Nibert M L, Chappell J D, Dermody T S
Institute for Molecular Virology, University of Wisconsin-Madison 53706, USA.
J Virol. 1995 Aug;69(8):5057-67. doi: 10.1128/JVI.69.8.5057-5067.1995.
Mammalian reoviruses exhibit differences in the capacity to grow in intestinal tissue: reovirus type 1 Lang (T1L), but not type 3 Dearing (T3D), can be recovered in high titer from intestinal tissue of newborn mice after oral inoculation. We investigated whether in vitro protease treatment of virions of T1L and T3D, using conditions to generate infectious subvirion particles (ISVPs) as occurs in the intestinal lumen of mice (D. K. Bodkin, M. L. Nibert, and B. N. Fields, J. Virol. 63:4676-4681, 1989), affects viral infectivity. Chymotrypsin treatment of T1L was associated with a 2-fold increase in viral infectivity, whereas identical treatment of T3D resulted in a 10-fold decrease in infectivity. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, we found that loss of T3D infectivity was correlated with cleavage of its sigma 1 protein. We used reassortant viruses to identify viral determinants of infectivity loss and sigma 1 cleavage and found that both phenotypes segregate with the sigma 1-encoding S1 gene. Comparable results were obtained when trypsin treatment of virions of T1L and T3D was used. In experiments to determine the fate of sigma 1 fragments following cleavage, the capacity of anti-sigma 1 monoclonal antibody G5 to neutralize infectivity of T3D ISVPs was significantly decreased in comparison with its capacity to neutralize infectivity of virions, suggesting that a sigma 1 domain bound by G5 is lost from viral particles after proteolytic digestion. In contrast to the decrease in infectivity, chymotrypsin treatment of T3D virions leading to generation of ISVPs resulted in a 10-fold increase in their capacity to produce hemagglutination, indicating that a domain of sigma 1 important for binding to sialic acid remains associated with viral particles after sigma 1 cleavage. Neuraminidase treatment of L cells substantially decreased the yield of T3D ISVPs in comparison with the yield of virions, indicating that a sigma 1 domain important for binding sialic acid also can mediate attachment of T3D ISVPs to L cells and lead to productive infection. These results suggest that cleavage of T3D sigma 1 protein following oral inoculation of newborn mice is at least partly responsible for the decreased growth of T3D in the intestine and provide additional evidence that T3D sigma 1 contains more than a single receptor-binding domain.
1型朗株呼肠孤病毒（T1L）经口服接种后，可从新生小鼠的肠道组织中高滴度回收，而3型迪林株（T3D）则不能。我们研究了在体外使用能产生感染性子病毒颗粒（ISVP）的条件（如同在小鼠肠腔中发生的那样，D.K.博德金、M.L.尼伯特和B.N.菲尔兹，《病毒学杂志》63:4676 - 4681，1989年）对T1L和T3D病毒粒子进行蛋白酶处理，是否会影响病毒感染性。用胰凝乳蛋白酶处理T1L会使病毒感染性增加2倍，而对T3D进行相同处理则导致感染性降低10倍。通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳，我们发现T3D感染性的丧失与其σ1蛋白的切割有关。我们使用重配病毒来确定感染性丧失和σ1切割的病毒决定因素，发现这两种表型均与编码σ1的S1基因相关。当用胰蛋白酶处理T1L和T3D病毒粒子时，也获得了类似的结果。在确定切割后σ1片段的命运的实验中，抗σ1单克隆抗体G5中和T3D ISVP感染性的能力与其中和病毒粒子感染性的能力相比显著降低，这表明在蛋白水解消化后，G5所结合的σ1结构域从病毒颗粒上丢失。与感染性降低相反，用胰凝乳蛋白酶处理T3D病毒粒子导致产生ISVP，其产生血凝的能力增加了10倍，这表明在σ1切割后，对结合唾液酸很重要的σ1结构域仍与病毒颗粒相关。用神经氨酸酶处理L细胞，与病毒粒子的产量相比，T3D ISVP的产量大幅降低，这表明对结合唾液酸很重要的σ1结构域也可以介导T3D ISVP与L细胞的附着并导致有效感染。这些结果表明，新生小鼠口服接种后T3D σ1蛋白的切割至少部分导致了T3D在肠道中生长的减少，并提供了额外的证据表明T3D σ1包含不止一个受体结合结构域。
