| A hallmark of alphaherpesvirus infection is the rapid shutoff of host macromolecular synthesis during infection. Shutoff of host protein synthesis in HSV-infected cells is mediated by a phosphoprotein encoded by the viral UL41 gene. The protein was named virion host shutoff (VHS) protein, which is also a tegument protein as the minor structural component of virus particle. During lytic infections, VHS enters into the cytosol as components of infecting virions and immediately initiates the degradation of cellular mRNAs. By interaction with the human translation initiation factor eIF4H, VHS is targeted to regions of translation initiation of mRNAs. The degradation of cellular mRNAs shifts the translation apparatus from host to viral mRNA and facilitates the expression of different classes of viral genes. During late infections, the tegument protein VP 16 (encoded by UL48 gene) may restrain VHS function and translocate VHS into the nucleus and/or into the viral assembly pathway. In addition, a number of studies suggest that VHS is an important determinant of virulence. It plays an important role in pathogenesis and multifunctional immune evasion mechanism of alphaherpesvirus by interfering with the IFN-mediated antiviral immune responses, down-regulating expression of major histocompatibility complex (MHC) class I and II molecules and reducing cytotoxic T-lymphocyte recognition.However, there are few studies on VHS of pseudorabies virus (PRV), and it is still unknown that whether PRV is similar to other alphaherpesvirus in VHS functions. Therefore, gene cloning, expression and structural analysis of VHS protein of PRV can be useful to identify the functions of UL41 gene of PRV and determine the precise mechanism of VHS activity. At the same time, it will not only play an important role in elucidating the exact mechanism of translational control of gene expression, pathogenesis and latent infection of PRV, but also be helpful in developing a new vaccine of VHS deletion mutant.In this study, a 1174 bp DNA fragment containing the complete coding sequence of UL41 gene of PRV strain Ea was amplified by PCR technique and cloned into downstream of the GST of an expression vector, pGEX-KG After induction by IPTG, a high expression of fusion protein was obtained. The result of denaturing polyacrylamide gels (SDS-PAGE) showed that the molecular weight of fusion protein of VHS and GST was about 66 KDa.The sequence analysis indicated that VHS had four conserved regions and the third conserved region was highly identical with the FEN-1 (1A76) domin of flap endonuclease 1. The three-dimensional structural prediction of VHS protein of PRV showed that the protein contained 10 helix and 22 p-sheet and was similar to the three-dimensional structure of VHS protein of human herpesvirus 1 by the software of PROSPECT.The prokaryotic expression plasmid of pGEX-UL48 was also transformed into Escherichia coli BL21 (DE3) and yielded a high expression of fusion protein of VP16 and GST. After renaturation and purification, VHS and VP16 proteins were separately injected into rabbits, and the effective antisera of VHS and VP16 were prepared.The UL41 and UL48 genes were again cloned into the eukaryotic expression vectors pcDNA3.1(+) and pCI-neo, resulting in eukaryotic plasmids expressing each or both UL41 and UL48 genes. These plasmids will be useful for further studying the functions of VHS protein of PRV, and determining whether VHS protein interacts with the tegument protein VP16. |
