Akkarawongsa Radeekorn, Pocaro Nina E, Case Gary, Kolb Aaron W, Brandt Curtis R
Department of Ophthalmology and Visual Sciences, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA.
Antimicrob Agents Chemother. 2009 Mar;53(3):987-96. doi: 10.1128/AAC.00793-08. Epub 2008 Dec 22.
The 773-residue ectodomain of the herpes simplex virus type 1 (HSV-1) glycoprotein B (gB) has been resistant to the use of mutagenic strategies because the majority of the induced mutations result in defective proteins. As an alternative strategy for the identification of functionally important regions and novel inhibitors of infection, we prepared a library of overlapping peptides homologous to the ectodomain of gB and screened for the ability of the peptides to block infection. Seven of 138 15-mer peptides inhibited infection by more than 50% at a concentration of 100 microM. Three peptides (gB94, gB122, and gB131) with 50% effective concentrations (EC(50)s) below 20 microM were selected for further studies. The gB131 peptide (residues 681 to 695 in HSV-1 gB [gB-1]) was a specific entry inhibitor (EC(50), approximately 12 microM). The gB122 peptide (residues 636 to 650 in gB-1) blocked viral entry (EC(50), approximately 18 microM), protected cells from infection (EC(50), approximately 72 microM), and inactivated virions in solution (EC(50), approximately 138 microM). We were unable to discern the step or steps inhibited by the gB94 peptide, which is homologous to residues 496 to 510 in gB-1. Substitution of a tyrosine in the gB122 peptide (Y640 in full-length gB-1) reduced the antiviral activity eightfold, suggesting that this residue is critical for inhibition. This peptide-based strategy could lead to the identification of functionally important regions of gB or other membrane proteins and identify novel inhibitors of HSV-1 entry.
单纯疱疹病毒1型（HSV-1）糖蛋白B（gB）的773个氨基酸残基的胞外域一直难以采用诱变策略进行研究，因为大多数诱导突变会导致蛋白质缺陷。作为鉴定功能重要区域和新型感染抑制剂的替代策略，我们制备了一个与gB胞外域同源的重叠肽库，并筛选这些肽阻断感染的能力。138个15肽中，有7个在100微摩尔浓度时抑制感染超过50%。选择了3个50%有效浓度（EC50）低于20微摩尔的肽（gB94、gB122和gB131）进行进一步研究。gB131肽（HSV-1 gB [gB-1]中的第681至695位氨基酸残基）是一种特异性进入抑制剂（EC50约为12微摩尔）。gB122肽（gB-1中的第636至650位氨基酸残基）阻断病毒进入（EC50约为18微摩尔），保护细胞免受感染（EC50约为72微摩尔），并使溶液中的病毒粒子失活（EC50约为138微摩尔）。我们无法确定gB94肽抑制的步骤，该肽与gB-1中的第496至510位氨基酸残基同源。gB122肽（全长gB-1中的Y640）中的酪氨酸替换使抗病毒活性降低了8倍，表明该残基对抑制作用至关重要。这种基于肽的策略可能会鉴定出gB或其他膜蛋白的功能重要区域，并识别出HSV-1进入的新型抑制剂。
