Published Ahead of Print 2 January 2013.
2013, 87(6):3196. DOI: 10.1128/JVI.02862-12.
and Rachel Fearns
Chadene Z. Tremaglio, Sarah L. Noton, Laure R. Deflubé
the Leader Promoter
ofCan Initiate Transcription from Position 3
Respiratory Syncytial Virus Polymerase
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Respiratory Syncytial Virus Polymerase Can Initiate Transcription
from Position 3 of the Leader Promoter
Chadene Z. Tremaglio, Sarah L. Noton, Laure R. Deflubé, Rachel Fearns
Department of Microbiology, Boston University School of Medicine, Boston, Massachusetts, USA
with an in vitro assay in which RSV RNA synthesis was reconstituted using purified RdRp and an RNA oligonucleotide showed
thatnt1to14oftheLepromoterweresufficienttosignalinitiationfrom ?3andthattheRdRpcouldaccessthe ?3initiation
ingly recognized as a significant pathogen of the elderly and im-
supportive care, and at present there are no approved vaccines.
RSV is a member of the family Paramyxoviridae in the order
Mononegavirales, the nonsegmented negative-strand (NNS) RNA
the template for mRNA transcription and genome replication,
(reviewed in reference 2).
The RSV genome is 15.2 kb and encodes mRNAs from 10 se-
quentially arranged genes (reviewed in reference 3). Two short
extragenic regions border the genome, a 3= 44-nt leader region
(Le) and a 5= 155-nt trailer (Tr) region (4, 5). The Le region con-
obligatorily sequential, polarized manner to generate 10 sub-
genomic mRNAs (6). During this process, the RdRp is controlled
by cis-acting gene start (GS) and gene end (GE) sequences that
sequences being identical and the tenth having only slight differ-
ences (9). In related viruses, the GS signals have been shown to be
cent transcript (10–12). Capping is thought to allow the RdRp to
enter into a stable elongation mode in which it can extend the
RNA to the GE signal, where the RdRp polyadenylates and then
releases the mRNA (13). The RdRp is then able to scan the tem-
plate to locate the next GS signal and reinitiate mRNA synthesis
(14). RNA replication involves synthesis of a replicative interme-
diate, the antigenome, which is a full-length positive-sense com-
plement of the genome. The trailer complement (TrC) region at
the 3= end of the antigenome contains a strong promoter for syn-
thesis of genome RNA (4, 15). In contrast to the viral mRNAs,
antigenome and genome RNAs are encapsidated in viral nucleo-
espiratory syncytial virus (RSV) is the leading cause of pneu-
monia and viral deaths in infants worldwide and is increas-
protein (N) as they are synthesized. Each N monomer binds 7
nucleocapsid structure (16), which can act as a template for the
sivity of the RdRp and is believed to enable the RdRp to read
through GE signals (17, 18).
Because transcription and RNA replication are both initiated
RdRp is coordinated between the two processes is poorly under-
RdRp initiates transcription. The mRNA for the first RSV gene
(NS1) is initiated at nt ?45 on the genome, but it is unclear how
this can occur. The first model is based largely on studies of other
paramyxoviruses and suggests that the RdRp initiates transcrip-
tion opposite nt ?1 of the Le, in the same way as replication
initiation. However, during transcription, the RdRp does not
form a stable elongation complex and so releases the nascent Le
transcript and scans to locate the first GS signal and reinitiate
transcription (19). The second model is based on studies with
template directly at the first GS signal (20, 21). It has also been
proposed that both mechanisms might come into play during in-
governed by the level of available N protein (19).
Studies to investigate the mechanisms of transcription and
replication initiation in RSV have mapped the sequence elements
Received 14 October 2012 Accepted 26 December 2012
Published ahead of print 2 January 2013
Address correspondence to Rachel Fearns, firstname.lastname@example.org.
Copyright © 2013, American Society for Microbiology. All Rights Reserved.
jvi.asm.orgJournal of Virology p. 3196–3207March 2013 Volume 87 Number 6
on June 10, 2014 by guest
once the N:RNA structure is opened by the RdRp engaging with
the Le promoter, all subsequent initiations take place directly at
ies do not distinguish between these two possible models, they do
offer a new perspective on how transcription is initiated in RSV.
of producing large amounts of small RNA. If the ?25-nt tran-
scripts are uncapped and unencapsidated, they have the potential
to be recognized by RIG-I and to alert the cell to the presence of
binds to RSV LeC-containing RNA to subvert activation of RIG-I
(37). It is also possible that these transcripts are not merely by-
products of transcription and replication but play a functional
the Tr RNA of Sendai virus can bind TIAR (and also possibly
study of the RSV Tr suggests it might play a similar role (39). In
addition, small RNAs generated from the influenza virus pro-
moter have been shown to bind the viral RdRp complex and in-
fluence transcription and replication (40, 41). An intriguing fea-
ture of the small RNAs generated from the RSV Le (and TrC)
processed microRNA; therefore, in future studies it will be of sig-
nificant interest to determine if they are able to function in the
cellular microRNA pathway.
assays illustrated in Fig. 5, as well as for making the initial observation
that the 4C minigenome makes a Le-CAT1 readthrough RNA, Bernard
Moss for providing MVA-T7, Peter Collins for providing the plasmids
required for minigenome expression, and Sean Whelan and members
of his group for helpful discussion.
This work was funded by NIH grant AI074903 to R.F. and NIH T32
grant AI07642 to the Department of Microbiology at Boston University
School of Medicine to support C.Z.T.
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RNA Synthesis Initiation within the RSV Leader Region
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