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

Chang, Stewart T.; Thomas, Matthew J.; Sova, Pavel; Green, Richard R.; Palermo, Robert E.; Katze, Michael G.

Time-specific, phased patterns of microRNA expression in HIV-infected cells. Because many microRNAs were detected with low read counts, we performed further statistical analysis using DESeq, a negative binomial-based test that treats expression as discrete counts (13) . By applying DESeq, we found that small numbers of microRNAs were DE at 5 and 12 hpi (numbers of microRNAs are in Table 1 ; expression values and microRNA IDs are in Fig. S2A to C in the supplemental material; full information is in Table S1 in the supplemental material). Furthermore, similar numbers of microRNAs were detected as DE in HIV-infected cells and HIV UV -infected cells at these time points (Table 1; see also Fig. S2D to F and Table S1 ). In contrast, at 24 hpi, a larger number of microRNAs were found DE, approximately 7-fold the number DE at 5 and 12 hpi (Table 1; see also Fig. S2C and Table S1 ). This expansion was specific to infection with live virus, as cells infected with HIV UV continued to show small numbers of DE genes at 24 hpi (Table 1; see also Fig. S2F and Table S1 ). This expansion in the number of DE microRNAs was similar to the expansion observed for DE mRNAs in the same samples (1), indicating that microRNA and mRNA expression were similarly affected by HIV-1 infection. However, out of those microRNAs found DE in HIV UV -infected cells at 24 hpi, the majority (13 out of 18) were also found DE in HIV-infected cells ( lated relative to their levels in mock-infected cells (Table 1 ; Fig. 2A and B). However, by 24 hpi, the majority of these microRNAs were found upregulated relative to mock-infected cells ( Fig. 2A and B ). This pattern was observed in the majority of DE microRNAs at earlier time points (five out of five downregulated at 5 hpi, and four out of eight downregulated at 12 hpi; Fig. 2A and B). Likewise, many of the microRNAs found DE at 24 hpi but not at earlier time points nonetheless displayed directions of changes in earlier and later time points (see Fig. S2G in the supplemental material). This phased time point specificity suggests that changes in mi-croRNAs tracked specific stages of HIV infection in vitro. Interestingly many of the trends observed in HIV-infected samples were also observed in HIV UV -infected samples, though with reduced magnitudes of change and/or not at statistical significance, indicating that viral attachment or internalization induced a similar but attenuated response, relative to live virus, even later in infection (see Fig. S2G ).