Model for polymerase access to the overlapped L gene of respiratory syncytial virus

The last two genes of respiratory syncytial virus (RSV), M2 and L, overlap by 68 nucleotides, an arrangement which has counterparts in a number of non segmented negative-strand RNA viruses. Thus, the gene-end (GE) signal of M2 lies downstream of the L gene start (CS) signal, separated by 45 nucleotid es. Since RSV transcription ostensibly is sequential and unidirectional fro m a single promoter within the 3' leader region, it was unclear how the pol ymerase accesses the L GS signal. Furthermore, it was previously shown that 90% of transcripts which are initiated at the L GS signal are polyadenylat ed and terminated at the M2 GE signal, yielding a short, truncated L mRNA a s the major transcription product of the L gene. Despite these apparent dow n-regulatory features, we show that the accumulation of full-length L mRNA during RSV infection is only sixfold less than that of its upstream neighbo r, M2, We used cDNA-encoded genome analogs in an intracellular transcriptio n assay to investigate the mechanism of transcription of the overlapped gen es. Expression of L was found to be dependent on sequential transcription f rom the 3' end of the genome. Apart from the L GS signal, the only other st rict requirement for initiation at L was the M2 GE signal. This implies tha t the polymerase accesses the L GS signal only following arrival at the M2 GE signal. Thus, polymerase which terminates at the M2 GE signal presumably scans upstream to initiate at the L GS signal. This also would provide a m echanism whereby polymerase which terminates prematurely during transcripti on of L could recycle from the M2 GE signal to the L GS signal, thereby acc ounting for the unexpectedly high level of synthesis of full length L mRNA. The sequence and spacing between the two signals were not critical. Furthe rmore, the polymerase also was capable of efficiently transcribing from an L GS signal placed downstream of the M2 CE signal, implying that the overla pping arrangement is not obligatory. When copies of the L GS signal were pl aced concurrently upstream and downstream of the M2 GE signal, both were ut ilized. This finding indicates that a polymerase situated at a GE signal is capable of scanning for a GS signal in either the upstream or downstream d irection and thereafter initiating transcription.