HSV-2 Triggers Upregulation of MALAT1 in CD4+ T cells and Promotes HIV Latency Reversal (Bulk RNA-Seq)

HSV-2 coinfection is associated with increased HIV-1 viral loads and expanded tissue reservoirs, but the mechanisms are not well-defined. HSV-2 recurrences result in an influx of activated CD4+ T cells to sites of viral replication and an increase in activated CD4+ T cells in peripheral blood. We hypothesized that HSV-2 induces changes in these cells that facilitate HIV-1 reactivation and replication and tested this hypothesis in human CD4+ T cells and 2D10 cells, a model of HIV-1 latency. HSV-2 promoted latency reversal in HSV-2 infected and bystander 2D10 cells. Bulk and single-cell RNA sequencing studies of activated primary human CD4+ T cells identified decreased expression of HIV-1 restriction factors and increased expression of transcripts including MALAT1 that could drive HIV replication in both the HSV-2-infected and bystander cells. Transfection of 2D10 cells with VP16, an HSV-2 protein that regulates transcription, significantly upregulated MALAT1 expression, decreased trimethylation of lysine 27 on histone H3 protein, and triggered HIV latency reversal. Knockout of MALAT1 from 2D10 cells abrogated the response to VP16 and reduced the response to HSV-2 infection. These results demonstrate that HSV-2 contributes to HIV-1 reactivation through diverse mechanisms including upregulation of MALAT1 to release epigenetic silencing. CD4 T cells from 5 healthy donor leukopaks were stimulated with ImmunoCultTM Human CD3/CD28 T Cell Activator (Cat. 10971, STEMCELL Technologies) for 72 h. Stimulated cells were mock infected or infected with HSV-2(333 ZAG) at MOI = 10 as described above. 24 hours post-infection cells were sorted on GFP by FACS, and RNA was isolated using the miRNeasy Micro Kit (Qiagen Cat. no. 217084) per manufacturer’s instructions.