![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
Previous Article | Next Article 
Journal of Virology, February 1999, p. 976-984, Vol. 73, No. 2
Department of Cancer Immunology/AIDS,
Received 14 July 1998/Accepted 14 October 1998
In vivo passage of a poorly replicating, nonpathogenic simian-human
immunodeficiency virus (SHIV-HXBc2) generated an efficiently replicating virus, KU-1, that caused rapid CD4+
T-lymphocyte depletion and AIDS-like illness in monkeys (S. V. Joag, Z. Li, L. Foresman, E. B. Stephens, L.-J. Zhao, I. Adany, D. M. Pinson, H. M. McClure, and O. Narayan, J. Virol.
70:3189-3197, 1996). The env gene of the KU-1 virus was
used to create a molecularly cloned virus, SHIV-HXBc2P 3.2, that
differed from a nonpathogenic SHIV-HXBc2 virus in only 12 envelope
glycoprotein residues. SHIV-HXBc2P 3.2 replicated efficiently and
caused rapid and persistent CD4+ T-lymphocyte depletion in
inoculated rhesus macaques. Compared with the envelope glycoproteins of
the parental SHIV-HXBc2, the SHIV-HXBc2P 3.2 envelope glycoproteins
supported more efficient infection of rhesus monkey peripheral blood
mononuclear cells. Both the parental SHIV-HXBc2 and the pathogenic
SHIV-HXBc2P 3.2 used CXCR4 but none of the other seven transmembrane
segment receptors tested as a second receptor. Compared with the
parental virus, viruses with the SHIV-HXBc2P 3.2 envelope glycoproteins
were more resistant to neutralization by soluble CD4 and antibodies.
Thus, changes in the envelope glycoproteins account for the ability of
the passaged virus to deplete CD4+ T lymphocytes rapidly
and specify increased replicative capacity and resistance to neutralization.
The human immunodeficiency viruses
type 1 (HIV-1) and HIV-2 cause AIDS by depleting host CD4+
T lymphocytes (2, 11, 17, 20). HIV-1 infection represents a
major public health problem, afflicting an estimated 30 million people
worldwide (according to UNAIDS and The World Health Organization). Efforts to understand HIV-induced disease and to develop an effective vaccine against HIV-1 will require animal models. The infection of
Asian macaques by simian immunodeficiency viruses (SIV) can result in
AIDS-like disease and therefore has been extremely useful for studies
of the pathogenesis of primate immunodeficiency viruses (27). However, differences between the HIV-1 and SIV
envelope glycoproteins limit the utility of the SIV-macaque model for
studying envelope glycoprotein determinants of pathogenicity and for
testing vaccine strategies directed against the viral glycoproteins.
To address these limitations, chimeric simian-human immunodeficiency
viruses (SHIVs) containing the tat, rev,
vpu, and env genes of HIV-1 have been constructed
and shown to infect macaques (21, 28, 31). The efficiency of
SHIV replication in macaques is greatly influenced by the sequence of
the HIV-1 envelope glycoproteins, which have been shown to specify
viral tropism and sensitivity to neutralizing antibodies (7-9,
26, 36, 41, 44-46, 48, 52, 55). These properties differ between
HIV-1 viruses that are primary (for example, those that were passaged
only in peripheral blood mononuclear cells [PBMC]) and viruses that
were adapted to replicate in immortalized cell lines. The latter,
laboratory-adapted viruses are typically more sensitive to neutralizing
antibodies than are primary viruses (52). All HIV-1 isolates
utilize CD4 as a receptor; primary viruses use the CCR5 chemokine
receptor as a second receptor, while laboratory-adapted viruses
typically use CXCR4 (1, 10, 13-15, 18).
SHIV chimerae constructed with the env gene from a
laboratory-adapted HIV-1 isolate, HXBc2, replicated efficiently in
rhesus monkey PBMC in culture. However, SHIV-HXBc2 viruses replicated poorly in rhesus monkeys, and no pathogenic consequences were observed
up to 2 years after infection (28). Although SHIV constructs expressing some primary virus envelope glycoproteins replicated more
efficiently in rhesus monkeys, these infections were also without
pathogenic consequences (40).
Serial passage of nonpathogenic SHIVs in vivo has generated viruses
that cause rapid depletion of CD4+ T lymphocytes and
AIDS-like illness in macaques (24, 40). SHIV (KU-1) was
generated by serial bone marrow transfer from animals originally
infected with the nonpathogenic SHIV-HXBc2. Infection with KU-1
resulted in dramatic CD4+ T-lymphocyte depletion within 4 weeks and AIDS-like illness in 70% of infected pig-tailed macaques
(24, 49). The replication level of the KU-1 virus in
infected macaques was significantly increased compared with that of the
SHIV-HXBc2 virus. Analysis of the uncloned KU-1 virus revealed several
changes in the vpr, tat, rev,
env, and nef genes and in the long terminal
repeats (LTRs) compared with the parental SHIV-HXBc2 virus (reference 50 and unpublished observations). In addition, the
altered initiation codon in the vpu gene of the parental
SHIV-HXBc2 virus was found to be restored in the KU-1 virus
(50). A functional vpu gene is not sufficient for
rendering the SHIV-HXBc2 virus pathogenic (29), suggesting
that some combination of vpr, tat,
rev, env, nef, and/or LTR changes
might contribute to the pathogenicity of the KU-1 virus. Here we test
the hypothesis that changes in the KU-1 envelope glycoproteins
contribute to the pathogenicity of the virus, and we investigate the
functional consequences of the observed env sequence changes.
Viruses and cells.
The KU-1 virus stock was obtained from
Opendra Narayan through the National Institutes of Allergy and
Infectious Diseases AIDS Research and Reference Reagent Program.
SIVmac239 and SIVmac316 viruses were provided
by Ronald Desrosiers at the New England Regional Primate Research Center.
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Changes in Human Immunodeficiency Virus Type 1 Envelope Glycoproteins Responsible for the Pathogenicity of a
Multiply Passaged Simian-Human Immunodeficiency Virus
(SHIV-HXBc2)
ABSTRACT
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
-mercaptoethanol, 10 mM HEPES, and 50 U of human
granulocyte-macrophage colony stimulating factor [GM-CSF] and 4 U of
macrophage colony-stimulating factor [M-CSF] per ml [Genzyme,
Inc.]), replenishing with fresh MDM every third day for 2 weeks prior
to infection.
Plasmids. The env fragment of the KU-1 virus was cloned as follows. Proviral DNA from KU-1-infected CEMx174 cells was purified by using the MicroTurboGen Genomic DNA Isolation Kit (Invitrogen, San Diego, Calif.) kit. A 3.64-kb PCR fragment containing unique Asp718 and NotI sites was generated using Pfu DNA polymerase and forward GBK1009 (5'-TTATATAAATAAGCGGCCGCGTACCGAGTTGACCAGGCG-3') and reverse GBK1010 primers (5'-ATTGTGGGTCACAGTCTATTAT-3'). To obtain the consensus KU-1 sequence in the env region, the 3.64-kb fragment was purified from an agarose gel with the Gene Clean II kit (Bio 101, San Diego, Calif.) and sequenced by automated DNA sequencing at the Dana-Farber Cancer Institute Molecular Biology Core Facility. The following primers were used for DNA sequencing: GBK96-3 (5'-GTTTGGGCCACACATGCC-3'), 96-4.HXBc2 (5'-GGTGCAGAAAGAATATGC-3'), GBK96-5 (5'-GCAGTCTAGCAGAAGAAG-3'), GBK96-6 (5'-CAATCCTCAGGAGGGGAC-3'), GBK96-7.HXBc2 (5'-CGAGATCTTCAGACTTGG-3'), GBK96-8 (5'-GTTGCAACTCACAGTCTG-3'), GBK96-9 (5'-GGCAAGTTTGTGGAATTGG-3'), and GBK96-47.HXBC2 (5'-GCATATTCTTTCTGCACC-3').
For cloning the KU-1 env gene, the purified 3.64-kb PCR fragment was digested with Asp718 and BamHI. The resulting fragment was cloned into the pSVIIIenv expression vector (22). The inserts from three independent clones were sequenced. The 3.2 clone, which contained all of the consensus changes found in the KU-1 env sequence (50), was selected for this study. The plasmids containing the 5' and 3' halves of the SHIV-HXBc2 provirus have been previously described (28). The Vpu+ 3' proviral clone (p3'u+SHIV) was used in these studies. The Asp718-BamHI fragment from the KU-1 3.2 env clone in pSVIIIenv was subcloned into the p3'u+SHIV proviral plasmid to create the p3'SHIV-HXBc2P 3.2 plasmid. The pHXBH10
envCAT plasmid contains an env-defective,
Vpu-expressing provirus with a chloramphenicol acetyltransferase (CAT) gene replacing the natural nef gene (53). The
pSHIVenvCAT plasmid contains an env-defective SHIV
provirus that expresses CAT. The pSHIVenvCAT plasmid was made by
cloning an env-deleted, HIV-1 HXBc2
SalI-BamHI fragment containing tat and
rev sequences into a plasmid containing an
SIVmac239 provirus. The CAT gene-containing BamHI fragment was cloned into the BamHI site,
inactivating the rev gene.
Plasmids encoding human CD4 (pCD4) and the human chemokine receptors
CCR5 and CXCR4 have been previously described (5, 10).
The pSVTat plasmid expressing the HIV-1 Tat was previously described
(47) and was kindly provided by Heinrich Göttlinger.
Antibodies, SDF-1, and soluble CD4. Purified human soluble CD4 (sCD4) was donated by Raymond Sweet (SmithKline Beecham, King of Prussia, Pa.). Human stromal cell-derived factor 1 (SDF-1) was supplied by Timothy Springer and Ian Clarke-Lewis (3, 36). The IgG1b12 antibody, which recognizes the CD4 binding site of the HIV-1 gp120 envelope glycoprotein, was donated by Dennis Burton and Carlos Barbas III (6). The F105 antibody, which also recognizes the CD4 binding site of the HIV-1 gp120 envelope glycoprotein, was a kind gift from Marshall Posner (39). The AG1121 antibody, which recognizes the V3 loop of the HIV-1 gp120 envelope glycoprotein, was purchased from AGMED, Inc., Bedford, Mass. Human sera recognizing HIV-1 envelope glycoproteins were pooled from HIV-1-infected individuals.
Virus replication assays. To generate the Vpu+ SHIV-HXBc2 and SHIV-HXBc2P 3.2 viruses, the p5'SHIV and either p3'u+SHIV or p3'SHIV-HXc2P 3.2 plasmids, respectively, were digested with SphI and then ligated. The ligated DNA was transfected into CEMx174 cells, and viruses were harvested at the peak of viral replication.
Equivalent amounts of virus, measured in reverse transcriptase units, were used to infect rhesus PBMC and macrophages. To detect virus production, cell-free supernatants from infected rhesus PBMC were harvested and assayed for reverse transcriptase activity as previously described (28). An aliquot of the cell-free supernatants from SHIV-infected rhesus macrophage cultures was harvested daily and replaced with fresh MDM. GM-CSF and M-CSF were added every third day. The supernatants were then assayed for the presence of the SIV p27 core protein by utilizing an SIV p27 enzyme-linked immunoassay kit (Coulter Corp., Hialeah, Fla.).env complementation assays.
To measure a single
round of infection, recombinant viruses expressing CAT were produced by
cotransfecting the pSVIIIenv plasmid expressing the envelope
glycoproteins of interest with either the pSHIV-CAT plasmid (for monkey
and canine target cells) or the pHXBH10envCAT plasmid (for
other target cells). Recombinant viruses made with pSHIVenvCAT
were generated in HEK 293 cells, while recombinant viruses made
with pHXBH10envCAT were generated in either HeLa or COS-1 cells.
Recombinant viruses were normalized for reverse transcriptase activity
and used to infect target cells. The target cells were (i) Cf2Th canine
thymocytes transfected with plasmids expressing CD4 and chemokine
receptors, (ii) CEMx174 cells, or (iii) rhesus PBMC. The Cf2Th cells
were cotransfected either with pCD4 and an empty pcDNA3.1 control or
with pCD4 and a pcDNA3.1 plasmid expressing either human CXCR4 or CCR5
by the calcium phosphate method (12). CAT activity was
measured in the target cells as previously described (22).
Animal inoculation. Three rhesus monkeys (Macaca mulatta) were inoculated intravenously with approximately 106 reverse transcriptase units of the SHIV-HXBc2P 3.2 virus generated in CEMx174 cells as described above. The rhesus monkeys were maintained in accordance with the guidelines of the Committee on Animals for the Harvard Medical School and the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, 1985). The monkeys were anesthetized with ketamine-HCl for all blood sampling, biopsies, and inoculations.
Lymphocyte phenotyping. Peripheral blood lymphocytes obtained from the infected animals were phenotyped for CD4 (OKT4-FITC; Ortho Diagnostics Systems, Raritan, N.J.), CD8 (T8-phycoerythrin; Dako, Inc., Carpinteria, Calif.), and CD20 (B1-fluorescein isothiocyanate; Coulter Corp.), subsets by using a commercial whole-blood lysis kit (Coulter), as previously described (42). Absolute lymphocyte counts in blood were determined on an automated hematology analyzer (T540; Coulter) that provided a partial differential count.
Measurement of viremia. The concentration of SIVmacp27 core antigen in heparinized plasma from the infected monkeys was determined by using a commercial kit (SIV Core [p27] Antigen EIA kit; Coulter).
Virus neutralization assay. Recombinant viruses containing either the HXBc2 or HXBc2P 3.2 envelope glycoproteins were generated as described above. Viruses were incubated with different concentrations of antibodies or sCD4 for 1 h at 37°C before the addition of CEMx174 target cells. Infected cells were harvested 3 days later, washed once in PBS, lysed in 0.25 M Tris-HCl (pH 7.5), and assayed for CAT activity as previously described (22).
SDF-1 inhibition assays were carried out using the same protocol, except that the rhesus PBMC target cells were incubated with 5 µg of SDF-1 per ml for 1 h at 37°C and 5% CO2 prior to the addition of the recombinant viruses.Radioimmunoprecipitation of envelope glycoproteins. Approximately 106 COS-1 cells were cotransfected with the pSVTat plasmid and the pSVIIIenv plasmid expressing either the HXBc2 or the HXBc2P 3.2 envelope glycoproteins. At 48 h after transfection, the cells were labeled with [35S]cysteine for 12 h and then lysed in 0.5% Nonidet P-40, 0.5 M NaCl, and 10 mM Tris-HCl (pH 7.5). Cell lysates were used for immunoprecipitation by a mixture of sera from HIV-1 infected individuals or by monoclonal antibodies, as previously described (39, 54).
GenBank/EMBL accession number. The full proviral sequence of the SHIV-HXBc2P 3.2 clone has been submitted to GenBank under accession no. AF041850.
| |
RESULTS |
|---|
|
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
|
|---|
Amino acid changes in the KU-1 envelope glycoproteins. To examine the possible contribution of changes in the viral envelope glycoproteins to the evolution of a pathogenic SHIV, an env fragment was cloned from cells infected with the pathogenic SHIV isolate, KU-1 (24). The cloned env fragment encodes the gp120 exterior glycoprotein and the ectodomain and transmembrane segments of the gp41 glycoprotein. Figure 1 shows the predicted amino acid sequence encoded by the cloned env fragment and compares that sequence to the parental HXBc2 sequence and to the consensus sequence of the KU-1 virus. The cloned env fragment (clone 3.2) encodes envelope glycoproteins identical in sequence to that of the KU-1 consensus. With respect to the parental HXBc2 envelope glycoproteins, 12 amino acid differences were seen. Most of these changes were located in the variable regions (V1 to V5) of the gp120 glycoprotein. Interestingly, a new potential N-linked glycosylation site in the KU-1 V1 variable loop was created, while a glycosylation site at asparagine-276 was lost. Three amino acid changes in gp41, two of which are encoded by the cloned env fragment, were found. Both changes in the gp41 ectodomain alter potential N-linked glycosylation sites, although the more C terminal of these sites is near or within the membrane-spanning region of gp41 and therefore is not utilized.
|
Replication of SHIV-HXBc2P 3.2 in activated rhesus PBMC and macrophages. To study the functional consequences of changes in the envelope glycoproteins found in the KU-1 virus, Vpu+ SHIV variants that differed only in env sequences were created (Fig. 1B). The replication of the parental SHIV-HXBc2 and SHIV-HXBc2P 3.2 viruses in rhesus monkey PBMC was examined. The uncloned KU-1 virus was also included in the experiment. As shown in Figure 2A, the KU-1 virus exhibited faster replication kinetics than did the molecularly cloned viruses. The SHIV-HXBc2P 3.2 virus replicated faster and achieved higher levels of virus production than did the SHIV-HXBc2 virus. Thus, changes in the envelope glycoproteins of the KU-1 virus are associated with faster replication kinetics in activated rhesus monkey PBMC.
|
Chemokine receptor use by the HXBc2 and HXBc2P 3.2 envelope glycoproteins. To date, the only second receptor known to be utilized by the HXBc2 envelope glycoproteins, which are derived from a laboratory-adapted virus, is CXCR4 (10, 18). Since a broadened range of chemokine receptor use might increase the efficiency of virus entry, we examined the ability of recombinant viruses containing the HXBc2 and HXBc2P 3.2 envelope glycoproteins to enter Cf2Th canine thymocytes expressing CD4 and a number of known second receptors for primate immunodeficiency viruses. Figure 3A shows that the HXBc2P 3.2 envelope glycoproteins utilize CXCR4 as a second receptor. By contrast, the entry of viruses with either the HXBc2 or the HXBc2P 3.2 envelope glycoproteins into cells expressing CD4 and CCR5 was inefficient. As expected, viruses with SIVmac239 envelope glycoproteins entered cells expressing CD4 and CCR5 but not those expressing CD4 and CXCR4 (32). The ability of viruses with the HXBc2 and HXBc2P 3.2 envelope glycoproteins to infect Cf2Th cells expressing CD4 and other seven transmembrane segment receptors (CCR2b, CCR3, STRL33, gpr 15, and gpr 1) that have been shown to act as efficient second receptors for primate immunodeficiency viruses (10, 14, 16, 30) was also examined. None of these proteins allowed the entry of viruses with either the HXBc2 or the HXBc2P 3.2 envelope glycoproteins into CD4-expressing Cf2Th cells (data not shown). Thus, we did not find evidence of qualitative differences in chemokine receptor use between the HXBc2 and the HXBc2P 3.2 envelope glycoproteins.
|
Infection of rhesus macaques with SHIV-HXBc2P 3.2. SHIV-HXBc2 replicates at only low levels in rhesus or pig-tailed macaques and does not cause CD4+ T-lymphocyte depletion in infected animals (29, 49). However, the KU-1 virus replicates more efficiently and causes rapid CD4+ T-lymphocyte depletion in pig-tailed and rhesus macaques (24). To investigate whether the KU-1-associated changes in env sequences contribute to these in vivo phenotypes, three rhesus macaques were inoculated intravenously with the molecularly cloned SHIV-HXBc2P 3.2 virus. The pattern of acute viremia in these animals was typical for SIV- or SHIV-infected monkeys, with a peak at approximately 14 days after inoculation (Fig. 4). The level of p27 antigenemia was approximately 50 to 100 times that previously seen (41) in four rhesus monkeys infected with the SHIV-HXBc2 virus (Fig. 4).
|
Neutralization of viruses with the HXBc2 and HXBc2P 3.2 envelope glycoproteins. SHIV-infected monkeys generate neutralizing antibodies against HIV-1 (29). Because the laboratory-adapted HXBc2 parent virus is highly susceptible to neutralization by sCD4 and antibodies (52), some of the envelope glycoprotein changes associated with the pathogenic KU-1 virus might modulate neutralization sensitivity. To test this, recombinant viruses with the HXBc2 or HXBc2P 3.2 envelope glycoproteins were incubated with sCD4 and neutralizing antibodies prior to infection of CEMx174 lymphocytes. CEMx174 lymphocytes were chosen as target cells in this experiment because the basal level of infectivity of recombinant viruses with HXBc2 and HXBc2P 3.2 envelope glycoproteins did not significantly differ in these cells (Fig. 5A). As shown in Figure 5B, viruses with the HXBc2P 3.2 envelope glycoproteins were less sensitive to sCD4 than were viruses with the HXBc2 envelope glycoproteins. The sensitivity of these viruses to neutralization by monoclonal antibodies was examined. Two antibodies against the CD4 binding site, F105 and the unusually potent IgG1b12 antibody, were tested, as well as the AG1121 antibody against the gp120 third variable (V3) loop. Recombinant viruses containing the HXBc2 envelope glycoproteins were, as expected, highly susceptible to neutralization by all three antibodies (Fig. 5C to E). By contrast, viruses containing the HXBc2P 3.2 envelope glycoproteins were less efficiently neutralized by all three antibodies. The epitopes for all three antibodies were retained on the HXBc2P 3.2 envelope glycoproteins, since these antibodies precipitated the HXBc2P 3.2 envelope glycoproteins with an efficiency comparable to that seen for the HXBc2 envelope glycoproteins (Fig. 5F). Thus, the HXBc2P 3.2 envelope glycoproteins are less sensitive to neutralization than are the HXBc2 envelope glycoproteins. It is likely that this altered sensitivity is mediated by changes in the envelope glycoproteins outside of the gp120 epitopes for these antibodies.
|
| |
DISCUSSION |
|---|
|
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
|
|---|
The rapidity with which CD4+ T lymphocytes are depleted in monkeys infected with pathogenic SHIV variants is remarkable. This model offers unique opportunities to understand the genetic elements that determine in vivo virus replication and CD4+ T-lymphocyte depletion. Previous studies revealed multiple differences in LTR, vpr, tat, rev, vpu, env, and nef sequences between the parent SHIV-HXBc2 and the uncloned, pathogenic SHIV (KU-1) (49, 50). Here, using a molecularly cloned virus, we show that changes in the SHIV envelope glycoproteins are sufficient to confer on an otherwise nonpathogenic virus the ability to replicate efficiently in vivo and to cause rapid and persistent CD4+ T-lymphocyte depletion. Twelve amino acid differences in the envelope glycoproteins distinguish the parent SHIV-HXBc2, which does not cause CD4+ T-lymphocyte depletion in infected animals (24, 29), from SHIV-HXBc2P 3.2, which caused profound loss of CD4+ T lymphocytes within 2 weeks of infection.
The basis for the ability of the SHIV-HXBc2P 3.2 virus to cause acute CD4+ T-lymphocyte depletion likely lies in the env-mediated increase in virus replication, which is manifest both in vivo and in tissue-cultured rhesus PBMC. Numerous studies in HIV-1-infected humans and SIV-infected monkeys have suggested that efficient virus replication is a prerequisite for CD4+ T-lymphocyte destruction and pathogenicity. The poor in vivo replication of the parental SHIV-HXBc2 (28, 29, 41) limits the possibility of pathogenic consequences, at least within the 2-year period after infection. The 50- to 100-fold increase in acute viremia seen for the SHIV-HXBc2P 3.2 may allow the pathogenic process to impact a large percentage of the CD4+ T-lymphocyte population. Further work will be required to understand the mechanistic basis of this replication increase. It does not, however, appear to result from a broadened use of chemokine receptors by the SHIV-HXBc2P 3.2 virus compared with the parental SHIV-HXBc2. Our results also indicate that the use of CXCR4 as a major coreceptor is sufficient to allow depletion of the vast majority of the CD4+ T-lymphocyte population. This is consistent with the reported expression of CXCR4 on resting as well as activated T lymphocytes (4).
The uncloned pathogenic KU-1 virus replicated more efficiently than the SHIV-HXBC2P 3.2 virus in rhesus PMBC and macrophages, suggesting the possibility that genetic elements other than env contribute in a positive way to the replication of the KU-1 virus. Our results indicate, however, that such KU-1 elements and their associated phenotypes, including enhanced replication in primary macrophages, are not necessary for acute CD4+ T-lymphocyte depletion in vivo.
In addition to mediating an increase in virus replication, the HXBc2P 3.2 envelope glycoproteins might contribute to the intrinsic ability of the virus to deplete CD4+ T lymphocytes. A comparison of the plasma antigenemia and CD4+ T-lymphocyte counts in rhesus macaques infected with SHIV-HXBc2P 3.2 or with SHIV-89.6 supports this possibility. Although SHIV-HXBc2P 3.2 did not achieve as high a level of viremia in one monkey as did SHIV-89.6 in rhesus monkeys (41), a precipitous decline in CD4+ T-lymphocyte counts was seen only in the monkey infected with the former virus. Since the differences between SHIV-HXBc2P 3.2 and SHIV-89.6 reside within the env gene, this observation implies that the envelope glycoproteins may dictate the efficiency of CD4+ T-lymphocyte destruction in this model. More carefully controlled in vivo studies in conjunction with in vitro characterization of the viral envelope glycoproteins may provide insights into the mechanistic basis of SHIV pathogenesis.
The envelope glycoproteins of the pathogenic SHIV-HXBc2P 3.2 render the virus less susceptible to neutralization by antibodies. In this respect, the laboratory-adapted HXBc2 envelope glycoproteins have evolved during in vivo passage to resemble the envelope glycoproteins of primary clinical isolates of HIV-1. It has been previously shown that monkeys infected with SHIV-HXBc2 initially generate strain-specific neutralizing antibodies that, with time, broaden to inhibit a variety of HIV-1 isolates (29). Indeed, some of the KU-1-associated changes in the envelope glycoproteins were previously seen in viruses isolated from SHIV-HXBc2-infected monkeys after several months of infection (29). While the advantage of neutralization resistance during acute infection is unclear, such resistance probably facilitates the establishment of persistent SHIV infections. Studies with antiviral therapy in HIV-1-infected humans and SIV-infected monkeys suggest that persistent virus replication is necessary for chronic CD4+ T lymphopenia (23, 35, 38).
As has been documented for primary HIV-1 isolates (52), SHIV-HXBc2P 3.2 resistance to neutralizing antibodies is mediated by envelope glycoprotein changes outside of the antibody epitopes. The antibodies to the gp120 epitopes in the third variable loop or near the CD4 binding site recognized the HXBc2P 3.2 envelope glycoproteins. This result is consistent with the observation that most of the gp120 changes associated with conversion to a pathogenic virus (Fig. 1A) are outside of the regions thought to contribute to the epitopes for the antibodies examined (54). The gp120 variable loops, where most of the KU-1-associated changes occur, have been suggested to contribute to the relative resistance of some primary HIV-1 isolates to neutralization (51). This resistance is typically mediated by a decrease in the antibody accessibility of the epitopes in the context of the oligomeric envelope glycoprotein complex (19, 33, 43, 52).
The availability of a molecularly cloned pathogenic SHIV-HXBc2P 3.2 should assist future studies of viral determinants of in vivo replication and pathogenicity. The HXBc2P 3.2 envelope glycoproteins represent a defined antigen that should be useful in exploring the nature of protective immune responses against this pathogenic virus.
| |
ACKNOWLEDGMENTS |
|---|
We acknowledge Opendra Narayan and the AIDS Research and Reference Reagent Program for providing the KU-1 virus. We thank Dennis Burton, Marshall Posner, and Raymond Sweet for reagents. We thank Yvette McLaughlin for manuscript preparation.
This work was supported by NIH grants AI-20729, CA-50139, AI-33832, RR-07000, RR-00163, and RR-00168 and Center for AIDS Research grant AI-28691; support from the G. Harold and Leila Mathers Foundation; support from the Friends 10; and a gift from the late William F. McCarty-Cooper. Mark Cayabyab is a recipient of a Ford Foundation Predoctoral Fellowship. Gunilla Karlsson was supported by a Fellowship from Douglas and Judith Krupp.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Department of Cancer Immunology/AIDS, Dana-Farber Cancer Institute, Harvard Medical School, 44 Binney St., Boston, MA 02115. Phone: (617) 632-3371. Fax: (617) 632-4338. E-mail: joseph_sodroski{at}dfci.harvard.edu.
| |
REFERENCES |
|---|
|
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
|
|---|
| 1. |
Alkhatib, G.,
C. Combadiere,
C. C. Broder,
Y. Feng,
P. E. Kennedy,
P. M. Murphy, and E. A. Berger.
1996.
CC CKR5: a RANTES, MIP-I , MIP-1 receptor as a fusion cofactor for macrophage-tropic HIV-1.
Science
272:1955-1958[Abstract].
|
| 2. |
Barre-Sinoussi, F.,
J. C. Chermann,
F. Rey,
M. T. Nugeyre,
S. Chamaret,
J. Gruest,
C. Dauguet,
C. Axler-Blin,
F. Vezinet-Brun,
C. Rouzioux,
W. Rozenbaum, and L. Montagnier.
1983.
Isolation of a T-lymphotropic retrovirus from a patient at risk for acquired immune deficiency syndrome (AIDS).
Science
220:868-871 |
| 3. | Bleul, C. C., M. Farzan, H. Choe, C. Parolin, I. Clark-Lewis, J. Sodroski, and T. A. Springer. 1996. The lymphocyte chemoattractant SDF-1 is a ligand for LESTR/fusion and blocks HIV-1 entry. Nature 382:829-833[Medline]. |
| 4. |
Bleul, C. C.,
L. Wu,
J. A. Hoxie,
T. A. Springer, and C. R. Mackay.
1997.
The HIV coreceptors CXCR4 and CCR5 are differentially expressed and regulated on human T lymphocytes.
Proc. Natl. Acad. Sci. USA
94:1925-1930 |
| 5. | Brand, D., K. Srinivasan, and J. Sodroski. 1995. Determinants of human immunodeficiency virus type 1 entry in the CDR2 loop of the CD4 glycoprotein. J. Virol. 69:166-171[Abstract]. |
| 6. |
Burton, D. R.,
J. Pyati,
R. Koduri,
S. J. Sharps,
G. B. Thornton,
P. W. Parren,
L. S. Sawyer,
R. M. Hendry,
N. Dunlop,
P. L. Nara, et al.
1994.
Efficient neutralization of primary isolates of HIV-1 by a recombinant human monoclonal antibody.
Science
266:1024-1027 |
| 7. |
Cann, A. J.,
M. J. Churcher,
M. Boyd,
W. O'Brien,
J. Q. Zhao,
J. Zack, and I. S. Chen.
1992.
The region of the envelope gene of human immunodeficiency virus type 1 responsible for determination of cell tropism.
J. Virol.
66:305-309 |
| 8. | Carrillo, A., and L. Ratner. 1996. Human immunodeficiency virus type 1 tropism for T-lymphoid cell lines: role of the V3 loop and C4 envelope determinants. J. Virol. 70:1301-1309[Medline]. |
| 9. |
Chesebro, B.,
J. Nishio,
S. Perryman,
A. Cann,
W. O'Brien,
I. S. Chen, and K. Wehrly.
1991.
Identification of human immunodeficiency virus envelope gene sequences influencing viral entry into CD4-positive HeLa cells, T-leukemia cells, and macrophages.
J. Virol.
65:5782-5789 |
| 10. | Choe, H., M. Farzan, Y. Sun, N. Sullivan, B. Rollins, P. D. Ponath, L. Wu, C. R. Mackay, G. LaRosa, W. Newman, N. Gerard, C. Gerard, and J. Sodroski. 1996. The beta-chemokine receptors CCR3 and CCR5 facilitate infection by primary HIV-1 isolates. Cell 85:1135-1148[Medline]. |
| 11. |
Clavel, F.,
D. Guetard,
F. Brun-Vezinet,
S. Chamaret,
M. A. Rey,
M. O. Santos-Ferreira,
A. G. Laurent,
C. Dauguet,
C. Katlama,
C. Rouzioux, et al.
1986.
Isolation of a new human retrovirus from West African patients with AIDS.
Science
233:343-346 |
| 12. | Cullen, B. R. 1989. Use of eukaryotic expression technology in the functional analysis of clones genes. Methods Enzymol. 152:64-73. |
| 13. | Deng, H., R. Liu, W. Ellmeier, S. Choe, D. Unutmaz, M. Burkhart, P. Di Marzio, S. Marmon, R. E. Sutton, C. M. Hill, C. B. Davis, S. C. Peiper, T. J. Schall, D. R. Littman, and N. R. Landau. 1996. Identification of a major co-receptor for primary isolates of HIV-1. Nature 381:661-666[Medline]. |
| 14. | Doranz, B. J., J. Rucker, Y. Yi, R. J. Smyth, M. Samson, S. C. Peiper, M. Parmentier, R. G. Collman, and R. W. Doms. 1996. A dual-tropic primary HIV-1 isolate that uses fusin and the beta-chemokine receptors CKR-5, CKR-3, and CKR-2b as fusion cofactors. Cell 85:1149-1158[Medline]. |
| 15. | Dragic, T., V. Litwin, G. P. Allaway, S. R. Martin, Y. Huang, K. A. Nagashima, C. Cayanan, P. J. Maddon, R. A. Koup, J. P. Moore, and W. A. Paxton. 1996. HIV-1 entry into CD4+ cells is mediated by the chemokine receptor CC-CKR-5. Nature 381:667-673[Medline]. |
| 16. |
Farzan, M.,
H. Choe,
K. Martin,
L. Marcon,
W. Hofmann,
G. Karlsson,
Y. Sun,
P. Barrett,
N. Marchand,
N. Sullivan,
N. Gerard,
C. Gerard, and J. Sodroski.
1997.
Two orphan seven-transmembrane segment receptors which are expressed in CD4-positive cells support simian immunodeficiency virus infection.
J. Exp. Med.
186:405-411 |
| 17. | Fauci, A. S., A. M. Macher, D. L. Longo, H. C. Lane, A. H. Rook, H. Masur, and E. P. Gelmann. 1984. NIH conference. Acquired immunodeficiency syndrome: epidemiologic, clinical, immunologic, and therapeutic considerations. Ann. Intern. Med. 100:92-106. |
| 18. | Feng, Y., C. C. Broder, P. E. Kennedy, and E. A. Berger. 1996. HIV-1 entry cofactor: functional cDNA cloning of a seven-transmembrane, G protein-coupled receptor. Science 272:872-877[Abstract]. |
| 19. | Fouts, T. R., J. M. Binley, A. Trkola, J. E. Robinson, and J. P. Moore. 1997. Neutralization of the human immunodeficiency virus type 1 primary isolate JR-FL by human monoclonal antibodies correlates with antibody binding to the oligomeric form of the envelope glycoprotein complex. J. Virol. 71:2779-2785[Abstract]. |
| 20. |
Gallo, R. C.,
S. Z. Salahuddin,
M. Popovic,
G. M. Shearer,
M. Kaplan,
B. F. Haynes,
T. J. Palker,
R. Redfield,
J. Okeske,
B. Safai,
G. White,
P. Foster, and P. D. Markham.
1984.
Frequent detection and isolation of cytopathic retroviruses (HTLV-III) from patients with AIDS and at risk for AIDS.
Science
224:500-503 |
| 21. | Hayami, M., and T. Igarashi. 1997. SIV/HIV-1 chimeric viruses having HIV-1 env gene: a new animal model and a candidate for attenuated live vaccine. Leukemia 11(Suppl. 3):95-97. |
| 22. |
Helseth, E.,
M. Kowalski,
D. Gabuzda,
U. Olshevsky,
W. Haseltine, and J. Sodroski.
1990.
Rapid complementation assays measuring replicative potential of human immunodeficiency virus type 1 envelope glycoprotein mutants.
J. Virol.
64:2416-2420 |
| 23. | Ho, D. D., A. U. Neumann, A. S. Perelson, W. Chen, J. M. Leonard, and M. Markowitz. 1995. Rapid turnover of plasma virions and CD4 lymphocytes in HIV-1 infection. Nature 373:123-126[Medline]. |
| 24. | Joag, S. V., Z. Li, L. Foresman, E. B. Stephens, L. J. Zhao, I. Adany, D. M. Pinson, H. M. McClure, and O. Narayan. 1996. Chimeric simian/human immunodeficiency virus that causes progressive loss of CD4+ T cells and AIDS in pig-tailed macaques. J. Virol. 70:3189-3197[Abstract]. |
| 25. | Karlsson, G. B., M. Halloran, J. Li, I. W. Park, R. Gomila, K. A. Reimann, M. K. Axthelm, S. A. Iliff, N. L. Letvin, and J. Sodroski. 1997. Characterization of molecularly cloned simian-human immunodeficiency viruses causing rapid CD4+ lymphocyte depletion in rhesus monkeys. J. Virol. 71:4218-4225[Abstract]. |
| 26. |
Koito, A.,
G. Harrowe,
J. A. Levy, and C. Cheng-Mayer.
1994.
Functional role of the V1/V2 region of human immunodeficiency virus type 1 envelope glycoprotein gp120 in infection of primary macrophages and soluble CD4 neutralization.
J. Virol.
68:2253-2259 |
| 27. | Letvin, N. L. 1990. Animal models for AIDS. Immunol. Today 11:322-326[Medline]. |
| 28. | Li, J., C. I. Lord, W. Haseltine, N. L. Letvin, and J. Sodroski. 1992. Infection of cynomolgus monkeys with a chimeric HIV-1/SIVmac virus that expresses the HIV-1 envelope glycoproteins. J. Acquired Immune Defic. Syndr. 5:639-646. |
| 29. | Li, J. T., M. Halloran, C. I. Lord, A. Watson, J. Ranchalis, M. Fung, N. L. Letvin, and J. Sodroski. 1995. Persistent infection of macaques with simian-human immunodeficiency viruses. J. Virol. 69:7061-7067[Abstract]. |
| 30. |
Liao, F.,
G. Alkhatib,
K. W. Peden,
G. Sharma,
E. A. Berger, and J. M. Farber.
1997.
STRL33, A novel chemokine receptor-like protein, functions as a fusion cofactor for both macrophage-tropic and T cell line-tropic HIV-1.
J. Exp. Med.
185:2015-2023 |
| 31. |
Luciw, P. A.,
E. Pratt-Lowe,
K. E. Shaw,
J. A. Levy, and C. Cheng-Mayer.
1995.
Persistent infection of rhesus macaques with T-cell-line-tropic and macrophage-tropic clones of simian/human immunodeficiency viruses (SHIV).
Proc. Natl. Acad. Sci. USA
92:7490-7904 |
| 32. | Marcon, L., H. Choe, K. A. Martin, M. Farzan, P. D. Ponath, L. Wu, W. Newman, N. Gerard, C. Gerard, and J. Sodroski. 1997. Utilization of C-C chemokine receptor 5 by the envelope glycoproteins of a pathogenic simian immunodeficiency virus, SIVmac239. J. Virol. 71:2522-2527[Abstract]. |
| 33. | Moore, J. P., Y. Cao, L. Qing, Q. J. Sattentau, J. Pyati, R. Koduri, J. Robinson, C. F. R. Barbas, D. R. Burton, and D. D. Ho. 1995. Primary isolates of human immunodeficiency virus type 1 are relatively resistant to neutralization by monoclonal antibodies to gp120 and their neutralization is not predicted by studies with monomeric gp120. J. Virol. 69:101-109[Abstract]. |
| 34. |
Mori, K.,
D. J. Ringler,
T. Kodama, and R. C. Desrosiers.
1992.
Complex determinants of macrophage tropism in env of simian immunodeficiency virus.
J. Virol.
66:2067-2075 |
| 35. | Nowak, M. A., A. L. Lloyd, G. M. Vasquez, T. A. Wiltrout, L. M. Wahl, N. Bischofberger, J. Williams, A. Kinter, A. S. Fauci, V. M. Hirsch, and J. D. Lifson. 1997. Viral dynamics of primary viremia and antiretroviral therapy in simian immunodeficiency virus infection. J. Virol. 71:7518-2755[Abstract]. |
| 36. | O'Brien, W. A., Y. Koyanagi, A. Namazie, J. Q. Zhao, A. Diagne, K. Idler, J. A. Zack, and I. S. Chen. 1990. HIV-1 tropism for mononuclear phagocytes can be determined by regions of gp120 outside the CD4-binding domain. Nature 348:69-73[Medline]. |
| 37. | Oberlin, E., A. Amara, F. Bachelerie, C. Bessia, J. L. Virelizier, F. Arenzana-Seisdedos, O. Schwartz, J. M. Heard, I. Clark-Lewis, D. F. Legler, M. Loetscher, M. Baggiolini, and B. Moser. 1996. The CXC chemokine SDF-1 is the ligand for LESTR/fusin and prevents infection by T-cell-line-adapted HIV-1. Nature 382:833-835[Medline]. |
| 38. | Phillips, A. N., J. Eron, J. Bartlett, D. R. Kuritzkes, V. A. Johnson, C. Gilbert, J. Johnson, A. Keller, and A. M. Hill. 1997. Correspondence between the effect of zidovudine plus lamivudine on plasma HIV level/CD4 lymphocyte count and the incidence of clinical disease in infected individuals. North American Lamivudine HIV Working Group. AIDS 11:169-175[Medline]. |
| 39. | Posner, M. R., L. A. Cavacini, C. L. Emes, J. Power, and R. Byrn. 1993. Neutralization of HIV-1 by F105, a human monoclonal antibody to the CD4 binding site of gp120. J. Acquired Immune Defic. Syndr. 67:7-14. |
| 40. |
Reimann, K. A.,
J. T. Li,
R. Veazey,
M. Halloran,
I. W. Park,
G. B. Karlsson,
J. Sodroski, and N. L. Letvin.
1996.
A chimeric simian/human immunodeficiency virus expressing a primary patient human immunodeficiency virus type 1 isolate env causes an AIDS-like disease after in vivo passage in rhesus monkeys.
J. Virol.
70:6922-6928 |
| 41. | Reimann, K. A., J. T. Li, G. Voss, C. Lekutis, K. Tenner-Racz, P. Racz, W. Lin, D. C. Montefiori, D. E. Lee-Parritz, Y. Lu, R. G. Collman, J. Sodroski, and N. L. Letvin. 1996. An env gene derived from a primary human immunodeficiency virus type 1 isolate confers high in vivo replicative capacity to a chimeric simian/human immunodeficiency virus in rhesus monkeys. J. Virol. 70:3198-3206[Abstract]. |
| 42. | Reimann, K. A., B. C. Waite, D. E. Lee-Parritz, W. Lin, B. Uchanska-Ziegler, M. J. O'Connell, and N. L. Letvin. 1994. Use of human leukocyte-specific monoclonal antibodies for clinically immunophenotyping lymphocytes of rhesus monkeys. Cytometry 17:102-108[Medline]. |
| 43. |
Sattentau, Q. J., and J. P. Moore.
1995.
Human immunodeficiency virus type 1 neutralization is determined by epitope exposure on the gp120 oligomer.
J. Exp. Med.
182:185-196 |
| 44. |
Sharpless, N. E.,
W. A. O'Brien,
E. Verdin,
C. V. Kufta,
I. S. Chen, and M. Dubois-Dalcq.
1992.
Human immunodeficiency virus type 1 tropism for brain microglial cells is determined by a region of the env glycoprotein that also controls macrophage tropism.
J. Virol.
66:2588-2593 |
| 45. | Shioda, T., J. A. Levy, and C. Cheng-Mayer. 1991. Macrophage and T cell-line tropisms of HIV-1 are determined by specific regions of the envelope gp120 gene. Nature 349:167-169[Medline]. |
| 46. |
Shioda, T.,
J. A. Levy, and C. Cheng-Mayer.
1992.
Small amino acid changes in the V3 hypervariable region of gp120 can affect the T-cell-line and macrophage tropism of human immunodeficiency virus type 1.
Proc. Natl. Acad. Sci. USA
89:9434-9438 |
| 47. | Sodroski, J., W. C. Goh, C. Rosen, K. Campbell, and W. A. Haseltine. 1986. Role of the HTLV-III/LAV envelope in syncytium formation and cytopathicity. Nature 322:470-474[Medline]. |
| 48. |
Stamatatos, L.,
A. Werner, and C. Cheng-Mayer.
1994.
Differential regulation of cellular tropism and sensitivity to soluble CD4 neutralization by the envelope gp120 of human immunodeficiency virus type 1.
J. Virol.
68:4973-4979 |
| 49. | Stephens, E. B., S. V. Joag, D. Sheffer, Z. Q. Liu, L. Zhao, S. Mukherjee, L. Foresman, I. Adany, Z. Li, D. Pinson, and O. Narayan. 1996. Initial characterization of viral sequences from a SHIV-inoculated pig-tailed macaque that developed AIDS. J. Med. Primatol. 25:175-185[Medline]. |
| 50. | Stephens, E. B., S. Mukherjee, M. Sahni, W. Zhuge, R. Raghavan, D. K. Singh, K. Leung, B. Atkinson, Z. Li, S. V. Joag, Z. Q. Liu, and O. Narayan. 1997. A cell-free stock of simian-human immunodeficiency virus that causes AIDS in pig-tailed macaques has a limited number of amino acid substitutions in both SIVmac and HIV-1 regions of the genome and has altered cytotropism. Virology 231:313-321[Medline]. |
| 51. |
Sullivan, N.,
Y. Sun,
J. Binley,
J. Lee,
C. F. Barbas,
P. W. H. I. Parren,
D. R. Burton, and J. Sodroski.
1998.
Determinants of HIV-1 envelope glycoprotein activation by soluble CD4 and monoclonal antibodies.
J. Virol.
72:6332-6338 |
| 52. | Sullivan, N., Y. Sun, J. Li, W. Hofmann, and J. Sodroski. 1995. Replicative function and neutralization sensitivity of envelope glycoproteins from primary and T-cell line-passaged human immunodeficiency virus type 1 isolates. J. Virol. 69:4413-4422[Abstract]. |
| 53. |
Thali, M.,
M. Charles,
C. Furman,
L. Cavacini,
M. Posner,
J. Robinson, and J. Sodroski.
1994.
Resistance to neutralization by broadly reactive antibodies to the human immunodeficiency virus type 1 gp120 glycoproteins conferred by a gp41 amino acid change.
J. Virol.
68:674-680 |
| 54. |
Thali, M.,
M. Charles,
C. Furman,
L. Cavacini,
M. Posner,
J. Robinson, and J. Sodroski.
1992.
Discontinuous, conserved neutralization epitopes overlapping the CD4-binding region of human immunodeficiency virus type 1 gp120 envelope glycoprotein.
J. Virol.
66:5635-5641 |
| 55. |
Westervelt, P.,
H. E. Gendelman, and L. Ratner.
1991.
Identification of a determinant within the human immunodeficiency virus 1 surface envelope glycoprotein critical for productive infection of primary monocytes.
Proc. Natl. Acad. Sci. USA
88:3097-3101 |
This article has been cited by other articles:
| |||||||||||||||||||||||||||
