BIOSYNTHESIS AND BIOCHEMICAL-PROPERTIES OF THE HEPATITIS-C VIRUS COREPROTEIN

The biosynthesis and biochemical properties of the putative nucleocaps id protein of hepatitis C virus (HCV) were investigated. RNA transcrip ts for cell-free translation were prepared from truncated forms of the cDNA construct encoding the structural proteins of HCV. Processing of the translation products was dependent on microsomal membranes and si gnal recognition particle, suggesting that release of the 21-kDa core protein from the polyprotein precursor is mediated solely by the signa l peptidase of the endoplasmic reticulum (ER) and is achieved by the r emoval of a putative signal sequence of approximately 18 residues loca ted at its C terminus. The core protein was found to bind membranes in vitro and in transfected cells, as shown by centrifugation analysis o f in vitro translation products and transfected-cell lysates. Immunofl uorescence of transfected cells showed that the core protein colocaliz ed with the E2 glycoprotein as well as with a cellular ER membrane mar ker. The nucleocapsid protein expressed by in vitro translation in rab bit reticulocyte lysates cosedimented with the large ribosomal subunit in sucrose gradients. The ribosome binding domain was mapped to the N -terminal region of the core protein. Moreover, the same region was sh own to bind RNA in vitro, suggesting that cosedimentation of core prot ein with ribosomes mag be mediated by the RNA binding of the nucleocap sid protein of HCV. These studies indicate that the HCV core protein i s a cytoplasmic protein associated with the ER membranes and possesses RNA binding activity.