Background: Cell-associated (CA) HIV-1 unspliced RNA is an important marker of the viral reservoir and the response to antiretroviral therapy (ART). Recently it has been used in clinical trials as a measure of virus activation by latency-reversing agents. Primers specific for the HIV gag regions are frequently used in PCR-based assays that quantify unspliced RNA. However, because HIV-1 integrates within actively transcribed host genes, it has been suggested that some of the transcripts detected by the gag-specific assays may not represent genuine HIV RNA but rather chimeric host-HIV readthrough transcripts. To properly interpret the results of the gag assays, it is necessary to determine the relative contribution of such readthrough transcripts to the HIV gag RNA in ART-treated patients.
Methods: We developed a sensitive nested real-time PCR assay that amplifies the 5′ LTR-encoded U3 – packaging signal region (U3-Psi) of HIV-1. This assay specifically measures host-HIV readthrough transcripts but does not detect genuine HIV-1 unspliced RNA (Fig. 1). Total DNA and total RNA were isolated from PBMC samples of 48 ART-treated patients whose plasma viremia had been undetectable (<40 copies/ml) for ≥1 year prior to the study. CA HIV-1 DNA and RNA were separately quantified in these samples using both the U3-Psi assay and the seminested real-time PCR assay specific for the HIV-1 gag region. The sensitivity of both assays is 4 copies/reaction. The same inputs of DNA or RNA were used for both assays.
Results: As expected, both U3-Psi and gag assays detected HIV-1 DNA in >90% of the patients (44/48 and 46/48, respectively) with no significant quantitative bias between the assays, demonstrating the functionality of the U3-Psi assay. HIV-1 gag RNA was detected in 44/48 of these patients (92%) with the median copy number of 590 (interquartile range, 217-1194) copies/µg total RNA. However, the detectability of readthrough RNA was only 40% (19/48 patients). In the 19 patients where the readthrough RNA was detected, its copy number was 49 (41-122) copies/µg total RNA, representing only 8.3% (2.4%-11.2%) of the HIV-1 gag RNA. Notably, the real readthrough/gag RNA ratio is much lower, as patients with undetectable readthrough RNA (60% of all patients) were excluded from this calculation.
Conclusions: We observed only a minor contribution of host-HIV-1 readthrough transcripts to the level of HIV-1 gag RNA. The vast majority of HIV-1 gag RNA transcripts in ART-treated patients represent genuine HIV-1 unspliced RNA.
Figure 1. Schematic representation of real-time PCR assays for the detection of readthrough and gag RNA. LTR, long terminal repeat; ORF, open reading frame; Ψ, HIV packaging signal (Psi).