Synergistic effects of gene-end signal mutations and the M2-1 protein on transcription termination by respiratory syncytial virus

Individual mononegavirus genes terminate with a short cis-acting element, t he gene-end (GE) signal, that directs polyadenylation and termination and m ight also influence the efficiency of reinitiation at the next downstream g ene. The 12-13 nucleotide (nt) GE signals of human respiratory syncytial vi rus (RSV) consist of a conserved pentanucleotide (3'-UCAAU, negative sense) , followed by a 3-nt middle region that is AU-rich but otherwise not conser ved, followed by a 4-or 5-nt poly(U) region that is thought to generate the poly(A) tail of the encoded mRNA by reiterative copying. Most of the natur ally occurring differences in the GE signals of the various RSV genes occur in the "middle" and "poly(U)" regions. We mutated a copy of the fusion pro tein (F) GE signal that was positioned at the end of the promoter-proximal gene of a tricistronic minigenome and evaluated the effect of these mutatio ns on RSV transcription in a plasmid-initiated, intracellular assay. Mutati ons confirmed the importance of the middle region's AU-rich nature and 3-nt length, and the poly(U) tract's 4-nt minimum functional length, with maxim al termination efficiency observed at five U residues. Nt assignments other than U at position 13 also affected the efficiency of termination, showing that this position is part of the functional 13-nt GE signal. These result s indicate that differences in nt assignments in the middle and poly(U) reg ions of the GE signal, which occur frequently In nature, affect the efficie ncy of termination. Unexpectedly, the ability of certain mutations to inhib it termination was completely dependent on coexpression of the M2-1 protein , and in many other cases the inhibitory effect of the mutation was greatly enhanced in the presence of M2-1. Thus, M2-1 appears to have the effect of altering the polymerase such that it ignores suboptimal GE signals. Intere stingly, certain mutations that greatly decreased the efficiency of termina tion in the absence of M2-1 did not have much effect on the expression of t he second gene, implying that correct termination and/or polyadenylation at the upstream gene is not obligatory for reinitiation at the next downstrea m gene. (C) 2001 Academic Press.