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. 1998 Jan;72(1):520-6.
doi: 10.1128/JVI.72.1.520-526.1998.

The product of the respiratory syncytial virus M2 gene ORF1 enhances readthrough of intergenic junctions during viral transcription

R W Hardy  1 , G W Wertz
Affiliations

The product of the respiratory syncytial virus M2 gene ORF1 enhances readthrough of intergenic junctions during viral transcription

R W Hardy et al. J Virol. 1998 Jan.

Abstract

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.

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Figures

FIG. 1
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 (HδV). 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.
FIG. 2
FIG. 2
Products of RNA synthesis from WT5 subgenomic replicon. Cells were infected with vTF7-3, transfected with cDNAs pWT5, pN, pP, pL, and pM2 (as indicated), and exposed to [3H]uridine in the presence of actinomycin D. Total RNA (lanes 1 to 3) was analyzed by agarose-urea gel electrophoresis. RNA extracted from cells transfected with pWT5, pN, pP, pL, and pM2 was subjected to oligo(dT) chromatography (lanes 4 and 5) or immunoprecipitation with anti-RS virus serum (lane 7) before agarose-urea gel electrophoresis; 2.5 times more cellular material was subjected to immunoprecipitation than was used for the total samples. A 3H-labeled T7 in vitro transcription product from pWT5 was run as a marker for the position of WT5 genomic RNA (lane 6). The − and + beside lane 7 signify the polarity of the replication products (rep.) in lanes 4 and 7 identified by the comigration of the negative-sense product with the in vitro transcript of WT5 in lane 6. UB, unbound fraction of oligo(dT) chromatography; B, bound fraction; IV, WT5 in vitro transcript; IP, RNA immunoprecipitated by anti-RSV serum.
FIG. 3
FIG. 3
Identification of RNAs produced from the WT5 template. (A) Labeled RNAs from transfections excluding (upper panel) or including (lower panel) 0.3 μg of pM2 (encoding ORF1 and ORF2 of M2 mRNA) were incubated with specific oligonucleotides (oligo.) as indicated, digested with RNase H, and separated by agarose-urea gel electrophoresis. Labels below the lanes designate which oligonucleotide was present during RNase H digestion. rep., replication products. (B) Diagrammatic representation of oligonucleotide binding sites and major products of RNase H digestion corresponding to RNAs A to H in panel A. +ve, positive; −ve, negative.
FIG. 4
FIG. 4
(A) Diagram of pM/SH and the RNAs synthesized from the dicistronic subgenomic replicon that it encodes. pM/SH was generated by inserting a DNA fragment encompassing the M/SH intergenic junction into the BsmI site of pWT5. The location of the oligonucleotide 3′M, used for RNase H analysis, is shown by the arrow. le, leader; tr, trailer; nt, nucleotides. (B) Products of RNA synthesis from M/SH genome analog in the presence of pN, pP, and pM2 but absence of pL (lane 1), presence of pN, pP, and pL but absence of pM2 (lane 2), or presence of pN, pP, pL, and pM2 (lane 3). Lanes 4 and 5 show the products of RNase H digestion of RNAs synthesized in the presence of pM2. RNAs in lane 4 were digested in the presence of oligo(dT); RNAs in lane 5 were digested in the presence of 3′M and oligo(dT). The asterisk indicates the 5′ products of RNase H digestion of mRNA1, r/t.B and r/t.C in the presence of 3′M, which comigrate.
FIG. 5
FIG. 5
Effects of ORF1 or ORF2, expressed individually, on RNA synthesis from the WT5 subgenomic replicon. (A) Diagram of pM2, pORF1, and pORF2 cDNAs described in Materials and Methods. (B) Increasing concentrations of plasmids pM2, pORF1, and pORF2 were included in transfections. Labeled, actinomycin D-resistant RNAs were analyzed by agarose-urea gel electrophoresis and visualized by fluorography. rep., replication product.
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