Rassa J C, Wilson G M, Brewer G A, Parks G D
Department of Microbiology and Immunology, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157-1064, USA.
Virology. 2000 Sep 1;274(2):438-49. doi: 10.1006/viro.2000.0494.
The paramyxovirus gene end U tracts are thought to serve as templates for the addition of a 3' polyA tail to viral mRNAs. The goal of the work described here was to determine the function in transcription of the naturally occurring variability in length of the gene end U tracts of the paramyxovirus simian virus 5 (SV5). An anchored RT-PCR assay was developed to test the hypothesis that the variable U tracts template the addition of variable lengths of polyA tails to mRNAs. The results showed that although the SV5 NP, M, and SH genes encode U tracts of seven, four, and six U residues, respectively, their mRNAs contain similar polyA tails of approximately 250-290 bases. These results indicate that the variable gene end U tracts are functionally equivalent in directing polyadenylation. A reverse genetics system based on a dicistronic minigenome containing the SH-HN gene junction was used to test the hypothesis that the variable U tracks affect the efficiency of transcription termination. Minigenome templates containing an SH gene end with a long U tract of six residues (U6) directed efficient transcription termination and reinitiation at the downstream HN start site with no nucleotide preference for the downstream intergenic region. Surprisingly, truncating the SH gene end U tract to four residues (U4) did not affect SH termination but, rather, reduced downstream HN reinitiation to 20-30% of wild-type levels. Efficient HN reinitiation could be restored to mutant U4 templates in either of two ways: by increasing the U-tract length from four to six residues or by increasing the length of the intergenic region. Efficient HN reinitiation required a minimum of six bases between the last nucleotide in SH and the first nucleotide in HN. We propose that for some paramyxoviruses, the gene end U tract serves a previously unrecognized role as a spacer region between the gene end and gene start sites.
副粘病毒基因末端的U序列被认为是病毒mRNA 3'多聚腺苷酸尾添加的模板。本文所述工作的目的是确定副粘病毒猿猴病毒5（SV5）基因末端U序列长度自然变异在转录中的功能。开发了一种锚定RT-PCR检测方法，以检验可变U序列为mRNA添加可变长度多聚腺苷酸尾的假设。结果表明，尽管SV5的NP、M和SH基因分别编码含7个、4个和6个U残基的U序列，但其mRNA含有长度约为250 - 290个碱基的相似多聚腺苷酸尾。这些结果表明，可变的基因末端U序列在指导多聚腺苷酸化方面功能等效。基于包含SH - HN基因连接区的双顺反子微型基因组的反向遗传学系统，用于检验可变U序列影响转录终止效率的假设。含有6个残基长U序列（U6）的SH基因末端的微型基因组模板可指导高效的转录终止，并在下游HN起始位点重新起始转录，对下游基因间区域无核苷酸偏好。令人惊讶的是，将SH基因末端U序列截短至4个残基（U4）并不影响SH的终止，但会将下游HN的重新起始降低至野生型水平的20 - 30%。可以通过两种方式之一将高效的HN重新起始恢复到突变的U4模板：将U序列长度从4个残基增加到6个残基，或增加基因间区域的长度。高效的HN重新起始需要SH的最后一个核苷酸与HN的第一个核苷酸之间至少有6个碱基。我们提出，对于某些副粘病毒，基因末端U序列在基因末端和基因起始位点之间起着以前未被认识到的间隔区作用。
