Chen M, Harty R N, Zhao Y, Holden V R, O'Callaghan D J
Department of Microbiology and Immunology, Louisiana State University Medical Center, Shreveport 71130-3932, USA.
J Virol. 1996 Jan;70(1):313-20. doi: 10.1128/JVI.70.1.313-320.1996.
Defective interfering (DI) particles of equine herpesvirus type 1 (EHV-1) are capable of mediating persistent infection (S. A. Dauenhauer, R. A. Robinson, and D. J. O'Callaghan, J. Gen. Virol. 60:1-14, 1982; R. A. Robinson, R. B. Vance, and D. J. O'Callaghan, J. Virol. 36:204-219, 1980). Sequence analysis of cloned DI particle DNA revealed that portions of two regulatory genes, ICP22 (IR4) and ICP27 (UL3), are linked in frame to form a unique hybrid open reading frame (ORF). This hybrid ORF, designated as the IR4/UL3 gene, encodes the amino-terminal 196 amino acids of the IR4 protein (ICP22 homolog) and the carboxy-terminal 68 amino acids of the UL3 protein (ICP27 homolog). Portions of DNA sequences encoding these two regulatory proteins, separated by more than 115 kbp in the standard virus genome, were linked presumably by a homologous recombination event between two identical 8-bp sequences. Reverse transcriptase-PCR and S1 nuclease analyses revealed that this unique ORF is transcribed by utilizing the transcription initiation site of ICP22 and the polyadenylation signal of ICP27 in DI particle-enriched infection. Immunoprecipitation and Western blot (immunoblot) analyses with antisera to the ICP22 and ICP27 proteins demonstrated that a 31-kDa hybrid protein was synthesized in the DI particle-enriched infection but not in standard virus infection. This 31-kDa hybrid protein was expressed at the same time as the ICP22 protein in DI particle-enriched infection and migrated at the same location on polyacrylamide gel electrophoresis as the protein expressed from a cloned IR4/UL3 expression vector. These observations suggested that the unique IR4/UL3 hybrid gene is expressed from the DI particle genome and may play a role in DI particle-mediated persistent infection.
1型马疱疹病毒（EHV-1）的缺陷干扰（DI）颗粒能够介导持续性感染（S. A. 道恩豪尔、R. A. 罗宾逊和D. J. 奥卡拉汉，《普通病毒学杂志》60:1 - 14，1982；R. A. 罗宾逊、R. B. 万斯和D. J. 奥卡拉汉，《病毒学杂志》36:204 - 219，1980）。对克隆的DI颗粒DNA进行序列分析发现，两个调控基因ICP22（IR4）和ICP27（UL3）的部分序列以读框相连，形成一个独特的杂交开放阅读框（ORF）。这个杂交ORF被命名为IR4/UL3基因，编码IR4蛋白（ICP22同源物）的氨基末端196个氨基酸和UL3蛋白（ICP27同源物）的羧基末端68个氨基酸。在标准病毒基因组中相隔超过115 kbp的编码这两种调控蛋白的DNA序列部分，大概是通过两个相同的8 bp序列之间的同源重组事件相连的。逆转录酶 - PCR和S1核酸酶分析表明，在富含DI颗粒的感染中，这个独特的ORF利用ICP22的转录起始位点和ICP27的聚腺苷酸化信号进行转录。用针对ICP22和ICP27蛋白的抗血清进行免疫沉淀和蛋白质印迹（免疫印迹）分析表明，在富含DI颗粒的感染中合成了一种31 kDa的杂交蛋白，而在标准病毒感染中则没有。在富含DI颗粒的感染中，这种31 kDa的杂交蛋白与ICP22蛋白同时表达，并且在聚丙烯酰胺凝胶电泳上迁移到与从克隆的IR4/UL3表达载体表达的蛋白相同的位置。这些观察结果表明，独特的IR4/UL3杂交基因从DI颗粒基因组中表达，并且可能在DI颗粒介导的持续性感染中发挥作用。
