Journal
ACS INFECTIOUS DISEASES
Volume 2, Issue 11, Pages 839-851Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acsinfecdis.6b00143
Keywords
HCV; NS5A domain II; NS5B; CypA; mass spectrometry
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Funding
- Canadian Institutes of Health Research (CIHR)
- Fonds de Recherche du Quebec Nature et Technologie (FRQNT)
- Canadian Foundation for Innovation (CFI)
- National Institutes of Health [AI062520]
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Domain II of the nonstructural protein 5 (NS5A) of the hepatitis C virus (HCV) is involved in intermolecular interactions with the viral RNA genome, the RNA-dependent RNA polymerase NS5B, and the host factor cyclophilin A (CypA). However, domain II of NS5A (NS5A(DII)) is largely disordered, which makes it difficult to characterize the protein-protein or protein-nucleic acid interfaces. Here we utilized a mass spectrometry-based protein footprinting approach in attempts to characterize regions forming contacts between NS5A(DII) and its binding partners. In particular, we compared surface topologies of lysine and arginine residues in the context of free and bound NS5A(DII). These experiments have led to the identification of an RNA binding motif ((RSRKFPR311)-R-305) in an arginine-rich region of NS5A(DII). Furthermore, we show that K308 is indispensable for both RNA and NS5B binding, whereas W316, further downstream, is essential for protein-protein interactions with CypA and NS5B. Most importantly, NS5A(DII) binding to NS5B involves a region associated with RNA binding within NS5B. This interaction down-regulated RNA synthesis by NS5B, suggesting that NS5A(DII) modulates the activity of NS5B and potentially regulates HCV replication.
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