c 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 Tloop-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.

BackgroundProcessive elongation of the integrated HIV-1 provirus is dependent on recruitment of P-TEFb by the viral Tat protein to the viral TAR RNA element. P-TEFb kinase activity requires phosphorylation of Thr186 in the T-loop of the CDK9 subunit. In resting CD4+T cells, low levels of T-loop phosphorylated CDK9 are found, which increase significantly upon activation. This suggests that the phosphorylation status of the T-loop is actively regulated through the concerted actions of cellular proteins such as Ser/Thr phosphatases. We investigated the role of phosphatase PPM1A in regulating CDK9 T-loop phosphorylation and its effect on HIV-1 proviral transcription.ResultsWe found that overexpression of PPM1A inhibits HIV-1 gene expression during viral infection and this required PPM1A catalytic function. Using an artificial CDK tethering system, we further found that PPM1A inhibits CDK9, but not CDK8 mediated activation of the HIV-1 LTR. SiRNA depletion of PPM1A in resting CD4+T cells increased the level of CDK9 T-loop phosphorylation and enhanced HIV-1 gene expression. We also observed that PPM1A protein levels are relatively high in resting CD4+T cells and are not up-regulated upon T cell activation.ConclusionsOur results establish a functional link between HIV-1 replication and modulation of CDK9 T-loop phosphorylation by PPM1A. PPM1A represses HIV-1 gene expression by inhibiting CDK9 T-loop phosphorylation, thus reducing the amount of active P-TEFb available for recruitment to the viral LTR. We also infer that PPM1A enzymatic activity in resting and activated CD4+ T cells are likely regulated by as yet undefined factors.

The HIV/AIDS field is gaining momentum in the goal of finding a functional cure for HIV infection by utilizing strategies that specifically reactivate the latent viral reservoir in combination with the HAART regimen to prevent further viral spread. Small-molecule inhibitors such as histone deacetylase (HDAC) and bromodomain and extraterminal (BET) inhibitors can successfully activate HIV transcription and reverse viral latency in clonal cell lines. However, in resting CD4+ T cells, thought to be the principal physiological reservoir of latent HIV, their effect in reactivating the viral reservoir is more variable. It is possible that the discrepant responsiveness of quiescent primary CD4+ T cells to HDAC and BET inhibitors could be attributed to the limiting levels of P-TEFb, a key viral transcription host cofactor, in these cells. In this review, we discuss the role of P-TEFb and the necessity for its mobilization in stimulating viral reactivation from latency upon treatment with HDAC and BET inhibitors.

By recruiting the host protein XPO1 (CRM1), the HIV-1 Rev protein mediates the nuclear export of incompletely spliced viral transcripts. We mined data from the recently described human nuclear complexome to identify a host protein, RBM14, which associates with XPO1 and Rev and is involved in Rev function. Using a Rev-dependent p24 reporter plasmid, we found that RBM14 depletion decreased Rev activity and Rev-mediated enhancement of the cytoplasmic levels of unspliced viral transcripts. RBM14 depletion also reduced p24 expression during viral infection, indicating that RBM14 is limiting for Rev function. RBM14 has previously been shown to localize to nuclear paraspeckles, a structure implicated in retaining unspliced HIV-1 transcripts for either Rev-mediated nuclear export or degradation. We found that depletion of NEAT1 RNA, a long noncoding RNA required for paraspeckle integrity, abolished the ability of overexpressed RBM14 to enhance Rev function, indicating the dependence of RBM14 function on paraspeckle integrity. Our study extends the known host cell interactome of Rev and XPO1 and further substantiates a critical role for paraspeckles in the mechanism of action of Rev. Our study also validates the nuclear complexome as a database from which viral cofactors can be mined. IMPORTANCEThis study mined a database of nuclear protein complexes to identify a cellular protein named RBM14 that is associated with XPO1 (CRM1), a nuclear protein that binds to the HIV-1 Rev protein and mediates nuclear export of incompletely spliced viral RNAs. Functional assays demonstrated that RBM14, a protein found in paraspeckle structures in the nucleus, is involved in HIV-1 Rev function. This study validates the nuclear complexome database as a reference that can be mined to identify viral cofactors. Since the HIV-1 genome encodes only 15 proteins, the virus must exploit the function of host cofactors at every step in its replication cycle (1). A meta-analysis of genome-wide small interfering RNA (siRNA) screens suggests that more than 2,410 proteins, or 9.5% of human genes, may be involved in HIV-1 replication (2). While data from siRNA screens provide insight into virus-host interactions and provide an opportunity to identify "druggable" targets to inhibit virus replication, these screens have limitations. Comparison of different genome-wide siRNA screens reveal minimal overlap in the host genes involved in HIV-1 replication, and these screens may frequently yield false-positive results due to siRNA off-target effects (3).In the present study, we utilized a novel strategy to identify host factors involved in HIV-1 replication. The human nuclear "complexome" was recently described in a high-throughput immunoprecipitation/mass spectrometry (IP/MS) study (4). This complexome describes endogenous protein complexes defined by identification of proteins that coimmunoprecipitated in more than 3,000 immunoprecipitations of HeLa cell nuclear extracts. We mined the nuclear complexome to identify cellular proteins found in complexe...

Productive transcription of the integrated HIV-1 provirus is restricted by cellular factors that inhibit RNA polymerase II elongation. The viral Tat protein overcomes this by recruiting a general elongation factor, P-TEFb, to the TAR RNA element that forms at the 5’ end of nascent viral transcripts. P-TEFb exists in multiple complexes in cells, and its core consists of a kinase, Cdk9, and a regulatory subunit, either Cyclin T1 or Cyclin T2. Tat binds directly to Cyclin T1 and thereby targets the Cyclin T1/P-TEFb complex that phosphorylates the CTD of RNA polymerase II and the negative factors that inhibit elongation, resulting in efficient transcriptional elongation. P-TEFb is tightly regulated in cells infected by HIV-1—CD4 + T lymphocytes and monocytes/macrophages. A number of mechanisms have been identified that inhibit P-TEFb in resting CD4 + T lymphocytes and monocytes, including miRNAs that repress Cyclin T1 protein expression and dephosphorylation of residue Thr186 in the Cdk9 T-loop. These repressive mechanisms are overcome upon T cell activation and macrophage differentiation when the permissivity for HIV-1 replication is greatly increased. This review will summarize what is currently known about mechanisms that regulate P-TEFb and how this regulation impacts HIV-1 replication and latency.