Caccuri Francesca, Messali Serena, Bortolotti Daria, Di Silvestre Dario, De Palma Antonella, Cattaneo Chiara, Bertelli Anna, Zani Alberto, Milanesi Maria, Giovanetti Marta, Campisi Giovanni, Gentili Valentina, Bugatti Antonella, Filippini Federica, Scaltriti Erika, Pongolini Stefano, Tucci Alessandra, Fiorentini Simona, d'Ursi Pasqualina, Ciccozzi Massimo, Mauri Pierluigi, Rizzo Roberta, Caruso Arnaldo
Section of Microbiology, Department of Molecular and Translational Medicine, University of Brescia, P.le Spedali Civili, 1, Brescia 25123, Italy.
Department of Chemical, Pharmaceutical and Agricultural Sciences, University of Ferrara, Via Luigi Borsari, 46, Ferrara 44121, Italy.
Virus Evol. 2022 May 21;8(1):veac042. doi: 10.1093/ve/veac042. eCollection 2022.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) emerge for their capability to better adapt to the human host aimed and enhance human-to-human transmission. Mutations in spike largely contributed to adaptation. Viral persistence is a prerequisite for intra-host virus evolution, and this likely occurred in immunocompromised patients who allow intra-host long-term viral replication. The underlying mechanism leading to the emergence of variants during viral persistence in the immunocompromised host is still unknown. Here, we show the existence of an ensemble of minor mutants in the early biological samples obtained from an immunocompromised patient and their dynamic interplay with the master mutant during a persistent and productive long-term infection. In particular, after 222 days of active viral replication, the original master mutant, named MB61, was replaced by a minor quasispecies (MB61) expressing two critical mutations in spike, namely Q493K and N501T. Isolation of the two viruses allowed us to show that MB61 entry into target cells occurred mainly by the fusion at the plasma membrane (PM), whereas endocytosis characterized the entry mechanism used by MB61. Interestingly, coinfection of two human cell lines of different origin with the SARS-CoV-2 isolates highlighted the early and dramatic predominance of MB61 over MB61 replication. This finding may be explained by a faster replicative activity of MB61 as compared to MB61 as well as by the capability of MB61 to induce peculiar viral RNA-sensing mechanisms leading to an increased production of interferons (IFNs) and, in particular, of IFN-induced transmembrane protein 1 (IFITM1) and IFITM2. Indeed, it has been recently shown that IFITM2 is able to restrict SARS-CoV-2 entry occurring by endocytosis. In this regard, MB61 may escape the antiviral activity of IFITMs by using the PM fusion pathway for entry into the target cell, whereas MB61 cannot escape this host antiviral response during MB61 coinfection, since it has endocytosis as the main pathway of entry. Altogether, our data support the evidence of quasispecies fighting for host dominance by taking benefit from the cell machinery to restrict the productive infection of competitors in the viral ensemble. This finding may explain, at least in part, the extraordinary rapid worldwide turnover of VOCs that use the PM fusion pathway to enter into target cells over the original pandemic strain.
严重急性呼吸综合征冠状病毒2（SARS-CoV-2）的关注变异株（VOCs）因其能更好地适应人类宿主并增强人际传播能力而出现。刺突蛋白的突变在很大程度上促成了这种适应性。病毒持续存在是宿主内病毒进化的先决条件，这可能发生在免疫功能低下的患者中，这类患者允许病毒在宿主体内进行长期复制。免疫功能低下宿主中病毒持续存在期间导致变异株出现的潜在机制仍然未知。在此，我们展示了在一名免疫功能低下患者的早期生物样本中存在一组次要突变体，以及它们在持续性和高效性长期感染期间与主要突变体的动态相互作用。特别是，在活跃的病毒复制222天后，最初的主要突变体，命名为MB61，被一个在刺突蛋白中表达两个关键突变即Q493K和N501T的次要准种（MB61）所取代。对这两种病毒的分离使我们能够表明，MB61进入靶细胞主要通过质膜（PM）融合，而内吞作用是MB61使用的进入机制的特征。有趣的是，用SARS-CoV-2分离株对两种不同来源的人类细胞系进行共感染，突出了MB61在复制方面比MB61更早且显著占优势。这一发现可以解释为与MB61相比，MB61具有更快的复制活性，以及MB61能够诱导特殊的病毒RNA感应机制，导致干扰素（IFNs）尤其是干扰素诱导跨膜蛋白1（IFITM1）和IFITM2的产量增加。确实，最近已表明IFITM2能够限制通过内吞作用发生的SARS-CoV-2进入。在这方面，MB61可能通过利用PM融合途径进入靶细胞来逃避IFITMs的抗病毒活性，而在与MB61共感染期间，MB61不能逃避这种宿主抗病毒反应，因为它以内吞作用作为主要进入途径。总之，我们的数据支持了准种通过利用细胞机制来限制病毒群体中竞争者的有效感染从而争夺宿主主导地位的证据。这一发现至少可以部分解释使用PM融合途径进入靶细胞的VOCs在全球范围内相对于原始大流行毒株异常快速的更新换代。
