IDENTIFICATION OF A MEMBRANE-FUSION DOMAIN AND AN OLIGOMERIZATION DOMAIN IN THE BACULOVIRUS GP64 ENVELOPE FUSION PROTEIN
IDENTIFICATION OF A MEMBRANE-FUSION DOMAIN AND AN OLIGOMERIZATION DOMAIN IN THE BACULOVIRUS GP64 ENVELOPE FUSION PROTEIN
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DOI：
10.1128/jvi.69.4.2583-2595.1995
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发表时间：
1995-04-01
影响因子：
5.4
通讯作者：
BLISSARD, GW
中科院分区：
文献类型：
--
作者：
MONSMA, SA;BLISSARD, GW
杆状病毒GP 64包膜融合蛋白（GP 64 EFP）是出芽病毒粒子的主要包膜糖蛋白，并且已显示在病毒粒子中和在转染细胞中单独表达时介导酸触发的膜融合。使用定点突变和功能分析寡聚化，运输，和膜融合，我们本地化的GP 64 EFP的两个功能域。为了鉴定伪杉毒蛾核型多角体病毒（Orgyia pseudotsugata multiple nuclear polyhedrosis virus，OpMNPV）GP 64 EFP的融合结构域，我们检测了GP 64 EFP胞外域的两个疏水区。疏水区I（氨基酸223至228）是在胞外域中表现出最高局部疏水性的6个疏水氨基酸的簇。疏水区II（氨基酸330至338）位于GP 64 EFP的保守区域内，其含有亮氨酸残基的七肽重复序列，并被预测形成两亲性α-螺旋。在区域I中，Leu-226和Leu-227（在疏水簇的中心）处的非保守氨基酸取代完全废除融合活性，但不阻止GP 64 EFP寡聚化或表面定位。为了确认区域I在膜融合活性中的作用，我们使用合成的21个氨基酸的肽来产生针对区域I的多克隆抗体，并证明抗肽抗体能够中和膜融合活性并降低病毒的感染性。在疏水区II，突变被设计为破坏几个结构特征：亮氨酸的七肽重复，预测的α-螺旋，或局部疏水性沿着一面的螺旋。单丙氨酸取代七肽亮氨酸没有阻止寡聚化，运输，或融合活性。然而，多个丙氨酸取代或脯氨酸（螺旋不稳定）取代破坏了寡聚化和运输的GP 64 EFP。此外，删除区域II和预测的α-螺旋的缺失对于寡聚化是有缺陷的，而保留区域II和预测的螺旋的较大缺失是寡聚化的。这些结果表明，区域II是所需的寡聚化和运输，并建议，该区域的预测螺旋结构可能是重要的这一功能。因此，通过使用诱变、功能测定和抗体抑制，两个功能结构域位于杆状病毒GP 64 EFP内：位于氨基酸223至228的融合结构域和位于预测的两亲性α-螺旋内的氨基酸327至335的寡聚化结构域。
The baculovirus GP64 envelope fusion protein (GP64 EFP) is the major envelope glycoprotein of the budded virion and has been shown to mediate acid-triggered membrane fusion both in virions and when expressed alone in transfected cells. Using site-directed mutagenesis and functional assays for oligomerization, transport, and membrane fusion, we localized two functional domains of GP64 EFP. To identify a fusion domain in the GP64 EFP of the Orgyia pseudotsugata multiple nuclear polyhedrosis virus (OpMNPV), we examined two hydrophobic regions in the GP64 EFP ectodomain. Hydrophobic region I (amino acids 223 to 228) is a cluster of 6 hydrophobic amino acids exhibiting the highest local hydrophobicity in the ectodomain. Hydrophobic region II (amino acids 330 to 338) lies within a conserved region of GP64 EFP that contains a heptad repeat of leucine residues and is predicted to form an amphipathic alpha-helix. In region I, nonconservative amino acid substitutions at Leu-226 and Leu-227 (at the center of the hydrophobic cluster) completely abolished fusion activity but did not prevent GP64 EFP oligomerization or surface localization. To confirm the role of region I in membrane fusion activity, we used a synthetic 21-amino-acid peptide to generate polyclonal antibodies against region I and demonstrated that antipeptide antibodies were capable of both neutralizing membrane fusion activity and reducing infectivity of the virus. In hydrophobic region II, mutations were designed to disrupt several structural characteristics: a heptad repeat of leucine, a predicted alpha-helix, or the local hydrophobicity along one face of the helix. Single alanine substitutions for heptad leucines did not prevent oligomerization, transport, or fusion activity. However, multiple alanine substitutions or proline (helix-destabilizing) substitutions disrupted both oligomerization and transport of GP64 EFP. In addition, a deletion that removed region II and the predicted alpha-helix was defective for oligomerization, whereas a larger deletion that retained region II and the predicted helix was oligomerized. These results indicate that region II is required for oligomerization and transport and suggest that the predicted helical structure of this region may be important for this function. Thus, by using mutagenesis, functional assays, and antibody inhibition, two functional domains were localized within the baculovirus GP64 EFP: a fusion domain located at amino acids 223 to 228 and an oligomerization domain located at amino acids 327 to 335 within a predicted amphipathic alpha-helix.