Both viral transcription and replication are reduced when the rabies virus nucleoprotein is not phosphorylated
Both viral transcription and replication are reduced when the rabies virus nucleoprotein is not phosphorylated
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DOI：
10.1128/jvi.76.9.4153-4161.2002
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发表时间：
2002-05-01
影响因子：
5.4
通讯作者：
Fu, ZF
中科院分区：
文献类型：
--
作者：
Wu, XF;Gong, XM;Fu, ZF
狂犬病病毒核蛋白（N）通过对新生基因组RNA的修饰，在病毒RNA转录和复制的调控中起重要作用。狂犬病病毒N是磷酸化的，并且先前的研究表明，在位置389处的磷酸化丝氨酸突变为丙氨酸导致狂犬病病毒微型基因组的病毒转录和/或复制的减少。在本研究中，我们将389位的丝氨酸（S）突变为丙氨酸（A）、甘氨酸（G）、天冬氨酸（D）、天冬酰胺（N）、谷氨酸（E）和谷氨酰胺（Q），并检查这些突变对狂犬病病毒在微型基因组中转录和复制的影响。此外，从S到A、S到D和S到E的突变也被掺入全长感染性病毒中。丝氨酸突变为其他氨基酸中的每一个导致未磷酸化的N的合成和在微型基因组中的病毒转录和复制的减少。从S到A和S到D的突变也导致全长感染性病毒中病毒转录和复制的减少。生长曲线研究表明，具有S到A突变的突变病毒（L16 A）的产量比野生型病毒（L16）的产量低10，000倍。与狂犬病病毒基因探针的北方印迹杂交显示，当N不被磷酸化时，突变病毒的病毒转录和复制速率降低多达10倍。对来自微型基因组系统和全长感染性病毒的数据的解释表明，狂犬病病毒N的磷酸化是复制所必需的。进一步的研究涉及放线菌酮处理感染的细胞显示，当N不磷酸化时，病毒转录也减少。总而言之，这些结果提供了明确的证据，证明N磷酸化在狂犬病毒转录和复制过程中发挥着重要作用。
Rabies virus nucleoprotein (N) plays vital roles in regulation of viral RNA transcription and replication by encapsidation of the nascent genomic RNA. Rabies virus N is phosphorylated, and previous studies demonstrated that mutation of the phosphorylated serine at position 389 to alanine resulted in reduction of viral transcription and/or replication of a rabies virus minigenome. In the present study, we mutated the serine (S) at position 389 to alanine (A), glycine (G), aspartic acid (D), asparagine (N), glutamic acid (E), and glutamine (Q) and examined the effects of these mutations on rabies virus transcription and replication in the minigenome. Furthermore, mutations from S to A, S to D, and S to E were also incorporated into the full-length infectious virus. Mutation of the serine to each of the other amino acids resulted in the synthesis of an unphosphorylated N and reduction of viral transcription and replication in the minigenome. Mutations from S to A and S to D also resulted in reduction of both viral transcription and replication in full-length infectious viruses. Growth curve studies indicated that production of the mutant virus with the S-to-A mutation (L16A) was as much as 10,000-fold less than that of the wild-type virus (L16). Northern blot hybridization with rabies virus gene probes revealed that the rates of viral transcription and replication were reduced by as much as 10-fold in the mutant viruses when the N was not phosphorylated. Interpretation of the data from the minigenome system and the full-length infectious virus indicates that phosphorylation of rabies virus N is necessary for replication. Further studies involving cycloheximide treatment of infected cells revealed that viral transcription was also reduced when the N was not phosphorylated. Taken together, these results provide definitive evidence that N phosphorylation plays an important role in the processes of rabies virus transcription and replication.