Analysis of the Hepatitis C virus dimerization process

BIOL 17

Sumangala S Shetty, shettys@duq.edu1, Ruszkowski Kailey, ruszkowskik@duq.edu1, and Mihaela Rita Mihailescu, mihailescum@duq.edu2. (1) Department of Chemistry and Biochemistry, Duquesne University, 308 Mellon Hall, 600 Forbes Avenue, Pittsburgh, PA 15282, (2) Department of Chemistry & Biochemistry, Duquesne University, 308 Mellon Hall, 600 Forbes Ave, Pittsburgh, PA 15282
Hepatitis C, a life threatening disease, is caused by the hepatitis C virus (HCV) which is replicated by an error-prone polymerase, therefore undergoing rapid evolution. Considering that 170 million people are chronically infected and over half of the total HCV infected patients are refractory to current therapy, there is an urgent need for an efficacious immunoprophylactic and therapeutic agent. HCV is an enveloped virus with a positive sense RNA genome of ~9.6kb. This RNA carries a large open reading frame (ORF) flanked by highly conserved 5'- and 3'- untranslated regions (UTRs). The ORF encodes for a single polypeptide which is cleaved to generate at least 10 different HCV proteins. Since HCV has a high mutational rate, the study of its UTR conserved regions would contribute towards the development of new therapeutic agents. The HCV 3' UTR contains a 98 nt sequence, named X RNA, which has been shown to be important in the virus replication cycle. Within X-RNA, there is a 16 nucleotide palindromic sequence (called dimer linkage sequence-DLS) that is responsible for the in vitro RNA dimerization, in the presence of the HCV core protein. In this study, we use biophysical techniques like fluorescence and NMR spectroscopy, to analyze the dimerization properties of a 55 nt X RNA fragment (containing the DLS) and its interactions with the HCV core protein. Our results strongly suggest that this dimerization process occurs through a kissing complex intermediate, which then gets converted to a more stable duplex structure in the presence of the HCV core protein.