Mutational analysis of the functional sites in porcine reproductive and respiratory syndrome virus non-structural protein 10.

Porcine reproductive and respiratory syndrome virus (PRRSV) is prevalent throughout the world and has caused major economic losses to the pig industry. Arterivirus non-structural protein 10 (nsp10) is a superfamily 1 helicase participating in multiple processes of virus replication. PRRSV nsp10, however, has not yet been well characterized. In this study, a series of nsp10 mutants were constructed and analysed for functional sites of different enzymic activities. We found that nsp10 could bind both ssDNA and dsDNA, and this binding activity could be inactivated by mutations at Cys25 and His32. These two mutations also abolished unwinding activity without affecting ATPase activity. In addition, substitution of Ala227 by Ser eliminated helicase activity, whilst substitution by Val enhanced unwinding activity. Taken together, our results showed that Cys25 and His32 in PRRSV nsp10 were critical for nucleic acid binding and unwinding, and that Ala227 played an important role in helicase activity.

[1]  Lei Zhou,et al.  Nsp9 and Nsp10 Contribute to the Fatal Virulence of Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus Emerging in China , 2014, PLoS pathogens.

[2]  Alexander E. Gorbalenya,et al.  Structural basis for the regulatory function of a complex zinc-binding domain in a replicative arterivirus helicase resembling a nonsense-mediated mRNA decay helicase , 2013, Nucleic acids research.

[3]  Huan Li,et al.  Methods for detecting ATP hydrolysis and nucleic acid unwinding of Japanese encephalitis virus NS3 helicase. , 2013, Journal of virological methods.

[4]  M. Kikkert,et al.  Arterivirus molecular biology and pathogenesis. , 2013, The Journal of general virology.

[5]  Hua Wu,et al.  Mutations in the genome of the highly pathogenic porcine reproductive and respiratory syndrome virus potentially related to attenuation. , 2012, Veterinary microbiology.

[6]  E. M. De La Cruz,et al.  ATP utilization and RNA conformational rearrangement by DEAD-box proteins. , 2012, Annual review of biophysics.

[7]  Yong‐Joo Jeong,et al.  Identification of myricetin and scutellarein as novel chemical inhibitors of the SARS coronavirus helicase, nsP13 , 2012, Bioorganic & Medicinal Chemistry Letters.

[8]  A. Pyle,et al.  Mechanism of Mss116 ATPase reveals functional diversity of DEAD-Box proteins. , 2011, Journal of molecular biology.

[9]  E. Snijder,et al.  The PRRSV replicase: Exploring the multifunctionality of an intriguing set of nonstructural proteins , 2010, Virus Research.

[10]  C. Gagnon,et al.  The role of porcine reproductive and respiratory syndrome (PRRS) virus structural and non-structural proteins in virus pathogenesis , 2010, Animal Health Research Reviews.

[11]  W. Cao,et al.  Pathway of ATP utilization and duplex rRNA unwinding by the DEAD-box helicase, DbpA , 2010, Proceedings of the National Academy of Sciences.

[12]  F. Chen,et al.  Genomic analysis of two Chinese strains of porcine reproductive and respiratory syndrome viruses with different virulence , 2010, Virus Genes.

[13]  Lei Zhou,et al.  The 30-Amino-Acid Deletion in the Nsp2 of Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus Emerging in China Is Not Related to Its Virulence , 2009, Journal of Virology.

[14]  W. Cao,et al.  The ATPase cycle mechanism of the DEAD-box rRNA helicase, DbpA. , 2008, Journal of molecular biology.

[15]  Di Liu,et al.  Emergence of Fatal PRRSV Variants: Unparalleled Outbreaks of Atypical PRRS in China and Molecular Dissection of the Unique Hallmark , 2007, PloS one.

[16]  J. Ziebuhr,et al.  A Complex Zinc Finger Controls the Enzymatic Activities of Nidovirus Helicases , 2005, Journal of Virology.

[17]  K. Faaberg,et al.  Functional Properties of the Predicted Helicase of Porcine Reproductive and Respiratory Syndrome Virus , 2002, Virology.

[18]  J. Ziebuhr,et al.  Biochemical Characterization of the Equine Arteritis Virus Helicase Suggests a Close Functional Relationship between Arterivirus and Coronavirus Helicases , 2000, Journal of Virology.

[19]  A. Gorbalenya,et al.  The Predicted Metal-Binding Region of the Arterivirus Helicase Protein Is Involved in Subgenomic mRNA Synthesis, Genome Replication, and Virion Biogenesis , 2000, Journal of Virology.

[20]  E. Snijder,et al.  Characterization of an Equine Arteritis Virus Replicase Mutant Defective in Subgenomic mRNA Synthesis , 1999, Journal of Virology.

[21]  J. D. den Boon,et al.  An infectious arterivirus cDNA clone: identification of a replicase point mutation that abolishes discontinuous mRNA transcription. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[22]  S. Ho,et al.  Site-directed mutagenesis by overlap extension using the polymerase chain reaction. , 1989, Gene.

[23]  M. Dillingham,et al.  Superfamily 1 helicases . , 2013, Frontiers in bioscience.

[24]  E. Jankowsky.  RNA helicases at work: binding and rearranging. , 2011, Trends in biochemical sciences.