Keil G M, Engelhardt T, Karger A, Enz M
Institute for Molecular and Cellular Virology, Insel Riems, Germany.
J Virol. 1996 May;70(5):3032-8. doi: 10.1128/JVI.70.5.3032-3038.1996.
Sequence analysis of the short unique (Us) segment of the bovine herpesvirus 1 (BHV-1) genome predicted that the Us open reading frame (ORF) 4 encodes a protein with homology to glycoprotein G (gG) of other alpha-herpesviruses (P. Leung-Tack, J.-C. Audonnet, and M. Riviere, Virology 199:409-421, 1994). RNA analysis showed that the Us ORF4 is contained within two transcripts of 3.5 and 1.8 kb. The 3.5 kb RNA represents a structurally bicistronic RNA which encompasses the Us ORF3 and Us ORF4, whereas the 1.8-kb RNA constitutes the monocistronic Us ORF4 mRNA. To identify the predicted BHV-I gG, recombinant vaccinia virus expressing the Us ORF4 was used to raise specific antibodies in rabbits. The antiserum recognized a 65-kDa polypeptide and a very diffusely migrating species of proteins with an apparent molecular mass of between 90 and greater than 240 kDa in supernatants of BHV-1-infected cells which was also precipitated together with 61- and 70-kDa polypeptides from cell-associated proteins. The specificity of the reaction was demonstrated by the absence of these proteins from the supernatant of cells infected with the Us ORF4 deletion mutant BHV-l/gp1-8. Treatment of the immunoprecipitated proteins with glycosidases and chondroitinase AC showed that the 65-kDa protein constitutes gG, which contains both N- and O-linked carbohydrates, and that the high-molecular-mass proteins contain glycosaminoglycans linked to a 65-kDa glycoprotein that is antigenically related to gG. These molecules were therefore named glycoproteoglycan C (gpgG). Pulse chase experiments indicated that gG and gpgG were processed from a common precursor molecule with an apparent molecular mass of 61 kDa via a 70-kDa intermediate. Both gG and gpgG could not be found associated with purified virions. In summary, our results identify the BHV-I gG protein and demonstrate the presence of a form of posttranslational modification, glycosamino-glycosylation, that has not yet been described for a herpesvirus-encoded protein.
牛疱疹病毒1型（BHV-1）基因组短独特（Us）片段的序列分析预测，Us开放阅读框（ORF）4编码一种与其他α-疱疹病毒的糖蛋白G（gG）具有同源性的蛋白质（P. Leung-Tack、J.-C. Audonnet和M. Riviere，《病毒学》199:409 - 421，1994）。RNA分析表明，Us ORF4包含在3.5 kb和1.8 kb的两种转录本中。3.5 kb的RNA代表一种结构上的双顺反子RNA，它包含Us ORF3和Us ORF4，而1.8 kb的RNA构成单顺反子Us ORF4 mRNA。为了鉴定预测的BHV-1 gG，使用表达Us ORF4的重组痘苗病毒在兔中产生特异性抗体。抗血清识别出一种65 kDa的多肽以及BHV-1感染细胞上清液中一种迁移非常弥散的蛋白质条带，其表观分子量在90至大于240 kDa之间，并且还与细胞相关蛋白中的61 kDa和70 kDa多肽一起沉淀。用缺失Us ORF4的突变体BHV-1/gp1-8感染的细胞上清液中不存在这些蛋白质，从而证明了反应的特异性。用糖苷酶和软骨素酶AC处理免疫沉淀的蛋白质表明，65 kDa的蛋白质构成gG，其同时含有N-连接和O-连接的碳水化合物，并且高分子量蛋白质含有与一种与gG抗原相关的65 kDa糖蛋白相连的糖胺聚糖。因此，这些分子被命名为糖蛋白聚糖C（gpgG）。脉冲追踪实验表明，gG和gpgG是通过一个70 kDa的中间体从一个表观分子量为61 kDa的共同前体分子加工而来的。在纯化的病毒粒子中未发现gG和gpgG。总之，我们的结果鉴定了BHV-1 gG蛋白，并证明了一种翻译后修饰形式——糖胺聚糖糖基化的存在，这种修饰尚未在疱疹病毒编码的蛋白质中被描述过。
