NDV has the potential for further development as a pretested, highly attenuated, intranasal vector to be available for expedited vaccine development for humans, who generally lack preexisting immunity against NDV.severe acute respiratory syndrome (SARS) ͉ respiratory tract ͉ monkey

Respiratory syncytial virus (RSV) is

BackgroundHIV-1 transcription is activated by the viral Tat protein that recruits host positive transcription elongation factor-b (P-TEFb) containing CDK9/cyclin T1 to the HIV-1 promoter. P-TEFb in the cells exists as a lower molecular weight CDK9/cyclin T1 dimer and a high molecular weight complex of 7SK RNA, CDK9/cyclin T1, HEXIM1 dimer and several additional proteins. Our previous studies implicated CDK2 in HIV-1 transcription regulation. We also found that inhibition of CDK2 by iron chelators leads to the inhibition of CDK9 activity, suggesting a functional link between CDK2 and CDK9. Here, we investigate whether CDK2 phosphorylates CDK9 and regulates its activity.ResultsThe siRNA-mediated knockdown of CDK2 inhibited CDK9 kinase activity and reduced CDK9 phosphorylation. Stable shRNA-mediated CDK2 knockdown inhibited HIV-1 transcription, but also increased the overall level of 7SK RNA. CDK9 contains a motif (90SPYNR94) that is consensus CDK2 phosphorylation site. CDK9 was phosphorylated on Ser90 by CDK2 in vitro. In cultured cells, CDK9 phosphorylation was reduced when Ser90 was mutated to an Ala. Phosphorylation of CDK9 on Ser90 was also detected with phospho-specific antibodies and it was reduced after the knockdown of CDK2. CDK9 expression decreased in the large complex for the CDK9-S90A mutant and was correlated with a reduced activity and an inhibition of HIV-1 transcription. In contrast, the CDK9-S90D mutant showed a slight decrease in CDK9 expression in both the large and small complexes but induced Tat-dependent HIV-1 transcription. Molecular modeling showed that Ser 90 of CDK9 is located on a flexible loop exposed to solvent, suggesting its availability for phosphorylation.ConclusionOur data indicate that CDK2 phosphorylates CDK9 on Ser 90 and thereby contributes to HIV-1 transcription. The phosphorylation of Ser90 by CDK2 represents a novel mechanism of HIV-1 regulated transcription and provides a new strategy for activation of latent HIV-1 provirus.

The cyclin-dependent kinase CDK9/cyclin T1 induces HIV-1 transcription by phosphorylating the carboxyterminal domain (CTD) of RNA polymerase II (RNAPII). CDK9 activity is regulated by protein phosphatase-1 (PP1) which was previously shown to dephosphorylate CDK9 Thr186. Here, we analyzed the effect of PP1 on RNAPII phosphorylation and CDK9 activity. The selective inhibition of PP1 by okadaic acid and by NIPP1 inhibited phosphorylation of RNAPII CTD in vitro and in vivo. Expression of the central domain of NIPP1 in cultured cells inhibited the enzymatic activity of CDK9 suggesting its activation by PP1. Comparison of dephosphorylation of CDK9 phosphorylated by (32P) in vivo and dephosphorylation of CDK9's Thr186 analyzed by Thr186 phospho-specific antibodies, indicated that a residue other than Thr186 might be dephosphorylated by PP1. Analysis of dephosphorylation of phosphorylated peptides derived from CDK9's T-loop suggested that PP1 dephosphorylates CDK9 Ser175. In cultured cells, CDK9 was found to be phosphorylated on Ser175 as determined by combination of Hunter 2D peptide mapping and LC-MS analysis. CDK9 S175A mutant was active and S175D – inactive, and dephosphorylation of CDK9's Ser175 upregulated HIV-1 transcription in PP1-dependent manner. Collectively, our results point to CDK9 Ser175 as novel PP1-regulatory site which dephosphorylation upregulates CDK9 activity and contribute to the activation of HIV-1 transcription.

The NS1 and NS2 proteins of human respiratory syncytial virus (HRSV) have been shown to antagonize the type I interferon (IFN) response, an effect subject to host range constraints. We have now found that the HRSV NS2 protein strongly controls IFN induction in mouse cells in vitro, validating the use of the mouse model to study the consequences of these gene deletions on host immunity. We evaluated the effects of deleting the NS1 and/or NS2 gene on the induction of HRSV-specific pulmonary cytotoxic T lymphocytes (CTL) in BALB/c and 129S6 mice in response to intranasal infection with HRSV lacking the NS1 and/or NS2 gene and subsequent challenge with wild-type (wt) HRSV. In mice infected with HRSV lacking the NS2 gene (⌬NS2) or lacking the NS2 gene in combination with the NS1 gene (⌬NS1/2 HRSV), the magnitude of the pulmonary CTL response was substantially elevated compared to that of mice infected with wt HRSV or the ⌬NS1 mutant, whether measured by binding of CD8 ؉ cells to an HRSV-specific major histocompatibility complex class I tetramer, by measurement of CD8 ؉ cells secreting gamma interferon (IFN-␥) in response to specific in vitro stimulation, or by a standard chromium release cell-killing assay. In contrast, in STAT1 knockout mice, which lack responsiveness to type I IFN, the level of IFN-␥-secreting CD8 ؉ cells was not significantly different for HRSV lacking the NS2 gene, suggesting that the increase in CTL observed in IFN-responsive mice is type I IFN dependent. Thus, the NS2 protein of HRSV suppresses the CTL component of the adaptive immune response, and this appears to be a consequence of its suppression of type I IFN.The interferon (IFN) system is an important component of host defense against viral pathogens. Type I IFNs include at least 14 species of alpha interferon (IFN-␣) and a single species of IFN-␤, and type II IFN is represented by a single species of IFN-␥. More recently, type III IFNs have been described, represented by IFN-1, -2, and -3 (also known as interleukin 29 , 28A, and 28B, respectively), which appear to have a number of similarities with type I IFNs (29). Type I and III IFNs are produced in response to viral infections or other stimuli by many types of cells, with type I IFN being produced most abundantly by plasmacytoid dendritic cells (4), whereas type II IFN is mostly secreted by activated T cells and NK cells. Type I IFNs bind to a specific cell surface receptor that activates JAK-STAT signal transduction, resulting in transcriptional activation of a broad panel of cellular genes. Some of these activated genes encode proteins such as Mx, PKR, and OAS that inhibit viral replication by various mechanisms, such as inhibition of intracellular trafficking of viral nucleocapsids, blocking of translation of viral proteins, and enzymatic degradation of viral RNA (reviewed in references 15 and 37). Type I IFNs are also involved in regulation of adaptive immune responses, including CD8 ϩ cytotoxic T lymphocytes (CTL) (reviewed in reference 45).Viruses exhibit a number of mechanisms t...