Yu Q, Davis P J, Brown T D, Cavanagh D
Division of Molecular Biology, Houghton Laboratory, Huntingdon, Cambridgeshire, U.K.
J Gen Virol. 1992 Jun;73 ( Pt 6):1355-63. doi: 10.1099/0022-1317-73-6-1355.
Negative-stranded virion RNA and oligonucleotide primers complementary to fusion (F) protein gene sequences were used to generate cDNA clones, revealing that the gene 5'-proximal to the F protein corresponded to the M2 (22K) gene, as in respiratory syncytial (RS) virus. The transcription start signal, GGGACAAGU, was identical to that of the F and matrix (M) proteins of turkey rhinotracheitis virus (TRTV). There were two sequences with the potential to function as transcription termination/poly(A) signals, located at nucleotides 751 to 762 and 777 to 787; 15 clones derived from mRNA indicated that the first of these sequences formed the major signal. Part of the next downstream (5') gene was sequenced; unlike mammalian pneumoviruses the TRTV M2 gene did not overlap the beginning of the 5'-proximal gene. Northern blotting indicated that infected Vero cells contained less M2 mRNA than F mRNA and that about half of the M2 mRNA was present as a F-M2 dicistronic mRNA. The M2 gene contained two overlapping open reading frames (ORFs 1 and 2), as with RS virus. ORF 1 comprised 558 nucleotides with the coding potential for a 186 amino acid polypeptide, M(r) 20959, eight or nine residues shorter than for human RS virus strains. The overall amino acid identity was 40%, the N-terminal one-third of the proteins sharing 62% of residues, the remainder 29%. A hydropathy plot of the TRTV M2 protein had close similarity to that of the M2 or RS virus. The protein was predicted to have a basic character with no N-terminal signal sequence or other major highly hydrophobic sequences. In vitro translation of a transcript comprising both ORFs 1 and 2 produced a single product of apparent M(r) 23000, corresponding to the M2 product of ORF 1. Site-directed mutagenesis confirmed that this product was derived from ORF 1 and that frameshifting was not involved. The second ORF was expressed only from a transcript which lacked the AUG codons of ORF 1 and, although occupying a similar position to that in the RS virus M2 gene, had virtually no amino acid identity in its 73 residue length and was approximately 25% shorter than the corresponding RS virus ORF 2. The hydropathy plot of the potential products of the second ORFs of TRTV and RS virus showed little resemblance. Taken together these results suggest that ORF 2 is unlikely to be expressed in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)
负链病毒粒子RNA和与融合（F）蛋白基因序列互补的寡核苷酸引物被用于生成cDNA克隆，结果显示，与F蛋白5'近端相邻的基因对应于M2（22K）基因，如同在呼吸道合胞（RS）病毒中一样。转录起始信号GGGACAAGU与火鸡鼻气管炎病毒（TRTV）的F蛋白和基质（M）蛋白的转录起始信号相同。有两个序列有可能作为转录终止/聚腺苷酸化信号，分别位于核苷酸751至762以及777至787处；从mRNA衍生而来的15个克隆表明，其中第一个序列形成主要信号。对下一个下游（5'）基因的部分进行了测序；与哺乳动物肺病毒不同，TRTV的M2基因不与5'近端基因的起始部分重叠。Northern印迹表明，受感染的Vero细胞中M2 mRNA的含量低于F mRNA，并且大约一半的M2 mRNA以F-M2双顺反子mRNA的形式存在。与RS病毒一样，M2基因包含两个重叠的开放阅读框（ORF 1和ORF 2）。ORF 1由558个核苷酸组成，具有编码186个氨基酸多肽的潜力，M（r）为20959，比人类RS病毒株的相应多肽短八到九个残基。总体氨基酸同一性为40%，蛋白质的N端三分之一共有62%的残基相同，其余部分为29%。TRTV M2蛋白的亲水性图谱与RS病毒的M2蛋白的亲水性图谱非常相似。预测该蛋白具有碱性特征，没有N端信号序列或其他主要的高度疏水序列。对包含ORF 1和ORF 2的转录本进行体外翻译产生了一个表观M（r）为23000的单一产物，对应于ORF 1的M2产物。定点诱变证实该产物源自ORF 1，且不涉及移码。第二个ORF仅从缺少ORF 1的AUG密码子的转录本中表达，尽管其位置与RS病毒M2基因中的位置相似，但其73个残基长度的氨基酸序列几乎没有同一性，并且比相应的RS病毒ORF 2短约25%。TRTV和RS病毒第二个ORF潜在产物的亲水性图谱几乎没有相似之处。综合这些结果表明，ORF 2在体内不太可能表达。（摘要截短至400字）
