Interactions of the Disordered Domain II of Hepatitis C Virus NS5A with Cyclophilin A, NS5B, and Viral RNA Show Extensive Overlap.
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 (NS5ADII) 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 NS5ADIIand its binding partners. In particular, we compared surface topologies of lysine and arginine residues in the context of free and bound NS5ADII. These experiments have led to the identification of an RNA binding motif (305RSRKFPR311) in an arginine-rich region of NS5ADII. 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, NS5ADIIbinding to NS5B involves a region associated with RNA binding within NS5B. This interaction down-regulated RNA synthesis by NS5B, suggesting that NS5ADIImodulates the activity of NS5B and potentially regulates HCV replication.
Department of Medical Microbiology and Immunology, University of Alberta , 6-020 Katz Group Centre, Edmonton, Alberta T6G 2E1, Canada.|Department of Microbiology and Immunology, McGill University , 3775 University Street, Montréal, Quebec H3A 2B4, Canada.
Marianne
M
Ngure
Department of Microbiology and Immunology, McGill University , 3775 University Street, Montréal, Quebec H3A 2B4, Canada.
Moheshwarnath
M
Issur
Center for Retrovirus Research and College of Pharmacy, The Ohio State University , Columbus, Ohio 43210, United States.
Nikoloz
N
Shkriabai
Department of Microbiology and Immunology, McGill University , 3775 University Street, Montréal, Quebec H3A 2B4, Canada.
Hsiao-Wei
HW
Liu
Department of Chemistry, McGill University , 801 Sherbrooke Street West, Montréal, Quebec H3A 0B8, Canada.
Gonzalo
G
Cosa
Center for Retrovirus Research and College of Pharmacy, The Ohio State University , Columbus, Ohio 43210, United States.
Mamuka
M
Kvaratskhelia
Department of Medical Microbiology and Immunology, University of Alberta , 6-020 Katz Group Centre, Edmonton, Alberta T6G 2E1, Canada.|Department of Microbiology and Immunology, McGill University , 3775 University Street, Montréal, Quebec H3A 2B4, Canada.|Department of Biochemistry, McGill University , 3655 Sir William Osler Promenade, Montréal, Quebec H3G 1Y6, Canada.
Matthias
M
Götte
D020816:Amino Acid Motifs; D021984:Cyclophilin A / Q000235:genetics / Q000378:metabolism*; D015967:Gene Expression Regulation, Viral; D016174:Hepacivirus / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006526:Hepatitis C / Q000201:enzymology* / Q000235:genetics / Q000821:virology*; D054884:Host-Pathogen Interactions; D006801:Humans; D011485:Protein Binding; D000072417:Protein Domains; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism*; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012324:RNA-Dependent RNA Polymerase
D021984:Cyclophilin A; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase
CypA; HCV; NS5A domain II; NS5B; mass spectrometry