The Product of the Respiratory Syncytial Virus M2 Gene ORF1 Enhances Readthrough of Intergenic Junctions during Viral Transcription

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.

呼吸道合胞（respiratory syncytial, RS）病毒M2蛋白的编码mRNA包含两个开放阅读框（open reading frames, ORFs）。ORF1可编码分子量为22 kDa的结构蛋白M2，ORF2则具备编码10 kDa蛋白（含90个氨基酸）的潜能。本研究借助痘苗病毒T7表达系统（vaccinia virus T7 expression system），通过在有无ORF1与ORF2产物的条件下对RNA进行直接代谢标记，分析了RS病毒单顺反子及双顺反子亚基因组复制子（subgenomic replicons）的RNA合成活性。在未添加ORF1和ORF2产物的情况下，可合成基因组RNA复制（genomic RNA replication）产生的负义链与正义链产物，以及多聚腺苷酸化mRNA（polyadenylated mRNA）。当完整的M2转录单元（包含ORF1与ORF2）或仅ORF1得以表达时，多聚腺苷酸化mRNA的合成量显著提升，其中多数源于病毒RNA聚合酶未能在基因终止信号（gene end signals）处终止，进而生成大量多顺反子mRNA（polycistronic mRNAs）。与此前的研究报道相符，ORF2的表达产物可抑制病毒RNA复制与mRNA转录。上述实验数据表明，M2蛋白作为转录抗终止因子（transcriptional antiterminator），能够增强病毒RNA聚合酶通读基因间连接区（intergenic junctions）的能力。本文最后讨论了该功能在病毒生命周期中的作用。