Large-Plaque Mutants of Sindbis Virus Show Reduced Binding to Heparan Sulfate, Heightened Viremia, and Slower Clearance from the Circulation

Laboratory strains of Sindbis virus must bind to the negatively charged glycosaminoglycan heparan sulfate in order to efficiently infect cultured cells. During infection of mice, however, we have frequently observed the development of large-plaque viral mutants with a reduced ability to bind to heparan sulfate. Sequencing of these mutants revealed changes of positively charged amino acids in putative heparin-binding domains of the E2 glycoprotein. Recombinant viruses were constructed with these changes as single amino acid substitutions in a strain Toto 1101 background. All exhibited decreased binding to heparan sulfate and had larger plaques than Toto 1101. When injected subcutaneously into neonatal mice, large-plaque viruses produced higher-titer viremia and often caused higher mortality. Because circulating heparin-binding proteins are known to be rapidly sequestered by tissue heparan sulfate, we measured the kinetics of viral clearance following intravenous injection. Much of the parental small-plaque Toto 1101 strain of Sindbis virus was cleared from the circulation by the liver within minutes, in contrast to recombinant large-plaque viruses, which had longer circulating half-lives. These findings indicate that a decreased ability to bind to heparan sulfate allows more efficient viral production in vivo, which may in turn lead to increased mortality. Because Sindbis virus is only one of a growing number of viruses from many families which have been shown to bind to heparan sulfate, these results may be generally applicable to the pathogenesis of such viruses.

辛德毕斯病毒（Sindbis virus）实验室毒株需结合带负电荷的糖胺聚糖（glycosaminoglycan）类分子硫酸乙酰肝素（heparan sulfate），方能高效感染体外培养的细胞。然而在小鼠感染实验中，我们常观察到大噬斑病毒突变株的出现，这类突变株结合硫酸乙酰肝素的能力有所下降。对这些突变株的测序结果显示，其E2糖蛋白（E2 glycoprotein）的推定肝素结合结构域内，带正电荷的氨基酸发生了变异。本研究以托托1101（Toto 1101）毒株为骨架，通过单点氨基酸突变引入上述变异，构建了重组病毒。所有重组病毒均表现出对硫酸乙酰肝素的结合能力下降，且噬斑尺寸较托托1101毒株更大。将大噬斑病毒皮下注射至新生小鼠体内后，可引发滴度更高的病毒血症，且通常会导致更高的死亡率。由于已知循环中的肝素结合蛋白会被组织中的硫酸乙酰肝素快速捕获，我们通过静脉注射方式检测了病毒清除动力学。亲本的小噬斑辛德毕斯病毒托托1101毒株可在数分钟内被肝脏从循环系统中清除；与之形成鲜明对比的是，重组大噬斑病毒的循环半衰期更长。上述研究结果表明，结合硫酸乙酰肝素能力的下降可提升病毒在体内的增殖效率，进而可能导致死亡率升高。目前已有来自多个病毒科的、数量持续增长的病毒被证实可结合硫酸乙酰肝素，而辛德毕斯病毒仅是其中一员，因此本研究结果或可推广至这类病毒的致病机制研究中。