Deep Sequencing of HIV-Infected Cells: Insights into Nascent Transcription and Host-Directed Therapy

Polyadenylated mature mRNAs are the focus of standard transcriptome analyses. However, the profiling of nascent transcripts, which often include nonpolyadenylated RNAs, can unveil novel insights into transcriptional regulation. Here, we separately se-quenced total RNAs (Total RNAseq) andmRNAs (mRNAseq) from the same HIV-1-infected human CD4 T cells. We found that many nonpolyadenylated RNAs were differentially expressed upon HIV-1 infection, and we identified 8 times more differentially expressed genes at 12 h postinfection by Total RNAseq than bymRNAseq. These expression changes were also evident by con-current changes in introns and were recapitulated by later mRNA changes, revealing an unexpectedly significant delay between transcriptional initiation andmature mRNA production early after HIV-1 infection. We computationally derived and validated the underlying regulatory programs, and we predicted drugs capable of reversing these HIV-1-induced expression changes fol-lowed by experimental confirmation. Our results show that combined total andmRNA transcriptome analysis is essential for fully capturing the early host response to virus infection and provide a framework for identifying candidate drugs for host-di-rected therapy against HIV/AIDS. IMPORTANCE In this study, we used mass sequencing to identify genes differentially expressed in CD4 T cells during HIV-1 infection. To our surprise, we foundmany differentially expressed genes early after infection by analyzing both newly transcribed unprocesse