Chromatography of mouse hepatitis virus replicative intermediate and replicative form RNA.

Abstract

One of our goals is to obtain large amounts of selected size classes of the Coronavirus replicative intermediates (RI) and native replicative form (RF) RNA that contain the templates for viral genome and messenger RNA syntheses. The two replicative molecules differ in their relative amounts of single-stranded and double-stranded character, with the RF RNA considered essentially or completely double-stranded. It is known that there are seven species of RI RNA, each of which is proportional in size and abundance to one of the seven species of viral messenger RNA produced in infected cells (Sawicki and Sawicki 1990; 1998; Schaad and Baric 1994; Baric and Yount 2000). The presence in coronavirus-infected cells of seven viral minus strand templates was first reported by Sethna and Brian (1989). To assist in this effort, we undertook an analysis of how mouse hepatitis virus (MHV-A59) RI and RF RNA fractionate on three gel filtration matrices, Sepharose 2B, Superose 6 and Sephacryl S-1000. A second goal was to attempt to explain the failure by others to find subgenomic RI and their RF cores after ribonuclease treatment and gel filtration chromatography.