Feng Guozhong, Krell Peter J
State Key Laboratory of Rice Biology, China National Rice Research Institute, Hangzhou, China Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada.
Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada
J Virol. 2014 Sep;88(18):10918-33. doi: 10.1128/JVI.01167-14. Epub 2014 Jul 9.
The DNA polymerase (DNApol) of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is essential for viral DNA replication. The DNApol exonuclease and polymerase domains are highly conserved and are considered functional in DNA replication. However, the role of the DNApol C terminus has not yet been characterized. To identify whether only the exonuclease and polymerase domains are sufficient for viral DNA replication, several DNApol C-terminal truncations were cloned into a dnapol-null AcMNPV bacmid with a green fluorescent protein (GFP) reporter. Surprisingly, most of the truncation constructs, despite containing both exonuclease and polymerase domains, could not rescue viral DNA replication and viral production in bacmid-transfected Sf21 cells. Moreover, GFP fusions of these same truncations failed to localize to the nucleus. Truncation of the C-terminal amino acids 950 to 984 showed nuclear localization but allowed for only limited and delayed viral spread. The C terminus contains a typical bipartite nuclear localization signal (NLS) motif at residues 804 to 827 and a monopartite NLS motif at residues 939 to 948. Each NLS, as a GFP fusion peptide, localized to the nucleus, but both NLSs were required for nuclear localization of DNApol. Alanine substitutions in a highly conserved baculovirus DNApol sequence at AcMNPV DNApol amino acids 972 to 981 demonstrated its importance for virus production and DNA replication. Collectively, the data indicated that the C terminus of AcMNPV DNApol contains two NLSs and a conserved motif, all of which are required for nuclear localization of DNApol, viral DNA synthesis, and virus production.
The baculovirus DNA polymerase (DNApol) is a highly specific polymerase that allows viral DNA synthesis and hence virus replication in infected insect cells. We demonstrated that the exonuclease and polymerase domains of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) alone are insufficient for viral DNA synthesis and virus replication. Rather, we identified three features, including two nuclear localization signals and a highly conserved 10-amino-acid sequence in the AcMNPV DNApol C terminus, all three of which are important for both nuclear localization of DNApol and for DNApol activity, as measured by viral DNA synthesis and virus replication.
苜蓿银纹夜蛾多核多角体病毒（AcMNPV）的DNA聚合酶（DNApol）对病毒DNA复制至关重要。DNApol的核酸外切酶和聚合酶结构域高度保守，被认为在DNA复制中发挥功能。然而，DNApol C末端的作用尚未得到表征。为了确定是否只有核酸外切酶和聚合酶结构域足以进行病毒DNA复制，将几种DNApol C末端截短体克隆到带有绿色荧光蛋白（GFP）报告基因的dnapol缺失型AcMNPV杆粒中。令人惊讶的是，尽管大多数截短构建体都包含核酸外切酶和聚合酶结构域，但它们无法挽救杆粒转染的Sf21细胞中的病毒DNA复制和病毒产生。此外，这些相同截短体的GFP融合蛋白未能定位于细胞核。截短C末端氨基酸950至984显示出核定位，但仅允许有限且延迟的病毒传播。C末端在804至827位残基处含有一个典型的双分型核定位信号（NLS）基序，在939至948位残基处含有一个单分型NLS基序。每个NLS作为GFP融合肽都定位于细胞核，但两个NLS都是DNApol核定位所必需的。在AcMNPV DNApol氨基酸972至981处高度保守的杆状病毒DNApol序列中的丙氨酸替代证明了其对病毒产生和DNA复制的重要性。总体而言，数据表明AcMNPV DNApol的C末端包含两个NLS和一个保守基序，所有这些都是DNApol核定位、病毒DNA合成和病毒产生所必需的。
杆状病毒DNA聚合酶（DNApol）是一种高度特异性的聚合酶，可在受感染的昆虫细胞中进行病毒DNA合成，从而实现病毒复制。我们证明，苜蓿银纹夜蛾多核多角体病毒（AcMNPV）单独的核酸外切酶和聚合酶结构域不足以进行病毒DNA合成和病毒复制。相反，我们确定了三个特征，包括AcMNPV DNApol C末端的两个核定位信号和一个高度保守的10个氨基酸序列，这三个特征对于DNApol的核定位以及通过病毒DNA合成和病毒复制测量的DNApol活性都很重要。
