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.

Several of the microRNAs that we observed as DE, particularly at early time points, were either poorly characterized or not previously associated with HIV, e.g., as reviewed by Houzet and Jeang (20) or Swaminathan et al. (21) . By identifying target mRNAs with anticorrelated expression patterns, we implicated one of these DE microRNAs, miR-3607-3p, in the regulation of cell cycle-and transcription-associated mRNAs. HIV infection is known to downregulate cell cycle-related genes (1, 22) , and differing levels of regulation with time may reflect evolving requirements of the virus following entry. In particular, the upregulation of cell cyclerelated transcription factors prior to replication, mediated by the downregulation of microRNAs such as miR-3607-3p, may result in the internal production of raw materials needed for viral replication or convert host chromatin to an open configuration. In particular, the NFATC3 (nuclear factor of activated T cells, cytoplasmic 3) gene, a target of miR-3607-3p in our analysis, encodes a transcription factor that has recently been shown to be involved in T-cell activation (23) . Likewise, the TCF7 gene, another predicted target of miR-3607-3p, encodes a T-cell-specific transcription factor (24) . Relieving the inhibition of these transcription factors via the suppression of miR-3607-3p and other microRNAs may represent an early event of HIV infection which is reversed later in HIV infection. Consistent with this interpretation, the MYH9 gene (myosin, heavy chain 9, nonmuscle), a target mRNA of miR-3607-3p found downregulated later in infection, was also found to be downregulated in HIV-infected human glomeruli, which may contribute to HIV-associated pathology (25) . While we cannot discern the mechanism by which viral entry or internalization might suppress miR-3607-3p and other early DE mi-croRNAs from our data, we speculate that a surface protein such as gp120 initiates a signaling cascade that destabilizes the available pool of miR-3607-3p. In general, we observed agreement between our results and those from previous studies, particularly for other, highly expressed microRNAs. For example, Hayes et al. measured changes in CEMx174 lymphocytes infected with HIV-1 strain NL4-3 (26) . Out of the 42 DE microRNAs observed by Hayes et al., 11 were also found to be DE at 24 hpi in our data set, perhaps indicating cell type specificity (26) . Of these, eight displayed concordant changes between the two data sets, with complete concordance observed for the most highly expressed microRNAs (let-7e, miR-92, and miR-106a, all with Ͼ10,000 reads per sample in uninfected SUP-T1 cells) (26) . A similar concordance among highly expressed microRNAs was observed for clinical samples as well. For example, among the DE microRNAs observed in PBMCs from HIV-infected individuals by Houzet et al., four out of the five mostly highly expressed microRNAs (let-7e, miR-20a, miR-20b, miR-92, and miR-182, again with Ͼ10,000 reads per sample in uninfected SUP-T1 cells) displayed directional concordance (6) . Likewise, concordance with the clinical data set from Witwer et al. was evident for highly expressed microRNAs, such as miR-155 (upregulated by 1.5-fold in PBMCs from viremic individuals and by 1.8-fold in our study at 24 hpi) and miR-181b (upregulated by 1.8-fold in PBMCs from viremic individuals and in our study at 24 hpi) (11) . Highly expressed microRNAs may therefore represent cell type-robust signatures.