Middle East Respiratory Syndrome Coronavirus Gene 5 Modulates Pathogenesis in Mice
Middle East Respiratory Syndrome Coronavirus Gene 5 Modulates Pathogenesis in Mice
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DOI：
10.1128/jvi.01172-20
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发表时间：
2021-02-01
影响因子：
5.4
通讯作者：
Enjuanes, Luis
中科院分区：
文献类型：
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作者：
Gutierrez-Alvarez, Javier;Wang, Li;Enjuanes, Luis
中东呼吸综合征冠状病毒（MERS-CoV）导致2012年出现的高致命性肺炎。关于MERS-CoV发病机制的信息有限，因为来自患者的数据很少，并且复制MERS临床表现的动物模型的生成一直具有挑战性。最近描述了人二肽基肽酶4敲入（hDPP 4-KI）小鼠和小鼠适应的MERS-CoV株（MERSMA-6-1-2）。感染MERSMA-6-1-2的hDPP 4-KI小鼠显示呼吸道疾病、肺中高病毒滴度和死亡的病理学体征。在这项工作中，通过将MERSMA-6-1-2基因组中所含的非同义突变引入MERS-CoV感染性cDNA中来工程化小鼠适应性MERS-CoV感染性cDNA，从而产生在hDDP 4-KI小鼠中具有毒性的重组小鼠适应性病毒（rMERS-MA）。MERS-CoV对细胞培养或小鼠肺的适应导致了属特异性基因5的突变和缺失，从而阻止了全长蛋白质的表达。相反，对476个MERS CoV野外分离株的分析表明，基因5在人类和骆驼体内都高度稳定。为了研究蛋白质5的作用，另外两种病毒被工程化以表达全长基因5（rMERS-MA-5 FL）或含有完整的基因5缺失（rMERS-MA-Delta 5）。rMERS-MA-5 FL病毒不稳定，因为在不同组织培养细胞中传代期间出现缺失，突出了MERS-CoV的不稳定性。在亚致死hDPP 4-KI小鼠模型中分析rMERS-MA-Delta 5的毒力。出乎意料的是，所有小鼠在感染rMERS-MA-Delta 5后死亡，与感染亲本病毒的小鼠相反，亲本病毒在蛋白质5的108位含有17个核苷酸（nt）缺失和终止密码子。在rMERS-MA-05感染小鼠的肺中，干扰素和促炎细胞因子的表达延迟和失调。总体而言，这些数据表明，rMERS-MA-Delta 5病毒比亲本病毒更具毒性，并表明小鼠适应的亲本病毒中存在的残留基因5序列具有改善严重MERS-CoV发病机制的功能。重要性中东呼吸综合征冠状病毒（MERS-CoV）是一种人畜共患病毒，可引起人类感染，死亡率高（接近35%）。动物模型和反向遗传学系统对于了解MERS-CoV发病机制至关重要。我们开发了一种小鼠适应MERS-CoV的反向遗传学系统，该系统复制了在人类中观察到的病毒行为。该系统对于研究特定病毒基因在发病机制中的作用非常有用。此外，我们描述了一种缺乏基因5表达的病毒，其毒性比亲本病毒更强。这些数据提供了在病毒感染的背景下IFN对基因5的调节的新功能，并将有助于开发新的抗病毒策略。
Middle East respiratory syndrome coronavirus (MERS-CoV) causes a highly lethal pneumonia that emerged in 2012. There is limited information on MERS-CoV pathogenesis, as data from patients are scarce and the generation of animal models reproducing MERS clinical manifestations has been challenging. Human dipeptidyl peptidase 4 knock-in (hDPP4-KI) mice and a mouse-adapted MERS-CoV strain (MERSMA-6-1-2) were recently described. hDPP4-KI mice infected with MERSMA-6-1-2 show pathological signs of respiratory disease, high viral titers in the lung, and death. In this work, a mouse-adapted MERS-CoV infectious cDNA was engineered by introducing nonsynonymous mutations contained in the MERSMA-6-1-2 genome into a MERS-CoV infectious cDNA, leading to a recombinant mouse-adapted virus (rMERS-MA) that was virulent in hDDP4-KI mice. MERS-CoV adaptation to cell culture or mouse lungs led to mutations and deletions in genus-specific gene 5 that prevented full-length protein expression. In contrast, analysis of 476 MERS-CoV field isolates showed that gene 5 is highly stable in vivo in both humans and camels. To study the role of protein 5, two additional viruses were engineered expressing a full-length gene 5 (rMERS-MA-5FL) or containing a complete gene 5 deletion (rMERS-MA-Delta 5). rMERS-MA-5FL virus was unstable, as deletions appeared during passage in different tissue culture cells, highlighting MERS-CoV instability. The virulence of rMERS-MA-Delta 5 was analyzed in a sublethal hDPP4-KI mouse model. Unexpectedly, all mice died after infection with rMERS-MA-Delta 5, in contrast to those infected with the parental virus, which contains a 17-nucleotide (nt) deletion and a stop codon in protein 5 at position 108. Expression of interferon and proinflammatory cytokines was delayed and dysregulated in the lungs of rMERS-MA-05-infected mice. Overall, these data indicated that the rMERS-MA-Delta 5 virus was more virulent than the parental one and suggest that the residual gene 5 sequence present in the mouse-adapted parental virus had a function in ameliorating severe MERS-CoV pathogenesis.IMPORTANCE Middle East respiratory syndrome coronavirus (MERS-CoV) is a zoonotic virus causing human infections with high mortality rate (similar to 35%). Animal models together with reverse-genetics systems are essential to understand MERS-CoV pathogenesis. We developed a reverse-genetics system for a mouse-adapted MERS-CoV that reproduces the virus behavior observed in humans. This system is highly useful to investigate the role of specific viral genes in pathogenesis. In addition, we described a virus lacking gene 5 expression that is more virulent than the parental one. The data provide novel functions in IFN modulation for gene 5 in the context of viral infection and will help to develop novel antiviral strategies.