McNabb D S, Courtney R J
Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, Shreveport 71130-3932.
J Virol. 1992 May;66(5):2653-63. doi: 10.1128/JVI.66.5.2653-2663.1992.
The UL35 open reading frame (ORF) of herpes simplex virus type 1 (HSV-1) has been predicted from DNA sequence analysis to encode a small polypeptide with a molecular weight of 12,095. We have investigated the protein product of the UL35 ORF by using a trpE-UL35 gene fusion to produce a corresponding fusion protein in Escherichia coli. The TrpE-UL35 chimeric protein was subsequently isolated and used as a source of immunogen for the production of rabbit polyclonal antiserum directed against the UL35 gene product. The TrpE-UL35 antiserum was found to recognize a 12-kDa protein which was specifically present in HSV-1-infected cells. By utilizing the TrpE-UL35 antiserum, the kinetics of synthesis of the UL35 gene product was examined, and these studies indicate that UL35 is expressed as a gamma 2 (true late) gene. The 12-kDa protein recognized by the TrpE-UL35 antiserum was associated with purified HSV-1 virions and type A and B capsids, suggesting that the UL35 ORF may encode the 12-kDa capsid protein variably designated p12, NC7, or VP26. To confirm this assignment, immunoprecipitation and immunoblotting studies were performed to demonstrate that the TrpE-UL35 antiserum reacts with the same polypeptide as an antiserum directed against the purified p12 capsid protein (anti-NC7) (G.H. Cohen, M. Ponce de Leon, H. Diggelmann, W.C. Lawrence, S.K. Vernon, and R.J. Eisenberg, J. Virol. 34:521-531, 1980). Furthermore, the anti-NC7 serum was also found to react with the TrpE-UL35 chimeric protein isolated from E. coli, providing additional evidence that the UL35 gene encodes p12. On the basis of these studies, we conclude that UL35 represents a true late gene which encodes the 12-kDa capsid protein of HSV-1.
通过DNA序列分析预测，单纯疱疹病毒1型（HSV-1）的UL35开放阅读框（ORF）编码一种分子量为12,095的小多肽。我们利用trpE-UL35基因融合在大肠杆菌中产生相应的融合蛋白，对UL35 ORF的蛋白质产物进行了研究。随后分离出TrpE-UL35嵌合蛋白，并将其用作免疫原，以制备针对UL35基因产物的兔多克隆抗血清。发现TrpE-UL35抗血清能识别一种12 kDa的蛋白，该蛋白特异性存在于HSV-1感染的细胞中。利用TrpE-UL35抗血清，检测了UL35基因产物的合成动力学，这些研究表明UL35作为γ2（真正晚期）基因表达。TrpE-UL35抗血清识别的12 kDa蛋白与纯化的HSV-1病毒粒子以及A型和B型衣壳相关，这表明UL35 ORF可能编码可变命名为p12、NC7或VP26的12 kDa衣壳蛋白。为了证实这一归属，进行了免疫沉淀和免疫印迹研究，以证明TrpE-UL35抗血清与针对纯化的p12衣壳蛋白的抗血清（抗NC7）（G.H. Cohen、M. Ponce de Leon、H. Diggelmann、W.C. Lawrence、S.K. Vernon和R.J. Eisenberg，J. Virol. 34:521-531，1980）反应的是同一种多肽。此外，还发现抗NC7血清也能与从大肠杆菌中分离出的TrpE-UL35嵌合蛋白反应，这进一步证明UL35基因编码p12。基于这些研究，我们得出结论，UL35是一个真正的晚期基因，它编码HSV-1的12 kDa衣壳蛋白。
