A change in coreceptor preference from CCR5 to CXCR4 late in infection has been well documented in some HIV-1 infected individuals since the early days of the AIDS epidemics, but the reasons and mechanisms for this tropism switch remain elusive. Because X4 emergence is strongly associated with rapid CD4+ T–cell loss and disease progression, and concerns that the introduction of CCR5 entry inhibitors as anti-HIV therapeutics could facilitate X4 emergence and exacerbate disease, there is an increasing need to improve our understanding of the selection pressures which favor CCR5-to-CXCR4 switch. Using a simian model of HIV-1 coreceptor switch, we tested in this study the hypothesis that an early selective force in the evolutionary pathway of tropism switch is the need for viruses to increase the efficiency of CD4 binding for infection of CD4low-expressing cells such as tissue macrophages. The adoption of a less constrained and more “open” envelope conformation that exposes the CD4 binding site for enhanced CD4 binding, in turn, releases or reduces envelope structural constraints that have been suggested to limit the pathways available for change in coreceptor preference. We show that R5 viruses evolved early in two rapid progressor macaques to become sCD4-sensitive, and this correlated with better gp120 binding to CD4 and with efficient infection of CD4low cells such as primary macrophages and the HeLa RC49 cells. Furthermore, significant changes in neutralization sensitivity to agents and antibodies directed against functional domains of both gp120 and gp41, including the V3 loop that is important for coreceptor binding were seen for R5 viruses present close to the time of X4 emergence in these rapid progressing macaques, consistent with global changes in envelope conformation and structural plasticity that facilitate the remodeling needed to expand or switch to CXCR4 usage. These observations in two R5 SHIVSF162P3N-infected macaques therefore support our proposed mechanistic model for coreceptor switching.
Several mechanisms can explain sCD4 sensitivity of HIV/SIV. For the early R5 viruses in macaques BR24 (w8, w12) and CA28 (w4), we showed that increase sCD4 sensitivity correlated with better CD4-Ig binding ( and
), consistent with exposure of the CD4 binding site and adoption of an “open” envelope conformation. For R5 viruses close to the time of switch (w16 for BR24 and w7 for CA28), however, changes in the CD4 binding site and/or alteration in the conformational changes induced by CD4 binding appeared to be the underlying basis ( and
). Interestingly, we observed, in both macaques, that sCD4-induced gp120 shedding decreased for Envs evolving prior to the time of switch ( and
), suggesting that a tighter interaction between the gp120 and gp41 may be necessary during the process of envelope remodeling to acquire CXCR4 use. Alternatively, it has been proposed that an increased number of virion-associated Env complexes available for receptor interaction might facilitate infection of CD4low
. Thus, it is conceivable that a more stable gp120-gp41 interaction, in particular for BR24 w8 and w12 and CA28 w4 Envs, increases gp120 retention by Env complexes for infection of CD4low
cells. Genetic studies to determine if virion-gp120 retention and infection of CD4low
cells of these early viruses in BR24 and CA28 are linked will be required to examine this latter possibility.
We show that acquisition of increased sCD4 sensitivity occurred in the presence of high amounts of CD4+ T cells, implying that paucity of CD4+ target T cells is not the driving force for viruses to expose their CD4 binding site and to increase CD4 binding. Moreover, we recently reported that viruses did not evolve early to become sCD4 sensitive in macaques that were depleted of B cells to abrogate or diminish antiviral antibody responses prior to infection with SHIVSF162P3N
, implying that the reduced antibody-driven pressure in the RPs was also not sufficient to select for viruses with an “open” Env conformation 
. Rather, the tight association between CD4 binding and infection of CD4low
cells of the evolving R5 viruses in both BR24 and CA28, and the finding that primary macrophages are the principle virus-producing cells at end-stage disease in these two macaques with coreceptor switch suggest that adoption of an “open” Env is in response to the need to use low levels of CD4 receptor more efficiently. However, increased sCD4 sensitivity and CD4 binding were seen as early as 4–8 wpi, a time when CD4+ T cells and not tissue macrophages are the preferred targets of HIV/SIV infection 
. This then raises the intriguing possibility that a selective pressure for altered CD4 affinity of the early R5 viruses in BR24 and CA28 could be decreasing CD4 expression levels on target T cells. Although direct evidence in support is lacking, infectivity of HIV-1 primary isolates in vitro is strongly dependent on the level of CD4 expression 
. Moreover, transmitted and founder viruses in acute HIV-1 infection have been reported to replicate poorly in monocyte-derived macrophages 
and to require high receptor levels for entry 
. Our finding that the ability of the acute viruses (w2 for BR24 and w1 for CA28) to bind CD4 and to infect CD4low
cells in both macaques is decreased is consistent with these reports in human, and suggests that CD4+ T cells expressing high amounts of the receptor may be the earliest and preferred targets of virus infection and depletion in vivo, leaving only cells with lower CD4 levels available during the post-acute phase of infection. Nevertheless, CD4 and CCR5 concentration requirements for R5 HIV-1 infections in vitro have been shown to be interdependent, with viruses being highly dependent on the CD4 concentrations or strength of the initial virus-CD4 bond when cell surface CCR5 density is low 
. Thus, it is possible that the selection factor for better CD4 usage we observed in the RP macaques following acute R5 SHIVSF162P3N
infection could be due to initially low CCR5 and not CD4 expressions on T lymphocytes. Studies to monitor variations in CD4 and CCR5 cell surface densities on target T cells during the course of SHIVSF162P3N
infection and to examine their relationship to macrophage infection and tropism switch in RP macaques will be needed to more clearly address the selection factors for viruses to evolve early to use low levels of the CD4 receptor more efficiently.
Because most HIV-1-infected individuals have developed neutralizing antibodies, less constrained and “open” envelopes are selected against and not commonly found. This then raises the question as to what extent the observed changes associated with the coreceptor switch in rapid progressor macaques that did not develop or maintain a strong antiviral antibody response reflect what occurs in humans. In this regard, it is noteworthy that X4 dominance is seen only towards end-stage disease in HIV-1 infected individuals, when the immune system is impaired 
. And, although rare, rapid progressor status has been documented in HIV-1 infected individuals 
, with phenotypic switch reported in cases where this was examined 
. Moreover, emergence of sCD4 neutralization-sensitive X4 viruses in the presence of neutralizing antibodies has been reported 
, suggesting that X4 virus evolution is in anatomical compartments with lower antibody pressure than in the plasma, and/or that these viruses spread via cell-cell, a mode of transmission that is less susceptible to antibody neutralization. Indeed, we have shown that peripheral lymph nodes that are enriched in target cells for X4 viruses are the preferred sites of their evolution and amplification 
, and the syncytium-inducing/fusion capacity of X4 viruses has been well documented 
. Thus, the observations made in the SHIV-infected macaques studied here are likely to represent an important step toward our understanding of HIV-1 coreceptor switch in humans.
In summary, our findings provide evidence that adoption of an “open” Env by R5 viruses in response to the selection pressure for better CD4 usage and infection of CD4low cells represents an early step in the chain of events leading to R5-to-X4 evolution, allowing other selection factors such as virus replication-associated mutational events that are required for tropism switch, but which usually come with costs to viral fitness because of structural constraints, to be manifested. Studies of coreceptor switch in RPs are useful for they allow examination of the process of R5 envelope evolution required for a generalized switch uncomplicated by the selection pressure of antiviral antibody responses. Although our studies were limited with respect to the number of animals, the similarity of the evolutionary pattern in structure and function of R5 envelope variants seen in the two outbred RP macaques that differed in the kinetics and levels of virus replication prior to the time of coreceptor switch support a shared mechanism and selective pressure(s) for the change in coreceptor preference. Further research will be required to determine if acquisition of an “open” Env conformation to increase CD4 affinity is a property unique to the early R5 viruses in R5-SHIVSF162P3N-infected RPs with coreceptor switch, and how broadly our findings in the SHIV-rhesus model relate to HIV infection of humans. Additionally, it will be of interest to examine coreceptor switching in SHIVSF162P3N-infected macaques that have developed a neutralizing antibody response, to discern the impact of humoral immune selection forces on the tempo and molecular pathways available for tropism switch.