Charité-Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health (BIH), Institute of Biochemistry, Berlin, Germany
DZHK (German Centre for Cardiovascular Research), Berlin, Germany.
J Virol. 2020 Sep 15;94(19). doi: 10.1128/JVI.00952-20.
Coxsackie B viruses (CVB) cause a wide spectrum of diseases, ranging from mild respiratory syndromes and hand, foot, and mouth disease to life-threatening conditions, such as pancreatitis, myocarditis, and encephalitis. Previously, we and others found that the soluble virus receptor trap sCAR-Fc strongly attenuates CVB3 infection in mice. In this study, we investigated whether treatment with sCAR-Fc results in development of resistance by CVB3. Two CVB3 strains (CVB3-H3 and CVB3 Nancy) were passaged in HeLa cells in the presence of sCAR-Fc. The CVB3-H3 strain did not develop resistance, whereas two populations of CVB3 Nancy mutants emerged, one with complete (CVB3) and one with partial (CVB3) resistance. DNA sequence alignment of the resistant virus variant CVB3 with CVB3 Nancy revealed an amino acid exchange from Asn(N) to Ser(S) at position 139 of the CVB3 capsid protein VP2 (N2139S), an amino acid predicted to be involved in the virus's interaction with its cognate receptor CAR. Insertion of the N2139S mutation into CVB3-H3 by site-directed mutagenesis promoted resistance of the engineered CVB3-H3 to sCAR-Fc. Interestingly, development of resistance by CVB3-H3 and the exemplarily investigated CVB3-clone 2 (CVB3) against soluble CAR did not compromise the use of cellular CAR for viral infection. Infection of HeLa cells showed that sCAR-Fc resistance, however, negatively affected both virus stability and viral replication compared to that of the parental strains. These data demonstrate that during sCAR-Fc exposure, CVB3 can develop resistance against sCAR-Fc by single-amino-acid exchanges within the virus-receptor binding site, which, however, come at the expense of viral fitness. The emergence of resistant viruses is one of the most frequent obstacles preventing successful therapy of viral infections, representing a significant threat to human health. We investigated the emergence of resistant viruses during treatment with sCAR-Fc, a well-studied, highly effective antiviral molecule against CVB infections. Our data show the molecular aspects of resistant CVB3 mutants that arise during repetitive sCAR-Fc usage. However, drug resistance comes at the price of lower viral fitness. These results extend our knowledge of the development of resistance by coxsackieviruses and indicate potential limitations of antiviral therapy using soluble receptor molecules.
柯萨奇 B 病毒（CVB）可引起广泛的疾病，从轻度呼吸道综合征和手足口病到危及生命的胰腺炎、心肌炎和脑炎等疾病。此前，我们和其他研究人员发现可溶性病毒受体陷阱 sCAR-Fc 可显著减弱 CVB3 感染小鼠的感染性。在这项研究中，我们研究了 sCAR-Fc 是否会导致 CVB3 产生耐药性。将两种 CVB3 株（CVB3-H3 和 CVB3 Nancy）在存在 sCAR-Fc 的情况下在 HeLa 细胞中传代。CVB3-H3 株未产生耐药性，而 CVB3 Nancy 的两种突变株出现了，一种具有完全（CVB3）耐药性，另一种具有部分（CVB3）耐药性。与 CVB3 Nancy 相比，抗性病毒变体 CVB3 的 DNA 序列比对揭示了 CVB3 衣壳蛋白 VP2 中第 139 位天冬酰胺（N）到丝氨酸（S）的氨基酸替换（N2139S），该氨基酸预测与病毒与其同源受体 CAR 的相互作用有关。通过定点突变将 N2139S 突变插入 CVB3-H3 中，促进了工程化的 CVB3-H3 对 sCAR-Fc 的抗性。有趣的是，CVB3-H3 和代表性研究的 CVB3 克隆 2（CVB3）对可溶性 CAR 的耐药性发展并未损害细胞 CAR 用于病毒感染。HeLa 细胞的感染表明，与亲本株相比，sCAR-Fc 耐药性会降低病毒稳定性和病毒复制。这些数据表明，在 sCAR-Fc 暴露期间，CVB3 可以通过病毒受体结合部位的单个氨基酸交换产生对 sCAR-Fc 的耐药性，但这是以病毒适应性为代价的。耐药病毒的出现是阻止病毒感染成功治疗的最常见障碍之一，对人类健康构成重大威胁。我们研究了在使用 sCAR-Fc 治疗期间耐药病毒的出现，sCAR-Fc 是一种针对 CVB 感染的研究充分、非常有效的抗病毒分子。我们的数据显示了在重复使用 sCAR-Fc 期间出现的耐药性 CVB3 突变体的分子方面。然而，耐药性是以较低的病毒适应性为代价的。这些结果扩展了我们对柯萨奇病毒产生耐药性的认识，并表明使用可溶性受体分子进行抗病毒治疗的潜在局限性。
