Next-Generation Sequencing of Small RNAs from HIV-Infected Cells Identifies Phased microRNA Expression Patterns and Candidate Novel microRNAs Differentially Expressed upon Infection

ABSTRACT HIV infection of CD4+ T cells induces a range of host transcriptional changes in mRNAs as well as microRNAs that may coordinate changes in mRNAs. To survey these dynamic changes, we applied next-generation sequencing, analyzing the small RNA fraction of HIV-infected cells at 5, 12, and 24 h postinfection (RNA-Seq). These time points afforded a view of the transcriptomic changes occurring both before and during viral replication. In the resulting small RNA-Seq data set, we detected a phased pattern of microRNA expression. Largely distinct sets of microRNAs were found to be suppressed at 5 and 12 h postinfection, and both sets of changes rebounded later in infection. A larger set of microRNA changes was observed at 24 h postinfection. When integrated with mRNA expression data, the small RNA-Seq data indicated a role for microRNAs in transcriptional regulation, T cell activation, and cell cycle during HIV infection. As a unique benefit of next-generation sequencing, we also detected candidate novel host microRNAs differentially expressed during infection, including one whose downregulation at 24 h postinfection may allow full replication of HIV to proceed. Collectively, our data provide a uniquely comprehensive view of the changes in host microRNAs induced by HIV during cellular infection. IMPORTANCE New sequencing technologies allow unprecedented views into changes occurring in virus-infected cells, including comprehensive and largely unbiased measurements of different types of RNA. In this study, we used next-generation sequencing to profile dynamic changes in cellular microRNAs occurring in HIV-infected cells. The sensitivity afforded by sequencing allowed us to detect changes in microRNA expression early in infection, before the onset of viral replication. A phased pattern of expression was evident among these microRNAs, and many that were initially suppressed were later overexpressed at the height of infection, providing unique signatures of infection. By integrating additional mRNA data with the microRNA data, we identified a role for microRNAs in transcriptional regulation during infection and specifically a network of microRNAs involved in the expression of a known HIV cofactor. Finally, as a distinct benefit of sequencing, we identified candidate nonannotated microRNAs, including one whose downregulation may allow HIV-1 replication to proceed fully.