Li Kejun, Zhang Shujing, Kronqvist Malin, Wallin Michael, Ekström Maria, Derse David, Garoff Henrik
Department of Biosciences and Nutrition, Karolinska Institute, S-141 57 Huddinge, Sweden.
J Virol. 2008 Jul;82(14):7135-43. doi: 10.1128/JVI.00448-08. Epub 2008 May 14.
Human T-cell leukemia virus (HTLV-1) Env carries a typical disulfide isomerization motif, C(225)XXC, in the C-terminal domain SU. Here we have tested whether this motif is used for isomerization of the intersubunit disulfide of Env and whether this rearrangement is required for membrane fusion. We introduced the C225A and C228A mutations into Env and found that the former but not the latter mutant matured into covalently linked SU-TM complexes in transfected cells. Next, we constructed a secreted Env ectodomain and showed that it underwent incubation-dependent intersubunit disulfide isomerization on target cells. However, the rearrangement was blocked by the C225A mutation, suggesting that C(225) carried the isomerization-active thiol. Still, it was possible to reduce the intersubunit disulfide of the native C225A ectodomain mutant with dithiothreitol (DTT). The importance of the CXXC-mediated disulfide isomerization for infection was studied using murine leukemia virus vectors pseudotyped with wild-type or C225A HTLV-1 Env. We found that the mutant Env blocked infection, but this could be rescued with DTT. The fusion activity was tested in a fusion-from-within assay using a coculture of rat XC target and transfected BHK-21 effector cells. We found that the mutation blocked polykaryon formation, but this could be reversed with DTT. Similar DTT-reversible inhibition of infection and fusion was observed when a membrane-impermeable alkylator was present during the infection/fusion incubation. We conclude that the fusion activity of HTLV-1 Env is controlled by an SU CXXC-mediated isomerization of the intersubunit disulfide. Thus, this extends the applicability of the isomerization model from gammaretroviruses to deltaretroviruses.
人类T细胞白血病病毒1型（HTLV-1）包膜糖蛋白（Env）在其C末端结构域SU中带有典型的二硫键异构化基序C(225)XXC。在此，我们测试了该基序是否用于Env亚基间二硫键的异构化，以及这种重排是否是膜融合所必需的。我们将C225A和C228A突变引入Env，发现前者而非后者突变体在转染细胞中成熟为共价连接的SU-TM复合物。接下来，我们构建了一种分泌型Env胞外结构域，并表明它在靶细胞上经历了依赖孵育的亚基间二硫键异构化。然而，C225A突变阻断了这种重排，表明C(225)携带了具有异构化活性的巯基。尽管如此，仍可用二硫苏糖醇（DTT）还原天然C225A胞外结构域突变体的亚基间二硫键。使用野生型或C225A HTLV-1 Env假型化的鼠白血病病毒载体研究了CXXC介导的二硫键异构化对感染的重要性。我们发现突变型Env阻断了感染，但这可用DTT挽救。在使用大鼠XC靶细胞和转染的BHK-21效应细胞共培养的“自内而外融合”试验中测试了融合活性。我们发现该突变阻断了多核体形成，但这可用DTT逆转。当在感染/融合孵育期间存在膜不透性烷化剂时，观察到类似的DTT可逆性感染和融合抑制。我们得出结论，HTLV-1 Env的融合活性由SU CXXC介导的亚基间二硫键异构化控制。因此，这将异构化模型的适用性从γ逆转录病毒扩展到了δ逆转录病毒。
