Optimization of targeted RNA recombination and mapping of a novel nucleocapsid gene mutation in the coronavirus mouse hepatitis virus

Abstract

We have recently described a method of introducing site-specific mutations into the genome of the coronavirus mouse hepatitis virus (MHV) by RNA recombination between cotransfected genomic RNA and a synthetic subgenomic mRNA (C. A. Koetzner, M. M. Parker, C. S. Ricard, L. S. Sturman, and P. S. Masters, J. Virol. 66:1841-1848. By using a thermolabile N protein mutant of MHV (Alb4) as the recipient virus and synthetic RNA7 (the mRNA for the nucleocapsid protein N) as the donor, we selected engineered recombinant viruses as heat-stable progeny resulting from cotransfection. We have now been able to greatly increase the efficiency of targeted recombination in this process by using a synthetic defective interfering (DI) RNA in place of RNA7. The frequency of recombination is sufficiently high that, with Alb4 as the recipient, recombinants can be directly identified without using thermal selection. The synthetic DI RNA has been used to demonstrate that the lesion in another temperature-sensitive and thermolabile MHV mutant, Albl, maps to the N gene. Sequencing of the Albl N gene revealed two closely linked point mutations that fall in a region of the N molecule previously noted as being the most highly conserved region among all of the coronavirus N proteins. Analysis of revertants of the Albl mutant revealed that one of the two mutations is critical for the temperature-sensitive phenotype; the second mutation is phenotypically silent.

Recombination between North American strains of porcine reproductive and respiratory syndrome virus

“…The arterivirus family belongs to the order Nido6irales, along with coronaviruses and toroviruses (Cavanagh, 1997). Coronaviruses undergo high frequency viral recombination (Lai et al, 1985;Keck et al, 1988;Masters et al, 1994;Jia et al, 1995;reviewed in Lai, 1996) and toroviruses appear to have undergone non-homologous recombination events (Snijder, et al, 1991). Although putative recombination events among arteriviruses have been reported (Chirnside et al, 1994;Godeny et al, 1998), we provide the first experimental evidence that arteriviruses undergo homologous recombination.…”

Reverse Genetics of The Largest RNA Viruses

“…Subsequently, Masters et al (1994) directly compared the rates of targeted recombination with RNA7 versus DI donor RNAs and noted frequencies with the latter comparable to those that had been observed with MIDI-C RNA. For this purpose a vector template for synthesis of a pseudo-DI RNA, pB36 ( Fig.…”

“…3). Although pB36 RNA was shown to be an authentically replicating DI RNA (Masters et al, 1994), it has never been determined if the larger constructs are replicationcompetent because they serve well as donor RNAs. Moreover, one particular variant smaller construct, pBL34 RNA (Hsue and Masters, 19971, was found to not replicate despite being an efficient donor RNA.…”

“…Moreover, one particular variant smaller construct, pBL34 RNA (Hsue and Masters, 19971, was found to not replicate despite being an efficient donor RNA. It was originally thought that the ability of the donor RNA to replicate was the crucial feature that led to an enhanced efficiency of recombination (Masters et al, 1994), but this may not be the case. Another observation, possibly related, is that many targeted recombinants were isolated that were found to have been generated by multiple crossovers (Hsue and Masters, 1997;Peng et al, 1995b;de Haan et al, 1998; L. Kuo and P. S. Masters, unpublished results, 1998).…”

“…In a few instances another N mutant, Albl, also has proved a suitable recipient (Fischer et al, 1997a;Peng et al, 1995b). Albl is a point mutant that, like Alb4, is both temperature sensitive and thermolabile (Masters et al, 1994). In contrast, an S gene mutant, Alb18 (Ricard et al, 1995), thus far has not been a useful recipient, perhaps because there is significant selective pressure favoring accumulation of revertants even at the supposedly permissive temperature for this virus.…”