CHARACTERIZATION OF THE INFECTIONS OF PERMISSIVE AND NONPERMISSIVE CELLS BY HOST RANGE MUTANTS OF VESICULAR STOMATITIS-VIRUS DEFECTIVE IN RNA METHYLATION
CHARACTERIZATION OF THE INFECTIONS OF PERMISSIVE AND NONPERMISSIVE CELLS BY HOST RANGE MUTANTS OF VESICULAR STOMATITIS-VIRUS DEFECTIVE IN RNA METHYLATION
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DOI：
10.1016/0042-6822(84)90142-9
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发表时间：
1984-01-01
期刊：
影响因子：
3.7
通讯作者：
MOYER, SA
中科院分区：
文献类型：
--
作者：
HORIKAMI, SM;DEFERRA, F;MOYER, SA
与野生型病毒不同，VSV [水泡性口炎病毒]的两种宿主范围突变体hr 1和hr 8具有mRNA甲基化缺陷，并指导全长加帽但未甲基化的病毒mRNA的体外合成。结果表明，在体内非允许感染的HEp-2细胞的这2个突变体的特征是减少合成的全长mRNA的水平上的主要转录特征和基因组长度的RNA的合成的总缺乏。在HEp-2细胞中由任一突变体合成的VSV mRNA在mRNA依赖性兔网织红细胞裂解物中体内或体外均不翻译。随后从感染的HEp-2细胞中分离和分析mRNA，结果表明信使的5“末端含有一个鸟苷酰化但未甲基化的帽结构（GpppA），这一发现可能是缺乏可翻译性的原因。因此，这些突变体不能正确地甲基化mRNA，无论它们是在体外合成还是在非允许感染的细胞内体内合成。它还表明，不像hr 1，由hr 8合成的mRNA的甲基化不足是部分可逆的，通过在体外添加高水平的蛋氨酸。允许性幼仓鼠肾（BHK）细胞的内源性HDMet水平比非允许性HEp-2细胞高10倍。与单独感染的细胞不同，HEp-2细胞与hr突变体和痘病毒的共感染产生对这2种宿主范围突变体的容许感染。在拯救条件下体内产生的VSV mRNA的分析揭示了完全甲基化的帽（7 mGppp（m）Am）的存在，表明痘病毒可以通过将VSV mRNA转化为由于细胞质痘病毒mRNA甲基转移酶的甲基化而具有免疫活性的形式来拯救突变体。两种突变体都能够在允许的BHK细胞中正常生长。对来自感染的容许细胞的抑制活性mRNA的分析显示主要存在5“-单甲基化帽，7 mGpppA。研究了VSV的另外2个宿主范围突变体（hr 5和hr 7）的非允许感染。与上述突变体hr 1和hr 8不同，这2种突变体在HEp-2细胞中合成mRNA，其在体内和体外都被翻译。hr 5和hr 7也不能在HEp-2细胞中体内合成基因组长度的RNA，因此似乎在病毒基因组RNA的复制水平上被直接阻断。
Two host range mutants of VSV [vesicular stomatitis virus], hr 1 and hr 8, which, unlike the wild-type virus, have a mRNA methylation defect and direct the in vitro synthesis of full-length capped but unmethylated viral mRNA have been described previously. It is shown that the in vivo nonpermissive infection of HEp-2 cells by either of these 2 mutants is characterized by the reduced synthesis of full-length mRNA at levels characteristic of primary transcription and the total lack of synthesis of genome-length RNA. The VSV mRNA synthesized by either mutant in HEp-2 cells are not translated either in vivo or in vitro in mRNA-dependent rabbit reticulocyte lysates. Subsequent isolation and analysis of the mRNA from infected HEp-2 cells has shown that the 5'' termini of the messages contain a cap structure which is guanylylated, but unmethylated (GpppA), a finding that might account for the lack of translatability. Hence these mutants are unable to properly methylate mRNA whether they are synthesized in vitro or in vivo within nonpermissively infected cells. It is also shown that unlike hr 1, the undermethylation of mRNA synthesized by hr 8 is partially reversible by the addition of high levels of AdoMet in vitro. Permissive baby hamster kidney (BHK) cells have a 10-fold higher level of endogenous AdoMet than the nonpermissive HEp-2 cells. Unlike singly infected cells, the coinfection of HEp-2 cells with either hr mutant and a poxvirus yields a permissive infection for these 2 host range mutants. Analysis of the VSV mRNA produced in vivo under the conditions of rescue reveals the presence of fully methylated caps (7mGppp(m)Am), suggesting that poxvirus may rescue the mutants by converting the VSV mRNA to a translationally active form due to methylation by the cytoplasmic poxvirus mRNA methyltransferase enzymes. Both mutants are able to grow normally in permissive BHK cells. An analysis of the translationally active mRNA from infected permissive cells shows the presence primarily of a 5''-monomethylated cap, 7mGpppA. The nonpermissive infections of 2 other host range mutants of VSV (hr 5 and hr 7) were studied. Unlike mutants hr 1 and hr 8 described above, these 2 mutants synthesize mRNA in HEp-2 cells which is translated both in vivo and in vitro. hr 5 and hr 7 also fail to synthesize genome-length RNA in vivo in HEp-2 cells and, hence, appear to be blocked directly at the level of the replication of the viral genome RNA.