Rikkonen M, Peränen J, Kääriäinen L
Institute of Biotechnology, University of Helsinki, Finland.
Arch Virol Suppl. 1994;9:369-77. doi: 10.1007/978-3-7091-9326-6_37.
The Semliki Forest virus-specific nonstructural protein nsP2 is transported into the nuclei of both infected and transfected BHK cells. The pentapeptide sequence P 648R RRV is an essential part of the nuclear localization signal (NLS) of nsP2, the middle arginine being the most critical residue for nuclear targeting. Host DNA and RNA syntheses are rapidly inhibited in virus-infected cells, and nsP2 could be involved in these processes. It has been postulated that the inhibition of cellular replication could be due to viral NTPase activity. We have expressed and purified nsP2 in E. coli using the highly efficient T7 based expression system. Purified nsP2 was shown to have ATPase and GTPase activities, and these specific activities were increased in the presence of single-stranded RNA, a typical feature of RNA helicases. The role of nsP2 in the nucleus was studied by creating a mutant virus SFV-RDR, which contained an altered NLS (PRDRV). The mutation affected neither the processing nor the stability of nsP2, but it did render nsP2 completely cytoplasmic. SFV-RDR was shown to be fully infectious, and no difference could be seen in the expression of viral proteins. In addition, the inhibition of host DNA synthesis was almost equally efficient in both wild-type and mutant-infected cells. The pathogenic properties of the mutant will be further studied.
塞姆利基森林病毒特异性非结构蛋白nsP2可转运至受感染和转染的BHK细胞的细胞核中。五肽序列P 648RRV是nsP2核定位信号（NLS）的重要组成部分，中间的精氨酸是核靶向所必需的关键残基。在病毒感染的细胞中，宿主DNA和RNA的合成会迅速受到抑制，nsP2可能参与了这些过程。据推测，细胞复制的抑制可能是由于病毒的NTPase活性。我们使用基于高效T7的表达系统在大肠杆菌中表达并纯化了nsP2。纯化后的nsP2显示具有ATPase和GTPase活性，并且在单链RNA存在的情况下这些比活性会增加，这是RNA解旋酶的典型特征。通过构建一种含有改变的NLS（PRDRV）的突变病毒SFV-RDR来研究nsP2在细胞核中的作用。该突变既不影响nsP2的加工过程也不影响其稳定性，但确实使nsP2完全定位于细胞质中。SFV-RDR被证明具有完全的感染性，并且在病毒蛋白的表达上没有差异。此外，在野生型和突变型感染的细胞中，宿主DNA合成的抑制效率几乎相同。该突变体的致病特性将进一步研究。
