Markosyan Ruben M, Leung Michael Y, Cohen Fredric S
Department of Molecular Biophysics and Physiology, Rush University Medical Center, Chicago, IL 60612, USA.
J Virol. 2009 Oct;83(19):10048-57. doi: 10.1128/JVI.00316-09. Epub 2009 Jul 22.
Residues that create the grooves of the human immunodeficiency virus type 1 (HIV-1) Env triple-stranded coiled coil (HR1) and the residues that pack into the grooves (HR2) to complete the formation of the six-helix bundle (6HB) were mutated. The extent and kinetics of fusion as well as pore enlargement were measured for each mutant. Mutations near the hairpin turns of each monomer of the 6HB were more important than those far from the turn, for both HR1 and HR2. This result is consistent with the idea that binding of HR2 to the HR1 grooves is initiated near the hairpin turn of each monomer. Mutations at the distal portions also reduced fusion, albeit to a smaller extent. An intermediate of fusion (temperature-arrested state [TAS]) was formed, and the consequences of mutation were compared; a mutant that exhibited less fusion also showed slower kinetics from TAS. This suggests that formation of the bundle is a rate-limiting step downstream of the intermediate state. The rate of enlargement of a fusion pore also correlated with the extent and kinetics of fusion. The rate of pore enlargement was severely reduced by mutation. This supports our prior conclusion that formation of the 6HB occurs after pore creation and strongly suggests that the free energy released by bundle formation is directly used to promote pore growth.
对构成人类免疫缺陷病毒1型（HIV-1）包膜三聚体卷曲螺旋（HR1）凹槽的残基以及填充到凹槽中以完成六螺旋束（6HB）形成的残基（HR2）进行了突变。对每个突变体的融合程度和动力学以及孔扩大情况进行了测量。对于HR1和HR2而言，6HB每个单体发夹转弯附近的突变比远离转弯处的突变更为重要。这一结果与HR2与HR1凹槽的结合在每个单体的发夹转弯附近开始的观点一致。远端部分的突变也会降低融合程度，尽管程度较小。形成了融合中间体（温度阻滞状态 [TAS]），并比较了突变的后果；融合程度较低的突变体从TAS开始的动力学也较慢。这表明束的形成是中间状态下游的限速步骤。融合孔扩大的速率也与融合程度和动力学相关。突变会严重降低孔扩大的速率。这支持了我们之前的结论，即6HB的形成发生在孔形成之后，并强烈表明束形成释放的自由能直接用于促进孔的生长。
