Stochastic HIV Gene Expression is a Novel Mechanism for HIV Persistence

Abstract

Abstract In HIV-infected people, the virus persists in resting memory CD4 T cells as a latent provirus integrated into transcribed genes. HIV has 9 gene products that are generated from 7 alternatively spliced mRNAs whose expression is regulated by the cell’s transcriptional state. Furthermore, transcription of genes accessible by RNA pol II occurs in bursts, giving rise to the stochastic expression of mRNAs in cells. The HIV protein Nef allows infected cells to evade CD8 T cell recognition through the downregulating of peptide-MHC complexes (pMHCs). The precise kinetics of pMHC downregulation by Nef upon reactivation of a latent provirus remains unclear. We explored this question by direct infection and longitudinal analysis of primary resting CD4 T cells with a CCR5-tropic intact replication-competent virus in which GFP reports the expression of Nef. We detected GFP+ cells 3 to 4 days after infection. These GFP+ cells were resting memory CD4 T cells and had downregulated CD4 and pMHCs. We then performed single-cell RNA-seq on sorted GFP+ cells to examine both cellular and virus gene expression, as well as HIV integration sites. Our dataset identified various transcribed host genes that had an integrated provirus. Strikingly we found GFP+ cells either (1) had no HIV mRNAs or (2) had HIV mRNAs encoding Nef, Vpr, Vif or Vpu-Env, but never HIV mRNAs encoding Gag-Pol, Tat or Rev. We conclude that in resting memory CD4 T cells, an integrated HIV genome is transcribed and alternatively spliced for mRNAs encoding Nef, Vif, Vpr or Vpu-Env. HIV expression is stochastic because infected cells didn’t always express HIV mRNAs. Therefore we propose stochastic HIV gene expression is a novel mechanism for HIV persistence by hiding the infected cells from the immune system.