Rauddi María L, Mac Donald Cecilia L, Affranchino José L, González Silvia A
Laboratorio de Virología, CONICET-Facultad de Ciencias Exactas y Naturales, Universidad de Belgrano, Buenos Aires, Argentina.
AIDS Res Hum Retroviruses. 2011 Mar;27(3):303-16. doi: 10.1089/aid.2010.0137. Epub 2010 Oct 23.
To gain a better understanding of the assembly process in simian immunodeficiency virus (SIV), we first established the conditions under which recombinant SIV Gag lacking the C-terminal p6 domain (SIV GagΔp6) assembled in vitro into spherical particles. Based on the full multimerization capacity of SIV GagΔp6, and to identify the Gag sequences involved in homotypic interactions, we next developed a pull-down assay in which a panel of histidine-tagged SIV Gag truncation mutants was tested for its ability to associate in vitro with GST-SIVGagΔp6. Removal of the nucleocapsid (NC) domain from Gag impaired its ability to interact with GST-SIVGagΔp6. However, this Gag mutant consisting of the matrix (MA) and capsid (CA) domains still retained 50% of the wild-type binding activity. Truncation of SIV Gag from its N-terminus yielded markedly different results. The Gag region consisting of the CA and NC was significantly more efficient than wild-type Gag at interacting in vitro with GST-SIVGagΔp6. Notably, a small Gag subdomain containing the C-terminal third of the CA and the entire NC not only bound to GST-SIVGagΔp6 in vitro at wild-type levels, but also associated in vivo with full-length Gag and was recruited into extracellular particles. Interestingly, when the mature Gag products were analyzed, the MA and NC interacted with GST-SIVGagΔp6 with efficiencies representing 20% and 40%, respectively, of the wild-type value, whereas the CA failed to bind to GST-SIVGagΔp6, despite being capable of self-associating into multimeric complexes.
为了更好地理解猿猴免疫缺陷病毒（SIV）的组装过程，我们首先确定了重组SIV Gag（缺失C末端p6结构域，即SIV GagΔp6）在体外组装成球形颗粒的条件。基于SIV GagΔp6的完全多聚化能力，并为了鉴定参与同型相互作用的Gag序列，我们接下来开发了一种下拉实验，其中测试了一组组氨酸标签的SIV Gag截短突变体在体外与GST-SIVGagΔp6结合的能力。从Gag中去除核衣壳（NC）结构域会损害其与GST-SIVGagΔp6相互作用的能力。然而，这个由基质（MA）和衣壳（CA）结构域组成的Gag突变体仍保留了50%的野生型结合活性。从SIV Gag的N末端进行截短产生了明显不同的结果。由CA和NC组成的Gag区域在体外与GST-SIVGagΔp6相互作用时比野生型Gag效率显著更高。值得注意的是，一个包含CA C末端三分之一和整个NC的小Gag亚结构域不仅在体外以野生型水平与GST-SIVGagΔp6结合，而且在体内与全长Gag相互作用并被募集到细胞外颗粒中。有趣的是，当分析成熟的Gag产物时，MA和NC与GST-SIVGagΔp6相互作用的效率分别为野生型值的20%和40%，而CA尽管能够自组装成多聚体复合物，但却未能与GST-SIVGagΔp6结合。
