Coronavirus Gene 7 Counteracts Host Defenses and Modulates Virus Virulence

Transmissible gastroenteritis virus (TGEV) genome contains three accessory genes: 3a, 3b and 7. Gene 7 is only present in members of coronavirus genus a1, and encodes a hydrophobic protein of 78 aa. To study gene 7 function, a recombinant TGEV virus lacking gene 7 was engineered (rTGEV-Δ7). Both the mutant and the parental (rTGEV-wt) viruses showed the same growth and viral RNA accumulation kinetics in tissue cultures. Nevertheless, cells infected with rTGEV-Δ7 virus showed an increased cytopathic effect caused by an enhanced apoptosis mediated by caspase activation. Macromolecular synthesis analysis showed that rTGEV-Δ7 virus infection led to host translational shut-off and increased cellular RNA degradation compared with rTGEV-wt infection. An increase of eukaryotic translation initiation factor 2 (eIF2α) phosphorylation and an enhanced nuclease, most likely RNase L, activity were observed in rTGEV-Δ7 virus infected cells. These results suggested that the removal of gene 7 promoted an intensified dsRNA-activated host antiviral response. In protein 7 a conserved sequence motif that potentially mediates binding to protein phosphatase 1 catalytic subunit (PP1c), a key regulator of the cell antiviral defenses, was identified. We postulated that TGEV protein 7 may counteract host antiviral response by its association with PP1c. In fact, pull-down assays demonstrated the interaction between TGEV protein 7, but not a protein 7 mutant lacking PP1c binding motif, with PP1. Moreover, the interaction between protein 7 and PP1 was required, during the infection, for eIF2α dephosphorylation and inhibition of cell RNA degradation. Inoculation of newborn piglets with rTGEV-Δ7 and rTGEV-wt viruses showed that rTGEV-Δ7 virus presented accelerated growth kinetics and pathology compared with the parental virus. Overall, the results indicated that gene 7 counteracted host cell defenses, and modified TGEV persistence increasing TGEV survival. Therefore, the acquisition of gene 7 by the TGEV genome most likely has provided a selective advantage to the virus.

猪传染性胃肠炎病毒（Transmissible gastroenteritis virus, TGEV）的基因组包含3个附属基因：3a、3b与7。基因7仅存在于冠状病毒α1属成员中，编码一条含78个氨基酸的疏水性蛋白。为研究基因7的功能，科研人员构建了缺失基因7的重组TGEV（rTGEV-Δ7）。该突变重组病毒与亲本野生型重组病毒（rTGEV-wt）在细胞培养物中展现出一致的增殖与病毒RNA积累动力学特征。然而，感染rTGEV-Δ7的细胞所出现的细胞病变效应更为显著，这一现象由半胱天冬酶（caspase）激活介导的细胞凋亡增强所导致。大分子合成分析结果显示，相较于rTGEV-wt感染组，rTGEV-Δ7感染会引发宿主翻译关闭，并加剧细胞RNA降解。在rTGEV-Δ7感染的细胞中，研究人员观察到真核翻译起始因子2α（eukaryotic translation initiation factor 2α, eIF2α）的磷酸化水平升高，且核酸酶（大概率为核糖核酸酶L，RNase L）的活性增强。上述结果表明，缺失基因7会强化双链RNA（double-stranded RNA, dsRNA）激活的宿主抗病毒应答。在蛋白7中，研究人员鉴定出一段保守序列基序，其可能介导蛋白7与细胞抗病毒防御的关键调控因子——蛋白磷酸酶1催化亚基（protein phosphatase 1 catalytic subunit, PP1c）的结合。据此我们推测，TGEV蛋白7可能通过与PP1c结合来拮抗宿主的抗病毒应答。事实上，下拉实验（pull-down assay）证实，TGEV蛋白7可与PP1结合，而缺失PP1c结合基序的蛋白7突变体则无此结合能力。此外，在病毒感染过程中，蛋白7与PP1的结合是eIF2α去磷酸化以及抑制细胞RNA降解所必需的。用rTGEV-Δ7与rTGEV-wt病毒接种新生仔猪的实验显示，相较于亲本病毒，rTGEV-Δ7的增殖动力学与致病进程均有所加快。综上，实验结果表明，基因7可拮抗宿主细胞防御机制，改变TGEV的持续感染状态，从而提升病毒的存活能力。因此，TGEV基因组获得基因7很可能为病毒带来了选择优势。