To elucidate the basis for the unusually strong resistance of macaque 359 to in vivo and in vitro SIV infection, we investigated early events in the viral infection and replication process. Initially, we examined the coding sequences of coreceptors used by SIV for mutations. We found no alterations in CCR3, Bonzo, BOB(GPR15), or CXCR4 genes in either resistant or susceptible macaques. On the other hand, genetic polymorphisms, confirmed by sequencing, were detected in the CCR5 gene; however, none of the changes led to amino acid substitutions. Thus, coreceptor alterations that might prevent viral entry could not account for the resistance of macaque 359 to SIV infection. Further, CD4 cells of macaque 359 in comparison to those of the highly susceptible macaque 660 expressed similar levels of CCR5 on the cell surface, both in terms of the percent positive cells and the fluorescence intensity as a measure of coreceptor density (Table ). Thus, a block at the level of cell entry seemed unlikely.
We therefore investigated postentry events in the viral replication cycle, focusing on early DNA intermediates of reverse transcription. The reverse transcription process is highly complex (55
), involving two strand transfer reactions. We focused on transcription of the minus strand, including the initial minus-strand strong-stop DNA, the DNA product made just after the first-strand transfer, and the DNA product at the end of minus-strand synthesis. All three of these steps were significantly inhibited after in vitro infection of macaque 359 CD4 T cells with SIVmac251
, indicating an early block in viral replication. The inhibition of early reverse transcription events would occur if viral entry were inhibited, as well as from a postentry mechanism. We therefore revisited the question of viral entry. A cell-cell fusion assay was used since it specifically evaluates entry. Other systems, such as pseudotyped viruses carrying the luciferase gene, involve several stages of the viral life cycle in addition to entry for expression of the marker gene. Here, the cell-cell fusion assays effectively demonstrated that SIV can enter cells of the macaque 359 as efficiently as cells of macaques highly susceptible to SIV infection. Therefore, the inhibition in viral replication is due to a postentry block in the replication cycle.
Since the replication of our SIVmac251
stock proceeded efficiently in the cells of control macaques, our results implicate a host cell mechanism in inhibition of viral replication in macaque 359 cells. Others have previously shown that the susceptibility of the macaque CD4+
T cells to in vitro SIV infection reliably predicts viral replication in vivo (18
). Importantly, these earlier studies suggested that the susceptibility phenotype was an intrinsic property of the CD4 T cells themselves rather than a consequence of immune responses acquired after viral infection. The cellular basis for the various susceptibilities was not elucidated, although Goldstein et al. suggested that postentry mechanisms might be involved (18
). Here the investigation of a highly resistant macaque which was able to clear its viral infection allowed us to demonstrate that at least one mechanism of innate host resistance is a postentry block in early viral reverse transcription events. It will be critically important to determine the prevalence of this phenomenon in the rhesus macaque population and also to identify the cellular inhibitory mechanism.
A number of cellular host factors that participate in the retroviral replication process have been identified. Several of these interact at the level of integration of proviral DNA. The product of the well-known murine Fv-1
gene, a derivative of an endogenous retroviral gag
), limits the replication of certain murine leukemia virus strains to mice that carry particular Fv-1
). Its inhibitory effect is associated with inefficient viral genome integration. Other cellular proteins positively influence the integration of proviral DNA, including the barrier to autointegration factor protein (26
); INI1, a homolog to a yeast transcription factor (22
); and proteins associated with integration-competent nucleoprotein complexes such as HMG I (2
) and HMG I(Y) (15
Other host proteins that function earlier in the viral replication cycle include components of the cytoskeleton and cyclophilin A. The latter, although not required for SIV replication (6
), binds the HIV type 1 capsid protein (31
) and is necessary for viral entry or uncoating (17
). A comparable cellular protein could fulfill the role of this host protein with chaperone-like activity for the SIV capsid. The early interaction of HIV with the cellular cytoskeleton is also intriguing. Actin microfilaments have been implicated in the formation of reverse transcription complexes necessary for initiating and completing the process of HIV reverse transcription (9
). Other unidentified cellular factors in the cytoskeleton compartment may also be important for reverse transcription (9
). Recently, a double-stranded RNA-binding protein, NF90, was shown to inhibit early events in HIV reverse transcription in GHOST cells transiently transfected with NF90 or stably expressing the protein (E. Agbottah, A. Spruill, and A. Kumar, unpublished data). Although NF90 is a nuclear protein, this observation suggests another category of cellular proteins that could potentially influence viral replication events.
The postentry block in SIV infection observed in vitro provides an explanation for the significant resistance of macaque 359 seen in vivo as well. The initial intravaginal SIV exposures which did not result in infection nevertheless elicited a low-level cellular immune response (B. Peng et al., unpublished data). Similar observations have been described for highly exposed but persistently seronegative women (23
). Although macaque 359 became infected after intrarectal SIV exposure, its low-level immunity, presumably helped by the host resistance mechanism inhibiting SIV replication, was sufficient to control infection and eventually clear the virus, as demonstrated by the transfusion experiment.
Our observation that a host cell mechanism inhibits early SIV replication events, thereby contributing to resistance to SIV infection, has several important implications. At a fundamental level, identification of the mechanism will expand our knowledge and understanding of the biochemical steps involved in reverse transcription. For clinical applications, identification of a natural cellular inhibitor of the reverse transcription process may be able to be exploited in novel therapeutic regimens. Finally, in a practical sense, an enhanced ability to screen rhesus macaques for cellular resistance to infection will greatly improve the macaque model for study of SIV pathogenesis and for vaccine development. It will be important to determine the prevalence of macaques that possess this type of cellular resistance. Subsequent identification of the mechanism in an outbred population will be difficult. However, macaques could be sorted based on the outcomes of mucosal challenges and subsequently assessed for levels of early reverse transcription products in vitro. Ultimately, for purposes of vaccine studies, macaques could be selected for susceptibility to infection, reducing the viral dose necessary to ensure the infection of all control animals and bringing challenge experiments to more realistic levels that are comparable to the natural exposures that occur in the human population.