| As obligate intracellular microorganism, virus completely depends on the host biosynthetic systems to achieve its replication, thus virus-host interaction determine the infection and pathogenesis of host. Upon virus infection, infected cells initiate the programmed cell death(apoptosis) through activation of caspases to interfere viral replication and even clear viruses. On the other hand, many viral proteins can imitate the substrates of caspases and can be cleaved by specific caspases to produce truncated proteins for promoting viral replication. For instance, caspase-3,-6 and-7 simultaneously cleave non-structural protein NS5 A of Hepatitis C virus(HCV) to generate truncated proteins to modify intracellular microenvironment, then enhancing HCV replication. Classical swine fever virus(CSFV) is also a member of the Family Flaviridae as HCV, but it’s unknown whether NS5 A cleavage is necessary for CSFV replication. Therefore, cleavage of CSFV NS5 A and related molecular mechanism was performed in the present study.To test if CSFV NS5 A is cleaved, swine testicle cells(ST) were infected with CSFV highly virulent strain Shimen and attenuated vaccine C-strain respectively. As a result, a truncated protein of 40 k Da was observed for Shimen NS5 A except the intact protein of 56 k Da, and only intact NS5 A protein occurred for C-strain, this probably because the expression levels of C-stain NS5 A is too low to be detected. To further analyze the CSFV NS5 A cleavage, NS5 As derived from Shimen and C-strain were expressed as EGFP fusion proteins in ST cells. Western blotting results indicated that Shimen NS5 A has one truncated protein of 68 k Da(truncated protein b) in addition to the intact protein about 83 k Da. For C-strain, one more truncated protein of 63 k Da(truncated protein c) was observed except truncated protein b and the intact form.To determine the role of caspases and calpains in NS5 A cleavage of CSFV, which were previously reported to cleave HCV NS5 A, inhibitors against caspases and calpains were subjected to treat ST cells stably expressing Shimen NS5A(P-E-S) and C-strain NS5A(P-E-H). Unexpected, caspases inhibitor can only block the production of truncated protein c of C-strain and calpain inhibitors have no effect on the NS5 A cleavage of Shimen and C-strain. On the contrast, NS5 A cleavage of both Shimen and C-strain were protected by calpains. Alignment of amino acids of NS5 A showed that amino acid residues at 272 to 275 of C-strain NS5A(D272TTD275) is potential binding motif of caspases and valine at 271 of C-strain NS5 A is the addicted amino acid residue of caspase-6, but the amino acid residue at that site of Shimen NS5 A is not. Thus, we speculate that caspase-6 may mediate the production of truncated protein c of C-strain. To confirm this, P-E-H cells was treated with caspase-6 specific inhibitor(Z-VEID-FMK), in which the expression of caspase-6 was also downregulated. As expect, production of truncated protein c was inhibited by the inhibitor and downregulation of caspase-6. Moreover, ST cells stably expressing caspase-6 or with downregulation of caspase-6 were constructed and then infected with C-strain or Shimen. Results showed that downregulation of caspase-6 significantly inhibited the viral replication of C-strain, but overexpression of caspase-6 has no effect. As for Shimen, both upregulation and downregulation of caspase-6 did not impair the viral amplification. In addition, calpain I inhibitor(ALLN) and calpain II inhibitor(ALLM) can significantly suppress the viral replication of Shimen and C-strain, indicating that calpain I and calpain II play an important role in the replication of CSFV Shimen and C-strain.To reveal the cleavage site at C-strain NS5 A for caspase-6, valine at 271 and analine at 277 of C-strain NS5 A were replaced with alanine at 271 and threonine at 277 of Shimen NS5 A, aspartic acids at 272 and 275 of C-strain NS5 A were replaced with glutamic acid, and ST cells stably expressing these mutated NS5 As were constructed. Western blotting indicated that aspartic acid at 275 and alanine at 277 of C-strain NS5 A are critical for the production of truncated protein c, while alanine at 271 and aspartic acid at 272 have no effect on truncated protein c production. Further, Shimen NS5 A can be cleaved to produce truncated protein c when threonine at 277 was replaced with alanine, and similar case is also occurred for GD53 NS5 A only when glutamic acid at 275 of GD53 NS5 A was replaced with aspartic acid. Therefore, aspartic acid at 275 and alanine at 277 of CSFV NS5 A are the critical amino acid residues for the production of truncated protein c by caspase-6.To further analyze the cleavage site for the production of truncated protein b, valine at 371 and aspartic acid at 373 of C-strain and Shimen NS5 A were replaced with leucine at 371 and glutamic acid at 373 of GD53 NS5 A according to the multiple alignment result of CSFV NS5 A and the molecular weight of truncated protein b, and related ST cell lines were established. As a result, valine at 371 and aspartic acid at 373 of C-strain and Shimen NS5 A are not associated with the production of truncated protein b. In addition, aspartic acid at 275 and 373, alanine at 335, valine at 362 and 371 of C-strain NS5 A were replaced with glutamic acid at 275 and 373, valine at 335, isoleucine at 362 and leucine at 371 of GD53 NS5 A, and ST cells stably expressing C-strain NS5 A with 2, 3 or 4 mutations were established. Western blotting showed that aspartic acid at 275 and 373, alanine at 335, valine at 362 and 371 are not the determinant for truncated protein b production.This study first report the cleavage of non-structural protein NS5 A of CSFV and caspase-6-mediated production of truncated protein c is critical for the viral replication, the cleavage site of C-strain NS5 A by caspase-6 is also determined. The above results will be useful for revealing the molecular mechanism of posttranslational modification of viral proteins and viral replication. |
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