Background

Over thirty years ago, HIV infection and its clinical manifestation, Acquired Immunodeficiency Syndrome (AIDS), emerged as a worldwide epidemic. Since then, significant understanding of HIV pathogenesis has occurred and the development of drug treatments now significantly extend patients’ lives. Current cART regimens encompass a variety of drugs that inhibit viral replication in several ways, which allows for the almost complete suppression of viral particles found in the blood and recovery of a healthy CD⁴⁺ T-cell population (CD⁴⁺) (Autran et al., 1997). However, the persistence of very low levels of HIV in plasma of cART treated patients, even those treated for decades, suggests the presence of a cell based ‘viral reservoir’. Viral reservoirs contain infected cells that do not release infectious virus (i.e., are latently infected), but can do so following activation, which may occur under a variety of conditions (Chun et al., 1995 and 1997). HIV latency is primarily attributed to proviral HIV DNA in resting memory CD4⁺ T cells (Anderson et al., 2011; Ho et al., 2013), although recent reviews highlight a breadth of research into other potential reservoirs (Abbas et al., 2015; Kandathil et al., 2016; Rothenberger et al., 2016; Sacha and Ndhlovu, 2016). The resting memory CD4⁺ T cells can live for long periods of time, contribute to low-level persistent viremia during cART and viral rebound after treatment interruption, and produce viral variants with escape mutations (Chun et al., 1997; Finzi et al., 1997). Methods to determine the effectiveness of antiretroviral therapy and latency-reversing agents by measuring the circulating resting memory CD4⁺ T cells have been developed and evaluated (Ericksson et al., 2013; Crooks et al., 2015). However, it is pertinent to consider that less than 2% of the total body lymphocyte population resides in peripheral blood (Svincher et al., 2014), making the evaluation of HIV persistence of tissue-resident lymphocyte populations in anatomical reservoirs critically important.

The use of single genome sequencing or SGS (also known as single genome amplification or SGA) has become the routine way to generate sequences for examination of HIV intrahost evolution (Kearney et al., 2014; Lamers et al., 2016; Rose et al., 2016), compartmentalization (Sturdevant et al., 2012; Evering et al., 2014), phyloanatomy (Salemi and Rife, 2016), persistence (Josephsson et al., 2013; Buzon et al., 2014; Boritz et al., 2016), and rebound dynamics (Kearney et al., 2015; Bednar et al., 2016). In contrast to bulk PCR methods wherein many targets are amplified together in the same tube, SGS uses end-point dilution to amplify from only one template. While some studies have demonstrated that bulk PCR and SGS produce sequences that are similar by certain metrics and the techniques can be used interchangeably (Jordan et al., 2010; Etemad et al., 2015), some analyses can only yield accurate results with sequences generated from SGS. These include identifying identical HIV sequences that may arise from clonally-expanding cells rather than PCR resampling (Wagner et al., 2013; Simonetti et al., 2016), determining proportions of viral variants in a sample through sequencing (Iyer et al., 2015), estimating evolutionary rate from point-mutations that occur only from viral reverse-transcriptase rather than PCR Taq errors (Novitsky et al., 2013), and evaluating recombination rates in vivo without including PCR-mediated recombination (Brown et al., 2011; Sanborn et al., 2015).

We used SGS to generate linked gp120 envelope and nef gene sequences from single starting templates to assess viral expression, compartmentalization and evolution in RNA and DNA extracted from a collection of fresh frozen tissues obtained from HIV-infected patients on cART who died with no detectable viral load in their plasma or cerebral spinal fluid at the time of death (Lamers et al., 2016a and 2016b; Rose et al., 2016). Our data demonstrated that a privileged environment exists in some tissues of these patients wherein expression of HIV continues; however, in other tissues, only unexpressed proviral DNA copies were identified. The inferred evolutionary rate of the tissue-based HIV sequences was not significantly different than previously reported rates of replicating virus in cART-negative subjects, suggesting on-going evolution.