| Avian leukosis virus (ALV), which belongs to the family Retroviridae, induces neoplasms in chicken and leads to severe economic losses in the poultry industry. The subgroup J became widespread in recent years and caused enormous economic losses. But up to now, there is no effective vaccine and treatment for ALV infection.A critical step in retroviral life cycle is the integration of the reverse-transcribed viral cDNA into the host cell genome. The viral integrase (IN) plays a key role in this process. Besides integration, IN is also involved in other aspects of viral life cycle such as uncoating, reverse transcription, viral protein expression and viral particle packaging. Therefore, cellular factors should be involved in the multiple functions. Indeed, HIV integrase has been found to interact with many host proteins, and become the new target for HIV treatment. However, the interactions between ALV integrase and host proteins have not been well studied. In this dissertation, we investigated the interaction between host proteins and ALV integrase.CSN6(COP9signalosome subunit6) was identified as a potential binding protein of ALV IN by Yeast two-hybrid screening using the integrase of ALV as bait and cDNAs generated from the chicken bursa of Fabricius as a library. To confirm whether ALV-J IN interacts with CSN6in cells, we overexpressed IN and CSN6in human293T cells, and then performed a co-immunoprecipitation analysis. The result verified the interaction. We also found that overexpressed IN could interact with the endogenous CSN6in chicken DF-1cells and overexpressed CSN6was capable of binding to IN when ALV-J infection. A domain mapping experiment showed that C terminal region of CSN6could bind to the catalytic motif of integrase directly. Laser confocal microscopy showed that both CSN6and integrase were diffused throughout the cells with some aggregated patches accumulated in the cytoplasm. When co-overexpressed, these two proteins were colocalized.In the viral replication experiments, we found overexpression of CSN6in DF-1cells inhibited whereas knockdown of CSN6promoted ALV-J production. We further manually isolated genomic DNA and compared the relative extent of ALV-J viral DNA integration between the control and CSN6siRNA-transfected cells. Both PCR and qPCR results showed that knockdown of CSN6increased viral DNA integration.Ubiquitination is an important protein post-translational modification and takes part in many cellular processes. We found ALV viral protein Gag, IN, RT can be modified by ubiquitination, and verified the modified region of ALV IN was localized in its catalytic motif. We also found IN was ubiquitinated independent on lysine residues, and the same phenomenon also occured in HIV IN. MS detection verified the ubiquitinated sites of ALV IN were at T10and C38.In conclusion, we identified CSN6as a restriction factor of ALV-J in its host chicken cells with IN being its primary target. Our results may provide a new strategy for the development of anti-retroviral drugs. |
