Role of the M2-1 transcription antitermination protein of respiratory syncytial virus in sequential transcription

M2-1 protein of human respiratory syncytial virus (RSV) is a transcription antitermination factor that is important fur the efficient synthesis of ful l-length mRNAs as well as for the synthesis of polycistronic readthrough mR NAs, which are characteristic of nonsegmented negative-strand RNA viruses. The contributions of these effects to RSV sequential transcription were inv estigated with minigenomes which contained one to five genes which were eit her foreign marker genes or authentic RSV genes. When evaluated on a promot er-proximal gene, the effect of M2-1 on the synthesis of full-length mRNA w as much greater for a long (1,212- or 1,780-nucleotide) gene (up to a 615-f old increase) than for a short (274-nucleotide) gene (less than a 2-fold in crease). This was independent of whether the gene contained non-RSV or RSV- specific sequence. Once the polymerase had terminated prematurely, it was u nable to reinitiate at a downstream gene. These studies also confirmed that M2-1 enhances the synthesis of polycistronic mRNAs; and that the magnitude of this effect varied greatly among different naturally occurring gene jun ctions, The synthesis of polycistronic mRNAs, which presumably involves ant itermination at the gene-end signal, required a higher level of M2-1 than d id the synthesis of the corresponding monocistronic mRNAs. M2-1 did not hav e a comparable antitermination effect at the junction between the leader re gion and the first gene, In a minigenome containing the NS1 and NS2 genes i n their authentic sequence context, synthesis of full-length NS1 and NS2 mR NAs in the absence of M2-1 was remarkably high (36 and 57%, respectively, o f the maximum levels observed in the presence of M2-1), In contrast, synthe sis of mRNA from additional downstream genes was highly dependent on M2-1. Thus, RSV has the potential for two transcription programs: one in the abse nce of M2-1, in which only the NS1 and NS2 genes are transcribed, and one i n the presence of M2-1, in which sequential transcription of the complete g enome occurs, The dependence on M2-1 for transcription was greater for a ge ne in the fifth position from the promoter than for one in the third positi on. This indicates that under conditions where M2-1 is limiting its concent ration affects the gradient of transcription. Although M2-1 was found to ha ve profound effects on transcription, it had no effect on replication of an y minigenome tested, suggesting that it is not an active participant in RNA replication or regulation of RNA replication. Finally, since a permissive RSV infection is marked by a gradual increase in the intracellular accumula tion of viral proteins including M2-1, we examined the relative abundances of various mRNAs during RSV infection for evidence of temporal regulation o f transcription. None was found, implying that the availability of M2-1 dur ing a permissive infection is sufficient at all times such that its concent ration does not mediate temporal regulation of gene transcription.