Background: A thorough understanding of the distribution of replication-competent virus will be needed to design future HIV eradication therapies. We studied the distribution of replication defective viral genomes in memory T cell subsets in blood and lymph nodes of long-term treated subjects. We also examined the distribution of clonal sequences in order to define the role of homeostatic proliferation as a cause of persistence.
Methodology: Using single-proviral sequencing we isolated intracellular HIV-1 genomes derived from defined subsets of CD4+ T cells (naive, central (CM)-, transitional (TM)-, and effector (EM)-memory) from peripheral blood and lymph node tissue. Samples were collected from 8 subjects on suppressive therapy (4-12 years): 5 who initiated therapy during early infection and 3 during chronic infection. G-to-A hypermutants were identified using the Hypermut program within the Los Alamos HIV Database. Levels of APOBEC 3F and 3G (A3F and A3G) were measured in T cells (CM, TM and EM) from blood and lymph node tissue using a gene expression sybr green method. Additional defective viral genomes were determined by stop codons or large deletions. Identical sequences were identified by phylogenetic analysis.
Results: The percent of cells infected with hypermutants was estimated to be 31% lower in CM cells than it was in EM cells (p=0.54), and it was estimated to be 35% higher in TM cells than in EM cells (p=0.51) but the percent of cells infected with other defective viral genomes tended to be higher for EM than CM (38-fold, p=0.0024) and TM (11-fold, p=0.0028). In both blood and lymph node tissue the gene expression of A3G and 3F was highest in EM T cells (EM>TM>CM). The estimated proportion of viral genomes that were hypermutants in subjects treated during early versus chronic infection was similar for all three cell types (<1.6-fold differences), but in EM cells the proportion of all viruses that had other defects was estimated to be >20-fold higher for chronic than early (p=0.018). Phylogenetic analyses showed expansions of identical HIV-1 sequences in all subjects treated during chronic infection; these expansions were predominantly found in EM T cells.
Conclusions: We found that the estimated proportion of HIV genomes during long-term ART that were hypermutants was similar in cells from subjects treated during early and chronic infection, suggesting that these genomes are established soon after infection. We also found >10-fold higher rates of non-hypermutant defective viral genomes in EM cells than either CM or TM cells in blood. These defective genomes and identical HIV-1 sequences predominated in EM T cells of specific subjects, suggesting that they were maintained by clonal expansion of T cells as they differentiated into more terminal cell populations.