Our recent studies have been directed at defining the roles of host and virus factors in the development of rapid progression. In the present study, we generated full-length infectious molecular clones that were representative of the spectrum of genetic RP-specific variants in RP macaques. Despite their obvious growth advantage in vivo, as evidenced by high plasma viremia and high levels of viral RNA expression in tissues, these mutations were disadvantageous for growth of virus in tissue culture. Thus, propagation of uncloned virus from an RP macaque resulted in the loss of RP-specific mutations. However, sequence analysis of sequential variants in an RP macaque inoculated with this isolate demonstrated reselection of RP mutations in vivo. This result indicates that RP mutations are a characteristic of end-stage viruses in RP macaques, but the dominance of the RP genotype is not correlated with the ability to replicate in human cell lines or primary macaque cells in vitro. The reselection of RP mutations, which were a minor population within the inoculum, suggests a strong positive selection pressure for this genotype in RP macaques.
One of the potent selective pressures on viruses is immune responses, but immune responses in RP macaques have generally waned by 4 weeks postinfection and thus are unlikely to be responsible for the selection of RP-specific mutations (22
). Actually, the pattern of sequence variation of gp120 in RP macaques differed from that previously observed in conventional SIV-infected macaques (8
). Variation was concentrated to variable regions, designated V1, V2, V3, V4, and V5, in conventional SIV infection that induces immune responses (5
), but these variable regions were conserved in viruses from RP macaques. In addition, some of the RP mutations were found in highly conserved regions, such as the GDPE motif and the V3 loop analog, which is the region homologous to the V3 loop in HIV-1 (7
). Taken together, these RP mutations do not appear to be the consequence of immune pressure.
A few prior studies have characterized infectious clones with RP-specific mutations that were derived from SIV-infected macaques (2
), with a specific focus on analyzing the viral determinants of macrophage tropism and neurotropism. Three such infectious clones are SIVmac316 (41
), SIVmac17E-Fr (2
), and SIVsm62d (21
). Due to the propensity for macrophage-associated disease in RP macaques, these clones were derived from RP macaques. However, although RP mutations were dominant in the tissues of these macaques, many of the RP mutations we have observed were not found in these infectious clones. Thus, SIVmac316 did not show the loss of the glycosylation site in V1/V2, P334L/R in the V3 analog, and D385N in the GDPE motif, although these mutations were highly dominant in macaque 316 (29
). Anderson et al. constructed molecular clones from two SIVmac239-infected macaques with SIV encephalitis in which loss of the glycosylation site in V1/V2 and D385N in the GDPE motif were dominant. However, all the clones with the D385N mutation were not infectious, and only SIVmac17E was shown to be infectious (2
). Finally, eight infectious clones were obtained from a SIVsm-infected RP macaque and designated as SIVsm62A through -J (21
). Although scattered mutations in the V1/V2 glycosylation site, V3 analog, and GDPE motif were observed, these specific clones showed low infectivity and narrow in vitro tropism. These previous studies indicated that RP mutations, especially D388N (D385N in SIVmac239) in the GDPE motif, were selectively excluded, because of extremely low in vitro infectivity. In the present study, we successfully constructed infectious clones with the constellation of characteristic RP-specific mutations, i.e., loss of the potential glycosylation site in the V1/V2 region (N158D), substitutions in the V3 analog (P337T, E340K, and R348W), and the D388N substitution in the GDPE motif. Although these viruses replicated inefficiently in vitro, they were still infectious in primary rhesus PBMC and will allow us to evaluate their properties in vivo.
Two of the motifs that are altered in RP viruses are highly conserved in SIV and/or HIV. Although the V3 loop is variable in HIV-1, it is highly conserved among SIVmac/sm viruses; it is involved in coreceptor recognition (7
), and changes in this region alter the tropism of both of these viruses (21
). The GDPE motif is highly conserved in both HIV-1 and SIV. Previous studies have shown that the GDPE motif is important for CD4 binding (45
), appearing to bind to the CD4 receptor molecule directly (31
). Mutational analyses revealed that D368N in the HIV-1 gp120 (GGN368
PE) and the analogous D385N substitutions in SIVmac impaired CD4 binding and viral infectivity (43
). Another related mutation in this motif, G383R (R383
GDPE) in SIVmac, also reduced CD4 binding and viral infectivity (47
). These two mutations were also suggested to be critical for CD4-independent use of CCR5 as a coreceptor for SIV (47
), which is a characteristic of RP variants (8
). RP clones constructed in this study had the D388N substitution in the GDPE motif (GGN388
PE) but retained infectivity in rhesus PBMC, although the replication was low and variable depending on the donor macaque. Therefore, the loss of infectivity of the GGN385
PE mutant of SIVmac239 suggests that other compensatory mutations may be required for replication of viruses with the GGN385
Despite changes in the V3 loop analog, all of our clones retained the ability to use CCR5 as their major coreceptor. This was not unexpected, since envelopes of SIVsm62 clones with similar substitutions in the V3 loop retained the ability to use CCR5 in cell fusion assays but demonstrated relative CD4 independence (12
). CD4 independence of the clones from H635 was not examined, but since the substitutions are similar to those observed in previous studies (8
), we assume that this is a conserved property of RP clones. The RP clones in the present study lost the ability to use GPR15 as a coreceptor, a conserved property of many SIVsm and SIVmac viruses, and had gained the ability to replicate (albeit to low levels) in parental GHOST cells that expressed CD4 but lacked other known coreceptors of HIV and SIV. This finding suggested the use of an alternative coreceptor in addition to CCR5. In terms of in vitro properties, the ability to use an alternative coreceptor was the only potential advantageous feature of these RP clones that was observed in this study. The use of an alternative coreceptor may be related to the donor-dependent replication in PBMC, which was observed in this study, and may be advantageous for in vivo replication after depletion of memory CD4+
cell subsets, in which most of CCR5+
cells are included. However, the significance of this finding will be unclear until the actual molecule is identified. Further analysis is required to identify the cell types which express this unknown coreceptor and whether cells infected in vivo express this coreceptor. The implication of this finding is that RP variants may be capable of infecting CD4+
T cells that do not coexpress CCR5, in addition to their ability to infect cells that only express CCR5 in the absence of CD4, thus potentially broadening the range of susceptible cells in macaques.
In the present study, full-length infectious clones of SIV that are representative of the consensus sequence of SIV in the plasma of an RP macaque were generated. These clones are thus likely to be characteristic of replicating virus in this macaque. We then evaluated the replication properties in primary macaque cells in vitro as an indirect measure of their in vivo tropism and replicative efficiency. In the absence of immune pressure, the efficiency of replication and the breadth of cellular tropism may be critical for the success of the virus. Thus, we expected an advantage of RP variants in the replication efficacy or cell tropism that would explain the high viral load in plasma and tissues of RP macaques (22
). In terms of tropism, macrophage tropism would be highly advantageous in a host after depletion of the classic target cells, CD4+
memory T cells. Consistent with this theory, SIV-producing macrophages were predominant in H635 and other RP macaques (18
). SIV-producing macrophages and multinucleated giant cells were commonly observed in the lungs and brains of RP macaques by immunohistochemistry and in situ hybridization analyses (18
; C. R. Brown, unpublished data). Accordingly, we predicted that RP-specific envelope mutations might be responsible for the enhanced replication in tissue macrophages at the expense of replication in CD4+
T cells. However, the cloned viruses from H635 replicated less efficiently in macaque macrophages (MDM and AM) in vitro than the parental strain. Thus, the selection of RP mutations in vivo did not correlate with enhanced replication of these variants in macrophages in vitro. This lack of correlation between in vitro and in vivo tropism was not entirely unanticipated, since studies with SIVmac239 and SIVmac316 have demonstrated that viral tropism for macrophages in vitro was not reflective of the subsequent replication in macrophages in vivo (4
). However, it is clear that the dominance of the RP genotype in vivo is not correlated with the ability to replicate in macrophages in vitro.
RP clones replicated less efficiently in primary macaque PBMC and macrophages than their parent, SIVsmE543-3. The low in vitro infectivity of RP clones is an enigma, since these viruses replicate to high levels in the RP macaques from which they were derived, as evidenced by high plasma viral RNA loads and large numbers of SIV RNA-expressing cells in tissues (22
). One possible key to this discrepancy between in vitro and in vivo infectivity may be the profound early immune disorder in RP macaques (22
), which may create a specific microenvironment that promotes the growth of RP variants. Supporting this hypothesis, high levels of proinflammatory cytokines such as IL-6 or chemokines such as monocyte chemoattractant protein-1 have been observed in RP macaques with SIV encephalitis (35
). Although these may be the product of infected macrophages, alternatively they may be driving the activation of macrophages and potentially promoting the growth of RP variants. The in vivo target cells for SIV infection, including macrophages, may exhibit altered susceptibility compared with that observed in culture systems in vitro. We consider it likely that tissue culture systems may not be reflective of the in vivo replication potential of RP variants.
The predominance of the RP-specific genotype in different RP macaques suggests a direct role of these variants in the pathogenesis of rapid progression. However, the poor replicative ability of these variants in primary cells in vitro was unexpected. If growth in primary macrophages and PBMC in vitro is reflective of in vivo replication potential, these variants may not play a direct role in inducing the early immune failure that characterizes RP macaques. Instead, these variants may have evolved specifically to replicate efficiently in the absence of immune pressure in a host with a paucity of memory CD4+
T cells. We consider it likely that the early immune damage observed in RP macaques may be the direct result of the parental virus, which efficiently replicates in memory CD4+
T cells. Such early immune dysfunction may be critical for the subsequent evolution and development of RP-specific variants. This theory is consistent with the result of genotype analysis of sequential plasma samples in H635, since the parental virus predominated early in infection coincident with the loss of memory cells and RP variants did not appear until 4 weeks. RP-specific variants may be linked with the development of macrophage-specific disorders such as SIV encephalitis but could play little direct role in the early immune events. This two-step process is reminiscent of the rapid, consistent SIV encephalitis model system using SIVsmB670 and the neurotropic SIVmac17E strain (59
), which essentially produces a high frequency of rapid progressors. Future studies will address the role of RP variants cloned from H635 in vivo in naive macaques, alone or in combination with the parental strain.
In summary, RP variants appear to be differentially selected in vivo in RP macaques specifically but are relatively unfit for replication in primary macaque cells in vitro. The infectious molecular clones obtained in the present study will be useful for in vivo studies with macaques to investigate the contribution of the RP variants to rapid progression and to understand the mechanisms underlying SIV encephalitis and other SIV-associated macrophage disorders.