Pisil Yalcin, Yazici Zafer, Shida Hisatoshi, Matsushita Shuzo, Miura Tomoyuki
Laboratory of Primate Model, Research Center for Infectious Diseases, Institute for Frontier Life and Medical Science, Kyoto University, Kyoto 615-8530, Japan.
Department of Virology, Faculty of Veterinary Medicine, 19 Mayis University, Samsun 55270, Turkey.
Pathogens. 2020 Mar 3;9(3):181. doi: 10.3390/pathogens9030181.
A tier 2 SHIV-MK38 strain was obtained after two in vivo passages of tier 1 SHIV-MK1. SHIV-MK38#818, cloned from the MK38 strain, was neutralisation-resistant, like the parental MK38 strain, to SHIV-infected monkey plasma (MP), HIV-1-infected human pooled plasma (HPP), and KD247 monoclonal antibody (mAb) (anti-V3 gp120 ). We investigated the mechanisms underlying the resistance of #818, specifically the amino acid substitutions that confer resistance to MK1. We introduced amino acid substitutions in the MK1 envelope by in vitro mutagenesis and then compared the neutralisation resistance to MP, HPP, and KD247 mAb with #818 in a neutralisation assay using TZM-bl cells. We selected 11 substitutions in the V1, V2, C2, V4, C4, and V5 regions based on the alignment of of MK1 and #818. The neutralisation resistance of the mutant MK1s with 7 of 11 substitutions in the V1, C2, C4, and V5 regions did not change significantly. These substitutions did not alter any negative charges or N-glycans. The substitutions N169D and K187E, which added negative charges, and S190N in the V2 region of gp120 and A389T in V4, which created sites for N-glycan, conferred high neutralisation resistance. The combinations N169D+K187E, N169D+S190N, and N169D+A389T resulted in MK1 neutralisation resistance close to that of #818. The combinations without 169D were neutralisation-sensitive. Therefore, N169D is the most important substitution for neutralisation resistance. This study demonstrated that although the V3 region sequences of #818 and MK1 are the same, V3 binding antibodies cannot neutralise #818 pseudovirus. Instead, mutations in the V2 and V4 regions inhibit the neutralisation of anti-V3 antibodies. We hypothesised that 169D and 190N altered the MK1 Env conformation so that the V3 region is buried. Therefore, the V2 region may block KD247 from binding to the tip of the V3 region.
在1级SHIV-MK1进行两次体内传代后获得了2级SHIV-MK38毒株。从MK38毒株克隆的SHIV-MK38#818，与亲代MK38毒株一样，对SHIV感染的猴血浆（MP）、HIV-1感染的人混合血浆（HPP）和KD247单克隆抗体（mAb）（抗V3 gp120）具有中和抗性。我们研究了#818抗性的潜在机制，特别是赋予对MK1抗性的氨基酸取代。我们通过体外诱变在MK1包膜中引入氨基酸取代，然后在使用TZM-bl细胞的中和试验中比较对MP、HPP和KD247 mAb的中和抗性与#818。基于MK1和#818的比对，我们在V1、V2、C2、V4、C4和V5区域选择了11个取代。在V1、C2、C4和V5区域有11个取代中的7个取代的突变型MK1的中和抗性没有显著变化。这些取代没有改变任何负电荷或N-聚糖。在gp120的V2区域添加负电荷 的取代N169D和K187E，以及在V4区域产生N-聚糖位点的S190N和A389T，赋予了高中和抗性。组合N169D+K187E、N169D+S190N和N169D+A389T导致MK1的中和抗性接近#818。没有169D的组合对中和敏感。因此，N169D是中和抗性最重要的取代。这项研究表明，尽管#818和MK1的V3区域序列相同，但V3结合抗体不能中和#818假病毒。相反，V2和V4区域的突变抑制了抗V3抗体的中和作用。我们假设169D和190N改变了MK1 Env构象，使得V3区域被掩埋。因此，V2区域可能会阻止KD247与V3区域的尖端结合。
