IDENTIFICATION OF A SPECIFIC INTERACTION BETWEEN THE CORONAVIRUS MOUSE HEPATITIS-VIRUS A59 NUCLEOCAPSID PROTEIN AND PACKAGING SIGNAL

The coronavirus mouse hepatitis virus (MHV) is an enveloped positive s tranded RNA virus. In infected cells MHV produces a 3' coterminal nest ed se; of subgenomic messenger RNAs. Only the genomic RNA, however, is encapsidated by the nucleocapsid protein and incorporated in infectio us MHV virions. II is believed that an RNA packaging signal (Ps), pres ent only in the genomic RNA, is responsible for this selectivity. Earl ier studies mapped this signal to a 69-nt stem-loop structure position ed in the 3' end of ORF1b., The selective encapsidation mechanism prob ably initiates by specific interaction of the packaging signal with th e nucleocapsid protein. In this study we demonstrate the in vitro inte raction of the MHV-A59 nucleocapsid protein with the packaging signal of: MHV using gel retardation and UV cross-linking assays. This intera ction was observed not only with the nucleocapsid protein from infecte d cells but also with that from purified virions and from cells expres sing a recombinant nucleocapsid protein. The specificity of the intera ction was demonstrated by competition experiments with nonlabeled Ps c ontaining RNAs, tRNA, and total cytoplasmic RNA. The results indicated that no virus specific modification of the N-protein or the presence of other viral proteins are required for this in vitro interaction. Th e assays described in this report provide us With a powerful tool for studying encapsidation (initiation) in more detail. (C) 1997 Academic Press.