Temporal Regulation of Baculovirus Gene Expression

瓜里诺杆状病毒在真核DNA病毒中是独一无二的，因为早期基因由宿主RNA聚合酶II转录，而晚期和非常晚期基因由病毒编码的RNA聚合酶转录。病毒RNA聚合酶含有四个等摩尔亚基，Lef-4、-8、-9和P47。Lef-8是四个亚基中最大的一个，它含有一个13个氨基酸的基序，该基序在RNA聚合酶的β亚基之间保守。Lef-9含有一个7个氨基酸的序列，与一个高度保守的β-质数序列同源。这些相似性表明Lef-8和Lef-9是RNA聚合酶的催化亚基。然而，与RNA聚合酶相比，这些同源性区域受到的限制要大得多。为了检验这些序列的功能相关性，将对保守的基序进行丙氨酸突变，并确定突变对转录活性的影响。此外，还将检测单个亚基的RNA聚合酶、启动子结合和NTP结合活性。更多的晚期转录因子将被检测刺激病毒RNA聚合酶以及DNA和RNA结合活性的能力。病毒蛋白VLF-1是差异调节晚期和非常晚期基因表达的因子的最佳候选者。VLF-1与整合酶家族有很强的同源性，但整合酶在杆状病毒的生命周期中没有明显的作用。因此，VLF-1可能以类似于整合酶的方式作用于DNA，并以序列特异性的方式与DNA结合，并以某种方式对其进行修饰。纯化的VLF-1将在DNA/RNA结合、拓扑异构酶活性和体外转录刺激试验中的活性方面进行表征。这一研究将有助于理解杆状病毒基因的时间表达调控机制，并对生物技术具有实际影响。杆状病毒表达系统广泛用于生产用作疫苗、诊断工具或治疗剂的蛋白质。然而，杆状病毒系统有局限性：它是暂时的，因为病毒载体杀死细胞；当细胞处理途径受到损害时，重组蛋白表达较晚。了解控制杆状病毒多角体蛋白表达的机制可能有助于设计在没有病毒感染的情况下工作的载体，从而不会杀死宿主或破坏蛋白质加工。

GuarinoBaculoviruses are unique among eukaryotic DNA viruses in that early genes are transcribed by the host RNA polymerase II while late and very late genes are transcribed by a viral-encoded RNA polymerase. The viral RNA polymerase contains four equimolar subunits, LEF-4, -8, -9, and p47. LEF-8, the largest of the four subunits, contains a 13 amino acid motif conserved among beta subunits of RNA polymerases. LEF-9 contains a 7 amino acid sequence with homology to a highly conserved beta-prime sequence. These similarities suggest that LEF-8 and LEF-9 are the catalytic subunits of RNA polymerase. However, these regions of homology are considerably more restricted than is common among RNA polymerases. To examine the functional relevance of these sequences, alanine mutagenesis will be conducted on the conserved motifs, and the effects of the mutations on transcription activity will be determined. In addition, the individual subunits will be assayed for RNA polymerase, promoter binding, and NTP binding activities. Additional late transcription factors will be assayed for the ability to stimulate viral RNA polymerase and for DNA and RNA binding activities.The viral protein VLF-1 is the best candidate for the factor that differentially regulates expression of late and very late genes. VLF-1 shows strong homology to a family of proteins that function as integrases, yet there is no obvious role for an integrase in the life cycle of baculoviruses. Thus, VLF-1 probably acts on DNA in a manner analogous to integrases and binds to DNA in a sequence-specific manner and modifies it in some way. Purified VLF-1 will be characterized with respect to its activity in assays for DNA/RNA binding, topoisomerase activity, and stimulation of in vitro transcription.This research should aid in understanding the mechanisms that regulate temporal expression of baculovirus genes, and have practical impacts on biotechnology. The baculovirus expression system is widely used for the production of proteins for use as vaccines, diagnostic tools, or therapeutic agents. However, the baculovirus system has limitations: it is transient because the viral vectors kill the cells; and recombinant proteins are expressed late when cellular processing pathways are compromised. A knowledge of the mechanisms that control baculovirus polyhedrin expression may aid in the design of vectors that work in the absence of virus infection and thus do not kill the host or impair protein processing.