- Email: [email protected]
Immune escape and tropism of HIV
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31Martin, D.W. et al. (1993) PYOC. Nat/ Acad. Sci. USA 90, 8377-8381 32 Martin, D.W. et al. (1993)J. Bacterial. 175,1153-1164 33 Martin, D.W. et al. (1994)]. Bacterial. 176,6688-6696 34 DeVries,C.A. and Ohman, D.E. (1994)J. Bacterial. 176,
47 Hatano, K., Goldberg,J.B. and Pier, G.B. (1995)Infect. Immun. 63,21-26 48 Goldberg,J.B. et al. (1992) Proc. Nat/ Acad. Sci. USA. 89,
6677-6687 35 Yu, H., Schurr, M.J. and Deretic,V. (1995)j. Bacterial. 177, 3259-3268 36 Goldberg,J.B. et al. (1993)I. Bacterial. 175,1303-1308 37 Gamer, J., Bujard, H. and Bnkau, B. (1992) Cell 69,833-842 38 Hughes, K.T. et al. (1993) Science 262,1277-1280 39 Lonetto, M.A. et al. (1994) Proc. Natl Acad. Sci. USA 86,
7573-7577 40 Erickson,J.W. and Gross, C.A. (1989) Genes Deu. 3,1462-1471 41 Johnson, K. et al. (1991) Mol. Microbial. 5,401-407 42 Roop, R.M., II et al. (1994) Infect. lmmun. 62,1000-1007 43 Cameron, R.M. et al. (1994)Microbiology 140,1977-1994 44 Terry, J.M., Pina, S.E.and Mattingly, S.J. (1991) Infect. lmmun. 59,471-477 45 Knirel, Y.A. (1990) CRC Crit. Rev. Microbial. 17,273-304 46 Arsenault, T.L. et al. (1991) Can. 1. Chem. 69,1273-1280
10616-10720 49 Evans, D,J. et al. (1994) Mol. Microbial. 13,427-434 50 Lightfoot,J. and Lam, J.S. (1993) Mol. Microbial. 8,771-782 51 Goldberg,J.B., Hatano, K. and Pier, G.B. (1993)1. Bacterial. 175,1605-1611 52 Coyne, M.J. et al. (1994)J. Bacterial. 176,3500-3507 53 Ye, R.W., Zielinski, N. and Chakrabarty, A.M. (1994) 1. Bacterial. 176,4851-4857 54 Hatano, K., Goldberg,J.B. and Pier, G.B. (1993)J. Bacterial.
175,5117-5128 55 Totten, P.A., Lara, J.C. and Lory, S. (1990)]. Bacterial. 172, 389-396 56 Stambach, M.N. and Lory, S. (1992) Mol. Microbial. 6,459-469 57 Simpson,D.A., Ramphal, R.R. and Lory, S. (1992) Infect.
Immun.60,3771-3779 58 Mork,T. and Hancock,R.E.W.(1993)Infect.Immun.61, 3287-3293 59 Saiman,L. andPrince,A. (1993)J.Clin. Invest. 92, 1875-1880
Immuneescapeand tropismof HIV Aine McKnight and Paul R. Clapham
H
IV-l was identified in 1983 as the agent that causes AIDS in humans. CD4 was shown to be the primary cell-surface receptor, reflecting the tropism of the virus for CD4expressing cells. In viva, HIV-l replication has been demonstrated in CD4+ T helper cells and specifically differentiated tissue macrophages, such as brain microglia, spinalcord macrophages, alveolar macrophages of the lung and blood monocytes. Dendritic cells, including Langerhans cells of the skin, can also be infected. Macrophages could be important in transporting the virus to different organs, thus seeding replication in new niches. As well as these tissues, CD4+lymphocytes, macrophages and dendritic cells in the secondary lymphoid organs, including the lymph nodes, adenoids, tonsils and spleen, are heavily infected (see the review by Levy, Ref. 1).Once established, HIV infection persists for life. In this article, we examine how HIV-l, by changing its phenotype (by shifting tropism and escaping from neutralizing antibodies), may evade the host immune response.
HIV-l cell tropisms are partly determined by the hypervariable loops VU-V2 and V3 in gp120, which also contain epitopes for neutralizing antibodies. Mutations conferring tropism changes can result in escape from neutralization and vice versa. We examine whether variant viruses that can colonize new cell types and simultaneously escape neutralizing antibodies have an enhanced advantage in tiuo. A. McKnight and P.R. Clapham * are in the Virology Laboratory, Chester Beatty Laboratories, Institute of Cancer Research, 237 F&am Road, London, UK SW3 6JB. ‘tel: +44 171 352 8133, fax: +44 171 352 3299, e-mail: [email protected], [email protected]
Cell tropism and envelope glycoproteins Some isolates of HIV-l have a preferential tropism for either T helper cells or macrophages, suggesting that efficient crossinfection of these cell types could be limited. However, the tropism is not absolute, and analysis of virus iso0
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lates in vitro reveals an array of phenotypes between the two extremes. It is still unclear whether or not viruses tropic for particular subsets of T cells or macrophages exist (Box 1). In addition to their tropism for macrophages or T cells, primary isolates of HIV-l can be categorized according to their ability to induce syncytia (large multinucleated cells) in primary peripheral blood lymphocytes (PBLs) or in the MT-2 T cell line (that is, as syncytium inducing or nonsyncytium inducing), as well as according to their replication rate. Virus isolates that replicate readily in CD4’ cell lines are described as ‘rapid/high’, whereas strains that fail to infect established cell lines and are relatively noncytopathic are termed ‘slow/ low’. Repeated passage of HIV-l strains in CD4’ T cell lines in vitro selects for syncytium-inducing strains that are more tropic for T cell lines and have a markedly reduced ability to infect macrophages. However, a minority of such strains can still infect macrophages efficiently, and are described as ‘dual tropic’.
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The viral sequences that determine these phenotypes lie mainly in the outer-envelope glycoprotein of HIV-l, gp120, which is attached noncovalently to the membranespanning glycoprotein, gp41. The gp120 glycoprotein mediates binding to CD4, which initiates events that lead to the fusion of the viral and cell membranes and the subsequent entry into the cell cytoplasm. Five distinct hypervariable regions (Vl-VS) and five relatively conserved regions (Cl-CS) can be distinguished in gp120, based on sequence analysis of different strains of HIV-l (Fig. 1). Determinants of tropism for macrophages or T cell lines, and determinants of syncytium-inducing ability, have been mapped to the variable loops Vl, V2 and particularly V3. For example, an increase in the number of charged amino acids in V3 correlates with a switch in phenotype from nonsyncytium inducing to syncytium inducing (Fig. 2). The role or function of the Vl, V2 and V3 loops during the virus-cell fusion process that leads to the distinct phenotypes described above is not clear. However, their critical involvement in such processes suggests that antibodies to these regions are likely to be neutralizing, which has been shown to be true for V2 and V3.
Box 1. Am changes in tropism advantageous for HIV-1In vh? *Do HIV variants that can colonize new cell types have an advantage other strains? l
Do slow-replicating variants that can colonize macrophages evade immunity and prevail?
*Late in infection, emerging syncytium-inducing strains often show an expanding tropism for CD4+ T cell lines in vitro. Could this wider host range reflect a broadening tropism for different T cell subsets in vivo?
epitopes that include the more variable amino acids flanking this motif are usually strain specific, while mAbs with epitopes that include the conserved tip are more broadly reactive. The first neutralizing antibodies to develop after infection and seroconversion are generally effective against the infecting strain or closely related isolates4. Most of these antibodies are directed against V3 (Ref. 5). Over time, the response broadens and antibodies that neutralize diverse laboratory isolates develop6. The exact epitopes that are recognized by these crossreactive antibodies are not clear, but they are likely to be situated in the gp120 regions that make up the conformationally dependent CDCbinding region, as
Regions of gpl20 targeted by neutralizingantibodies V2 and V3 sequences are hyper-
variable between strains, hence neutralizing antibodies to these regions are likely to be strain specific, reacting preferentially with the inducing amino acid sequence. Indeed, monoclonal antibodies (mAbs) to V2 are generally strain specific, although antibodies that are more broadly reactive do occur2. Sequences within V2 form both linear and conformationally dependent epitopes that induce neutralizing antibodies3. The V3 loop was the first region of gp120 to be defined as a target for neutralizing antibodies. V3 is less variable than is Vl or V2, and contains a relatively conserved motif, Gly-Pro-Gly, at the central tip (Fig. 2). Neutralizing mAbs that target the V3 loop and have
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over
well as an epitope in gp41. Monoclonal Abs to these regions can neutralize diverse HIV-l strains in vitro.
Escape from neutralizing antibodies The tolerance of variation in the V2 and V3 regions of gp120 suggests that mutants that can escape neutralizing antibodies might easily arise. In fact, HIV-l escape mutants that resist neutralization by mAbs against V2 (Refs 2,7) or V3 can be selected in vitro8>9.Mutations that result in escape from anti-V3 mAbs can occur inside or outside the V3 loop. Amino acid substitutions inside V3 probably alter the antibody-binding site, whereas distant mutations probably
-i
y
COOH
c5 Fig. 1. HIV-1 envelope glycoprotein gpl20 based on the HXlO clone of HIV-1 IAI strain. Lines joining discontinuous sequences represent disulphide bonds and the numbers denote amino acids. (Redrawn from Ref. 27.)
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results in viruses that escape antibody neutralization.
flg. 2. The V3 loop of HIV-1 is 32-35 amino acids long and is delimited by a disulphide bond between two cysteines at the base. It is a major determinant of viral tropism, and amino acid substitutions have been shown to affect the biclogical properties and tropism of HIV-l. The sequence shown is derived from HIV-1 lAI strain. An asterisk indicates amino acids that are likely to have positively charged side chains in syncytium-inducing isolatesZ8. A filled circle indicates that a single amino acid change at this position changes the tropism of the Gun-l strain of HIV-1 (Ref. 29; see text and Table 1). A filled square indicates the position of an amino acid change shown by Shioda et aL30 to allow the SF-2 strain HIV-1 to infect the CD4+ T cell line MT-4.
AF w “I
T G
K*
alter the conformation or render it inaccessible to antibody. Strains of HIV-l passaged in vitro without selection pressure from neutralizing antibodies are generally more susceptible to neutralization by anti-V3-loop antibodies than are primary strains, which have morecryptic V3 loopsi”. Perhaps the presence of a more exposed V3 loop results in more-efficient virus-cell fusion and increases the replication rate. During primary infection, there is often massive replication before neutralizing antibodies are produced and, during this stage of infection, it might be speculated that a faster-replicating virus with an exposed V3 loop would be selected. Exposure to newly synthesized anti-V3-loop antibodies after seroconversion might then select variant viruses with mutations favouring a cryptic V3 that
Escape in viva
Unequivocal confirmation of escape from neutralization in vivo is difficult to obtain. Arendrup et all3 studied the neutralization of sequential isolates from HIV-l-infected individuals by homologous serum. Initially, strain-specific neutralizing antibodies developed and preceded the emergence of virus isolates that could escape. Subsequently, neutralizing antibodies that were effective against the escaped virus developed. The first neutralizing antibodies arising after seroconversion are thought to be directed against the V3 100~~. Escape from such V3-loop neutralization has been shown to occur in experimentally infected chimpanzees14, where resistance to neutralization was found to be conferred by changes outside V3. Subsequently, escape mutants with amino acid substitutions at the crown of V3 were isolated. Sequential isolates from a laboratory worker accidentally infected with HIV-l LA1 were shown to contain an amino acid substitution that conferred resistance in vitro to an anti-V3-loop mAb (Ref. 15).
limit antibody accessibility and allow the virus to escape antibody neutralization. Escape mutants of the LA1 strain of HIV-l that resist neutralization by a serum from an HIV-l-positive individual have been selected in vitro1’J2. These variants were found to have mutations either in gp41 or in the C3 region of gp120. Both types of mutation conferred directed resistance to mAbs against the CD4-binding region of gp120. These observations suggest that the dominant crossneutralizing antibodies in the human serum samples used were to the CD4binding region, and that envelope changes in the C3 region of gp120 or in gp41 altered the conformation of this region to allow escape. Clearly, it is not difficult to demonstrate in vitro that variation generated by virus replication
Virus escape and tropism changes So far we have discussed how the
variable regions V2 and V3 of gp120 are not only determinants of tropism, but also targets for neutralizing antibodies. In the V3 region in particular some, but certainly not all, amino acid substitutions result in a change in viral tropism. It is also true that some amino acid substitutions in the V3 loop can result in escape from neutralizing antibodies. Recently, we tested to what extent these phenotypes affect each other using Gun1, an isolate of HIV-l that is dual tropic, that is, it can infect both T cell lines and macrophages (Table 1)9. A variant of Gun-l selected for growth in a brain-derived cell line not only lost infectivity for macrophages, but also escaped neutralization by a mAb recognizing the Gun-l V3 loop. This change in phenotype is due to a single amino
Table 1. Amino acid substitutions in the HIV-l strain Guwl resulting from in v/&o selection for tropism change and neutralization escapee Resulting tropism Selectlon None Tropism change Neutralization escape
V34oop sequence
Macrophage T cell line Brain glioma
GPGRAFHAI GSGRAFHAI GPGRAFHAI GSGRAINAI GSGRTFQAI GSGRALHAI
+ + + +
+ + + + + +
+ + +/+/-
The HIV-1 strain Gun-l infects macrophages and T cells, but not CD4+ brain glioma Cells. Substitution of serine for proline at the tip of the V3 loop of gp120 switches the viral tropism so that the virus can infect CD4+ brain glioma cells, but not macrophages 9.29. Selection for four viruses to escape neutralization results in altered tropism and amino acid substitutions in the V3 loop. The bold letters denote the substituted amino acids.
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Seroconversion
Infection
+4
I 1 1000 xl
/
Minor or no symptoms
*-4
AIDS
+
------
Neutralizing antibodies
n
HIV-specific CTLs
Viraemia
4
e
*4
F_(
4-8
weeks
2-3 years
Up to 12 years Time
Fig. 3. Within 3 weeks of primary infection, about 40% of individuals show some symptoms of viral infection, including headache, muscle ache, tender lymph nodes, sore throat and fever. These symptoms usually last up to 3 weeks. High levels of virus can be detected in the blood, and CD4+ cell numbers decline. This phase lasts about 4-8weeks, after which viraemia is reduced dramatically because of the initiation of the immune response. Specific cytotoxic T cells (CTLs) are detected before neutralizing antibodies appear. In the asymptomatic phase after seroconversion, the individual is apparently healthy, although occasionally symptoms, such as diarrhoea and night sweats, can occur. The viral load varies, but remains much reduced. Within 12years the majority of individuals succumb to AIDS, which is characterized by opportunistic infections, neoplasms, Kaposi’s sarcoma, severe weight loss and dementia. After the diagnosis of AIDS, most individuals die within 3years. Abbreviation: PBL, peripheral blood lymphocyte.
acid substitution at the crown of the V3 loop (Gly-Pro-Gly to GlySer-Gly). We also tested the reciprocal situation. Does escape from neutralizing antibodies affect tropism? We produced further neutralizing mAbs that were specific for the variant V3 loop, and used them to select for neutralization escape mutants. The four escape mutants that we analysed each had an altered tropism. Two gained infectivity for primary macrophages, while one reverted to the wild-type V3 loop (Gly-Pro-Gly) and tropism, losing infectivity for the brainderived ceils and regaining tropism for macrophages. The tropism of a fourth escape mutant became restricted, losing infectivity for brain cells, without regainin macrophage infectivity. Amino acid changes
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located at or near the crown of the V3 loop are likely to cause these phenotypic changes. Thus, escape from neutralizing antibodies resulted in an altered viral phenotype, which gave three of four mutants the ability to colonize macrophages, while restricting tropism for a fourth. Such escape from neutralizing anti-V3 antibodies would be an advantage to HIV in z&o, as it would endow a new swarm of variants with expanded or new cell tropisms. Furthermore, V3-loop peptides have been shown to be presented by the major histocompatibility complex (MHC) class I and class II in humans and are recognized by cytotoxic T cells (CTLs) and by T helper cells. Thus, amino acid changes in V3 that confer escape from neutralization or tropism changes might
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also result in escape from cellular immunity. Timing of immune pressure The selective pressures on HIV-l vary throughout the course of clinical infection, which can be divided into three distinct phases considering mainly viral load16 (Fig. 3). The virus that becomes successfully established in the recipient is only a small subset of the virus population in the blood of the donor. After transmission, in the initial phase, the viral replication rate is high and the plasma load increases rapidly. The adaptive immune response has yet to feature and the viruses that replicate fastest in the host cells dominate. Little is understood about the effect of the innate immune system (for example, natural killer cells) on selection at this
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stage. Nevertheless, variants are produced at this stage because the reverse transcriptase of HIV makes errors during each replication cycle. However, these variants are obscured by dominating faster-replicating clones, so that sampling of viral sequences at this stage seems to show the presence of a homogeneous population”. The second phase starts at seroconversion, and is characterized by the initiation of the cellular and humoral immune responses, which results in a dramatic reduction in the viral load and replication rate. Specific CTLs can be detected before antibodies, and are likely to eliminate most virus-producing cells during this phaseIs. Nevertheless, antibodies aid clearance by forming complexes that are removed, in part, by macrophages lining the liver and spleen sinusoids. Neutralizing antibodies contained in the complexes may protect macrophages from infection during this task. Virus strains surviving after seroconversion must somehow evade this immune onslaught, and are likely to include variants generated during the primary burst of virus replication. Indeed, variation detected after seroconversion is sharply increased, indicating that variant viruses do prevail at this stage. Such strains are usually nonsycytium inducing, slow/low and macrophage tropic. It is not clear whether or not macrophages provide a safer haven than do T cells for viruses to evade immune attack. Infection of macrophages may result in escape from CTLs if the virus remains in a dormant or latent state. Certainly in vitro, antigen production in macrophages is not usually apparent for 2-3 weeks after infection, whereas antigen is detected in PBLs within a few days of infection. Slowerreplicating forms are thus likely to persist at this stage, and viruses isolated usually have a slow/low phenotype. Macrophages may also offer a safe haven by ushering the genome into an ‘immunoprivileged’ site, such as the brain. Viral replication in secondary lymphoid organs, such as the spleen and lymph nodes, is high and continues at all stages of infec-
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tion19. Even during the asymptomatic period, CD4+ cell counts in the blood are continually being eroded. However, the immune system must continue to pressurize the replicating virus population during the asymptomatic stage. The last stage is characterized by the onset of AIDS. There is a sharp increase in viral replication and load, and a further depletion of CD4’ cell numbers. Faster-replicating and more-cytopathic viruses can be isolated from many individuals who progress to AIDS and, in about half of these, the virus isolated is syncytium inducing20*21. This switch of phenotype precedes the accelerated loss in CD4+ cell count, and is associated with a more rapid onset of AIDS than that occurring in those who maintain nonsyncytium-inducing strains. The emergence of faster-replicating variants later on may reflect the inability of the immune response to restrain such viruses. Studies of V3-loop sequences suggest that, as time progresses, the selective pressure for amino acid changes fallsz2. Eventually, dominant clones emerge, reflecting the exhaustion of immune responses. Immune selection and virus evolution
Recent estimates of virus production are of the order of 5 1O9virions per day, and complete turnover of CD4’ cells occurs in about 2d (Refs 23,24). This represents an enormous replicative activity and, together with the error-prone nature of the HIV reverse transcriptase, contributes to the tremendous diversity of HIV. This diversity surely leads to competition for survival, the main driving force of darwinian selection, and the consequent evolution of viruses that are best adapted to their changing environment. An alternative idea proposes that HIV-l diversity is driven by the random activation of infected cells in the lymph node25, and that immune selection may not significantly affect HIV diversity. Thus,
an infected T cell is activated in the lymph node when it comes into contact with antigen presented by the MHC and recognized by its
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specific T cell receptor. The expansion of a particular genome by the activation of a T cell is thus random, depending on the antigen presented, and is not because the virus is adapted for survival. Indeed, the virus variability generated within the lymph node and spleen is enormous, and it would be surprising if this did not contribute to the diversity of HIV. Viruses activated by this mechanism must then be subject to immune selective pressures, as well as to opportunities to colonize new cell types. Thus, a combination of random activation, followed by selection of the fittest, contributes to variation. It is important to bear in mind that ‘There are many unknown laws of correlation of growth, which, when one part of the organisation is modified through variation, and the modifications are accumulated by natural selection for the good of the being, will cause other modifications, often of the most unexpected nature’ Darwin, 185926. Acknowledgements We are grateful to Robin Weiss, David Wilkinson and the two refereesfor critically reading this manuscript, and to Graham Simmons for advice. References 1 Levy,J.A. (1993) Microbial.Rev. 57, 183-289 2 Shotton, C. et al. (1995)1. Viral.69, 222-230 3 Fung, M.S. etal. (1992)]. Virol.66, 848-856 4 Albert, J. et al. (1990)AIDS 4, 107-112 5 Goudsmit, J. et al. (1988)Proc. Nat1 Acad. Sci. USAg&4478-4482 6 McKnight, A.et al. (1992) AIDS 6, 799-802 7 Yoshiyama,H. et al. (1994)1. Virol.68, 974978 8 McKeating,J.A. et al. (1989)AIDS 3, 777-784 9 M&night, A.et al. (1995)1. Viral.69, 3167-3170 10 Bou Habib, D.C. et al. (1994)1. Virol. 68,6006-6013 11 Reitz, M.S.J. et al. (1988) Cell 54, 57-63 12 McKeating,J.A. et al. (1993)J. Virol. 67,5216-5225 13 Arendrup, M. et al. (1992)J. AlDS 5, 303-307 14 Nara, P.L. et al. (1990)1. Virol.64, 3779-3791
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21 Tersmette, M. et al. (1988)1, Viral. 62,2026-2032 22 Bonhoeffer,S., Holmes, EC. and Nowak, M.A. (1995) Nature 376,125 23 Wei, X.P. et al. (1995) Nature 373, 117-122 24 Ho, D.D. et al. (1995) Nature 373, 123-126 2.5 Delassus,S., Cheynier, R. and Wain-Hobson, S. (1992)1. Virol. 66,5642-5645
15 di Marzo Veronese,F. et al. (1993) 1. Biol. Chem. 268,25894-25901 16 Weiss,R.A. (1993) Science260, 1273-1279 17 Zhang, L.Q. et al. (1993)1. Virol. 67,3345-3356 18 Koup, R.A. (1994)I. Viral. 68, 4650-4655 19 Embretson,J. et al. (1993) Proc. Nut1 Acad. Sci. USA90,357-361 20 kj6, B. et al. (1986) Lancet ii, 660-662
26 Darwin, C. (1859) The Origin of the Species, John Murray 27 Leonard, C.K. et al. (1990)I. Biol. Chem. 265,10373-10382 28 Fouchier, R.A. et al. (1992)1. Viral. 66,3183-3187 29 Take&i, Y. et al. (1991)1. ViroL 65,1710-1718 30 Shioda,T., Levy,J.A. and Cheng Mayer, C. (1992) Proc. Nut1 Acad. Sci. USA89,9434-9438
Measlesvirusreplicationin neuralceh Ian CD. Johnston, Lee M. Dunster, Jiirgen Schneider-SchauEles and Sibylle Schneider-Schaulies Measles virus gene expression is attenuated in neural cells by mechanisms that affect both viral transcription and translation. Host enzymes that hypermutate viral genes, and those induced by cytokines, may act cooperatively to slow viral replication and to favor persistent measles virus infections in the human central nervous system.
ersistent measles virus infections of the central nervous system (CNS), such as subacute sclerosing panencephalitis (SSPE) and measles inclusion body encephalitis (MIBE; which is
P
confined to immunocompromised hosts), occur at low frequency months or years after primary measles virus infection. Massive numbers of viral core particles are seen in neurons and glial cells in both the gray and white matter, without infectious virus being released*, Persistent measles virus infections in the human CNS are characterized by defective viral replication, which mainly affects the envelope genes. This allows the virus to survive in a cell-associated form for long periods, while remaining inaccessible to host immune surveillance. Restrictions to measles virus gene expression have been defined directly in brain material from autopsies. Expression of the viral M, F and H genes, which encode envelope proteins, is attenuated by the presence of remarkably low frequencies of the corresponding mRNAs and/or the incorporation of sequence mutations within these reading frames, leading to defective protein expression or to complete abolition of translation (summarized in Table 1)2. Consequently, late in persistence, the virus lacks components that are essential to
I.C.D. Johnston, L..M. Dunster, J. Schneider-kbaulies and S. ScbneiderSchaulies l are in the Institute for Virology, Versbacher Str, 7,97078 Wiirzburg, Germany. *tel: +49 931201 5965, far: +49 9312013934
the assembly and budding of mature infectious particles. Moreover, the low expression or the complete absence of major viral antigenic determinants on the cell surface interferes with the recognition of infected cells by the host immune system. These restrictions in measles virus gene expression seem to explain how measles virus persistence is maintained late in infection. However, they do not explain how measles virus persistence is initially established in the CNS, considering that measles virus is normally highly cytolytic, and increasing evidence suggests that measles viruses entering the CNS are primarily not defective. In this article, we aim to give some insight into the mechanisms 0
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that govern the primary interaction of measles virus with cells of the CNS. In particular, we con-
sider whether and how factors that are intrinsic to the infected neural cell (or that are induced in the course of the infection) are involved in attenuating measles virus gene expression, by slowing down viral gene functions and interfering with rapid productive infection of these cells. We suggest that this host-cell-mediated control is essential for the establishment of persistent infection and makes a major contribution to the pathogenesis of measles-virus-induced human diseases of the CNS. Viral gene expression in neural cells As a consequence of the transcriptional strategy common to most paramyxoviruses, individual measles-virus-specific mRNAs are synthesized with decreasing efficiency according to their location along the viral genome, in vivo and in vitro3(Box 1). In brain material from SSPE cases, mRNAs that are transcribed from the 5’ end of the genome, in particular those encoding the fusion protein and the hemagglutinin, accumulate to lower levels than those that occur during an acute infection. This provides an initial efficient means of downregulating these gene products’. This particular transcriptional
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31Martin, D.W. et al. (1993) PYOC. Nat/ Acad. Sci. USA 90, 8377-8381 32 Martin, D.W. et al. (1993)J. Bacterial. 175,1153-1164 33 Martin, D.W. et al. (1994)]. Bacterial. 176,6688-6696 34 DeVries,C.A. and Ohman, D.E. (1994)J. Bacterial. 176,
47 Hatano, K., Goldberg,J.B. and Pier, G.B. (1995)Infect. Immun. 63,21-26 48 Goldberg,J.B. et al. (1992) Proc. Nat/ Acad. Sci. USA. 89,
6677-6687 35 Yu, H., Schurr, M.J. and Deretic,V. (1995)j. Bacterial. 177, 3259-3268 36 Goldberg,J.B. et al. (1993)I. Bacterial. 175,1303-1308 37 Gamer, J., Bujard, H. and Bnkau, B. (1992) Cell 69,833-842 38 Hughes, K.T. et al. (1993) Science 262,1277-1280 39 Lonetto, M.A. et al. (1994) Proc. Natl Acad. Sci. USA 86,
7573-7577 40 Erickson,J.W. and Gross, C.A. (1989) Genes Deu. 3,1462-1471 41 Johnson, K. et al. (1991) Mol. Microbial. 5,401-407 42 Roop, R.M., II et al. (1994) Infect. lmmun. 62,1000-1007 43 Cameron, R.M. et al. (1994)Microbiology 140,1977-1994 44 Terry, J.M., Pina, S.E.and Mattingly, S.J. (1991) Infect. lmmun. 59,471-477 45 Knirel, Y.A. (1990) CRC Crit. Rev. Microbial. 17,273-304 46 Arsenault, T.L. et al. (1991) Can. 1. Chem. 69,1273-1280
10616-10720 49 Evans, D,J. et al. (1994) Mol. Microbial. 13,427-434 50 Lightfoot,J. and Lam, J.S. (1993) Mol. Microbial. 8,771-782 51 Goldberg,J.B., Hatano, K. and Pier, G.B. (1993)1. Bacterial. 175,1605-1611 52 Coyne, M.J. et al. (1994)J. Bacterial. 176,3500-3507 53 Ye, R.W., Zielinski, N. and Chakrabarty, A.M. (1994) 1. Bacterial. 176,4851-4857 54 Hatano, K., Goldberg,J.B. and Pier, G.B. (1993)J. Bacterial.
175,5117-5128 55 Totten, P.A., Lara, J.C. and Lory, S. (1990)]. Bacterial. 172, 389-396 56 Stambach, M.N. and Lory, S. (1992) Mol. Microbial. 6,459-469 57 Simpson,D.A., Ramphal, R.R. and Lory, S. (1992) Infect.
Immun.60,3771-3779 58 Mork,T. and Hancock,R.E.W.(1993)Infect.Immun.61, 3287-3293 59 Saiman,L. andPrince,A. (1993)J.Clin. Invest. 92, 1875-1880
Immuneescapeand tropismof HIV Aine McKnight and Paul R. Clapham
H
IV-l was identified in 1983 as the agent that causes AIDS in humans. CD4 was shown to be the primary cell-surface receptor, reflecting the tropism of the virus for CD4expressing cells. In viva, HIV-l replication has been demonstrated in CD4+ T helper cells and specifically differentiated tissue macrophages, such as brain microglia, spinalcord macrophages, alveolar macrophages of the lung and blood monocytes. Dendritic cells, including Langerhans cells of the skin, can also be infected. Macrophages could be important in transporting the virus to different organs, thus seeding replication in new niches. As well as these tissues, CD4+lymphocytes, macrophages and dendritic cells in the secondary lymphoid organs, including the lymph nodes, adenoids, tonsils and spleen, are heavily infected (see the review by Levy, Ref. 1).Once established, HIV infection persists for life. In this article, we examine how HIV-l, by changing its phenotype (by shifting tropism and escaping from neutralizing antibodies), may evade the host immune response.
HIV-l cell tropisms are partly determined by the hypervariable loops VU-V2 and V3 in gp120, which also contain epitopes for neutralizing antibodies. Mutations conferring tropism changes can result in escape from neutralization and vice versa. We examine whether variant viruses that can colonize new cell types and simultaneously escape neutralizing antibodies have an enhanced advantage in tiuo. A. McKnight and P.R. Clapham * are in the Virology Laboratory, Chester Beatty Laboratories, Institute of Cancer Research, 237 F&am Road, London, UK SW3 6JB. ‘tel: +44 171 352 8133, fax: +44 171 352 3299, e-mail: [email protected], [email protected]
Cell tropism and envelope glycoproteins Some isolates of HIV-l have a preferential tropism for either T helper cells or macrophages, suggesting that efficient crossinfection of these cell types could be limited. However, the tropism is not absolute, and analysis of virus iso0
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lates in vitro reveals an array of phenotypes between the two extremes. It is still unclear whether or not viruses tropic for particular subsets of T cells or macrophages exist (Box 1). In addition to their tropism for macrophages or T cells, primary isolates of HIV-l can be categorized according to their ability to induce syncytia (large multinucleated cells) in primary peripheral blood lymphocytes (PBLs) or in the MT-2 T cell line (that is, as syncytium inducing or nonsyncytium inducing), as well as according to their replication rate. Virus isolates that replicate readily in CD4’ cell lines are described as ‘rapid/high’, whereas strains that fail to infect established cell lines and are relatively noncytopathic are termed ‘slow/ low’. Repeated passage of HIV-l strains in CD4’ T cell lines in vitro selects for syncytium-inducing strains that are more tropic for T cell lines and have a markedly reduced ability to infect macrophages. However, a minority of such strains can still infect macrophages efficiently, and are described as ‘dual tropic’.
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The viral sequences that determine these phenotypes lie mainly in the outer-envelope glycoprotein of HIV-l, gp120, which is attached noncovalently to the membranespanning glycoprotein, gp41. The gp120 glycoprotein mediates binding to CD4, which initiates events that lead to the fusion of the viral and cell membranes and the subsequent entry into the cell cytoplasm. Five distinct hypervariable regions (Vl-VS) and five relatively conserved regions (Cl-CS) can be distinguished in gp120, based on sequence analysis of different strains of HIV-l (Fig. 1). Determinants of tropism for macrophages or T cell lines, and determinants of syncytium-inducing ability, have been mapped to the variable loops Vl, V2 and particularly V3. For example, an increase in the number of charged amino acids in V3 correlates with a switch in phenotype from nonsyncytium inducing to syncytium inducing (Fig. 2). The role or function of the Vl, V2 and V3 loops during the virus-cell fusion process that leads to the distinct phenotypes described above is not clear. However, their critical involvement in such processes suggests that antibodies to these regions are likely to be neutralizing, which has been shown to be true for V2 and V3.
Box 1. Am changes in tropism advantageous for HIV-1In vh? *Do HIV variants that can colonize new cell types have an advantage other strains? l
Do slow-replicating variants that can colonize macrophages evade immunity and prevail?
*Late in infection, emerging syncytium-inducing strains often show an expanding tropism for CD4+ T cell lines in vitro. Could this wider host range reflect a broadening tropism for different T cell subsets in vivo?
epitopes that include the more variable amino acids flanking this motif are usually strain specific, while mAbs with epitopes that include the conserved tip are more broadly reactive. The first neutralizing antibodies to develop after infection and seroconversion are generally effective against the infecting strain or closely related isolates4. Most of these antibodies are directed against V3 (Ref. 5). Over time, the response broadens and antibodies that neutralize diverse laboratory isolates develop6. The exact epitopes that are recognized by these crossreactive antibodies are not clear, but they are likely to be situated in the gp120 regions that make up the conformationally dependent CDCbinding region, as
Regions of gpl20 targeted by neutralizingantibodies V2 and V3 sequences are hyper-
variable between strains, hence neutralizing antibodies to these regions are likely to be strain specific, reacting preferentially with the inducing amino acid sequence. Indeed, monoclonal antibodies (mAbs) to V2 are generally strain specific, although antibodies that are more broadly reactive do occur2. Sequences within V2 form both linear and conformationally dependent epitopes that induce neutralizing antibodies3. The V3 loop was the first region of gp120 to be defined as a target for neutralizing antibodies. V3 is less variable than is Vl or V2, and contains a relatively conserved motif, Gly-Pro-Gly, at the central tip (Fig. 2). Neutralizing mAbs that target the V3 loop and have
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well as an epitope in gp41. Monoclonal Abs to these regions can neutralize diverse HIV-l strains in vitro.
Escape from neutralizing antibodies The tolerance of variation in the V2 and V3 regions of gp120 suggests that mutants that can escape neutralizing antibodies might easily arise. In fact, HIV-l escape mutants that resist neutralization by mAbs against V2 (Refs 2,7) or V3 can be selected in vitro8>9.Mutations that result in escape from anti-V3 mAbs can occur inside or outside the V3 loop. Amino acid substitutions inside V3 probably alter the antibody-binding site, whereas distant mutations probably
-i
y
COOH
c5 Fig. 1. HIV-1 envelope glycoprotein gpl20 based on the HXlO clone of HIV-1 IAI strain. Lines joining discontinuous sequences represent disulphide bonds and the numbers denote amino acids. (Redrawn from Ref. 27.)
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results in viruses that escape antibody neutralization.
flg. 2. The V3 loop of HIV-1 is 32-35 amino acids long and is delimited by a disulphide bond between two cysteines at the base. It is a major determinant of viral tropism, and amino acid substitutions have been shown to affect the biclogical properties and tropism of HIV-l. The sequence shown is derived from HIV-1 lAI strain. An asterisk indicates amino acids that are likely to have positively charged side chains in syncytium-inducing isolatesZ8. A filled circle indicates that a single amino acid change at this position changes the tropism of the Gun-l strain of HIV-1 (Ref. 29; see text and Table 1). A filled square indicates the position of an amino acid change shown by Shioda et aL30 to allow the SF-2 strain HIV-1 to infect the CD4+ T cell line MT-4.
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alter the conformation or render it inaccessible to antibody. Strains of HIV-l passaged in vitro without selection pressure from neutralizing antibodies are generally more susceptible to neutralization by anti-V3-loop antibodies than are primary strains, which have morecryptic V3 loopsi”. Perhaps the presence of a more exposed V3 loop results in more-efficient virus-cell fusion and increases the replication rate. During primary infection, there is often massive replication before neutralizing antibodies are produced and, during this stage of infection, it might be speculated that a faster-replicating virus with an exposed V3 loop would be selected. Exposure to newly synthesized anti-V3-loop antibodies after seroconversion might then select variant viruses with mutations favouring a cryptic V3 that
Escape in viva
Unequivocal confirmation of escape from neutralization in vivo is difficult to obtain. Arendrup et all3 studied the neutralization of sequential isolates from HIV-l-infected individuals by homologous serum. Initially, strain-specific neutralizing antibodies developed and preceded the emergence of virus isolates that could escape. Subsequently, neutralizing antibodies that were effective against the escaped virus developed. The first neutralizing antibodies arising after seroconversion are thought to be directed against the V3 100~~. Escape from such V3-loop neutralization has been shown to occur in experimentally infected chimpanzees14, where resistance to neutralization was found to be conferred by changes outside V3. Subsequently, escape mutants with amino acid substitutions at the crown of V3 were isolated. Sequential isolates from a laboratory worker accidentally infected with HIV-l LA1 were shown to contain an amino acid substitution that conferred resistance in vitro to an anti-V3-loop mAb (Ref. 15).
limit antibody accessibility and allow the virus to escape antibody neutralization. Escape mutants of the LA1 strain of HIV-l that resist neutralization by a serum from an HIV-l-positive individual have been selected in vitro1’J2. These variants were found to have mutations either in gp41 or in the C3 region of gp120. Both types of mutation conferred directed resistance to mAbs against the CD4-binding region of gp120. These observations suggest that the dominant crossneutralizing antibodies in the human serum samples used were to the CD4binding region, and that envelope changes in the C3 region of gp120 or in gp41 altered the conformation of this region to allow escape. Clearly, it is not difficult to demonstrate in vitro that variation generated by virus replication
Virus escape and tropism changes So far we have discussed how the
variable regions V2 and V3 of gp120 are not only determinants of tropism, but also targets for neutralizing antibodies. In the V3 region in particular some, but certainly not all, amino acid substitutions result in a change in viral tropism. It is also true that some amino acid substitutions in the V3 loop can result in escape from neutralizing antibodies. Recently, we tested to what extent these phenotypes affect each other using Gun1, an isolate of HIV-l that is dual tropic, that is, it can infect both T cell lines and macrophages (Table 1)9. A variant of Gun-l selected for growth in a brain-derived cell line not only lost infectivity for macrophages, but also escaped neutralization by a mAb recognizing the Gun-l V3 loop. This change in phenotype is due to a single amino
Table 1. Amino acid substitutions in the HIV-l strain Guwl resulting from in v/&o selection for tropism change and neutralization escapee Resulting tropism Selectlon None Tropism change Neutralization escape
V34oop sequence
Macrophage T cell line Brain glioma
GPGRAFHAI GSGRAFHAI GPGRAFHAI GSGRAINAI GSGRTFQAI GSGRALHAI
+ + + +
+ + + + + +
+ + +/+/-
The HIV-1 strain Gun-l infects macrophages and T cells, but not CD4+ brain glioma Cells. Substitution of serine for proline at the tip of the V3 loop of gp120 switches the viral tropism so that the virus can infect CD4+ brain glioma cells, but not macrophages 9.29. Selection for four viruses to escape neutralization results in altered tropism and amino acid substitutions in the V3 loop. The bold letters denote the substituted amino acids.
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Fig. 3. Within 3 weeks of primary infection, about 40% of individuals show some symptoms of viral infection, including headache, muscle ache, tender lymph nodes, sore throat and fever. These symptoms usually last up to 3 weeks. High levels of virus can be detected in the blood, and CD4+ cell numbers decline. This phase lasts about 4-8weeks, after which viraemia is reduced dramatically because of the initiation of the immune response. Specific cytotoxic T cells (CTLs) are detected before neutralizing antibodies appear. In the asymptomatic phase after seroconversion, the individual is apparently healthy, although occasionally symptoms, such as diarrhoea and night sweats, can occur. The viral load varies, but remains much reduced. Within 12years the majority of individuals succumb to AIDS, which is characterized by opportunistic infections, neoplasms, Kaposi’s sarcoma, severe weight loss and dementia. After the diagnosis of AIDS, most individuals die within 3years. Abbreviation: PBL, peripheral blood lymphocyte.
acid substitution at the crown of the V3 loop (Gly-Pro-Gly to GlySer-Gly). We also tested the reciprocal situation. Does escape from neutralizing antibodies affect tropism? We produced further neutralizing mAbs that were specific for the variant V3 loop, and used them to select for neutralization escape mutants. The four escape mutants that we analysed each had an altered tropism. Two gained infectivity for primary macrophages, while one reverted to the wild-type V3 loop (Gly-Pro-Gly) and tropism, losing infectivity for the brainderived ceils and regaining tropism for macrophages. The tropism of a fourth escape mutant became restricted, losing infectivity for brain cells, without regainin macrophage infectivity. Amino acid changes
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located at or near the crown of the V3 loop are likely to cause these phenotypic changes. Thus, escape from neutralizing antibodies resulted in an altered viral phenotype, which gave three of four mutants the ability to colonize macrophages, while restricting tropism for a fourth. Such escape from neutralizing anti-V3 antibodies would be an advantage to HIV in z&o, as it would endow a new swarm of variants with expanded or new cell tropisms. Furthermore, V3-loop peptides have been shown to be presented by the major histocompatibility complex (MHC) class I and class II in humans and are recognized by cytotoxic T cells (CTLs) and by T helper cells. Thus, amino acid changes in V3 that confer escape from neutralization or tropism changes might
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also result in escape from cellular immunity. Timing of immune pressure The selective pressures on HIV-l vary throughout the course of clinical infection, which can be divided into three distinct phases considering mainly viral load16 (Fig. 3). The virus that becomes successfully established in the recipient is only a small subset of the virus population in the blood of the donor. After transmission, in the initial phase, the viral replication rate is high and the plasma load increases rapidly. The adaptive immune response has yet to feature and the viruses that replicate fastest in the host cells dominate. Little is understood about the effect of the innate immune system (for example, natural killer cells) on selection at this
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stage. Nevertheless, variants are produced at this stage because the reverse transcriptase of HIV makes errors during each replication cycle. However, these variants are obscured by dominating faster-replicating clones, so that sampling of viral sequences at this stage seems to show the presence of a homogeneous population”. The second phase starts at seroconversion, and is characterized by the initiation of the cellular and humoral immune responses, which results in a dramatic reduction in the viral load and replication rate. Specific CTLs can be detected before antibodies, and are likely to eliminate most virus-producing cells during this phaseIs. Nevertheless, antibodies aid clearance by forming complexes that are removed, in part, by macrophages lining the liver and spleen sinusoids. Neutralizing antibodies contained in the complexes may protect macrophages from infection during this task. Virus strains surviving after seroconversion must somehow evade this immune onslaught, and are likely to include variants generated during the primary burst of virus replication. Indeed, variation detected after seroconversion is sharply increased, indicating that variant viruses do prevail at this stage. Such strains are usually nonsycytium inducing, slow/low and macrophage tropic. It is not clear whether or not macrophages provide a safer haven than do T cells for viruses to evade immune attack. Infection of macrophages may result in escape from CTLs if the virus remains in a dormant or latent state. Certainly in vitro, antigen production in macrophages is not usually apparent for 2-3 weeks after infection, whereas antigen is detected in PBLs within a few days of infection. Slowerreplicating forms are thus likely to persist at this stage, and viruses isolated usually have a slow/low phenotype. Macrophages may also offer a safe haven by ushering the genome into an ‘immunoprivileged’ site, such as the brain. Viral replication in secondary lymphoid organs, such as the spleen and lymph nodes, is high and continues at all stages of infec-
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tion19. Even during the asymptomatic period, CD4+ cell counts in the blood are continually being eroded. However, the immune system must continue to pressurize the replicating virus population during the asymptomatic stage. The last stage is characterized by the onset of AIDS. There is a sharp increase in viral replication and load, and a further depletion of CD4’ cell numbers. Faster-replicating and more-cytopathic viruses can be isolated from many individuals who progress to AIDS and, in about half of these, the virus isolated is syncytium inducing20*21. This switch of phenotype precedes the accelerated loss in CD4+ cell count, and is associated with a more rapid onset of AIDS than that occurring in those who maintain nonsyncytium-inducing strains. The emergence of faster-replicating variants later on may reflect the inability of the immune response to restrain such viruses. Studies of V3-loop sequences suggest that, as time progresses, the selective pressure for amino acid changes fallsz2. Eventually, dominant clones emerge, reflecting the exhaustion of immune responses. Immune selection and virus evolution
Recent estimates of virus production are of the order of 5 1O9virions per day, and complete turnover of CD4’ cells occurs in about 2d (Refs 23,24). This represents an enormous replicative activity and, together with the error-prone nature of the HIV reverse transcriptase, contributes to the tremendous diversity of HIV. This diversity surely leads to competition for survival, the main driving force of darwinian selection, and the consequent evolution of viruses that are best adapted to their changing environment. An alternative idea proposes that HIV-l diversity is driven by the random activation of infected cells in the lymph node25, and that immune selection may not significantly affect HIV diversity. Thus,
an infected T cell is activated in the lymph node when it comes into contact with antigen presented by the MHC and recognized by its
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specific T cell receptor. The expansion of a particular genome by the activation of a T cell is thus random, depending on the antigen presented, and is not because the virus is adapted for survival. Indeed, the virus variability generated within the lymph node and spleen is enormous, and it would be surprising if this did not contribute to the diversity of HIV. Viruses activated by this mechanism must then be subject to immune selective pressures, as well as to opportunities to colonize new cell types. Thus, a combination of random activation, followed by selection of the fittest, contributes to variation. It is important to bear in mind that ‘There are many unknown laws of correlation of growth, which, when one part of the organisation is modified through variation, and the modifications are accumulated by natural selection for the good of the being, will cause other modifications, often of the most unexpected nature’ Darwin, 185926. Acknowledgements We are grateful to Robin Weiss, David Wilkinson and the two refereesfor critically reading this manuscript, and to Graham Simmons for advice. References 1 Levy,J.A. (1993) Microbial.Rev. 57, 183-289 2 Shotton, C. et al. (1995)1. Viral.69, 222-230 3 Fung, M.S. etal. (1992)]. Virol.66, 848-856 4 Albert, J. et al. (1990)AIDS 4, 107-112 5 Goudsmit, J. et al. (1988)Proc. Nat1 Acad. Sci. USAg&4478-4482 6 McKnight, A.et al. (1992) AIDS 6, 799-802 7 Yoshiyama,H. et al. (1994)1. Virol.68, 974978 8 McKeating,J.A. et al. (1989)AIDS 3, 777-784 9 M&night, A.et al. (1995)1. Viral.69, 3167-3170 10 Bou Habib, D.C. et al. (1994)1. Virol. 68,6006-6013 11 Reitz, M.S.J. et al. (1988) Cell 54, 57-63 12 McKeating,J.A. et al. (1993)J. Virol. 67,5216-5225 13 Arendrup, M. et al. (1992)J. AlDS 5, 303-307 14 Nara, P.L. et al. (1990)1. Virol.64, 3779-3791
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21 Tersmette, M. et al. (1988)1, Viral. 62,2026-2032 22 Bonhoeffer,S., Holmes, EC. and Nowak, M.A. (1995) Nature 376,125 23 Wei, X.P. et al. (1995) Nature 373, 117-122 24 Ho, D.D. et al. (1995) Nature 373, 123-126 2.5 Delassus,S., Cheynier, R. and Wain-Hobson, S. (1992)1. Virol. 66,5642-5645
15 di Marzo Veronese,F. et al. (1993) 1. Biol. Chem. 268,25894-25901 16 Weiss,R.A. (1993) Science260, 1273-1279 17 Zhang, L.Q. et al. (1993)1. Virol. 67,3345-3356 18 Koup, R.A. (1994)I. Viral. 68, 4650-4655 19 Embretson,J. et al. (1993) Proc. Nut1 Acad. Sci. USA90,357-361 20 kj6, B. et al. (1986) Lancet ii, 660-662
26 Darwin, C. (1859) The Origin of the Species, John Murray 27 Leonard, C.K. et al. (1990)I. Biol. Chem. 265,10373-10382 28 Fouchier, R.A. et al. (1992)1. Viral. 66,3183-3187 29 Take&i, Y. et al. (1991)1. ViroL 65,1710-1718 30 Shioda,T., Levy,J.A. and Cheng Mayer, C. (1992) Proc. Nut1 Acad. Sci. USA89,9434-9438
Measlesvirusreplicationin neuralceh Ian CD. Johnston, Lee M. Dunster, Jiirgen Schneider-SchauEles and Sibylle Schneider-Schaulies Measles virus gene expression is attenuated in neural cells by mechanisms that affect both viral transcription and translation. Host enzymes that hypermutate viral genes, and those induced by cytokines, may act cooperatively to slow viral replication and to favor persistent measles virus infections in the human central nervous system.
ersistent measles virus infections of the central nervous system (CNS), such as subacute sclerosing panencephalitis (SSPE) and measles inclusion body encephalitis (MIBE; which is
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confined to immunocompromised hosts), occur at low frequency months or years after primary measles virus infection. Massive numbers of viral core particles are seen in neurons and glial cells in both the gray and white matter, without infectious virus being released*, Persistent measles virus infections in the human CNS are characterized by defective viral replication, which mainly affects the envelope genes. This allows the virus to survive in a cell-associated form for long periods, while remaining inaccessible to host immune surveillance. Restrictions to measles virus gene expression have been defined directly in brain material from autopsies. Expression of the viral M, F and H genes, which encode envelope proteins, is attenuated by the presence of remarkably low frequencies of the corresponding mRNAs and/or the incorporation of sequence mutations within these reading frames, leading to defective protein expression or to complete abolition of translation (summarized in Table 1)2. Consequently, late in persistence, the virus lacks components that are essential to
I.C.D. Johnston, L..M. Dunster, J. Schneider-kbaulies and S. ScbneiderSchaulies l are in the Institute for Virology, Versbacher Str, 7,97078 Wiirzburg, Germany. *tel: +49 931201 5965, far: +49 9312013934
the assembly and budding of mature infectious particles. Moreover, the low expression or the complete absence of major viral antigenic determinants on the cell surface interferes with the recognition of infected cells by the host immune system. These restrictions in measles virus gene expression seem to explain how measles virus persistence is maintained late in infection. However, they do not explain how measles virus persistence is initially established in the CNS, considering that measles virus is normally highly cytolytic, and increasing evidence suggests that measles viruses entering the CNS are primarily not defective. In this article, we aim to give some insight into the mechanisms 0
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that govern the primary interaction of measles virus with cells of the CNS. In particular, we con-
sider whether and how factors that are intrinsic to the infected neural cell (or that are induced in the course of the infection) are involved in attenuating measles virus gene expression, by slowing down viral gene functions and interfering with rapid productive infection of these cells. We suggest that this host-cell-mediated control is essential for the establishment of persistent infection and makes a major contribution to the pathogenesis of measles-virus-induced human diseases of the CNS. Viral gene expression in neural cells As a consequence of the transcriptional strategy common to most paramyxoviruses, individual measles-virus-specific mRNAs are synthesized with decreasing efficiency according to their location along the viral genome, in vivo and in vitro3(Box 1). In brain material from SSPE cases, mRNAs that are transcribed from the 5’ end of the genome, in particular those encoding the fusion protein and the hemagglutinin, accumulate to lower levels than those that occur during an acute infection. This provides an initial efficient means of downregulating these gene products’. This particular transcriptional
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