The Molecular Mechanism Of Host Vimentin Protein Contributing To Reticuloendotheliosis Virus Infection

Reticuloendotheliosis(RE), caused by Reticuloendotheliosis virus(REV), is an important oncological disease mainly including short stature syndrome, tumors, and immunosuppression. In addition, REV contamination of some live vaccines, resulting in the reduction of the immune effect, has previously been reported for Marek’s disease and fowl pox. It has been reported that the co-infection of REV with other immunosuppressive viruses, such as Marek’s disease virus, avian leukemia virus, chicken infectious anemia virus, and infectious bursal disease virus becomes more and more severe, which causes severe economic loses in chicken flocks in recent years. So, much more attention should be paid on the infection and pathogenic mechnism of REV.Recently, researches about REV mainly focuse on virus genome insertion, co-infection, and vaccine contamination. However, the data on the molecular mechanism of REV infection are still limited. Identification of the interactions between REV and cellular proteins may be helpful for understanding of the viral replication mechanisms, since interactions between viral and host proteins play a central role in the infection process. In our previous studies, we utilized the two-dimensional electrophoresis method to identify the differentially expreesed proteins that are associated with REV infection. From several candidate proteins, we initially selected vimentin for further study. Some research have been reported that vimentin plays an important role in virus infection. The aim of this study was to explore the role of vimentin in REV infection.It was shown that vimentin was expresssed in different chicken tissues, especially the thymus, bursa, and spleen, and was upregulated in the spleen and cells after REV infection usine Real time PCR. Silencing of vimentin with small interfering RNAs inhibited REV replication and gp90 expression using Western blotting, Real time PCR, and the detection of viral titer. Meanwhile, disruption of vimentin filaments by acrylamide caused a reduction of both REV replication and gp90 expression. The results indicated that vimentin promoted REV replication.In order to explore the mechnism of the influence of viemtin protein on REV replication, the recombinant protease(PR) fused with the His-tag was expressed in Escherichia coli system in soluble type. Then the enzymatic activity of PR were analyzed, and the results indicated that the prokaryotic expressed protease has enzyme activity, which has a condition dependent, including the temperature, p H, ions. Further studies showed that the prokaryotic expressed PR protein cleaved vimentin protein, which was expressed in Escherichia coli system or in CEF cells. Furthermore, the cleavage of vimentin dependents on PR protein structure and the active center DTG triplet. On the other hand, after expressing PR in cells, vimentin were also cleaved. Confocal laser scanning microscopy assays indicated that the distribution of vimentin was changed after expressing PR in cells or REV infection, and then the vimentin surrounded to the nucleus and colocalized with REV antigen.In summary, REV infection enhances vimentin expression; REV replication dependents on host vimentin. REV PR cleavages vimentin protein in vitro and vivo, and the cleavage activity dependents on PR protein structure and DTG triplet. REV infection can change vimentin distribution by the interaction between PR and host. Vimentin surrounding the nucleus may serve a cytoprotective function to concentrate virus structural proteins and establish a virus replication factory, which may be one of the molecular mechnism for vimentin involving in REV replication and infection.