Lu M, Swevers L, Iatrou K
Department of Medical Biochemistry, The University of Calgary, Calgary, Alberta T2N 4N1, Canada.
J Virol. 1998 Jun;72(6):4789-97. doi: 10.1128/JVI.72.6.4789-4797.1998.
As part of our effort to identify baculovirus proteins acting as transcriptional regulators, we have characterized a gene, p95, of Bombyx mori nuclear polyhedrosis virus (BmNPV) that encompasses an open reading frame for a putative 95-kDa polypeptide (P95). The N-terminal half of the conceptually translated P95 contains two zinc finger-type DNA-binding motifs, and its C terminus contains a proline-rich region reminiscent of transcriptional activation regions. Northern blot analysis indicates that two mRNA species, 3.5 and 1.7 kb in size, are transcribed from the p95 gene at different times postinfection. These two mRNA species are produced by differential polyadenylation site usage. While the longer transcript can encode the P95 protein, the shorter one may encode a prematurely terminated version of the P95 polypeptide produced by ribosome frameshifting occurring at heptanucleotide "slippage" sites located near the relevant polyadenylation site. Transcription of the p95 gene is initiated at a proximal site located 70 nucleotides upstream of the translation start codon of P95, a middle site located 170 nucleotides from the start codon, and a set of three closely spaced distal sites located 385, 390, and 409 nucleotides from the translation start codon. The middle and distant initiation sites are utilized before and after BmNPV DNA replication, while transcripts initiated at the proximal site occur largely during the late and very late stages of viral infection. Transient-expression assays indicate that P95 can stimulate gene expression driven by the promoter of its own gene and the promoter of the cytoplasmic actin gene of B. mori. The P95-mediated trans activation can be further augmented by BmIE1, an immediate-early gene product of BmNPV. In contrast to the case with the actin promoter, however, the promoter of the p95 gene can be trans activated by the product of its own gene only in the presence of BmIE1. Our data suggest that proteins P95 and BmIE1 of BmNPV and, by analogy, those of other baculoviruses may interact with each other and synergize to potentiate transcription.
作为我们鉴定杆状病毒转录调节蛋白工作的一部分，我们对家蚕核型多角体病毒（BmNPV）的一个基因p95进行了表征，该基因包含一个假定的95 kDa多肽（P95）的开放阅读框。从概念上翻译的P95的N端一半包含两个锌指型DNA结合基序，其C端包含一个富含脯氨酸的区域，让人联想到转录激活区域。Northern印迹分析表明，在感染后不同时间从p95基因转录出两种大小分别为3.5 kb和1.7 kb的mRNA。这两种mRNA是通过不同的聚腺苷酸化位点使用产生的。较长的转录本可以编码P95蛋白，而较短的转录本可能编码由核糖体移码产生的P95多肽的过早终止版本，核糖体移码发生在相关聚腺苷酸化位点附近的七核苷酸“滑动”位点。p95基因的转录起始于P95翻译起始密码子上游70个核苷酸处的近端位点、起始密码子170个核苷酸处的中间位点以及翻译起始密码子385、390和409个核苷酸处的一组三个紧密间隔的远端位点。中间和远端起始位点在BmNPV DNA复制之前和之后被利用，而在近端位点起始的转录本主要发生在病毒感染的晚期和极晚期。瞬时表达分析表明，P95可以刺激由其自身基因的启动子和家蚕细胞质肌动蛋白基因的启动子驱动的基因表达。P95介导的反式激活可以被BmNPV的立即早期基因产物BmIE1进一步增强。然而，与肌动蛋白启动子的情况不同，p95基因的启动子仅在存在BmIE1的情况下才能被其自身基因的产物反式激活。我们的数据表明，BmNPV的蛋白P95和BmIE1，以及类似地其他杆状病毒的蛋白，可能相互作用并协同增强转录。
