The Gene 10 (UL49.5) Product of Equine Herpesvirus 1 Is Necessary and Sufficient for Functional Processing of Glycoprotein M

The functional cooperation of equine herpesvirus 1 (EHV-1) glycoprotein M (gM) and the gene 10 (UL49.5) product was analyzed. Transient-transfection experiments using gM and UL49.5 expression plasmids as well as RK13 cell lines constitutively expressing UL49.5 (RK49.5) or gM (RKgM) demonstrated that the endo-β-N-acetylglucosaminidase H (endo H)-resistant mature form of gM was detectable only after coexpression of the two proteins. Deletion of the EHV-1 UL49.5-homologous gene 10 in strain KyA resulted in a small-plaque phenotype and up to 190-fold-reduced virus titers. The growth defects of the mutant KyAΔ49.5 virus, which were very similar to those of a gM-negative KyA virus, could be completely compensated for by growth of the mutant virus on RK49.5 cells or by repairing the deletion of gene 10 in the revertant virus KyAΔ49.5R. Analysis of cells infected with the UL49.5-negative EHV-1 demonstrated that gM was not transported to the trans-Golgi network in the absence of the UL49.5 product. In contrast, gM was efficiently transported and processed to the endo H-resistant mature form in KyAΔ49.5-infected RK49.5 cells. Furthermore, radioimmunoprecipitation experiments demonstrated that gM maturation was observed only if a 10,000-M(r) protein was coprecipitated with gM in KyA- or KyAΔ49.5R-infected cells or virions. This protein was absent in cells infected with KyΔ49.5 or KyAΔgM, suggesting that it was the EHV-1 UL49.5 product. Taken together, our results demonstrate that the expression of the EHV-1 UL49.5 product is necessary and sufficient for gM processing and that it is required for efficient virus replication.本研究分析了马疱疹病毒1型（equine herpesvirus 1, EHV-1）糖蛋白M（glycoprotein M, gM）与基因10（UL49.5）编码产物的功能协同作用。采用gM与UL49.5表达质粒，以及稳定表达UL49.5（RK49.5）或gM（RKgM）的RK13细胞系开展瞬时转染实验，结果显示，仅当两种蛋白共表达时，方可检测到gM的内切β-N-乙酰葡糖胺糖苷酶H（endo-β-N-acetylglucosaminidase H, endo H）抗性成熟形式。敲除KyA毒株中EHV-1 UL49.5同源基因10后，病毒呈现小噬斑表型，病毒滴度最高可降至原水平的1/190。KyAΔ49.5突变病毒的生长缺陷与gM缺失型KyA毒株极为相似，该缺陷可通过在RK49.5细胞中培养突变病毒，或在回复突变病毒KyAΔ49.5R中修复基因10的缺失完全得到补偿。对UL49.5阴性EHV-1感染细胞的分析显示，在缺乏UL49.5编码产物的情况下，gM无法被转运至反式高尔基网络。与之相反，在KyAΔ49.5感染的RK49.5细胞中，gM可被有效转运并加工为endo H抗性的成熟形式。此外，放射免疫沉淀实验结果显示，仅在KyA或KyAΔ49.5R感染的细胞或病毒粒子中，当分子量为10 kDa的蛋白与gM共沉淀时，才可观察到gM的成熟过程。该蛋白在KyAΔ49.5或KyAΔgM感染的细胞中缺失，提示其即为EHV-1 UL49.5编码产物。综上，本研究结果证实，EHV-1 UL49.5编码产物的表达对gM的加工过程既是必需的，也是充分的，且其对于病毒的高效复制不可或缺。