HIV-1 provirus transcription and translation in macrophages differs from pre-integrated cDNA complexes and requires E2F transcriptional programs

HIV-1 cDNA pre-integration complexes have been shown to persist for weeks in macrophages and to be transcriptionally active. Early and late gene transcripts are produced, along with some viral proteins, yet whole virus is not. Previous work has focused on the transcription and translation of HIV-1 genes; however, our understanding of cellular milieu that accompanies viral production is incomplete. We have used an in vitro system to model HIV-1 infection of macrophages, and single cell RNA sequencing to compare the transcriptomes of uninfected cells, cells harboring pre-integration HIV-1 complexes and those actively making late HIV proteins. These are also compared to control cells, not exposed to virus. Several observations provide new perspective on the effects of HIV-1 transcription from pre-integrated cDNA versus from integrated provirus. First, HIV-1 transcript levels do not necessarily correlate with virus production, many cells harboring pre-integrated complexes have transcript loads comparable to cells making late virus proteins. Second, early and late transcripts are easily detectable in abundance in from pre-integration cDNA transcription, although the cells containing integrated provirus and actively making late proteins are more likely to be expressing gag and accessory protein transcripts at higher levels. Third, the background transcriptomes of cells harboring pre-integration HIV-1 cDNA are not otherwise detectably altered from cells not containing any HIV-1 transcript. Fourth, integration and production of late viral proteins is accompanied by a switch from transcriptomes dominated by NFkB and AP-1 promoted transcription to a transcriptome dominated by E2F family transcription products. Even though NFkB and AP-1 are transcription factors in the HIV-1 LTR, and E2F has been reported to suppress HIV-1 transcription, it is clear that when cells in this model are making late HIV-1 gene proteins the transcriptome of the cell is dominated by E2F regulated transcripts. While some of these observations may seem heretical, single cell analysis provides a more nuanced understanding of pre-integration cDNA transcription and the transcriptome changes that support late HIV-1 protein production.