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Previous Article | Next Article 
J Virol, January 1998, p. 520-526, Vol. 72, No. 1
Department of Microbiology, University of
Alabama School of Medicine at Birmingham, Birmingham, Alabama 35294
Received 3 September 1997/Accepted 9 October 1997
The mRNA encoding the M2 protein of respiratory syncytial (RS)
virus contains two open reading frames (ORFs). ORF1 encodes the 22-kDa
structural protein, M2, and ORF2 has the potential to encode a 10-kDa
protein (90 amino acids). Using a vaccinia virus T7 expression system,
we examined the RNA synthetic activities of mono- and dicistronic
subgenomic replicons of RS virus by direct metabolic labeling of RNA in
the presence and absence of the products of ORF1 and ORF2. In the
absence of ORF1 and ORF2, the negative- and positive-sense products of
genomic RNA replication and positive-sense polyadenylated mRNA(s) were
synthesized. Expression of the whole M2 transcription unit (containing
ORF1 and ORF2) or ORF1 alone caused an increase in the synthesis of
polyadenylated mRNA, the majority of which was due to a substantial
increase in the quantity of polycistronic mRNAs generated by the
polymerase failing to terminate at gene end signals. In agreement with
previous reports, the ORF2 product was found to inhibit viral RNA
replication and mRNA transcription. These data show that the M2 protein
functions as a transcriptional antiterminator that enhances the ability of the viral RNA polymerase to read through intergenic junctions. The
role of such a function during the viral life cycle is discussed.
Human respiratory syncytial (RS)
virus is a member of the pneumovirus genus of the
Paramyxoviridae and is the leading viral cause of pediatric
pneumonia and bronchiolitis. During transcription of the negative-sense
RNA genome (15,222 nucleotides), 10 mRNAs that encode at least 11 proteins are made (10, 13). The genomic RNA is present in
infected cells and virions as a ribonucleocapsid complex, in which the
RNA is tightly bound by the nucleocapsid (N) protein (21).
Also associated with the ribonucleocapsid are the phosphoprotein (P)
and large polymerase protein (L) (21). These two proteins
are the viral components required for transcription of the RS virus
mRNAs and, along with the N protein, for replication of the genome
(20, 34). Recent work indicates that the RS virus M2 protein
is also involved in transcription (9).
The genome of RS virus possesses a single polymerase entry site at or
near the 3' end, and UV mapping has shown transcription of the genes
occurs in a sequential polar fashion from 3' to 5' (15).
Each gene is flanked by conserved gene start and gene end signals, and
the genes are separated by intergenic regions of varying size and
sequence which are not represented in the mRNAs (7, 24). The
exact mechanisms of initiation and termination of transcription for
nonsegmented negative-strand RNA viruses are not understood. However,
the gene start is believed to signal transcription initiation and mRNA
capping, and the gene end signals transcription termination and
polyadenylation (23, 25). The level of each RS virus mRNA
correlates with its distance from the 3' promoter in the genome; i.e.,
the 3'-most gene gives rise to the greatest amount of mRNA (2,
15). By analogy with other nonsegmented negative-strand RNA
viruses, this regulation is thought to be achieved by transcriptional
attenuation at each gene junction. It is postulated that some of the
polymerase molecules which have terminated transcription of the
upstream gene do not reinitiate transcription of the downstream gene
(22). In vesicular stomatitis virus, where this has been
examined directly (3, 22), transcriptional attenuation has
been measured at approximately 30% at each junction.
The M2 gene, found only in the pneumoviruses, encodes an mRNA
containing two open reading frames (ORFs). ORF1 encodes the 22-kDa
structural protein designated M2, whereas ORF2 has the capacity to
produce a 90-amino-acid polypeptide (5, 14), detection of
which has not been reported. The M2 protein contains a
Cys3-His1 motif near the amino terminus which
is highly conserved in the M2 proteins of the members of the
pneumovirus genus (1, 5, 14, 27, 35). This motif in other
proteins has been shown to bind zinc ions (33). Also,
interaction between the M2 and N proteins has been observed upon
coexpression and in infected cells (17). During dissociation
of virions with detergent and high salt concentrations, M2 was removed
from the nucleocapsid cores under similar conditions to the matrix (M)
protein and was therefore termed matrix-like (21). Recent
reports have shown that the M2 protein is required for recovery of
infectious RS virus from a cDNA clone (8), and it has been
proposed that the M2 protein enhances the processivity of the
polymerase and thus acts as a transcription elongation factor (9,
20).
Here we report an analysis of the roles played by the products of ORF1
and ORF2 of the M2 mRNA in RS virus RNA transcription and replication
by direct observation of the RNA, using metabolic labeling. Results
presented here show that expression of the product of ORF1, the M2
protein, led to an enhancement in transcriptional readthrough at gene
end signals. This finding demonstrates yet another role of the M2
protein in addition to that previously reported (9). The
implications of a transcriptional antitermination activity for the
replication cycle of the virus are discussed.
cDNA constructs.
A cDNA encoding a subgenomic replicon of RS
virus A-2 strain was generated as previously described by Yu et al.
(34). The subgenomic replicon WT5 shown in Fig.
1 was derived from the previously described WT (wild-type) analog (34). The cDNA encoding the subgenomic WT replicon was generated by fusing the 5' 1,182 nucleotides of the RS virus genome (nucleotides 14040 to 15222 in the positive sense; a single nucleotide deletion is present in the WT replicon at a
position corresponding to 15211 in the RS virus genome) to the 3' 413 nucleotides of the genome (nucleotides 1 to 413), using MunI
sites located in the cDNA clones of the NS1 and L genes. The cDNA
encoding WT5 (pWT5 [Fig. 1]) was generated by digestion of the WT
cDNA with BclI and BglII. This removed a 860-bp
fragment of L sequence (nucleotides 14146 to 15005 of the RS virus
genome). Upon religation, this construct contained 745 nucleotides of
RS virus sequence. It was predicted that the subgenomic replicon RNA
synthesized from this cDNA by T7 RNA polymerase followed by autocatalytic cleavage by the hepatitis delta ribozyme would contain two non-RS virus G residues at the 5' end preceding the trailer sequence and would therefore be 747 nucleotides in length. This subgenomic replicon contained a G-to-C change at position 4 in the
leader sequence which causes enhanced RNA synthesis from this template
(11). A cDNA encoding a subgenomic replicon containing two
transcription units, pM/SH, was made by the insertion of a BsmI fragment containing the M/SH gene junction (nucleotides
3895 to 4497 of the RS virus genome) into a unique BsmI site
(nucleotide 319 of the WT5 sequence) in pWT5 (see Fig. 4A).
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
The Product of the Respiratory Syncytial Virus M2
Gene ORF1 Enhances Readthrough of Intergenic Junctions during
Viral Transcription
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
View larger version (13K):
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FIG. 1.
Diagram of plasmid pWT5 and the RNAs predicted to be
synthesized from the monocistronic subgenomic replicon that it encodes.
The subgenomic replicon transcribed from pWT5 includes the RS virus 3'
and 5' termini flanking a fused partial NS1 and L gene. The 3' leader
(le) sequence includes the G-to-C mutation previously described
(11). These sequences were inserted between a T7 RNA
polymerase promoter (T7) and a copy of the self-cleaving hepatitis
delta ribozyme (H 
V). Thus, transcription by T7 polymerase followed
by self-cleavage gave rise to a negative-sense copy of the subgenomic
replicon RNA. This RNA can act as a template from which the RS virus
RNA polymerase can synthesize a 547-nucleotide (nt) mRNA [not
including the poly(A) tail] or a positive-sense product of
replication. tr, trailer.
DNA transfections, radioactive labeling, and electrophoretic analysis of RNA. HEp-2 cells were grown in minimum essential medium (GIBCO Laboratories) supplemented with 5% heat-inactivated fetal bovine serum in 60-mm-diameter dishes. HEp-2 cells, infected with recombinant vaccinia virus expressing T7 RNA polymerase (vTF7-3), were transfected with plasmids expressing the subgenomic replicons and plasmids that expressed the trans-acting proteins necessary for RNA encapsidation, replication, and transcription (6, 34). For the RNA synthesis assays, cells were transfected with 5 µg of pWT5 or pM/SH, 5 µg of pN, 2 µg of pP, 2 µg of pL, and 0 or 0.1 to 2 µg of pM2, pORF1, or pORF2. Sixteen hours posttransfection, RS virus-specific RNAs were metabolically labeled with [3H]uridine (33 µCi/ml) for 5 h in the presence of actinomycin D (10 µg/ml).
Cells were harvested and cytoplasmic extracts were prepared as previously described (29). RNA was purified either directly or after immunoprecipitation of ribonucleocapsids, using RS virus-specific antiserum (34). Purified RNAs were analyzed by electrophoresis in 1.75% agarose-urea gels (31) and detected by fluorography (26).Immunoprecipitation of encapsidated RNA. Ribonucleocapsid complexes were immunoprecipitated from cytoplasmic extracts with goat anti-RS virus antiserum (Chemicon) and protein G-Sepharose (Pharmacia). RNA was purified by phenol extraction of precipitated complexes and analyzed as described above.
Oligo(dT) chromatography. RNA purified by phenol extraction was subjected to oligo(dT) chromatography [oligo(dT)-cellulose; New England Biolabs]. Polyadenylated RNA was bound to the oligo(dT)-cellulose in a high-salt buffer (10 mM Tris-HCl [pH 7.5], 0.4 M NaCl, 1 mM EDTA, 0.02% sodium dodecyl sulfate) and eluted in low salt (100 mM Tris-HCl [pH 7.5], 1 mM EDTA). The bound and unbound fractions were recovered by ethanol precipitation, analyzed by 1.75% agarose-urea gel electrophoresis, and detected by fluorography.
In vitro transcription. RNA was generated in vitro by using T7 RNA polymerase (GIBCO-BRL) according to the manufacturer's instructions, with minor modifications (32). The ribonucleoside triphosphate concentrations were increased to 2.5 mM and supplemented with [3H]UTP (80 µCi/ml). RNA products of in vitro synthesis were resolved in 1.75% agarose-urea gels.
RNase H analysis of RNA products.
Phenol-extracted RNAs were
incubated at 25°C for 20 min with an equal volume (20 µl) of 2×
RNase H buffer (3, 4) and 10 µl of 1× RNase H buffer with
or without 1 µg of an appropriate oligonucleotide. RNase H digestions
were performed by the addition of 2 U of RNase H (GIBCO-BRL) and
incubation for 30 min at 37°C. Digested RNAs were recovered by
ethanol precipitation. RNAs were identified by electrophoretic mobility
shifts in 1.75% agarose-urea gels following RNase H digestion. The
oligonucleotides used are as follows (all designations are given from
5' to 3' of the positive or negative replication product of the
subgenomic replicon WT5): oligo(dT), T12-15 (anneals to
polyadenylated RNA); 3'L, 5'-GTTAGTGTATAGCTATGGG-3' (anneals
to positive-sense WT5 RNA from nucleotides 546 to 564); 
tr,
5'-AACCAATTAGATTAGGG-3' (anneals to positive-sense WT5 RNA
from nucleotides 675 to 691); 5'NS1, 5'-GAATTGCTGCCCATCTC-3'
(anneals to positive-sense WT5 RNA from nucleotides 96 to 112);
and +tr, 5'-CATTTTAATCTTGGAGG-3' (anneals to negative-sense
WT5 RNA from nucleotides 84 to 100).
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RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
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Effect of M2 mRNA products on RNA synthesis from the WT5 template. The effect of the M2 protein(s) on RNA synthesis was analyzed initially by using a subgenomic replicon, WT5, containing a single transcription unit (Fig. 1). This was generated by fusing the 3' end of the RS virus genome, containing the leader and NS1 gene start signal, to the 5' end of the genome, including the L gene end signal and the trailer sequence (745 nucleotides in total [Fig. 1]). Thus, the entire central portion of the RS virus genome was deleted, and a single transcription unit designed to express an mRNA of 547 nucleotides not including poly(A) was left (Fig. 1).
Expression of this genomic analog in vTF7-3-infected HEp-2 cells in the presence of N, P, and L proteins resulted in the actinomycin D-resistant synthesis of three RNA species (Fig. 2, lane 2). Upon addition of pM2 (encoding ORF1 and ORF2 of the M2 mRNA) to transfections, a significant increase in the quantity of slower-migrating RNA was observed (Fig. 2, lane 3). The RNA species were preliminarily identified by (i) immunoprecipitation of N encapsidated genomic RNA replication products, (ii) comigration of RNA with a negative-sense in vitro transcript of WT5, and (iii) oligo(dT) chromatography. The two slower-migrating RNAs (seen readily in lane 2 but masked in lane 3 by another RNA species [see below]) were identified as products of replication (negative and positive sense), as they were immunoprecipitated with anti-RS virus serum in the presence (Fig. 2, lane 7) and absence (data not shown) of pM2. This result indicated that they were encapsidated to form a ribonucleoprotein complex (28, 34). The upper band of the doublet was provisionally identified as the negative-sense product of replication, as it comigrated with an in vitro transcript of the negative-sense subgenomic replicon WT5 (Fig. 2, lane 6). While the negative- and positive-sense replication products are the same size, they can be separated by agarose-urea gel electrophoresis due to the difference in base composition (28).
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Identification of RNA products by RNase H analysis. The similar electrophoretic mobilities of the genomic RNAs and the polyadenylated r/t RNA synthesized in greater abundance upon addition of pM2 prevented the use of Northern blot analysis to definitively identify these RNAs. Therefore, digestion with RNase H following annealing of oligonucleotides specific for regions of the genome, antigenome, or mRNA was used to identify the products of RNA synthesis (3, 4). These analyses were performed on RNAs harvested from transfected cells in which neither M2 ORF1 nor ORF2 was present, or in which both ORFs of the M2 mRNA (pM2) were present, in order to determine their effect on viral RNA synthesis.
Figure 3 shows the identification of the products of RNA synthesis from the WT5 subgenomic replicon in the presence and absence of pM2 (encoding ORF1 and ORF2 of the M2 mRNA) and confirms the effect of pM2 on the pattern of RNA synthesis (Fig. 3A, lanes 1 and 7). These data also show that the products of RNA synthesis are not digested by RNase H in the absence of an oligonucleotide (Fig. 3A, lanes 1 and 7). However, in the presence of specific oligonucleotides, certain RNAs became susceptible to RNase H digestion, which allowed them to be identified.
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tr). Digestion in the presence of tr caused no
shift in migration of the mRNA band (Fig. 3A, lanes 3 and 9),
indicating that the positive-sense trailer sequence was not present in
this RNA. Thus, we concluded that this RNA is the expected
547-nucleotide, polyadenylated mRNA product of transcription.
The positive-sense replication product is predicted to contain
positive-sense leader, NS1 gene, L gene, and trailer sequences. Sensitivity to RNase H digestion following annealing of the 3'L and
tr oligonucleotides was used initially to identify this RNA. Following annealing with 3'L, all products of RNA synthesis except the
negative-sense replication product (provisionally identified above)
were sensitive to RNase H digestion, which produced bands A and B (Fig.
3A, lanes 2 and 8). Digestion in the presence of tr did not alter the
migration of the negative-sense replication product or the mRNA but did
give rise to bands C and D (Fig. 3A, lanes 3 and 9). These products of
RNase H digestion were of sizes consistent with the predicted products
outlined in Fig. 3B. It was postulated that bands A and C migrated more
slowly than B and D due to the presence of leader sequence (Fig. 3B)
and, hence, represented the RNase H digestion products of the
positive-sense replication product. To determine if this supposition
was correct, a digestion was performed in the presence of
oligonucleotides 3'L and 5'NS1 in order to remove both the 3' and 5'
ends of all positive-sense RNAs. This digestion gave rise to a single
band, G (Fig. 3A, lanes 5 and 11), indicating that the slower migration of bands A and C was due to the presence of leader sequence at their 5'
ends.
The r/t RNA, synthesized in greater abundance in the presence of pM2,
and which migrated near the genomic RNAs but was retained by
oligo(dT)-cellulose, was identified by RNase H digestion following annealing with oligonucleotide tr. This digestion caused the loss of
the positive replication product and r/t (Fig. 3A, lanes 3 and 9) and
gave rise to bands C and D, indicating the presence of trailer sequence
in these RNAs. The fact that band D migrated faster than band C
indicated that it did not contain leader sequence; hence, it was
concluded that band D was a digestion product of a readthrough
transcript (band D migrated more slowly than the mRNA, as 83 nucleotides of trailer sequence still remain at its 3' end following
RNase H digestion). The level of band D, but not C, was increased when
the cDNA encoding the M2 mRNA was included in transfections, thus
indicating that a product encoded by the M2 mRNA was enhancing
transcriptional readthrough at the L/trailer junction.
The final RNA species identified was the negative-sense product of
replication. Following incubation with the oligonucleotide +tr, which
anneals to negative-sense trailer sequence (Fig. 3B), RNase H digestion
led to the disappearance of the slower-migrating replication product
and the appearance of band H (Fig. 3A, lanes 6 and 12). No change in
the migration of the positive-sense product of replication, r/t RNA, or
the mRNA was seen. This result confirmed the slower-migrating product
of replication was negative sense.
These analyses allowed us to identify the negative- and positive-sense
products of replication, the predicted mRNA, and a readthrough
product of transcription which failed to terminate at the L gene end
and read through into trailer. This readthrough product was
synthesized primarily in the presence of the M2 mRNA ORF1 and ORF2
(pM2). Although the trailer sequence does not contain an RS virus
polyadenylation signal, the data show that the readthrough transcript was polyadenylated by its ability to bind to
oligo(dT)-cellulose and its sensitivity to RNase H digestion in the
presence of oligo(dT) (band E [lanes 4 and 10]). We postulate that
the vaccinia virus poly(A) polymerase adds poly(A) to the 3'-terminal
hydroxyl group of this readthrough transcript (18, 19).
Encapsidation of the replication products probably prevents
polyadenylation of these RNAs by this enzyme. Polyadenylation at the
L/trailer junction by the RS virus polymerase followed by
readthrough rather than termination can be eliminated as a
possibility because if this were the case, exposure to RNase H in the
presence of oligo(dT) would cause the digestion of the poly(A) tract
and removal of the 3' trailer sequence; therefore, product E (Fig. 3A,
lanes 4 and 10) would not be observed.
Readthrough at an internal gene junction. These results with a subgenomic replicon encoding one transcription unit demonstrated that the presence of M2 mRNA (ORF1 and ORF2) caused significant readthrough of the L gene transcription termination signal to give rise to an mRNA containing the trailer sequence. However, the L/trailer junction is not representative of the other RS virus gene junctions, as the L gene end signal is not followed by a downstream gene start signal. This may affect the efficiency of transcriptional termination and the behavior of the viral transcriptase. Therefore, the effect of the products of the M2 mRNA on transcription was analyzed by using a subgenomic replicon encoding two transcription units, separated by the authentic M/SH gene junction (Fig. 4A).
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Assignment of readthrough function to ORF1 product. Increased transcriptional readthrough of gene end signals was observed when the products of the M2 mRNA (ORF1 and ORF2) were present in transfections. To determine which of the two ORF products was responsible for causing this readthrough, cDNAs that expressed either ORF1 or ORF2 were generated (Fig. 5A). The cDNAs pM2 (encoding ORF1 and ORF2), pORF1, and pORF2 were added incrementally to transfections in order to determine the effects of increasing levels of ORF1 and ORF2 products. Figure 5B shows that the presence of pM2 caused an increase in readthrough when it was present at low levels in transfections. However, as the concentration of cDNA in transfections was increased, a corresponding decrease in viral RNA synthesis resulted. In contrast, expression of ORF1 alone resulted in an increase in readthrough transcription, but increasing the amount of cDNA, which led to a corresponding increase in protein expression (9, 20a, 29), did not have an inhibitory effect on RNA synthesis (Fig. 5B). ORF2 alone, however, significantly inhibited viral RNA synthesis even when expressed at low levels, as previously observed by Collins et al. (9). These data show that the product of ORF1, the M2 protein, caused the RS virus polymerase to read through transcription termination signals.
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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We have used an approach whereby the effects of trans-acting factors on replication and transcription of RS virus can be analyzed by direct metabolic labeling in the absence of helper virus (34). The products of transcription and replication of RS virus subgenomic replicons containing one or two transcription units were compared in the absence and presence of the cDNA pM2, encoding both ORF1 and ORF2 of the M2 mRNA. It was found that the expression of ORF1 and ORF2 caused a change in the pattern of RNA synthesis. Analysis of the RNA products by RNase H digestion in the presence of specific oligonucleotides demonstrated that the observed change in RNA synthesis was due to an increase in the production of readthrough transcripts (Fig. 3 and 4). Further, it was shown, using the M/SH dicistronic subgenomic replicon, that a product of either ORF1 or ORF2 allowed the viral polymerase to read through intergenic junctions as well as the L/trailer junction. Also, readthrough could occur at more than one termination signal to produce a transcription product containing mRNA1, mRNA2, and trailer sequence. This effect has not been reported in previous work using similar systems (9, 24).
Using cDNAs encoding only ORF1 or ORF2 of the M2 mRNA, we assigned the readthrough function to the product of ORF1, the 22-kDa M2 protein which is also a structural component of the RS virus virion. In contrast, the product of ORF2 dramatically downregulated both replication and transcription as previously reported (9).
Our conclusions concerning the function of the M2 protein differ from previous reports that M2 functions to enhance processive transcription but does not affect readthrough of transcription termination signals (9). We postulate that our different results may, in part, be due to the differences in the systems used: (i) these studies used direct RNA analysis rather than Northern blotting, (ii) the time of analysis posttransfection (a direct 5-h label at 16 h posttransfection in this report, compared to Northern blotting of total RNA harvested 42 h posttransfection in previous reports); and (iii) the agarose-urea gel system used here gave a higher resolution. Our finding that the M2 protein acts primarily as a transcription antiterminator does not conflict with but rather extends the basic finding by Collins et al. (9) that M2 enhances polymerase processivity. It has been demonstrated in other biological systems that factors which give rise to antitermination also increase polymerase processivity as part of the antitermination process (16).
During a natural RS virus infection, readthrough transcription occurs approximately 10% of the time (13, 24). This is significantly higher than observed with the prototypic nonsegmented negative-sense RNA virus, vesicular stomatitis virus, where readthrough occurs approximately 3% of the time (22). In fact, the readthrough transcripts of RS virus are so abundant that they were used to develop the first map of the gene order (13). Indirect evidence has also suggested that the efficiency of transcription termination at the end of the NS1 and NS2 genes is lower than for other RS virus genes (2, 23). These observations lend circumstantial support to the concept of RS virus encoding a protein which enhances transcriptional readthrough.
Examining the role played by transcription antitermination factors in other systems may help us understand why RS virus requires such a factor. Proteins such as N and Q of phage lambda allow the Escherichia coli RNA polymerase to access genes downstream of a termination site where the polymerase normally dissociates from the template (16). These factors play an essential role in balancing gene expression during the life cycle of the phage. The RS virus genome contains 10 transcriptional units (excluding the leader gene), and so the polymerase encounters nine transcription termination signals before it reaches the polymerase gene start site. Assuming that transcriptional attenuation occurs at each gene junction (15, 22, 24), this will lead to a diminishingly small number of polymerase molecules which reach the polymerase (L) gene start site. An antitermination factor that allows readthrough of transcription termination signals would counteract the severity of attenuation. This would allow more polymerase molecules to access promoter-distal genes as transcriptional attenuation is reduced, which may explain the requirement of the M2 protein in order to obtain recovery of virus from a full-length cDNA clone (8). We postulate that the M2 protein may function to allow more polymerase molecules deeper access into the genome, thus maintaining an optimal ratio of proteins by regulating transcription. A protein equivalent to M2 has not been identified in members of the Mononegavirales, which possess only five or six transcriptional units, and if our hypothesis is correct, one would not be required since transcriptional attenuation would not as severely restrict polymerase access to promoter distal genes.
The gene start signal for the L gene occurs prior to the gene end signal for the M2 gene, which means the two transcription units overlap. Previous reports have suggested that this overlap downregulates transcription of the L mRNA (12) in order to prevent overexpression of the L protein, which is believed to be detrimental to the virus (30). This would appear to be in direct contradiction to the theory we propose in which the M2 protein increases polymerase access to the L gene start. It is possible that the two regulatory mechanisms described above work as a check and balance to temporally control the level of L protein expression.
The finding that M2 enhances transcriptional readthrough suggests that RS virus possesses a second level of transcription regulation in addition to attenuation at the gene junctions. There are a number of possible mechanisms by which the M2 protein could prevent the polymerase from terminating transcription. We are currently investigating the specific manner by which the M2 protein causes transcriptional antitermination.
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ACKNOWLEDGMENTS |
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We thank members of our laboratory and the L. A. Ball laboratory for advice and constructive criticism.
This work was supported by Public Health Service grants AI12464 and AI20181 from the NIH and NIAID to G.W.W.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Microbiology, University of Alabama School of Medicine at Birmingham, BBRB Box 17, Room 366, 845 19th St. South, Birmingham, AL 35294. Phone: (205) 934-0877. Fax: (205) 934-1636. E-mail: gail_wertz{at}microbio.uab.edu.
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REFERENCES |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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