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Small Amino Acid Changes in the V3 Loop of Human Immunodeficiency Virus Type 2 Determines the Coreceptor Usage for CXCR4 and CCR5
Virology 264, 237–243 (1999) Article ID viro.1999.0006, available online at http://www.idealibrary.com on
Small Amino Acid Changes in the V3 Loop of Human Immunodeficiency Virus Type 2 Determines the Coreceptor Usage for CXCR4 and CCR5 Yoshitaka Isaka,* Akihiko Sato,* ,1 Shigeru Miki,* Shinobu Kawauchi,* Hitoshi Sakaida,† Toshiyuki Hori,† Takashi Uchiyama,† Akio Adachi,‡ Masanori Hayami,† Tamio Fujiwara,* and Osamu Yoshie§ *Shionogi Institute for Medical Science, Osaka 566-0022; †Institute of Virus Research, Kyoto University, Kyoto; ‡Department of Virology, Tokushima University School of Medicine, Tokushima 770; and §Department of Bacteriology, Kinki University School of Medicine, Osaka 589-8511, Japan Received April 8, 1999; returned to author for revision May 20, 1999; accepted September 13, 1999 HIV-2 GH-1 is a molecular clone derived from an AIDS patient from Ghana. In contrast to the prototypic molecular clone ROD, GH-1 exhibits a narrow range of target cell specificity. By an infectious assay using HeLa-CD4 cells stably transfected with an HIV-1 LTR-b-galactosidase reporter gene and transiently expressing various cloned chemokine receptors, we have examined the coreceptor usage of GH-1. In contrast to ROD, which uses principally CXCR4, GH-1 was found to use mainly if not exclusively CCR5 but not CXCR4. The distinct coreceptor usage of these two molecular clones allowed us to further map the region of gp120 that is important for the coreceptor specificity. By constructing a series of chimeric viruses between GH-1 and ROD, we have demonstrated that the C-terminal half of the V3 loop region of gp120 determines the differential coreceptor usage between GH-1 and ROD, and only a few amino acid differences in this region appear to be able to shift the specificity between CCR5 and CXCR4. Notably, the shift in the coreceptor usage from CCR5 to CXCR4 is associated with an increase in the net positive charge in the V3 region. © 1999 Academic Press
CD4 and either CCR5, CCR3 or CXCR4 (Sol et al., 1997). Most HIV-2 strains that used CCR5 also could use CCR3. As observed for HIV-1, HIV-2 strains derived from asymptomatic individuals generally use CCR5 or CCR3, whereas those derived from AIDS patients often use CXCR4 (Sol et al., 1997). Recently the list of chemokine receptors that could be used by primary isolates of HIV-2 were further expanded to include CCR1, CCR2b, CCR3, CCR4, and CXCR4 (McKnight et al., 1998). Some HIV-2 strains also were able to use orphan 7-transmembrane G-protein-coupled receptors STRL33/Bob and GPR15/ Bonzo, which were initially identified as entry coreceptors for some SIV strains (Deng et al., 1997). Thus the coreceptor usage of HIV-2 appears to be much more promiscuous than those of HIV-1 and SIV. The cell tropism of HIV-1 is mainly determined by the V3 loop region of the envelope protein gp120 and amino acid changes in the V3 loop affect the coreceptor usage (Choe et al., 1996; Cocchi et al., 1996; Bieniasz et al., 1997). The role of V3 loop of HIV-2 in the coreceptor usage, however, remains to be seen. In this report, we have demonstrated that HIV-2 GH-1 strain derived from an AIDS patient in Ghana (Hasegawa et al., 1989) mainly if not exclusively uses CCR5 for virus entry in contrast to HIV-2 ROD (Clavel et al., 1986) that principally uses CXCR4. The distinct coreceptor usage of these two HIV-2 molecular clones has allowed us to map the regions of gp120 that determine the coreceptor usage of HIV-2. We have shown that, like HIV-1, the V3 loop region of gp120 is critically important for the coreceptor usage of HIV-2
INTRODUCTION Since the discovery of the chemokine receptors CCR5 and CXCR4 as the major entry coreceptors for macrophage-tropic (M-tropic or R5) and T-cell-line tropic (Ttropic or X4) strains of HIV-1, respectively, the list of the chemokine receptors capable of functioning as entry coreceptors for different strains of HIV-1 is rapidly expanding (Cairns and D’Souza, 1998). Recently, several reports have been published on the coreceptor usage of HIV-2 and SIV (Endres et al., 1996; Bron et al., 1997; Chen et al., 1997; Marcon et al., 1997; Reeves et al., 1997; Sol et al., 1997; McKnight et al., 1998). While genetically divergent SIV strains all use CCR5 and not CXCR4 for entry coreceptor (Chen et al., 1997), a broad range of chemokine receptors has been shown to be used as coreceptor for HIV-2. CD4-independent infection by several HIV-2 strains was shown to be directly mediated by CXCR4 (Endres et al., 1996; Reeves et al., 1997). HIV-2 ROD/A (the prototype) and ROD/B (CD4-independent variant) also were able to use CCR3 and V28/CX3CR1 efficiently when facilitated by soluble CD4 (Reeves et al., 1997). When studied in a cell-to-cell fusion assay, the envelope protein of HIV-2 ROD was further found to be capable of using CCR1, CCR2, CCR3, CCR4, CCR5, CXCR2, and CXCR4 (Bron et al., 1997). Primary HIV-2 isolates also were shown to infect cells coexpressing 1 To whom reprint requests should be addressed. Fax: 181-6-63822598. E-mail: [email protected].
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0042-6822/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
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FIG. 1. Growth of HIV-2 GH-1 and ROD in human T cell lines. Molt-4 and Jurkat were infected with GH-1 and ROD. RT activity in culture supernatants was determined at indicated time points postinfection. All assays were done in triplicate and mean 6 SD was obtained. Representative results from two separate experiments are shown.
those transiently transfected with one of the following chemokine receptors, CCR1, CCR2b, CCR3, and CCR5, were infected with GH-1 and ROD as well as control X4 and R5 HIV-1 strains. Viral infection was measured by tat-induced transactivation of b-gal. As shown in Fig. 2, HIV-2 GH-1 as well as R5 type HIV-1 NL162 (Tachibana et al., 1997) infected only HeLa-CD4 expressing CCR5. On the other hand, HIV-2 ROD and X4 type HIV-1 NL432 (Adachi et al., 1986) were capable of infecting even parental HeLa-CD4 cells apparently by using the endogenous CXCR4 (Endres et al., 1996; Reeves et al., 1997). This was supported by inhibition with IVR7, a monoclonal antibody to CXCR4 (Hori et al., 1998). As shown in Fig. 3, infection of ROD as well as that of X4 type HIV-1 NL432 to HeLa-CD4 or HeLa-CD4 expressing CCR5 was effectively blocked by IVR7. On the other hand, infection of GH-1 as well as that of R5 type HIV-1 NL162 to HeLa-CD4 expressing CCR5 was not affected by IVR7 at all. Thus GH-1 uses mainly if not exclusively CCR5 but not CXCR4, whereas ROD efficiently uses CXCR4 as reported previously (Endres et al., 1996; Reeves et al., 1997). The distinct coreceptor usage of GH-1 and ROD with
and only a few amino acid differences especially in the C-terminal half of the V3 loop region correlate the shift of coreceptor usage between GH-1 and ROD. RESULTS AND DISCUSSION HIV-2 ROD, a molecularly cloned and T-cell-lineadapted HIV-2 strain (Clavel et al., 1986), was shown to use CXCR4 as an entry coreceptor in cell-free virus infection assays (Endres et al., 1996; Reeves et al., 1997). Furthermore HIV-2 ROD was able to use CCR3 and V28/ CX3CR1 when infection was facilitated by soluble CD4 (Reeves et al., 1997). Another molecular clone HIV-2 GH-1 (Hasegawa et al., 1989) shows ;85% homology with HIV-2 ROD in the nucleotide sequence but seems to have a narrower target cell specificity than ROD (Kawamura et al., 1998). Availability of a molecular clone with a coreceptor usage different from ROD would be useful to map the region of gp120 involved in the coreceptor usage of HIV-2. We therefore compared the infectivity of ROD and GH-1 by using human T cell lines. As shown in Fig. 1, ROD grew well in both Molt-4 and Jurkat, whereas GH-1 grew only in Molt-4. Thus the infectivity of HIV-2 GH-1 is more restricted than that of HIV-2 ROD, suggesting the difference in their coreceptor usage. Furthermore infection of GH-1 to Molt-4 was found to be significantly suppressed by RANTES but not by SDF-1, whereas infection of ROD to Jurkat was suppressed by SDF-1 (data not shown, but see Fig. 6). We therefore examined the coreceptor usage of HIV-2 GH-1 by a cell-free infection assay using HeLa-CD4 cells stably transfected with an LTR-b-gal reporter gene (Tachibana et al., 1997). Parental HeLa-CD4 cells or
FIG. 2. Coreceptor usage of HIV-2 GH-1. HeLa-CD4 cells stably transformed with LTR-b-gal were transiently transfected with an expression vector without or with the indicated chemokine receptors. Cells were infected with HIV-2 ROD, HIV-2 GH-1, HIV-1 NL432 (X4), and HIV-1 NL162 (R5). After 4 days, transactivation of the LTR-b-gal reporter gene was measured. All assays were done in triplicate and mean 6 SD was obtained. Representative results from three separate experiments are shown.
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FIG. 3. Distinct coreceptor usage between HIV-2 ROD and GH-1. HeLa-CD4 cells stably transformed with LTR-b-gal were transiently transfected with a control or a CCR5-expression vector and infected with indicated viruses. The treatment of cells with IVR-7, an monoclonal anti-CXCR4 (Hori et al., 1998), or control mouse IgG was carried out for 1 h before infection. After 4 days, transactivation of the LTR-b-gal reporter gene was measured. All assays were done in triplicate and mean 6 SD was obtained. Representative results from three separate experiments are shown.
regard to CCR5 and CXCR4 allowed us to map the critical regions of gp120 in HIV-2 that determine the coreceptor selectivity for CCR5 and CXCR4. We therefore generated a series of gp120 chimeric viruses between GH-1 and ROD in the background of GH-1 (Fig. 4) and tested their infectivity to HeLa-CD4 or HeLa-CD4 transiently expressing CCR5. As shown in Fig. 5, GH-1(RODNN) in which the
FIG. 4. Schematic depiction of the chimeric viruses between GH-1 and ROD. Open and closed boxes indicate sequences derived from GH-1 and ROD, respectively.
FIG. 5. Coreceptor usage of the chimeric HIV-2 viruses. HeLa-CD4 cells stably transformed with LTR-b-gal were transiently transfected with a control or a CCR5-expression vector and infected with indicated viruses. The treatment of cells with IVR-7, an monoclonal anti-CXCR4 (Hori et al., 1998), or control mouse IgG was carried out for 1 h at 37°C before infection. After 4 days, transactivation of the LTR-b-gal reporter gene was measured. All assays were done in triplicate and mean 6 SD was obtained. Representative results from three separate experiments are shown.
entire gp120 was derived from ROD was capable of infecting parental HeLa-CD4. Similarly, GH-1(RODNA) in which the N-terminal half of gp120 including the V3 loop region was derived from ROD-infected parental HeLaCD4. The infection of these chimeric viruses was effectively blocked by IVR7, a monoclonal antibody to CXCR4 (Hori et al., 1998). In contrast, GH-1(RODAN) in which the N-terminal half of gp120 including the V3 loop region was derived from GH-1 infected only CCR5-expressing HeLaCD4 and was not affected by IVR7. The critical role of the V3 loop region in the coreceptor usage was demonstrated further by GH-1(V3ROD) in which only the V3 loop was derived from ROD in the background of GH-1. This chimeric virus infected parental HeLa-CD4 and was inhibited by IVR7. To further map the subregion of the V3 loop that determines the coreceptor usage, we tested two chimeric viruses GH-1 (V3RG) and GH-1 (V3GR) in which the N-terminal half and the C-terminal half of the V3 loop were derived from ROD, respectively (Fig. 4). Whereas GH-1(V3RG) infected only CCR5-expressing HeLa-CD4 and was not inhibited by IVR7, GH-1(V3GR) infected parental HeLa-CD4 and was inhibited by IVR7. Thus the C-terminal half of the V3 loop region is critical, at least in the context of gp120 of GH-1, in determining the coreceptor usage between CCR5 and CXCR4.
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FIG. 6. Inhibition of infection of the parental and chimeric HIV-2 viruses to human T cell lines by SDF-1 or RANTES. Jurkat and Molt-4 were infected with ROD, GH-1, and indicated chimeric viruses. In some cultures, RANTES or SDF-1 was included at 5 mg/ml. RT activity in culture supernatants was determined at indicated days postinfection. All assays were done in triplicate and mean 6 SD was obtained. Representative results from three separate experiments are shown.
We also wished to examine the coreceptor usage of the parental and chimeric GH-1 viruses by using natural target cells. However, even the parental ROD and especially GH-1 gew only poorly in PHA-activated PBMC. This was probably due to their long-term adaptation to established cell lines (Clavel et al., 1986; Hasegawa et al., 1989). We therefore infected these viruses to Jurkat and Molt-4 (Fig. 1). As shown in Fig. 6, infection of ROD to Jurkat was effectively blocked by SDF-1 but not by RANTES, whereas that of GH-1 to Molt-4 was inhibited by RANTES but not by SDF-1. The relative inefficiency of RANTES in inhibiting infection of GH-1 to Molt-4 might be due to its utilization of multiple coreceptors expressed on Molt-4. Among the chimeric viruses, only V3ROD and V3GR, both of which were capable of using CXCR4 but not CCR5 (Fig. 4), grew well in Jurkat and Molt-4. As expected, their infections were effectively blocked by SDF-1 but not by RANTES. Thus these results were consistent to those obtained by using HeLa-CD4 transfectants and supported in part the important role of the C-terminal half of the V3-loop region in the coreceptor usage even using natural T cell lines. In Fig. 7, the amino acid alignment of the V3 loop regions of ROD, GH-1, and two V3-loop chimeric viruses is shown. Furthermore HIV-2 and SIV clones with known
coreceptor usage are shown for comparison (Deng et al., 1997; Hill et al., 1997; McKnight et al., 1998). In comparison to ROD, GH-1 has six amino acid differences (positions 8, 10, 11, 13, 18, and 29) and two amino acid deletions (position 23 and 24). The C-terminal half of the V3 loop region that is critical in determining the coreceptor usage corresponds to the sequence between aa-18 and aa-36. In this region, histidine at position 18 and lysine at position 29 in ROD are substituted by leucine and threonine, respectively, in GH-1 and histidine and tyrosine at positions 23 and 24 are absent in GH-1. The two amino acid substitutions at positions 18 and 29 as well as the deletion at position 23 all result in decrease in the net positive charge. A similar correlation between the coreceptor usage and the net charge of the V3 loop is seen among other HIV-2 and SIV clones. This observation is also consistent to the reported correlation between the coreceptor usage and the net charge of the V3 loop region in HIV-1 strains (Shioda et al., 1992). Furthermore the N-terminal half (aa-1 to aa-17) of UC2 is identical to that of GH-1 even though UC2 uses CXCR4. This is quite in keeping with the present observation that the C-terminal half (aa-18 to aa-36) of the V3 loop is critical for determining the coreceptor usage between CCR5 and CXCR4. The net charge differences between the X4 and
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FIG. 7. Amino acid alignment of the V3 loop regions of HIV-2 and SIV clones. A gap is indicated by 2.The net charge and coreceptor usage are shown on the right.
R5 clones are also more pronounced when their Cterminal halves are compared. It is also notable that the two X4 clones have histidine at aa-18, whereas all the R5 clones have leucine at this position. In conclusion, HIV-2 GH-1, despite its isolation from an AIDS patient in Ghana and its long-term adaptation to established cell lines, uses mainly if not exclusively CCR5 as the entry coreceptor instead of CXCR4. Furthermore GH-1 is unable to use CCR1, CCR2b, or CCR3 even though R5 strains of HIV-2 are usually highly promiscuous and able to use, apart from CCR5, one or more of the CCR1, CCR2b, and CCR3 (Morner et al., 1999). It is not known whether GH-1 clone with such a restricted coreceptor usage was representative of the viral quasispecies in the original patient. Nevertheless, the availability of two HIV-2 molecular clones GH-1 and ROD with distinct coreceptor usage allowed us for the first time to map the region of gp120 of HIV-2 that critically determines the coreceptor usage. Like that of HIV-1, the V3 loop region of gp120 of HIV-2 has been shown to be critical for the coreceptor usage and only a few amino acid changes especially in the C-terminal half of this region dramatically shift the coreceptor usage between CCR5 and CXCR4. The availability of GH-1, ROD, and their chimeric molecular clones thus may be useful to further analyze the interaction of HIV-2 gp120 proteins, which tend to be less dependent on CD4 and more structurally stable than those of HIV-1 (Ross et al., 1999), with CD4 and an entry coreceptor. MATERIALS AND METHODS Cells and chemokines SW480 (Adachi et al., 1986) and HeLa-CD4 cells stably transfected with a LTR-b-gal reporter gene [HIV-1 NL432 long terminal repeat (LTR)-b-galactosidase] (Tachibana et al., 1997) were maintained in DMEM (GIBCO–BRL)
supplemented with 10% heat-inactivated fetal calf serum (FCS). CD4-positive human cell lines M8166, Jurkat, and Molt-4 (Shibata et al., 1990; Kawamura et al., 1998) were maintained in RPMI 1640 supplemented with 10% heatinactivated FCS. Recombinant chemokines were purchased from Peprotech. Viruses The following molecular infectious clones were used; pNL432 for HIV-1 NL432 (Adachi et al., 1986), pNL162env for HIV-1 NL162 (Tachibana et al., 1997), pGH-123 for HIV-2 GH-1 (Shibata et al., 1990), and pLA317 for HIV-2 ROD (Kawamura et al., 1998). Six GH-1 clones with the region of gp120 chimeric between ROD and GH-1 were generated as follows. The full-length coding region of HIV-2 ROD gp120 was amplified from pLA317 by using primers 159-CATTTTGCTAGCTAGTGCTTGCTTAGTA and 259-CTGCTGTCGCGAGAAAACCCAAGAACCC and ligated to the NheI and NruI sites of pGH123 to generate GH-1(RODNN). The N-terminal half of HIV-2 ROD gp120 including the V3 loop was amplified from pLA317 by using primers 159-CATTTTGCTAGCTAGTGCTTGCTTAGTA and 259-TGCTTGCCTAGGTCTTTTATTGATCGGCTG and ligated to the NheI and AvrII sites of pGH123 to generate GH-1(RODNA). The C-terminal half of HIV-2 ROD gp120 without the V3 loop was amplified from pLA317 by using primers 159-AAAAGACCTAGGCAAGCATGGTGCTGGTTCAAA and 259-CTGCTGTCGCGAGAAAACCCAAGAACCC and ligated to the AvrII and NruI sites of pGH123 to generate GH-1(RODAN). The V3 loop region of HIV-2 ROD was synthesized and ligated to the SmaI site (created by PCR subcloning) and AvrII site of pGH123 to generate GH-1(V3ROD). The N-terminal half of the V3 loop region of HIV-2 ROD was synthesized and ligated to the SmaI and BalI/StuI sites of pGH123 to generate GH-1(V3RG). The C-terminal half of the V3 loop
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region of HIV-2 ROD was synthesized and ligated to the BalI/StuI and AvrII sites of pGH123 to generate GH1(V3GR). SW480 cells were transfected with the plasmids by modified calcium phosphate method (Adachi et al., 1986) and cultured for 48–72 h. The culture supernatants were then used to infect M8166. The culture supernatants were harvested after the appearance of cytopathic effects, clarified by centrifugation at 3000 rpm for 15 min at 4°C, and stored in aliquots as virus stocks at 280°C until use. Infectious assay HeLa-CD4 cells stably transfected with an LTR-b-gal reporter gene were seeded into 6-cm dishes at 2 3 10 6 cells per dish. After overnight culture, cells were transfected with 20 mg of an expression vector for CCR1, CCR2b, CCR3, or CCR5 (Kitaura et al., 1996; Tachibana et al., 1997) by using a modified calcium phosphate method (Adachi et al., 1986). After 48 h culture, cells were washed with PBS, resuspended by using PBS containing 0.5 mM EDTA, and seeded into 24-well plates. Cells were infected with virus in 500 ml of culture medium (RT activity, 1 3 10 6 cpm/ml) for 2 h at 37°C and supplied with 2 ml of culture medium. After 4 d, cells were lyzed by freezing and thawing in 250 ml of the reporter lysis buffer (Promega). After centrifugation, each sample was assayed in triplicate by using Luminescent b-gal Detection kit (CLONTECH). To determine the role of endogenous CXCR4, cells were pretreated with a mAb IVR7 (Hori et al., 1998) or control mouse IgG at 500 mg/ml for 1 h at 37°C and infected with the virus. To determine HIV replication in T cell lines, 2 3 10 5 cells of Molt-4 or Jurkat were mixed with 1 ml of HIV-2 GH-1 (RT activity, 1 3 10 6 cpm/ml), ROD (RT activity, 0.5 3 10 6 cpm/ml), or chimeric viruses (RT activity, 2 3 10 6 cpm/ml). After incubation at 37°C for 1 h, cells were washed, seeded into 96-well plates (2 3 10 4 cells/well) and cultured at 37°C in 5% CO 2 air. In some cultures, chemokines were included at 5 mg/ml. After 3, 5, and 7 d p.i., RT activities in the culture supernatants were determined as described previously (Sato et al., 1995). ACKNOWLEDGMENTS We are grateful to Drs. Yorio Hinuma and Masakazu Hatanaka for constant support and encouragement. We also thank Akemi Suyama for excellent technical assistance. This work was supported in part by grants from the Ministry of Health and Welfare and the Ministry of Education, Science, Culture, and Sports of Japan.
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Ishimoto, A., and Adachi, A. (1990). Mutation analysis of the human immunodeficiency virus type 2 (HIV-2) genome in relation to HIV-1 and Simian Immunodeficiency virus SIV AGM. J. Virol. 64, 742–747. Shioda, T., Levy, J. A., and Cheng-Meyer, C. (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. Sol, N., Ferchal, F., Braun, J., Pleskoff, O., Treboute, C., Ansart, I., and Alizon, M. (1997). Usage of the coreceptors CCR-5, CCR-3, and CXCR-4 by primary and cell line-adapted human immunodeficiency virus type 2. J. Virol. 71, 8237–8244. Tachibana, K., Nakajima, T., Sato, A., Igarashi, K., Shida, H., Iizawa, H., Yoshida, N., Yoshie, O., Kishimoto, T., and Nagasawa, T. (1997). CXCR-4/fusin is not a species-specific barrier in murine cells for HIV-1 entry. J. Exp. Med. 185, 1865–1870.
Small Amino Acid Changes in the V3 Loop of Human Immunodeficiency Virus Type 2 Determines the Coreceptor Usage for CXCR4 and CCR5 Yoshitaka Isaka,* Akihiko Sato,* ,1 Shigeru Miki,* Shinobu Kawauchi,* Hitoshi Sakaida,† Toshiyuki Hori,† Takashi Uchiyama,† Akio Adachi,‡ Masanori Hayami,† Tamio Fujiwara,* and Osamu Yoshie§ *Shionogi Institute for Medical Science, Osaka 566-0022; †Institute of Virus Research, Kyoto University, Kyoto; ‡Department of Virology, Tokushima University School of Medicine, Tokushima 770; and §Department of Bacteriology, Kinki University School of Medicine, Osaka 589-8511, Japan Received April 8, 1999; returned to author for revision May 20, 1999; accepted September 13, 1999 HIV-2 GH-1 is a molecular clone derived from an AIDS patient from Ghana. In contrast to the prototypic molecular clone ROD, GH-1 exhibits a narrow range of target cell specificity. By an infectious assay using HeLa-CD4 cells stably transfected with an HIV-1 LTR-b-galactosidase reporter gene and transiently expressing various cloned chemokine receptors, we have examined the coreceptor usage of GH-1. In contrast to ROD, which uses principally CXCR4, GH-1 was found to use mainly if not exclusively CCR5 but not CXCR4. The distinct coreceptor usage of these two molecular clones allowed us to further map the region of gp120 that is important for the coreceptor specificity. By constructing a series of chimeric viruses between GH-1 and ROD, we have demonstrated that the C-terminal half of the V3 loop region of gp120 determines the differential coreceptor usage between GH-1 and ROD, and only a few amino acid differences in this region appear to be able to shift the specificity between CCR5 and CXCR4. Notably, the shift in the coreceptor usage from CCR5 to CXCR4 is associated with an increase in the net positive charge in the V3 region. © 1999 Academic Press
CD4 and either CCR5, CCR3 or CXCR4 (Sol et al., 1997). Most HIV-2 strains that used CCR5 also could use CCR3. As observed for HIV-1, HIV-2 strains derived from asymptomatic individuals generally use CCR5 or CCR3, whereas those derived from AIDS patients often use CXCR4 (Sol et al., 1997). Recently the list of chemokine receptors that could be used by primary isolates of HIV-2 were further expanded to include CCR1, CCR2b, CCR3, CCR4, and CXCR4 (McKnight et al., 1998). Some HIV-2 strains also were able to use orphan 7-transmembrane G-protein-coupled receptors STRL33/Bob and GPR15/ Bonzo, which were initially identified as entry coreceptors for some SIV strains (Deng et al., 1997). Thus the coreceptor usage of HIV-2 appears to be much more promiscuous than those of HIV-1 and SIV. The cell tropism of HIV-1 is mainly determined by the V3 loop region of the envelope protein gp120 and amino acid changes in the V3 loop affect the coreceptor usage (Choe et al., 1996; Cocchi et al., 1996; Bieniasz et al., 1997). The role of V3 loop of HIV-2 in the coreceptor usage, however, remains to be seen. In this report, we have demonstrated that HIV-2 GH-1 strain derived from an AIDS patient in Ghana (Hasegawa et al., 1989) mainly if not exclusively uses CCR5 for virus entry in contrast to HIV-2 ROD (Clavel et al., 1986) that principally uses CXCR4. The distinct coreceptor usage of these two HIV-2 molecular clones has allowed us to map the regions of gp120 that determine the coreceptor usage of HIV-2. We have shown that, like HIV-1, the V3 loop region of gp120 is critically important for the coreceptor usage of HIV-2
INTRODUCTION Since the discovery of the chemokine receptors CCR5 and CXCR4 as the major entry coreceptors for macrophage-tropic (M-tropic or R5) and T-cell-line tropic (Ttropic or X4) strains of HIV-1, respectively, the list of the chemokine receptors capable of functioning as entry coreceptors for different strains of HIV-1 is rapidly expanding (Cairns and D’Souza, 1998). Recently, several reports have been published on the coreceptor usage of HIV-2 and SIV (Endres et al., 1996; Bron et al., 1997; Chen et al., 1997; Marcon et al., 1997; Reeves et al., 1997; Sol et al., 1997; McKnight et al., 1998). While genetically divergent SIV strains all use CCR5 and not CXCR4 for entry coreceptor (Chen et al., 1997), a broad range of chemokine receptors has been shown to be used as coreceptor for HIV-2. CD4-independent infection by several HIV-2 strains was shown to be directly mediated by CXCR4 (Endres et al., 1996; Reeves et al., 1997). HIV-2 ROD/A (the prototype) and ROD/B (CD4-independent variant) also were able to use CCR3 and V28/CX3CR1 efficiently when facilitated by soluble CD4 (Reeves et al., 1997). When studied in a cell-to-cell fusion assay, the envelope protein of HIV-2 ROD was further found to be capable of using CCR1, CCR2, CCR3, CCR4, CCR5, CXCR2, and CXCR4 (Bron et al., 1997). Primary HIV-2 isolates also were shown to infect cells coexpressing 1 To whom reprint requests should be addressed. Fax: 181-6-63822598. E-mail: [email protected].
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0042-6822/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
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FIG. 1. Growth of HIV-2 GH-1 and ROD in human T cell lines. Molt-4 and Jurkat were infected with GH-1 and ROD. RT activity in culture supernatants was determined at indicated time points postinfection. All assays were done in triplicate and mean 6 SD was obtained. Representative results from two separate experiments are shown.
those transiently transfected with one of the following chemokine receptors, CCR1, CCR2b, CCR3, and CCR5, were infected with GH-1 and ROD as well as control X4 and R5 HIV-1 strains. Viral infection was measured by tat-induced transactivation of b-gal. As shown in Fig. 2, HIV-2 GH-1 as well as R5 type HIV-1 NL162 (Tachibana et al., 1997) infected only HeLa-CD4 expressing CCR5. On the other hand, HIV-2 ROD and X4 type HIV-1 NL432 (Adachi et al., 1986) were capable of infecting even parental HeLa-CD4 cells apparently by using the endogenous CXCR4 (Endres et al., 1996; Reeves et al., 1997). This was supported by inhibition with IVR7, a monoclonal antibody to CXCR4 (Hori et al., 1998). As shown in Fig. 3, infection of ROD as well as that of X4 type HIV-1 NL432 to HeLa-CD4 or HeLa-CD4 expressing CCR5 was effectively blocked by IVR7. On the other hand, infection of GH-1 as well as that of R5 type HIV-1 NL162 to HeLa-CD4 expressing CCR5 was not affected by IVR7 at all. Thus GH-1 uses mainly if not exclusively CCR5 but not CXCR4, whereas ROD efficiently uses CXCR4 as reported previously (Endres et al., 1996; Reeves et al., 1997). The distinct coreceptor usage of GH-1 and ROD with
and only a few amino acid differences especially in the C-terminal half of the V3 loop region correlate the shift of coreceptor usage between GH-1 and ROD. RESULTS AND DISCUSSION HIV-2 ROD, a molecularly cloned and T-cell-lineadapted HIV-2 strain (Clavel et al., 1986), was shown to use CXCR4 as an entry coreceptor in cell-free virus infection assays (Endres et al., 1996; Reeves et al., 1997). Furthermore HIV-2 ROD was able to use CCR3 and V28/ CX3CR1 when infection was facilitated by soluble CD4 (Reeves et al., 1997). Another molecular clone HIV-2 GH-1 (Hasegawa et al., 1989) shows ;85% homology with HIV-2 ROD in the nucleotide sequence but seems to have a narrower target cell specificity than ROD (Kawamura et al., 1998). Availability of a molecular clone with a coreceptor usage different from ROD would be useful to map the region of gp120 involved in the coreceptor usage of HIV-2. We therefore compared the infectivity of ROD and GH-1 by using human T cell lines. As shown in Fig. 1, ROD grew well in both Molt-4 and Jurkat, whereas GH-1 grew only in Molt-4. Thus the infectivity of HIV-2 GH-1 is more restricted than that of HIV-2 ROD, suggesting the difference in their coreceptor usage. Furthermore infection of GH-1 to Molt-4 was found to be significantly suppressed by RANTES but not by SDF-1, whereas infection of ROD to Jurkat was suppressed by SDF-1 (data not shown, but see Fig. 6). We therefore examined the coreceptor usage of HIV-2 GH-1 by a cell-free infection assay using HeLa-CD4 cells stably transfected with an LTR-b-gal reporter gene (Tachibana et al., 1997). Parental HeLa-CD4 cells or
FIG. 2. Coreceptor usage of HIV-2 GH-1. HeLa-CD4 cells stably transformed with LTR-b-gal were transiently transfected with an expression vector without or with the indicated chemokine receptors. Cells were infected with HIV-2 ROD, HIV-2 GH-1, HIV-1 NL432 (X4), and HIV-1 NL162 (R5). After 4 days, transactivation of the LTR-b-gal reporter gene was measured. All assays were done in triplicate and mean 6 SD was obtained. Representative results from three separate experiments are shown.
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FIG. 3. Distinct coreceptor usage between HIV-2 ROD and GH-1. HeLa-CD4 cells stably transformed with LTR-b-gal were transiently transfected with a control or a CCR5-expression vector and infected with indicated viruses. The treatment of cells with IVR-7, an monoclonal anti-CXCR4 (Hori et al., 1998), or control mouse IgG was carried out for 1 h before infection. After 4 days, transactivation of the LTR-b-gal reporter gene was measured. All assays were done in triplicate and mean 6 SD was obtained. Representative results from three separate experiments are shown.
regard to CCR5 and CXCR4 allowed us to map the critical regions of gp120 in HIV-2 that determine the coreceptor selectivity for CCR5 and CXCR4. We therefore generated a series of gp120 chimeric viruses between GH-1 and ROD in the background of GH-1 (Fig. 4) and tested their infectivity to HeLa-CD4 or HeLa-CD4 transiently expressing CCR5. As shown in Fig. 5, GH-1(RODNN) in which the
FIG. 4. Schematic depiction of the chimeric viruses between GH-1 and ROD. Open and closed boxes indicate sequences derived from GH-1 and ROD, respectively.
FIG. 5. Coreceptor usage of the chimeric HIV-2 viruses. HeLa-CD4 cells stably transformed with LTR-b-gal were transiently transfected with a control or a CCR5-expression vector and infected with indicated viruses. The treatment of cells with IVR-7, an monoclonal anti-CXCR4 (Hori et al., 1998), or control mouse IgG was carried out for 1 h at 37°C before infection. After 4 days, transactivation of the LTR-b-gal reporter gene was measured. All assays were done in triplicate and mean 6 SD was obtained. Representative results from three separate experiments are shown.
entire gp120 was derived from ROD was capable of infecting parental HeLa-CD4. Similarly, GH-1(RODNA) in which the N-terminal half of gp120 including the V3 loop region was derived from ROD-infected parental HeLaCD4. The infection of these chimeric viruses was effectively blocked by IVR7, a monoclonal antibody to CXCR4 (Hori et al., 1998). In contrast, GH-1(RODAN) in which the N-terminal half of gp120 including the V3 loop region was derived from GH-1 infected only CCR5-expressing HeLaCD4 and was not affected by IVR7. The critical role of the V3 loop region in the coreceptor usage was demonstrated further by GH-1(V3ROD) in which only the V3 loop was derived from ROD in the background of GH-1. This chimeric virus infected parental HeLa-CD4 and was inhibited by IVR7. To further map the subregion of the V3 loop that determines the coreceptor usage, we tested two chimeric viruses GH-1 (V3RG) and GH-1 (V3GR) in which the N-terminal half and the C-terminal half of the V3 loop were derived from ROD, respectively (Fig. 4). Whereas GH-1(V3RG) infected only CCR5-expressing HeLa-CD4 and was not inhibited by IVR7, GH-1(V3GR) infected parental HeLa-CD4 and was inhibited by IVR7. Thus the C-terminal half of the V3 loop region is critical, at least in the context of gp120 of GH-1, in determining the coreceptor usage between CCR5 and CXCR4.
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FIG. 6. Inhibition of infection of the parental and chimeric HIV-2 viruses to human T cell lines by SDF-1 or RANTES. Jurkat and Molt-4 were infected with ROD, GH-1, and indicated chimeric viruses. In some cultures, RANTES or SDF-1 was included at 5 mg/ml. RT activity in culture supernatants was determined at indicated days postinfection. All assays were done in triplicate and mean 6 SD was obtained. Representative results from three separate experiments are shown.
We also wished to examine the coreceptor usage of the parental and chimeric GH-1 viruses by using natural target cells. However, even the parental ROD and especially GH-1 gew only poorly in PHA-activated PBMC. This was probably due to their long-term adaptation to established cell lines (Clavel et al., 1986; Hasegawa et al., 1989). We therefore infected these viruses to Jurkat and Molt-4 (Fig. 1). As shown in Fig. 6, infection of ROD to Jurkat was effectively blocked by SDF-1 but not by RANTES, whereas that of GH-1 to Molt-4 was inhibited by RANTES but not by SDF-1. The relative inefficiency of RANTES in inhibiting infection of GH-1 to Molt-4 might be due to its utilization of multiple coreceptors expressed on Molt-4. Among the chimeric viruses, only V3ROD and V3GR, both of which were capable of using CXCR4 but not CCR5 (Fig. 4), grew well in Jurkat and Molt-4. As expected, their infections were effectively blocked by SDF-1 but not by RANTES. Thus these results were consistent to those obtained by using HeLa-CD4 transfectants and supported in part the important role of the C-terminal half of the V3-loop region in the coreceptor usage even using natural T cell lines. In Fig. 7, the amino acid alignment of the V3 loop regions of ROD, GH-1, and two V3-loop chimeric viruses is shown. Furthermore HIV-2 and SIV clones with known
coreceptor usage are shown for comparison (Deng et al., 1997; Hill et al., 1997; McKnight et al., 1998). In comparison to ROD, GH-1 has six amino acid differences (positions 8, 10, 11, 13, 18, and 29) and two amino acid deletions (position 23 and 24). The C-terminal half of the V3 loop region that is critical in determining the coreceptor usage corresponds to the sequence between aa-18 and aa-36. In this region, histidine at position 18 and lysine at position 29 in ROD are substituted by leucine and threonine, respectively, in GH-1 and histidine and tyrosine at positions 23 and 24 are absent in GH-1. The two amino acid substitutions at positions 18 and 29 as well as the deletion at position 23 all result in decrease in the net positive charge. A similar correlation between the coreceptor usage and the net charge of the V3 loop is seen among other HIV-2 and SIV clones. This observation is also consistent to the reported correlation between the coreceptor usage and the net charge of the V3 loop region in HIV-1 strains (Shioda et al., 1992). Furthermore the N-terminal half (aa-1 to aa-17) of UC2 is identical to that of GH-1 even though UC2 uses CXCR4. This is quite in keeping with the present observation that the C-terminal half (aa-18 to aa-36) of the V3 loop is critical for determining the coreceptor usage between CCR5 and CXCR4. The net charge differences between the X4 and
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FIG. 7. Amino acid alignment of the V3 loop regions of HIV-2 and SIV clones. A gap is indicated by 2.The net charge and coreceptor usage are shown on the right.
R5 clones are also more pronounced when their Cterminal halves are compared. It is also notable that the two X4 clones have histidine at aa-18, whereas all the R5 clones have leucine at this position. In conclusion, HIV-2 GH-1, despite its isolation from an AIDS patient in Ghana and its long-term adaptation to established cell lines, uses mainly if not exclusively CCR5 as the entry coreceptor instead of CXCR4. Furthermore GH-1 is unable to use CCR1, CCR2b, or CCR3 even though R5 strains of HIV-2 are usually highly promiscuous and able to use, apart from CCR5, one or more of the CCR1, CCR2b, and CCR3 (Morner et al., 1999). It is not known whether GH-1 clone with such a restricted coreceptor usage was representative of the viral quasispecies in the original patient. Nevertheless, the availability of two HIV-2 molecular clones GH-1 and ROD with distinct coreceptor usage allowed us for the first time to map the region of gp120 of HIV-2 that critically determines the coreceptor usage. Like that of HIV-1, the V3 loop region of gp120 of HIV-2 has been shown to be critical for the coreceptor usage and only a few amino acid changes especially in the C-terminal half of this region dramatically shift the coreceptor usage between CCR5 and CXCR4. The availability of GH-1, ROD, and their chimeric molecular clones thus may be useful to further analyze the interaction of HIV-2 gp120 proteins, which tend to be less dependent on CD4 and more structurally stable than those of HIV-1 (Ross et al., 1999), with CD4 and an entry coreceptor. MATERIALS AND METHODS Cells and chemokines SW480 (Adachi et al., 1986) and HeLa-CD4 cells stably transfected with a LTR-b-gal reporter gene [HIV-1 NL432 long terminal repeat (LTR)-b-galactosidase] (Tachibana et al., 1997) were maintained in DMEM (GIBCO–BRL)
supplemented with 10% heat-inactivated fetal calf serum (FCS). CD4-positive human cell lines M8166, Jurkat, and Molt-4 (Shibata et al., 1990; Kawamura et al., 1998) were maintained in RPMI 1640 supplemented with 10% heatinactivated FCS. Recombinant chemokines were purchased from Peprotech. Viruses The following molecular infectious clones were used; pNL432 for HIV-1 NL432 (Adachi et al., 1986), pNL162env for HIV-1 NL162 (Tachibana et al., 1997), pGH-123 for HIV-2 GH-1 (Shibata et al., 1990), and pLA317 for HIV-2 ROD (Kawamura et al., 1998). Six GH-1 clones with the region of gp120 chimeric between ROD and GH-1 were generated as follows. The full-length coding region of HIV-2 ROD gp120 was amplified from pLA317 by using primers 159-CATTTTGCTAGCTAGTGCTTGCTTAGTA and 259-CTGCTGTCGCGAGAAAACCCAAGAACCC and ligated to the NheI and NruI sites of pGH123 to generate GH-1(RODNN). The N-terminal half of HIV-2 ROD gp120 including the V3 loop was amplified from pLA317 by using primers 159-CATTTTGCTAGCTAGTGCTTGCTTAGTA and 259-TGCTTGCCTAGGTCTTTTATTGATCGGCTG and ligated to the NheI and AvrII sites of pGH123 to generate GH-1(RODNA). The C-terminal half of HIV-2 ROD gp120 without the V3 loop was amplified from pLA317 by using primers 159-AAAAGACCTAGGCAAGCATGGTGCTGGTTCAAA and 259-CTGCTGTCGCGAGAAAACCCAAGAACCC and ligated to the AvrII and NruI sites of pGH123 to generate GH-1(RODAN). The V3 loop region of HIV-2 ROD was synthesized and ligated to the SmaI site (created by PCR subcloning) and AvrII site of pGH123 to generate GH-1(V3ROD). The N-terminal half of the V3 loop region of HIV-2 ROD was synthesized and ligated to the SmaI and BalI/StuI sites of pGH123 to generate GH-1(V3RG). The C-terminal half of the V3 loop
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region of HIV-2 ROD was synthesized and ligated to the BalI/StuI and AvrII sites of pGH123 to generate GH1(V3GR). SW480 cells were transfected with the plasmids by modified calcium phosphate method (Adachi et al., 1986) and cultured for 48–72 h. The culture supernatants were then used to infect M8166. The culture supernatants were harvested after the appearance of cytopathic effects, clarified by centrifugation at 3000 rpm for 15 min at 4°C, and stored in aliquots as virus stocks at 280°C until use. Infectious assay HeLa-CD4 cells stably transfected with an LTR-b-gal reporter gene were seeded into 6-cm dishes at 2 3 10 6 cells per dish. After overnight culture, cells were transfected with 20 mg of an expression vector for CCR1, CCR2b, CCR3, or CCR5 (Kitaura et al., 1996; Tachibana et al., 1997) by using a modified calcium phosphate method (Adachi et al., 1986). After 48 h culture, cells were washed with PBS, resuspended by using PBS containing 0.5 mM EDTA, and seeded into 24-well plates. Cells were infected with virus in 500 ml of culture medium (RT activity, 1 3 10 6 cpm/ml) for 2 h at 37°C and supplied with 2 ml of culture medium. After 4 d, cells were lyzed by freezing and thawing in 250 ml of the reporter lysis buffer (Promega). After centrifugation, each sample was assayed in triplicate by using Luminescent b-gal Detection kit (CLONTECH). To determine the role of endogenous CXCR4, cells were pretreated with a mAb IVR7 (Hori et al., 1998) or control mouse IgG at 500 mg/ml for 1 h at 37°C and infected with the virus. To determine HIV replication in T cell lines, 2 3 10 5 cells of Molt-4 or Jurkat were mixed with 1 ml of HIV-2 GH-1 (RT activity, 1 3 10 6 cpm/ml), ROD (RT activity, 0.5 3 10 6 cpm/ml), or chimeric viruses (RT activity, 2 3 10 6 cpm/ml). After incubation at 37°C for 1 h, cells were washed, seeded into 96-well plates (2 3 10 4 cells/well) and cultured at 37°C in 5% CO 2 air. In some cultures, chemokines were included at 5 mg/ml. After 3, 5, and 7 d p.i., RT activities in the culture supernatants were determined as described previously (Sato et al., 1995). ACKNOWLEDGMENTS We are grateful to Drs. Yorio Hinuma and Masakazu Hatanaka for constant support and encouragement. We also thank Akemi Suyama for excellent technical assistance. This work was supported in part by grants from the Ministry of Health and Welfare and the Ministry of Education, Science, Culture, and Sports of Japan.
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multiple regions within both the viral envelope and the CCR5 coreceptor. EMBO J. 16, 2599–2609. Bron, R., Klasse, P. J., Wilkinson, D., Clapham, P. R., Pelchen-Matthews, A., Power, C., Wells, T. N. C., Kim, J., Peiper, S. C., Hoxie, J. A., and Marsh, M. (1997). Promiscuous use of CC and CXC chemokine receptors in cell-to-cell fusion mediated by a human immunodeficiency virus type 2 envelope protein. J. Virol. 71, 8405–8415. Cairns, J. S., and D’Souza, M. P. (1998). Chemokines and HIV-1 second receptors: the therapeutic connection. Nat. Med. 4, 563–568. Chen, Z., Zhou, P., Ho, D. D., Landau, N. R., and Marx, P. A. (1997). Genetically divergent strains of simian immunodeficiency virus use CCR5 as a coreceptor for entry. J. Virol. 71, 2705–2714. Choe, H., Farzan, M., Sun, Y., Sullivan, N., Rollins, B., Ponath, P. D., Wu, L., Mackay, C. R., LaRosa, G., Newman, W., Gerard, N., Gerard, C., and Sodroski, J. (1996). The b-chemokine receptors CCR3 and CCR5 facilitate infection by primary HIV-1 isolates. Cell 85, 1135–1148. Clavel, F., Guetard, D., Brun-Vezinet, F., Chamaret, S., Rey, M. -A., Santos-Ferreira, M. O., Laurent, A. G., Dauguet, C., Katlama, C., Rouzioux, C., Klatzmann, D., Champalimaud, J. L., and Montagnier, L. (1986). Isolation of a new human retrovirus from west African patients with AIDS. Science 233, 343–346. Cocchi, F., DeVico, A. L., Garzino-Demo, A., Cara, A., Gallo, R. C., and Lusso, P. (1996). The V3 domain of the HIV-1 gp120 envelop glycoprotein is critical for chemokine-mediated blockade of infection. Nat. Med. 2, 1244–1247. Deng, H. K., Unutmaz, D., KewalRamani, V. N., and Littman, D. R. (1997). Expression cloning of new receptors used by simian and human immunodeficiency viruses. Nature 388, 296–300. Endres, M. J., Clapham, P. R., Marsh, M., Ahuja, M., Turner, J. D., McKnight, A., Thomas, J. F., Stoebenau-Haggarty, B., Choe, S., Vance, P. J., Wells, T. N. C., Power, C. A., Sutterwala, S. S., Doms, R. W., Landau, N. R., and Hoxie, J. A. (1996). CD4-independent infection by HIV-2 is mediated by fusin/CXCR4. Cell 87, 745–756. Hasegawa, A., Tsujimoto, H., Maki, N., Ishikawa, K., Miura, T., Fukasawa, M., Miki, K., and Hayami, M. (1989). Genomic divergence of HIV-2 from Ghana. 1989. AIDS Res. Hum. Retroviruses 5, 593–604. Hill, C. M., Deng, H., Unutmaz, D., KewalRamani, V. N., Bastiani, L., Gorny, M. K., Zolla-Pazner, S., and Littman, D. R. (1997). Envelope glycoproteins from human immunodeficiency virus types 1 and 2 and simiam immunodeficiency virus can use human CCR5 as a coreceptor for viral entry and make direct CD4-dependent interactions with this chemokine receptor. J. Virol. 71, 6296–6304. Hori, T., Sakaida, H., Sato, A., Nakajima, T., Shida, H., Yoshie, O., and Uchiyama, T. (1998). Detection and delineation of CXCR-4 (fusin) as an entry and fusin cofactor for T-tropic HIV-1 by three different monoclonal antibodies. J. Immunol. 160, 180–188. Kawamura, M., Shimano, R., Ogasawara, T., Inubushi, R., Amano, K., Akari, H., and Adachi, A. (1998). Mapping the genetic determinants of human immunodeficiency virus type 2 for cell tropism and replication efficiency. Arch. Virol. 143, 1–9. Kitaura, M., Nakajima, T., Imai, T., Harada, S., Combadiere, C., Tiffany, H. L., Murphy, P. M., and Yoshie, O. (1996). Molecular cloning of human eotaxin, an eosinophil-selective CC chemokine, and identification of a specific eosinophil eotaxin receptor, CC chemokine receptor 3. J. Biol. Chem. 271, 7725–7730. Marcon, L., Choe, H., Martin, K. A., Farzan, M., Ponath, P. D., Wu, L., Newman, W., Gerard, N., Gerard, C., and Sodroski, J. (1997). Utilization of C-C chemokine receptor 5 by the envelope glycoproteins of a pathogenic simian immunodeficiency virus, SIVmac 239. J. Virol. 71, 2522–2527. McKnight, A., Dittmar, M. T., Moniz-Periera, J., Ariyoshi, K., Reeves, J. D., Hibbitts, S., Whitby, D., Aarons, E., Proudfoot, A. E. I., Whittle, H., and Clapham, P. R. (1998). A broad range of chemokine receptors are used by primary isolates of human immunodeficiency virus type 2 as coreceptors with CD4. J. Virol. 72, 4065–4071. Morner, A., Bjorndal, A., Albert, J., KewalRamani, V. N., Littman, D. R., Inoue, R., Thorstensson, R., Fenyo, E. M., and Bjorling, E. (1999).
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