Center for Veterinary Health Sciences, Oklahoma State University, Stillwater, Oklahoma, USA.
J Virol. 2012 Apr;86(8):4432-43. doi: 10.1128/JVI.06744-11. Epub 2012 Feb 8.
An experimental system was developed to generate infectious human respiratory syncytial virus (HRSV) lacking matrix (M) protein expression (M-null virus) from cDNA. The role of the M protein in virus assembly was then examined by infecting HEp-2 and Vero cells with the M-null virus and assessing the impact on infectious virus production and viral protein trafficking. In the absence of M, the production of infectious progeny was strongly impaired. Immunofluorescence (IF) microscopy analysis using antibodies against the nucleoprotein (N), attachment protein (G), and fusion protein (F) failed to detect the characteristic virus-induced cell surface filaments, which are believed to represent infectious virions. In addition, a large proportion of the N protein was detected in viral replication factories termed inclusion bodies (IBs). High-resolution analysis of the surface of M-null virus-infected cells by field emission scanning electron microscopy (SEM) revealed the presence of large areas with densely packed, uniformly short filaments. Although unusually short, these filaments were otherwise similar to those induced by an M-containing control virus, including the presence of the viral G and F proteins. The abundance of the short, stunted filaments in the absence of M indicates that M is not required for the initial stages of filament formation but plays an important role in the maturation or elongation of these structures. In addition, the absence of mature viral filaments and the simultaneous increase in the level of the N protein within IBs suggest that the M protein is involved in the transport of viral ribonucleoprotein (RNP) complexes from cytoplasmic IBs to sites of budding.
我们构建了一个实验系统，可从 cDNA 生成缺乏基质 (M) 蛋白表达的感染性人呼吸道合胞病毒 (HRSV) (M 缺失病毒)。然后，通过用 M 缺失病毒感染 HEp-2 和 Vero 细胞，并评估其对感染性病毒产生和病毒蛋白运输的影响，来研究 M 蛋白在病毒组装中的作用。在缺乏 M 的情况下，产生感染性后代的能力受到严重损害。使用针对核蛋白 (N)、附着蛋白 (G) 和融合蛋白 (F) 的抗体进行免疫荧光 (IF) 显微镜分析未能检测到特征性的病毒诱导细胞表面丝状结构，这些结构被认为代表感染性病毒颗粒。此外，大量 N 蛋白在称为包涵体 (IB) 的病毒复制工厂中被检测到。通过场发射扫描电子显微镜 (SEM) 对 M 缺失病毒感染细胞的表面进行高分辨率分析，发现存在大片区域，这些区域内存在大量密集且均匀的短丝状结构。尽管这些丝状结构异常短，但与包含 M 的对照病毒诱导的丝状结构相似，包括存在病毒 G 和 F 蛋白。在缺乏 M 的情况下，这些短而发育不良的丝状结构大量存在表明，M 蛋白对于丝状结构形成的初始阶段不是必需的，但在这些结构的成熟或伸长中发挥重要作用。此外，由于缺乏成熟的病毒丝状结构以及 IB 内 N 蛋白水平的同时增加，表明 M 蛋白参与了将病毒核糖核蛋白 (RNP) 复合物从细胞质 IB 运输到出芽部位。
