Abstract
BackgroundThe membrane topology and molecular mechanisms for endoplasmic reticulum (ER) localization of classical swine fever virus (CSFV) non-structural 2 (NS2) protien is unclear. We attempted to elucidate the subcellular localization, and the molecular mechanisms responsible for the localization of this protein in our study. The NS2 gene was amplified by reverse transcription polymerase chain reaction, with the transmembrane region and hydrophilicity of the NS2 protein was predicted by bioinformatics analysis. Twelve cDNAs of the NS2 gene were amplified by the PCR deletion method and cloned into a eukaryotic expression vector, which was transfected into a swine umbilical vein endothelial cell line (SUVEC). Subcellular localization of the NS2 protein was characterized by confocal microscopy, and western blots were carried out to analyze protein expression.ResultsOur results showed that the -NH2 terminal of the CSFV NS2 protein was highly hydrophobic and the protein localized in the ER. At least four transmembrane regions and two internal signal peptide sequences (amino acids103-138 and 220-262) were identified and thought to be critical for its trans-localization to the ER.ConclusionsThis is the first study to identify the internal signal peptide sequences of the CSFV NS2 protein and its subcellular localization, providing the foundation for further exploration of this protein's function of this protein and its role in CSFV pathogenesis.
Highlights
The membrane topology and molecular mechanisms for endoplasmic reticulum (ER) localization of classical swine fever virus (CSFV) non-structural 2 (NS2) protien is unclear
Construction of recombinant expressing plasmid Using designed primers, 12 amino-terminal truncated fragments of the NS2 genes were amplified by polymerase chain reaction (PCR) and the sizes of these amplified products were verified by electrophoresis (Figure 2)
Expression and subcellular localization of CSFV NS2 protein Western blot analysis showed that all the target protein were correctly expressed and displayed the expected molecular weight (Figure 4)
Summary
The membrane topology and molecular mechanisms for endoplasmic reticulum (ER) localization of classical swine fever virus (CSFV) non-structural 2 (NS2) protien is unclear. We attempted to elucidate the subcellular localization, and the molecular mechanisms responsible for the localization of this protein in our study. The causative agent of CSF is classical swine fever virus (CSFV), a member of the Pestivirus genus within the Flaviviridae family of viruses, which contains the genera Flavivirus and Hepacivirus (hepatitis C viruses, HCV)[1]. Our previous study demonstrated that CSFV NS2 was a hydrophobic protein and localized in the endoplasmic reticulum (ER) membrane, independently of CSFV p7 peptides. Our results indicated that CSFV NS2 protein contains two internal signal peptide sequences, which are critical for trans-localization to the ER, and this protein probably possesses at least four transmembrane regions. The findings are crucial for elucidating the function of CSFV NS2 protein, and have potentially important implications for understanding the molecular mechanisms of pathogenesis for this economically important agricultural disease
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