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 nonsegmented negative-strand RNA viruses. Thus, the gene-end (GE) signal of M2 lies downstream of the L gene-start (GS) signal, separated by 45 nucleotides. Since RSV transcription ostensibly is sequential and unidirectional from a single promoter within the 3' leader region, it was unclear how the polymerase accesses the L GS signal. Furthermore, it was previously shown that 90% of transcripts which are initiated at the L GS signal are polyadenylated and terminated at the M2 GE signal, yielding a short, truncated L mRNA as the major transcription product of the L gene. Despite these apparent down-regulatory features, we show that the accumulation of full-length L mRNA during RSV infection is only sixfold less than that of its upstream neighbor, M2. We used cDNA-encoded genome analogs in an intracellular transcription assay to investigate the mechanism of transcription of the overlapped genes. Expression of L was found to be dependent on sequential transcription from the 3' end of the genome. Apart from the L GS signal, the only other strict requirement for initiation at L was the M2 GE signal. This implies that 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 mechanism whereby polymerase which terminates prematurely during transcription of L could recycle from the M2 GE signal to the L GS signal, thereby accounting for the unexpectedly high level of synthesis of full-length L mRNA. The sequence and spacing between the two signals were not critical. Furthermore, the polymerase also was capable of efficiently transcribing from an L GS signal placed downstream of the M2 GE signal, implying that the overlapping arrangement is not obligatory. When copies of the L GS signal were placed concurrently upstream and downstream of the M2 GE signal, both were utilized. 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 direction and thereafter initiating transcription.
Fearns
R
R
Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892-0720, USA.
Collins
P L
PL
D001483:Base Sequence; D012321:DNA-Directed RNA Polymerases / Q000235:genetics* / Q000502:physiology; D005814:Genes, Viral; D015707:HN Protein; D008969:Molecular Sequence Data; D018113:Respiratory Syncytial Virus, Human / Q000235:genetics*; D014158:Transcription, Genetic; D014759:Viral Envelope Proteins; D014764:Viral Proteins / Q000235:genetics
D015707:HN Protein; D014759:Viral Envelope Proteins; D014764:Viral Proteins; C116204:attachment protein G; D012321:DNA-Directed RNA Polymerases