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Vpx and Vpr proteins of HIV-2 up-regulate the viral infectivity by a distinct mechanism in lymphocytic cells
Microbes and Infection 5 (2003) 387–395 www.elsevier.com/locate/micinf
Original article
Vpx and Vpr proteins of HIV-2 up-regulate the viral infectivity by a distinct mechanism in lymphocytic cells Fumiko Ueno a, Hiroshi Shiota a, Maki Miyaura b, Akiko Yoshida b, Akiko Sakurai b, Junko Tatsuki b, A. Hajime Koyama b, Hirofumi Akari c, Akio Adachi b, Mikako Fujita b,* a b
Department of Ophthalmology and Visual Neuroscience, The University of Tokushima School of Medicine, Tokushima 770-8503, Japan Department of Virology, The University of Tokushima Graduate School of Medicine, 3-18-15 Kuramoto-cho, Tokushima 770-8503, Japan c Tsukuba Primate Center for Medical Sciences, National Institute of Infectious Diseases, Ibaraki 305-0843, Japan Received 6 November 2002; accepted 27 December 2002
Abstract Mutants of human immunodeficiency virus type 2 (HIV-2) carrying a frame-shift mutation in vpx, vpr, and in both genes were monitored for their growth potentials in a newly established lymphocytic cell line, HSC-F. Worthy of note, the replication of a vpx single mutant, but not vpr, was severely impaired in these cells, and that of a vpx-vpr double mutant was more damaged. Defective replication sites of the vpx single and vpx-vpr double mutants were demonstrated to be mapped, respectively, to the nuclear import of viral genome, and to both, this process and the virus assembly/release stage. While the mutational effect of vpr was small, the replication efficiency in one cycle of the vpx mutant relative to that of wild-type virus was estimated to be 10%. The growth phenotypes of the vpx, vpr, and vpx-vpr mutant viruses in HSC-F cells were essentially repeated in primary human lymphocytes. In primary human macrophages, whereas the vpx and vpx-vpr mutants did not grow at all, the vpr mutant grew equally as well as the wild-type virus. These results strongly suggested that Vpx is critical for up-regulation of HIV-2 replication in natural target cells by enhancing the genome nuclear import, and that Vpr promotes HIV-2 replication somewhat, at least in lymphocytic cells, at a very late replication phase. © 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Keywords: HIV-2; Vpx; Vpr
1. Introduction Five independent subgroups of primate lentiviruses are now generally recognized [1,2]. They all possess a number of auxiliary genes that are not present in the classic and typical retroviruses [3,4]. Of these genes, some are nonessential or accessory because they can be deleted without abrogating the ability of virus to replicate at least in certain types of target cells [3–5]. The viruses that cause AIDS in humans, designated human immunodeficiency virus types 1 and 2 (HIV-1 and 2), are related but distinct members of the lentiviral subfamily of retroviruses. HIV-2 and simian immunodeficiency viruses isolated from the rhesus (SIVmac) and sooty mangabey (SIVsm) monkeys constitute an independent subgroup and carry a unique set of accessory genes different from that of HIV-1. The accessory gene specific to HIV* Corresponding author. Tel.: +81-886-33-9232; fax: +81-886-33-7080. E-mail address: [email protected] (M. Fujita). © 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. DOI: 1 0 . 1 0 1 6 / S 1 2 8 6 - 4 5 7 9 ( 0 3 ) 0 0 0 4 2 - X
2/SIVmac/SIVsm is vpx. While vpx is dispensable for virus replication in established cell lines [6–18], it is essential for the replication of viruses of the HIV-2 group in monocytederived macrophages (MDMs) and important for replication in vivo [10,13,18–22]. In MDMs, Vpx facilitates the nuclear localization of viral reverse transcription complex [19,22]. It has also been noted that, in peripheral blood mononuclear cells (PBMCs) or lymphocytes (PBLs), vpx-defective viruses grow to a lesser extent than does wild-type (wt) virus [6,10–14,21]. Although not studied extensively, one report raised a possibility that Vpx enhances the viral DNA synthesis in PBMCs [8]. Another unique feature of viruses of the HIV-2 group in the five primate lentiviruses is that they contain both vpx and vpr genes in their genomes. Vpx and vpr share considerable sequence similarity and are juxtaposed in the genome, and therefore, it has been suggested that they arose from a gene-duplication event [23,24]. However, although the importance of vpr has been shown by in vivo study [25], its role in viral replication in natural target cells
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Fig. 1. Structure of HIV-2 proviral clones used in this study. Locations of mutations and insertions introduced into the proviral genome are shown schematically. Frame-shift mutations (black triangles) in vpx, vpr, env, and rev are located at StyI, EcoRI, NsiI, PstI sites, respectively, as previously described [13,17,45]. The first group (from pGL-AN to pGL-St/Ec) includes infectious clones carrying single or double frame-shift mutations in the vpx and vpr genes. The second group (from pGLnCAT to pGLnCAT-Ns) includes clones carrying CAT (black boxes) in nef as previously described [13]. The third group (from pGL-Ns to pGLDrev-Ns) includes env-minus clones as previously described [13]. Clone pGLDrev-Ns in the third group and all clones in the fourth group (from pGLDrev to pGLDrev-St/Ec) lack an initiation codon for rev (white triangles).
such as PBMCs and MDMs has been a subject of controversy. Some studies showed that vpr is essential or important for virus replication in these cells [14,26,27]. The viral replication events responsible for these observations have not yet been determined. Other studies indicated that vpr mutant and wt viruses replicated equally well in natural target cells [11,12,16,21,25,28]. On the other hand, it has been reported that vpx-vpr double mutant was severely attenuated in vitro and in vivo [12,29,30]. The virological and molecular basis underlying these results is currently unknown. As summarized above, little is known about the functional role of HIV-2 Vpx in virus replication in lymphocytic cells. In addition, no extensive functional studies on HIV-2 Vpr have been carried out yet. In this report, we have performed a systemic functional analysis of HIV-2 Vpx and Vpr in a newly established lymphocytic cell line, HSC-F, PBLs and MDMs using a variety of proviral mutant constructs. We demonstrate here that HIV-2 Vpx is critical for the nuclear import of viral reverse transcription complex in lymphocytic cells, and that HIV-2 Vpr is required for the optimal production of progeny virions in these cells. We also show that HIV-2 Vpr is completely dispensable, in sharp contrast to Vpx, for virus replication in MDMs.
2. Materials and methods 2.1. Cells Lymphocytic cell lines HSC-F [31,32] and M8166 [33] were maintained in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS). Monolayer cell lines multinuclear activation of galactosidase indicator (MAGI) [34] and 293T [35] were maintained in Eagle’s minimal essential medium containing 10% heat-inactivated FBS. Human PBLs and MDMs were separated from PBMCs by adhesion to the plastic essentially as previously described [36]. Nonadherent cells (PBLs) were then stimulated with 10 mg/ml phytohemagglutinin P (Difco Laboratories, Detroit, MI) in RPMI-1640 medium supplemented with 10% heat-inactivated FBS for 3 d, and cultured in the presence of 50 µg/ml of recombinant human interleukin 2 for infection experiments. Adherent cells (monocytes) were cultured in RPMI-1640 medium supplemented with 10% heatinactivated human serum AB (Nabi, Boca Raton, FL) for 8–11 d in the presence or absence of 5 ng/ml of granulocytemacrophage colony stimulating factor (GM-CSF) (PeproTech EC Ltd, London, England) to obtain differentiated MDMs for infection experiments.
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2.2. Transfection For transfection, uncleaved plasmid DNA was introduced into 293T and HSC-F cells by the calcium-phosphate coprecipitation and electroporation methods, respectively [37]. 2.3. Infection for determination of viral growth kinetics Target cells were infected with cell-free viruses obtained by transfection of 293T cells with various infectious clones (first group in Fig. 1) as previously described [38]. 2.4. Virus titer Virus titers were determined by reverse transcriptase (RT) activity [39], MAGI cell infectivity [34], chloramphenicol acetyltransferase (CAT) activity [40], and enzyme immunoassay (EIA) of SIV Gag p27 (Coulter Corp., Miami, FL). 2.5. Analysis of early and late viral replication phases To monitor the progression of early viral replication phase, proviral CAT-constructs (second group in Fig. 1) were used. An HIV-2 Rev-expression vector prev2 [41] and various CAT-constructs were co-transfected into 293T cells, and 48 h later, cell-free viruses were prepared from the culture supernatants. Equal RT units of CAT-viruses were then inoculated into HSC-F cells, and CAT activity in cells harvested at 48 h post-infection was determined as indicative of early phase. To monitor the progression of late viral replication phase, env-minus proviral clones (third group in Fig. 1) were used. HSC-F cells were transfected with these clones, and 72 h later, culture supernatants were harvested and concentrated by centrifugation as previously described [42] to obtain virions. Amounts of virions as judged by EIA of Gag p27 were then determined as indicative of late phase. 2.6. Analysis of the synthesis and nuclear localization of viral DNA To determine whether viral DNA is synthesized and imported into nucleus, rev-minus proviral clones (fourth group in Fig. 1) were used. An HIV-2 Rev-expression vector prev2 [41] and various rev-minus clones were co-transfected into 293T cells, and 48 h later, cell-free viruses were prepared from the culture supernatants. Equal RT units of the viruses were then inoculated into HSC-F cells as previously described [43], and DNA was extracted from infected cells at intervals. DNA samples (0.5 µg) were subjected to polymerase chain reaction (PCR) analysis to monitor the reverse transcription and nuclear import processes. Primers used and conditions for PCR were as follows. (i) Early reverse transcription product (R/U5 region): 5’-CAGCACTAGCAGGTAGAGCC-3’ (sense) and 5’-AAGCGGAAGGGTCCTAACAG-3’ (antisense), 94 °C for 3 min, 23 cycles of 94 °C for 45 s, 58 °C for 45 s, and 72 °C for 1 min, and finally 72 °C for 10 min. (ii) Late reverse transcription product (U5/5’-end
Fig. 2. Infectivity of DVpx, DVpr, and DVpx·DVpr for MAGI and M8166 cells. (A) MAGI infectivity of the mutants. Virus samples were prepared from 293T cells transfected with 20 µg of the clones indicated (first group in Fig. 1), and their infectivity was determined by MAGI assay [34]. MAGI infectivity relative to that of wt virus is shown. No blue foci were observed in mock-infected cells. (B) Growth kinetics in M8166 cells of the mutants. Cells (1 × 106) were infected with 1 × 106 RT units of cell-free viruses, and virus replication was monitored at intervals by RT production in the culture supernatants. Input viruses were prepared from 293T cells transfected with 20 µg of the clones indicated on the right (first group in Fig. 1). Mock, pUC19.
noncoding region): 5’-CTGTTAGGACCCTTCCGCTT-3’ (sense) and 5’-CTTTCACTCCCGCTCCACAC-3’ (antisense), 94 °C for 3 min, 24 cycles of 94 °C for 45 s, 58 °C for 45 s, and 72 °C for 1 min, and finally 72 °C for 10 min.
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Fig. 3. Replication ability in HSC-F cells of DVpx, DVpr, and DVpx·DVpr at the early and late phases. (A) Growth kinetics in HSC-F cells of the mutants. HSC-F cells (1 × 107) were infected with 1 × 106 RT units of cell-free viruses, and virus replication was monitored at intervals by RT production in the culture supernatants. Input viruses were prepared from 293T cells transfected with 20 µg of various clones, indicated on the right (first group in Fig. 1). Mock, pUC19. (B) Analysis of early replication phase of the mutants in HSC-F cells. Input viruses were prepared by co-transfection of a Rev-expression vector, prev2 [41], and the clones indicated on the right (second group in Fig. 1) (20 µg for each) into 293T cells. HSC-F cells (1 × 107) were infected with 1 × 106 RT units of these viruses, and CAT activity in cells 48 h later was determined. Percent conversions of chloramphenicol to its acetylated forms, after subtracting those by a negative control (GLnCAT-Ns in Fig. 1), vs. reaction hours for CAT assay are plotted in the figure. (C) Analysis of late replication phase of the mutants in HSC-F cells. HSC-F cells (5 × 106) were electroporated with 10 µg of proviral clones indicated (third group in Fig. 1), and the culture supernatants were harvested 72 h later. Virions were pelleted through centrifugation, and lysates were prepared as previously described [42]. Viral amounts in the lysates were determined by EIA of Gag p27, and the level of virus production was calculated by subtraction of that by a negative control (GLDrev-Ns in Fig. 1). Virus production relative to that of wt clone is shown.
(iii) Two-long terminal repeat (LTR) circular product (U5/U3 region): 5’-CTGTTAGGACCCTTCCGCTT-3’ (sense) and 5’-CATAGCCACCCGAAACACAT-3’ (antisense), 94 °C for 3 min, 28 cycles of 94 °C for 45 s, 64 °C for 45 s, and 72 °C for 1 min, and finally 72 °C for 10 min. As a control for PCR, b-globin gene was amplified using the primers GH20 and PC04 [44] under the same conditions, for early reverse transcription product. For DNA standards, pGL-AN (first group in Fig. 1) solutions diluted stepwise were amplified by PCR using early and late primers as described above. 2.7. Proviral DNA constructs An infectious DNA clone of HIV-2, designated pGL-AN, was constructed from pGH123 [45] by replacing a region of tat and env genes with that of an HIV-2ROD clone [13,17]. Four accessory genes of pGH123 are all functional as judged by our mutational analyses [11,13,16,17,45]. All proviral mutant clones used in this report were constructed from pGL-AN, and are shown in Fig. 1. Construction and characterization of some of the clones in Fig. 1, designated pGL-St [13], pGL-Ec [17], pGLnCAT [13], pGLnCAT-St [13], and pGLnCAT-Ns [13], have been described previously. The other mutant clones in Fig. 1 were constructed by combination of recombinant DNA techniques routinely used [13,17,33,45–47]. As for pGLDrev constructs in Fig. 1, the initiation codon for rev was first converted into ACG by
QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA), and the DNA fragment containing the mutation was then inserted into proviral clones. 3. Results 3.1. Replication potentials of GL mutant viruses We recently reported that macaque monkey cell lines susceptible to various SIVs could be established by infection with Herpesvirus saimiri [31,32,48]. One of the cell lines, designated HSC-F, was interleukin-2-independent, CD4- and CXCR4-positive, and highly susceptible to viruses of the HIV-2 group but not to those of the HIV-1 group [49]. No H. saimiri production was observed in HSC-F cells [31]. We were interested in monitoring the mutational effects of HIV-2 vpx and vpr genes on viral replication in this new lymphocytic cell line using proviral mutant clones derived from wt pGL-AN (first group in Fig. 1). Wt GL-AN virus grows exquisitely well in a wide variety of lymphocytic cell lines, PBLs and MDMs of human and simian origins [13,17]. Before doing the infection experiments in HSC-F cells, we confirmed the replication ability of mutant viruses (referred to as D hereafter) in MAGI and M8166 cells. Input sample viruses were prepared by transfection of the proviral clones into 293T cells, and then were inoculated into MAGI and
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M8166 cells. As shown in Fig. 2, the three mutant viruses were similarly infectious for these cells with wt virus. Growth kinetics of the mutant viruses in HSC-F cells were then similarly determined. As clearly seen in Fig. 3A, the replication of DVpr was as efficient as that of wt virus. In contrast, DVpx grew in the cells with delayed kinetics. The replication of DVpx·DVpr was severely impaired.
3.2. Determination of defective replication sites of DVpx and DVpx·DVpr in HSC-F cells The defective replication phase of DVpx and DVpx·DVpr in HSC-F cells was determined by the single-round replication assay [13,46,47] using rev-minus CAT (second group in Fig. 1) and env-minus (third group in Fig. 1) constructs. The early replication phase of DVpx and DVpx·DVpr was examined by measuring CAT activity in cells infected with CATviruses. Input CAT viruses were prepared by co-transfection of a Rev-expression vector prev2 and various CAT constructs into 293T cells, and were inoculated into HSC-F cells. At 48 h post-infection, cells were harvested for CAT assay. As shown in Fig. 3B, CAT activity was determined in a timecourse experiment. It was readily observed that CAT activity in cells infected with DVpx or DVpx·DVpr was much lower than that in cells infected with wt or DVpr. From the slopes of individual reaction curves, an approximately 10-fold reduction in CAT activity occurred when HSC-F cells were infected with DVpx or DVpx·DVpr. These results were reproduced in the two other independent experiments (data not shown). The late replication phase of DVpx and DVpx·DVpr was examined by determining virion production after transfection. HSC-F cells were electroporated with env-minus proviral clones, and 72 h later, amounts of progeny virions were measured by EIA of Gag p27. As shown in Fig. 3C, while DVpx produced a similar level of progeny virions with that of wt clone upon transfection, an approximately twofold reduction in the yield of progenies was observed for DVpr and DVpx·DVpr. We then monitored whether virus DNA is synthesized and imported into the nucleus of cells infected with DVpx or DVpx·DVpr. Input viruses were prepared by co-transfection of a Rev-expression vector, prev2, and rev-minus clones (fourth group in Fig. 1) into 293T cells, and inoculated into HSC-F cells. At 24 h post-infection, cells were harvested for PCR analysis. As shown in Fig. 4, a comparable level of virus DNA was synthesized in cells infected with DVpx, DVpr, DVpx·DVpr, or wt virus. In contrast, no circular form of virus DNA with two LTRs, which is known to be present only in the nucleus of cells infected with retroviruses [50], was detected in cells infected with DVpx or DVpx·DVpr. These data were reproduced in the two other independent experiments (data not shown). Furthermore, similar results were obtained for DNA samples prepared from cells harvested at 12 and 48 h post-infection (data not shown).
Fig. 4. Analysis of the viral reverse transcription and nuclear import processes in HSC-F cells infected with DVpx, DVpr, or DVpx·DVpr. (A) Analysis of viral DNAs by PCR. Input viruses were prepared by co-transfection of a Rev-expression vector, prev2 [41], and the clones indicated at the top (fourth group in Fig. 1) (20 µg for each) into 293T cells. HSC-F cells (1.5 × 107) were infected with 6 × 106 RT units of these viruses, and DNA was extracted from the cells 24 h later. DNA samples (0.5 µg) were subjected to PCR to detect early (R/U5 region) and late (U5/5’-end noncoding region) reverse transcription products, and to detect two-LTR circle in the nucleus. To ascertain an approximate equality of DNA amount in each sample, b-globin gene was amplified by PCR. All products amplified by PCR were resolved on a 3% agarose gel. (B) DNA standards for PCR. Solutions of wt clone pGL-AN (first group in Fig. 1), prepared by stepwise dilutions, were subjected to PCR using primers for early and late reverse transcription products for standards. Numbers at the top represent relative DNA amount. The WT samples here contained the same contents as those in (A).
3.3. Replication of DVpx, DVpr and DVpx·DVpr in human PBLs and MDMs Finally, we determined the growth kinetics of DVpx, DVpr, and DVpx·DVpr in human PBLs and MDMs. PBL and MDM preparations were made independently from several HIV-seronegative persons, and used as targets for virus infection. Input mutant viruses were prepared by transfection of proviral clones (first group in Fig. 1) into 293T cells, and inoculated into the primary PBL and MDM cultures.
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Fig. 5. Growth kinetics in PBLs of DVpx, DVpr, and DVpx·DVpr. PBLs (1 × 106) were infected with equal RT units of cell-free viruses (1 × 104 for A and C–E, and 1 × 105 for B), and virus replication was monitored at intervals by RT production in the culture supernatants. PBLs were separated from five independently prepared PBMC samples. Input viruses were prepared from 293T cells transfected with the clones indicated on the right (first group in Fig. 1). Mock, pUC19.
Fig. 5 shows the viral growth kinetics in PBLs. In the five PBL preparations, essentially the same results were obtained. While DVpr displayed growth kinetics similar to those of wt virus, DVpx and DVpx·DVpr, particularly DVpx·DVpr, exhibited a severe growth defect in good agreement with the results in HSC-F cells (Fig. 3A). Fig. 6 shows the viral growth kinetics in MDMs. Out of the three MDM preparations, two were made and infected with viruses in the absence of GM-CSF, and another was in the presence of the factor. As is clear in Fig. 6, essentially the same results were obtained. In the three MDM preparations, no replication of DVpx and DVpx·DVpr was detected, consistent with previous reports [10,13,18,19,21,22]. In sharp contrast, DVpr grew considerably well in MDMs, and no consistently significant growth defect was observed, in agreement with the results previously reported [12,21]. In summary, DVpx and DVpx·DVpr but not DVpr displayed a severe growth defect in PBLs and MDMs.
4. Discussion The new and key findings in this report are that HIV-2 Vpx augments virus replication in lymphocytic cells by enhancing the nuclear import of viral DNA (Figs. 3 and 4), and that
HIV-2 Vpr is necessary for the efficient virion release from these cells (Fig. 3). Vpx and Vpr contributed to efficient HIV-2 replication to a different degree. In our assays, Vpx and Vpr enhanced virus replication in a single-round by 10and 2-fold, respectively (Fig. 3). In any case, the replication of HIV-2 in lymphocytic cells was greatly impaired when both Vpx and Vpr were absent (Figs. 3 and 5) as reported previously [12]. In vivo studies coincide with this result. While SIVmac without either vpx or vpr induces AIDS in monkeys, without vpx and vpr, it cannot induce the disease [29,30]. It has been well established that HIV-2 Vpx is essential for nuclear localization of viral DNA in infected MDMs [19,22]. Although HIV-2 Vpr has been reported to induce cell cycle arrest [19,51] and apoptosis (our unpublished observation), its direct role in virus replication is presently unknown. We demonstrated here the nuclear transfer of viral DNA by HIV-2 Vpx in infected lymphocytic cells, and no appreciable contribution of HIV-2 Vpr to this process, in contrast to HIV-1 Vpr [52]. Instead, HIV-2 Vpr was found to act at a very late step in the replication cycle in lymphocytic cells. However, no functional role of HIV-2 Vpr in the virus replication in MDMs was found in our assay (Fig. 6).
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Fig. 6. Growth kinetics in MDMs of DVpx, DVpr, and DVpx·DVpr. MDMs in a well of 24-well tissue culture plates were infected with 1 × 106 RT units of cell-free viruses, and virus replication was monitored at intervals by RT production in the culture supernatants. MDMs were separated from three independently prepared PBMC samples. MDMs in (A) and (B) were cultured in the absence of GM-CSF throughout the experiment (from the start of cell culture, virus infection, and to the end of observation period), whereas MDMs in (C) were cultured in the presence of GM-CSF. Input viruses were prepared from 293T cells transfected with the clones indicated on the right (first group in Fig. 1). Replication of T-tropic HIV-1 NL432 virus [11,16,33,37,42,46,47,49] was not detected at all in any cell preparations here (data not shown). Mock, pUC19.
Not only in nondividing MDMs but also in dividing lymphocytic cells, Vpx is required for the optimal nuclear transport of HIV-2 DNA. In contrast, HIV-1 Vpr, the functional counterpart of HIV-2 Vpx, is not necessary at all for the nuclear import in dividing cells of HIV-1 DNA. The molecular basis for this difference, probably related to the structure of viral pre-integration complex, is intriguing but currently unknown. Further study is required to clarify the nuclear transfer process of viral DNAs of HIV-1 and HIV-2. The precise replication process of HIV-2 at which Vpr is required is also unclear at present. On the basis of the results shown in Fig. 3, the step is predicted to be present around the viral assembly/release stage. The molecular event of the Vpr action at this step remains to be elucidated.
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Acknowledgements We thank Tokushima Red Cross Blood Center, Tokushima 770-0044, Japan, for buffy coats of HIV-seronegative blood donors. We are indebted to Kazuko Yoshida for editorial assistance. This work was supported by a Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science and Technologyof Japan (14021078), a Grant-in-Aid for Scientific Research (B) from the Japan Society for the Promotion of Science (14370103), and a Health Sciences Research Grant (Research on HIV/AIDS 13110201) from the Ministry of Health, Labor and Welfare of Japan.
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Original article
Vpx and Vpr proteins of HIV-2 up-regulate the viral infectivity by a distinct mechanism in lymphocytic cells Fumiko Ueno a, Hiroshi Shiota a, Maki Miyaura b, Akiko Yoshida b, Akiko Sakurai b, Junko Tatsuki b, A. Hajime Koyama b, Hirofumi Akari c, Akio Adachi b, Mikako Fujita b,* a b
Department of Ophthalmology and Visual Neuroscience, The University of Tokushima School of Medicine, Tokushima 770-8503, Japan Department of Virology, The University of Tokushima Graduate School of Medicine, 3-18-15 Kuramoto-cho, Tokushima 770-8503, Japan c Tsukuba Primate Center for Medical Sciences, National Institute of Infectious Diseases, Ibaraki 305-0843, Japan Received 6 November 2002; accepted 27 December 2002
Abstract Mutants of human immunodeficiency virus type 2 (HIV-2) carrying a frame-shift mutation in vpx, vpr, and in both genes were monitored for their growth potentials in a newly established lymphocytic cell line, HSC-F. Worthy of note, the replication of a vpx single mutant, but not vpr, was severely impaired in these cells, and that of a vpx-vpr double mutant was more damaged. Defective replication sites of the vpx single and vpx-vpr double mutants were demonstrated to be mapped, respectively, to the nuclear import of viral genome, and to both, this process and the virus assembly/release stage. While the mutational effect of vpr was small, the replication efficiency in one cycle of the vpx mutant relative to that of wild-type virus was estimated to be 10%. The growth phenotypes of the vpx, vpr, and vpx-vpr mutant viruses in HSC-F cells were essentially repeated in primary human lymphocytes. In primary human macrophages, whereas the vpx and vpx-vpr mutants did not grow at all, the vpr mutant grew equally as well as the wild-type virus. These results strongly suggested that Vpx is critical for up-regulation of HIV-2 replication in natural target cells by enhancing the genome nuclear import, and that Vpr promotes HIV-2 replication somewhat, at least in lymphocytic cells, at a very late replication phase. © 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Keywords: HIV-2; Vpx; Vpr
1. Introduction Five independent subgroups of primate lentiviruses are now generally recognized [1,2]. They all possess a number of auxiliary genes that are not present in the classic and typical retroviruses [3,4]. Of these genes, some are nonessential or accessory because they can be deleted without abrogating the ability of virus to replicate at least in certain types of target cells [3–5]. The viruses that cause AIDS in humans, designated human immunodeficiency virus types 1 and 2 (HIV-1 and 2), are related but distinct members of the lentiviral subfamily of retroviruses. HIV-2 and simian immunodeficiency viruses isolated from the rhesus (SIVmac) and sooty mangabey (SIVsm) monkeys constitute an independent subgroup and carry a unique set of accessory genes different from that of HIV-1. The accessory gene specific to HIV* Corresponding author. Tel.: +81-886-33-9232; fax: +81-886-33-7080. E-mail address: [email protected] (M. Fujita). © 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. DOI: 1 0 . 1 0 1 6 / S 1 2 8 6 - 4 5 7 9 ( 0 3 ) 0 0 0 4 2 - X
2/SIVmac/SIVsm is vpx. While vpx is dispensable for virus replication in established cell lines [6–18], it is essential for the replication of viruses of the HIV-2 group in monocytederived macrophages (MDMs) and important for replication in vivo [10,13,18–22]. In MDMs, Vpx facilitates the nuclear localization of viral reverse transcription complex [19,22]. It has also been noted that, in peripheral blood mononuclear cells (PBMCs) or lymphocytes (PBLs), vpx-defective viruses grow to a lesser extent than does wild-type (wt) virus [6,10–14,21]. Although not studied extensively, one report raised a possibility that Vpx enhances the viral DNA synthesis in PBMCs [8]. Another unique feature of viruses of the HIV-2 group in the five primate lentiviruses is that they contain both vpx and vpr genes in their genomes. Vpx and vpr share considerable sequence similarity and are juxtaposed in the genome, and therefore, it has been suggested that they arose from a gene-duplication event [23,24]. However, although the importance of vpr has been shown by in vivo study [25], its role in viral replication in natural target cells
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Fig. 1. Structure of HIV-2 proviral clones used in this study. Locations of mutations and insertions introduced into the proviral genome are shown schematically. Frame-shift mutations (black triangles) in vpx, vpr, env, and rev are located at StyI, EcoRI, NsiI, PstI sites, respectively, as previously described [13,17,45]. The first group (from pGL-AN to pGL-St/Ec) includes infectious clones carrying single or double frame-shift mutations in the vpx and vpr genes. The second group (from pGLnCAT to pGLnCAT-Ns) includes clones carrying CAT (black boxes) in nef as previously described [13]. The third group (from pGL-Ns to pGLDrev-Ns) includes env-minus clones as previously described [13]. Clone pGLDrev-Ns in the third group and all clones in the fourth group (from pGLDrev to pGLDrev-St/Ec) lack an initiation codon for rev (white triangles).
such as PBMCs and MDMs has been a subject of controversy. Some studies showed that vpr is essential or important for virus replication in these cells [14,26,27]. The viral replication events responsible for these observations have not yet been determined. Other studies indicated that vpr mutant and wt viruses replicated equally well in natural target cells [11,12,16,21,25,28]. On the other hand, it has been reported that vpx-vpr double mutant was severely attenuated in vitro and in vivo [12,29,30]. The virological and molecular basis underlying these results is currently unknown. As summarized above, little is known about the functional role of HIV-2 Vpx in virus replication in lymphocytic cells. In addition, no extensive functional studies on HIV-2 Vpr have been carried out yet. In this report, we have performed a systemic functional analysis of HIV-2 Vpx and Vpr in a newly established lymphocytic cell line, HSC-F, PBLs and MDMs using a variety of proviral mutant constructs. We demonstrate here that HIV-2 Vpx is critical for the nuclear import of viral reverse transcription complex in lymphocytic cells, and that HIV-2 Vpr is required for the optimal production of progeny virions in these cells. We also show that HIV-2 Vpr is completely dispensable, in sharp contrast to Vpx, for virus replication in MDMs.
2. Materials and methods 2.1. Cells Lymphocytic cell lines HSC-F [31,32] and M8166 [33] were maintained in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS). Monolayer cell lines multinuclear activation of galactosidase indicator (MAGI) [34] and 293T [35] were maintained in Eagle’s minimal essential medium containing 10% heat-inactivated FBS. Human PBLs and MDMs were separated from PBMCs by adhesion to the plastic essentially as previously described [36]. Nonadherent cells (PBLs) were then stimulated with 10 mg/ml phytohemagglutinin P (Difco Laboratories, Detroit, MI) in RPMI-1640 medium supplemented with 10% heat-inactivated FBS for 3 d, and cultured in the presence of 50 µg/ml of recombinant human interleukin 2 for infection experiments. Adherent cells (monocytes) were cultured in RPMI-1640 medium supplemented with 10% heatinactivated human serum AB (Nabi, Boca Raton, FL) for 8–11 d in the presence or absence of 5 ng/ml of granulocytemacrophage colony stimulating factor (GM-CSF) (PeproTech EC Ltd, London, England) to obtain differentiated MDMs for infection experiments.
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2.2. Transfection For transfection, uncleaved plasmid DNA was introduced into 293T and HSC-F cells by the calcium-phosphate coprecipitation and electroporation methods, respectively [37]. 2.3. Infection for determination of viral growth kinetics Target cells were infected with cell-free viruses obtained by transfection of 293T cells with various infectious clones (first group in Fig. 1) as previously described [38]. 2.4. Virus titer Virus titers were determined by reverse transcriptase (RT) activity [39], MAGI cell infectivity [34], chloramphenicol acetyltransferase (CAT) activity [40], and enzyme immunoassay (EIA) of SIV Gag p27 (Coulter Corp., Miami, FL). 2.5. Analysis of early and late viral replication phases To monitor the progression of early viral replication phase, proviral CAT-constructs (second group in Fig. 1) were used. An HIV-2 Rev-expression vector prev2 [41] and various CAT-constructs were co-transfected into 293T cells, and 48 h later, cell-free viruses were prepared from the culture supernatants. Equal RT units of CAT-viruses were then inoculated into HSC-F cells, and CAT activity in cells harvested at 48 h post-infection was determined as indicative of early phase. To monitor the progression of late viral replication phase, env-minus proviral clones (third group in Fig. 1) were used. HSC-F cells were transfected with these clones, and 72 h later, culture supernatants were harvested and concentrated by centrifugation as previously described [42] to obtain virions. Amounts of virions as judged by EIA of Gag p27 were then determined as indicative of late phase. 2.6. Analysis of the synthesis and nuclear localization of viral DNA To determine whether viral DNA is synthesized and imported into nucleus, rev-minus proviral clones (fourth group in Fig. 1) were used. An HIV-2 Rev-expression vector prev2 [41] and various rev-minus clones were co-transfected into 293T cells, and 48 h later, cell-free viruses were prepared from the culture supernatants. Equal RT units of the viruses were then inoculated into HSC-F cells as previously described [43], and DNA was extracted from infected cells at intervals. DNA samples (0.5 µg) were subjected to polymerase chain reaction (PCR) analysis to monitor the reverse transcription and nuclear import processes. Primers used and conditions for PCR were as follows. (i) Early reverse transcription product (R/U5 region): 5’-CAGCACTAGCAGGTAGAGCC-3’ (sense) and 5’-AAGCGGAAGGGTCCTAACAG-3’ (antisense), 94 °C for 3 min, 23 cycles of 94 °C for 45 s, 58 °C for 45 s, and 72 °C for 1 min, and finally 72 °C for 10 min. (ii) Late reverse transcription product (U5/5’-end
Fig. 2. Infectivity of DVpx, DVpr, and DVpx·DVpr for MAGI and M8166 cells. (A) MAGI infectivity of the mutants. Virus samples were prepared from 293T cells transfected with 20 µg of the clones indicated (first group in Fig. 1), and their infectivity was determined by MAGI assay [34]. MAGI infectivity relative to that of wt virus is shown. No blue foci were observed in mock-infected cells. (B) Growth kinetics in M8166 cells of the mutants. Cells (1 × 106) were infected with 1 × 106 RT units of cell-free viruses, and virus replication was monitored at intervals by RT production in the culture supernatants. Input viruses were prepared from 293T cells transfected with 20 µg of the clones indicated on the right (first group in Fig. 1). Mock, pUC19.
noncoding region): 5’-CTGTTAGGACCCTTCCGCTT-3’ (sense) and 5’-CTTTCACTCCCGCTCCACAC-3’ (antisense), 94 °C for 3 min, 24 cycles of 94 °C for 45 s, 58 °C for 45 s, and 72 °C for 1 min, and finally 72 °C for 10 min.
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Fig. 3. Replication ability in HSC-F cells of DVpx, DVpr, and DVpx·DVpr at the early and late phases. (A) Growth kinetics in HSC-F cells of the mutants. HSC-F cells (1 × 107) were infected with 1 × 106 RT units of cell-free viruses, and virus replication was monitored at intervals by RT production in the culture supernatants. Input viruses were prepared from 293T cells transfected with 20 µg of various clones, indicated on the right (first group in Fig. 1). Mock, pUC19. (B) Analysis of early replication phase of the mutants in HSC-F cells. Input viruses were prepared by co-transfection of a Rev-expression vector, prev2 [41], and the clones indicated on the right (second group in Fig. 1) (20 µg for each) into 293T cells. HSC-F cells (1 × 107) were infected with 1 × 106 RT units of these viruses, and CAT activity in cells 48 h later was determined. Percent conversions of chloramphenicol to its acetylated forms, after subtracting those by a negative control (GLnCAT-Ns in Fig. 1), vs. reaction hours for CAT assay are plotted in the figure. (C) Analysis of late replication phase of the mutants in HSC-F cells. HSC-F cells (5 × 106) were electroporated with 10 µg of proviral clones indicated (third group in Fig. 1), and the culture supernatants were harvested 72 h later. Virions were pelleted through centrifugation, and lysates were prepared as previously described [42]. Viral amounts in the lysates were determined by EIA of Gag p27, and the level of virus production was calculated by subtraction of that by a negative control (GLDrev-Ns in Fig. 1). Virus production relative to that of wt clone is shown.
(iii) Two-long terminal repeat (LTR) circular product (U5/U3 region): 5’-CTGTTAGGACCCTTCCGCTT-3’ (sense) and 5’-CATAGCCACCCGAAACACAT-3’ (antisense), 94 °C for 3 min, 28 cycles of 94 °C for 45 s, 64 °C for 45 s, and 72 °C for 1 min, and finally 72 °C for 10 min. As a control for PCR, b-globin gene was amplified using the primers GH20 and PC04 [44] under the same conditions, for early reverse transcription product. For DNA standards, pGL-AN (first group in Fig. 1) solutions diluted stepwise were amplified by PCR using early and late primers as described above. 2.7. Proviral DNA constructs An infectious DNA clone of HIV-2, designated pGL-AN, was constructed from pGH123 [45] by replacing a region of tat and env genes with that of an HIV-2ROD clone [13,17]. Four accessory genes of pGH123 are all functional as judged by our mutational analyses [11,13,16,17,45]. All proviral mutant clones used in this report were constructed from pGL-AN, and are shown in Fig. 1. Construction and characterization of some of the clones in Fig. 1, designated pGL-St [13], pGL-Ec [17], pGLnCAT [13], pGLnCAT-St [13], and pGLnCAT-Ns [13], have been described previously. The other mutant clones in Fig. 1 were constructed by combination of recombinant DNA techniques routinely used [13,17,33,45–47]. As for pGLDrev constructs in Fig. 1, the initiation codon for rev was first converted into ACG by
QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA), and the DNA fragment containing the mutation was then inserted into proviral clones. 3. Results 3.1. Replication potentials of GL mutant viruses We recently reported that macaque monkey cell lines susceptible to various SIVs could be established by infection with Herpesvirus saimiri [31,32,48]. One of the cell lines, designated HSC-F, was interleukin-2-independent, CD4- and CXCR4-positive, and highly susceptible to viruses of the HIV-2 group but not to those of the HIV-1 group [49]. No H. saimiri production was observed in HSC-F cells [31]. We were interested in monitoring the mutational effects of HIV-2 vpx and vpr genes on viral replication in this new lymphocytic cell line using proviral mutant clones derived from wt pGL-AN (first group in Fig. 1). Wt GL-AN virus grows exquisitely well in a wide variety of lymphocytic cell lines, PBLs and MDMs of human and simian origins [13,17]. Before doing the infection experiments in HSC-F cells, we confirmed the replication ability of mutant viruses (referred to as D hereafter) in MAGI and M8166 cells. Input sample viruses were prepared by transfection of the proviral clones into 293T cells, and then were inoculated into MAGI and
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M8166 cells. As shown in Fig. 2, the three mutant viruses were similarly infectious for these cells with wt virus. Growth kinetics of the mutant viruses in HSC-F cells were then similarly determined. As clearly seen in Fig. 3A, the replication of DVpr was as efficient as that of wt virus. In contrast, DVpx grew in the cells with delayed kinetics. The replication of DVpx·DVpr was severely impaired.
3.2. Determination of defective replication sites of DVpx and DVpx·DVpr in HSC-F cells The defective replication phase of DVpx and DVpx·DVpr in HSC-F cells was determined by the single-round replication assay [13,46,47] using rev-minus CAT (second group in Fig. 1) and env-minus (third group in Fig. 1) constructs. The early replication phase of DVpx and DVpx·DVpr was examined by measuring CAT activity in cells infected with CATviruses. Input CAT viruses were prepared by co-transfection of a Rev-expression vector prev2 and various CAT constructs into 293T cells, and were inoculated into HSC-F cells. At 48 h post-infection, cells were harvested for CAT assay. As shown in Fig. 3B, CAT activity was determined in a timecourse experiment. It was readily observed that CAT activity in cells infected with DVpx or DVpx·DVpr was much lower than that in cells infected with wt or DVpr. From the slopes of individual reaction curves, an approximately 10-fold reduction in CAT activity occurred when HSC-F cells were infected with DVpx or DVpx·DVpr. These results were reproduced in the two other independent experiments (data not shown). The late replication phase of DVpx and DVpx·DVpr was examined by determining virion production after transfection. HSC-F cells were electroporated with env-minus proviral clones, and 72 h later, amounts of progeny virions were measured by EIA of Gag p27. As shown in Fig. 3C, while DVpx produced a similar level of progeny virions with that of wt clone upon transfection, an approximately twofold reduction in the yield of progenies was observed for DVpr and DVpx·DVpr. We then monitored whether virus DNA is synthesized and imported into the nucleus of cells infected with DVpx or DVpx·DVpr. Input viruses were prepared by co-transfection of a Rev-expression vector, prev2, and rev-minus clones (fourth group in Fig. 1) into 293T cells, and inoculated into HSC-F cells. At 24 h post-infection, cells were harvested for PCR analysis. As shown in Fig. 4, a comparable level of virus DNA was synthesized in cells infected with DVpx, DVpr, DVpx·DVpr, or wt virus. In contrast, no circular form of virus DNA with two LTRs, which is known to be present only in the nucleus of cells infected with retroviruses [50], was detected in cells infected with DVpx or DVpx·DVpr. These data were reproduced in the two other independent experiments (data not shown). Furthermore, similar results were obtained for DNA samples prepared from cells harvested at 12 and 48 h post-infection (data not shown).
Fig. 4. Analysis of the viral reverse transcription and nuclear import processes in HSC-F cells infected with DVpx, DVpr, or DVpx·DVpr. (A) Analysis of viral DNAs by PCR. Input viruses were prepared by co-transfection of a Rev-expression vector, prev2 [41], and the clones indicated at the top (fourth group in Fig. 1) (20 µg for each) into 293T cells. HSC-F cells (1.5 × 107) were infected with 6 × 106 RT units of these viruses, and DNA was extracted from the cells 24 h later. DNA samples (0.5 µg) were subjected to PCR to detect early (R/U5 region) and late (U5/5’-end noncoding region) reverse transcription products, and to detect two-LTR circle in the nucleus. To ascertain an approximate equality of DNA amount in each sample, b-globin gene was amplified by PCR. All products amplified by PCR were resolved on a 3% agarose gel. (B) DNA standards for PCR. Solutions of wt clone pGL-AN (first group in Fig. 1), prepared by stepwise dilutions, were subjected to PCR using primers for early and late reverse transcription products for standards. Numbers at the top represent relative DNA amount. The WT samples here contained the same contents as those in (A).
3.3. Replication of DVpx, DVpr and DVpx·DVpr in human PBLs and MDMs Finally, we determined the growth kinetics of DVpx, DVpr, and DVpx·DVpr in human PBLs and MDMs. PBL and MDM preparations were made independently from several HIV-seronegative persons, and used as targets for virus infection. Input mutant viruses were prepared by transfection of proviral clones (first group in Fig. 1) into 293T cells, and inoculated into the primary PBL and MDM cultures.
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Fig. 5. Growth kinetics in PBLs of DVpx, DVpr, and DVpx·DVpr. PBLs (1 × 106) were infected with equal RT units of cell-free viruses (1 × 104 for A and C–E, and 1 × 105 for B), and virus replication was monitored at intervals by RT production in the culture supernatants. PBLs were separated from five independently prepared PBMC samples. Input viruses were prepared from 293T cells transfected with the clones indicated on the right (first group in Fig. 1). Mock, pUC19.
Fig. 5 shows the viral growth kinetics in PBLs. In the five PBL preparations, essentially the same results were obtained. While DVpr displayed growth kinetics similar to those of wt virus, DVpx and DVpx·DVpr, particularly DVpx·DVpr, exhibited a severe growth defect in good agreement with the results in HSC-F cells (Fig. 3A). Fig. 6 shows the viral growth kinetics in MDMs. Out of the three MDM preparations, two were made and infected with viruses in the absence of GM-CSF, and another was in the presence of the factor. As is clear in Fig. 6, essentially the same results were obtained. In the three MDM preparations, no replication of DVpx and DVpx·DVpr was detected, consistent with previous reports [10,13,18,19,21,22]. In sharp contrast, DVpr grew considerably well in MDMs, and no consistently significant growth defect was observed, in agreement with the results previously reported [12,21]. In summary, DVpx and DVpx·DVpr but not DVpr displayed a severe growth defect in PBLs and MDMs.
4. Discussion The new and key findings in this report are that HIV-2 Vpx augments virus replication in lymphocytic cells by enhancing the nuclear import of viral DNA (Figs. 3 and 4), and that
HIV-2 Vpr is necessary for the efficient virion release from these cells (Fig. 3). Vpx and Vpr contributed to efficient HIV-2 replication to a different degree. In our assays, Vpx and Vpr enhanced virus replication in a single-round by 10and 2-fold, respectively (Fig. 3). In any case, the replication of HIV-2 in lymphocytic cells was greatly impaired when both Vpx and Vpr were absent (Figs. 3 and 5) as reported previously [12]. In vivo studies coincide with this result. While SIVmac without either vpx or vpr induces AIDS in monkeys, without vpx and vpr, it cannot induce the disease [29,30]. It has been well established that HIV-2 Vpx is essential for nuclear localization of viral DNA in infected MDMs [19,22]. Although HIV-2 Vpr has been reported to induce cell cycle arrest [19,51] and apoptosis (our unpublished observation), its direct role in virus replication is presently unknown. We demonstrated here the nuclear transfer of viral DNA by HIV-2 Vpx in infected lymphocytic cells, and no appreciable contribution of HIV-2 Vpr to this process, in contrast to HIV-1 Vpr [52]. Instead, HIV-2 Vpr was found to act at a very late step in the replication cycle in lymphocytic cells. However, no functional role of HIV-2 Vpr in the virus replication in MDMs was found in our assay (Fig. 6).
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Fig. 6. Growth kinetics in MDMs of DVpx, DVpr, and DVpx·DVpr. MDMs in a well of 24-well tissue culture plates were infected with 1 × 106 RT units of cell-free viruses, and virus replication was monitored at intervals by RT production in the culture supernatants. MDMs were separated from three independently prepared PBMC samples. MDMs in (A) and (B) were cultured in the absence of GM-CSF throughout the experiment (from the start of cell culture, virus infection, and to the end of observation period), whereas MDMs in (C) were cultured in the presence of GM-CSF. Input viruses were prepared from 293T cells transfected with the clones indicated on the right (first group in Fig. 1). Replication of T-tropic HIV-1 NL432 virus [11,16,33,37,42,46,47,49] was not detected at all in any cell preparations here (data not shown). Mock, pUC19.
Not only in nondividing MDMs but also in dividing lymphocytic cells, Vpx is required for the optimal nuclear transport of HIV-2 DNA. In contrast, HIV-1 Vpr, the functional counterpart of HIV-2 Vpx, is not necessary at all for the nuclear import in dividing cells of HIV-1 DNA. The molecular basis for this difference, probably related to the structure of viral pre-integration complex, is intriguing but currently unknown. Further study is required to clarify the nuclear transfer process of viral DNAs of HIV-1 and HIV-2. The precise replication process of HIV-2 at which Vpr is required is also unclear at present. On the basis of the results shown in Fig. 3, the step is predicted to be present around the viral assembly/release stage. The molecular event of the Vpr action at this step remains to be elucidated.
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Acknowledgements We thank Tokushima Red Cross Blood Center, Tokushima 770-0044, Japan, for buffy coats of HIV-seronegative blood donors. We are indebted to Kazuko Yoshida for editorial assistance. This work was supported by a Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science and Technologyof Japan (14021078), a Grant-in-Aid for Scientific Research (B) from the Japan Society for the Promotion of Science (14370103), and a Health Sciences Research Grant (Research on HIV/AIDS 13110201) from the Ministry of Health, Labor and Welfare of Japan.
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