van Bokhoven H, Verver J, Wellink J, van Kammen A
Department of Molecular Biology, Agricultural University, Wageningen, The Netherlands.
J Gen Virol. 1993 Oct;74 ( Pt 10):2233-41. doi: 10.1099/0022-1317-74-10-2233.
In order to identify the viral polymerase involved in cowpea mosaic virus (CPMV) RNA replication the 87K, 110K and 170K proteins as well as the complete 200K polyprotein of CPMV B-RNA have been produced in cowpea protoplasts, using expression vectors based on the 35S promoter of cauliflower mosaic virus. CPMV-specific proteins were obtained that were indistinguishable from proteins found in CPMV-infected protoplasts. Proteolytic processing of precursor proteins synthesized from the expression vectors proved that the 24K protease contained within these proteins is active. Moreover, it was established that protoplasts transfected with the expression vector containing the entire 200K coding sequence, but not those transfected with vectors containing the 170K, 110K or 87K coding sequences, were able to support replication of co-inoculated M-RNA. Despite the ability to support replication of M-RNA for protoplasts transiently expressing the 200K coding region, CPMV-specific RNA polymerase activity dependent on exogenous added template RNA could not be detected in extracts of these protoplasts in assays using poly(A).oligo(U) or other template/primer combinations. In contrast, extracts of protoplasts in which poliovirus polymerase was produced exhibited RNA polymerase activity in such assays. These results indicate that the CPMV polymerase, unlike the poliovirus polymerase, is not able to use oligo(U) as a primer or cannot function on exogenous template and primer RNA.
为了鉴定参与豇豆花叶病毒（CPMV）RNA复制的病毒聚合酶，利用基于花椰菜花叶病毒35S启动子的表达载体，在豇豆原生质体中表达了CPMV B-RNA的87K、110K和170K蛋白以及完整的200K多聚蛋白。获得了与CPMV感染的原生质体中发现的蛋白无法区分的CPMV特异性蛋白。对从表达载体合成的前体蛋白进行蛋白水解加工证明，这些蛋白中包含的24K蛋白酶具有活性。此外，还确定用含有完整200K编码序列的表达载体转染的原生质体能够支持共接种的M-RNA的复制，而用含有170K、110K或87K编码序列的载体转染的原生质体则不能。尽管瞬时表达200K编码区的原生质体能够支持M-RNA的复制，但在使用聚（A）·寡聚（U）或其他模板/引物组合的检测中，在这些原生质体的提取物中未检测到依赖于外源添加模板RNA的CPMV特异性RNA聚合酶活性。相比之下，在这些检测中，产生脊髓灰质炎病毒聚合酶的原生质体提取物表现出RNA聚合酶活性。这些结果表明，与脊髓灰质炎病毒聚合酶不同，CPMV聚合酶不能使用寡聚（U）作为引物，也不能在外源模板和引物RNA上发挥作用。
