Masunaga K, Mizumoto K, Kato H, Ishihama A, Toyoda T
Department of Virology, Kurume University School of Medicine, Kurume, Fukuoka, 830-0011, Japan.
Virology. 1999 Mar 30;256(1):130-41. doi: 10.1006/viro.1999.9625.
Influenza virus RNA polymerase with the subunit structure PB1-PB2-PA is involved in both transcription and replication of the RNA genome, including the unique cap-I-dependent RNase activity. To map the important domains for RNA polymerization, cap-I-dependent RNase, and cap-I-binding activity, we generated site-specific antibodies against overlapping 150-amino-acid peptides that cover each entire subunit. Monospecific antibodies against each subunit inhibited RNA synthesis in vitro. Those against PB1 and PB2 inhibited the cap-I-dependent RNase activity, but those against PB2 alone slightly inhibited the cap-I-binding activity. Antibodies against the N-terminal amino acids 1-159 of PB2 that overlap the PB1-binding site on PB2 and the C-terminal amino acids 501-617 of PA that overlap the putative nucleotide-binding site and PB1-binding site on PA inhibited RNA polymerizing activity as well as monospecific antibodies. Those against the N-terminal (amino acids 1-159); the central region (amino acids 305-559) of PB2, where a part of the cap-binding domain predicted previously is localized; the N-terminal (amino acids 1-222) of PB1; and amino acids 301-517 and 601-716 of PA inhibited the cap-I-dependent RNase activity. The cap-binding domain on PB2 could be mapped in amino acids 402-559, where one of the cap-binding domains mapped previously overlapped.
具有PB1 - PB2 - PA亚基结构的流感病毒RNA聚合酶参与RNA基因组的转录和复制，包括独特的帽依赖型核糖核酸酶活性。为了绘制RNA聚合、帽依赖型核糖核酸酶和帽结合活性的重要结构域，我们针对覆盖每个完整亚基的重叠150个氨基酸的肽段产生了位点特异性抗体。针对每个亚基的单特异性抗体在体外抑制RNA合成。针对PB1和PB2的抗体抑制帽依赖型核糖核酸酶活性，但仅针对PB2的抗体略微抑制帽结合活性。针对PB2上与PB1结合位点重叠的N端氨基酸1 - 159以及PA上与推定的核苷酸结合位点和PB1结合位点重叠的C端氨基酸501 - 617的抗体，与单特异性抗体一样抑制RNA聚合活性。针对PB2的N端（氨基酸1 - 159）、PB2的中央区域（氨基酸305 - 559，先前预测的帽结合结构域的一部分位于此处）、PB1的N端（氨基酸1 - 222）以及PA的氨基酸301 - 517和601 - 716的抗体抑制帽依赖型核糖核酸酶活性。PB2上的帽结合结构域可定位在氨基酸402 - 559处，此处与先前绘制的一个帽结合结构域重叠。
