Cyclin T1 and CDK9 T-Loop Phosphorylation Are Downregulated during Establishment of HIV-1 Latency in Primary Resting Memory CD4 + T Cells

Abstract

ABSTRACT P-TEFb, a cellular kinase composed of Cyclin T1 and CDK9, is essential for processive HIV-1 transcription. P-TEFb activity is dependent on phosphorylation of Thr186 in the CDK9 T loop. In resting CD4 + T cells which are nonpermissive for HIV-1 replication, the levels of Cyclin T1 and T-loop-phosphorylated CDK9 are very low but increase significantly upon cellular activation. Little is known about how P-TEFb activity and expression are regulated in resting central memory CD4 + T cells, one of the main reservoirs of latent HIV-1. We used an in vitro primary cell model of HIV-1 latency to show that P-TEFb availability in resting memory CD4 + T cells is governed by the differential expression and phosphorylation of its subunits. This is in contrast to previous observations in dividing cells, where P-TEFb can be regulated by its sequestration in the 7SK RNP complex. We find that resting CD4 + T cells, whether naïve or memory and independent of their infection status, have low levels of Cyclin T1 and T-loop-phosphorylated CDK9, which increase upon activation. We also show that the decrease in Cyclin T1 protein upon the acquisition of a memory phenotype is in part due to proteasome-mediated proteolysis and likely also to posttranscriptional downregulation by miR-150. We also found that HEXIM1 levels are very low in ex vivo - and in vitro -generated resting memory CD4 + T cells, thus limiting the sequestration of P-TEFb in the 7SK RNP complex, indicating that this mechanism is unlikely to be a driver of viral latency in this cell type.