Christine H. Herrmann

Induction of TAK (Cyclin T1/P-TEFb) in Purified Resting CD4+ T Lymphocytes by Combination of Cytokines

ABSTRACT Combinations of cytokines are known to reactivate transcription and replication of latent human immunodeficiency virus type 1 (HIV-1) proviruses in resting CD4+ T lymphocytes isolated from infected individuals. Transcription of the HIV-1 provirus by RNA polymerase II is strongly stimulated by the viral Tat protein. Tat function is mediated by a cellular protein kinase known as TAK (cyclin T1/P-TEFb) that is composed of Cdk9 and cyclin T1. We have found that treatment of peripheral blood lymphocytes and purified resting CD4+ T lymphocytes with the combination of interleukin-2 (IL-2), IL-6, and tumor necrosis factor alpha resulted in an increase in Cdk9 and cyclin T1 protein levels and an increase in TAK enzymatic activity. The cytokine induction of TAK in resting CD4+ T lymphocytes did not appear to require proliferation of lymphocytes. These results suggest that induction of TAK by cytokines secreted in the microenvironment of lymphoid tissue may be involved in the reactivation of HIV-1 in CD4+ T lymphocytes harboring a latent provirus.

Transient Induction of Cyclin T1 during Human Macrophage Differentiation Regulates Human Immunodeficiency Virus Type 1 Tat Transactivation Function

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) Tat protein is essential for viral replication and stimulates transcription of the integrated provirus by recruiting the kinase complex TAK/P-TEFb, composed of cyclin T1 (CycT1) and Cdk9, to the viral TAR RNA element. TAK/P-TEFb phosphorylates the RNA polymerase II complex and stimulates transcriptional elongation. In this report, we investigated the regulation of TAK/P-TEFb in primary human macrophages, a major target cell of HIV infection. While Cdk9 levels remained constant, CycT1 protein expression in freshly isolated monocytes was very low, increased early during macrophage differentiation, and, unexpectedly, decreased to very low levels after about 1 week in culture. The kinase activity of TAK/P-TEFb paralleled the changes in CycT1 protein expression. RNA analysis indicated that the transient induction of CycT1 protein expression involves a posttranscriptional mechanism. In transient transfection assays, the ability of Tat to transactivate the HIV long terminal repeat (LTR) in the late differentiated macrophages was greatly diminished relative to its ability to transactivate the HIV LTR in early differentiated cells, strongly suggesting that CycT1 is limiting for Tat function in late differentiated macrophages. Interestingly, lipopolysaccharide, a component of the cell wall of gram-negative bacteria, reinduced CycT1 expression late in macrophage differentiation. These results raise the possibility that regulation of CycT1 expression may be involved in establishing latent infection in macrophages and that opportunistic infection may reactivate the virus by inducing CycT1 expression.

Regulation of TAK / P-TEFb in CD4+ T Lymphocytes and Macrophages

HIV replication occurs principally in activated CD4+ T cells and macrophages. The HIV-1 Tat protein is essential for HIV replication and requires a cellular protein kinase activity termed TAK/P-TEFb, composed of CDK9 and cyclin T1, for its transactivation function. This article reviews recent work indicating that under some circumstances TAK/P-TEFb is likely to be limiting for HIV replication in CD4+ T cells and macrophages, and discusses mechanisms of regulation of the TAK/P-TEFb subunits in these cell types. In resting CD4+ T lymphocytes, TAK/P-TEFb function is low. Following lymphocyte activation, even under conditions of minimal activation in which activation markers and cellular proliferation are not induced, both CDK9 and cyclin T1 mRNA and protein levels are increased, leading to an induction of TAK/P-TEFb kinase activity that correlates with increased viral replication. In macrophages, regulation of TAK/P-TEFb involves mechanisms distinct from those in lymphocytes. In freshly isolated monocytes, CDK9 protein levels are high, while cyclin T1 protein levels are low to undetectable. Cyclin T1 protein expression is up-regulated during early macrophage differentiation by a mechanism that involves post-transcriptional regulation. Later during differentiation, cyclin T1 expression becomes shut off by a post-transcriptional mechanism, and this correlates with a decrease in Tat transactivation. Interestingly, cyclin T1 can be re-induced with lipopolysaccharide (LPS). These findings suggest that changes in cyclin T1 expression can influence HIV-1 replication levels in monocytes and macrophages. Important areas for future research on Tat and TAK/P-TEFb function are discussed.

Differential localization and expression of the Cdk9 42k and 55k isoforms

Cdk9, a member of the cyclin‐dependent kinase family, is the catalytic subunit of P‐TEFb, a protein kinase complex that stimulates transcriptional elongation. Cdk9, complexed with its regulatory partner cyclin T1, serves as the cellular mediator of the transactivation function of the HIV Tat protein. There are two known isoforms of Cdk9: a 42 kDa protein (42k, originally identified as PITALRE) and a more recently identified 55 kDa form (55k). To investigate possible functional differences between the two isoforms, we examined their kinase activities, their subcellular distributions, and their expression levels in primary cells relevant to HIV infection. Both isoforms were found to hyper‐phosphorylate the carboxyl‐terminal domain of the largest subunit of RNA polymerase II and displayed identical phosphorylation patterns with 144 peptide substrates. Epitope‐tagged transiently‐expressed Cdk9 42k localized diffusely in the nucleoplasm, while Cdk9 55k accumulated in the nucleolus. In primary undifferentiated monocytes, Cdk9 55k expression was not detected although 42k was present at high levels; however, 55k expression was induced upon macrophage differentiation. In primary lymphocytes, the levels of 55k decreased or remained steady following activation, while the levels of 42k increased. The promoter for 42k was significantly stronger than that of 55k in HeLa cells, and only the 42k promoter was responsive to activation signals in primary lymphocytes. These results indicate that expression of the 42k and 55k isoforms is differentially regulated and suggest that functional differences between the 42k and 55k isoforms of Cdk9 are likely to depend on access to substrates based on their differential subcellular localization and expression patterns.

Human Immunodeficiency Virus Type 1 Infection Induces Cyclin T1 Expression in Macrophages

ABSTRACT The Tat protein of human immunodeficiency virus type 1 (HIV-1) is essential for viral replication and activates RNA polymerase II transcriptional elongation through the association with a cellular protein kinase composed of Cdk9 and cyclin T1. Tat binds to this kinase complex through a direct protein-protein interaction with cyclin T1. Monocytes/macrophages are important targets of HIV-1 infection, and previous work has shown that cyclin T1 but not Cdk9 protein expression is low in monocytes isolated from blood. While Cdk9 expression is expressed at a high level during monocyte differentiation to macrophages in vitro, cyclin T1 expression is induced during the first few days of differentiation and is shut off after 1 to 2 weeks. We show here that the shutoff of cyclin T1 expression in late-differentiated macrophages involves proteasome-mediated proteolysis. We also show that cyclin T1 can be reinduced by a number of pathogen-associated molecular patterns that activate macrophages, indicating that up-regulation of cyclin T1 is part of an innate immune response. Furthermore, we found that HIV-1 infection early in macrophage differentiation results in sustained cyclin T1 expression, while infection at late times in differentiation results in the reinduction of cyclin T1. Expression of the viral Nef protein from an adenovirus vector suggests that Nef contributes to the HIV-1 induction of cyclin T1. These findings suggest that HIV-1 infection hijacks a component of the innate immune response in macrophages that results in enhancement rather than inhibition of viral replication.

siRNA depletion of 7SK snRNA induces apoptosis but does not affect expression of the HIV‐1 LTR or P‐TEFb‐dependent cellular genes

P‐TEFb is a general transcriptional elongation factor composed of Cdk9 and either cyclin T1, T2, or K. A substantial portion of P‐TEFb is associated with the 7SK small nuclear RNA (7SK) and the HEXIM1 or HEXIM2 proteins; this complex has reduced kinase activity in vitro relative to free P‐TEFb. Here we report that 7SK and HEXIM1 levels are induced in activated lymphocytes concomitantly with increased P‐TEFb activity and global transcription. We used siRNA‐mediated depletion to probe the function of 7SK in HeLa cells. Depletion of 7SK caused a large reduction in the association of HEXIM1 with Cdk9 and cyclin T1, and greatly reduced the amount of the cyclin T1 present in the 7SK/HEXIM1/P‐TEFb complex. Similar to previous studies, siRNA‐mediated depletion of 7SK resulted in increased expression of several reporter plasmids tested, including a plasmid lacking promoter elements. However, in contrast to previous studies, which did not examine the effects of 7SK depletion on endogenous gene expression, depletion of 7SK did not appear to affect the expression of the corresponding endogenous genes. Moreover, 7SK depletion had no effect on expression from the integrated HIV‐1 provirus or the c‐myc and MCL‐1 genes, three transcription units known to be highly dependent upon P‐TEFb. Importantly, depletion of 7SK was found to cause apoptosis by 72 h post‐transfection in HeLa cells. These results suggest that 7SK may provide an essential cellular function whose relation to P‐TEFb function is unclear.

Cyclin T1 but not cyclin T2a is induced by a post-transcriptional mechanism in PAMP-activated monocyte-derived macrophages

Positive transcription elongation factor b (P‐TEFb) is an RNA polymerase II elongation factor which exists as multiple complexes in human cells. These complexes contain cyclin‐dependent kinase 9 as the catalytic subunit and different cyclin subunits—cyclin T1, T2a, T2b, or K. Cyclin T1 is targeted by the human immunodeficiency virus (HIV) Tat protein to activate transcription of the HIV provirus. Expression of this P‐TEFb subunit is highly regulated in monocyte‐derived macrophages (MDMs). Cyclin T1 is induced early during differentiation and is shut off later by proteasome‐mediated proteolysis. Cyclin T1 can be reinduced by pathogen‐associated molecular patterns (PAMPs) or HIV infection. In this study, we analyzed regulation of P‐TEFb in MDMs by examining 7SK small nuclear RNA and the HEXIM1 protein; these factors associate with P‐TEFb and are thought to regulate its function. 7SK and HEXIM1 were induced early during differentiation, and this correlates with increased overall transcription. 7SK expression remained high, but HEXIM1 was shut off later during differentiation by proteasome‐mediated proteolysis. Significantly, the cyclin T2a subunit of P‐TEFb was not shut off during differentiation, and it was not induced by activation. Induction of cyclin T1 by PAMPs was found to be a slow process and did not involve an increase in cyclin T1 mRNA levels. Treatment of MDMs with PAMPs or a proteasome inhibitor induced cyclin T1 to a level equivalent to treatment with both agents together, suggesting that PAMPs and proteasome inhibitors act at a similar rate‐limiting step. It is therefore likely that cyclin T1 induction by PAMPs is the result of a reduction in proteasome‐mediated proteolysis.

55K isoform of CDK9 associates with Ku70 and is involved in DNA repair

Positive elongation factor b (P-TEFb) is a cellular protein kinase that is required for RNA polymerase II (RNAP II) transcriptional elongation of protein coding genes. P-TEFb is a set of different molecular complexes, each containing CDK9 as the catalytic subunit. There are two isoforms of the CDK9 protein - the major 42KDa CDK9 isoform and the minor 55KDa isoform that is translated from an in-frame mRNA that arises from an upstream transcriptional start site. We found that shRNA depletion of the 55K CDK9 protein in HeLa cells induces apoptosis and double-strand DNA breaks (DSBs). The levels of apoptosis and DSBs induced by the depletion were reduced by expression of a 55K CDK9 protein variant resistant to the shRNA, indicating that these phenotypes are the consequence of depletion of the 55K protein and not off-target effects. We also found that the 55K CDK9 protein, but not the 42K CDK9 protein, specifically associates with Ku70, a protein involved in DSB repair. Our findings suggest that the 55K CDK9 protein may function in repair of DNA through an association with Ku70.