, but it seems in- expressed in the infected monocytes - but here the herently unlikely they play more than an adjunctive virus may be illuminating an aspect of normal cell biology. Whether HCMV exerts immunosuppressive role to specific immunity. effects by direct infection of T cells is uncertain, and further experiments are needed which exclude any HCMV specific cytotoxic T cells Our approach to analysing the Tc response to effects from monocytes. There is a single recent report that HCMV can act HCMV, has been to study HCMV specific T cells in normal seropositive subjects (who have never had as a polyclonal activator of B cells36, which is of symptomatic HCMV infection), using the now usual course a property of EBV-this awaits confirmation. technique of secondary in vitro stimulation with antigen, and expansion of responding cells in IL-229. Clinical aspects All the above work bears directly on the clinical We found that when free virus was used as antigen, T4 + proliferative lines were generated but we were problem of HCMV. Although not the main focus of unable to generate HCMV specific Tc (using MHC this review, several points warrant emphasis. matched HCMV infected fibroblasts as target cells). Increased numbers of T8 ÷ T cells are detectable However, when autologous HCMV infected fibro- in peripheral blood during primary HCMV infection blasts were used as stimulator cells, the resultant and reactivation episodes, as in other herpes virus T-cell lines were predominantly T8 ÷ and displayed infections 37. However their precise function is unHCMV specific and MHC class I - restricted lysis. certain as it still is in the case of EBV, although there These T-cell lines killed cells which had only been is some evidence for their having suppressor infected with HCMV for 6 h or which had been activity38. In their study of bone marrow transplant treated with phosphonoformate - this indicates recipients with HCMV infection Quinnan and colthat the virus determinant being recognized is leagues observed a positive correlation between the expressed only in the infected cell, coincident with direct detection of MHC restricted lysis of HCMV either the immediate early or early proteins. Thus at infected cells by PBL and recovery from the infecleast a proportion of the HCMV specific memory Tc tion. Rook et aL 39 have also investigated patients in normal persistently infected individuals appear to with AIDS, in whom HCMV is a major cause of be directed at antigens expressed at early times in morbidity and mortality: they report that HCMV the virus replicative cycle; whether they are directed specific Tc cannot be directly detected in AIDS specifically to immediate early antigens as for patients with HCMV infection, but can be induced in vitro by treating their peripheral blood lymphoMCMV remains to be shown. MHC restricted lysis of HCMV infected cells has cytes with exogenous IL-2 alone. This suggests also been directly demonstrated using peripheral activated Tc are present but cannot expand which is blood lymphocytes obtained in vivo during active obviously consistent with the depletion of T4 ÷ cells. HCMV infection (in bone marrow transplant However, the therapeutic (in vivo) administration of
Immunology Today, vol. 7, No. 2, 1986
reviews
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IL-2 to AIDS patients did not result in the development of detectable Tc activity 4°. In disseminated HCMV infection the virus can be detected in many different organs by DNA hybridization, in apparent contrast to the very limited range of cells it can infect in v i t r o 41. This again emphasises the need to understand the cellular factors required for replication. The prevention of primary infection will presumably depend more on antibody recognizing neutralizing determinants on the virion, and this would be the logical basis for a vaccine. Any vaccine should preferably be a subunit vaccine - the use of live HCMV as a vaccine has been reported 42, but the extent of attenuation of the strains used is uncertain; theoretical objections have also been raised to the use of live virus which may establish persistence and whose biological properties are uncertain. Passive immunization (using human IgG with a high titre of anti-CMV antibody) may confer some protection against primary infection in seronegative subjects at risk 43, but might not be expected to confer much protection against reactivation. There is still no proven effective chemotherapy for limiting HCMV disease, but the new antiviral DHPG (9-(1,3dihydroxy-2-propoxymethyl) guanine) seems promising; however experience to date in patients with AIDS still suggests that in the absence of an effective T-cell response any chemotherapy will only be 'virostatic', and CMV disease will recrudesce as soon as treatment is stopped.
1636 1651 5 Gonczol, E., Andrews, P.W. and Plotkin, S.A. (1984)Science 224, 159-161 6 Nelson, J. and Groudine, M. J. Mol. Cell. Biol. (in press) ? Liebowitz, J.L. and Oefinger, P.E.(1985) ImmunoL Today 6, 82-83 8 Rice, G.P.A, Schrier, R.D. and Oldstone, M.B.A. (1984) Proc. NatlAcad. Sci. USA 81, 6134 6138 9 Einhorn, L. and Ost, A. (1984) J. Infect. Dis. 149, 207-214 10 Ruger, R., Bornkamm, G.W. and Fleckenstein, B. (1984) J. Gen. ViroL 65, 1351-1364 11 Schrier, R., Nelson, J.A. and Oldstone, M.B.A. (1985) Science 230, 1048-1050 12 Rickinson, A.B., Yao, Q.Y. and Wallace, V.E. (1985) Brit. Med. Bull. 41,75-79 13 Bancroft, G.J., Shellam, G.R. and Chalmer, J.E. (1981) J. ImmunoL 126, 98~994 14 Sheltam, G.R., Allen, J.E., Papadimitriou, J.M. etaL (1981) Proc. NatlAcad. Sci. USA 78, 5104-5108 15 Bukowski, J.F., Woda, B.A. and Welsh, R.M (1984)J. Virol. 52, 119-128 16 Bukowski, J.F, Warner, J.F., Dennert, G. etaL (1985) J. Exp. Med. 161,40 52 17 Quinnan, G.V., Manischewitz, J.E. and Ennis, E.A. (1980) J. Gen. Virol. 47, 503-508 ,18 Reddehase, MJ., Keil, G.M and Koszinowski, U.H. (1984) J. Immunol. 132, 482-489 19 Reddehase, M.J., Keil, GM. and Koszinowski, U.H. (1984) Eur. J. ImmunoL 14, 56-61. 20 Townsend, A.R. and Skehel, J~(1984) Ce1139, 13 25 21 Nash, A.A. (1985) Brit. Med. Bull. 41,41-45 22 Olding, L.B., Jensen, F.C. and OIdstone, M.B.A. (1975) J. Exp. Med. 141,561-572 23 Loh, L. and Hudson, J.B. (1980) Infect. Immun. 27, 54-60 24 Grundy, J.E., Shanley, J.D. and Shearer, GM. (1985) Transplantation 39, 548 553 25 Borysiewicz, L.K., Rodgers, B., Graham, S. etaL (1985) Conclusion Clearly, despite this new information, the picture J. Immunol. 134, 2695-2701 Vodinelich, L., Sutherland, R., Schreider, C. etaL (1983) is still incomplete. Insofar as any general rules 26 Proc. Natl Acad. Sci. USA 80,835-839 emerge about the immunology of the herpes 27 Bishop, G.A., Marlin, S.D., Schwartz, S.A. etal. (1984) viruses, it appears that events occurring very early in J. Immunol. 133, 2206-2214 the virus cycle will repay further study - both as 28 Borysiewicz, L.K., Graham, S. and Sissons, J.G.P. Eur. targets for the immune response and for possible J. Immunol (in press) subtle pathogenic effects they may produce. This 29 Borysiewicz, L.K., Morris, S.M., Page,J. etal. (1983)Eur. J. ImmunoL 13,804-809 applies particularly during persistent infection, 30 Quinnan, G.V., Kirmani, N., Esber, E. etal. (1981) when T-cell surveillance against early events in cells J. Immunol. 126, 2036-2041 ' supporting replication or in which reactivation has 31 Rinaldo, C.R. and Hirsch, M.S. (1980)J. Infect. Dis. 141, occurred (for CMV), or against transformed cells 488-495 (for EBV), appears cruciak Further information on 32 Carney, W.P. and Hirsch, M.S. (1981)J. Infect. Dis. 144, the molecular biology of the virus is obviously an 47-54 essential prerequisite to a full understanding of its 33 Rodgers, B.C., Scott, D.M., Mundin, J. etal. (1985) J. Virol. 55, 527-532 pathogenesis. 34 Amento, E.P., Kurnick, J.T. and Krane, SM. (1985) We and the herpes viruses have obviously been J. ImmunoL 134, 350-357 learning to live together for a long time, and have 35 Scala, G., Kuang, Y.D., Hall, R.E.etal. (1984)J. Exp. Med. evolved a complex relationship with each other; like 159, 1637-1652 all such relationships, the fact that it works so well 36 Hutt Fletcher, L.M., Bakachandran, N. and Elkins, M.H. most of the time is perhaps just as impressive as the (1983)./. Exp. Med. 158, 2171-2176 37 Schooley, R.T. (1983) N. Eng. J. Med. 308, 307-313 dramatic consequences of its breakdown. 38 Carney, W.P., Incoviello, V., Hirsch, M.S. (1983)./. Immunol. The work was supported by the MRC and Wellcome 130, 390-393 Trust. JGPS is a Wellcome Senior Lecturer and LKB a 39 Rook, A.H., Masur, H., Lane, H.C. etal. (1983)J. Clin. Invest. 72,398-403 Lister Research Fellow. 40 Lane, H.C., Siegel, J.P., Rook, A.H. etal. (1984) J. BioL Resp. Mod. 3, 512-516 References 41 Myerson, D., Hackman, R.C., Nelson, S.A. etal. (1983) 1 Everett, R.D. (1984). EMBOJ. 3, 3135-3141 Human PathoL 15,430-439 2 Kingston, R.E., Baldwin, A.S. and Sharp, P.A. (1985). Ce1141, 42 Plotkin, S.A., Friedman, HM., Fleisher, G.R. etal. (1984) 3-5 Lanceti, 528 531 3 Ho, D.D., Rota, T.R., Andrews, C.A., etaL (1984) J. Infect. Dis. 150, 956-957 43 Winston, D.J., Pollard, R.B., Ho, W.E. etal. (1982)Ann. 4 Dutko, F. and Oldstone, M.B.A. (1981)J. Exp. Med. 159, Intern. Med. 97, 11-18
61
reviewsCytomegalovirus-its cellular immunologyand biology
The biology of persistent virus infections presents many intriguing questions, which extend well beyond classical virology into molecular and cell biology and immunology. Here, Patrick Sissonsand his colleagues review the current state of knowledge of the biology and immunology of one particular medically important persistent human virus - human cytomegalovirus (HCMV) and they discuss insights into the normal and pathological virus-host relationship provided by recent work both on HCMV itself, and on its murine counterpart mouse cytomegalovirus (MCMV). HCMV is one of five human herpes viruses (the others being herpes simplex (HSV) 1 and 2, varicella zoster (VZV) and Epstein-Barr (EBV) viruses). Like the others HCMV persists following primary infection and may subsequently reactivate, and is widely prevalent, infecting 50-100% of adults depending on the particular population studied. EBV has, of course, long been of interest to immunologists because of its tropism for, and ability to transform, B cells, and because it appears to illustrate the importance of T-cell surveillance in containing ~iraiiy induced tumours. Of the other four HCMV as a particular propensity to produce severe morbidity and death in immunosuppressed subjects - principally allograft recipients (especially of bone marrow transplants) and more recently those with acquired immunodeficiency syndrome (AIDS). Current evidence suggests that HCMV displays a twosided relationship with the immune response, being controlled by it and yet capable of itself producing immunosuppression. Although clearly not unique among viruses in this respect (this may be something of a general strategy for persistent viruses with human T-cell leukaemia virus (HTLV)-III perhaps the ultimate example), the widespread distribution, and complex biology of HCMV, and the frequency with which it produces clinical problems, make it a challenging example to study. Molecular virology
HCMV is the largest herpes virus with a linear double stranded DNA genome of about 235 kilobase pairs (kbp), divided into long and short unique regions each flanked by inverted terminal repeat sequences (Fig. 1). The genome has the capacity to code for about 150 proteins, but their characterization is as yet incomplete. Although the molecular biology of HCMV has previously received less attention than that of HSV, the situation is now changing rapidly: the complete nucleotide sequence of FICMV will probably be known soon - this should accelerate characterization of the virus proteins by recognition of open reading frames, and prediction
MRCClinicalImmunologyResearchGroup, RoyalPostgraduateMedical School,London W12 OHS,UK ~) 1986, Elsevier Science Pubtishers B.V., Amsterdam
0167
49191861502.00
J.G.P. Sissons, L.K. Borysiewicz, Brian Rodgersand Diane Scott of the proteins from the nucleotide sequence. The virus genome is transcribed in a regulated sequence, broadly analogous to that of HSV, with serial transcription of three different classes of viral mRNAs. These are the immediate-early, early and late RNAs - the proteins they code for go by the same designation. The majority of the HCMV immediate-early (IE) genes, including the gene coding for the major 72kDa IE polypeptide, are transcribed from a restricted region of the long unique sequence of the virat genome, contained within the Hind IIIE fragment (Fig. 1). In the case of HSV it is known that the major immediate early proteins can act as trans acting factors - that is, they are capable of binding to and activating transcription from transcription units on independent duplex DNA molecules-their prime function being to activate transcription of the early virus proteins. It is now clear that the IE proteins of HCMV can also activate genes in trans~ : for instance, in short term transfection assays, the Hind IIIE fragment of HCMV DNA produced trans activation of transcription from both viral and cellular promoters. There is considerable current interest in the trans activating properties of other viral proteins, including the adenovirus Ela and HTLV tat proteins, and of oncogenes 2, and it is tempting to speculate that alteration in transcription of host cell genes might be responsible for some of the pathogenic effects of HCMV. The early viral mRNAs are then transcribed, virus DNA replication occurs, and following this the virus late RNAs are transcribed the proteins they code for are mainly the structural proteins of the virus particle. Expression of these individual sets of CMV proteins can be manipulated experimentally by the use of metabolic inhibitors, as shown in Fig. 2. This ability to manipulate expression of the stagespecific proteins, and the fact that the immediateearly and early proteins are only present in the infected cell and not in the virus particle itself, are both pertinent to the experiments discussed below. The replication strategy and sequence of protein expression of MCMV appear to be essentially similar to HCMV and its detailed molecular biology is also under study. However there is very little DNA homology between the two viruses. Cellular tropisms
The cytomegaloviruses (unlike HSV) show a high degree of species specificity, and will only productively infect cells of their particular species in vitro.
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IRL IRS TRS Thus HCMV cannot be studied in T,, UL /l~ US W r.tJ I~ ~ 235kb the mouse, and whilst there are. r.~ II Hind III E ~ J undoubtedly major similarities in m ECoRI J the biology of MCMV and HCMV, / / it may not necessarily always be Hind III e - region of major ~ ~i~ correct to extrapolate direct from IE transcription of HC R1 the mouse to the human virus. The fibroblast is the only cell that i i is permissive for a full cycle of 1.9skb HCMV replication in vitro, 5kb 2,2kb 1~75 / although one recent report 3 sug- Ilkb 2.15 / gests endothelial cells may also support virus replication (which major 72K IE protein might be relevant to patho~ - ~ Cellular homologies genesis). Other cell types may be Fig.1.6enomestructureand immediateearlytranscriptsof HCMV(Ad 169strain). infected but then presumably lack some host cell factor(s) required for further gene expression. For example MCMV and HCMV can latently bone marrow cells, following their infection with ~infect teratoma cell lines but only then enter a full HCMV in vitro 8'9 - about 10% of PBM expressed replicative cycle when the cell is induced to the major 72kDa HCMV immediate-early protein differentiate by chemical agents4's - t h e block here (assessed with a specific monoclonal antibody), and is at the level of transcription of the IE genes6. It is one study suggested this was mainly in monocytes8. not known whether this occurs in vivo but it is an It can be envisaged that even expression of the IE interesting possibility, perhaps particularly in the proteins alone might produce pathogenic effects context of haemopoietic cell differentiation. (by virtue of their trans activating properties discusThe important question of the sites at which sed above). Interestingly this expression was only CMV persists in vivo still awaits a definitive answer. readily apparent when the infecting virus was a MCMV persists in the salivary gland, and can also recent clinical isolate, as opposed to the usually latently infect mouse macrophages and possibly B used laboratory strains which have been passaged cells. HCMV can be identified in many different cells in fibrobtasts for years, such as AD169. This raises during disseminated clinical infection; however, it is the problem for other workers that although they much less certain in which cell(s) HCMV normally may be working on a molecularly better defined persists in the silently infected host - possible virus, its biology may be of uncertain clinical relecandidates are epithelial cells in salivary gland and vance! renal tubule. Particular uncertainty has surrounded Hybridization studies for virus DNA and for the question of the extent to which HCMV infects, mRNA transcripts would help resolve these quesand can replicate in, monocytes and lymphocytes tions. It is now known that HCMV DNA has regions (discussed in this journal in a recent Compass showing extensive sequence homology with normal article7). Although it is quite widely assumed that cellular DNA 10 , which makes selection of appropriHCMV does infect these cells, this is partly based on ate probes particularly important and interpretation the fact that the virus can be isolated from buffy of most previous studies difficult. In a very recent coat in patients, is transmitted by blood transfusion, report the EcoR1J fragment (from the region of IE and on a measure of extrapolation from the mouse. transcription see Fig. 1.) was used as a probe in a However it is difficult or impossible to establish sensitive in situ hybridization assay which detected productive infection in any peripheral blood mono- virus mRNA: Schrier et aL found that 0.5-2% of nuclear cell (PBM) in vitro. Two recent reports have PBM from normal subjects seropositive for HCMV suggested that limited virus gene expression may showed hybridization (this was without infection in occur in a small proportion of PBM, and also in vitro), and their preliminary evidence suggests most of the positive cells were T cells and monocytes 11. This is probably the hardest evidence yet for PBM being a normal site of persistence for HCMV, and further results using this approach will be i/ of great interest. Immediate early proteins rly proteins late proteins / Like the other herpes viruses, HCMV has been proposed as a Inhibitors Cycloheximide phosphono formate candidate oncogenic virus - one and actinomycin D tumour with which an associaFig.2. Sequentialsynthesisof HCMVproteins and thor r~etabolicinhibition. Inhibitionof protein syntion has particularly been sugthesis with cydofleximideallowsaccumulationof IEgene transcripts, and its removalin the presenceof gested is Kaposrs sarcoma (both the transcriptioninhibitoractinomycinD allowstheir expressionwithout latergene transcription.Expression of the late proteins can be prevented by the use of the virus DNA polymerase inhibithe African endemic and more
tor, phosphonoformate.
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recent AIDS associated forms), which is thought to antigen, and that two independent populations of be of endothelial cell origin. This area cannot be MCMV specific Tc can then be detected. One is specific for structural (late) viral proteins, and the reviewed here, but it is worth noting that recent attempts to demonstrate HCMV DNA in the cells of other detects an antigen present on target cells AIDS associated Kaposi's sarcoma have been un- treated with cycloheximide and actinomycin D successful. Any causal association thus remains very which express only the MCMV IE proteins. Tc, showing similar specificity for cells expressing late much unproven. antigens and IE proteins, could be generated in Immunoloqical control vitro from adult mice which had been neonatally It seems increasingly likely that of the other four herpes viruses only HSV and VZV manifest classical infected with MCMV, although this required restimulation with antigen as well as IL-219: thus latency, and perhaps persist without replicating because they are latent in neuronal cells, which are memory Tc are maintained during latent or persislong-lived and do not divide. In contrast EBV, which tent infection. The specificity for cells expressing IE probably persists in oropharyngeal epithelial cells proteins has also been shown with Tc clones. The (which have a rapid turnover), seems to replicate at MCMV antigen recognized by the Tc which correlow level for much of the time in the normal lates with IE antigen expression awaits structural carrier 12. It is in this latter situation that the host definition - especially as the IE proteins have hitherto been thought of primarily as nuclear proteins. response may be crucial in keeping this replication Reddehase and colleagues term the antiqen LYDIEA in check at local sites and preventing dissemination. (lymphocyte detected IE antigen) and point to its How then does the persistently infected normal potential similarity with the lymphocyte determined host manage to live with CMV, (a lytic rather than a transforming herpes virus), and what happens membrane antigen (LYDMA) recognized by EBV when the normal relationship breaks down? As yet . specific To. This correlation of specificity with exthere is no complete answer to the question, but pression of a viral nuclear protein is particularly interesting in the context of the recent reported recent work provides some intriguing clues. recognition of the influenza nucleoprotein by cross MCMV and NK cells reactive 'flu specific Tc 20 - and its implication that Natural killer (NK) cells have a postulated role in viral determinants recognised by Tc do not necesvivo against tumours and virus infection; probably sarily have to be the conventional surface glycoprothe most convincing evidence for their playing a teins. role in controlling any virus infection comes from Studies of the relative protection against MCMV studies on MCMV. Genetically determined resist- conferred by transfer of these two different populaance to MCMV correlates with NK cell activity 13, tions of Tc would obviously be of interest. The role and homozygous beige mice (which have defective of delayed type hypersensitivity in MCMV infection NK cell function) are very susceptible to MCMV 14. still remains to be determined despite its demonEspecially persuasive evidence comes from the strated protective role in primary HSV infection in recent studies of Bukowski, Welsh and col- the mouse 21. leagues ls'16. They showed that mice depleted of NK cells with anti-asialo GM1 developed more serious MCMV and immunosuppression There is fairly clear evidence that MCMV can disease from MCMV is. Then using adoptiye transfer experiments they found that spleen cells con- establish persistent infection in immunocompetent ferred protection from lethal MCMV infection in cells: this definitely includes macrophages from suckling mice, and the phenotype of the protective which the virus can be recovered by co-cultivation, cell was that of an NK cell rather than a T cell; and probably a proportion of mouse B cells22, finally they conferred protection by transfer of a MCMV infection also impairs macrophage depencloned NK cell line. This protective effect of NK cells dent T-cell proliferation; this immunosuppressive did not apply to LCM virus infection and so cannot effect is apparently mediated through infection of be assumed to apply to all virus infections ~6, but the macrophage 23, and seems to have its counterthese experiments do suggest NK cells play an part in the human situation. There is also evidence important role at least in limiting the initial infection that MCMV can increase alloreactive respones to major histocompatibility (MHC) antigens and enwith MCMV. hance graft versus host reactions in transplanted MCMV specific cytotoxic T cells m i c e 24. It was shown some time ago that, just as for many other viruses, virus specific cytotoxic T cells Immunology of human CMV infection (To) are generated following MCMV infection, and There are obvious constraints on the approaches their presence correlates with recovery from which can be used in investigating human CMV infection ~7. However, recently Reddehase, Koszin- infection, but there have been recent studies of the owski and colleagues have analysed the T~ response NK and Tc killing of HCMV infected cells both in to MCMV in more detail, with particular reference vitro and in clinical studies. to its specificity for the staged expression of virus proteins ~8'19. They report that during primary HCMV and NK cells MCMV infection, T cells from lymph nodes can be As with other viruses, HCMV infected cells are expanded in vitro in interleukin 2 (IL-2) without more susceptible to NK lysis. A component of this
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enhanced susceptibility is independent of interferon recipients) 3°. This was without any restimulation in acting on the effector population, and related to a vitro, making it difficult to examine specificity in any detail (see below). virus induced change in the cell. HCMV infected cells show increased susceptibility to NK killing at the stage when the virus early antigens are expressed: the effector cells are Leu HCMV and immunosuppression 7 +, Leu 1 1+ large granular lymphocytes - the same Immunosuppressive properties have been attricells which mediate NK lysis of tumour cells25. In all buted to HCMV at a rather anecdotal clinical level systems in which NK cells have been studied there is patients with HCMV disease often have other continuing uncertainty about the target structures opportunistic infections - but there is accumulatwhich the NK cells actually recognize. Proposed ing experimental evidence to give weight to this candidates have included glycolipids, sugars and impression. It was shown some years ago that various membrane glycoproteins, including recently monocytes from patients with primary HCMV the transferrin receptor 26. In the case of virus mononucleosis were less able to support mitogen infected cells there is little general evidence to induced T-cell responses31, as were normal monosuggest that viral proteins themselves are NK cell cytes infected with HCMV in vitro 32. We have target structures, and it seems more likely that any recently shown that the ability of monocytes to such structure is also present on normal cells (a produce interleukin-I (IL-1) activity is abrogated by possible exception is HSV where one group 27 have HCMV infection in vitro; this is associated with the evidence for its glycoproteins being recognized by release of an inhibitor of IL-1 from the monocyte NK cells). We recently found a direct correlation cultures 33. This inhibitor is almost certainly a host between the susceptibility of virus infected cells to cell protein acting at the level of the IL-1 responding NK lysis, and their expression of transferrin cell, and shows similarities to inhibitors of IL-1 and receptor 28. In the case of HCMV the increase in thymocyte proliferation described by others 34"3s. Its transferrin receptor temporally associated with EA release is presumably induced by HCMV - other expression, but we were unable to inhibit NK lysis viruses we studied did not produce this effect. The with purified transferfin receptor. Thus despite character of this inhibitor, and whether it plays any these close correlations it seemed unlikely to be the normal regulatory role in vivo, remain to be detertarget structure for NK cells. The role of human NK mined, as does the extent to which HCMV is cells in vivo remains unknown, but it seems in- expressed in the infected monocytes - but here the herently unlikely they play more than an adjunctive virus may be illuminating an aspect of normal cell biology. Whether HCMV exerts immunosuppressive role to specific immunity. effects by direct infection of T cells is uncertain, and further experiments are needed which exclude any HCMV specific cytotoxic T cells Our approach to analysing the Tc response to effects from monocytes. There is a single recent report that HCMV can act HCMV, has been to study HCMV specific T cells in normal seropositive subjects (who have never had as a polyclonal activator of B cells36, which is of symptomatic HCMV infection), using the now usual course a property of EBV-this awaits confirmation. technique of secondary in vitro stimulation with antigen, and expansion of responding cells in IL-229. Clinical aspects All the above work bears directly on the clinical We found that when free virus was used as antigen, T4 + proliferative lines were generated but we were problem of HCMV. Although not the main focus of unable to generate HCMV specific Tc (using MHC this review, several points warrant emphasis. matched HCMV infected fibroblasts as target cells). Increased numbers of T8 ÷ T cells are detectable However, when autologous HCMV infected fibro- in peripheral blood during primary HCMV infection blasts were used as stimulator cells, the resultant and reactivation episodes, as in other herpes virus T-cell lines were predominantly T8 ÷ and displayed infections 37. However their precise function is unHCMV specific and MHC class I - restricted lysis. certain as it still is in the case of EBV, although there These T-cell lines killed cells which had only been is some evidence for their having suppressor infected with HCMV for 6 h or which had been activity38. In their study of bone marrow transplant treated with phosphonoformate - this indicates recipients with HCMV infection Quinnan and colthat the virus determinant being recognized is leagues observed a positive correlation between the expressed only in the infected cell, coincident with direct detection of MHC restricted lysis of HCMV either the immediate early or early proteins. Thus at infected cells by PBL and recovery from the infecleast a proportion of the HCMV specific memory Tc tion. Rook et aL 39 have also investigated patients in normal persistently infected individuals appear to with AIDS, in whom HCMV is a major cause of be directed at antigens expressed at early times in morbidity and mortality: they report that HCMV the virus replicative cycle; whether they are directed specific Tc cannot be directly detected in AIDS specifically to immediate early antigens as for patients with HCMV infection, but can be induced in vitro by treating their peripheral blood lymphoMCMV remains to be shown. MHC restricted lysis of HCMV infected cells has cytes with exogenous IL-2 alone. This suggests also been directly demonstrated using peripheral activated Tc are present but cannot expand which is blood lymphocytes obtained in vivo during active obviously consistent with the depletion of T4 ÷ cells. HCMV infection (in bone marrow transplant However, the therapeutic (in vivo) administration of
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IL-2 to AIDS patients did not result in the development of detectable Tc activity 4°. In disseminated HCMV infection the virus can be detected in many different organs by DNA hybridization, in apparent contrast to the very limited range of cells it can infect in v i t r o 41. This again emphasises the need to understand the cellular factors required for replication. The prevention of primary infection will presumably depend more on antibody recognizing neutralizing determinants on the virion, and this would be the logical basis for a vaccine. Any vaccine should preferably be a subunit vaccine - the use of live HCMV as a vaccine has been reported 42, but the extent of attenuation of the strains used is uncertain; theoretical objections have also been raised to the use of live virus which may establish persistence and whose biological properties are uncertain. Passive immunization (using human IgG with a high titre of anti-CMV antibody) may confer some protection against primary infection in seronegative subjects at risk 43, but might not be expected to confer much protection against reactivation. There is still no proven effective chemotherapy for limiting HCMV disease, but the new antiviral DHPG (9-(1,3dihydroxy-2-propoxymethyl) guanine) seems promising; however experience to date in patients with AIDS still suggests that in the absence of an effective T-cell response any chemotherapy will only be 'virostatic', and CMV disease will recrudesce as soon as treatment is stopped.
1636 1651 5 Gonczol, E., Andrews, P.W. and Plotkin, S.A. (1984)Science 224, 159-161 6 Nelson, J. and Groudine, M. J. Mol. Cell. Biol. (in press) ? Liebowitz, J.L. and Oefinger, P.E.(1985) ImmunoL Today 6, 82-83 8 Rice, G.P.A, Schrier, R.D. and Oldstone, M.B.A. (1984) Proc. NatlAcad. Sci. USA 81, 6134 6138 9 Einhorn, L. and Ost, A. (1984) J. Infect. Dis. 149, 207-214 10 Ruger, R., Bornkamm, G.W. and Fleckenstein, B. (1984) J. Gen. ViroL 65, 1351-1364 11 Schrier, R., Nelson, J.A. and Oldstone, M.B.A. (1985) Science 230, 1048-1050 12 Rickinson, A.B., Yao, Q.Y. and Wallace, V.E. (1985) Brit. Med. Bull. 41,75-79 13 Bancroft, G.J., Shellam, G.R. and Chalmer, J.E. (1981) J. ImmunoL 126, 98~994 14 Sheltam, G.R., Allen, J.E., Papadimitriou, J.M. etaL (1981) Proc. NatlAcad. Sci. USA 78, 5104-5108 15 Bukowski, J.F., Woda, B.A. and Welsh, R.M (1984)J. Virol. 52, 119-128 16 Bukowski, J.F, Warner, J.F., Dennert, G. etaL (1985) J. Exp. Med. 161,40 52 17 Quinnan, G.V., Manischewitz, J.E. and Ennis, E.A. (1980) J. Gen. Virol. 47, 503-508 ,18 Reddehase, MJ., Keil, G.M and Koszinowski, U.H. (1984) J. Immunol. 132, 482-489 19 Reddehase, M.J., Keil, GM. and Koszinowski, U.H. (1984) Eur. J. ImmunoL 14, 56-61. 20 Townsend, A.R. and Skehel, J~(1984) Ce1139, 13 25 21 Nash, A.A. (1985) Brit. Med. Bull. 41,41-45 22 Olding, L.B., Jensen, F.C. and OIdstone, M.B.A. (1975) J. Exp. Med. 141,561-572 23 Loh, L. and Hudson, J.B. (1980) Infect. Immun. 27, 54-60 24 Grundy, J.E., Shanley, J.D. and Shearer, GM. (1985) Transplantation 39, 548 553 25 Borysiewicz, L.K., Rodgers, B., Graham, S. etaL (1985) Conclusion Clearly, despite this new information, the picture J. Immunol. 134, 2695-2701 Vodinelich, L., Sutherland, R., Schreider, C. etaL (1983) is still incomplete. Insofar as any general rules 26 Proc. Natl Acad. Sci. USA 80,835-839 emerge about the immunology of the herpes 27 Bishop, G.A., Marlin, S.D., Schwartz, S.A. etal. (1984) viruses, it appears that events occurring very early in J. Immunol. 133, 2206-2214 the virus cycle will repay further study - both as 28 Borysiewicz, L.K., Graham, S. and Sissons, J.G.P. Eur. targets for the immune response and for possible J. Immunol (in press) subtle pathogenic effects they may produce. This 29 Borysiewicz, L.K., Morris, S.M., Page,J. etal. (1983)Eur. J. ImmunoL 13,804-809 applies particularly during persistent infection, 30 Quinnan, G.V., Kirmani, N., Esber, E. etal. (1981) when T-cell surveillance against early events in cells J. Immunol. 126, 2036-2041 ' supporting replication or in which reactivation has 31 Rinaldo, C.R. and Hirsch, M.S. (1980)J. Infect. Dis. 141, occurred (for CMV), or against transformed cells 488-495 (for EBV), appears cruciak Further information on 32 Carney, W.P. and Hirsch, M.S. (1981)J. Infect. Dis. 144, the molecular biology of the virus is obviously an 47-54 essential prerequisite to a full understanding of its 33 Rodgers, B.C., Scott, D.M., Mundin, J. etal. (1985) J. Virol. 55, 527-532 pathogenesis. 34 Amento, E.P., Kurnick, J.T. and Krane, SM. (1985) We and the herpes viruses have obviously been J. ImmunoL 134, 350-357 learning to live together for a long time, and have 35 Scala, G., Kuang, Y.D., Hall, R.E.etal. (1984)J. Exp. Med. evolved a complex relationship with each other; like 159, 1637-1652 all such relationships, the fact that it works so well 36 Hutt Fletcher, L.M., Bakachandran, N. and Elkins, M.H. most of the time is perhaps just as impressive as the (1983)./. Exp. Med. 158, 2171-2176 37 Schooley, R.T. (1983) N. Eng. J. Med. 308, 307-313 dramatic consequences of its breakdown. 38 Carney, W.P., Incoviello, V., Hirsch, M.S. (1983)./. Immunol. The work was supported by the MRC and Wellcome 130, 390-393 Trust. JGPS is a Wellcome Senior Lecturer and LKB a 39 Rook, A.H., Masur, H., Lane, H.C. etal. (1983)J. Clin. Invest. 72,398-403 Lister Research Fellow. 40 Lane, H.C., Siegel, J.P., Rook, A.H. etal. (1984) J. BioL Resp. Mod. 3, 512-516 References 41 Myerson, D., Hackman, R.C., Nelson, S.A. etal. (1983) 1 Everett, R.D. (1984). EMBOJ. 3, 3135-3141 Human PathoL 15,430-439 2 Kingston, R.E., Baldwin, A.S. and Sharp, P.A. (1985). Ce1141, 42 Plotkin, S.A., Friedman, HM., Fleisher, G.R. etal. (1984) 3-5 Lanceti, 528 531 3 Ho, D.D., Rota, T.R., Andrews, C.A., etaL (1984) J. Infect. Dis. 150, 956-957 43 Winston, D.J., Pollard, R.B., Ho, W.E. etal. (1982)Ann. 4 Dutko, F. and Oldstone, M.B.A. (1981)J. Exp. Med. 159, Intern. Med. 97, 11-18
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