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A SMALL FRACTION OF PROVIRUSES IN EXPANDED CLONES EXPRESS UNSPLICED HIV RNA IN VIVO
Andrew Musick1, Jonathan Spindler1, Brandon Keele2, Michael J. Bale1, Wei Shao2, Ann Wiegand1, John Mellors3, John Coffin4, Frank Maldarelli1, Mary F. Kearney1
1NCI, Frederick, MD, USA,2Leidos Biomed, Inc, Frederick, MD, USA,3Univ of Pittsburgh, Pittsburgh, PA, USA,4Tufts Univ, Boston, MA, USA
The vast majority of proviruses that persist on ART are defective. Of the minority that are intact (~2%), the fractions that are latent or transcriptionally active are not known. To address this question, we determined the fraction of proviruses that express HIV RNA in vivo in cell populations carrying either intact or defective proviruses.
PBMC were obtained from Patient #1 in Maldarelli, et al. (Science, 2014). This donor had multiple clones of cells that contain intact or defective proviruses. Proviral expression was determined by single-genome pro-pol sequencing (SGS) of HIV DNA and RNA from multiple aliquots of PBMC diluted to an endpoint such that each aliquot contained one to a few HIV RNA expressing cells. Intact proviruses were identified using viral outgrowth assays (VOA). The levels and fractions of cells expressing HIV RNA were determined for probable clones (identified by identical sequence matches) carrying intact and defective proviruses.
A total of 77 million PBMC were analyzed, of which 10,450 contained HIV pro-pol sequences. Fourteen percent of the infected cells expressed HIV RNA. The median levels of expression in single cells was 1 RNA/cell (ranging from 1-16). We identified 412 different WT or hypermutant RNA species in infected cells. Of these, 3 were from expression of intact proviruses, 81 from obviously defective proviruses, and 328 from proviruses that were likely defective (did not grow out in VOA but did not contain stop codons in the region analyzed). The median fraction of cells in the probable clones (those with matching DNA and RNA) that carried intact proviruses (N=3) and expressed HIV RNA was 2.3% (1.2%-8.8%). For clones carrying defective proviruses (N=5), the median expressing was 3.5% (0.9%-7.0%), and for presumptive clones carrying likely defective proviruses (N=26), the median was 6.6% (1.3%-66.7%) (p =0.51 for a difference across groups).
The large majority (>80%) of infected cells that persist on ART are either latent or incapable of HIV RNA expression. A small fraction of proviruses within infected clones expressed unspliced HIV RNA, but this fraction was not significantly different between clones carrying intact proviruses from clones containing obviously defective proviruses, indicating that HIV RNA expression appears similarly detrimental (or non-detrimental) for infected cells regardless of whether the provirus they carry is intact.