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Research Article
zishu pan
State Key Laboratory of Virology
Corresponding Author
ORCiD: https://orcid.org/0000-0002-5326-3264
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Pestivirus nonstructural protein 3 (NS3) is a multifunctional protein with protease and helicase activities that are essential for the virus replication. In this study, we used a combination of biochemical and genetic approaches to investigate the relationship between the positively charged patch on protease module and NS3 function. The surface patch was composed of four basic residues R50, K74 and K94 in NS3 protease domain and H24 in the structurally integrated cofactor NS4A PCS . Single residue or simultaneous four-residue substitution in the patch to alanine or aspartic acid hardly affect ATPase activity. However, the single R50, K94 or H24 residue or simultaneous four-residue substitution resulted in the apparent changes of the helicase activity and RNA-binding ability of NS3. When these mutations were introduced into a classical swine fever virus (CSFV) cDNA clone, the single K94 residue or simultaneous four-residue substitution (Qua_A or Qua_D) impaired the infectious virus production. Furthermore, the replication efficiency of the CSFV variants was partially correlated to the helicase activity of NS3 in vitro . Our results suggest that the conserved positively charged patch on the NS3 plays an important role in modulating the NS3 helicase activity in vitro and CSFV production.
Keywords
Pestivirus, classical swine fever virus, nonstructural protein NS3, helicase, protease
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This preprint is under consideration at Archives of Virology. A preprint is a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Learn more about In Review
Research Article
zishu pan
State Key Laboratory of Virology
Corresponding Author
ORCiD: https://orcid.org/0000-0002-5326-3264
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
Peer Review Timeline
CURRENT STATUS: Under Review
Version 1
Posted 24 Mar, 2021
No community comments so far
Editor assigned
On 08 Feb, 2021
First submitted
On 11 Jan, 2021
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
Subject Areas
Virology
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Pestivirus nonstructural protein 3 (NS3) is a multifunctional protein with protease and helicase activities that are essential for the virus replication. In this study, we used a combination of biochemical and genetic approaches to investigate the relationship between the positively charged patch on protease module and NS3 function. The surface patch was composed of four basic residues R50, K74 and K94 in NS3 protease domain and H24 in the structurally integrated cofactor NS4A PCS . Single residue or simultaneous four-residue substitution in the patch to alanine or aspartic acid hardly affect ATPase activity. However, the single R50, K94 or H24 residue or simultaneous four-residue substitution resulted in the apparent changes of the helicase activity and RNA-binding ability of NS3. When these mutations were introduced into a classical swine fever virus (CSFV) cDNA clone, the single K94 residue or simultaneous four-residue substitution (Qua_A or Qua_D) impaired the infectious virus production. Furthermore, the replication efficiency of the CSFV variants was partially correlated to the helicase activity of NS3 in vitro . Our results suggest that the conserved positively charged patch on the NS3 plays an important role in modulating the NS3 helicase activity in vitro and CSFV production.
Figure 1
Figure 2
Figure 3
Figure 4
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