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Journal of Virology, January 1999, p. 388-397, Vol. 73, No. 1
Laboratory of Infectious Diseases, National
Institute of Allergy and Infectious Diseases, Bethesda, Maryland
20892-0720
Received 21 April 1998/Accepted 23 September 1998
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.
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Model for Polymerase Access to the Overlapped L
Gene of Respiratory Syncytial Virus
*
Corresponding author. Mailing address: LID, NIAID, NIH,
7 Center Dr., MSC 0720, Bethesda, MD 20892-0720. Phone: (301) 496-3481. Fax: (301) 496-8312. E-mail:
pcollins{at}atlas.niaid.nih.gov.
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