Pathobiology 1992;60:225-233
Division of Infectious Diseases, West Los Angeles VA Medical Center and Department of Medicine, UCLA School of Medicine, Los Angeles. Calif., USA
Key Words HIV-1 Tropism Variation Mononuclear phagocyte Polymerase chain reaction CD4 V3 loop Neutralization
Viral Determinants of Cellular Tropism
Abstract
Cells of mononuclear phagocyte lineage are the predominant cell type produc ing human immunodeficiency virus type 1 (HIV-1) in extravascular tissues; HIV-1 infection of mononuclear phagocytes may be directly related to pri mary disease manifestations and also appears to contribute to the immune deficiency of AIDS. Whereas peripheral blood lymphocytes are permissive for nearly all strains of HIV-1, only some HIV-1 strains replicate efficiently in mononuclear phagocytes. Recombinant virus strains have been used to iden tify a 157-amino acid region of gpl20 that can confer macrophage tropism. This region is distinct from the principal CD4 binding domain of gpl20 and includes the major type-specific neutralizing epitope located in the third hypervariable domain. V3. Quantitative assay of HIV-1-specific DNA by polymerase chain reaction early after infection suggests that HIV-1 strain dif ferences in macrophage tropism are determined at the level of entry. These studies suggest that target cell interactions with gpl20 in addition to or in conjunction with the CD4 binding domain are necessary for efficient entry into mononuclear phagocytes.
The Role of HIV Infection of Mononuclear Phagocytes in Clinical A ID S
Infection by human immunodeficiency virus (HIV) can result in a broad spectrum of disease, caused in part by development of progressive immune deficiency. The CD4+T-lymphocyte was the first cell identified as a target for HIV infection in vivo. This cell is susceptible to infec tion by all strains of HIV-1, with rare exception. There is an inexorable decline in CD4+ T cells following acute infection; loss of these cells leads to development of cellu lar immune deficiency and subsequent opportunistic in fection and other diseases. However, it is cells of mononu
Received: July 5. 1991 Accepted: August 1 ,1991
clear phagocyte lineage that are the predominant cell type producing HIV-1 in extravascular tissues [12, 13.21,45], Disease manifestations believed to be a direct result of HIV-1 infection (and not due to the resulting immunode ficiency) have been associated with mononuclear phago cyte infection; for example, AIDS dementia, neuropathy, pneumonitis and dermatitis have been associated with infection of mononuclear phagocytes in the central ner vous system (CNS), spinal cord, lung and skin, respec tively [30]. In addition, HIV-1 infection of mononuclear phago cytes appears to contribute to the immune deficiency of AIDS. Mononuclear phagocytes are immunologically ac-
Dr. William A. O’Brien 691/WI1 IF West LA VA Medical Center Los Angeles, CA 90073 (USA)
© 1992 S. Karger AG, Basel 1015-2008/92/0604-0225 $2.75/0
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William A. O'Brien
Quantitation of HIV-1
Early attempts to detect viral proteins or RNA in peripheral blood mononuclear cells (PBMC) of patients with symptomatic HIV infection demonstrated a fre quency of infected cells of 1/10,000 or less [ 16]. However, the viral load in patients is much higher than previously thought. Quantitative cultures using endpoint dilution were used to measure infectious virus titer both in PBMC and plasma [ 18], Although there was a wide range of virus titer between patients, in symptomatic HIV-positive patients with low CD4 cell number, mean plasma titers were 3.500 tissue culture infectious doses (TCID)/ml, or 1 TCID/0.3 pi. and the PBMC viral titers were 2.200 TCID/106 cells or 1 TCID/400 PBMC. In patients without symptoms, infectious virus titer was 100-fold lower. Virus titer ap pears to be higher in patients with more advanced disease [6,18], Recently, quantitative polymerase chain reaction (PCR) methods were used to determine viral load. HIV1-specific DNA is readily detected in PBMC by PCR. Schnittman et al. [38] showed a frequency of infection of 1/100 or greater in CD4-positive lymphocytes of patients with AIDS. Lower frequencies of infection (1/1,000 to less than 1/10.000) were seen in asymptomatic seropositive patients. In this study, the highest viral burden was seen in patients having a deteriorating clinical course. Thus, increasing viral burden appears to be associated with dis
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ease progression and development of virus-induced dis ease. Schnittman et al. [39] separated mononuclear phago cytes, CD4+ lymphocytes and CD8+ lymphocytes by fluo rescence-activated cell sorting to identify which blood mononuclear cells are infected by HIV-1. HIV-specific DNA was present in T4 cells in 10 of 10 patients; HIVspecific DNA was detected in blood mononuclear phago cytes from only 2 of 10 of these patients. The authors con clude that T cells and not mononuclear phagocytes are the principal reservoir for HIV, at least in the blood. How ever, mononuclear phagocytes are only transiently present in the blood. HIV-specific DNA can be consis tently demonstrated in seropositive patients in mononu clear phagocytes purified by adherence [D. Ho, pers. commun.]. In addition, they migrate to extravascular tissues, where they differentiate in response to local factors. Lev els of viral DNA in the brain tissue of patients who died with AIDS dementia, where most viral nucleic acids are associated with mononuclear phagocytes, is over ten times greater than in paired samples taken from patient blood [34], Although few studies have measured viral load in extravascular tissues other than brain [40], it is likely that productive infection in extravascular tissues associated with clinical disease is of great magnitude.
HIV-1 Infection of Primary Blood Cells
Viremia is typically present during acute infection [4, 8], suggesting that infection of blood cells is involved. Nearly all HIV-l strains replicate in lectin-stimulated peripheral blood lymphocytes (PBL), whether the virus was derived from PBL, transformed cell lines, mononu clear phagocytes or infected tissues. HIV-l infection of stimulated PBL results in virus production within 24 h. Replication occurs rapidly, with a peak in virus produc tion at day 5-7 (fig. la). There is no significant virus pro duction following infection of quiescent PBL, but effi cient entry does occur [49], Initiation of reverse tran scription is equivalent following infection of resting and stimulated PBL. This was demonstrated by a quantita tive PCR assay using oligonucleotide primer pairs de signed to amplify DNA in the HIV-l R/U5 region of the LTR, the first region formed in retroviral reverse tran scription. However, reverse transcription is not com pleted in these cells; there is little full-length viral DNA formed in quiescent cells, as measured by PCR primer pairs designed to amplify HIV-l LTR/^a^-specific DNA, which is not formed until near completion of reverse
Viral Determinants o f Cellular Tropism
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tive cells [46], First, they internalize and process antigen for presentation on the cell surface, in association with major histocompatibility gene complex class II molecules. Second, they secrete cytokines, proteases and comple ment proteins. Third, they bear receptors for lympho kines, which are secretory products of lymphocytes that can activate these cells and modify their function. Fourth, they are involved in complex interactions with T and B lymphocytes in the host immune response. HIV-1 binding to the CD4 molecule on mononuclear phagocytes can induce release of interleukin 1 and tumor necrosis factor [28], Infected mononuclear phagocytes have also been shown to be impaired in immune function and response to cytokines [2]. Therefore, insight into the mechanism of infection of mononuclear phagocytes is important for improved understanding of disease caused by HIV-1. Characterization of virus determinants important for effi cient HIV-1 infection of mononuclear phagocytes will be the topic of this review.
Fig. 1. Kinetics of HIV-l replication in cultured primary blood mononuclear cells. Normal human blood mononuclear cells were recovered by Ficoll-Hypaque density centrifugation. PBL and blood mononuclear phagocytes were separated by adherence. PBL (2 X I07) stimulated with PHA (Wellcome. 0.8 pg/ml) for 48 h (a) were infected with cell-free virus containing 500 ng of HIV-1 p24 [Abbott. ELISA; H1V-1 (A ). IIIV-1jr ^ ’sp ( a ), HIV-1sp2 (o) and HIV-1 m .4-,1 (o)[. Medium was changed every 2 days and assayed for virus at days 3, 7. 9 and 11 after infection. Blood mononuclear phagocytes (5 X 105) (b) were infected after 6 days of adherence with 10 ng of HIV-1 p24. Medium was changed every 3-4 days and assayed for virus at days 7, 14, 17 and 21. Experiments a and b used the same virus stocks.
cytes with y-interferon, a potent activator of mononuclear phagocytes, does not affect proliferation and inhibits virus production. However, y-interferon treatment prior to infection has been shown to enhance virus production [24], Effects of cytokine treatment are complex, but it appears that activation of these cells generally enhances virus replication.
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transcription. If these cells are stimulated following infec tion. the full-length viral DNA structure is formed, and virus production follows. Although the partially reversetranscribed retroviral structure appears to persist in quiescent cells in a latent form, it is labile and degrades with a half-life of approximately I day. Therefore, infec tion of quiescent cells can result in short-lived latent infection, but the viral DNA will be degraded unless the cells are stimulated shortly after infection. In vivo, most circulating T lymphocytes are in a quiescent state and are activated to proliferate only in response to specific anti gen. The labile HIV-1 DNA in quiescent cells could con tribute to the prolonged duration of clinical latency. The degradation of viral DNA in quiescent cells may explain the relatively low percentage of infected cells in the circu lation of seropositive patients, particularly early in the disease course. HIV-1 replication in blood mononuclear phagocytes occurs with delayed kinetics compared to stimulated PBL. There is little extracelluar virus production seen until 4-5 days after in vitro infection. This gradually rises, with a peak of virus production at approximately 3 weeks (fig. lb). One possible explanation for differences in the kinetics of virus production in these cell types is delayed extracellular release of virus. Orenstein et al. [32] have observed intracellular budding of virions into cytoplas mic vacuoles by electron microscopy. Very little virus is seen extracellularly early after infection. Thus, following infection, mononuclear phagocytes can produce large amounts of virus, and, because these cells are resistant to killing by HIV-1, can slowly release virus over prolonged periods of time. In addition to impaired extracellular bud ding of virus, other events in the retroviral life cycle may affect kinetics of virus production, including entry, re verse transcription, integration, virus expression or virus assembly. Using PCR primers to detect DNA at different stages of reverse transcription in HIV-1-infected mono nuclear phagocytes, we observed delayed kinetics of re verse transcription compared to that of T lymphocytes [O’Brien et al., in preparation]. Like quiescent PBL. mononuclear phagocytes gener ally do not proliferate, although, unlike quiescent PBL. they can be productively infected. Virus production can be dramatically increased in mononuclear phagocytes ac tivated by treatment with cytokines. Treatment of HIV1-infected mononuclear phagocytes with granulocytemacrophage colony-stimulating factor (GM-CSF), macro phage colony-stimulating factor (M-CSF) or interleukin 3 is associated with a dose-dependent increase in cell prolif eration [24], Treatment of infected mononuclear phago-
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Viral Determinants o f Cellular Tropism
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HIV-1 Variation
Identification of Virus Determinants Important for Mononuclear Phagocyte Tropism
Virus strains more distantly related to the JR strains, such as HIV-1 hxb2 and HIV-1 NL4-3, which are derived from serial passage in transformed T-cell lines, are very inefficient at replicating in peripheral blood mononuclear phagocytes, and there is usually no significant virus pro duction following infection of these cells. These labora tory strains are also unable to replicate in primary brain microglial cells [48], which are resident macrophages of the brain. Primary blood microglial cells can be produc tively infected by virus strains that replicate in blood mononuclear phagocytes [48], Because of the marked dif ferences in the ability of HIV-1JR.|.-L and HIV-INL4-3 to productively infect mononuclear phagocytes, molecular clones of these strains were used to generate molecular recombinant viruses in order to identify viral regions involved in determining mononuclear phagocyte cell tro pism. For initial recombinant virus studies, purified DNA fragments from these cloned virus strains were ligated prior to electroporation of chimeric proviral DNA into 729-6 cells. This transfection technique utilizes a pulsed
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Mononuclear phagocyte and T cell line tropism
Fig. 3. The HIV-1 envelope gene. Conserved regions are depicted with dark shading, and variable regions are shown in white. Regions encoding the extracellular glycoprotein. gp!20, and the transmem brane glycoprotein, gp4l, are shown above. Functional domains identified for neutralization (V3 loop), CD4 binding, fusion, and mononuclear phagocyte and T-cell line tropism are shown.
electrical current to nonspecifically introduce DNA into human cells. Recombinant virus is formed in these cells, and high titer virus can be produced following short-term coculture with PHA-stimulated PBL. Recombinant viruses (fig. 2) composed of progres sively smaller regions of HIV-lJR.FL at the 3' end joined at Sst I (nt 5999), Dra III (nt 6591), or Stu I (nt 6822) still replicated efficiently in mononuclear phagocytes. The recombinant virus, HIV-1nF.xiio, which contains the Nef and U3/R LTR elements from H IV -l^ .^ , does not pro ductively infect mononuclear phagocytes. By substituting internal fragments from molecular clones of HIV-1jR_fi into the infectious clone, pNL4-3, a region conferring mononuclear phagocyte tropism was localized to the env gene by the recombinant, HIV-lNFN-sx, and particularly, the recombinant, HIV-1NFn-sm- The latter recombinant contains only 157 amino acids from the HIV-l JR.FLgpl20 region (residues 202-358) and can efficiently infect mononuclear phagocytes. Of note, this region of gpl20 includes the major neutralization domain and does not include the originally defined CD4 binding domain (amino acids 404-447) [25] (fig. 3). The recombinant virus, HIV-1 nfn-mx> which contains the CD4 binding domain and all of gp41 encoded by HIV-1 JR.FL, does not replicate in mononuclear phagocytes. Mononuclear phagocyte cell tropism has been mapped to the same region of gp!20 using recombinant viruses containing sequences from HIV-1SF|62, another macro
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There is extensive variation of HIV-1 strains at the nucleotide sequence level, even between strains recovered from the same individual [ 15, 23,29, 37], Despite the cor relation of HIV-1 infection of mononuclear phagocytes with clinical disease, different HIV-1 isolates vary mark edly in their ability to productively infect mononuclear phagocytes. Distinct differences in target cell tropism have been identified for different HIV-1 isolates obtained from the same individual [23], HIV-1JR.CSF was isolated from cell-free cerebrospinal fluid (CSF) of a patient who died with severe AIDS encephalopathy. This strain repli cates efficiently in phytohemagglutinin (PHA)-stimulated PBL. but does not replicate as well in blood mononuclear phagocytes from most donors. In contrast, the strain HIV-1 jR.[7L, recovered from frontal lobe brain tissue of the same patient, replicates efficiently and to high titer in stimulated PBL and blood mononuclear phagocytes from all donors tested (over 200). Although these strains are genetically distinct, they are more closely related than are strains isolated from different individuals [29]. Thus, dif ferences in cellular tropism appear to be related to viral genetic variation.
phage-tropic strain [41]. Cell tropism for transformed Tcell lines appears to map to a similar region of gpl20: however, viral determinants of tropism for these cell types appears to be mutually exclusive [41]. In general, recom binant viruses that replicate efficiently in mononuclear phagocytes do not productively infect transformed T-cell lines, and the converse is also true. We have seen similar results using recombinant viruses from H I V - 1 j F. f l and HIV-l NL4.3.
tion appears to be affected by LTR sequence variation [14], Variation in the pol gene could affect reverse tran scription. integration or protease activity. Virus strains exhibiting AZT resistance have been shown to have reduced infectivity [47], Other viral genes whose func tions are poorly defined, such as vpr, vif vpu and nef, may also contribute to differences in virus replication by affecting events following entry.
Neutralization of HIV Infection Mechanism of Mononuclear Phagocyte Tropism
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Neutralization studies with monoclonal antibodies generated from gpl20-derived recombinant peptides have identified four highly immunogenic domains (pre sumed to be exposed on the surface of nascent envelope glycoprotein), only one of which is neutralizing [8], The major type-specific neutralizing epitope is located in the third hypervariable domain, V3, which is thought to form a 35-amino acid loop structure (amino acids 293-327) by disulfide linkage of two cysteine residues. Antibody bind ing to this loop or site-directed mutagenesis of this region disturbs infectivity without affecting CD4 binding [33, 36, 42], A nucleotide change resulting in a single amino acid substitution at the tip of the loop (amino acid 311) conferred the ability to replicate in other host cells, in addition to PBL [43], The V3 region is contained within the 157-amino acid region important for mononuclear phagocyte infectivity [30, 41]. Interaction of viral gpl20 with CD4 on the surface of mononuclear phagocytes, PBL or transformed T-cell lines is a critical step for HIV-l infection, since anti-Leu 3A monoclonal antibody (which blocks interaction of gp 120 with the CD4 molecule) can block infection of all three cell types with permissive strains [9. 26], This suggests that an additional post-bind ing event is involved in virus entry for mononuclear phagocytes, as well as other cell types. The second conserved region of gp!20, C2, located within the region important for cell tropism. has also been implicated in post-binding events. Antisera directed against gpl20 amino acids 244-264 blocked infection of three different strains without affecting virus binding to CD4-positive cells [17], A point mutation in HIV-lNL4-3 (equivalent to amino acid 259 of H I V - 1 j r . p l ) did not block HIV-l binding to CD4, but abrogated infectivity of T cells. Revertants appeared during in vitro passage that retained the 259 mutation, but contained compensatory amino acid changes in distant sites in gp 120 in the first conserved domain (amino acid 127) and in V3 (amino acid 301) [49], This suggests that binding and entry
Viral Determinants o f Cellular Tropism
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Insight into the step of the retroviral life cycle responsi ble for differences in cell tropism can be inferred from quantitative assay of HIV-specific DNA early after infec tion of mononuclear phagocytes. Total cellular DNA was analyzed by PCR 48 h after infection with HIV-1 strains that markedly varied in ability to productively infect these cells. Using a multiplicity of infection of 0.01 (infectivity determined by limiting dilution in PBL), there was over 100-fold less HIV-1-specific DNA with poorly tropic HIV-1 strains compared with the macrophage-tropic strains. HIV-1jr.pl and HIV-lNFN.Sx. Similar differences in HIV-1-specific DNA levels are seen in transformed Tcell lines by PCR analysis following infection with HIVI j r -c s f or HIV-1 NL4.3 strains that vary in ability to pro ductively infect these cells [4]. Taken together, the map ping of mononuclear phagocyte tropism to gpl20 and the low levels of viral DNA seen in cells in nonproductive infection suggest that HIV-l strain differences in the abil ity to infect mononuclear phagocytes can result from differences in the ability to enter these cells, at least for HIV-l strains from JR and strain HIV-l NL4-3A possible explanation for the observed differences in viral entry is differences in virus binding to target cells. Envelope glycoproteins derived from isolates of HIV and simian immunodeficiency virus (SIV) that differ broadly in host cell range and cytopathicity have been shown to differ in affinity for CD4 by nearly 300-fold [20], How ever. using the same assay, there was no detectable differ ence in CD4 binding using gp 120 derived from HIV1j r . p l , HIV-l j r . c s f or HIV-lNI4-3 [3]. Thus, restriction in cell entry for these strains appear to occur after binding. Cell tropism may also be determined by viral genetic differences at several other steps in the retroviral life cycle. Highly related cloned virus strains from the same individual that differed in ability to productively infect the T-cell line, CEM. also differed in both basal and Tatinduced LTR-linked gene expression. In this case, replica
involved interaction of distinct domains of gpl20. Dele tion and mutagenesis studies of V3 and the CD4 binding domain [22] suggest that these interactions are conforma tion dependent. Further support for conformational in teractions of gpi20 during initiation of infection was demonstrated by reductions in CD4 binding by muta tions in distinct regions of gpl20 [7, 31]. Soluble CD4 (sCD4) can also block infection, but with broad differences in efficiency for different virus types [10]. Laboratory strains passaged extensively in cell lines, such as HIV-1|| xb-2. arc inhibited by relatively low con centrations of sCD4 (less than 0.1 pg/ml). In contrast, pri mary strains, particularly those that replicate in mononu clear phagocytes, require 100- to 1,000-fold higher con centrations to inhibit infection in PBL; comparable levels of sCD4 are required to inhibit infection with these strains in mononuclear phagocytes. Dependence on alter nate CD4-independent mechanisms for entry into mono nuclear phagocytes is unlikely, since anti-Leu 3A neutral izes infection of PBL, mononuclear phagocytes and trans formed T-cell lines with permissive strains at similar anti body concentrations [10, 11]. Surprisingly, sCD4 appears to enhance infection with SIV [1], Conformational changes following interactions of sCD4 with cither HIV-1 or SIV are likely to be important for post-binding events.
HIV-1 cell tropism Primary T cell
Antibody-Dependent Enhancement Fig. 4. Proposed model for HIV entry into target cells (figure by R. Duggan).
Model of HIV-1 Infection of Mononuclear Phagocytes
Recombinant virus studies have identified a region of gp120 that can be important in determining highly effi cient virus entry into mononuclear phagocytes. Although CD4 binding appears to be involved in most cases, this region is distinct from sequences previously identified as important for CD4 binding. Based on studies with Leu 3A and sCD4, CD4 is clearly a critical determinant of infec tion of mononuclear phagocytes and T cells, but we pro pose that target cell interactions with the region of gpl20 at amino acids 202-358, in addition to or in conjunction with the CD4 binding domain, are necessary for efficient entry into mononuclear phagocytes (fig. 4). In trans formed T-cell lines, gpl 20 interactions with other cell sur
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Other cell-virus interactions that do not involve CD4 may be important for initiation of infection. CD4-independent entry appears to occur in cells that bear Fc recep tors (FcR) following opsonization of the virion. Cells bearing the FcR include mononuclear phagocytes, granu locytes, natural killer cells and fibroblasts. Infection of T cells or mononuclear phagocytes can be enhanced by diluted sera from HIV-infected patients [19. 35. 43], Enhanced infection of these cells is blocked by mono clonal antibody to FcR III (CD 16), but is not blocked by monoclonal anti-FcR I (CD64), anti-FcR II (CD32), antiLeu 3A. or sCD4 [19]. CMV infection of fibroblasts induces expression of these FcR and allows entry of opsonized virion particles [27], Thus, any cell expressing FcR may be infectable by HIV-1 in the presence of HIV-l -specific antibodies. The relevance of this mecha nism in vivo is not known.
face molecules would be involved. Since essentially all virus strains replicate efficiently in PBL, a full repertoire of surface molecules are expressed on these cells that allows entry of a wide variety of HIV-1 strains. A second cellular protein present on the surface of mononuclear phagocytes may function as a second receptor or facilitate entry for HIV-1 strains that efficiently infect these cells. In transformed T-cell lines another protein may be in volved that allows entry of a distinct and exclusive group of HIV-1 strains. This second interaction may involve other domains of CD4 rather than distinct molecules. Alternatively, rather than facilitating cell entry, tropism may be determined by gpl 20-target cell interactions that block virus internalization. In this case, nonproductive infection of either mononuclear phagocytes or trans formed T-cell lines may occur following interaction of inhibitory molecules with g p l20 domains other than the originally defined CD4 binding domain. Internalization is likely to be more complex than sug gested by this model. Although we and others have identi fied one domain important for efficient virus entry into cells, viral genetic variation in multiple domains of the env gene may be involved in cell tropism. In addition.
human genetic variation will result in differences in the pattern and structure of molecules expressed on the cell surface. Mononuclear phagocytes obtained from different donors show marked differences in susceptibility to infec tion with the same virus strain [30].
Summary HIV infection of mononuclear phagocytes appears to be impor tant for genesis of AIDS-related diseases. The ability to productively infect these cells in vitro is determined by a 157-amino acid region in some viral strains that is located upstream of the CD4 binding domain and includes the V3 neutralization domain. More precise definition of viral determinants of mononuclear phagocyte tropism. and. particularly, accessory cell proteins and their interaction with gpl 20 and gp41 will enhance our understanding of pathogenesis of HIV disease and may allow development of new therapeutic strate gies.
Acknowledgements 1 thank Irvin Chen. Patrick Green. David Camerini. Paul Krogstad and Jerome Zack for helpful comments and discussions, and Wendy Aft for preparation of this manuscript.
1 Allan JS. Strauss J. Buck DW: Enhancement of SIV infection with soluble receptor molecules. Science 1990:247:1084-1088. 2 Baldwin GC, Fleischmann J, Chung G. Koyanagi Y, Chen ISY. Golde DW: Human immu nodeficiency virus causes mononuclear phago cyte dysfunction. Proc Natl Acad Sei USA 1990;87:3933-3937. 3 Brighty DW. Rosenberg M, Chen ISY, IveyHoyle M: Envelope proteins from clinical iso lates o f human immunodeficiency virus type I that are refractory to neutralization by soluble CD4 possess high affinity for the CD4 receptor. Proc Natl Acad Sei USA 1991;88:7802-7805. 4 Cann AJ, Zack JA, Go AS, ArrigoSJ. Koyanagi Y, Green PL, Koyanagi Y. PangS. Chen ISY: Human immunodeficiency virus type I T-cell tropism is determined by events prior to provi rus formation. J Virol 1990;64:4735-4742. 5 Clark SJ, Saag MS, Decker WD. Campbell-Hill S. Roberson JL. Veldkamp PJ, Kappes JC, Hahn BH, Shaw GM: High titers of cytopathic virus in plasma of patients with symptomatic primary HIV-1 infection. N Engl J Med 1991: 324:954-960.
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6 Coombs RW, Collier AC, Allain JP, Nikora B. Leuther M, Gjerset GF. Corey L: Plasma viremia in human immunodeficiency virus infec tion. N Engl J Med 1989:321: 1626-1631. 7 Cordonnier A, Riviere Y, Montagnier L, Emerman M: Effects of mutations in hyperconserved regions of the extracellular glycoprotein of human immunodeficiency virus type I on receptor binding. J Virol 1989:63:4464-4468. 8 Daar ES, Ho DD: The structure and functions of retroviral envelope glycoproteins. Semin Vi rol 1990:1:205-214. 9 Daar ES, Li XL. Moudgil T. Ho DD: High con centrations of recombinant soluble CD4 are required to neutralize primary HIV-I isolates. Proc Natl Acad Sei USA 1990:87:6574-6578. 10 Daar ES. Moudgil T. Meyer RD. Ho DD: Transient high levels of viremia in patients with pri mary human immunodeficiency virus type I infection. N Engl J Med 1990:324:961-964. 11 Dalgleish AG, Beverley PCL, Clapham PR, Crawford DH. Greaves MF, Weiss RA: The CD4 (T4) antigen is an essential component of the receptor for the AIDS retrovirus. Nature 1984;312:763-767.
12 Eilbott DJ, Peress N, Burger H. LaNeve D, Orenstein J. Gendelman HE, Seidman R. Weiser B: Human immunodeficiency virus type 1 in spinal cords of acquired immunodefi ciency syndrome in patients with myelopathy: Expression and replication in macrophages. Proc Natl Acad Sri USA 1989;86:3337-3341. 13 Gartner SP. Markovits P. Markovitz DM, Betts RF. Popovic M: Virus isolation from and identification of HTLV-Ill/LAV-producing cells in brain tissue from a patient with AIDS. JAMA 1986:256:2365-2371. 14 Golub El, Li G. Volsky DJ: Differences in the basal activity of the long terminal repeat deter mine different replicative capacities of two closely related human immunodeficiency virus type I isolates. J Virol 1990:64:3654-3660. 15 Hahn BH, Shaw GM. Taylor ME. Redfield RR. Markham PD, Salahuddin SZ, WongStaal F. Gallo RC, Parks ES. Parks WP: Ge netic variation in HTLV-I1I/LAV overtim e in patients with AIDS or at risk for AIDS. Science 1986;232:1548-1553. 16 Harper ME, Marsclle LM. Gallo RC. WongStaal F: Detection of lymphocytes expressing human T-lymphotropic virus type III in lymph nodes and peripheral blood from infected indi viduals by in situ hybridization. Proc Natl Acad Sri USA 1986:83:772-776.
O’Brien
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References
29 Myers G. Rabson AB. Josephs SF, Smith TF. Bcrzofsky JA, Wong-Staal F (eds): Human Re troviruses and AIDS 1990. Los Alamos. Los Alamos National Laboratory, 1990. 30 O'Brien WA. Koyanagi Y. Namazie A, Zhao JQ. Diagne A, Idler K. Zack JA. Chen ISY: HIV-I tropism for mononuclear phagocytes can be determined by regions of gp!20 which do not include the CD4 binding domain. Na ture 1990:348:69-73. 31 Olshevsky U. Helscth E. Furman C. Li J. Hascltine W, Sodroski J: Identification of individ ual human immunodeficiency virus type 1 gp 120 amino acids important forCD4 receptor binding. J Virol 1990;64:5701-5707. 32 Orenstein JM, Mcltzer MS, Phipps T. Gendclman HE: Cytoplasmic assembly and accumula tion of human immunodeficiency virus types I and 2 in recombinant human colony-stimulat ing factor-1-treated human monocytes: An ul trastructural study. J Virol 1988:62:2578— 2586. 33 Palker TJ, Matthews TJ. Clark ME. Cianciolo GJ. Randall RR. Langlois AJ. White GC. Safai B. Snyderntan R. Bologncsi DP. Haynes BF: A conserved region of the COOH terminus of human immunodeficiency virus gp!20 enve lope protein contains an immunodominant epitope. Proc Natl Acad Sci USA I987;842479-2483. 34 Pang S. Koyanagi Y. Miles S. Wiley C, Vinters H. Chen ISY: The structure of HIV DNA in brain and blood o f AIDS patients. Nature 1990;343:85-90. 35 Robinson WE Jr. Montefiori DC. Mitchell WM, Prince AM, Alter HJ, Dreesman GR, Eichberg JW: Antibody-dependent enhance ment of human immunodeficiency virus type 1 (HIV-I) infection in vitro by serum from HIVI-infected and passively immunized chimpan zees. Proc Natl Acad Sci USA 1989:86:47104714. 36 Ruschc JR. Javaherian K. McDanalC, PetroJ, Lynn DL. Grimaila R. Langlois AJ. Gallo RC. Arthur LO, Fischinger PJ, Bolognesi DP. Put ney SD, Matthews TJ: Antibodies that inhibit fusion of human immunodeficiency virus-in fected cells bind a 24-amino acid sequence of the viral envelope, gpl20. Proc Natl Acad Sci USA 1988;85:3198-3202. 37 Saag MS. Hahn BH. Gibbons J, Li Y. Parks F.S. Parks WP. Shaw GM: Extensive variation of human immunodeficiency virus type I in vivo. Nature 1988;334:440-444. 38 Schnittman SM. Greenhouse JJ. Psallidopoulos MC. Baseler M. Salzman NP. Fauci AS. Lane HC: Increasing viral burden in CD4+ T cells from patients with human immunodefi ciency' virus (HIV) infection reflects rapidly progressive immunosuppression and clinical disease. Ann Intern Med 1990:113:438-443.
39 Schnittman SM. Psallidopoulos MC, Lane HC, Thompson L, Baseler M. Massari F, Fox CH, Salzman N P, Fauci AS: The reservoir for H IVI in human peripheral blood is a T cell that maintains expression of CD4. Science 1989; 245:305-308. 40 Shaw GM, Harper ME. Hahn BH, Epstien LG, Gajdusek DC. Price RW. Navia BA. Pctito CK. O’Hara CJ.Groopm an JE.Cho ES, Oleske JM. Wong-Staal F. Gallo RC: HTLV-III infec tion in brains of children and adults with AIDS encephalopathy. Science 1985;227:177-181. 41 Shioda T, Levy JA, Cheng-Mayer C: Macro phage and T cell-line tropisms of HIV-1 are determined by specific regions of the envelope gpl20gcne. Nature 1991:349:167-169. 42 Skinner MA, Langlois AJ, McDanal CB. McDougal JS. Bolognesi DP. Matthews TJ: Neutralizing antibodies to an immunodomi nant envelope sequence do not prevent gpl20 binding to C D 4.J Virol 1988:62:4195-4200. 43 Takeda A. Tuazon CU, Ennis FA: Antibodyenhanced infection by HIV-I via Fc receptormediated entry. Science 1988;242:580-583. 44 Takeuchi Y. Akutsu M. Murayama K, Shimizu N, Hoshino H: Host range mutant of human immunodeficiency virus type 1: Modification of cell torpism by a single point mutation at the neutralization epitope in the env gene. J Virol 1991;65:1710-1718. 45 Tschachler E. Groh V. Popovic M, Mann DL, Konrad K, Safai B, Eron L. diMarzo Veronese. Wolff K. Slingl G: Epidermal Langerhans cells - A target for HTLV-II1/LAV infection. J In vest Dermatol 1987;88:233-237. 46 Unanue ER, Allen PM: The basis for the immunoregulatory role of macrophages and other accessory cells. Science 1987;236:551-557. 47 Wair.berg MA. Rookc R. Tremblay M, Yao XJ. Gao Q, Stem M, Belleau B: Characterization of AZT-resistant isolates of HIV-1: Susceptibility to deoxythiacytidine and other nucleosides (ab stract SB 87). Vlth Int Conf AIDS. San Francis co. 1990. 48 Watkins BA. Dorn HH. Kelly WB, Armstrong RC, Potts BJ. Michaels F, Kufta CV. DuboisDalcq M: Specific tropism ofHIV-I for micro glial cells in primary human brain cultures. Science 1990;249:549-553. 49 Willey RL. Ross EK. Buckler-White AJ. Theo dore TS. Martin MA: Functional interaction of constant and variable domains of human im munodeficiency virus type I gpl20. J Virol 1989;63:3595-3600. 50 Zack JA, Arrigo SJ, Weitsman SR. Go AS. Haislip A, Chen ISY: HIV-I entry into quies cent primary lymphocytes: Molecular analysis reveals a labile, latent viral structure. Cell 1990:61:213-222.
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17 Ho DD, Kaplan JC. Rackauskas IE, Gurney ME: Second conserved domain of gpI20 is important for HIV infectivity and antibody neutralization. Science 1988;239:1021-1023. 18 Ho DD, Moudgil T, Alam M: Quantitation of human immunodeficiency virus type I in the blood of infected persons. N Engl J Med 1989; 321:1621-1625. 19 HomsyJ. Meyer M .TatenoS, ClarksonS. Levy JA: The Fc and not CD4 receptor mediates antibody enhancement of HIV infection in hu man cells. Science 1989:244:1357-1360. 20 Ivey-Hoyle M. Culp JS. Chaikin MA. Hellmig BD. Matthews TJ, Sweet RW. Rosenberg M: Envelope glycoproteins from biologically di verse isolates of immunodeficiency viruses have widely different affinities for CD4. Proc Natl Acad Sci USA 1991;88:512-516. 21 Koenig S, Gendelman HE, Orenstein JM. Dal Canto MC. Pezeshkpour GH. Yungbluth M, Janotta F, Aksamit A, Martin MA, Fauci AS: Detection of AIDS virus in macrophages in brain tissue from AIDS patients with encepha lopathy. Science 1986;233:1089-1093. 22 Kowalski M. Potz J. Basiripour T. Dorfman T, Goh WC, Terwilliger E, Dayton A, Rosen C. Haseltinc W, Sodroski J: Functional regions of the envelope glycoprotein of human immuno deficiency virus tvpe 1. Science 1987:237: 1351-1355. 23 Koyanagi Y, Miles S, Mitsuyasu RT, Merrill JE, Vinters HV, Chen ISY: Dual infection of the central nervous system by AIDS viruses with distinct cellular tropisms. Science 1987; 236:819-822. 24 Koyanagi Y, O'Brien WA, Zhao JQ, Goldc DW, Gasson JC. Chen ISY: Cytokines alter production of HIV-I from primary’ mononu clear phagocytes. Science ! 988;241:1673— 1675. 25 Lasky LA. Nakamura G. Smith DH. Fennie C, Shimasaki C, Patzer E. Berman P. Gregory T. Capon DJ: Delineation of a region of the hu man immunodeficiency virus type I gpl20gly coprotein critical for interaction with the CD4 receptor. Cell 1987:50:975-985. 26 McDougal JS. Kennedy MS. Sligh JM, Cort SP, Mawle A. Nicholson JKA: Binding of HTLVIII/LAV to T4+ cells by a complex of the 11OK viral protein and the T4 molecule. Science 1986;231:382-385. 27 McKcating JA. Griffiths PD. Weiss RA: HIV susceptibility conferred to human fibroblasts by cytomegalovirus-induced Fc receptor. Na ture 1990:343:659-661. 28 Merrill JE. Koyanagi Y. Chen ISY: Interleukin I and tumor necrosis factor can be induced from mononuclear phagocytes by HIV-I bind ing to the CD4 receptor. J Virol 1989:63:44044408.
Division of Infectious Diseases, West Los Angeles VA Medical Center and Department of Medicine, UCLA School of Medicine, Los Angeles. Calif., USA
Key Words HIV-1 Tropism Variation Mononuclear phagocyte Polymerase chain reaction CD4 V3 loop Neutralization
Viral Determinants of Cellular Tropism
Abstract
Cells of mononuclear phagocyte lineage are the predominant cell type produc ing human immunodeficiency virus type 1 (HIV-1) in extravascular tissues; HIV-1 infection of mononuclear phagocytes may be directly related to pri mary disease manifestations and also appears to contribute to the immune deficiency of AIDS. Whereas peripheral blood lymphocytes are permissive for nearly all strains of HIV-1, only some HIV-1 strains replicate efficiently in mononuclear phagocytes. Recombinant virus strains have been used to iden tify a 157-amino acid region of gpl20 that can confer macrophage tropism. This region is distinct from the principal CD4 binding domain of gpl20 and includes the major type-specific neutralizing epitope located in the third hypervariable domain. V3. Quantitative assay of HIV-1-specific DNA by polymerase chain reaction early after infection suggests that HIV-1 strain dif ferences in macrophage tropism are determined at the level of entry. These studies suggest that target cell interactions with gpl20 in addition to or in conjunction with the CD4 binding domain are necessary for efficient entry into mononuclear phagocytes.
The Role of HIV Infection of Mononuclear Phagocytes in Clinical A ID S
Infection by human immunodeficiency virus (HIV) can result in a broad spectrum of disease, caused in part by development of progressive immune deficiency. The CD4+T-lymphocyte was the first cell identified as a target for HIV infection in vivo. This cell is susceptible to infec tion by all strains of HIV-1, with rare exception. There is an inexorable decline in CD4+ T cells following acute infection; loss of these cells leads to development of cellu lar immune deficiency and subsequent opportunistic in fection and other diseases. However, it is cells of mononu
Received: July 5. 1991 Accepted: August 1 ,1991
clear phagocyte lineage that are the predominant cell type producing HIV-1 in extravascular tissues [12, 13.21,45], Disease manifestations believed to be a direct result of HIV-1 infection (and not due to the resulting immunode ficiency) have been associated with mononuclear phago cyte infection; for example, AIDS dementia, neuropathy, pneumonitis and dermatitis have been associated with infection of mononuclear phagocytes in the central ner vous system (CNS), spinal cord, lung and skin, respec tively [30]. In addition, HIV-1 infection of mononuclear phago cytes appears to contribute to the immune deficiency of AIDS. Mononuclear phagocytes are immunologically ac-
Dr. William A. O’Brien 691/WI1 IF West LA VA Medical Center Los Angeles, CA 90073 (USA)
© 1992 S. Karger AG, Basel 1015-2008/92/0604-0225 $2.75/0
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William A. O'Brien
Quantitation of HIV-1
Early attempts to detect viral proteins or RNA in peripheral blood mononuclear cells (PBMC) of patients with symptomatic HIV infection demonstrated a fre quency of infected cells of 1/10,000 or less [ 16]. However, the viral load in patients is much higher than previously thought. Quantitative cultures using endpoint dilution were used to measure infectious virus titer both in PBMC and plasma [ 18], Although there was a wide range of virus titer between patients, in symptomatic HIV-positive patients with low CD4 cell number, mean plasma titers were 3.500 tissue culture infectious doses (TCID)/ml, or 1 TCID/0.3 pi. and the PBMC viral titers were 2.200 TCID/106 cells or 1 TCID/400 PBMC. In patients without symptoms, infectious virus titer was 100-fold lower. Virus titer ap pears to be higher in patients with more advanced disease [6,18], Recently, quantitative polymerase chain reaction (PCR) methods were used to determine viral load. HIV1-specific DNA is readily detected in PBMC by PCR. Schnittman et al. [38] showed a frequency of infection of 1/100 or greater in CD4-positive lymphocytes of patients with AIDS. Lower frequencies of infection (1/1,000 to less than 1/10.000) were seen in asymptomatic seropositive patients. In this study, the highest viral burden was seen in patients having a deteriorating clinical course. Thus, increasing viral burden appears to be associated with dis
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ease progression and development of virus-induced dis ease. Schnittman et al. [39] separated mononuclear phago cytes, CD4+ lymphocytes and CD8+ lymphocytes by fluo rescence-activated cell sorting to identify which blood mononuclear cells are infected by HIV-1. HIV-specific DNA was present in T4 cells in 10 of 10 patients; HIVspecific DNA was detected in blood mononuclear phago cytes from only 2 of 10 of these patients. The authors con clude that T cells and not mononuclear phagocytes are the principal reservoir for HIV, at least in the blood. How ever, mononuclear phagocytes are only transiently present in the blood. HIV-specific DNA can be consis tently demonstrated in seropositive patients in mononu clear phagocytes purified by adherence [D. Ho, pers. commun.]. In addition, they migrate to extravascular tissues, where they differentiate in response to local factors. Lev els of viral DNA in the brain tissue of patients who died with AIDS dementia, where most viral nucleic acids are associated with mononuclear phagocytes, is over ten times greater than in paired samples taken from patient blood [34], Although few studies have measured viral load in extravascular tissues other than brain [40], it is likely that productive infection in extravascular tissues associated with clinical disease is of great magnitude.
HIV-1 Infection of Primary Blood Cells
Viremia is typically present during acute infection [4, 8], suggesting that infection of blood cells is involved. Nearly all HIV-l strains replicate in lectin-stimulated peripheral blood lymphocytes (PBL), whether the virus was derived from PBL, transformed cell lines, mononu clear phagocytes or infected tissues. HIV-l infection of stimulated PBL results in virus production within 24 h. Replication occurs rapidly, with a peak in virus produc tion at day 5-7 (fig. la). There is no significant virus pro duction following infection of quiescent PBL, but effi cient entry does occur [49], Initiation of reverse tran scription is equivalent following infection of resting and stimulated PBL. This was demonstrated by a quantita tive PCR assay using oligonucleotide primer pairs de signed to amplify DNA in the HIV-l R/U5 region of the LTR, the first region formed in retroviral reverse tran scription. However, reverse transcription is not com pleted in these cells; there is little full-length viral DNA formed in quiescent cells, as measured by PCR primer pairs designed to amplify HIV-l LTR/^a^-specific DNA, which is not formed until near completion of reverse
Viral Determinants o f Cellular Tropism
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tive cells [46], First, they internalize and process antigen for presentation on the cell surface, in association with major histocompatibility gene complex class II molecules. Second, they secrete cytokines, proteases and comple ment proteins. Third, they bear receptors for lympho kines, which are secretory products of lymphocytes that can activate these cells and modify their function. Fourth, they are involved in complex interactions with T and B lymphocytes in the host immune response. HIV-1 binding to the CD4 molecule on mononuclear phagocytes can induce release of interleukin 1 and tumor necrosis factor [28], Infected mononuclear phagocytes have also been shown to be impaired in immune function and response to cytokines [2]. Therefore, insight into the mechanism of infection of mononuclear phagocytes is important for improved understanding of disease caused by HIV-1. Characterization of virus determinants important for effi cient HIV-1 infection of mononuclear phagocytes will be the topic of this review.
Fig. 1. Kinetics of HIV-l replication in cultured primary blood mononuclear cells. Normal human blood mononuclear cells were recovered by Ficoll-Hypaque density centrifugation. PBL and blood mononuclear phagocytes were separated by adherence. PBL (2 X I07) stimulated with PHA (Wellcome. 0.8 pg/ml) for 48 h (a) were infected with cell-free virus containing 500 ng of HIV-1 p24 [Abbott. ELISA; H1V-1 (A ). IIIV-1jr ^ ’sp ( a ), HIV-1sp2 (o) and HIV-1 m .4-,1 (o)[. Medium was changed every 2 days and assayed for virus at days 3, 7. 9 and 11 after infection. Blood mononuclear phagocytes (5 X 105) (b) were infected after 6 days of adherence with 10 ng of HIV-1 p24. Medium was changed every 3-4 days and assayed for virus at days 7, 14, 17 and 21. Experiments a and b used the same virus stocks.
cytes with y-interferon, a potent activator of mononuclear phagocytes, does not affect proliferation and inhibits virus production. However, y-interferon treatment prior to infection has been shown to enhance virus production [24], Effects of cytokine treatment are complex, but it appears that activation of these cells generally enhances virus replication.
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transcription. If these cells are stimulated following infec tion. the full-length viral DNA structure is formed, and virus production follows. Although the partially reversetranscribed retroviral structure appears to persist in quiescent cells in a latent form, it is labile and degrades with a half-life of approximately I day. Therefore, infec tion of quiescent cells can result in short-lived latent infection, but the viral DNA will be degraded unless the cells are stimulated shortly after infection. In vivo, most circulating T lymphocytes are in a quiescent state and are activated to proliferate only in response to specific anti gen. The labile HIV-1 DNA in quiescent cells could con tribute to the prolonged duration of clinical latency. The degradation of viral DNA in quiescent cells may explain the relatively low percentage of infected cells in the circu lation of seropositive patients, particularly early in the disease course. HIV-1 replication in blood mononuclear phagocytes occurs with delayed kinetics compared to stimulated PBL. There is little extracelluar virus production seen until 4-5 days after in vitro infection. This gradually rises, with a peak of virus production at approximately 3 weeks (fig. lb). One possible explanation for differences in the kinetics of virus production in these cell types is delayed extracellular release of virus. Orenstein et al. [32] have observed intracellular budding of virions into cytoplas mic vacuoles by electron microscopy. Very little virus is seen extracellularly early after infection. Thus, following infection, mononuclear phagocytes can produce large amounts of virus, and, because these cells are resistant to killing by HIV-1, can slowly release virus over prolonged periods of time. In addition to impaired extracellular bud ding of virus, other events in the retroviral life cycle may affect kinetics of virus production, including entry, re verse transcription, integration, virus expression or virus assembly. Using PCR primers to detect DNA at different stages of reverse transcription in HIV-1-infected mono nuclear phagocytes, we observed delayed kinetics of re verse transcription compared to that of T lymphocytes [O’Brien et al., in preparation]. Like quiescent PBL. mononuclear phagocytes gener ally do not proliferate, although, unlike quiescent PBL. they can be productively infected. Virus production can be dramatically increased in mononuclear phagocytes ac tivated by treatment with cytokines. Treatment of HIV1-infected mononuclear phagocytes with granulocytemacrophage colony-stimulating factor (GM-CSF), macro phage colony-stimulating factor (M-CSF) or interleukin 3 is associated with a dose-dependent increase in cell prolif eration [24], Treatment of infected mononuclear phago-
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Viral Determinants o f Cellular Tropism
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HIV-1 Variation
Identification of Virus Determinants Important for Mononuclear Phagocyte Tropism
Virus strains more distantly related to the JR strains, such as HIV-1 hxb2 and HIV-1 NL4-3, which are derived from serial passage in transformed T-cell lines, are very inefficient at replicating in peripheral blood mononuclear phagocytes, and there is usually no significant virus pro duction following infection of these cells. These labora tory strains are also unable to replicate in primary brain microglial cells [48], which are resident macrophages of the brain. Primary blood microglial cells can be produc tively infected by virus strains that replicate in blood mononuclear phagocytes [48], Because of the marked dif ferences in the ability of HIV-1JR.|.-L and HIV-INL4-3 to productively infect mononuclear phagocytes, molecular clones of these strains were used to generate molecular recombinant viruses in order to identify viral regions involved in determining mononuclear phagocyte cell tro pism. For initial recombinant virus studies, purified DNA fragments from these cloned virus strains were ligated prior to electroporation of chimeric proviral DNA into 729-6 cells. This transfection technique utilizes a pulsed
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Mononuclear phagocyte and T cell line tropism
Fig. 3. The HIV-1 envelope gene. Conserved regions are depicted with dark shading, and variable regions are shown in white. Regions encoding the extracellular glycoprotein. gp!20, and the transmem brane glycoprotein, gp4l, are shown above. Functional domains identified for neutralization (V3 loop), CD4 binding, fusion, and mononuclear phagocyte and T-cell line tropism are shown.
electrical current to nonspecifically introduce DNA into human cells. Recombinant virus is formed in these cells, and high titer virus can be produced following short-term coculture with PHA-stimulated PBL. Recombinant viruses (fig. 2) composed of progres sively smaller regions of HIV-lJR.FL at the 3' end joined at Sst I (nt 5999), Dra III (nt 6591), or Stu I (nt 6822) still replicated efficiently in mononuclear phagocytes. The recombinant virus, HIV-1nF.xiio, which contains the Nef and U3/R LTR elements from H IV -l^ .^ , does not pro ductively infect mononuclear phagocytes. By substituting internal fragments from molecular clones of HIV-1jR_fi into the infectious clone, pNL4-3, a region conferring mononuclear phagocyte tropism was localized to the env gene by the recombinant, HIV-lNFN-sx, and particularly, the recombinant, HIV-1NFn-sm- The latter recombinant contains only 157 amino acids from the HIV-l JR.FLgpl20 region (residues 202-358) and can efficiently infect mononuclear phagocytes. Of note, this region of gpl20 includes the major neutralization domain and does not include the originally defined CD4 binding domain (amino acids 404-447) [25] (fig. 3). The recombinant virus, HIV-1 nfn-mx> which contains the CD4 binding domain and all of gp41 encoded by HIV-1 JR.FL, does not replicate in mononuclear phagocytes. Mononuclear phagocyte cell tropism has been mapped to the same region of gp!20 using recombinant viruses containing sequences from HIV-1SF|62, another macro
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There is extensive variation of HIV-1 strains at the nucleotide sequence level, even between strains recovered from the same individual [ 15, 23,29, 37], Despite the cor relation of HIV-1 infection of mononuclear phagocytes with clinical disease, different HIV-1 isolates vary mark edly in their ability to productively infect mononuclear phagocytes. Distinct differences in target cell tropism have been identified for different HIV-1 isolates obtained from the same individual [23], HIV-1JR.CSF was isolated from cell-free cerebrospinal fluid (CSF) of a patient who died with severe AIDS encephalopathy. This strain repli cates efficiently in phytohemagglutinin (PHA)-stimulated PBL. but does not replicate as well in blood mononuclear phagocytes from most donors. In contrast, the strain HIV-1 jR.[7L, recovered from frontal lobe brain tissue of the same patient, replicates efficiently and to high titer in stimulated PBL and blood mononuclear phagocytes from all donors tested (over 200). Although these strains are genetically distinct, they are more closely related than are strains isolated from different individuals [29]. Thus, dif ferences in cellular tropism appear to be related to viral genetic variation.
phage-tropic strain [41]. Cell tropism for transformed Tcell lines appears to map to a similar region of gpl20: however, viral determinants of tropism for these cell types appears to be mutually exclusive [41]. In general, recom binant viruses that replicate efficiently in mononuclear phagocytes do not productively infect transformed T-cell lines, and the converse is also true. We have seen similar results using recombinant viruses from H I V - 1 j F. f l and HIV-l NL4.3.
tion appears to be affected by LTR sequence variation [14], Variation in the pol gene could affect reverse tran scription. integration or protease activity. Virus strains exhibiting AZT resistance have been shown to have reduced infectivity [47], Other viral genes whose func tions are poorly defined, such as vpr, vif vpu and nef, may also contribute to differences in virus replication by affecting events following entry.
Neutralization of HIV Infection Mechanism of Mononuclear Phagocyte Tropism
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Neutralization studies with monoclonal antibodies generated from gpl20-derived recombinant peptides have identified four highly immunogenic domains (pre sumed to be exposed on the surface of nascent envelope glycoprotein), only one of which is neutralizing [8], The major type-specific neutralizing epitope is located in the third hypervariable domain, V3, which is thought to form a 35-amino acid loop structure (amino acids 293-327) by disulfide linkage of two cysteine residues. Antibody bind ing to this loop or site-directed mutagenesis of this region disturbs infectivity without affecting CD4 binding [33, 36, 42], A nucleotide change resulting in a single amino acid substitution at the tip of the loop (amino acid 311) conferred the ability to replicate in other host cells, in addition to PBL [43], The V3 region is contained within the 157-amino acid region important for mononuclear phagocyte infectivity [30, 41]. Interaction of viral gpl20 with CD4 on the surface of mononuclear phagocytes, PBL or transformed T-cell lines is a critical step for HIV-l infection, since anti-Leu 3A monoclonal antibody (which blocks interaction of gp 120 with the CD4 molecule) can block infection of all three cell types with permissive strains [9. 26], This suggests that an additional post-bind ing event is involved in virus entry for mononuclear phagocytes, as well as other cell types. The second conserved region of gp!20, C2, located within the region important for cell tropism. has also been implicated in post-binding events. Antisera directed against gpl20 amino acids 244-264 blocked infection of three different strains without affecting virus binding to CD4-positive cells [17], A point mutation in HIV-lNL4-3 (equivalent to amino acid 259 of H I V - 1 j r . p l ) did not block HIV-l binding to CD4, but abrogated infectivity of T cells. Revertants appeared during in vitro passage that retained the 259 mutation, but contained compensatory amino acid changes in distant sites in gp 120 in the first conserved domain (amino acid 127) and in V3 (amino acid 301) [49], This suggests that binding and entry
Viral Determinants o f Cellular Tropism
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Insight into the step of the retroviral life cycle responsi ble for differences in cell tropism can be inferred from quantitative assay of HIV-specific DNA early after infec tion of mononuclear phagocytes. Total cellular DNA was analyzed by PCR 48 h after infection with HIV-1 strains that markedly varied in ability to productively infect these cells. Using a multiplicity of infection of 0.01 (infectivity determined by limiting dilution in PBL), there was over 100-fold less HIV-1-specific DNA with poorly tropic HIV-1 strains compared with the macrophage-tropic strains. HIV-1jr.pl and HIV-lNFN.Sx. Similar differences in HIV-1-specific DNA levels are seen in transformed Tcell lines by PCR analysis following infection with HIVI j r -c s f or HIV-1 NL4.3 strains that vary in ability to pro ductively infect these cells [4]. Taken together, the map ping of mononuclear phagocyte tropism to gpl20 and the low levels of viral DNA seen in cells in nonproductive infection suggest that HIV-l strain differences in the abil ity to infect mononuclear phagocytes can result from differences in the ability to enter these cells, at least for HIV-l strains from JR and strain HIV-l NL4-3A possible explanation for the observed differences in viral entry is differences in virus binding to target cells. Envelope glycoproteins derived from isolates of HIV and simian immunodeficiency virus (SIV) that differ broadly in host cell range and cytopathicity have been shown to differ in affinity for CD4 by nearly 300-fold [20], How ever. using the same assay, there was no detectable differ ence in CD4 binding using gp 120 derived from HIV1j r . p l , HIV-l j r . c s f or HIV-lNI4-3 [3]. Thus, restriction in cell entry for these strains appear to occur after binding. Cell tropism may also be determined by viral genetic differences at several other steps in the retroviral life cycle. Highly related cloned virus strains from the same individual that differed in ability to productively infect the T-cell line, CEM. also differed in both basal and Tatinduced LTR-linked gene expression. In this case, replica
involved interaction of distinct domains of gpl20. Dele tion and mutagenesis studies of V3 and the CD4 binding domain [22] suggest that these interactions are conforma tion dependent. Further support for conformational in teractions of gpi20 during initiation of infection was demonstrated by reductions in CD4 binding by muta tions in distinct regions of gpl20 [7, 31]. Soluble CD4 (sCD4) can also block infection, but with broad differences in efficiency for different virus types [10]. Laboratory strains passaged extensively in cell lines, such as HIV-1|| xb-2. arc inhibited by relatively low con centrations of sCD4 (less than 0.1 pg/ml). In contrast, pri mary strains, particularly those that replicate in mononu clear phagocytes, require 100- to 1,000-fold higher con centrations to inhibit infection in PBL; comparable levels of sCD4 are required to inhibit infection with these strains in mononuclear phagocytes. Dependence on alter nate CD4-independent mechanisms for entry into mono nuclear phagocytes is unlikely, since anti-Leu 3A neutral izes infection of PBL, mononuclear phagocytes and trans formed T-cell lines with permissive strains at similar anti body concentrations [10, 11]. Surprisingly, sCD4 appears to enhance infection with SIV [1], Conformational changes following interactions of sCD4 with cither HIV-1 or SIV are likely to be important for post-binding events.
HIV-1 cell tropism Primary T cell
Antibody-Dependent Enhancement Fig. 4. Proposed model for HIV entry into target cells (figure by R. Duggan).
Model of HIV-1 Infection of Mononuclear Phagocytes
Recombinant virus studies have identified a region of gp120 that can be important in determining highly effi cient virus entry into mononuclear phagocytes. Although CD4 binding appears to be involved in most cases, this region is distinct from sequences previously identified as important for CD4 binding. Based on studies with Leu 3A and sCD4, CD4 is clearly a critical determinant of infec tion of mononuclear phagocytes and T cells, but we pro pose that target cell interactions with the region of gpl20 at amino acids 202-358, in addition to or in conjunction with the CD4 binding domain, are necessary for efficient entry into mononuclear phagocytes (fig. 4). In trans formed T-cell lines, gpl 20 interactions with other cell sur
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Other cell-virus interactions that do not involve CD4 may be important for initiation of infection. CD4-independent entry appears to occur in cells that bear Fc recep tors (FcR) following opsonization of the virion. Cells bearing the FcR include mononuclear phagocytes, granu locytes, natural killer cells and fibroblasts. Infection of T cells or mononuclear phagocytes can be enhanced by diluted sera from HIV-infected patients [19. 35. 43], Enhanced infection of these cells is blocked by mono clonal antibody to FcR III (CD 16), but is not blocked by monoclonal anti-FcR I (CD64), anti-FcR II (CD32), antiLeu 3A. or sCD4 [19]. CMV infection of fibroblasts induces expression of these FcR and allows entry of opsonized virion particles [27], Thus, any cell expressing FcR may be infectable by HIV-1 in the presence of HIV-l -specific antibodies. The relevance of this mecha nism in vivo is not known.
face molecules would be involved. Since essentially all virus strains replicate efficiently in PBL, a full repertoire of surface molecules are expressed on these cells that allows entry of a wide variety of HIV-1 strains. A second cellular protein present on the surface of mononuclear phagocytes may function as a second receptor or facilitate entry for HIV-1 strains that efficiently infect these cells. In transformed T-cell lines another protein may be in volved that allows entry of a distinct and exclusive group of HIV-1 strains. This second interaction may involve other domains of CD4 rather than distinct molecules. Alternatively, rather than facilitating cell entry, tropism may be determined by gpl 20-target cell interactions that block virus internalization. In this case, nonproductive infection of either mononuclear phagocytes or trans formed T-cell lines may occur following interaction of inhibitory molecules with g p l20 domains other than the originally defined CD4 binding domain. Internalization is likely to be more complex than sug gested by this model. Although we and others have identi fied one domain important for efficient virus entry into cells, viral genetic variation in multiple domains of the env gene may be involved in cell tropism. In addition.
human genetic variation will result in differences in the pattern and structure of molecules expressed on the cell surface. Mononuclear phagocytes obtained from different donors show marked differences in susceptibility to infec tion with the same virus strain [30].
Summary HIV infection of mononuclear phagocytes appears to be impor tant for genesis of AIDS-related diseases. The ability to productively infect these cells in vitro is determined by a 157-amino acid region in some viral strains that is located upstream of the CD4 binding domain and includes the V3 neutralization domain. More precise definition of viral determinants of mononuclear phagocyte tropism. and. particularly, accessory cell proteins and their interaction with gpl 20 and gp41 will enhance our understanding of pathogenesis of HIV disease and may allow development of new therapeutic strate gies.
Acknowledgements 1 thank Irvin Chen. Patrick Green. David Camerini. Paul Krogstad and Jerome Zack for helpful comments and discussions, and Wendy Aft for preparation of this manuscript.
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