Discovery of an HIV-1-encoded circular RNA that enhances viral transcription

Circular RNAs (circRNAs) are a class of transcripts that are formed when portions of a pre-mRNA molecule are spliced together in a closed loop. Here, we discover a novel HIV-1-encoded viral circRNA present in patient-derived plasma and infected T-cell lines, demonstrating that an RNA retrovirus can generate circRNAs. This HIV-1-encoded circRNA is packaged within HIV-1 virions, binds the HIV-1 Tat protein, and enhances transcription from the HIV-1 promoter. Our study sheds lights on a previously overlooked aspect of HIV-1 transcription and presents interactions between viral proteins and viral circRNAs as novel mechanisms underlying viral pathogenesis. Overall design: Total RNA was isolated from uninfected Jurkat T-cells and two HIV-1 infected Jurkat-derived clones (8B10 and 5F9). Each clone contains stable integration of a modified HIV-1 provirus (NL4-3-d6-GFP) containing a GFP reporter gene and loss-of function mutations in all viral ORFs except for those encoding Tat and Rev. Following isolation, total RNA from each sample was further processed to yield three fractions with varying levels of circRNA enrichment: (i) poly(A)-enriched RNA, containing minimal circRNA enrichment, (ii) RNA depleted of both poly(A)-tailed and ribosomal RNAs, containing moderate circRNA enrichment (RR0), and (iii) RNA depleted of both poly(A)-tailed and ribosomal RNAs then further treated with the exonuribonuclease RNase R for for 2 hours (RR120), containing high circRNA enrichment. Of note, the RR0 fraction was also mock-digested with water in place of enzyme for 2 hours. Two replicates were generated for each RNA fraction for a total of 18 libraries (3 cell lines x 3 RNA fractions x 2 replicates). Libraries were prepared using the NEBNext Ultra II Directional RNA Library Prep Kit and sequenced on an Illumina Next-Seq Machine (1x150bp reads, single-end).