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.

On the basis of these observations, we selected two time points to analyze by NGS: 12 and 24 hpi concurrent with the beginning of viral RNA accumulation and the occurrence of near-peak RNA levels prior to cell death, respectively. We isolated RNA from infected and mock-infected replicate cell samples at these two time points, created cDNA libraries from polyadenylated RNA, and subjected the libraries to analysis by NGS. For each sample, we obtained on average over 21 million 75-nucleotide (nt) reads mapping to either HIV or human genomes. A large number of reads mapped to the viral genome (2.5 to 8 million depending on the time point, see Table S1 in the supplemental material). By 12 hpi, viral reads constituted~18% of the total mapped reads, and by 24 hpi, this proportion increased to~38%. Reads mapping to the viral genome indicated the presence of RNA splicing events. In addition to splicing events involving known splice sites, we also observed evidence of five splicing events involving one or more previously unobserved splice sites (see Fig. S2 in the supplemental material). We tested the presence of one of these sites (located 3= of the annotated splice acceptor site 2) by quantitative PCR (qPCR) and observed an amplicon size consistent with the usage of the site (Fig. S2A to S2C) .