Neeleman L, Bol J F
Gorlaeus Laboratories, Leiden University, Leiden, 2300 RA, The Netherlands.
Virology. 1999 Feb 15;254(2):324-33. doi: 10.1006/viro.1998.9568.
cDNA clones of RNAs 1 and 2 of alfalfa mosaic virus (AMV) were slightly modified to permit transcription of infectious RNAs with T7 RNA polymerase. Together with transcripts of an available clone of AMV RNA 3, these transcripts were used to study cis- and trans-acting functions of AMV proteins in protoplasts from nontransgenic tobacco plants and from plants transformed with the P1 and P2 genes, encoded by RNAs 1 and 2, respectively. Transgenic P1 was unable to complement mutations in the P1 gene in RNA 1, pointing to a cis-acting function of P1 in RNA 1 replication. A study of the replication of RNA 3 mutants in nontransgenic protoplasts revealed that coat protein (CP) expressed from RNA 3 in the inoculum is required in trans for replication and encapsidation of RNAs 1 and 2 but is required in cis for replication and encapsidation of RNA 3. CP is required in the inoculum to initiate infection of nontransgenic plants and protoplasts. When protoplasts expressing both P1 and P2 (P12 protoplasts) were infected with RNAs 1, 2, and 3, initiation of replication of RNAs 1 and 2 required the presence of CP in the inoculum, whereas the initiation of replication of RNA 3 did not. This demonstrated that CP expressed from RNA 3 cannot substitute for the early function of CP in the inoculum. The results showed that CP in the inoculum is required to permit viral minus-strand RNA synthesis, whereas CP expressed from RNA 3 after the initiation of infection is required for plus-strand RNA synthesis.
苜蓿花叶病毒（AMV）RNA 1和RNA 2的cDNA克隆经过轻微修饰，以便用T7 RNA聚合酶转录感染性RNA。这些转录本与AMV RNA 3的一个可用克隆的转录本一起，用于研究AMV蛋白在非转基因烟草植物以及分别由RNA 1和RNA 2编码的P1和P2基因转化的植物原生质体中的顺式和反式作用功能。转基因P1无法互补RNA 1中P1基因的突变，这表明P1在RNA 1复制中具有顺式作用功能。对RNA 3突变体在非转基因原生质体中的复制研究表明，接种物中由RNA 3表达的外壳蛋白（CP）对于RNA 1和RNA 2的复制和衣壳化是反式必需的，但对于RNA 3的复制和衣壳化是顺式必需的。接种物中的CP是启动非转基因植物和原生质体感染所必需的。当用RNA 1、RNA 2和RNA 3感染同时表达P1和P2的原生质体（P12原生质体）时，RNA 1和RNA 2复制的起始需要接种物中存在CP，而RNA 3复制的起始则不需要。这表明由RNA 3表达的CP不能替代接种物中CP的早期功能。结果表明，接种物中的CP是允许病毒负链RNA合成所必需的，而感染开始后由RNA 3表达的CP是正链RNA合成所必需的。
