Effect of macromolecular crowding agents on human immunodeficiency virus type 1 capsid protein assembly in vitro
Effect of macromolecular crowding agents on human immunodeficiency virus type 1 capsid protein assembly in vitro
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DOI：
10.1128/jvi.79.22.14271-14281.2005
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发表时间：
2005-11-01
影响因子：
5.4
通讯作者：
Mateu, MG
中科院分区：
文献类型：
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作者：
del Alamo, M;Rivas, G;Mateu, MG
人类免疫缺陷病毒1型（human immunodeficiency virus type 1，HIV-1）衣壳蛋白CA的体外自组装研究为HIV-1成熟衣壳的结构和组装提供了重要的信息。然而，以前观察到CA体外聚合仅在非常高的离子强度下发生。在这里，我们分析了CA组装在体外添加无关的，惰性的大分子（拥挤剂），旨在模仿拥挤（非常高的大分子有效浓度）的环境内的HIV-1病毒粒子的影响。拥挤剂诱导CA的快速和有效的聚合，即使在低（接近生理）离子强度。这样组装的中空圆柱体在形状和尺寸上与在高离子强度下在稀蛋白质溶液中形成的那些圆柱体没有区别。然而，注意到两个重要的差异：（i）在非常高的离子强度下，通过稀释的captase样颗粒的分解是不可检测的，但是在拥挤剂存在下，在低离子强度下迅速发生，和（ii）来自在CA二聚化界面处具有突变的假定感染性HIV-1的变体CA在没有拥挤剂的情况下不能在任何离子强度下组装;相反，当使用拥挤剂时，即使在低离子强度下，该突变也允许有效组装。使用低离子强度和惰性大分子来模拟HIV-1病毒粒子内的拥挤环境，可以更好地体外评估条件、突变或/和其他分子（包括潜在的抗病毒化合物）对HIV-1衣壳组装、稳定性和拆卸的影响。
Previous studies on the self-assembly of capsid protein CA of human immunodeficiency virus type 1 (HIV-1) in vitro have provided important insights on the structure and assembly of the mature HIV-1 capsid. However, CA polymerization in vitro was previously observed to occur only at very high ionic strength. Here, we have analyzed the effects on CA assembly in vitro of adding unrelated, inert macromolecules (crowding agents), aimed at mimicking the crowded (very high macromolecular effective concentration) environment within the HIV-1 virion. Crowding agents induced fast and efficient polymerization of CA even at low (close to physiological) ionic strength. The hollow cylinders thus assembled were indistinguishable in shape and dimensions from those formed in dilute protein solutions at high ionic strength. However, two important differences were noted: (i) disassembly by dilution of the capsid-like particles was undetectable at very high ionic strength, but occurred rapidly at low ionic strength in the presence of a crowding agent, and (ii) a variant CA from a presumed infectious HIV-1 with mutations at the CA dimerization interface was unable to assemble at any ionic strength in the absence of a crowding agent; in contrast, this mutation allowed efficient assembly, even at low ionic strength, when a crowding agent was used. The use of a low ionic strength and inert macromolecules to mimic the crowded environment inside the HIV-1 virion may lead to a better in vitro evaluation of the effects of conditions, mutations or/and other molecules, including potential antiviral compounds, on HIV-1 capsid assembly, stability and disassembly.