Date Published: August 5, 2016
Publisher: Public Library of Science
Author(s): Sarah B. Laskey, Christopher W. Pohlmeyer, Katherine M. Bruner, Robert F. Siliciano, Daniel C. Douek.
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.
The HIV-1 virion carries two copies of a 9.7 kb RNA viral genome, which is reverse transcribed to DNA and integrated into the genome of a host cell during infection. Although combination antiretroviral therapy (ART) can suppress HIV-1 plasma viremia indefinitely to a level below the clinical limit of detection, the virus persists for decades in a latent reservoir composed of resting memory CD4+ T cells carrying integrated viral genomes, known as proviruses [1–4]. The development, composition, and plasticity of this latent reservoir, which presents a major barrier to the cure of HIV-1 infection, are all active areas of investigation [5,6].
In this study, we defined the CPS as a metric for how well a subgenomic amplicon differentiates unique HIV-1 genomes. We calculated CPS values for hypothetical amplicons of varying sizes across the HIV-1 genome to investigate the contribution of size and location to the capacity of an amplicon to distinguish unique genomes. Finally, we calculated the CPS values for commonly used primer sets and used them to estimate the background level of clonality in phylogenetic trees generated with those primer sets.