Measuring Hepatitis C Virus Envelopment by Using a Proteinase K Protection Assay

Abstract

The infectious virion of hepatitis C virus (HCV) is made up of the viral nucleocapsid surrounded by an envelope that contains an ER-derived membrane bilayer, cellular lipids, and the viral E1 and E2 glycoproteins. Because the infectious HCV particle contains both protein and lipid layers, selective disruption of these layers and analysis for the presence or absence of resulting virion components can be used to study the virion assembly process. This chapter describes an experimental method to measure HCV virion envelopment, which can reveal the mechanisms of how specific viral protein-protein interactions and host factors contribute to the process of HCV envelopment.

Keywords: Electroporation; Envelopment; Hepatitis C virus; Immunoblot; Protection assay; Proteinase K.

Figure 1.

The HCV virion. The HCV virion contains a central Core/RNA complex known as the nucleocapsid, surrounded by lipids and an ER-derived membrane, referred to as the envelope. This envelope also contains the HCV E1 and E2 glycoproteins. Each of the these main virion layers can be antagonized (as noted) for studies of HCV virion formation, as described in the text.

Figure 2.

Immunoblot analysis for HCV Core protein of Huh7.5 cell lysates at 48 hours post electroporation with in vitro transcribed HCV RNA [Wild-type (WT) or with a deletion in the E1/E2 genes (ΔE1/E2)] subjected to the indicated treatments in a proteinase K protection assay.