Understanding the nature of persistent HIV-1 has important implications for the management of HIV-1 infection and for understanding the evolutionary dynamics of HIV-1 in vivo. We show here that, in infected children, replication-competent ancestral HIV-1 quasispecies are stored in latently infected resting CD4+
T cells despite successful HAART. Analysis of the genotypic mutational patterns of replication-competent viruses isolated from latently infected cells revealed that viruses persisting in this reservoir are diverse and primarily reflect selection by pre-HAART treatment regimens. Most strikingly, wild-type viruses lacking any known drug resistance mutations persisted in this compartment even in patients who had developed drug resistance and who were treated for many years with regimens that strongly favored these drug-resistant mutants. These results suggest that sequences entering the reservoir at early time points in infection are not completely replaced by the dynamic processes that affect the pool of latently infected cells. The persistence of viruses with widely varying levels of genotypic drug resistance is consistent with the idea that the viruses in the latent reservoir are under no active selective pressure from the drugs. Phylogenetic analysis of reservoir sequences showed a striking lack of temporal structure, in sharp contrast to the continuously divergent evolutionary pattern described for env
sequences in the main pool of actively replicating virus (33
). The time of sampling showed no consistent correlation with the degree of evolutionary divergence. This lack of temporal structure is characteristic of a stable reservoir (26
It is important to point out that our results do not exclude the possibility that the reservoir may have a very stable component, as well as a more labile component that is maintained by low-level ongoing viral replication. We detected in one patient (C11) with suboptimal suppression the appearance of new resistance mutations in viruses obtained from resting CD4+ T cells. Although additional studies will be required to determine whether these newly emergent viruses have become fixed in the stable pool of integrated virus in the latent reservoir, the results obtained with this patient suggest that the methods used can detect newly emergent resistant virus entering resting CD4+ T cells. What is particularly striking is that, even in this case, wild-type viruses could still be isolated. Thus, at least a component of the reservoir must be extremely stable.
The recovery of archival wild-type virus and of mutants with different levels of genotypic resistance, in concert with the lack of temporal structure on phylogenetic analysis, strongly supports the notion that resting CD4+
T cells provide a long-term reservoir for HIV-1 (2
). This reservoir acts as a stable archive for replication-competent viruses in infected individuals and has the potential to serve as a persistent source of infectious HIV-1, including wild-type virus, despite prolonged suppressive HAART, a conclusion that has important implications for the treatment of HIV-1 infection.
Alternative explanations for the persistence of wild-type virus in this compartment include the existence of a drug sanctuary site in which wild-type virus replicates without evolving drug resistance and from which seeding of the reservoir occurs. This sanctuary site would have to have limited permeability to the nucleoside analogues. Although such a site has not yet been identified, we cannot exclude this alternative. Another alternative explanation is that the presence of wild-type virus reflects poor compliance. This explanation is not consistent with the prolonged suppression of viremia that most of these patients have experienced. A final consideration is whether the wild-type viruses persist because they are more fit than resistant viruses even in the presence of drug selection. This alternative is not consistent with the dramatic effects of HAART on virus levels in plasma and the well-accepted observation that treatment failure generally involves resistant viruses.
It is important to emphasize that, in contrast to most genetic studies of viral persistence, only replication-competent viruses were analyzed here. This allowed us to avoid the analysis of defective viruses that can be detected in PCR-based studies of viral persistence. Thus, the persistence of wild-type virus does not reflect the survival of defective sequences not capable of mediating viral gene expression or virus production. In addition, the fact that all of the viruses analyzed have demonstrated potential for robust in vitro replication highlights the potential pathogenic significance of this archival reservoir.
The mechanism involved in the long-term maintenance of infectious archival HIV-1 in resting CD4+
T cells is a matter of great interest. One possibility is that the stability of the reservoir is a reflection of the establishment of HIV-1 latency in a cell type that is designed to survive individually or in the form of clonal progeny for an individual's lifespan. This mode of persistence is consistent with the fundamental biology of memory T cells (27
). Another factor contributing to the stability of the latent reservoir may be replenishment by ongoing virus release from other sources (10
). Previous studies of the evolution of HIV-1 in the resting CD4+
T-cell compartment in infected adults treated with suppressive HAART for 2 years have revealed two patterns of viral persistence. Sequence evolution in the C2 to V3 domains of HIV-1 env
gene suggestive of ongoing viral replication could be detected in some patients, whereas a striking arrest in the evolution was detected in others (16
). Similarly, studies of the evolutionary patterns of the pol
gene in infected adults treated with up to 2.3 years of HAART have largely demonstrated the lack of detectable evolution of genotypic drug resistance to the new drugs in the HAART regimen (13
). The occurrence of new drug resistance mutations in patients on HAART has been associated with suboptimal suppression of viral replication (25
). In the present study, we provide further longitudinal evidence that, in HIV-1-infected children who achieve suppression of viral replication, a stable pattern of archival wild-type and drug-resistant virus predominates in the resting CD4+
T-cell compartment despite up to 5 years of suppressive HAART and intermittent viremia. In patients with suboptimal suppression of viremia, newly arising drug-resistant viruses can enter the resting CD4+
T-cell compartment, but even in this situation the persistence of wild-type, drug-sensitive viruses is observed. Some investigators have suggested that the latent reservoir may turnover with a half-life as short as 6 months (31
). If this were the case, >98% of the original latent reservoir sequences should be replaced in 3 years. The finding that wild-type viruses persist despite many years of nonsuppressive antiretroviral therapy suggests that while replenishment of the latent reservoir may occur, at least a substantial portion of the reservoir behaves as an extremely stable archive that is not undergoing active selection by the drugs being used.
The contribution of the latent reservoir to the low level of virus production that continues in patients on HAART (10
) is a matter of great interest. In a recent study of the genotypic resistance profiles of virus present in plasma at low levels in children and adults who had prolonged suppression of viremia to <50 copies/ml on HAART, we found a similar archival pattern of viral persistence and demonstrated the presence in the plasma of sequences that were indistinguishable from sequences isolated from the latent reservoir at earlier time points (19
). The present study demonstrates that one potential source of archival virus found at low levels in the plasma of patients on HAART is release from cells in the latent reservoir that become activated. When antiretroviral therapy is discontinued or drug concentrations are lowered by nonadherence or compromised bioavailability, archival HIV-1 with the greatest replicative capacity can become the predominate species in plasma. Indeed, recent studies by Deeks et al. have shown that, in patients failing combination antiretroviral therapy with highly resistant virus, wild-type virus comes to predominate in plasma several weeks after therapy is discontinued (9
). This observation supports the notion of reemergence of archival HIV-1 from a reservoir that has an extremely long half-life or is not under drug selective pressure. Other groups have shown that following interruption of HAART, the viruses that appear in the circulation in some cases resemble viruses detected in the latent reservoir, while in other cases sequences not identified in the reservoir were seen (6
). In the present study, we demonstrated that archival wild-type HIV-1 persists in a replication-competent form in resting CD4+
T cells despite up to 10 years of continuous antiretroviral exposure. This replication-competent wild-type HIV-1 has the potential to reemerge when therapy is discontinued. Our results imply that the most likely mechanism for the reappearance of the wild-type virus after the interruption of therapy in patients with multidrug-resistant HIV-1 infection is not genetic reversion of multiple drug resistance mutations but rather the reemergence of archived wild-type virus from the latent reservoir in resting CD4+
T cells or some similarly stable reservoir. These findings support the idea that memory T cells or their progeny can survive for years and likely decades. Therapeutic decisions regarding changes in regimens, “recycling drugs” or treatment interruption must be made with the knowledge that all previously circulating wild-type and drug-resistant forms of the virus in a given patient can be archived in this reservoir and may emerge when conditions are favorable.