Manning John T, Seregin Alexey V, Yun Nadezhda E, Koma Takaaki, Huang Cheng, Barral José, de la Torre Juan C, Paessler Slobodan
Department of Pathology, University of Texas Medical Branch Galveston, TX, USA.
Department of Immunology and Microbial Science, Scripps Research Institute La Jolla, CA, USA.
Front Cell Infect Microbiol. 2017 Feb 6;7:20. doi: 10.3389/fcimb.2017.00020. eCollection 2017.
Junin virus (JUNV), a highly pathogenic New World arenavirus, is the causative agent of Argentine hemorrhagic fever (AHF). The live-attenuated Candid #1 (Can) strain currently serves as a vaccine for at-risk populations. We have previously shown that the Can glycoprotein (GPC) gene is the primary gene responsible for attenuation in a guinea pig model of AHF. However, the mechanisms through which the GPC contributes to the attenuation of the Can strain remain unknown. A more complete understanding of the mechanisms underlying the attenuation and immunogenicity of the Can strain will potentially allow for the rational design of additional safe and novel vaccines. Here, we provide a detailed comparison of both RNA and protein expression profiles between both inter- and intra-segment chimeric JUNV recombinant clones expressing combinations of genes from the Can strain and the pathogenic Romero (Rom) strain. The recombinant viruses that express Can GPC, which were shown to be attenuated in guinea pigs, displayed different RNA levels and GPC processing patterns as determined by Northern and Western blot analyses, respectively. Analysis of recombinant viruses containing amino acid substitutions selected at different mouse brain passages during the generation of Can revealed that altered Can GPC processing was primarily due to the T168A substitution within G1, which eliminates an N-linked glycosylation motif. Incorporation of the T168A substitution in the Rom GPC resulted in a Can-like processing pattern of Rom GPC. In addition, JUNV GPCs containing T168A substitution were retained within the endoplasmic reticulum (ER) and displayed significantly lower cell surface expression than wild-type Rom GPC. Interestingly, the reversion A168T in Can GPC significantly increased GPC expression at the cell surface. Our results demonstrate that recombinant JUNV (rJUNV) expressing Can GPC display markedly different protein expression and elevated genomic RNA expression when compared to viruses expressing Rom GPC. Additionally, our findings indicate that the N-linked glycosylation motif at amino acid positions 166-168 is important for trafficking of JUNV GPC to the cell surface, and the elimination of this motif interferes with the GPC release from the ER.
胡宁病毒（JUNV）是一种高致病性的新大陆沙粒病毒，是阿根廷出血热（AHF）的病原体。减毒活疫苗株Candid #1（Can）目前用作高危人群的疫苗。我们之前已经表明，Can糖蛋白（GPC）基因是AHF豚鼠模型中负责减毒的主要基因。然而，GPC导致Can株减毒的机制仍然未知。更全面地了解Can株减毒和免疫原性的潜在机制，可能有助于合理设计更多安全、新颖的疫苗。在此，我们详细比较了表达来自Can株和致病性罗梅罗（Rom）株基因组合的片段间和片段内嵌合JUNV重组克隆之间的RNA和蛋白质表达谱。表达Can GPC的重组病毒在豚鼠中显示出减毒，分别通过Northern印迹和Western印迹分析确定，其显示出不同的RNA水平和GPC加工模式。对在Can株产生过程中不同小鼠脑传代时选择的氨基酸替代的重组病毒进行分析发现，Can GPC加工改变主要是由于G1内的T168A替代，该替代消除了一个N-连接糖基化基序。在Rom GPC中引入T168A替代导致Rom GPC呈现类似Can的加工模式。此外，含有T168A替代的JUNV GPC保留在内质网（ER）中，并且与野生型Rom GPC相比，细胞表面表达显著降低。有趣的是，Can GPC中的回复突变A168T显著增加了GPC在细胞表面的表达。我们的结果表明，与表达Rom GPC的病毒相比，表达Can GPC的重组JUNV（rJUNV）显示出明显不同的蛋白质表达和升高的基因组RNA表达。此外，我们的研究结果表明，氨基酸位置166 - 168处的N-连接糖基化基序对于JUNV GPC转运到细胞表面很重要，并且该基序的消除会干扰GPC从ER释放。
