Murine coronavirus packaging signal confers packaging to nonviral RNA

Abstract

Studies of defective interfering (DI) RNAs of the murine coronavirus mouse hepatitis virus (MHV) suggest that a 69-nucleotide-long packaging signal is necessary for MHV genomic RNA packaging into MHV particles. In this study we showed that when RNA transcripts that consisted of a non-MHV sequence and the packaging signal were expressed in MHV-infected cells, they were packaged into MHV particles. Those RNA transcripts that lacked the packaging signal or those containing a mutated packaging signal did not package efficiently. Thus, the presence of the packaging signal was sufficient for RNA packaging into MHV particles. Packaging of viral genomic RNA into virus particles involves recognition and interaction of various molecules and is an essential step in the multiplication of any RNA virus. RNA packaging signals necessary for virus RNA packaging are described for several RNA viruses and hepadnaviruses (1, 5, 7, 11, 20, 27, 36, 37, 41, 42). Among these viruses, there are several examples in which the identified packaging signal is necessary and sufficient for viral RNA packaging (1, 7, 11, 42). The packaging signal(s) of some RNA viruses is mapped within more than one viral genomic region (20, 27, 36); for many viruses, it is not known whether a combination of multiple genomic regions or only one region is necessary for viral RNA packaging. Nor is it known if the packaging signal alone can suffice for viral RNA packaging. Coronaviruses are large enveloped viruses containing a long, single-stranded, positive-sense genomic RNA that cause a variety of diseases in humans and animals (35). A prototype coronavirus, mouse hepatitis virus (MHV), contains a 31 kblong MHV genomic RNA (17, 26). Other coronaviruses, such as infectious bronchitis virus (40), transmissible gastroenteritis virus (TGEV) (29), and bovine coronavirus (9), package virusspecific subgenomic RNAs as well as nonviral cellular RNA. Coronavirus contains four proteins, S, M, sM, and N protein; all except N protein are found in the virus envelope. S protein binds to host-cell receptors (38) and induces cell fusion (4). M protein, a triple-spanning transmembrane protein (2), is the most abundant glycoprotein. A minute amount of sM protein associates with MHV envelope (39). N protein and the genomic RNA form a helical nucleocapsid (21). Recently Risco et al. demonstrated that TGEV particles contain a spherical internal core, which seems to consist of mostly M protein and a lesser amount of N protein (28). They also showed that the helical nucleocapsid locates inside of the internal core (28). M protein and sM protein are necessary for packaging of viral nucleocapsid (12) and MHV envelope formation (3, 12, 34), while S protein is dispensable for both these functions (3, 12, 34). The MHV nonstructural proteins, includ

Coronavirus cis-Acting RNA Elements

“…PSs of alpha-and gammacoronaviruses were first identified for TGEV and IBV (Escors et al, 2003;Penzes et al, 1994). MHV DI RNA studies revealed a 69-nt SL structure that was (i) located in the 3 0 region of ORF1b, (ii) confirmed to be required for DI RNA packaging, and (iii) shown to interact with the viral N protein (Fosmire et al, 1992;Molenkamp and Spaan, 1997;Woo et al, 1997). Subsequent studies indicated that a larger PS element and, possibly, additional factors are required for optimal packaging efficiency (Bos et al, 1997;Cologna and Hogue, 2000;Narayanan and Makino, 2001).…”

Molecular Interactions in the Assembly of Coronaviruses

“…This interaction has so far been reported only for MHV, by Makino and co-workers. These workers had shown earlier that the 69-nucleotide packaging signal located in the pol1b gene could mediate the incorporation into virions of RNAs of even noncoronaviral origin (Woo et al, 1997). They subsequently showed that this incorporation is most likely effected by a direct and specific interaction of the signal with the M protein.…”

“…The signal appeared to be sufficient for RNA packaging as its inclusion allowed a synthetic subgenomic mRNA of MHV-A59 to be packaged specifically; the encapsidation efficiency of the mRNA was, however, significantly lower than that of the defective genomic RNA from which it was transcribed . Even a nonviral RNA was found to be packaged into MHV particles when provided with the packaging signal (Woo et al, 1997). Buoyant density analysis of the particles revealed that the RNA was not assembled separately but copackaged with helper virus RNA.…”

Group-specific structural features of the 5′-proximal sequences of coronavirus genomic RNAs

“…Studies of the genomic PSs in CoVs have been mainly focused on group IIa CoVs in the past, e.g. MHV and BCoV (Fosmire et al, 1992;Makino et al, 1990;van der Most et al, 1991;Woo et al, 1997;Chen et al, 2007;Cologna and Hogue, 2000). For other groups of CoVs, e.g.…”