Recombinant SARS-CoV-2 lacking initiating and internal methionine codons within ORF10 is attenuated in vivo-猫眼课题宝

DOI：

10.1101/2023.08.04.551973

发表时间：

2023-08

期刊：

bioRxiv

影响因子：

通讯作者：

中科院分区：

其他

文献类型：

作者：

Shichun Gu;Eleanor G. Bentley;R. Milligan;Abdulaziz M Almuqrin;Parul Sharma;Adam Kirby;D. F. Mega;A. Kipar;Maximilian Erdmann;J.-M. Bazire;K. Heesom;Philip A. Lewis;I. Donovan-Banfield;Charlotte Reston;Isobel Webb;Simon de Neck;Xaiofeng Dong;J. Hiscox;A. Davidson;James P. Stewart;D. Matthews

SARS-CoV-2的ORF 10位于病毒基因组的3 '末端。然而，功能性ORF 10产物的潜在作用甚至存在一直是争论的主题。在病毒基因组RNA中存在显著的结构特征，其本身可以解释ORF 10核苷酸序列的保留，而不需要功能性蛋白质产物。为了进一步探索这个问题，我们制备了两种重组病毒，首先是基于原始武汉分离株的基因组序列并在刺突蛋白中包含早期D614G突变的对照病毒（WT）。我们还制备了第二种病毒，除了两个额外的变化，即起始ORF 10起始密码子和终止密码子（ORF 10 KO）的内部甲硫氨酸密码子被替换外，与WT相同。在这里，我们表明，这两种病毒在过表达TMPRSS2的VeroE6细胞系（VTN细胞）中具有明显相同的生长动力学。然而，在过表达ACE2和TMPRSS2的A549细胞（A549-AT细胞）中，ORF10KO病毒似乎具有小的生长速率优势。使用生长竞争实验，其中将两种病毒混合，在VTN或A549-AT细胞中传代，并对所得输出病毒进行测序。我们发现在VTN细胞中WT病毒迅速占优势，而在A549-AT细胞中ORF10KO病毒占优势。然后，我们使用SARS-CoV-2感染的仓鼠模型，并确定ORF10KO病毒具有减弱的致病性（通过体重减轻来测量）。我们发现ORF10KO与WT相比，下呼吸道中的病毒滴度几乎降低了10倍。相比之下，WT和ORF10KO病毒在上呼吸道中具有相似的滴度。对感染ORF10KO病毒的仓鼠肺中的病毒RNA进行测序，结果显示该病毒经常回复为WT。我们的数据表明，保留一个功能性的ORF10序列是非常可取的SARS-CoV-2感染仓鼠和影响病毒的能力，在下呼吸道传播。

SARS-CoV-2 has been proposed to encode ORF10 as the 3’ terminal gene in the viral genome. However, the potential role and even existence of a functional ORF10 product has been the subject of debate. There are significant structural features in the viral genomic RNA that could, by themselves, explain the retention of the ORF10 nucleotide sequences without the need for a functional protein product. To explore this question further we made two recombinant viruses, firstly a control virus (WT) based on the genome sequence of the original Wuhan isolate and with the inclusion of the early D614G mutation in the Spike protein. We also made a second virus, identical to WT except for two additional changes that replaced the initiating ORF10 start codon and an internal methionine codon for stop codons (ORF10KO). Here we show that the two viruses have apparently identical growth kinetics in a VeroE6 cell line that over expresses TMPRSS2 (VTN cells). However, in A549 cells over expressing ACE2 and TMPRSS2 (A549-AT cells) the ORF10KO virus appears to have a small growth rate advantage. Growth competition experiments were used whereby the two viruses were mixed, passaged in either VTN or A549-AT cells and the resulting output virus was sequenced. We found that in VTN cells the WT virus quickly dominated whereas in the A549-AT cells the ORF10KO virus dominated. We then used a hamster model of SARS-CoV-2 infection and determined that the ORF10KO virus has attenuated pathogenicity (as measured by weight loss). We found an almost 10-fold reduction in viral titre in the lower respiratory tract for ORF10KO vs WT. In contrast, the WT and ORF10KO viruses had similar titres in the upper respiratory tract. Sequencing of viral RNA in the lungs of hamsters infected with ORF10KO virus revealed that this virus frequently reverts to WT. Our data suggests that the retention of a functional ORF10 sequence is highly desirable for SARS-CoV-2 infection of hamsters and affects the virus’s ability to propagate in the lower respiratory tract.

Recombinant SARS-CoV-2 lacking initiating and internal methionine codons within ORF10 is attenuated in vivo

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