Natural and Recombinant SARS-CoV-2 Isolates Rapidly Evolve In Vitro to Higher Infectivity through More Efficient Binding to Heparan Sulfate and Reduced S1/S2 Cleavage.
Natural and Recombinant SARS-CoV-2 Isolates Rapidly Evolve In Vitro to Higher Infectivity through More Efficient Binding to Heparan Sulfate and Reduced S1/S2 Cleavage.
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DOI：
10.1128/jvi.01357-21
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发表时间：
2021-10-13
影响因子：
5.4
通讯作者：
Frolov I
中科院分区：
文献类型：
--
作者：
Shiliaev N;Lukash T;Palchevska O;Crossman DK;Green TJ;Crowley MR;Frolova EI;Frolov I
严重急性呼吸综合征冠状病毒2型（SARS-CoV-2）的毒力因子之一是能够与ACE 2受体高亲和力相互作用，介导病毒进入细胞。我们的研究结果表明，在细胞培养的几个通道内，无论是天然分离的SARS-CoV-2和重组cDNA衍生的变体获得额外的能力，结合硫酸乙酰肝素（HS）。这促进了病毒颗粒在与ACE 2进一步相互作用之前与细胞的初级附着。通过多种机制获得与HS的相互作用。这些包括（i）在S蛋白的N-末端结构域（NTD）中点突变的积累，其增加该结构域表面的正电荷，（ii）将含有带正电荷的氨基酸的异源肽插入NTD中，和（iii）弗林蛋白酶切割位点下游的第一个氨基酸的突变。最后一个突变影响S蛋白的加工，将未加工的弗林蛋白酶切割位点转化为肝素结合肽，并使病毒不太能够形成合胞体。这些病毒适应性导致病毒颗粒对肝素的亲和力更高，噬斑大小显著增加，病毒传播更有效，感染滴度更高，感染性更高2个数量级。检测到的适应也表明NTD在病毒附着和进入中的积极作用。与其他RNA阳性（RNA+）病毒一样，进化为HS结合可能导致体内病毒减毒。SARS-CoV-2的刺突蛋白是病毒致病的主要决定因素。它介导与ACE 2受体的结合，随后介导病毒包膜和细胞膜的融合。我们的研究结果表明，SARS-CoV-2在培养细胞中繁殖期间迅速进化。其刺突蛋白在NTD和弗林蛋白酶切割位点（FCS）的P1′位置获得突变。NTD中的氨基酸取代或短肽的插入紧密位于蛋白质表面并增加其正电荷。它们强烈增加病毒对硫酸乙酰肝素的亲和力，使其对培养细胞的感染性显著增加，并使基因组当量与PFU（GE/PFU）的比率降低几个数量级。S686 G突变还将FCS转化为肝素结合肽。因此，进化的SARS-CoV-2变体有效地利用细胞表面的糖胺聚糖，在刺突与ACE 2受体的高亲和力相互作用之前进行初级附着。
One of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virulence factors is the ability to interact with high affinity to the ACE2 receptor, which mediates viral entry into cells. The results of our study demonstrate that within a few passages in cell culture, both the natural isolate of SARS-CoV-2 and the recombinant cDNA-derived variant acquire an additional ability to bind to heparan sulfate (HS). This promotes a primary attachment of viral particles to cells before their further interactions with the ACE2. Interaction with HS is acquired through multiple mechanisms. These include (i) accumulation of point mutations in the N-terminal domain (NTD) of the S protein, which increases the positive charge of the surface of this domain, (ii) insertions into the NTD of heterologous peptides containing positively charged amino acids, and (iii) mutation of the first amino acid downstream of the furin cleavage site. This last mutation affects S protein processing, transforms the unprocessed furin cleavage site into the heparin-binding peptide, and makes viruses less capable of syncytium formation. These viral adaptations result in higher affinity of viral particles to heparin, dramatic increase in plaque sizes, more efficient viral spread, higher infectious titers, and 2 orders of magnitude higher infectivity. The detected adaptations also suggest an active role of NTD in virus attachment and entry. As in the case of other RNA-positive (RNA+) viruses, evolution to HS binding may result in virus attenuation in vivo. IMPORTANCE The spike protein of SARS-CoV-2 is a major determinant of viral pathogenesis. It mediates binding to the ACE2 receptor and, later, fusion of viral envelope and cellular membranes. The results of our study demonstrate that SARS-CoV-2 rapidly evolves during propagation in cultured cells. Its spike protein acquires mutations in the NTD and in the P1′ position of the furin cleavage site (FCS). The amino acid substitutions or insertions of short peptides in NTD are closely located on the protein surface and increase its positive charge. They strongly increase affinity of the virus to heparan sulfate, make it dramatically more infectious for the cultured cells, and decrease the genome equivalent to PFU (GE/PFU) ratio by orders of magnitude. The S686G mutation also transforms the FCS into the heparin-binding peptide. Thus, the evolved SARS-CoV-2 variants efficiently use glycosaminoglycans on the cell surface for primary attachment before the high-affinity interaction of the spikes with the ACE2 receptor.
影响因子：
3.7
作者：
Thackray LB;Holmes KV
通讯作者：
Holmes KV