Evaluating Clonal Expansion of HIV-Infected Cells: Optimization of PCR Strategies to Predict Clonality

Abstract

In HIV-infected individuals receiving suppressive antiretroviral therapy, the virus persists indefinitely in a reservoir of latently infected cells. The proliferation of these cells may contribute to the stability of the reservoir and thus to the lifelong persistence of HIV-1 in infected individuals. Because the HIV-1 replication process is highly error-prone, the detection of identical viral genomes in distinct host cells provides evidence for the clonal expansion of infected cells. We evaluated alignments of unique, near-full-length HIV-1 sequences to determine the relationship between clonality in a short region and clonality in the full genome. Although it is common to amplify and sequence short, subgenomic regions of the viral genome for phylogenetic analysis, we show that sequence identity of these amplicons does not guarantee clonality across the full viral genome. We show that although longer amplicons capture more diversity, no subgenomic region can recapitulate the diversity of full viral genomes. Consequently, some identical subgenomic amplicons should be expected even from the analysis of completely unique viral genomes, and the presence of identical amplicons alone is not proof of clonally expanded HIV-1. We present a method for evaluating evidence of clonal expansion in the context of these findings.

Paired quantitative and qualitative assessment of the replication-competent HIV-1 reservoir and comparison with integrated proviral DNA

“…To characterize the cultured viruses molecularly, we produced cDNA from culture supernatants and sequenced the env gene using primers that resulted in a clonal prediction score of 94 of 100 (silicianolab.johnshopkins.edu/cps) (21). Thus, there was a high probability that identical env sequences represented identical full-length genomes.…”

Longitudinal study reveals HIV-1–infected CD4+ T cell dynamics during long-term antiretroviral therapy

“…Clones were identified as proviruses with identical nucleotide sequences and, if deleted, with identical deletion junctions, as has been done previously (48,58,59). Although definitive assignment of clonality requires integration site analysis, full genome sequence identity provides very strong evidence for clonality (60). This is especially true for defective proviruses, given the underlying sequence diversity of HIV-1, the heterogeneity of defects, and the inability of these viruses to spread.…”

Proviruses with identical sequences comprise a large fraction of the replication-competent HIV reservoir

“…found identical env sequences from independent VOA cultures and proviruses that were detected longitudinally over a 4–6 month interval in four participants, suggesting the persistence of HIV-infected clones [14]. However, this study was limited by the small number of participants (N = 4) and the use of short amplicons (~2.9 kb) that limit the certainty of clonality [37]. …”