The Transcription Activator Of Foamy Virus Regulates Host Cell Cycle And Is Regulated By Host MicroRNA

Virus is a kind of pathogen that has to utilize cellular machinery to complete its proliferation and replication. Due to the limited package capacity, the virus could not encode all the proteins required for this process. In contrast, after viral infection, the host cell immediately initiates its defense mechanisms to counteract, reduce and even eliminate the viral invasion. In this thesis, foamy virus was used as a model system to study the interaction between this virus and the host cell, especially the impact of viral infection on host cell cycle progression. In addition, current study revealed that the RNA silencing repressor, the Tombusvirus p19 protein, regulated the expressions of cell cycle related genes in mammal cells.Borf1 is a transcription activator encoded by bovine foamy virus genome, and can activate the transcriptions of its own gene as well as viral structural genes. However, the effect of Borf1 on the expressions of cell cycle related genes has not been exploited. In current study, a human embryonic kidney 293 cell stably expressing Borf1 gene was established and used for the detection of the effect of Borf1 on the endogenous gene expressions in host cells. The results revealed that stably expressing Borf1 significantly caused G1/G0 arrest, and in the same time reduced S phase population. Semi-quantitative RT-PCR demonstrated that Borf1 down-regulated the mRNA levels of cyclin A2, cyclin B1, cyclin E1 and CDC2, and upregulated the mRNA levels of CDK2 and E2F-1. Western blot analysis on these cell cycle related genes further confirmed the above results. Additionally, Borf1 could reduce the endogenous p15 protein expression. Thus. Borf1 may cause G1 arrest by influencing the expressions of a number of cell cycle related genes which may play critical roles at the restriction points during G2-M and G1-S transition.Besides its function as a pathogenicity determinant, the Tombusvirus P19 also serves as a suppressor of RNA interference (RNAi) by sequestering intracellular small RNAs such as the small interfering RNAs (siRNAs) and microRNAs (miRNAs). However, the effect of P19 on mammalian cells has not been evaluated before. In this study, a human embryonic kidney 293 cell line that stably expressed p19 (HEK293-p19) was generated. Flow cytometric analysis revealed that over-expression of P19 caused a significant accumulation of G2/M phase cells. Cell proliferation assays demonstrated a reduced DNA replication and cell growth in HEK293-p19 cells. Moreover, p19 altered the expression profiles of a number of cell cycle regulators in HEK293 cells, such as upregulation of cyclin A1, CDK2, CDK4, CDK6, p27, cyclin D2, cyclin D3 and E2F1, and downregulation of p15, cyclin A2, cyclin B1 and cyclin E1. Thus, the data strongly indicate that p19 might influence multiple G2/M regulators to cause G2/M arrest.MicroRNA is a class of noncoding RNAs with 21-25nt in length that functions posttranscriptionally to repress targeted gene expression by either inhibiting protein translation or promoting mRNA degradation. Although a large number of microRNAs have been isolated, only a few of them have the known functions. Moreover, there are only limited reports on the roles of microRNA in HFV infection. To this end, we used bioinformatic approach to screen the potential microRNA targeting site(s) in the RNA genome of human foamy virus (HFV). We found one has-miR-491-5p targeting site in the coding region of Bell gene. The targeted sequence was isolated from HFV and inserted into the 3'UTR of pEGFP-C1. The EGFP expressions were significantly repressed by either the chemically synthesized or the vector expressed has-miR-491-5p. Further, the repression could be rescued with either the chemically modified miRNA inhibitor or the overexpression of p19. Study indicated that has-miR-491-5p mediated posttranscriptionally gene repression occurred at mRNA rather than protein level. Finally, has-miR-491-5p could effectively blocked Bell expressions from both expression vector and HFV viral genome. Thus, the established system might be useful for the study of HFV replication and gene regulation.In conclusion, some key regulatory proteins such as Borf1 and P19 produced from early viral infection have major influences on not only viral genome replication but also host cell cycle progression such as cell cycle arrest, which can be induced by the alterations of cell cycle related genes. In additoion, the host may exert its many defense mechanisms against viral invasion. One of the choices that the virus uses for self-defense might be to induce posttranscriptional gene silencing mediated by target site-specific microRNA. For example, the host miR-491 might inhibit HFV viral replication by miRNA-mediated posttranscriptional gene repression to down-regulate the expression of transcriptional activator Bell. This type of gene repression mediated by miR-491 may offer a good advantage for HFV to establish viral latency.