Kaptein Suzanne J F, Beisser Patrick S, Gruijthuijsen Yvonne K, Savelkouls Kim G M, van Cleef Koen W R, Beuken Erik, Grauls Gert E L M, Bruggeman Cathrien A, Vink Cornelis
Department of Medical Microbiology, Cardiovascular Research Institute Maastricht, University of Maastricht, 6202 AZ Maastricht, The Netherlands.
J Gen Virol. 2003 Sep;84(Pt 9):2517-2530. doi: 10.1099/vir.0.19227-0.
The rat cytomegalovirus (RCMV) R33 and R78 genes are conserved within members of the subfamily Betaherpesvirinae and encode proteins (pR33 and pR78, respectively) that show sequence similarity with G protein-coupled receptors. Previously, the biological relevance of these genes was demonstrated by the finding that R33- and R78-deleted RCMV strains are severely attenuated in vivo. In addition, R78-deleted strains were found to replicate less efficiently in cell culture. To monitor of the expression of R33- and R78-encoded proteins, recombinant RCMV strains, designated RCMV33G and RCMV78G, were generated. These recombinants expressed enhanced green fluorescent protein (EGFP)-tagged versions of pR33 and pR78 instead of native pR33 and pR78, respectively. Here it is reported that, although RCMV33G replicates as efficiently as wt virus in rat embryo fibroblast cultures, strain RCMV78G produces virus titres that are 3- to 4-fold lower than wt RCMV in the culture medium. This result indicates that the pR78-EGFP protein, as expressed by RCMV78G, does not completely functionally replace its native counterpart (pR78) in vitro. Interestingly, in infected rats, infectious RCMV33G was produced in significantly lower amounts than infectious wt RCMV, as well as RCMV78G, in the salivary glands. Conversely, although RCMV33G replicated to similar levels as wt virus in the spleen, both RCMV78G and an R78 knock-out strain (RCMV Delta R78a) replicated poorly in this organ. Together, these data indicate that R78 is crucial for the production of infectious RCMV in the spleen of infected rats.
大鼠巨细胞病毒（RCMV）的R33和R78基因在β疱疹病毒亚科成员中保守，编码的蛋白质（分别为pR33和pR78）与G蛋白偶联受体具有序列相似性。此前，这些基因的生物学相关性通过以下发现得以证明：缺失R33和R78的RCMV毒株在体内严重减毒。此外，发现缺失R78的毒株在细胞培养中复制效率较低。为监测R33和R78编码蛋白的表达，构建了重组RCMV毒株，命名为RCMV33G和RCMV78G。这些重组体分别表达增强型绿色荧光蛋白（EGFP）标记的pR33和pR78版本，而非天然的pR33和pR78。本文报道，尽管RCMV33G在大鼠胚胎成纤维细胞培养物中的复制效率与野生型病毒相同，但RCMV78G毒株在培养基中产生的病毒滴度比野生型RCMV低3至4倍。这一结果表明，RCMV78G表达的pR78 - EGFP蛋白在体外不能完全功能性替代其天然对应物（pR78）。有趣的是，在感染的大鼠中，唾液腺中产生的感染性RCMV33G的量明显低于感染性野生型RCMV以及RCMV78G。相反，尽管RCMV33G在脾脏中的复制水平与野生型病毒相似，但RCMV78G和R78基因敲除毒株（RCMV ΔR78a）在该器官中的复制能力较差。这些数据共同表明，R78对于感染大鼠脾脏中感染性RCMV的产生至关重要。
