Deng Wentao, Jin Ge, Lin Biing-Yuan, Van Tine Brian A, Broker Thomas R, Chow Louise T
Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, Alabama 35294-0005, USA.
J Virol. 2003 Oct;77(19):10213-26. doi: 10.1128/jvi.77.19.10213-10226.2003.
The papillomavirus replicative helicase E1 and the origin recognition protein E2 are required for efficient viral DNA replication. We fused the green fluorescent protein (GFP) to the human papillomavirus type 11 E1 protein either in a plasmid with the E1 coding region alone (nucleotides [nt] 832 to 2781) (pGFP-11E1) or in a plasmid containing both the E1 and E2 regions (nt 2723 to 3826) and the viral origin of replication (ori) (p11Rc). The former supported transient replication of an ori plasmid, whereas the latter was a self-contained replicon. Unexpectedly, these plasmids produced predominantly a cytoplasmic variant GFP or a GFP-E1 E4 protein, respectively. The majority of the mRNAs had an intragenic or intergenic splice from nt 847 to nt 2622 or from nt 847 to nt 3325, corresponding to the E2 or E1 E4 messages. pGFP-11E1dm and p11Rc-E1dm, mutated at the splice donor site, abolished these splices and increased GFP-E1 protein expression. Three novel, alternatively spliced, putative E2 mRNAs were generated in higher abundance from the mutated replicon than from the wild type. Relative to pGFP-11E1, low levels of pGFP-11E1dm supported more efficient replication, but high levels had a negative effect. In contrast, elevated E2 levels always increased replication. Despite abundant GFP-E1 protein, p11Rc-E1dm replicated less efficiently than the wild type. Collectively, these observations show that the E1/E2 ratio is as important as the E1 and E2 concentrations in determining the replication efficiency. These findings suggest that alternative mRNA splicing could provide a mechanism to regulate E1 and E2 protein expression and DNA replication during different stages of the virus life cycle.
乳头瘤病毒复制解旋酶E1和起始识别蛋白E2是病毒DNA高效复制所必需的。我们将绿色荧光蛋白（GFP）与人乳头瘤病毒11型E1蛋白融合，融合方式有两种，一种是仅在含有E1编码区（核苷酸[nt]832至2781）的质粒中（pGFP - 11E1），另一种是在含有E1和E2区域（nt 2723至3826）以及病毒复制起始位点（ori）的质粒中（p11Rc）。前者支持ori质粒的瞬时复制，而后者是一个独立的复制子。出乎意料的是，这些质粒分别主要产生一种细胞质变体GFP或一种GFP - E1 E4蛋白。大多数mRNA在nt 847至nt 2622或nt 847至nt 3325处有基因内或基因间剪接，分别对应E2或E1 E4信使RNA。在剪接供体位点发生突变的pGFP - 11E1dm和p11Rc - E1dm消除了这些剪接，并增加了GFP - E1蛋白的表达。与野生型相比，从突变的复制子中产生了三种新的、选择性剪接的、假定的E2 mRNA，其丰度更高。相对于pGFP - 11E1，低水平的pGFP - 11E1dm支持更有效的复制，但高水平则有负面影响。相反，E2水平升高总是会增加复制。尽管有大量的GFP - E1蛋白，p11Rc - E1dm的复制效率却低于野生型。总体而言，这些观察结果表明，在决定复制效率方面，E1/E2比率与E1和E2浓度同样重要。这些发现表明，选择性mRNA剪接可能提供一种机制，在病毒生命周期的不同阶段调节E1和E2蛋白表达以及DNA复制。
