Identification of Amino Acid Variants in the Hepatitis C Virus Non-Structural Protein 4A

Identification of Amino Acid Variants in the Hepatitis C Virus Non-Structural Protein 4A

ALEJANDRO DANIEL BERMÚDEZ-AGUIRRE,^1,2 LUIS PADILLA-NORIEGA,³ EDGAR ZENTENO^1,4 and JULIO REYES-LEYVA²

¹Departamento de Bioquímica, Facultad de Medicina, Universidad Nacional Autónoma de México, Mexico, DF, Mexico
²Laboratorio de Virología, Centro de Investigaciones Biomédicas de Oriente, Instituto Mexicano del Seguro Social, Hospital General de Zona No. 5, Puebla, Mexico
³Departamento de Biología Molecular y Biotecnología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Mexico, DF, Mexico
⁴Facultad de Medicina Humana, Universidad Ricardo Palma, Santiago de Surco, Peru

Chronic infections by hepatitis C virus (HCV) are a major cause of cirrhosis and hepatic cancer. The replication of HCV involves translation and proteolytic processing of polyproteins. The HCV single-stranded RNA encodes a single polyprotein of C/E1/E2/p7/NS2/NS3/NS4A/NS4B/NS5A/NS5B. The structural proteins, C, E1, E2, and p7, arise from the viral polyprotein by host proteases. Cleavage at the non-structural NS2/NS3 junction is performed by the NS2 protease. NS3 forms a complex with NS4A to cleave the rest of the viral polyprotein. The central 12-amino-acid sequence of NS4A, 21-GSVVIVGRIILS-32 (NS4Awt) is a determinant to enhance the NS3 protease activity at the NS5A/5B junction. We found that, from 13 blood donors infected with HCV, one sample showed five amino acid changes in the NS4A central region at V23I, I25C, I30S, L31T, and S32L, and another sample showed three changes at V23I, I25C, and I30V in this region. The other 11 samples showed the NS4Awt sequence. The effect of such amino acid variations on the NS3 proteolytic activity was evaluated in vitro using the central 12-amino-acid NS4Awt sequence with specific changes joined to NS3, and NS5A/5B as a substrate. Our results indicate that the amino acid changes of NS4A at V23I and I25C do not enhance the protease activity of NS3, whereas the amino acid changes at I30S, L31T, and S32L, as well as the NS4Awt sequence, enhance NS3 activity. Our results confirm that protease cofactor, encoded in NS4A, is of major regulatory relevance for the replication cycles of HCV.

Keywords —— Hepatitis C Virus; variant; NS4A; quasispecies; protease.

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Tohoku J. Exp. Med., 2009, 218, 165-175

Correspondence: Alejandro Daniel Bermúdez-Aguirre, Departamento de Bioquímica, Facultad de Medicina, PO Box 70159, Universidad Nacional Autónoma de México, 04510, México.

e-mail: dbamail@ymail.com, viro3b@yahoo.com.mx