The NS1 protein from influenza A viruses contains a four-amino-acid sequence at its carboxyl terminus that is termed the PDZ-binding motif (PBM). The NS1 PBM is predicted to bind to cellular PDZ proteins and functions as a virulence determinant in infected mice. ESEV is the consensus PBM sequence of avian influenza viruses, while RSKV is the consensus sequence of human viruses. Currently circulating highly pathogenic H5N1 influenza viruses encode an NS1 protein with the ESEV PBM. We identified cellular targets of the avian ESEV PBM and identified molecular mechanisms involved in its function. Using glutathione S-transferase (GST) pull-down assays, we found that the ESEV PBM enables NS1 to associate with the PDZ proteins Scribble, Dlg1, MAGI-1, MAGI-2, and MAGI-3. Because Scribble possesses a proapoptotic activity, we investigated the interaction between NS1 and Scribble. The association between NS1 and Scribble is direct and requires the ESEV PBM and two Scribble PDZ domains. We constructed recombinant H3N2 viruses that encode an H6N6 avian virus NS1 protein with either an ESEV or mutant ESEA PBM, allowing an analysis of the ESEV PBM in infections in mammalian cells. The ESEV PBM enhanced viral replication up to 4-fold. In infected cells, NS1 with the ESEV PBM relocalized Scribble into cytoplasmic puncta concentrated in perinuclear regions and also protected cells from apoptosis. In addition, the latter effect was eliminated by small interfering RNA (siRNA)-mediated Scribble depletion. This study shows that one function of the avian ESEV PBM is to reduce apoptosis during infection through disruption of Scribble's proapoptotic function.

The cellular kinase complex P-TEFb is composed of Cdk9 and cyclin T, and it is required for expression of most protein-coding genes by RNAP II. Cdk9 has been shown recently to be activated in cis by autophosphorylation of Thr186 in its T-loop. Using a phosphospecific Cdk9 antibody, we examined the level of Cdk9 T-loop phosphorylation in resting and activated CD4(+) T lymphocytes. Cdk9 T-loop phosphorylation was found to be low-to-undetectable in resting CD4(+) T lymphocytes, and upon activation by distinct stimuli, there is a rapid (<1 h) increase in pCdk9 that does not require protein synthesis. The low level of Cdk9 T-loop phosphorylation was not to be a result of the absence of an associated regulatory cyclin partner. These observations suggest that autophosphorylation of the Cdk9 T-loop is repressed in resting CD4(+) T lymphocytes. The low level of T-loop phosphorylation in resting cells is also reflected in a low level of phosphorylation of Ser2 in the carboxyl terminal domain of RNAP II, suggesting that lack of Cdk9 T-loop autophosphorylation may limit RNAP II elongation in quiescent CD4(+) T lymphocytes.

Cdk9 is the catalytic subunit of a general RNA polymerase II elongation factor known as positive transcription elongation factor b (P-TEFb). The kinase function of P-TEFb requires phosphorylation of Thr-186 in the T-loop of Cdk9 to allow substrates to access the catalytic core of the enzyme. To identify human phosphatases that dephosphorylate the T-loop of Cdk9, we used a Thr-186-phosphospecific antiserum to screen a phosphatase expression library. Overexpression of PPM1A and the related PPM1B greatly reduced Cdk9 T-loop phosphorylation in vivo. PPM1A and Cdk9 appear to associate in vivo as the proteins could be co-immunoprecipitated. The short hairpin RNA depletion of PPM1A resulted in an increase in Cdk9 T-loop phosphorylation. In phosphatase reactions in vitro, purified PPM1A could dephosphorylate Thr-186 both with and without the association of 7SK RNA, a small nuclear RNA that is bound to ϳ50% of total cellular P-TEFb. PPM1B only efficiently dephosphorylated Cdk9 Thr-186 in vitro when 7SK RNA was depleted from P-TEFb. Taken together, our data indicate that PPM1A and to some extent PPM1B are important negative regulators of P-TEFb function.The positive transcription elongation factor b (P-TEFb) 2 is a cellular kinase complex that regulates elongation of most mammalian protein-coding genes transcribed by RNA polymerase II (1, 2). P-TEFb enhances the processivity of RNA polymerase II through the phosphorylation of the carboxyl-terminal domain (CTD) of the polymerase as well as antagonizing the actions of negative factors such as negative elongation factor and 5,6-dichloro-1-␤-D-ribofuranosylbenzimidazole sensitivity-inducing factor. P-TEFb is comprised of cyclin-dependent kinase 9 (Cdk9), the kinase core, and its cyclin partner, either Cyclin T1, Cyclin T2, or Cyclin K. Two isoforms of Cdk9 exist, a major 42-kDa protein and a minor 55-kDa protein that contains 117 residues at the amino terminus that are absent in the 42-kDa protein (3). In most cells examined, Cyclin T1 is the predominant regulatory subunit for Cdk9. The Cyclin T1/P-TEFb complex has been studied extensively as this P-TEFb complex is targeted by the human immunodeficiency virus type 1 Tat protein to activate RNA polymerase II transcription of the integrated provirus, and this is essential for viral replication.As a key transcription factor, the activity of P-TEFb is carefully regulated within the cell. The first level of control is mediated by the regulated expression of its components, namely Cdk9 and its Cyclin partners. In primary human CD4 ϩ T lymphocytes and monocytes, Cdk9 and Cyclin T2 expression are relatively high, whereas that of Cyclin T1 is low. Upon T cell activation or monocyte differentiation, Cyclin T1 protein expression is strongly up-regulated by post-transcriptional mechanisms, whereas expression of Cdk9 and Cyclin T2 remains relatively constant (4 -9). Low levels of Cyclin T1 in resting CD4ϩ T cells and freshly isolated blood monocytes may function as one of the rate-limiting factors not only for transcription of many cellul...

P-TEFb functions to induce the elongation step of RNA polymerase II transcription by phosphorylating the carboxyl-terminal domain of the largest subunit of RNA polymerase II. Core PTEFb is comprised of Cdk9 and a cyclin regulatory subunit, with Cyclin T1 being the predominant Cdk9-associated cyclin. The kinase activity of P-TEFb is dependent on phosphorylation of the Thr186 residue located within the T-loop domain of the Cdk9 subunit. Here, we used immunofluorescence deconvolution microscopy to examine the subcellular distribution of phosphoThr186 Cdk9/Cyclin T1 P-TEFb heterodimers. We found that phospho-Thr186 Cdk9 displays a punctate distribution throughout the non-nucleolar nucleoplasm and it co-localizes with Cyclin T1 almost exclusively within nuclear speckle domains. Phospho-Thr186 Cdk9 predominantly colocalized with the hyperphosphorylated forms of RNA polymerase II. Transient expression of kinasedefective Cdk9 mutants revealed that neither is Thr186 phosphorylation or kinase activity required for Cdk9 speckle localization. Lastly, both the Brd4 and HEXIM1 proteins interact with P-TEFb at or very near speckle domains and treatment of cells with the Cdk9 inhibitor flavopiridol alters this distribution. These results indicate that the active form of P-TEFb resides in nuclear speckles and raises the possibility that speckles are sites of P-TEFb function and exchange between negative and positive P-TEFb regulatory complexes.RNA polymerase II (RNAPII) transcription is a highly regulated process involving several different stages that include pre-initiation, initiation, promoter clearance, elongation, and termination (Sims et al., 2004). The elongation step is of particular interest, as it has become increasingly clear that this stage is coordinately regulated for both the production of full-length transcripts and mRNA processing events (Sims et al., 2004;Bentley, 2005). Moreover, recent studies have shown that RNAPII elongation is the rate limiting step for expression of a large portions of cellular genes (Guenther et al., 2007;Hargreaves et al., 2009). The carboxylterminal domain (CTD) of the largest subunit of RNAPII is critical for the transition from transcriptional initiation to elongation and the integration of mRNA processing (Phatnani and Greenleaf, 2006). The CTD is an evolutionarily conserved domain and is comprised of multiple tandem copies of the consensus repeat heptad YSPTSPS (Corden, 1990). The phosphorylation pattern of the heptad repeats within the CTD change as the polymerase progresses through the stages of transcription and this appears to orchestrate the association of different cellular factors NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript involved in transcription and co-transcriptional mRNA processing (Buratowski, 2003;Sims et al., 2004;Bentley, 2005;Phatnani and Greenleaf, 2006).The transcription cycle begins with the formation of pre-initiation complexes in which the hypophosphorylated CTD form of RNAPII is recruited to gene promoters (Dahmus, 1995...

Previous analyses of human immunodeficiency virus type 1 (HIV-1) integration sites generated in infections in vitro or in patients in whom viral replication was repressed by antiviral therapy have demonstrated a preference for integration within protein-coding genes. We analyzed integration sites in peripheral blood mononuclear cells (PBMCs), spleen, lymph node, and cerebral cortex from patients with untreated HIV-1 infections. The great majority of integration sites in each tissue were within genes. Statistical analyses of the frequencies of integration in genes in PBMCs and lymph tissue demonstrated a strong preference for integration within genes. Although the sample size for brain tissue was too small to demonstrate a clear statistical preference for integration in genes, four of the five integration sites identified in brain were within genes. Taken together, our data indicate that HIV-1 preferentially integrates within genes during untreated infection.