There are several reasons to hypothesize that variants that appear in the blood are potentially compartmentalized. Previous studies have demonstrated compartmentalization of HIV-1 variants between different anatomical compartments, such as the central nervous system, genital tract, and different lymphoid tissues (13
), as well as tissue microenvironments (24
). The gut-associated lymphoid tissue, in particular, represents a major source of active replication of potentially compartmentalized CCR5-tropic virus populations (1
). It is possible that virus spatially compartmentalized in these anatomical sites may be represented as distinct variants in the peripheral blood. Because the initiation of HAART abruptly blocks new rounds of HIV-1 infection, presumably without impacting viral RNA production from cells already infected, different decay rates of compartmentalized variants following suppression of viral replication should reflect different life spans of the cells from which they are emerging. Viral populations compartmentalized in either cell types or tissues that experience differential drug exposure may also decay at different rates if viral replication continues at some level in the presence of a suboptimal drug concentration. However, our observation that all detectable HIV-1 genetic variants declined at comparable rates suggests that the vast majority of the coexisting HIV-1 subpopulations in the peripheral blood are not compartmentalized either in cell types with different life spans or in cells or tissues with various degrees of antiretroviral drug bioavailability.
Our use of the HTA allowed for relatively sensitive detection of variants potentially comprising as little as 1 to 3% of the population (33
). However, one important limitation of this study is the sensitivity for detection of minority viral populations below this 1% threshold, which may be produced by cells with different life spans. Such viral populations almost certainly exist, based on the biphasic decay kinetics of the bulk HIV-1 RNA load during HAART (4
). It has been hypothesized that the second phase of decay of the bulk HIV-1 RNA load represents virus produced by cells with a longer life span, presumably cells of the monocyte lineage. Compartmentalization of viral DNA populations between CD4+
T cells and monocytes has been reported (21
), and assuming that monocytes have longer half-lives relative to activated CD4+
T cells, variants compartmentalized in these different cell types would be expected to exhibit different rates of decay during HAART (11
). However, the proportion of variants compartmentalized in productively infected monocytes may be too small to be detected in our assay (62
), and monocytes may not be productively infected and only produce virus upon entry into tissue and differentiation (72
). Furthermore, the decay characteristics of HIV-1 upon initiation of therapy that includes an integrase inhibitor suggest that much of the second phase of decay observed in conventional therapy represents cells that are slowly undergoing integration and that the proportion of productively infected, long-lived cells is smaller than previously thought (53
). Another limitation of this study is that it depends on the assumption that compartmentalized subpopulations can be distinguished by their env
genotypes and, in particular, genotypes that can be resolved by HTA. However, in addition to its function in determining host cell tropism, the extreme genetic complexity of env
within infected individuals makes it a highly sensitive target for the detection of coexisting viral subpopulations, and any other genomic region that may drive HIV-1 compartmentalization would likely be linked to distinct env
variants as a result of founder effects, genetic isolation, or compartment-specific evolution. Furthermore, any compartmentalized variants would have likely diverged enough to be resolved by HTA analysis (30
). Thus, we can conservatively conclude that the lack of genetic compartmentalization and the differential decay rates observed in this study apply to the bulk of the HIV-1 population in the peripheral blood that represents primarily the first phase of viral RNA decay during HAART.
Another potential opportunity for cellular compartmentalization is between naïve and memory CD4+
T cells. While naïve and memory T cells express similar levels of CXCR4, CCR5 is expressed only in memory cells (5
). Previous studies have found a wide range of preferential infection by, and potential compartmentalization of, X4 and R5 variants in these cell types in a manner consistent with their coreceptor expression patterns (3
). If X4 and R5 variants are compartmentalized in these two cell types to a significant degree, then similarity in decay rates would indicate that the life spans of infected naïve and memory cells are similar when they become activated and produce virus. It is thought that the bulk of viral replication occurs in activated CD4+
memory T cells (34
), in which case any potential compartmentalization of R5 and X4 variants observed in resting memory and naïve cells may represent only a small fraction of the total population. Also, activated and previously activated T cells express both CCR5 and CXCR4 (5
), providing a potential source of mixing of R5 and X4 variants. There is evidence to suggest that the pool of cells supporting the bulk of virus replication is not homogeneous in its susceptibility to infection by X4 and R5 variants (22
) and that X4 and R5 variants may be differentially affected by antiretroviral therapy (22
). However, we found no difference in the decay rates of X4 and R5 variants upon initiation of therapy, and the decay rates of these variants are within the range reported in other studies for the first phase of decay, presumably reflecting the life spans of the activated memory cells supporting ~99% of the virus population (34
). This finding is consistent with a model where virus is emerging from a homogeneous pool of cells that is sufficiently susceptible to infection by both X4 and R5 variants to account for most of the production of these variants found in the periphery. This is also further supported by the finding of a lack of genetic compartmentalization between X4 and R5 populations in both subjects 101 and 109 for regions outside of env
, indicating some overlap of target cell types. However, the lack of data indicating differential decay rates of variants does not allow a definitive conclusion to be drawn regarding the half-lives of infected cells in different cellular subsets until the degree of cellular compartmentalization of X4 and R5 variants can be more fully and directly accounted for in studies of this type.
The divergent X4 and R5 lineages indicate some degree of genetic compartmentalization between these variants. This could be due to physical isolation in different cell types or to genetic linkage selected across env
for the ability to use different coreceptors. However, we detected an overall lack of compartmentalization and evidence of recombination between these populations in sequence regions increasingly distal of 3′ of V3, suggesting the potential for sequence mixing between R5 and X4 variants in a coinfected cell (Fig. and ). This observation is consistent with previous reports that have identified X4/R5 recombinants both within env
and between env
and other regions of the genome (51
). However, our use of the single genome amplification approach avoided the possibility of recombination during PCR, which may have created artificial recombinants in some previous studies. These data support the conclusion that while X4 and R5 variants may preferentially replicate in distinct cellular compartments, they are not genetically isolated and must with some frequency infect the same cell types. Still, the deep branch points in the phylogenetic trees suggest that the initial outgrowth of X4 variants is from a monoclonal genotype.
This study found little evidence for differential decay and compartmentalization of env
variants comprising the bulk of the virus in the peripheral blood, even in the case of divergent coreceptor phenotypes, indicating that HAART is equally effective on all the detectible variants making up the bulk virus in the peripheral blood. However, new technologies are becoming available that will allow sampling to below 1% (52
), and the application of these technologies may yet reveal minor populations that exhibit differential rates of decay upon initiation of therapy.