REGULATION OF SINDBIS VIRUS-RNA REPLICATION - UNCLEAVED P123 AND NSP4 FUNCTION IN MINUS-STRAND RNA-SYNTHESIS, WHEREAS CLEAVED PRODUCTS FROM P123 ARE REQUIRED FOR EFFICIENT PLUS-STRAND RNA-SYNTHESIS
REGULATION OF SINDBIS VIRUS-RNA REPLICATION - UNCLEAVED P123 AND NSP4 FUNCTION IN MINUS-STRAND RNA-SYNTHESIS, WHEREAS CLEAVED PRODUCTS FROM P123 ARE REQUIRED FOR EFFICIENT PLUS-STRAND RNA-SYNTHESIS
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DOI：
10.1128/jvi.68.3.1874-1885.1994
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发表时间：
1994-03-01
影响因子：
5.4
通讯作者：
STRAUSS, JH
中科院分区：
文献类型：
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作者：
SHIRAKO, Y;STRAUSS, JH
辛德毕斯病毒的非结构蛋白nsP 1、nsP 2、nsP 3和nsP 4以及中间多聚蛋白由两种前体多聚蛋白P123和P1234通过nsP 2的C-末端一半编码的蛋白水解酶产生。我们研究了辛德毕斯病毒RNA合成的中间和成熟加工产物的要求和功能，通过使用在加工1/2、2/3或3/4切割位点中具有缺陷的定点突变体，单独或以各种组合。在1/2和2/3位点都有切割缺陷的突变体，其仅产生不可切割的P123和成熟nsP 4作为最终产物，在30 ℃下10小时后产生的病毒是野生型病毒的10（-3）倍，并且在40 ℃下不能存活。在加工23位点方面有缺陷的突变体，其制造nsP 1、nsP 4和P23以及前体P123，在30 ℃下的生长效率为野生型病毒的10（-1），在40 ℃下的生长效率为野生型病毒的10（-3）。这些突变体的早期负链RNA合成与野生型病毒一样有效，而正链RNA合成与野生型病毒相比大幅下降。加工3/4位点有缺陷的突变体在30或40 ℃下不能存活。3/4位点突变体可以由不能切割1/2或2/3位点的突变体补充，这可以提供成熟的nsP 4。我们将这些结果解释为：（i）成熟nsP 4是RNA复制所必需的，（ii）nsP 4和未切割的P123在负链RNA合成中起作用，（iii）P123的切割是有效的正链RNA合成所必需的。我们建议，辛德毕斯病毒RNA复制的P123的差异蛋白水解调节。在感染早期，nsP 4和未切割的P123形成瞬时负链RNA复制复合物，其在P123切割后消失。在感染后期，病毒蛋白酶活性水平的升高消除了P123的从头合成，并且不可能进一步合成负链RNA。相比之下，nsP 4和P123的裂解产物形成正链RNA复制复合物，其在整个感染周期中稳定并保持活性。
Nonstructural proteins of Sindbis virus, nsP1, nsP2, nsP3, and nsP4, as well as intermediate polyproteins, are produced from two precursor poly-proteins, P123 and P1234, by a proteolytic enzyme encoded in the C-terminal half of nsP2. We studied the requirements for and the functions of the intermediate and mature processing products for Sindbis virus RNA synthesis by using site-directed mutants which have a defect(s) in processing the 1/2, 2/3, or 3/4 cleavage sites either singly or in various combinations. A mutant defective in cleaving both the 1/2 and 2/3 sites, which makes only uncleavable P123 and mature nsP4 as final products, produced 10(-3) as much virus as did the wild-type virus after 10 h at 30 degrees C and was nonviable at 40 degrees C. A mutant defective in processing the 2/3 site, which makes nsP1, nsP4, and P23 as well as precursor P123, grew 10(-1) as efficiently as wild-type virus at 30 degrees and 10(-3) as efficiently at 40 degrees C. Early minus-strand RNA synthesis by these mutants was as efficient as that by wild-type virus, whereas plus-strand RNA synthesis was substantially decreased compared with that by wild-type virus. A mutant defective in processing the 3/4 site was nonviable at either 30 or 40 degrees C. The 3/4 site mutant could be complemented by the mutant unable to cleave either the 1/2 or 2/3 site, which can provide mature nsP4. We interpret these results to signify that (i) mature nsP4 is required for RNA replication, (ii) nsP4 and uncleaved P123 function in minus-strand RNA synthesis, and (iii) cleavage of P123 is required for efficient plus-strand RNA synthesis. We propose that Sindbis virus RNA replication is regulated by differential proteolysis of P123. Early in infection, nsP4 and uncleaved P123 form transient minus-strand RNA replication complexes which vanish upon cleavage of P123. Later in infection, an elevated level of viral proteinase activity eliminates de novo synthesis of P123, and no further synthesis of minus-strand RNA is possible. In contrast, nsP4 and cleavage products from P123 form plus-strand RNA replication complexes which are stable and remain active throughout the infection cycle.