The Role Of CDK2 In The Replication Of Herpes Viruses

A high percentage of the world's population and animals are infected with Herpes virus and infection can cause a variety of disorders. Herpes virus can establish lifelong latent and recurrent infection after acute infection. The study on the mechanism of herpes virus replication will advance our knowledge of the virus-cell interactions and reduce the threat to he human health and the economical losses of cattle industry. Herpes viruses and other DNA viruses that possess large genomes encode many major and accessory DNA replication proteins and can replicate in arrested cells such as neurons, thus their replication was long thought to be independent from cell cycle and cellular proteins whose activities are regulated in a cell cycle dependent manner such as the cyclin-dependent kinase(CDK). But studies in 1998 show that replication of herpes virus (HSV) can be inhibited by pharmacological CDK inhibitors (PCI). Since then more attention has been drawn on the study on the role of CDK in the replication of viruses and HSV, again, becomes an important model for the study of virus-host cells interactions.There is not yet unanimous agreement on the identity of the CDKs whose activities are required in HSV replication. According to the paper review and our previous results, our hypothesis is that CDK2 plays pivotal role in the initiation of productive replication of HSV and reactivation from latency. To address this question systematically, we used doxycycline (Dox) inducible dominant negative CDK2 (dn-CDK2) cell lines and siRNA against CDK2, cyclin E and cyclin A to inhibit CDK2 activity and observe the relationship between the the replication of HSV and CDK2 activity. The results shows that the replication of HSV in CDK2 deficient cells was multiplicity of infection ( MOI ) dependent; The CDK2 activity of the Dox treated HT29 Tet-On dn-CDK2 cells was induced 9 hour post infection; The HSV went into rapid productive replication after CDK2 was induced. The discovery shows directly in the first time the relationship between HSV replication and CDK2 activity. Micrococcal nuclease (MCN) digestion experiment shows that the HSV genome was resistant to MCN 5 hours post infection (hpi) in Dox induced HT29 Tet-On dn-CDK2 cells, suggesting that CDK2 may promote the HSV genome to form "open" chromosome to regulate HSV gene expression.1. The replication of HSV in HT29 Tet-On dn-CDK2 cellsHT29 Tet-On dn-CDK2 cells incorporated a doxycycline (Dox) inducible Tet On promoter regulated dn-CDK2 gene and can express the dn-CDK2 protein which can down regulate the activity of CDK2. HT29 Tet-On dnCDK2 cells were treated or untreated with 5μg/ml Dox and infected with HSV 48 hours after treating. Virus was harvested 24 hpi and titrated. To our surprise that there is no obvious difference of the production of infectious HSV when infected with high MOI between the Dox induced and uninduced HT29 Tet-On dn-CDK2 cells, while the production of infectious HSV was 10 times higher in uninduced HT29 Tet-On dn-CDK2 cells than that of Dox induced HT29 Tet-On dn-CDK2 cells. The results suggest there is MOI dependency of the growth of HSV in the dn-CDK2 expressing HT29 Tet-On dn-CDK2 cells, which is similar with the growth of ICP0-null mutant HSV in cultured cells. Our results suggest that CDK2 or the substrate of CDK2 might be the replacer of the function of ICP0.2. The relationship between the HSV replication and the CDK2 activityTo analyze the reason of the replication of HSV in Dox induced HT29 Tet-On dn-CDK2 cells, we tested the activity of CDK2 in HSV infected dn-CDK2 expressing HT29 Tet-On dn-CDK2 cells by means of immunoprecipitation. The results shows that the CDK2 activity was increased 6 hpi and reached maximum 9 hpi and decrease 12 hpi in HSV infected HT29 Tet-On dn-CDK2 cells compared with that of mock infections. The one-step replication assay of HSV in HT29 Tet-On dn-CDK2 cells results shows that the production of infectious HSV in dn-CDK2 expressing HT29 Tet-On dn-CDK2 cells was at its lowest 6 hpi and increased 9 hpi while the production of infectious HSV was at its lowest 9 hpi and increased 12 hpi. The 9 hour delay in dn-CDK2 expressing HT29 Tet-On dn-CDK2 cells was just the time needed for the induction of CDK2 activity. Our results show that the HSV can induce the CDK2 activity of host cells and goes into fast productive replication after the CDK2 activity is induced. The CDK2 activity plays important role in the initiation of productive replication of HSV.3. The inhibition of siRNA against CDK2 on HSV replicationTo further test the importance of CDK2 in HSV replication, we developed siRNAs that specially target CDK2, cyclin A and cyclin E, co-transfected into Vero cells and observed the gene expression of infected HSV-GFP recombinant viruses when CDK2 activity has been inhibited by siRNA.The results shows that the gene expression of HSV was inhibited in siRNA against CDK2 transfected cells compared with that of untransfected cells, suggesting again that CDK2 is important in the gene expression of HSV. The best inhibition results came from the co-transfection of three siRNAs together.4. The effects of CDK2 on HSV genomic chromatinizationHSV genomes are mostly chromatinized during latent infections and non-chromatinized during lytic infections. We infer that the delay of HSV replication in CDK2 activity inhibited cells might be related to the chrominization of HSV genome. To test this hypothesis, we harvested the nuclei of HSV infected HT29 Tet-On dn-CDK2 cells induced and uninduced with Dox, digested the nuclei by micrococcal nuclease (MCN), southern blotted to the NC membrane after electrophoresis and hybridized by HSV probe. The results shows that the HSV genome were digested into smears 5 hpi in uninduced HT29 Tet-On dn-CDK2 cells while the HSV genome were resistant to MCN in Dox induced HT29 Tet-On dn-CDK2 cells, suggesting that HSV genome is chromatinized in Dox induced HT29 Tet-On dn-CDK2 cells. Our results indicate that CDK2 might promote the transcription of HSV by disrupting the chromatinization of the HSV genome.