Brindley Melinda A, Hughes Laura, Ruiz Autumn, McCray Paul B, Sanchez Anthony, Sanders David A, Maury Wendy
Department of Microbiology, Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA.
J Virol. 2007 Jul;81(14):7702-9. doi: 10.1128/JVI.02433-06. Epub 2007 May 2.
The filoviruses Ebola virus (EBOV) and Marburg virus (MARV) are responsible for devastating hemorrhagic fever outbreaks. No therapies are available against these viruses. An understanding of filoviral glycoprotein 1 (GP1) residues involved in entry events would facilitate the development of antivirals. Towards this end, we performed alanine scanning mutagenesis on selected residues in the amino terminus of GP1. Mutant GPs were evaluated for their incorporation onto feline immunodeficiency virus (FIV) particles, transduction efficiency, receptor binding, and ability to be cleaved by cathepsins L and B. FIV virions bearing 39 out of 63 mutant glycoproteins transduced cells efficiently, whereas virions bearing the other 24 had reduced levels of transduction. Virions pseudotyped with 23 of the poorly transducing GPs were characterized for their block in entry. Ten mutant GPs were very poorly incorporated onto viral particles. Nine additional mutant GPs (G87A/F88A, K114A/K115A, K140A, G143A, P146A/C147A, F153A/H154A, F159A, F160A, and Y162A) competed poorly with wild-type GP for binding to permissive cells. Four of these nine mutants (P146A/C147A, F153A/H154A, F159A, and F160A) were also inefficiently cleaved by cathepsins. An additional four mutant GPs (K84A, R134A, D150A, and E305/E306A) that were partially defective in transduction were found to compete effectively for receptor binding and were readily cleaved by cathepsins. This finding suggested that this latter group of mutants might be defective at a postbinding, cathepsin cleavage-independent step. In total, our study confirms the role of some GP1 residues in EBOV entry that had previously been recognized and identifies for the first time other residues that are important for productive entry.
丝状病毒埃博拉病毒（EBOV）和马尔堡病毒（MARV）可引发毁灭性的出血热疫情。目前尚无针对这些病毒的治疗方法。了解参与病毒进入过程的丝状病毒糖蛋白1（GP1）残基将有助于开发抗病毒药物。为此，我们对GP1氨基末端的选定残基进行了丙氨酸扫描诱变。对突变型GP进行了评估，包括其整合到猫免疫缺陷病毒（FIV）颗粒上的情况、转导效率、受体结合以及被组织蛋白酶L和B切割的能力。携带63种突变糖蛋白中39种的FIV病毒粒子能有效地转导细胞，而携带其他24种的病毒粒子转导水平降低。用23种转导效率低的GP假型化的病毒粒子，对其进入过程中的阻断情况进行了表征。10种突变型GP很难整合到病毒颗粒上。另外9种突变型GP（G87A/F88A、K114A/K115A、K140A、G143A、P146A/C147A、F153A/H154A、F159A、F160A和Y162A）与野生型GP竞争结合允许细胞的能力较差。这9种突变体中的4种（P146A/C147A、F153A/H154A、F159A和F160A）也不能被组织蛋白酶有效切割。另外4种在转导方面存在部分缺陷的突变型GP（K84A、R134A、D150A和E305/E306A）被发现能有效地竞争受体结合，并且很容易被组织蛋白酶切割。这一发现表明，后一组突变体可能在结合后、不依赖组织蛋白酶切割的步骤中存在缺陷。总的来说，我们的研究证实了一些GP1残基在EBOV进入过程中的作用，这些作用此前已被认识到，并且首次鉴定出了其他对有效进入很重要的残基。
