Wang T H, Rijnbrand R C, Lemon S M
Department of Microbiology and Immunology, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7290, USA.
J Virol. 2000 Dec;74(23):11347-58. doi: 10.1128/jvi.74.23.11347-11358.2000.
Among a myriad of putative functions assigned to the hepatitis C virus (HCV) core protein, several studies suggest that it may modulate internal ribosome entry site (IRES)-mediated initiation of translation. We compared the translational activity of dicistronic reporter transcripts containing the HCV IRES within the intercistronic space fused to downstream sequence encoding either 22 amino acids (aa) or 173 aa of the core protein. The inclusion of the nearly full-length core protein-coding sequence significantly suppressed translation in vitro and in transfected HepG2 cells. However, this suppression was not eliminated by frameshift mutations introduced into the core sequence, suggesting that it occurred at the RNA level and not as a result of core protein expression in cis. Similarly, the expression of core protein (aa 1 to 191) in trans from a recombinant baculovirus did not suppress IRES-directed translation from any of these transcripts in transfected Huh-7 cells. While core protein expression did decrease IRES activity in HepG2 cells (up to 79% suppression), the expression of beta-galactosidase from a control baculovirus also suppressed IRES activity (up to 56%), strongly suggesting that this suppression was nonspecific. Finally, the addition of purified recombinant core protein (aa 1 to 179) to in vitro translation reactions at concentrations up to a 10-fold molar excess over the RNA transcripts resulted in no significant reduction in IRES activity. Consistent with these results, a gel retention assay indicated no difference in the affinities of the recombinant HCV core protein and a recombinant Venezuelan equine encephalitis virus capsid protein for HCV IRES-containing RNA transcripts. We conclude that while the inclusion of core protein-coding sequence downstream of the IRES may reduce the efficiency of cap-independent translation on HCV RNA, the core protein itself has no biologically relevant activity in modulating HCV IRES activity.
在赋予丙型肝炎病毒（HCV）核心蛋白的众多假定功能中，多项研究表明它可能调节内部核糖体进入位点（IRES）介导的翻译起始。我们比较了双顺反子报告转录本的翻译活性，这些转录本在顺反子间空间包含与编码核心蛋白22个氨基酸（aa）或173个aa的下游序列融合的HCV IRES。包含几乎全长的核心蛋白编码序列在体外和转染的HepG2细胞中显著抑制翻译。然而，引入核心序列的移码突变并未消除这种抑制，这表明它发生在RNA水平，而非顺式核心蛋白表达的结果。同样，重组杆状病毒反式表达核心蛋白（aa 1至191）在转染的Huh-7细胞中并未抑制这些转录本中任何一个的IRES指导的翻译。虽然核心蛋白表达确实降低了HepG2细胞中的IRES活性（抑制高达79%），但对照杆状病毒表达的β-半乳糖苷酶也抑制了IRES活性（抑制高达56%），这强烈表明这种抑制是非特异性的。最后，在体外翻译反应中加入纯化的重组核心蛋白（aa 1至179），其浓度比RNA转录本高10倍摩尔过量，IRES活性没有显著降低。与这些结果一致，凝胶滞留试验表明重组HCV核心蛋白和重组委内瑞拉马脑炎病毒衣壳蛋白对含HCV IRES的RNA转录本的亲和力没有差异。我们得出结论，虽然在IRES下游包含核心蛋白编码序列可能会降低HCV RNA上不依赖帽的翻译效率，但核心蛋白本身在调节HCV IRES活性方面没有生物学相关活性。
