Proc. Nadl. Acad. Sci. USA Vol. 89, pp. 9434-9438, October 1992 Medical Sciences
Small amino acid changes in the V3 hypervariable region of gpl20 can affect the T-cell-line and macrophage tropism of human immunodeficiency virus type 1 TATSUO SHIODA*, JAY A. LEVY, AND CECILIA CHENG-MAYERt Cancer Research Institute, Department of Medicine, University of California, School of Medicine, San Francisco, CA 94143-0128
Communicated by Maurice R. Hilleman, July 9, 1992
ABSTRACT Human immunodeficiency virus type 1 (HIV-1) strains display a high degree of heterogeneity in their biological properties that correlate with in vivo pathogenesis of the virus. We previously demonstrated that overlapping regions encompassing the third hypervariable domain (V3), within the envelope glycoprotein gpl20 determine the tropisms of HIV-1 for T-cell lines and primary macrophages. Studies with mutant viruses carring one or more amino acid substitutions in the V3 loop have now identified this hypervariable domain as a major determinant for these cellular host range properties. Three to five amino acid changes in this domain, but rarely a single amino acid substitution, can confer macrophage tropism and alter T-cell-line tropism. These fings emphasize the effect on cell tropism of small amino acid differences in the viral envelope and suggest that the overall conformation of the V3 loop plays the major role in determining the ability of HIV-1 to infect T-cell lines and primary macrophages.
The V3 loop of gpl20 contains a principal neutralization domain (13-18) and the epitopes for major histocompatibility complex class I-restricted cytotoxic-T-lymphocyte (19) and helper-T-cell (20) recognition. This envelope region has been identified as a major determinant of macrophage tropism (21). Moreover, a single amino acid mutation in the highly conserved central portion of the V3 loop has been reported to affect dramatically the HIV-1 tropism for a brain-derived fibroblast cell line (22). To examine the role of the V3 loop sequences of HIVlsF2mc and HIV-lSF162mc in their T-cell-line and macrophage tropisms, respectively, we have introduced one or more amino acid substitutions in the V3 domains of these viruses. With these mutant viruses, we demonstrate that very limited amino acid changes in this domain can alter the T-cell-line and macrophage tropisms of these isolates.
Human immunodeficiency virus type 1 (HIV-1) isolates vary in their ability to grow in different cell types (1-3), to replicate rapidly and to high titers (4-6), and to cause cytopathic changes in infected cells (7-9). Some of these in vitro biological properties correlate with in vivo pathogenesis of HIV-1 (10, 11) and may also be linked to the different clinical manifestations of AIDS. Determination of the HIV-1 genes, or specific regions within the genes, that control these biological properties, is an important step toward understanding HIV-1 infection and in developing antiviral therapies. We previously generated a series of recombinant viruses between molecular clones of T-cell-line-tropic HIV-lsF2 (HIV-lsF2mc) and primary macrophage-tropic HIV-1SF162 (HIV-1SF162mc) and examined their tropism for these cell types (12). Results of those studies showed that a 0.49kilobase-pair (kb) Stu I-Mst II fragment containing sequences encoding the third hypervariable domain (V3) of the envelope glycoprotein gpl20 also encoded the major determinant for the macrophage tropism of HIV-1, whereas an overlapping 0.98-kb Dra III-Ssp I fragment was required for infection of the T-cell line Hut78 (12). However, to achieve infection efficiency equivalent to that of the parental viruses, additional regions of gpl20 are required. In the case of primary macrophages, a 0.25-kb Dra III-Stu I fragment containing sequences encoding the V1 and V2 domains of gpl20 is necessary. For efficient infection of the Hut78 T-cell line, a relatively large portion of the viral genome that includes the entire gp120 coding sequence and a part of the gp4l coding sequence is required (12). These observations indicate the involvement of multiple and discontinuous envelope determinants in determining HIV-1 tropism.
Virus and Cell Lines. HIV-1sF2 was isolated from the peripheral blood mononuclear cells (PBMCs) of a patient with oral candidiasis (23). This isolate can replicate well in PBMCs from uninfected individuals and in established T-cell lines such as Hut78, MT-4, and Jurkat but cannot productively infect primary peripheral blood macrophages (10, 23). HIV-1SF162, which was isolated from the brain of a patient with toxoplasmosis, can replicate well in PBMCs and macrophages but not in established T-cell lines (24). The proviral genomes of HIV-1sF2 and HIV-1SF162 have been molecularly cloned and sequenced (25-27). The Hut78, MT-4, and Jurkat T-cell lines, maintained in our laboratory (10), were grown in RPMI 1640 medium supplemented with 10%o heat-inactivated (560C, 30 min.) fetal bovine serum (FBS). PBMCs were obtained from normal seronegative blood provided by Irwin Memorial Blood Bank (San Francisco). The cells were stimulated with phytohemagglutinin (3 pug/ml) for 3 days before use and then were maintained in RPMI 1640 medium supplemented with 10%o FBS and 10%o IL-2 (Electro-Nucleonics) (10). Human rhabdomyosarcoma (RD4) cells were grown in Dulbecco's modified Eagle's medium supplemented with 10o FBS. Primary peripheral blood monocytes were obtained from PBMCs by the plastic adherent technique (10) and maintained in RPMI 1640 medium supplemented with 10%o FBS and 5% heat-inactivated human serum to allow their differentiation into macrophages. Generation of Mutant DNA and Viruses. Seven-hundredbase-pair Stu I-Ssp I fragments of HIV-1SF2mc and HIV1SF162mc env genes were subcloned into plasmid vector pTZ19U to generate single-stranded DNAs that were then used to generate mutant DNAs by site-directed mutagenesis
MATERIALS AND METHODS
Abbreviations: HIV, human immunodeficiency virus; PBMC, peripheral blood mononuclear cell; RT, reverse transcriptase. *Present address: Department of Viral Infection, Institute of Medical Sciences, University of Tokyo, Tokyo, Japan. tTo whom reprint requests should be addressed.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 9434
hr at 370C. Cells were washed once and maintained in the culture medium described above. One day after infection of primary macrophages, cells were treated with 0.05% trypsin for 10 min at 370C to remove residual input virus, washed once, and maintained in the culture medium. Culture supernatants of the infected cells were assayed for RT activity at 3- to 4-day intervals as described previously (12, 27). p25 core antigen levels in the culture fluids were determined by an enzyme-linked immunosorbent assay (Coulter) in cases where RT activity was very low.
pGRAF
31H-V 32oT-A
G
T-T- 31,1
T
G 3MR-D
S
K R
T
G
N N N
D
I
R 330x_
p
9435
Proc. Natl. Acad. Sci. USA 89 (1992)
Medical Sciences: Shioda et al.
A
R
IC cH .c I
*****001 N
N
FIG. 1. Comparison of the amino acid sequences ofthe V3 region of HIV-1sF2mc and HIV-lSF162.. The complete V3 sequence of HIV-1SF2,, is shown and the five HIV-1sFl62mc-specific amino acid residues are designated. Numbers indicate positions of these five amino acid changes in the V3 domain of HIV-415762m.c- The positions of amino acid residues are relative to the HIV-1sF2, referenced in the Los Alamos AIDS and Human Retroviruses data base (47).
with mutant oligodeoxynucleotides as primers (28). Threehundred-base-pair Bgi II-Mst II fragments carrying the mutations were then used to construct the mutant proviral DNAs. Mutant viruses were recovered by transfection of mutagenized proviral DNA into RD4 cells followed by cocultivation with PBMCs from seronegative individuals (12, 27). All recovered viruses were grown in PBMCs for a short time (
Small amino acid changes in the V3 hypervariable region of gpl20 can affect the T-cell-line and macrophage tropism of human immunodeficiency virus type 1 TATSUO SHIODA*, JAY A. LEVY, AND CECILIA CHENG-MAYERt Cancer Research Institute, Department of Medicine, University of California, School of Medicine, San Francisco, CA 94143-0128
Communicated by Maurice R. Hilleman, July 9, 1992
ABSTRACT Human immunodeficiency virus type 1 (HIV-1) strains display a high degree of heterogeneity in their biological properties that correlate with in vivo pathogenesis of the virus. We previously demonstrated that overlapping regions encompassing the third hypervariable domain (V3), within the envelope glycoprotein gpl20 determine the tropisms of HIV-1 for T-cell lines and primary macrophages. Studies with mutant viruses carring one or more amino acid substitutions in the V3 loop have now identified this hypervariable domain as a major determinant for these cellular host range properties. Three to five amino acid changes in this domain, but rarely a single amino acid substitution, can confer macrophage tropism and alter T-cell-line tropism. These fings emphasize the effect on cell tropism of small amino acid differences in the viral envelope and suggest that the overall conformation of the V3 loop plays the major role in determining the ability of HIV-1 to infect T-cell lines and primary macrophages.
The V3 loop of gpl20 contains a principal neutralization domain (13-18) and the epitopes for major histocompatibility complex class I-restricted cytotoxic-T-lymphocyte (19) and helper-T-cell (20) recognition. This envelope region has been identified as a major determinant of macrophage tropism (21). Moreover, a single amino acid mutation in the highly conserved central portion of the V3 loop has been reported to affect dramatically the HIV-1 tropism for a brain-derived fibroblast cell line (22). To examine the role of the V3 loop sequences of HIVlsF2mc and HIV-lSF162mc in their T-cell-line and macrophage tropisms, respectively, we have introduced one or more amino acid substitutions in the V3 domains of these viruses. With these mutant viruses, we demonstrate that very limited amino acid changes in this domain can alter the T-cell-line and macrophage tropisms of these isolates.
Human immunodeficiency virus type 1 (HIV-1) isolates vary in their ability to grow in different cell types (1-3), to replicate rapidly and to high titers (4-6), and to cause cytopathic changes in infected cells (7-9). Some of these in vitro biological properties correlate with in vivo pathogenesis of HIV-1 (10, 11) and may also be linked to the different clinical manifestations of AIDS. Determination of the HIV-1 genes, or specific regions within the genes, that control these biological properties, is an important step toward understanding HIV-1 infection and in developing antiviral therapies. We previously generated a series of recombinant viruses between molecular clones of T-cell-line-tropic HIV-lsF2 (HIV-lsF2mc) and primary macrophage-tropic HIV-1SF162 (HIV-1SF162mc) and examined their tropism for these cell types (12). Results of those studies showed that a 0.49kilobase-pair (kb) Stu I-Mst II fragment containing sequences encoding the third hypervariable domain (V3) of the envelope glycoprotein gpl20 also encoded the major determinant for the macrophage tropism of HIV-1, whereas an overlapping 0.98-kb Dra III-Ssp I fragment was required for infection of the T-cell line Hut78 (12). However, to achieve infection efficiency equivalent to that of the parental viruses, additional regions of gpl20 are required. In the case of primary macrophages, a 0.25-kb Dra III-Stu I fragment containing sequences encoding the V1 and V2 domains of gpl20 is necessary. For efficient infection of the Hut78 T-cell line, a relatively large portion of the viral genome that includes the entire gp120 coding sequence and a part of the gp4l coding sequence is required (12). These observations indicate the involvement of multiple and discontinuous envelope determinants in determining HIV-1 tropism.
Virus and Cell Lines. HIV-1sF2 was isolated from the peripheral blood mononuclear cells (PBMCs) of a patient with oral candidiasis (23). This isolate can replicate well in PBMCs from uninfected individuals and in established T-cell lines such as Hut78, MT-4, and Jurkat but cannot productively infect primary peripheral blood macrophages (10, 23). HIV-1SF162, which was isolated from the brain of a patient with toxoplasmosis, can replicate well in PBMCs and macrophages but not in established T-cell lines (24). The proviral genomes of HIV-1sF2 and HIV-1SF162 have been molecularly cloned and sequenced (25-27). The Hut78, MT-4, and Jurkat T-cell lines, maintained in our laboratory (10), were grown in RPMI 1640 medium supplemented with 10%o heat-inactivated (560C, 30 min.) fetal bovine serum (FBS). PBMCs were obtained from normal seronegative blood provided by Irwin Memorial Blood Bank (San Francisco). The cells were stimulated with phytohemagglutinin (3 pug/ml) for 3 days before use and then were maintained in RPMI 1640 medium supplemented with 10%o FBS and 10%o IL-2 (Electro-Nucleonics) (10). Human rhabdomyosarcoma (RD4) cells were grown in Dulbecco's modified Eagle's medium supplemented with 10o FBS. Primary peripheral blood monocytes were obtained from PBMCs by the plastic adherent technique (10) and maintained in RPMI 1640 medium supplemented with 10%o FBS and 5% heat-inactivated human serum to allow their differentiation into macrophages. Generation of Mutant DNA and Viruses. Seven-hundredbase-pair Stu I-Ssp I fragments of HIV-1SF2mc and HIV1SF162mc env genes were subcloned into plasmid vector pTZ19U to generate single-stranded DNAs that were then used to generate mutant DNAs by site-directed mutagenesis
MATERIALS AND METHODS
Abbreviations: HIV, human immunodeficiency virus; PBMC, peripheral blood mononuclear cell; RT, reverse transcriptase. *Present address: Department of Viral Infection, Institute of Medical Sciences, University of Tokyo, Tokyo, Japan. tTo whom reprint requests should be addressed.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 9434
hr at 370C. Cells were washed once and maintained in the culture medium described above. One day after infection of primary macrophages, cells were treated with 0.05% trypsin for 10 min at 370C to remove residual input virus, washed once, and maintained in the culture medium. Culture supernatants of the infected cells were assayed for RT activity at 3- to 4-day intervals as described previously (12, 27). p25 core antigen levels in the culture fluids were determined by an enzyme-linked immunosorbent assay (Coulter) in cases where RT activity was very low.
pGRAF
31H-V 32oT-A
G
T-T- 31,1
T
G 3MR-D
S
K R
T
G
N N N
D
I
R 330x_
p
9435
Proc. Natl. Acad. Sci. USA 89 (1992)
Medical Sciences: Shioda et al.
A
R
IC cH .c I
*****001 N
N
FIG. 1. Comparison of the amino acid sequences ofthe V3 region of HIV-1sF2mc and HIV-lSF162.. The complete V3 sequence of HIV-1SF2,, is shown and the five HIV-1sFl62mc-specific amino acid residues are designated. Numbers indicate positions of these five amino acid changes in the V3 domain of HIV-415762m.c- The positions of amino acid residues are relative to the HIV-1sF2, referenced in the Los Alamos AIDS and Human Retroviruses data base (47).
with mutant oligodeoxynucleotides as primers (28). Threehundred-base-pair Bgi II-Mst II fragments carrying the mutations were then used to construct the mutant proviral DNAs. Mutant viruses were recovered by transfection of mutagenized proviral DNA into RD4 cells followed by cocultivation with PBMCs from seronegative individuals (12, 27). All recovered viruses were grown in PBMCs for a short time (
