Department of Microbiology and Immunology, Jefferson Medical College, Thomas Jefferson University, 233 S. 10th Street, Philadelphia, PA 19107, USA.
J Virol. 2011 Jan;85(2):697-704. doi: 10.1128/JVI.01309-10. Epub 2010 Nov 10.
Previous comparisons of different rabies virus (RV) strains suggested an inverse relationship between pathogenicity and the amount of glycoprotein produced in infected cells. In order to provide more insight into this relationship, we pursued an experimental approach that allowed us to alter the glycoprotein expression level without altering the glycoprotein sequence, thereby eliminating the contribution of amino acid changes to differences in viral virulence. To this end, we constructed an infectious clone of the highly pathogenic rabies virus strain CVS-N2c and replaced its cognate glycoprotein gene with synthetic versions in which silent mutations were introduced to replace wild-type codons with the most or least frequently used synonymous codons. A recombinant N2c variant containing the fully codon-optimized G gene and three variants carrying a partially codon-deoptimized G gene were recovered on mouse neuroblastoma cells and shown to express 2- to 3-fold more and less glycoprotein, respectively, than wild-type N2c. Pathogenicity studies in mice revealed the WT-N2c virus to be the most pathogenic strain. Variants containing partially codon-deoptimized glycoprotein genes or the codon-optimized gene were less pathogenic than WT-N2c but still caused significant mortality. We conclude that the expression level of the glycoprotein gene does have an impact on pathogenicity but is not a dominant factor that determines pathogenicity. Thus, strategies such as changes in codon usage that aim solely at altering the expression level of the glycoprotein gene do not suffice to render a pathogenic rabies virus apathogenic and are not a viable and safe approach for attenuation of a pathogenic strain.
先前对不同狂犬病病毒 (RV) 株的比较表明，致病性与感染细胞中产生的糖蛋白量之间存在反比关系。为了更深入地了解这种关系，我们采用了一种实验方法，该方法允许我们在不改变糖蛋白序列的情况下改变糖蛋白表达水平，从而消除氨基酸变化对病毒毒力差异的贡献。为此，我们构建了高度致病性狂犬病病毒株 CVS-N2c 的感染性克隆，并将其糖蛋白基因替换为合成版本，在该版本中，沉默突变被引入以用最常用或最不常用的同义密码子替换野生型密码子。在小鼠神经母细胞瘤细胞上回收了含有完全密码子优化 G 基因的重组 N2c 变体和携带部分密码子去优化 G 基因的三个变体，分别显示出表达 2 到 3 倍更多和更少的糖蛋白，而野生型 N2c。在小鼠中的致病性研究表明，WT-N2c 病毒是最具致病性的菌株。含有部分密码子去优化糖蛋白基因或密码子优化基因的变体比 WT-N2c 的致病性要低，但仍会导致显着的死亡率。我们得出结论，糖蛋白基因的表达水平确实对致病性有影响，但不是决定致病性的主要因素。因此，旨在仅改变糖蛋白基因表达水平的密码子使用策略不足以使致病性狂犬病病毒无致病性，并且不是减弱致病性菌株的可行和安全方法。
