Margulies Barry J, Gibson Wade
Towson University Herpes Virus Lab, Department of Biological Sciences, Towson University, Towson, MD 21252, USA.
Virus Res. 2007 Jan;123(1):57-71. doi: 10.1016/j.virusres.2006.08.003. Epub 2006 Sep 8.
Human cytomegalovirus (HCMV), a member of the beta-herpesvirus family, encodes four homologues of cellular G protein-coupled receptors (GPCRs). One of these, the protein product of HCMV open reading frame (ORF) UL33, has been identified in HCMV-infected cells and virus particles and shown to be heat-aggregatable and N-glycosylated. Another, the product of ORF US28, has been functionally characterized as a beta-chemokine receptor. Here we report the use of RT-PCR, coupled in vitro transcription-translation, immunoprecipitation, and Western immunoassays to (i) show that RNA from the open reading frame US27 appears predominantly during the late phase of replication; (ii) identify the protein encoded by HCMV US27 in infected cells and enveloped virus particles; (iii) demonstrate that the US27-encoded protein is heterogeneously N-glycosylated and resolves as two species following treatment with peptide N-glycosidase F; and (iv) show that both the recombinant and deglycoylated infected cell US27 protein aggregate when heated in the presence of SDS prior to electrophoresis in polyacrylamide gels, a property which is abrogated with the addition of urea to sample buffer.
人巨细胞病毒（HCMV）是β疱疹病毒家族的成员，编码四种细胞G蛋白偶联受体（GPCR）的同源物。其中之一，HCMV开放阅读框（ORF）UL33的蛋白产物，已在HCMV感染的细胞和病毒颗粒中被鉴定出来，并显示出可热聚集和N-糖基化。另一个，ORF US28的产物，在功能上被表征为一种β趋化因子受体。在此，我们报告了使用逆转录聚合酶链反应（RT-PCR）、体外转录-翻译偶联、免疫沉淀和Western免疫分析来：（i）表明开放阅读框US27的RNA主要在复制后期出现；（ii）在感染细胞和包膜病毒颗粒中鉴定HCMV US27编码的蛋白；（iii）证明US27编码的蛋白是异质性N-糖基化的，在用肽N-糖苷酶F处理后解析为两种形式；以及（iv）表明重组的和去糖基化的感染细胞US27蛋白在聚丙烯酰胺凝胶电泳前于SDS存在下加热时会聚集，在样品缓冲液中加入尿素可消除这一特性。
