ASSEMBLED CORONAVIRUS FROM COMPLEMENTATION OF 2 DEFECTIVE INTERFERINGRNAS

In the presence of an RNA(-) temperature-sensitive (ts) mutant helper virus, two coronavirus mouse hepatitis virus (MHV) defective interferi ng (DI) RNAs complemented each other, resulting in the assembly of MHV particles; we used this ability to complement as a means to study cor onavirus assembly. One of the two DI RNAs was DISsA, a naturally occur ring self-replicating DI RNA encoding N protein and the gene 1 protein s that encode RNA polymerase function; DIssA supports the replication and transcription of other non self-replicating DI RNAs. The other DI was a genetically engineered DI RNA that encoded sM and M proteins. Co infection of these two DIs at the nonpermissive temperature for the ts helper virus resulted in replication and transcription of both DI RNA s but not in synthesis of the helper virus RNAs. MHV particles contain ing DI RNAs, N protein, and M protein, all of which were exclusively d erived from the two DI RNAs, were released from the coinfected cells; the amount of sM protein was below the limits of detection. Analyses o f DI RNAs with mutations in the two envelope protein genes demonstrate d that hi and sM proteins appeared to be required for assembly and rel ease of MHV particles that contained DI RNAs and N protein, while S pr otein was not required for assembly and release of MHV particles.