Persistence of episomal HIV-1 infection intermediates in patients on highly active anti-retroviral therapy

Abstract

Treatment of HIV-1-infected individuals with a combination of anti-retroviral agents results in sustained suppression of HIV-1 replication, as evidenced by a reduction in plasma viral RNA to levels below the limit of detection of available assays. However, even in patients whose plasma viral RNA levels have been suppressed to below detectable levels for up to 30 months, replication-competent virus can routinely be recovered from patient peripheral blood mononuclear cells and from semen. A reservoir of latently infected cells established early in infection may be involved in the maintenance of viral persistence despite highly active anti-retroviral therapy. However, whether virus replication persists in such patients is unknown. HIV-1 cDNA episomes are labile products of virus infection and indicative of recent infection events. Using episome-specific PCR, we demonstrate here ongoing virus replication in a large percentage of infected individuals on highly active anti-retroviral therapy, despite sustained undetectable levels of plasma viral RNA. The presence of a reservoir of 'covert' virus replication in patients on highly active anti-retroviral therapy has important implications for the clinical management of HIV-1-infected individuals and for the development of virus eradication strategies.

Fig. 1

Detection of 2-LTR replication intermediates of HIV-1. a, cDNA intermediates in HIV-1 reverse transcription. Dashed lines, viral RNA; solid lines, cDNA; *, primer binding site. Far left, subcellular location of the main stages in reverse transcription (the exact boundaries are not yet defined). After translocating to the nucleus, linear cDNA either undergoes integration to form the provirus or circularizes to form episomes containing 1-LTR or 2-LTRs. b, Strategy for 2-LTR-specific PCR amplification, with organization of the LTRs at the circle junction and binding sites for 2-LTR circle-specific primers. For amplification of all forms of viral cDNA, the 2-LTR circle-specific primers were reversed in orientation, such that amplification is internal of the LTR. c, Selective isolation of episomal forms of viral cDNA. Log dilutions of HIV-1-infected MT-4 cells (CD4⁺ T-cell line) were adjusted to 1 × 10⁶ total cells by the addition of uninfected cells. Total cellular DNA and extrachromosomal DNA were isolated and amplified with α-tubulin-specific primers and with 2-LTR-specific primers. The recovery of cellular DNA and 2-LTR circles was 0.6 and 32%, respectively, indicating selective enrichment for 2-LTR circle forms of viral cDNA. d, 2-LTR circle junction sequences amplified from extrachromosomal DNA of five HIV-1-infected individuals (left margin, P1–P5) using 2-LTR circle-specific primers.

Fig. 2

Stability of 2-LTR circles in vitro and in vivo. a, Instability of 2-LTR circle forms of viral cDNA in vitro. CD4⁺ MT-4 T cells or CD4⁺ Jurkat-CCR5 T cells were infected with the X4 variant, HIV-1_LAI or the R5 variant HIV-1_ADA and viral infection and cDNA synthesis were allowed to proceed for 24 h, then further rounds of infection were then prevented by the addition of 5 μM ZDV or 1 μM Nevirapine, respectively; cells were then maintained in the presence of these reverse transcriptase (RT) inhibitors. Upward arrows, sampling intervals. Total cellular DNA was isolated (horizontal axis, time in hours) and amplified with 2-LTR circle-specific primers and with internal (LTR U5–R) primers (Fig. 1b). b, Instability of 2-LTR circle forms of viral cDNA in vivo. PBMCs were obtained from four HIV-1-infected individuals (patients Gu, Sm, Za and Ha) who, after adjustment of their anti-retroviral regimens to more potent combinations, showed rapid decreases in plasma viral RNA levels. The frequency of viral genomes was determined relative to the amount of genomic cellular DNA (measured directly by absorbance at 260 nm); 100 ng cellular DNA represents approximately 1 × 10⁶ PBMCs.

Fig. 3

Relationship between 2-LTR circle frequency and interval during which plasma viral RNA was undetectable in plasma (fewer than 50 copies/ml). Data represent all 63 patients. Statistical analysis (r_s = −0.143; P = 0.26; Spearman’s rank correlation) indicates that within the time frame of the analysis, there was no correlation between 2-LTR circle frequency and duration of undetectable plasma viral RNA.

Fig. 4

High-input virus co-culture assays for replication-competent HIV-1. Virus co-culture results for replicate cultures (each containing 1 × 10⁷ CD8-depleted PBMCs): ■, positive; □, negative. Treatment regimens, CD4⁺ T-cell status and time of undetectable plasma viral RNA, Table 1. There is significant correlation between the frequency of positive virus co-cultures and levels of 2-LTR circles (r_s = 0.892, Spearman’s rank correlation; P < 0.01; P < 0.02, paired t-test).