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1 Division of Medical Microbiology, Department of Laboratory Medicine, Lund University, Sölvegatan 23, 223 62 Lund, Sweden
2 National Center for Epidemiology, Budapest, Hungary
3 Swedish Institute for Infectious Disease Control, Stockholm, Sweden
Correspondence
Anna Laurén
anna.lauren{at}med.lu.se
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Present address: Yerkes National Primate Research Center of Emory University, Atlanta, GA, USA. 
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Letvin et al., 1985) and the viruses enter cells through interaction of the viral envelope with CD4 and a coreceptor on the cell surface. The two major coreceptors used by HIV-1 are CCR5 and CXCR4 (Deng et al., 1996; Dragic et al., 1996; Feng et al., 1996). Viruses using the CCR5 coreceptor (R5 phenotype) predominate early in asymptomatic HIV-1 infection, whilst CXCR4-using viruses (X4 phenotype) can be isolated in approximately half of patients that progress to AIDS (Björndal et al., 1997; Karlsson et al., 1994). Thus, CXCR4-using HIV-1 isolates are regarded as more pathogenic. CXCR4-using viruses are often characterized by dual tropism (R5X4) or multitropism and may use CCR5, CCR3 and/or CCR2b in combination with CXCR4. CCR5 is also the major coreceptor for SIV (Chen et al., 1997; Edinger et al., 1997; Marcon et al., 1997), whereas CXCR4 use is rare and has been shown with virus isolated and grown on human peripheral blood mononuclear cells (hPBMCs) (Owen et al., 2000; Schols & de Clercq, 1998; Vödrös et al., 2001a). Instead, SIV isolates often use CXCR6 and the orphan receptor gpr15/BOB (Alkhatib et al., 1997; Deng et al., 1997; Farzan et al., 1997). Use of CXCR6 and gpr15 by HIV-1 is much less frequent. Furthermore, both HIV and SIV have been shown to use a wide set of alternative coreceptors, including CCR1, CCR2b, CCR3, CCR8, CX3CR1/V28, gpr1, APJ, ChemR23 and RDC1, but the in vivo role of these coreceptors is unknown (reviewed by Clapham & McKnight, 2002).
One important role for virus coreceptor usage could be at transmission, in determining the first cell to become infected. We know that R5 HIV-1 replicates preferentially in the asymptomatic phase of infection. Moreover, primary intestinal epithelial cells seem to transfer R5 HIV-1 variants selectively to CCR5+ cells (Meng et al., 2002). In addition, vaginal infection of rhesus macaques with a mixture of chimeric simian/human immunodeficiency viruses having either an X4 or an R5 HIV-1 envelope results in selective transmission of viruses containing the R5 envelope gene (Lu et al., 1996). Both intravaginal and intravenous infections of macaques with SIV conceivably lead to a major change in the intestinal lymphoid tissue. The CD4+ T cell in the gut and other tissues throughout the body is the primary target for virus replication and acute infection is accompanied by an extensive loss of CD4+ T cells within the first 2 weeks of infection (Hirsch et al., 1998; Li et al., 2005; Mattapallil et al., 2005; Stahl-Hennig et al., 1999; Veazey et al., 1998; Zhang et al., 1999). The initial steps of infection could conceivably be different depending on the route of transmission and this in turn could influence pathogenesis. Indeed, the influence of the route of transmission on pathogenic outcome has been widely discussed. Comparisons of disease progression in HIV-1 infection between injecting drug users and homosexual men have shown that the latter had a significantly accelerated progression rate (Eskild et al., 1997; Pehrson et al., 1997). On the other hand, Prins & Veugelers (1997) found little evidence for different disease progression between injecting drug users and homosexual men. Similarly, Hengge et al. (2003) found that the course of HIV disease does not depend on the mode of transmission. When looking at HIV-1 biological phenotype, however, Spijkerman et al. (1995) found a lower prevalence and incidence of the syncytium-inducing phenotype (now classified as CXCR4-using virus) among injecting drug users compared with homosexual men.
This prompted us to examine the possible influence of the route of infection on pathogenesis in a model system, using the same SIVsm inoculum virus (originating from a sooty mangabey), administered either intrarectally (IR) or intravenously (IV) to 30 cynomolgus macaques. The animals were monitored regularly for up to 5 years after infection, depending on the rate of disease progression. Sequential isolates were obtained on both hPBMCs and cynomolgus macaque PBMCs (mPBMCs) and tested for coreceptor use in the GHOST(3) and U87.CD4 cell lines, expressing CD4 and one coreceptor. We have shown here that particular coreceptor-usage patterns varied with the pathogenic outcome of SIV infection, but not according to the route of transmission. In addition, in a subgroup of monkeys, emergence of neutralization resistance was followed using both autologous and heterologous sera [see accompanying paper by Laurén et al. (2006) in this issue)].
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The virus stock used for infection was propagated in vitro in cultures of mPBMCs (Quesada-Rolander et al., 1996). Cell-free virus stocks (10 MID50) were used for infection, which meant that animals infected by the IR route received an approximately 103-fold higher virus dose than the IV-inoculated animals. The monkeys were monitored for general clinical status. Blood samples for virus isolation and for viral load and CD4+ T-cell count determinations were collected at regular intervals. Animals were kept until development of AIDS or, if asymptomatic, until the end of the study period, when they were euthanized (Table 1). In the present study, two to four isolates were tested from each monkey, depending on virus isolation frequency and the length of the monkey's survival time. The first isolate was usually obtained as early as 2 weeks post-infection (p.i.); the second isolate was obtained by 3 or 4 months; the third isolate was chosen at a time in between the second and the last isolate; and the last (fourth) isolate was collected at the time of euthanization (Table 2).
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). Reisolates were passaged no more than twice. Cell-free supernatants were screened for the amount of reverse transcriptase (RT) with a Cavidi HS kit Lenti RT (Cavidi Tech) and stored at –80 °C until use. SIV RNA levels in plasma were measured by using a highly sensitive quantitative competitive RT-PCR assay with a lower detection limit of 100 RNA equivalents (ml plasma)–1, as described in detail elsewhere (Ten Haaft et al., 1998). Animals were monitored for changes in their CD4+ cell counts by using two-colour flow-cytometric analysis as reported previously (Mäkitalo et al., 2000). CD4+ T-cell values were evaluated as the percentage of the total T-cell count. When observing the rate of change of CD4+ lymphocytes, values obtained before infection were set as 100 % for each animal and following values were calculated in relation to these set-point values. The rate of change as a percentage of the CD4+ lymphocyte population was fitted by linear-regression analysis.
HIV-2 strain 1010, known to use all of the coreceptors evaluated in this study (Mörner et al., 1999), was used as a positive control in tests for coreceptor usage.
Determination of coreceptor usage.
The GHOST(3) cell lines stably expressed the CD4 receptor and one of the chemokine receptors CCR3, CCR5, CXCR4, CXCR6 or the orphan receptor gpr15 (Mörner et al., 1999). As a control, the parental cell line had been engineered to express the CD4 receptor alone and none of the coreceptors. Notably, the clone 3 cell line used in the experiments expressed a low but detectable level of endogenous CXCR4. GHOST(3) cells are stably transfected with the green fluorescent protein (GFP) gene driven by the HIV-2ROD long terminal repeat. Upon infection, the viral Tat protein activates GFP expression. The GHOST(3) cell lines were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 7·5 % FBS and antibiotics (penicillin and streptomycin) in 25 cm2 cell-culture flasks.
U87.CD4 cell lines expressing no coreceptor (parental) or expressing CCR3, CXCR4 or CCR5 (Deng et al., 1997) were used in one experiment. U87.CD4 cells were maintained in DMEM supplemented with 10 % FBS and antibiotics (penicillin and streptomycin) in 25 cm2 cell-culture flasks. Cultures were incubated in a humidified atmosphere with 5 % CO2 at 37 °C.
Infection of GHOST(3) and U87.CD4 cells was carried out as described previously (Björndal et al., 1997; Vödrös et al., 2001b). Briefly, 1 day before infection, cells were seeded in 24- or 48-well plates. Before infection, medium was replaced with 200 µl fresh medium containing 2 µg polybrene ml–1 and 300 or 150 µl undiluted virus was added to two identical wells of 24- or 48-well plates, respectively. Cultures were incubated overnight, washed with PBS and further incubated with fresh medium. Three days after infection, the GHOST(3) cultures were observed with light and fluorescence microscopes and cells from one of the duplicate wells were treated with EDTA and harvested. The detached cells were fixed with paraformaldehyde at a final concentration of 2 %. Fixed cells were kept at 4 °C for at least 2 h before analysis by flow cytometry. Infected U87.CD4 cultures were screened for syncytia by using light microscopy at days 3 and 6 after infection. Culture supernatant from infected cultures was collected at day 6 from both U87.CD4 and GHOST(3) cells for viral-antigen detection. An in-house HIV-2/SIV capture ELISA was used to detect viral antigens produced by the infected cells (Thorstensson et al., 1991).
The infected GHOST(3) cells were analysed with a flow cytometer (FACScan; Becton Dickinson). The results from the flow-cytometric analysis gave us a ‘ratio to cell negative’ (RTCN) value, a quantitative measure of the coreceptor usage by the tested virus (Vödrös et al., 2001b). Briefly, RTCN=(FIx%)virus inf./(FIx%) neg. control, where % is the proportion of fluorescence-positive cells and FI is the mean fluorescence intensity of the fluorescence-positive cells. Negative controls were uninfected cultures from the corresponding coreceptor-expressing cell line. An RTCN value of >10 was considered positive, indicating use of that particular coreceptor; an RTCN value of <5 was considered negative; values of between 5 and 10 remained indeterminate.
In a set of experiments, the CXCR4 antagonist AMD3100 was used to block CXCR4 on the GHOST(3) cells. AMD3100 was added to cells 5 min prior to infection in 200 µl culture medium at a concentration of 1 µg ml–1.
Statistics.
The different groups of monkeys were compared by using the Mann–Whitney non-parametric test, calculated by using SPSS statistical software.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, Fig. 1). The three groups were named progressor (P), slow progressor (SP) and long-term non-progressor (LTNP). There was no difference in either the CD4 decline or the viral load when comparing monkeys inoculated by different routes of infection. As expected, CD4 decline was more pronounced in the first 3 months of infection, whilst the decline of CD4+ T cells was less thereafter. There was one exception to this general rule: monkey 56-3 did not seem to lose any CD4 cells for the first 3 months after infection, although CD4 counts declined thereafter. For the majority of animals, the pattern of viral load was consistent with the observations by Ten Haaft et al. (1998), in that a threshold plasma virus load that was greater than 105 RNA equivalents (ml plasma)–1 at 6–12 weeks after inoculation could predict a faster disease progression. A threshold virus load value that remained below 104 RNA equivalents (ml plasma)–1 was indicative of a non-pathogenic course of infection. Even though there was no difference in viral load according to route of infection, the isolates from IV-infected monkeys replicated to higher titres on hPBMCs than isolates from IR-infected monkeys (P=0·006, Mann–Whitney test). This was also reflected by the fact that virus isolations were more frequently successful in the animals infected by the IV route.
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). Median CD4 decline in the P group was –3·34 % CD4+ T cells per month (range, –8·05 to –0·69 % CD4+ T cells per month). Due to early disease symptoms, three of the monkeys (C87, D23 and D24) were euthanized 1 year after infection. Others developed symptoms of disease later, but all were euthanized by 27 months p.i. and the median time to AIDS in this group was 18 months. Virus isolation frequency was high, with values above 82 % (median, 91 %). Interestingly, all of the IV-infected monkeys had a virus isolation frequency of 100 %, even though their viral load was not exceptionally high. None of the IR-infected monkeys had a virus isolation frequency of 100 % and one monkey (C39) had an unusually low virus isolation frequency (58 %). Monkey C39 was also exceptional for having a different pattern of plasma viral load (Fig. 1). In most of the P monkeys, plasma viral load was initially high [>106 RNA copies (ml plasma)–1] and stayed high, whereas in monkey C39, viral load declined below 103 copies (ml plasma)–1 within 3 months and then increased slowly again to values of >104 copies (ml plasma)–1. Monkeys C20, C27 and C44 with the slowest CD4 decline in the P group also showed a drop in viral load pattern, but it increased again within 3 months; in the case of C20 and C44, viral load was above 104, whereas for monkey C27, the level was somewhat lower. Monkey C27 had to be euthanized at 18 months after infection because of extensive diarrhoea and weight loss and was therefore considered a P monkey.
Slow progressors (SP).
Median CD4 cell decline in the SP group was –0·98 % CD4+ T cells per month (range, –1·92 to –0·88 % cells per month) (Table 1). Four out of five monkeys in this group did not show disease symptoms during the study period (median 43 months), whilst one monkey developed lymphoma 47 months p.i. The other monkeys, although without signs of disease, showed a higher rate of CD4 cell decline (Table 1) and had a higher virus isolation frequency (median 75 %) than the monkeys in the LTNP group (median 31 %, Table 1). In the two SP monkeys (C54, 56-3) in which viral load was measured, initial viraemia declined sharply and remained low for many months (Fig. 1c). It should be remembered that monkey 56-3 was the only monkey with a stable CD4 count for the first 3 months after infection (Table 1). The SP group also fell between the P and LTNP groups with regard to the viral load parameter.
Long-term non-progressors.
With one exception, the monkeys in the LTNP group did not show any symptoms of disease over the observation period of 34–60 months. Monkey C1 showed lymphopenia and weight loss by the time of autopsy at 60 months p.i. (Table 1). Whilst LTNP monkeys also showed a substantial CD4 decline for the initial 3–4 months after infection, the overall decline in the percentage of CD4+ T cells was not particularly distinct (median CD4 decline of –0·26 % CD4+ T cells per month). Overall, the relative number of CD4+ cells in this group remained close to the CD4 values before infection for a long period of time. Late in infection, the CD4 values decreased to slightly below the CD4 values before infection. Virus isolation from LTNPs was unsuccessful at many time points and varied between 10 and 72 % for individual monkeys. The viral load of the LTNP group decreased after the early peak viraemia to values of between 103 and 104 RNA copies (ml plasma)–1 and remained stable over the entire study period (Fig. 1c).
Coreceptor use of sequential SIVsm reisolates
Isolation on hPBMCs.
Coreceptor usage of 105 isolates from 30 monkeys was tested on GHOST(3) cells (Table 2). All isolates, with one exception, infected cells via CCR5, and in general CCR5 was the most efficiently used coreceptor. CCR5 was defined as the main coreceptor used in most cases (70 out of 105 isolates), based on RTCN values at least twice as high as with other coreceptors. In another 20 cases, CCR5 was used as efficiently as CXCR6 or gpr15. The reisolates could infect CXCR6- or gpr15-expressing cells in 60 and 55 % of the cases, respectively, and 46 % of the isolates used all three receptors. The 12 month reisolate from monkey D24, which did not use CCR5 at all, used CXCR4 efficiently as the major coreceptor (RTCN ranged from 107 to 244 and was tested in three independent experiments). In addition, ten isolates derived from three P monkeys (five isolates), one SP (four isolates) and one LTNP (one isolate) used CXCR6 (six isolates), CXCR6 in combination with CCR3 or gpr15 (two isolates) or used the CXCR4 (two isolates) receptor more efficiently than CCR5 (Table 2).
The effect of virus quantity on infection was analysed in two different ways. We performed dilution experiments with three different viruses and found that, within a 16-fold dilution range, the most efficiently used coreceptors could not be diluted out (Fig. 2). We also plotted the virus dose used for infection of GHOST(3) cell lines, expressed as concentration of RT (pg ml–1) against the number of coreceptors used by a particular isolate (Fig. 3). Whilst the majority of isolates had RT activity corresponding to the 3 log10 pg RT ml–1 range (69 %), there were a few isolates containing <3 log10 pg RT (21 % had 2 log10 pg RT ml–1 and 7 % had 1 log10 pg RT ml–1). Such comparison was important, as some of the animals did not yield more virus than 2 log10 pg RT ml–1 in the virus stocks from PBMCs. Interestingly, all three monkeys diagnosed with lymphoma (C38, C39 and C54) and two out of seven LTNP monkeys (C82, C93) yielded low-titre virus. Characteristically, such viruses used CCR5 as the major, and most often the only, coreceptor.
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). In LTNP monkeys, coreceptor usage narrowed or stable CCR5 usage (R5 virus) dominated in six out of seven cases. The same pattern was seen in three out of five SP animals. A minority of P monkeys showed the narrowing pattern (7/18 cases), whereas the remaining animals showed broadening of coreceptor use (3/18), stable coreceptor use of several coreceptors (1/18) or fluctuation between the different coreceptor-usage patterns (7/18). We also compared monkeys infected by the mucosal or IV route of infection with regard to the number of coreceptors used. There was no difference in how many coreceptors were used according to route of infection (Mann–Whitney test).
Isolation on mPBMCs.
Wherever cells were available from the infected monkeys, viruses were reisolated on mPBMCs. A total of 11 isolations was carried out. Eight of these were from samples exactly matching the time of isolations on hPBMCs, whilst the remaining three samples were 2 or 4 months apart (with some samples from monkeys C73 and D24). Similar to isolates obtained on hPBMCs, the major coreceptor used was CCR5 (Table 3). CXCR6, less frequently CCR3 and in one case gpr15 were used as additional coreceptors. The majority of isolates induced GFP in parental, CCR3- and CXCR4-expressing cells to a similar extent. Therefore, first we inhibited the CXCR4 background on the GHOST(3) cells with AMD3100. Interestingly, AMD3100 could not inhibit GFP induction in these cases, suggesting that the cells might have expressed an unidentified receptor that could serve as an entry factor for many of the mPBMC isolates. To rule out the possible effect of the unidentified receptor on the results, we tested coreceptor usage of isolates on another indicator cell line, U87.CD4, expressing CCR3, CCR5, CXCR4 or no coreceptor (parental cells). When a low level of syncytium induction and viral-antigen production in U87.CD4 parental cells was observed, it was taken as background and compared with cells engineered to express coreceptors. In this way, we confirmed CCR3 use, but excluded CXCR4 use by the mPBMC isolates.
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). Both sets of isolates used CCR5 and CXCR6 regularly, whereas the use of CCR3 was more variable in both cases. Furthermore, only one mPBMC isolate included in the present comparison was able to use gpr15, whereas the hPBMC counterparts could use this coreceptor. In this comparison of exact matches of isolates, three of the hPBMC isolates could infect cells using CXCR4, whereas none of the viruses isolated on mPBMCs could use the CXCR4 coreceptor. Conversely, isolates derived from mPBMCs were able to use an additional, unidentified coreceptor on GHOST(3) cells more often than isolates from hPBMCs.
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). Whilst the first pattern has been encountered in an earlier work (Vödrös et al., 2001a, 2003), the late appearance of CXCR4 use, reminiscent of HIV-1 infection, has not been demonstrated in SIV infection. In this study, CXCR4-using virus could be isolated on hPBMCs from four animals with progressive disease (C38, C86, C87 and D24). Interestingly, all four animals were infected by the IR route. Sequential isolates from one macaque (C73) showed fluctuation in CXCR4 use: CXCR4-using virus was detected 2 weeks after infection and disappeared later on, but was isolated again at the time of immunodeficiency. There were monkeys in all three groups from which virus with CXCR4 usage could be isolated shortly after infection. Remarkably, three out of seven animals in the LTNP group harboured CXCR4-using virus initially. In all three cases, CXCR4 usage disappeared and we observed a general narrowing of coreceptor usage over time.
With regard to the endogenously expressed CXCR4 on GHOST(3) cells, it was important to confirm that isolates indeed used CXCR4 and that CXCR4 use of multitropic isolates did not interfere with the use and detection of other receptors. Therefore, we carried out a set of experiments in which we blocked CXCR4 on the cells by adding a CXCR4 antagonist, AMD3100, to the cultures prior to infection. CXCR4 use of early reisolates was inhibited by AMD3100 (86–97 % inhibition) in four out of five macaques tested (Table 4). In one monkey, the CXCR4 usage of the early isolate was inhibited by AMD3100 only to 54 %. The emerging CXCR4 use by late reisolates could also be verified and, in addition, the inhibition of CXCR4 on the cells did not interfere with the alternative coreceptor (CXCR6 and gpr15)-usage pattern. In line with our previous observations (Vödrös et al., 2001a, 2003), in some cases, we could not achieve complete inhibition of CXCR4 use by AMD3100 (B173, B174, C68; Table 4). This indicated that some of the viruses, similar to the viruses isolated on mPBMCs, may use an additional coreceptor also present on GHOST(3) cells or that the SIV envelope interaction with CXCR4 is different from the interaction between envelopes of HIV-1 and CXCR4, as suggested by Owen et al. (2000).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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CCR5 has been shown to be the major coreceptor for SIV and is essential for cell entry in combination with CD4 (Chen et al., 1997; Edinger et al., 1997; Marcon et al., 1997). In addition, CXCR6 and gpr15 have been shown to be common coreceptors for SIV (Alkhatib et al., 1997; Deng et al., 1997; Farzan et al., 1997). Our results are in agreement with previous studies on sequential SIV isolates where CCR5 has been shown to be an important coreceptor for both early and late SIVsm and SIVmne variants (Kimata et al., 1999a; Vödrös et al., 2001a). However, impaired ability to use gpr15 by SIVmac had no effect on infection of rhesus macaques, indicating that gpr15 plays a minor role in pathogenicity in vivo (Pöhlmann et al., 1999). Likewise, we found only one gpr15-using virus out of 11 when isolated on mPMBCs. CXCR4 use for SIV has been a rare phenomenon and in at least two studies was demonstrated with virus isolated and/or passaged on hPBMCs (Owen et al., 2000; Schols & de Clercq, 1998; Vödrös et al., 2001a).
The fast selection of CXCR4-using virus within one virus isolation and a maximum of two passages on hPBMCs is highly interesting. SIV adaptation to human cells has been described previously and was associated with a shorter form of gp41 (gp31) (Hirsch et al., 1989). In our case, SIV might use a coreceptor that is advantageous during growth on hPBMCs and, because of this, may acquire CXCR4 use during isolation on hPBMCs. This may not be surprising, as we know that CXCR4 is expressed at higher levels than CCR5 in hPBMC cultures (Bleul et al., 1997). However, there is no evidence that the in vivo distribution of CCR3, CCR5 and CXCR4 differs between humans and macaques (Zhang et al., 1998). Comparison of 11 rhesus macaque chemokine receptors with their human counterparts showed close similarities of 98·9 % (CCR5) and 99·4 % (CXCR4) (Margulies et al., 2001). The similarity of CXCR6 was somewhat lower, 96·8 %, but, in our hands, SIVsm envelopes fused with human CCR5, (CXCR6) and gpr15 as efficiently as with rhesus counterparts (Vödrös et al., 2003).
A considerable number of our isolates, from both hPBMCs (eight isolates) and mPBMCs (six isolates), could also infect the GHOST(3) parental cell line, even in the presence of the CXCR4 antagonist AMD3100. This is in line with our previous observations that complete inhibition of CXCR4 usage by SIVsm could not be achieved by AMD3100, indicating that these viruses may use an additional coreceptor also present on GHOST(3) cells (Vödrös et al., 2001a). Forte et al. (2003) have also suggested the possibility that SIVmne variants that are able to infect PBMCs lacking CCR5 use an unidentified coreceptor. Another possibility could be that the SIV envelope interaction with CXCR4 is different from the interaction of the HIV-1 envelope and CXCR4, as suggested by Owen et al. (2000).
It has been shown previously that different HIV-1 and SIV isolates can interact with receptors in different ways (Antonsson et al., 2003; Edinger et al., 1997; Karlsson et al., 2003). Macrophage-tropic SIV has been shown to depend on both the N-terminal and the second extracellular loop (ECL-2) of CCR5 for cell entry, whilst T cell-tropic SIV isolates require only CCR5 ECL-2 (Edinger et al., 1997). A general comparison of SIVsm and HIV-1 may be difficult. However, it has been suggested that ECL-2 is important for CXCR4-using HIV-1, whilst viruses of the R5 phenotype vary in their requirement for binding to the N terminus of CCR5 (Brelot et al., 1997; Doranz et al., 1999; Karlsson et al., 2004). By using receptor chimeras between CCR5 and CXCR4, Karlsson et al. (2004) showed that the R5 phenotype of HIV-1 undergoes evolution over time and changes the mode of CCR5 usage. Also, it has been shown that CXCR4-using HIV-1 seems to undergo evolution in vivo and shows decreasing sensitivity to CXCR4 antagonists (Stalmeijer et al., 2004), suggesting that a change in the binding capacity to ECL-2 occurs. In support of this, it is known that the CXCR4 antagonist AMD3100 binds to ECL-2 (Gerlach et al., 2001; Hatse et al., 2001; Labrosse et al., 1998). Conceivably, SIV may also change in the infected macaques over time and, as a result, late virus isolates may differ from early isolates and inoculum virus in the mode of receptor usage. Such a change may lead to altered tropism for different cell types. Indeed, late SIV isolates are often more cytopathic than early isolates, further supporting the possibility that SIV also changes throughout infection (Kimata et al., 1999a, b; Rudensey et al., 1995).
Although we could not isolate CXCR4-using viruses on mPBMCs, a significant number of SIVsm viruses isolated on hPBMCs used CXCR4 efficiently in this study. On the one hand, we were able to isolate CXCR4-using virus on hPBMCs from seven macaques 2 weeks after infection, but not beyond 3 months, confirming our previous findings (Vödrös et al., 2001a). On the other hand, the finding that virus with CXCR4 usage could be isolated from four macaques late in disease, at the time of immunodeficiency, was new and unexpected. In one P animal (monkey C73), CXCR4-using virus was isolated both early and late in infection, but not in between. The last isolate of monkey D24 had a unique phenotype, X4X6, using CXCR4 as the major coreceptor and not using CCR5 at all (Table 2). Viruses from the remaining three monkeys showed evolution to CXCR4 use, although use of CCR5 and CXCR6 was more efficient than CXCR4 use. When large populations of HIV-1 subtype B-infected individuals are considered, the appearance of CXCR4 usage has been estimated to involve 50 % of AIDS cases (Björndal et al., 1997; Karlsson et al., 1994). Large variations between groups and also according to subtype have been reported. For example, in HIV-1 subtype C infections, the X4 phenotype occurred less frequently (17 % of the AIDS cases; Cilliers et al., 2003). In our SIVsm material, overall 5/18 monkeys (28 %) in the P group yielded CXCR4-using viruses.
A further similarity between HIV-1 and SIVsm can be found by considering the overall pattern of coreceptor usage in relation to the pathogenic process. The majority of P monkeys yielded virus with a broadening of coreceptor usage, stable use of several coreceptors or fluctuation between different coreceptor-usage patterns over time. In LTNP monkeys, coreceptor usage of isolates narrowed from multitropic to CCR5 use only, or, when CCR5 was the only coreceptor used, this remained stable over the entire observation period. We have previously evaluated the coreceptor usage of sequential isolates from six SIVsm-infected cynomolgus macaques with progressive disease and found a narrowing pattern of coreceptor usage, in that late isolates obtained at the time of immunodeficiency often could not establish a productive infection in gpr15- or CXCR6-expressing cells (Vödrös et al., 2001a, 2003). In the present study, we found a similar pattern in seven P monkeys. However, if considering the large number of animals (18 P monkeys) used in the present study, the majority of monkeys with progressive disease could use multiple coreceptors effectively. Taken together, our results indicate that SIV may evolve in the infected host, resulting in changes in the mode of coreceptor usage to include the use of CXCR4 or a similar receptor.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Antonsson, L., Boketoft, A., Garzino-Demo, A., Olde, B. & Owman, C. (2003). Molecular mapping of epitopes for interaction of HIV-1 as well as natural ligands with the chemokine receptors, CCR5 and CXCR4. AIDS 17, 2571–2579.[CrossRef][Medline]
Björndal, Å., Deng, H., Jansson, M. & 7 other authors (1997). Coreceptor usage of primary human immunodeficiency virus type 1 isolates varies according to biological phenotype. J Virol 71, 7478–7487.[Abstract]
Bleul, C. C., Wu, L., Hoxie, J. A., Springer, T. A. & Mackay, C. R. (1997). The HIV coreceptors CXCR4 and CCR5 are differentially expressed and regulated on human T lymphocytes. Proc Natl Acad Sci U S A 94, 1925–1930.
Brelot, A., Heveker, N., Pleskoff, O., Sol, N. & Alizon, M. (1997). Role of the first and third extracellular domains of CXCR-4 in human immunodeficiency virus coreceptor activity. J Virol 71, 4744–4751.[Abstract]
Chen, Z., Zhou, P., Ho, D. D., Landau, N. R. & Marx, P. A. (1997). Genetically divergent strains of simian immunodeficiency virus use CCR5 as a coreceptor for entry. J Virol 71, 2705–2714.[Abstract]
Cilliers, T., Nhlapo, J., Coetzer, M., Orlovic, D., Ketas, T., Olson, W. C., Moore, J. P., Trkola, A. & Morris, L. (2003). The CCR5 and CXCR4 coreceptors are both used by human immunodeficiency virus type 1 primary isolates from subtype C. J Virol 77, 4449–4456.
Clapham, P. R. & McKnight, Á. (2002). Cell surface receptors, virus entry and tropism of primate lentiviruses. J Gen Virol 83, 1809–1829.
Deng, H., Liu, R., Ellmeier, W. & 12 other authors (1996). Identification of a major co-receptor for primary isolates of HIV-1. Nature 381, 661–666.[CrossRef][Medline]
Deng, H. K., Unutmaz, D., KewalRamani, V. N. & Littman, D. R. (1997). Expression cloning of new receptors used by simian and human immunodeficiency viruses. Nature 388, 296–300.[CrossRef][Medline]
Doranz, B. J., Orsini, M. J., Turner, J. D., Hoffman, T. L., Berson, J. F., Hoxie, J. A., Peiper, S. C., Brass, L. F. & Doms, R. W. (1999). Identification of CXCR4 domains that support coreceptor and chemokine receptor functions. J Virol 73, 2752–2761.
Dragic, T., Litwin, V., Allaway, G. P. & 8 other authors (1996). HIV-1 entry into CD4+ cells is mediated by the chemokine receptor CC-CKR-5. Nature 381, 667–673.[CrossRef][Medline]
Edinger, A. L., Amedee, A., Miller, K. & 11 other authors (1997). Differential utilization of CCR5 by macrophage and T cell tropic simian immunodeficiency virus strains. Proc Natl Acad Sci U S A 94, 4005–4010.
Eskild, A., Magnus, P., Brekke, T. & 7 other authors (1997). The impact of exposure group on the progression rate to acquired immunodeficiency syndrome. A comparison between intravenous drug users, homosexual men and heterosexually infected subjects. Scand J Infect Dis 29, 103–109.[Medline]
Farzan, M., Choe, H., Martin, K. & 10 other authors (1997). Two orphan seven-transmembrane segment receptors which are expressed in CD4-positive cells support simian immunodeficiency virus infection. J Exp Med 186, 405–411.
Feng, Y., Broder, C. C., Kennedy, P. E. & Berger, E. A. (1996). HIV-1 entry cofactor: functional cDNA cloning of a seven-transmembrane, G protein-coupled receptor. Science 272, 872–877.[Abstract]
Forte, S., Harmon, M.-E., Pineda, M. J. & Overbaugh, J. (2003). Early- and intermediate-stage variants of simian immunodeficiency virus replicate efficiently in cells lacking CCR5. J Virol 77, 9723–9727.
Fultz, P. N., McClure, H. M., Anderson, D. C., Swenson, R. B., Anand, R. & Srinivasan, A. (1986). Isolation of a T-lymphotropic retrovirus from naturally infected sooty mangabey monkeys (Cercocebus atys). Proc Natl Acad Sci U S A 83, 5286–5290.
Gerlach, L. O., Skerlj, R. T., Bridger, G. J. & Schwartz, T. W. (2001). Molecular interactions of cyclam and bicyclam non-peptide antagonists with the CXCR4 chemokine receptor. J Biol Chem 276, 14153–14160.
Hatse, S., Princen, K., Gerlach, L.-O., Bridger, G., Henson, G., De Clercq, E., Schwartz, T. W. & Schols, D. (2001). Mutation of Asp171 and Asp262 of the chemokine receptor CXCR4 impairs its coreceptor function for human immunodeficiency virus-1 entry and abrogates the antagonistic activity of AMD3100. Mol Pharmacol 60, 164–173.
Hengge, U. R., Franz, B., Hoersch, S. & Goos, M. (2003). Course of HIV disease does not depend on risk group: 7.5-year follow-up in 296 patients. Int J STD AIDS 14, 451–457.
Hirsch, V. M., Edmondson, P., Murphey-Corb, M., Arbeille, B., Johnson, P. R. & Mullins, J. I. (1989). SIV adaptation to human cells. Nature 341, 573–574.[CrossRef][Medline]
Hirsch, V. M., Sharkey, M. E., Brown, C. R. & 8 other authors (1998). Vpx is required for dissemination and pathogenesis of SIVSM PBj: evidence of macrophage-dependent viral amplification. Nat Med 4, 1401–1408.[CrossRef][Medline]
Karlsson, A., Parsmyr, K., Sandström, E., Fenyö, E. M. & Albert, J. (1994). MT-2 cell tropism as prognostic marker for disease progression in human immunodeficiency virus type 1 infection. J Clin Microbiol 32, 364–370.
Karlsson, I., Antonsson, L., Shi, Y., Karlsson, A., Albert, J., Leitner, T., Olde, B., Owman, C. & Fenyö, E. M. (2003). HIV biological variability unveiled: frequent isolations and chimeric receptors reveal unprecedented variation of coreceptor use. AIDS 17, 2561–2569.[CrossRef][Medline]
Karlsson, I., Antonsson, L., Shi, Y. & 7 other authors (2004). Coevolution of RANTES sensitivity and mode of CCR5 receptor use by human immunodeficiency virus type 1 of the R5 phenotype. J Virol 78, 11807–11815.
Kestler, H., Kodama, T., Ringler, D. & 8 other authors (1990). Induction of AIDS in rhesus monkeys by molecularly cloned simian immunodeficiency virus. Science 248, 1109–1112.
Kimata, J. T., Gosink, J. J., KewalRamani, V. N., Rudensey, L. M., Littman, D. R. & Overbaugh, J. (1999a). Coreceptor specificity of temporal variants of simian immunodeficiency virus Mne. J Virol 73, 1655–1660.
Kimata, J. T., Kuller, L., Anderson, D. B., Dailey, P. & Overbaugh, J. (1999b). Emerging cytopathic and antigenic simian immunodeficiency virus variants influence AIDS progression. Nat Med 5, 535–541.[CrossRef][Medline]
Labrosse, B., Brelot, A., Heveker, N., Sol, N., Schols, D., De Clercq, E. & Alizon, M. (1998). Determinants for sensitivity of human immunodeficiency virus coreceptor CXCR4 to the bicyclam AMD3100. J Virol 72, 6381–6388.
Laurén, A., Thorstensson, R. & Fenyö, E. M. (2006). Comparative studies on mucosal and intravenous transmission of simian immunodeficiency virus (SIVsm): the kinetics of evolution to neutralization resistance are related to progression rate of disease. J Gen Virol 87, 595–606.
Letvin, N. L., Daniel, M. D., Sehgal, P. K. & 7 other authors (1985). Induction of AIDS-like disease in macaque monkeys with T-cell tropic retrovirus STLV-III. Science 230, 71–73.
Li, Q., Duan, L., Estes, J. D. & 7 other authors (2005). Peak SIV replication in resting memory CD4+ T cells depletes gut lamina propria CD4+ T cells. Nature 434, 1148–1152.[Medline]
Lu, Y., Brosio, P., Lafaile, M., Li, J., Collman, R. G., Sodroski, J. & Miller, C. J. (1996). Vaginal transmission of chimeric simian/human immunodeficiency viruses in rhesus macaques. J Virol 70, 3045–3050.[Abstract]
Mäkitalo, B., Böttiger, P., Biberfeld, G. & Thorstensson, R. (2000). Cell-mediated immunity to low doses of SIVsm in cynomolgus macaques did not confer protection against mucosal rechallenge. Vaccine 19, 298–307.[CrossRef][Medline]
Marcon, L., Choe, H., Martin, K. A. & 7 other authors (1997). Utilization of C-C chemokine receptor 5 by the envelope glycoproteins of a pathogenic simian immunodeficiency virus, SIVmac239. J Virol 71, 2522–2527.[Abstract]
Margulies, B. J., Hauer, D. A. & Clements, J. E. (2001). Identification and comparison of eleven rhesus macaque chemokine receptors. AIDS Res Hum Retroviruses 17, 981–986.[CrossRef][Medline]
Mattapallil, J. J., Douek, D. C., Hill, B., Nishimura, Y., Martin, M. & Roederer, M. (2005). Massive infection and loss of memory CD4+ T cells in multiple tissues during acute SIV infection. Nature 434, 1093–1097.[CrossRef][Medline]
Meng, G., Wei, X., Wu, X. & 9 other authors (2002). Primary intestinal epithelial cells selectively transfer R5 HIV-1 to CCR5+ cells. Nat Med 8, 150–156.[CrossRef][Medline]
Mörner, A., Björndal, Å., Albert, J., KewalRamani, V. N., Littman, D. R., Inoue, R., Thorstensson, R., Fenyö, E. M. & Björling, E. (1999). Primary human immunodeficiency virus type 2 (HIV-2) isolates, like HIV-1 isolates, frequently use CCR5 but show promiscuity in coreceptor usage. J Virol 73, 2343–2349.
Nilsson, C., Thorstensson, R., Gilljam, G. & 7 other authors (1995). Protection against monkey-cell grown cell-free HIV-2 challenge in macaques immunized with native HIV-2 envelope glycoprotein gp125. Vaccine Res 4, 165–175.
Owen, S. M., Masciotra, S., Novembre, F., Yee, J., Switzer, W. M., Ostyula, M. & Lal, R. B. (2000). Simian immunodeficiency viruses of diverse origin can use CXCR4 as a coreceptor for entry into human cells. J Virol 74, 5702–5708.
Pehrson, P., Lindback, S., Lidman, C., Gaines, H. & Giesecke, J. (1997). Longer survival after HIV infection for injecting drug users than for homosexual men: implications for immunology. AIDS 11, 1007–1012.[CrossRef][Medline]
Pöhlmann, S., Stolte, N., Münch, J., Ten Haaft, P., Heeney, J. L., Stahl-Hennig, C. & Kirchhoff, F. (1999). Co-receptor usage of BOB/GPR15 in addition to CCR5 has no significant effect on replication of simian immunodeficiency virus in vivo. J Infect Dis 180, 1494–1502.[CrossRef][Medline]
Prins, M. & Veugelers, P. J. (1997). Comparison of progression and non-progression in injecting drug users and homosexual men with documented dates of HIV-1 seroconversion. European Seroconverter Study and the Tricontinental Seroconverter Study. AIDS 11, 621–631.[CrossRef][Medline]
Quesada-Rolander, M., Mäkitalo, B., Thorstensson, R., Zhang, Y.-J., Castaños-Velez, E., Biberfeld, G. & Putkonen, P. (1996). Protection against mucosal SIVsm challenge in macaques infected with a chimeric SIV that expresses HIV type 1 envelope. AIDS Res Hum Retroviruses 12, 993–999.[Medline]
Rudensey, L. M., Kimata, J. T., Benveniste, R. E. & Overbaugh, J. (1995). Progression to AIDS in macaques is associated with changes in the replication, tropism, and cytopathic properties of the simian immunodeficiency virus variant population. Virology 207, 528–542.[CrossRef][Medline]
Schols, D. & De Clercq, E. (1998). The simian immunodeficiency virus mnd(GB-1) strain uses CXCR4, not CCR5, as coreceptor for entry in human cells. J Gen Virol 79, 2203–2205.[Abstract]
Spijkerman, I. J., Koot, M., Prins, M., Keet, I. P., van den Hoek, A. J., Miedema, F. & Coutinho, R. A. (1995). Lower prevalence and incidence of HIV-1 syncytium-inducing phenotype among injecting drug users compared with homosexual men. AIDS 9, 1085–1092.[Medline]
Stahl-Hennig, C., Steinman, R. M., Tenner-Racz, K. & 7 other authors (1999). Rapid infection of oral mucosal-associated lymphoid tissue with simian immunodeficiency virus. Science 285, 1261–1265.
Stalmeijer, E. H. B., van Rij, R. P., Boeser-Nunnink, B., Visser, J. A., Naarding, M. A., Schols, D. & Schuitemaker, H. (2004). In vivo evolution of X4 human immunodeficiency virus type 1 variants in the natural course of infection coincides with decreasing sensitivity to CXCR4 antagonists. J Virol 78, 2722–2728.
Ten Haaft, P., Verstrepen, B., Überla, K., Rosenwirth, B. & Heeney, J. (1998). A pathogenic threshold of virus load defined in simian immunodeficiency virus- or simian-human immunodeficiency virus-infected macaques. J Virol 72, 10281–10285.
Thorstensson, R., Walther, L., Putkonen, P., Albert, J. & Biberfeld, G. (1991). A capture enzyme immunoassay for detection of HIV-2/SIV antigen. J Acquir Immune Defic Syndr 4, 374–379.
Veazey, R. S., DeMaria, M., Chalifoux, L. V. & 7 other authors (1998). Gastrointestinal tract as a major site of CD4+ T cell depletion and viral replication in SIV infection. Science 280, 427–431.
Vödrös, D., Thorstensson, R., Biberfeld, G., Schols, D., De Clercq, E. & Fenyö, E. M. (2001a). Coreceptor usage of sequential isolates from cynomolgus monkeys experimentally infected with simian immunodeficiency virus (SIVsm). Virology 291, 12–21.[CrossRef][Medline]
Vödrös, D., Tscherning-Casper, C., Navea, L., Schols, D., De Clercq, E. & Fenyö, E. M. (2001b). Quantitative evaluation of HIV-1 coreceptor use in the GHOST(3) cell assay. Virology 291, 1–11.[CrossRef][Medline]
Vödrös, D., Thorstensson, R., Doms, R. W., Fenyö, E. M. & Reeves, J. D. (2003). Evolution of coreceptor use and CD4-independence in envelope clones derived from SIVsm-infected macaques. Virology 316, 17–28.[CrossRef][Medline]
Zhang, L., He, T., Talal, A., Wang, G., Frankel, S. S. & Ho, D. D. (1998). In vivo distribution of the human immunodeficiency virus/simian immunodeficiency virus coreceptors: CXCR4, CCR3, and CCR5. J Virol 72, 5035–5045.
Zhang, Z.-Q., Schuler, T., Zupancic, M. & 21 other authors (1999). Sexual transmission and propagation of SIV and HIV in resting and activated CD4+ T cells. Science 286, 1353–1357.
Received 5 August 2005;
accepted 24 November 2005.
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, Individual isolates; |, median per group. For details of coreceptor usage, see Table 2