Next-Generation Sequencing Reveals HIV-1-Mediated Suppression of T Cell Activation and RNA Processing and Regulation of Noncoding RNA Expression in a CD4(+) T Cell Line

Chang, Stewart T.; Sova, Pavel; Peng, Xinxia; Weiss, Jeffrey; Law, G. Lynn; Palermo, Robert E.; Katze, Michael G.

HIV-1 infection results in a small set of differentially expressed host genes at 12 hpi. To characterize the host response to HIV infection, we mapped the remaining nonviral reads to RefSeq-annotated human gene loci and quantified them as FPKM (fragments per kilobase of exon per million mapped fragments). We selected a set of 34 host genes whose expression spanned a range of values and measured the expression levels directly by qPCR (see Fig. S3 in the supplemental material). Measurements by NGS showed a high degree of correlation with qPCR results at both time points (R Ï­ 0.97 and R Ï­ 0.98 at 12 and 24 hpi, respectively), indicating a close match between the two methods. We then identified specific host genes that were differentially expressed (DE) during infection. Comparing FPKM for each gene in infected cells to time-matched mock-infected cells, we found that a total of 106 genes were DE at 12 hpi (representing all fold changes with Benjamini-Hochberg-adjusted P values of less than 0.05; Table 1 ). By 24 hpi, the number of DE genes had increased to 5,006 representing over 50% of all 9,992 gene loci detected under stringent criteria (Table 1) . Closer examination of these values showed that a large proportion of DE genes occurred with smallmagnitude changes. In particular, 69% (73 of 106) and 49% (2,465 of 5,006) of the expressed genes exhibited 1-to 1.5-fold changes at 12 and 24 hpi, respectively ( Table 1 ). The observed precision of the sequencing-based measurements (Fig. S3 ) supported the use of low-fold change thresholds. Other values for the statistical threshold were also used, and for all but the most stringent thresholds (when fewer than 10 genes were observed to be up-or downregulated at 12 hpi), a similar predominance of small-magnitude changes was observed (see Table S2 in the supplemental material).