Proc. Natl. Acad. Sci. USA Vol. 87, pp. 8080-8084, October 1990 Medical Sciences The human immunodeficiency virus type 2 gene is essential for productive infection of human macrophages (nefgene/macrophage infection) NAOHIKO HATTORI, FRANK MICHAELS, KATHLEEN FARGNOLI, LuISA MARCON, ROBERT C. GALLO, AND GENOVEFFA FRANCHINI* Laboratory of Tumor Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 Contributed by Robert C. Gallo, June 28, 1990 ABSTRACT The human immunodeficiency virus (HIV) HIV-2 molecular clone (HIV-2sbl/isy) (6) that is infectious in genetic determinant(s) responsible for tropism in human T cells primary peripheral blood human T cells and macrophages in or macrophages are not well defined. We studied the role ofthe vitro and infects rhesus macaques in vivo (25) to study the HIV type 2 (HIV-2) nefand vpr genes in viral tropism. HIV-2 function of vpr and nef in viral tropism, infectivity, and mutants, lacking either vpr or nef genes, or both vpr and nef, cytopathogenicity in vitro. were obtained by site-specific mutagenesis of a biologically active HIV-2 proviral clone (HIV-2,w/iy), which is infectious in both human T cells and macrophages. Viral progeny carrying MATERIALS AND METHODS mutations of nef, vpr, or of both nef and vpr genes replicated Cloning, Mutagenesis, and DNA Analysis. To reconstitute a more efllciently than the parental virus in primary human replication-competent provirus in plasmids we used DNA peripheral blood cells and in the human Hut 78 T-cell line. In fragments from a recombinant A DNA clone containing a contrast, the HIV-2 nef mutant infected human macrophages complete provirus of HIV-2Sbl/iy. The EGP plasmid was as efficiently as the parental virus, whereas viruses lacking the obtained in two steps by subcloning a 4-kilobase (kb) EcoRI vpr gene either alone or in conjunction with the lack of the nef fragment and a 2.3-kb EcoRI-Sac I fragment in Bluescript gene did not replicate in macrophages. Thus, some lack of nef (Stratagene). The KF-3 plasmid was obtained by cloning a in HIV-2 enhances viral replication in T cells and does not 6-kb Xba I fragment at the Kpn I-Sac I site ofBluescript after interfere with viral replication in primary macrophages, treating both the vector and the insert with T4 polymerase to whereas vpr is essential for replication of HIV-2 in human generate blunt ends (26). The two plasmids were linearized by macrophages. Because the parental HIV-2.bI/iy cloned virus using Sac I. Linearized KF-3 was treated with bacterial also infects rhesus macaques, the use in animal studies of these alkaline phosphatase, and the DNAs of both plasmids were IIV-2 mutants with differences in cell tropism and rates of ligated. The ligated DNA was purified by phenol extraction replication will be highly useful in understanding the mecha- and transfected into Hut 78 cell line. To mutagenize nef, we nism of viral infectivity and possibly pathogenicity in vivo. linearized the KF-3 plasmid with EcoRV and introduced a Cla I linker (5'-GATCGATC-3'), which generated an in- AIDS is associated with at least two , human frame termination codon and truncated the nefopen reading immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) frame at amino acid 59. The vpr gene open reading frame was (1, 2). HIV-1 infection is prevalent in AIDS patients world- mutated (27) by using the mutagenic primer (5'-CTAIA- wide, while HIV-2 is prevalent in some West African coun- GiACTJAAGCACCAGC-3') that changed the AUG codon to tries (3). HIV-2 is genetically and structurally related to the TAG termination codon and introduced another in-frame HIV-1 (4-6) but appears far less pathogenic than HIV-1 (7). termination codon, TAA, three bases downstream. This In addition to the structural genes (gag, ) and the mutation was introduced in an M13 subclone containing a gene, HIV-1 and HIV-2 have similar accessory genes (nef, viral Bgl II-Sac I fragment of 900 base pairs (bp). The vif, vpr, , and ). One distinctive difference between the mutated Bgl II-Sac I fragment was substituted in the EGP two viruses is the presence of the vpu gene in HIV-1 (8) and plasmid. The plasmids containing the mutated vpr and nef the gene in HIV-2 (9-11). genes were ligated together as described above and trans- Various monkey species are naturally infected with lenti- fected into Hut 78 cells. viruses related to HIV-1 and HIV-2 (12-20). Simian immu- Southern blot analysis ofthe genomic DNA was performed nodeficiency virus (SIV) of rhesus macaque (SIVm,) and of as described (28). Polymerase chain reaction analysis (29) of sooty mangabey (SIVx,) are more closely related structurally genomic DNA (1 ,ug) was done by using the described primers and genetically to HIV-2 than to HIV-1 (21-24). SIV of (60 ng each), a denaturation step at 94°C for 20 min followed African green monkey (SIVag,), evolutionarily equally dis- by hybridizing at 55°C for 20 min, and extension at 72°C for tant from HIV-1 and HIV-2 (18, 19), differs structurally from 30 min for a total of 30 cycles. both because it lacks vpr, although it carries vpx. SIV Production of Viral Stocks. The plasmids containing the in the mandrill (SIVXd), on the other hand, lacks vpx but isogenic and mutated proviral DNAs were transfected into carries vpr (20). Interestingly, the nefgene is maintained in all the Hut 78 cell line, as described. Transfected cell lines were HIVs and SIVs. The biological implication of these genetic monitored for viral expression by detecting reverse transcrip- differences among the known primate immunodeficiency tase (RT) activity in the supernatants (30) and for production viruses could play a key role in viral tropism, virus-host of other viral . When an adequate level of viral interaction, and disease development, but at the current time this role is not known. We have used a replication-competent Abbreviations: PBMC, peripheral blood mononuclear cells; RT, ; HIV-1 and -2, human immunodeficiency virus type 1 and type 2, respectively; SIV, simian immunodeficiency virus; The publication costs ofthis article were defrayed in part by page charge vpr-, mutated vpr; nefr, mutated nef; vpr--nefr, mutated vpr plus payment. This article must therefore be hereby marked "advertisement" mutated nef. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 8080 Downloaded by guest on October 2, 2021 Medical Sciences: Hattori et al. Proc. Natl. Acad. Sci. USA 87 (1990) 8081

production was obtained, viral stocks were prepared from A A Clone HIV-2SBL/ISY supernatants of the infected cells. 5' E Infection of Peripheral Blood Mononuclear Cells (PBMCs) and Monocytes. PBMCs were isolated by isopycnic centrif- ugation of Lymphoprep (Organon Teknika-Cappel) and de- pleted of monocytes by overnight adherence. Nonadherent cells were isolated, stimulated with phytohemoagglutinin for 3 days, and infected with an amount of virus equivalent to 2 x 105 cpm of RT activity per ml per 1.5 x 106 cells. Hut 78 cells were infected identically. After 1-hr viral exposure cells were washed and seeded in 5 ml of RPMI 1640 medium containing 20%o fetal calf serum, penicillin at 50 units/ml, streptomycin at 50 ,ug/ml, and 5 AuM L-glutamine. At the Plasmid EGP Plasmid KF-3 indicated sampling times, the medium was replaced, and RT activity was assayed in the culture supernatants as described B 1~_I (30). Monocytes were isolated from PBMCs by adherence to Cleave with Sad plastic as described (31). Cells were infected while in sus- 5' ' pension by adding an amount of virus equivalent to 2 or 4 x LTRSS '2 105 cpm of RT to 1 x 107 monocytes for 1 hr with periodic agitation. The cells were washed once and seeded in RPMI s E 1640 medium/20% fetal calf serum containing penicillin at 50 Ligate units/ml, streptomycin at 50 and 5 pLM L-glutamine. gg/ml, 5' S 3' At the indicated sampling times, culture supernatants were LTR removed and cleared by low-speed centrifugation. The su- Transfect to Hut 78 cells pernatants were collected, and the pellet consisting of both by DEAE Dextran method viable and nonviable cells was returned to the original cultures; the amount of RT in the supernatants was deter- C 'I mined as described (30). Analysis. At the times chosen for protein analysis by radioimmunoprecipitation assay, the culture medium was removed, and the cells were washed once with RPMI 1640 Hut 78 cells medium lacking cysteine and methionine but containing 10% FIG. 1. Schematic representation of the strategy used to recon- dialyzed fetal calf serum. Cells were resuspended in 2 ml of stitute biologically active proviral DNA from two plasmids contain- methionine and cysteine-free medium containing [35S]- ing overlapping regions of HIV-2,bl/isy. E, EcoRl; S, Sac l; X, Xba methionine and [35S]cysteine at 100 pCi/ml (1 Ci = 37 GBq) I. each and incubated overnight. At the end of incubation, cells were washed and then lysed with 0.15 mM NaCl/0.05 mM Infectivity of HIV-2 Viral Mutants. To measure the relative Tris HCI, pH 7.2/1% Triton X-100/1% sodium deoxycho- infectivity of the mutated viruses we prepared cell-free late/0.1% NaDodSO4; the cell lysate was then immunopre- supernatants from the transfected Hut 78 cells, normalized cipitated (32) with human serum (1:100) from an individual them for equal content of viral RT activity (2 X 105 cpm/ml), infected with HIV-2. and applied them to phytohemagglutinin-stimulated (for 3 days) human PBMCs enriched for T lymphocytes. The ki- netics of viral infection were studied by following the amount RESULTS of viral RT activity in the medium and the synthesis of viral Generation of HIV-2 Mutants in nef and vpr Genes. Two p24 protein in the infected cells every 3 days after infection. overlapping pieces of proviral DNA from the recombinant The results of three experiments on three different blood phage were subcloned separately in a Bluescript vector (Fig. donors are presented in Fig. 2. In Fig. 2A, RT activity was 1) because plasmids containing the entire provirus were detected in both cultures infected with HIV-2 neff and HIV-2 unstable in Escherichia coli. Plasmid EGP DNA carrying the neff-vpr- mutants at day 9, but expression of p24 was seen vpr mutation was ligated either with plasmid KF-3 DNA or with the HIV-2 neff virus as early as day 6 after infection with plasmid KF-3 DNA carrying the mutated nef gene to (Fig. 2A). Similarly, in the HIV-2 vpr--infected culture, p24 generate an HIV-2 vpr- or an HIV-2 vpr--neff mutant, was evident at day 9, and RT activity was evident at day 12. respectively. The HIV-2 neff mutant was obtained by ligat- Wild-type HIV-2sbl/isy scored positive for RT at day 15, but ing plasmid EGP DNA and plasmid KF-3 DNA with the low levels of p24 were already detected at day 9. In Fig. 2B, mutated nefgene. The ligated DNA was transfected into Hut higher expression of viral RT was detected in both HIV-2 78 cell line, which is CD4+ and can support HIV-2 replica- vpr- and neff than in the wild type at day 11, although the tion. kinetics of viral expression were slightly different from Fig. To monitor viral production in Hut 78-transfected cells we 2A. Because use of equal amounts of viral RT activity in the followed RT activity and the presence of metabolically la- infectivity assay does not necessarily reflect the amount of beled viral in the medium ofthe transfected cells. RT biologically active virus in the viral stock used, we tested the activity was detectable, on the average, at 15 days after infectivity of serial dilutions (from 10-1 to 10-9) of each viral transfection, and labeled gpl20 and p24 were present in the stock in Hut 78 cell line. We followed the RT activity supernatant ofthe cells transfected with all HIV-2 mutants as produced in the medium of duplicate cell culture for each well as with HIV-2sbl/isy (data not shown), indicating that virus for 1 month. One TCID50 (tissue culture 50% infectious abolishing nefand vpr function did not impair production of dose) corresponded to the minimal amount ofeach viral stock extracellular virus in this human T-cell line. Furthermore, all that yielded a productive infection in 50% of the culture. The HIV-2 mutants were as cytopathic as the wild type and PBMCs of a third donor were then infected at a multiplicity induced very large syncytia in the transfected cells (data not of infection (moi) of 0.01 [one viral infectious dose (TCID50) shown). every 100 cells]. The results of the third experiment are Downloaded by guest on October 2, 2021 8082 Medical Sciences: Hattori et al. Proc. Natl. Acad. Sci. USA 87 (1990) aI A B c .

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V _ -. 5 10 15 20 21 DAYS 0 7 11 14 21 DAYS OF CULTURE DAYS OF CULTURE FIG. 2. Kinetics of viral infection of human PBMC (A, B, and D) and Hut 78 cells (C). A and C include radioimmunoprecivitation of viral proteins various days after infection. WT, wild type.

shown in Fig. 2D. Slightly higher expressions of the HIV-2 DNA with EcoRI and Cla I. The HIV-2 vpr- DNA, which is nef virus were seen at day 10 when compared with the wild isogenic in nef with wild-type HIV-2sbl/isy, lacks the 1.4-kb type. Taken together, the data indicate that the abrogation of internal band. neffunction conferred a slight growth advantage to HIV-2 in To monitor whether reversion of the nucleotide change primary human cells. Absence of the vpr gene per se ap- introduced in vpr occurred, we performed polymerase chain peared also to confer a slight increase in viral replication in reaction analyses (29) on the DNA of infected cells with two two of three experiments. Such an effect of vpr on viral HIV-2Wbl/isy-specific primers (5'-GTCCAAGGTACCGT- expression was even more evident when a similar experiment CAC-3'; 5'-CCCTAATGAGCTCTCTGGC-3') and obtained was conducted using Hut 78 as target cell. Fig. 2C shows that the nucleotide sequence ofthe mutated region in seven clones RT activity as well as p24 protein were detected at day 10 in of the amplified DNA fragment. No evidence of reversion the HIV-2 vpr- mutant, whereas wild-type HIV-2SbW/jSy be- was detected (data not shown). came positive for both parameters only at day 21. The Infection of Primary Macrophages. HIV-2,bl/,sy infects not kinetics of Hut 78 infection ofthe HIV-2 nef and vpr--nefr only human primary T cells but also human primary macro- mutants mirrored the kinetics seen when PBMCs (from donor phages. To investigate whether the abrogation of nefand vpr 1) were used as target cells (Fig. 2 A and C). function affected the capability of HIV-2 to infect both types Stability of the HIV-2 Mutant Genotypes. To ascertain of target cells, we infected human macrophages obtained whether the nef mutation introduced in the proviral DNAs from peripheral blood with the HIV-2SbW/iSy and the HIV-2 was retained after many cycles of viral replication, we mutants by using a described procedure (31). Parallel exper- cleaved the genomic DNAs of the infected Hut 78 cells with iments with PBMCs from the same donor were also done as EcoRI and Cla I. The Cla I recognition site is not present in a positive control on the biological activity of the viral stocks HIV-2sbi/isy DNA but was inserted in the proviral DNA ofthe used. RT activity in the supernatant of the infected macro- HIV-2 nefr (see Materials and Methods) and is therefore phages was measured every few days; results are shown in diagnostic for mutation in nef. Fig. 3 shows that the mutation Fig. 4. In Fig. 4A we used 2 x 105 cpm of RT activity for each in nefin both HIV-2 nefr and vpr--nef was readily detected virus. High levels of viral replication in macrophages were as a 1.4-kb band derived from the double digestion ofgenomic seen with the HIV-2sbl/isy and HIV-2 nefr mutant. Surpris- Downloaded by guest on October 2, 2021 Medical Sciences: Hattori et al. Proc. Natl. Acad. Sci. USA 87 (1990) 8083

EcoR EcoRI (Clal) To determine whether the low level of RT activity in the 5'LT R I I supernatants of the macrophage culture infected with the _I HIV-2 vpr- and the HIV-2 vpr -nef was from delayed virus release rather than from impaired viral expression, we met- 4.6 1.4 abolically labeled the macrophage culture 20 days after Probe infection and scored for viral proteins by immunoprecipita- B8 tion. Supernatants and total cellular lysates were immuno- HIV-2SBL precipitated by using a human serum positive for antibodies vpr- against HIV-2 proteins. Fig. 4B (Right) indicates that viral vpr- nef- nef- proteins could be readily detected in both cell lysate and Kb supernatant of HIV-2Sbl/iy and HIV-2 nefr mutant-infected macrophages but not in macrophages infected with HIV-2 vpr- or HIV-2 vpr -nef mutants. Thus, the defect in the vpr Ob-_0 4.6 gene appeared to impair viral expression rather than viral release from human macrophages. Viral entry and reverse transcription were apparently not inhibited by the mutation introduced in the vpr gene because we demonstrated a 1.4 specific fragment of viral DNA of 0.591 kb by polymerase chain reaction analysis of the DNAs of the macrophages infected with the HIV-2 vpr-, as well as in the wild type and the HIV-2 4 level of viral DNA was FIG. 3. Southern blot analysis ofthe DNA ofchronically infected nefr (Fig. Right). A low Hut 78 cells. Restriction enzyme and Southern blot (28) analyses of also detected in the macrophage infected with the HIV-2 the DNA of infected Hut 78 cells were done after several months of vpr-nefr (Fig. 4). Why a lower level of viral DNA and no culture. Because a Cla I site was introduced into the nef gene, we RT activity can be found in the culture infected with the restricted the DNAs of HIV-2 nef, HIV-2 vpr-, and HIV-2 vpr-- HIV-2 lacking both the vpr and the nef genes is not clear nefr with Cla I and EcoRI to generate an internal viral band of 1.4 because lack of the nef gene per se does not appear to kb in the proviruses carrying the mutation presented schematically interfere with HIV-2 expression in human macrophages. above. Filters carrying the genomic DNAs were hybridized accord- ing to described procedures (28) with B8 probe (6). LTR, long Understanding the mechanism of action of both vpr and nef terminal repeat. will clarify whether the two gene products interact on a common pathway in the HIV-2 replicative cycle in human macrophages. ingly, neither HIV-2 vpr- nor HIV-2 vpr--nef mutants produced significant levels of RT in the supernatant. Results of a parallel culture of the PBMCs of the same individual are DISCUSSION shown in Fig. 2B. As shown, the same viral stock of HIV-2 Our results show that HIV-2 defective in nef gene function vpr- and HIV-2 vpr-nef promptly replicated in PBMCs can replicate efficiently in human macrophages and yield but failed to induce significant RT activity in the supernatant extracellular viruses. In contrast, when a defect is introduced of the macrophage cultures. A second experiment was per- in the vpr gene, no viral replication can be detected in human formed with more virus (4 x 105 cpm of RT activity), and the macrophages. The importance of the vpr gene for viral results represented in Fig. 4B Left again indicated failure of replication in human macrophages is also further confirmed the HIV-2 vpr- and HIV-2 vpr--nefr viruses to productively by failure of the HIV-2 vpr--nef- mutant to productively replicate in human macrophages. infect primary macrophages.

105 A HIV-2s3L (nef-) HIV-2sBL ISV (WT) 104 C-,

HIV-2SBL (vpr-nef-) HVS_ 0 5 10 15 20 25 DAYS OF CULTURE FIG. 4. Viral infection of human primary mac- rophages. (A and B Left) RT activity in the super- 0-, natants of macrophage cultures after infection with H- B 1 2 3 4 Hr- CL SCL S CL S (CL S HIV-2sbl/isy or HIV-2 mutants of two different blood donor macrophages. *, HIV-2 nef; *, HIV- gp12O 2sbl/isy; 0, HIV-2 vpr-; I , HIV-2 vpr--neff; o, uninfected control. (B Right upper) Radioimmune * .. 0 precipitation of 20 days after infection E from the experiment of B was done as described. .f CL, cell lysate; S, supernatant; 1, HIV-2 vpr-; 2, HIV-2 vpr--nefr; 3, HIV-2 nefr; 4, HIV-2sbl/isy. 4w 4 + p24 (B Right) Autoradiography of hybridization with the B8 probe (6) of polymerase chain reaction Kb amplified 591 bp of HIV-2 sequence from infected 0591 macrophage DNA (24 days after infection) of the experiment of B. Primers used in the polymerase UC 1 2 3 4 chain reaction are as described in text. UC, unin- DAYS OF CULTURE fected control DNA. Downloaded by guest on October 2, 2021 8084 Medical Sciences: Hattori et al. Proc. Natl. Acad Sci. USA 87 (1990) The fact that HIV-2 nefr virus replicated slightly better Sehgal, P., Daniel, M. D., Desrosiers, R. C. & Essex, M. than did the parental virus in human T cells suggests that (1985) Science 228, 1199-1201. HIV-2,blf/sy carries a functional nef gene which, as has been 13. Ohta, Y., Masuda, T., Tsujimoto, H., Ishikawa, K.-I., suggested for HIV-1 (33, 34), exerts a negative effect on viral Kodama, T., Morikawa, S., Nakai, M., Honjo, S. & Hayami, expression in vitro. More importantly, a similar effect is M. (1988) Int. J. Cancer 41, 115-122. evident in primary human T cells. Considerable disagreement 14. Fultz, P. N., McClure, H. M., Anderson, D. C., Swenson, R. B., Anand, R. & Srinivasan, A. (1986) Proc. Natl. Acad. exists among different laboratories regarding the effect of the Sci. USA 83, 5286-5290. nef gene on viral growth (33-37) and viral transcription 15. Murphey-Corb, M., Martin, L. N., Rangan, S. R. S., Baskin, (35-38). Nevertheless, the HIV-2,bi/isy nef mutant under G. B., Gormus, B. J., Wolf, R. H., Andes, W. A., West, M. & our experimental conditions displays a slight growth advan- Montelaro, R. C. (1986) Nature (London) 321, 435-437. tage when compared with wild-type HIV-2sbl/isy. 16. Lowenstine, L. J., Pederson, N. C., Higgins, J., Pallis, K. C., The vpr gene of HIV-2 is dispensable for viral replication Uyeda, A., Marx, P., Lerche, N. W., Munn, R. J. & Gardner, in human T cells, as has been reported for HIV-1 (39, 40) and M. B. (1986) Int. J. Cancer 38, 563-574. HIV-2 (41), but our data indicate that its expression is 17. Benveniste, R. E., Arthur, L. O., Tsai, C.-C., Sowder, R., required for viral propagation in primary macrophages. Copeland, T. D., Henderson, L. E. & Oroszlan, S. (1986) J. The HIV-1 vpr gene has been shown to be a trans-activator Virol. 60, 483-490. of the HIV-1 long terminal repeats as well as of heterologous 18. Fukasawa, M., Miura, T., Hasegawa, A., Morikawa, S., Tsu- promoters (42). The HIV-2 vpr gene also trans-activates in jimoto, H., Miki, K., Kitamura, T. & Hayami, M. (1988) Nature (London) 333, 457-461. vitro (N.H., R.C.G., and G.F., unpublished observation), 19. Li, Y., Naidu, Y. M., Daniel, M. D. & Desrosiers, R. C. (1989) and this function might play an important role in viral J. Virol. 63, 1800-1802. replication in human macrophages. Because the molecularly 20. Tsujimoto, H., Hasegawa, A., Maki, N., Fukasawa, M., Miura, cloned HIV-2,bl/isy virus infects rhesus macaques (25), the T., Speidel, S., Cooper, R. W., Moriyama, E. N., Gojobori, T. use in animals of HIV-2 mutants that replicate efficiently in & Hayami, M. (1989) Nature (London) 341, 539-541. both macrophages and T cells (nef-) or in T cells only (vpr- 21. Franchini, G., Gallo, R. C., Guo, H. G., Gurgo, C., Collalti, and vpr--nef-) will hopefully provide useful insights into E., Fargnoli, K. A., Hall, L. F., Wong-Staal, F. & Reitz, viral tropism in the establishment ofpersistent infection in the M. S., Jr. (1987) Nature (London) 328, 539-542. host and allow evaluation of vaccine efficacy. 22. Hirsch, V., Riedel, N. & Mullins, J. (1987) Cell 49, 307-309. 23. Chakrabarti, L., Guyader, M., Alizon, M., Daniel, M. D., We are grateful to Dr. Sue Gartner for help in some of the Desrosiers, R. C., Tiollais, P. & Sonigo, P. (1987) Nature experiments; to Ersell Richardson, Randy Zicht, and Robert Zeman (London) 328, 543-547. for technical assistance; and to Dr. Oliviero Varnier for providing 24. Hirsch, V. M., Opmsted, R. A., Murphy-Corb, M., Purcell, HIV-2-positive human sera. We also thank Dr. Ronald Desrosier for R. H. & Johnson, P. R. (1989) Nature (London) 339, 389-392. helpful suggestions. 25. Franchini, G., Markham, P. 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