The Function Of Classical Swine Fever Virus NS2 Protein

Classical swine fever(CSF) is a highly contagious and often fatal disease of pigs that caused by classical swine fever virus (CSFV ) which is classified as a notifiable (previously List A) disease by the World Organization for Animal Health (OIE). Recently, almost all the researchers were focused on the development of the detection methods and novel vaccines, only few reports pay attention to the molecular pathology of CSF. To elucidate the function of CSFV nonstructure protein 2 (NS2), for which is poorly understand, in the present study, the degradation characterization, subcellular localization of CSFV NS2 protein, as well as the effect of CSFV NS2 protein on the cell proliferation and cell cycle, the regulation of CSFV NS2 protein on the inflammatory factors expressed in the swine vascular endothelial cells were extensively investigated.From the experiments, the following results were obtained:(1)The CSFV NS2 gene was successfully amplified by reverse transcription polymerase chain reaction (RT-PCR), the sequences analysis showed that, CSFV NS2 gene was 1371 bp encoding 457 amino acids (aa), of which, the molecular weight is 53 ku. Bioinformatics analysis showed that there were probably five trans-membrane regions in the N-terminal of CSFV NS2 protein which showed high possibility of hydrophobicity, however, the C-terminal showed great hydrophilicity. From the first to the 170th aa, the secondary structure is mainly ofβ-lap, to the C-terminal, it is mainly ofα-coil. The Ser223 and Ser224 showed great possibility of phosphorylation, as well as the Thr37, Thr47, Thy204, Thy332 and Thy392.(2)The CSFV NS2 protein was expressed as fused to the green fluorescent protein (GFP), Western blot analysis showed that the molecular weight of CSFV NS2 was approximately 53 ku, this protein was a short-live protein for its dynamically degradation through the proteasomal degradation pathway which was inhibited by proteasomal inhibitor MG132. Co-localization suggested that CSFV NS2 protein localized in the endoplasmic reticulum (ER), at least including 2 internal signal sequences and 4 trans-membrane regions.(3)Cell proliferation assay results showed that CSFV NS2 was capable of inhibiting of the host cells proliferation through inducing the cell cycle arrested in the S-phase rather than inducing apoptosis. The mRNA and protein level analysis of cyclinA revealed that CSFV NS2 expression promoted the proteasomal degradation of Cyclin A protein rather than suppressing the transcription of Cyclin A.(4)CSFV NS2 induced the ER stress and activated the nuclear factor kappaB (NF-кB) which may strongly contribute to the accelerated proteasomal degradation of Cylin A and CSFV NS2 protein.(5)The susceptibility and propagation of CSFV in swine vascular endothelial cells was investigated by indirect immunofluorescence assay and RT-PCR, the results showed that swine vascular endothelial cells was susceptible to CSFV which propagated efficiently. Further study showed that the infection of CSFV dramatically up-regulated the cell surface adhesion molecule integrinβ3 expressed in vascular endothelial cells, and the CSFV NS2 played an important role in this process.(6)The CSFV NS2 significantly induced the interleukin-8 (IL-8) expressed in vascular endothelial cells through the activation of NF-кB. The proteasomal inhibitor MG132 was able to suppress the NF-кB activity and subsequently inhibited the transcription of IL-8, however CSFV NS2-expressing cells showed antagonizes to the inhibition induced by MG132.(7)The proteasomal inhibitor MG132 induced the apoptosis of the normal vascular endothelial cells, however, CSFV NS2-expressing cells showed antagonizes to the apoptosis effect induced by MG132. Further study showed that CSFV NS2 protein up-regulated the expression of the anti-apoptotic protein Bcl-2(B-cell leukemia-lymphoma-2) in the host cells, which possibly contributed to the CSFV NS2-expressing cells showing anti-apoptosis effect.In conclusion, in the present study, the characterization of CSFV NS2 protein and the interaction between CSFV NS2 protein and vascular endothelial cells were mainly investigated. This is the first time to reveal the short-live characterization and the ER-localization of CSFV NS2 protein in the host cells. CSFV NS2 protein played an important role in the regulation of cell proliferation and cell cycle, as well as in the up-regulation of integrinβ3, chemotatic factor IL-8 and the anti-apoptotic protein Bcl-2 expressed in vascular endothelial cells. All the results from this study provided novel information to the function of CSFV NS2 protein, and, may be helpful for us to understand the molecular pathogenesis of CSFV.