Cdk2 catalytic activity is essential for meiotic cell division in vivo.

Cyclin-dependent kinases (Cdks) control the eukaryotic cell cycle by phosphorylating serine and threonine residues in key regulatory proteins, but some Cdk family members may exert kinase-independent functions that cannot easily be assessed using gene knockout approaches. While Cdk2-deficient mice display near-normal mitotic cell proliferation due to the compensatory activities of Cdk1 and Cdk4, they are unable to undergo meiotic generation of gametes and are consequently sterile. To investigate whether Cdk2 regulates meiosis via protein phosphorylation or by alternative kinase-independent mechanisms, we generated two different knockin mouse strains in which Cdk2 point mutations ablated enzyme activity without altering protein expression levels. Mice homozygous for the mutations Cdk2(D145N/D145N) or Cdk2(T160A/T160A) expressed only 'kinase-dead' variants of Cdk2 under the control of the endogenous promoter, and despite exhibiting normal expression of cell cycle regulatory proteins and complexes, both mutations rendered mice sterile. Mouse cells that expressed only 'kinase-dead' variants of Cdk2 displayed normal mitotic cell cycle progression and proliferation both in vitro and in vivo, indicating that loss of Cdk2 kinase activity exerted little effect on this mode of cell division. In contrast, the reproductive organs of Cdk2 mutant mice exhibited abnormal morphology and impaired function associated with defective meiotic cell division and inability to produce gametes. Cdk2 mutant animals were therefore comparable to gene knockout mice, which completely lack the Cdk2 protein. Together, our data indicate that the essential meiotic functions of Cdk2 depend on its kinase activity, without which the generation of haploid cells is disrupted, resulting in sterility of otherwise healthy animals.

The PI3K pathway acting on alternative HIV-1 pre-mRNA splicing.

HIV-1 mediates pro-survival signals and prevents apoptosis via the phosphatidylinositol-3-kinase (PI3K) pathway. This pathway, however, also affects phosphorylation of serine-arginine (SR) proteins, a family of splicing regulatory factors balancing splice site selection. We now show that pharmacologic inhibition of PI3K signalling alters the HIV-1 splicing pattern of both minigene- and provirus-derived mRNAs. This indicates that HIV-1 might also promote PI3K signalling to balance processing of its transcripts by regulating phosphorylation of splicing regulatory proteins.

Cloning the simian varicella virus genome in E. coli as an infectious bacterial artificial chromosome.

Simian varicella virus (SVV) is closely related to human varicella-zoster virus and causes varicella and zoster-like disease in nonhuman primates. In this study, a mini-F replicon was inserted into a SVV cosmid, and infectious SVV was generated by co-transfection of Vero cells with overlapping SVV cosmids. The entire SVV genome, cloned as a bacterial artificial chromosome (BAC), was stably propagated upon serial passage in E. coli. Transfection of pSVV-BAC DNA into Vero cells yielded infectious SVV (rSVV-BAC). The mini-F vector sequences flanked by loxP sites were removed by co-infection of Vero cells with rSVV-BAC and adenovirus expressing Cre-recombinase. Recombinant SVV generated using the SVV-BAC genetic system has similar molecular and in vitro replication properties as wild-type SVV. To demonstrate the utility of this approach, a SVV ORF 10 deletion mutant was created using two-step Red-mediated recombination. The results indicate that SVV ORF 10, which encodes a homolog of the HSV-1 virion VP-16 transactivator protein, is not essential for in vitro replication but is required for optimal replication in cell culture.