Summary auto-generated
This study examined internal proteolytic cleavage of the NS3 protein from dengue virus 2 (DEN-2), a multifunctional protein essential for virus replication. Using radioactive labeling and immunoprecipitation in transfected COS cells and DEN-2-infected cells, researchers identified four polypeptides: the precursor NS2B/3, full-length NS3, a truncated form NS3α, and a previously unidentified C-terminal fragment designated NS3β. The authors demonstrated that cleavage occurs at the conserved internal site R457-R/G460 within an RNA helicase sequence motif, producing NS3α (50 kDa) and NS3β (19 kDa). Importantly, this internal cleavage is mediated by the viral NS2B/3 protease complex rather than cellular proteases, and prior cleavage at the NS2B/NS3 junction is not required. The biological significance of this internal cleavage remains unclear but may regulate viral enzymatic activities, particularly the helicase and RNA triphosphatase functions associated with NS3. The findings provide molecular evidence for an important proteolytic processing event in dengue virus replication.
Key findings
- Internal cleavage of dengue virus 2 NS3 protein at residues R457/G460 produces two fragments: NS3α (50 kDa) and a previously unknown NS3β fragment (19 kDa)
- The viral NS2B/3 protease complex, not cellular proteases, is responsible for the internal NS3 cleavage
- Internal NS3 cleavage can occur independently of prior NS2B/NS3 junction cleavage, suggesting it is a separate proteolytic event
- Mutagenesis of the internal cleavage site abolishes both NS3α and NS3β production, confirming the specific proteolytic site
- The cleavage may regulate NS3-associated enzymatic activities including helicase and RNA triphosphatase functions
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Abstract
The NS3 protein of flaviviruses is a multifunctional polypeptide required for virus replication. Enzymic activities that have been demonstrated or predicted from the presence of sequence motifs include protease, NTPase, helicase and RNA triphosphatase. Both full-length and truncated forms of NS3 have been identified in infected cells. To examine internal cleavage of the NS3 protein of dengue virus 2 (DEN-2), infected cells or COS cells transfected with cDNA encoding NS2B/3 were radiolabelled and immunoprecipitated with antiserum against NS3 or hyperimmune mouse ascitic fluid. The polypeptides detected were NS2B/3 (Mr 83000), NS3 (Mr 69000), NS3' (Mr 50000) and NS3" (Mr 19000). The latter polypeptide has not been previously identified. For DEN-2, it has been proposed that NS3' results from cleavage at the site ...R457R / GR460... within an RNA helicase sequence motif of NS3. Our results demonstrated that cleavage occurred at this site, and that prior cleavage between NS2B/NS3 was not necessary.