Schlesinger R W, Bradshaw G L, Barbone F, Reinacher M, Rott R, Husak P
Department of Molecular Genetics and Microbiology, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway 08854-5635.
J Virol. 1989 Apr;63(4):1695-703. doi: 10.1128/JVI.63.4.1695-1703.1989.
The combined presence of WSN gene segments 6 (neuraminidase), 7 (M1 and M2), and 8 (NS1 and NS2) in reassortants of WSN with A/Aichi/2/68 (H3N2) has been found by others to be necessary for full expression of neurovirulence in mice. We are examining the expression of the analogous three gene segments in brains of mice after intracerebral infection with non-neuroadapted strains A/WS/33 (WS) (from which WSN was derived) and A/PR/8/34 (PR8). Our aim is to determine possible mechanisms by which one or more of the five gene products may restrict replication of these strains in mouse brain cells to a single cycle, yielding noninfectious hemagglutinating particles (incomplete growth cycle). We found that minority subsets of such particles did produce plaques, provided they were activated by trypsin (analogous to other abortive systems producing virions with uncleaved HA), a step obviated for some WSN virions by indirect promotion of hemagglutinin cleavage by the neuraminidase of that strain. The percentage of such potentially infectious virions, relative to total hemagglutinating particles, was significantly lower in WS- or PR8-infected than in WSN-infected brains, suggesting possible defects in synthesis or function of M1 protein in the former. Cells in immunostained sections and appropriate bands in Western blots (immunoblots) of viral proteins electrophoretically separated from lysates of PR8-infected brains reacted with antibody to nucleoprotein but not to M1 protein. Either method revealed the presence of both proteins in WSN-infected brains. In contrast, Western blot analyses of particles concentrated from PR8-, WS-, or WSN-infected brains by hemadsorption, elution, and pelleting did reveal NP and M1 bands with comparable relative peroxidase-antiperoxidase staining intensities. The findings suggest that availability of M1 protein is a factor influencing the extent or rate of assembly of potentially infectious (i.e., trypsin-activated) progeny virions in mouse brains and that in this respect the two non-neurovirulent strains differ from WSN quantitatively rather than qualitatively.
其他人已发现，在WSN与A/爱知/2/68（H3N2）的重配体中，WSN基因片段6（神经氨酸酶）、7（M1和M2）和8（NS1和NS2）共同存在是小鼠中神经毒力充分表达所必需的。我们正在研究用非神经适应株A/WS/33（WS）（WSN由此衍生而来）和A/PR/8/34（PR8）脑内感染小鼠后，小鼠脑中类似的三个基因片段的表达情况。我们的目的是确定五种基因产物中的一种或多种可能通过何种机制将这些毒株在小鼠脑细胞中的复制限制为单个周期，产生无感染性的血凝颗粒（不完全生长周期）。我们发现，此类颗粒的少数亚群确实能形成噬斑，前提是它们被胰蛋白酶激活（类似于其他产生带有未切割HA的病毒粒子的流产系统），对于某些WSN病毒粒子，该毒株的神经氨酸酶间接促进血凝素切割可避免这一步骤。相对于总血凝颗粒，此类潜在感染性病毒粒子的百分比在WS或PR8感染的脑中显著低于WSN感染的脑，这表明前者中M1蛋白的合成或功能可能存在缺陷。从PR8感染的脑裂解物中电泳分离的病毒蛋白的免疫染色切片中的细胞以及免疫印迹（Western印迹）中的适当条带与核蛋白抗体反应，但不与M1蛋白抗体反应。两种方法均显示WSN感染的脑中两种蛋白均存在。相比之下，通过血细胞吸附、洗脱和沉淀从PR8、WS或WSN感染的脑中浓缩的颗粒的Western印迹分析确实显示了具有可比相对过氧化物酶-抗过氧化物酶染色强度的NP和M1条带。这些发现表明，M1蛋白的可用性是影响小鼠脑中潜在感染性（即胰蛋白酶激活的）子代病毒粒子组装程度或速率的一个因素，并且在这方面，这两种非神经毒力毒株与WSN在数量上而非质量上存在差异。
