von Einem Jens, Wellington Janet, Whalley J Millar, Osterrieder Kerstin, O'Callaghan Dennis J, Osterrieder Nikolaus
Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853, USA.
J Virol. 2004 Mar;78(6):3003-13. doi: 10.1128/jvi.78.6.3003-3013.2004.
Most equine herpesvirus 1 (EHV-1) strains, including the naturally occurring virulent RacL11 isolate, encode a large glycoprotein, gp2 (250 kDa), which is expressed from gene 71. Besides other alterations in the viral genome, the avirulent strain KyA harbors an in-frame deletion of 1,242 nucleotides in gene 71. To examine the contributions of gp2 variation to virus growth and virulence, mutant RacL11 and KyA viruses expressing full-length or truncated gp2 were generated. Western blot analyses demonstrated expression of a 250-kDa gp2 in cells infected with RacL11 virus or a mutant KyA virus harboring full-length gene 71, whereas a 75- to 80-kDa gp2 was detected in cells infected with KyA or mutant RacL11 virus expressing KyA gp2. The RacL11 gp2 precursor of 250 kDa in size and its truncated KyA counterpart of 80 kDa, as well as the 42-kDa carboxy-terminal gp2 subunit, were incorporated into virus particles. Absence of gp2 in RacL11 resulted in a 6-fold reduction of extracellular virus titers and a 13% reduction of plaque diameters, whereas gp2-negative KyA exhibited a 55% reduction in plaque diameter and a 51-fold decrease in extracellular virus titers. The massive growth defects of gp2-negative KyA could be restored by reinsertion of the truncated but not the full-length gp2 gene. The virulence of the generated gp2 mutant viruses was compared to the virulence of KyA and RacL11 in a murine infection model. RacL11 lacking gp2 was apathogenic for BALB/c mice, and insertion of the truncated KyA gp2 gene into RacL11 was unable to restore virulence. Similarly, replacement in the KyA genome of the truncated with the full-length RacL11 gene 71 did not result in the generation of virulent virus. From the results we conclude that full-length and truncated EHV-1 gp2 are not functionally equivalent and cannot compensate for the action of their homologues in allogeneic virus backgrounds.
大多数马疱疹病毒1型（EHV-1）毒株，包括自然存在的强毒株RacL11分离株，都编码一种大型糖蛋白gp2（250 kDa），它由基因71表达。除了病毒基因组的其他改变外，无毒株KyA在基因71中有一个1242个核苷酸的框内缺失。为了研究gp2变异对病毒生长和毒力的影响，构建了表达全长或截短型gp2的突变型RacL11和KyA病毒。蛋白质免疫印迹分析表明，在感染RacL11病毒或携带全长基因71的突变型KyA病毒的细胞中表达了250 kDa的gp2，而在感染KyA或表达KyA gp2的突变型RacL11病毒的细胞中检测到75至80 kDa的gp2。大小为250 kDa的RacL11 gp2前体及其截短的80 kDa的KyA对应物，以及42 kDa的羧基末端gp2亚基，都被整合到病毒颗粒中。RacL11中缺乏gp2导致细胞外病毒滴度降低6倍，噬斑直径减小13%，而gp2阴性的KyA噬斑直径减小55%，细胞外病毒滴度降低51倍。gp2阴性的KyA的大量生长缺陷可以通过重新插入截短的而非全长的gp2基因来恢复。在小鼠感染模型中，将产生的gp2突变病毒的毒力与KyA和RacL11的毒力进行了比较。缺乏gp2的RacL11对BALB/c小鼠无致病性，将截短的KyA gp2基因插入RacL11中无法恢复其毒力。同样，在KyA基因组中用全长的RacL11基因71替换截短的基因也不会产生强毒病毒。从结果我们得出结论，全长和截短的EHV-1 gp2在功能上不等同，并且不能在异源病毒背景中补偿其同源物的作用。
