Transcriptomic changes in primary CD4+ T cells upon infection with HIV-1 harboring an intact or defective vpr open-reading frame
We performed unbiased RNA sequencing (RNA-seq) analyses of primary CD4+ T cells infected with HIV-1 harboring an intact or defective Vpr open reading frame and analyzed which genes are deregulated in a Vpr-dependent manner. Our transcriptome analyses showed that NFAT controls 46.5% of 1083 Vpr-deregulated genes in primary CD4+ T cells. Gene set enrichment analyses revealed that Vpr upregulated processes related to T cell signaling, proliferation, and immune function, while downregulating cell cycle progression, ribosome activity, and cytoskeleton organization. qRT-PCR confirmed Vpr-mediated modulation of specific genes, including the upregulation of NEIL1, TNFS4 and CXCL10, and the downmodulation of CCNB1, CDC20, CENPA and PLK1. Hence, a significant portion of Vpr-deregulated genes in primary CD4+ T cells are controlled by NFAT, affecting pathways related to T cell activation, cell cycle progression, cytoskeleton, and chromosome organization. Overall design: PHA-prestimulated CD4+ T cells were infected with purified and concentrated HIV-1 stocks via spinoculation and analyzed at 48 hours post infection by flow cytometry to assess infection rates. Upon confirmation of similarly high infection rates, CD4+ T cells of three healthy donors were subjected to RNA extraction and RNA-seq. Sequencing yielded 21 to 51 million reads per sample.