Simian virus 40 Vp1 DNA-binding domain is functionally separable from the overlapping nuclear localization signal and is required for effective virion formation and full viability
Simian virus 40 Vp1 DNA-binding domain is functionally separable from the overlapping nuclear localization signal and is required for effective virion formation and full viability
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DOI：
10.1128/jvi.75.16.7321-7329.2001
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发表时间：
2001-08-01
影响因子：
5.4
通讯作者：
Kasamatsu, H
中科院分区：
文献类型：
--
作者：
Li, PP;Nakanishi, A;Kasamatsu, H
在猿猴病毒 40 (SV40) 主要衣壳蛋白 Vp1 上鉴定出 DNA 结合结构域 (DBD)，并检查了该结构域在 SV40 生命周期中的功能。通过在体外测定各种重组 Vp1 蛋白的 DNA 结合来绘制 DBD。羧基末端58残基截短的Vp1 Delta C58五聚体结合DNA，就蛋白质五聚体而言K-d为1.8 X 10(-9) M，而全长Vp1和羧基末端17残基截短的Vp1 Delta C17具有相当的表观K(d)s，为5.3 X 10(-9)至7.3 X就蛋白质单体而言，为10(-9) M。先前在 Vp1 上鉴定出核定位信号 (NLS)，由两个 N 末端基本簇 NLS1 (4-KRK-6) 和 NLS2 (15-KKPK-18) 组成。在 NLS1 (NLSm1)、NLS2 (NLSm2) 或两个基本簇 (NLSm1.2) 中含有多点突变的 Vp1 Delta C58 五聚体的 DNA 结合活性逐渐降低，降至 NLSm1 的 Vp1 Delta C58 水平的 0.7%。 2 Vp1。这些数据与 N 末端截短蛋白质的数据一起，将 DBD 与二分 NLS 重叠。通过利用 NLS 表型互补来研究 Vp1 DBD 在感染期间的作用（N. Ishii、A. Nakanishi、M. Yamada、M. H. Macalalad 和 H. Kasamatsu, J. Virol. 68:8209-8216, 1994），其中 NLS 缺陷的 Vp1 在存在野生型次要衣壳蛋白的情况下可以定位于细胞核Vp2 和 Vp3。这种方法使得剖析双功能 Vp1 NLS-DBD 在细胞核中病毒粒子组装中的作用成为可能。活的非重叠SV40 (NO-SV40) DNA NLSm1、NLSm2 和NLSm1-2 的突变体在转染至宿主细胞后正常复制，并产生正常水平的衣壳蛋白。所有突变体 Vp1 都能在体外与 Vp3 相互作用。突变体 NLSm1 和 NLSm1-2 无法存活，并且突变体 Vp1s 意外地未能定位到细胞核，而 Vp2 和 Vp3 却定位到细胞核，这表明突变的 NLS1 充当了 Vp1 细胞质定位的显性信号。突变体 NLSm2（其中突变体 Vp1 的核定位缺陷由 Vp2 和 Vp3 补充）显示出活力降低了 5,000 倍。对 NLSm2 DNA 转染的细胞裂解物的分析显示，受 DNase I 保护的病毒 DNA 水平降低了 10 倍，但在 DNase I 抗性材料中发现了病毒粒子样颗粒。集体结果支持 Vp1 NLS2-DBD2 在病毒粒子组装中的作用。结果还表明，该决定因素可以在新细胞的感染中发挥作用。
A DNA-binding domain (DBD) was identified on simian virus 40 (SV40) major capsid protein Vp1, and the domain's function in the SV40 life cycle was examined. The DBD was mapped by assaying various recombinant Vp1 proteins for DNA binding in vitro. The carboxy-terminal 58-residue truncated Vp1 Delta C58 pentamer bound DNA,with a K-d of 1.8 X 10(-9) M in terms of the protein pentamer, while full-length Vp1 and carboxy-terminal-17-truncated Vp1 Delta C17 had comparable apparent K(d)s of 5.3 X 10(-9) to 7.3 X 10(-9) M in terms of the protein monomers. Previously identified on Vp1 was a nuclear localization signal (NLS) consisting of two N-terminal basic clusters, NLS1 (4-KRK-6) and NLS2 (15-KKPK-18). Vp1 Delta C58 pentamers harboring multiple-point mutations in NLS1 (NLSm1), NLS2 (NLSm2), or both basic clusters (NLSm1 . 2) had progressively decreased DNA-binding activity, down to 0.7% of the Vp1 Delta C58 level for NLSm1 . 2 Vp1. These data, along with those of N-terminally truncated proteins, placed the DBD in overlap with the bipartite NLS. The role of the Vp1 DBD during infection was investigated by taking advantage of NLS phenotypic complementation (N. Ishii, A. Nakanishi, M. Yamada, M. H. Macalalad, and H. Kasamatsu, J. Virol. 68:8209-8216, 1994), in which an NLS-defective Vp1 could localize to the nucleus in the presence of wild-type minor capsid proteins Vp2 and Vp3. This approach made it possible to dissect the role of the bifunctional Vp1 NLS-DBD in virion assembly in the nucleus. Mutants of the viable nonoverlaping SV40 (NO-SV40) DNA NLSm1, NLSm2, and NLSm1 - 2 replicated normally following transfection into host cells and produced capsid proteins at normal levels. All mutant Vp1s were able to interact with Vp3 in vitro. The mutants NLSm1 and NLSm1 - 2 were nonviable, and the mutant Vp1s unexpectedly failed to localize to the nucleus though Vp2 and Vp3 did, suggesting that the mutated NLS1 acted as a dominant signal for the cytoplasmic localization of Vp1. Mutant NLSm2, for which the mutant Vp1's nuclear localization defect was complemented by Vp2 and Vp3, displayed a 5,000-fold reduced viability. Analysis of NLSm2 DNA-transfected cell lysate revealed a 10-fold reduction in the level of DNase I-protected viral DNA, and yet virion-like particles were found among the DNase I-resistant material. Collective results support a role for Vp1 NLS2-DBD2 in the assembly of virion particles. The results also suggest that this determinant can function in the infection of new cells.