Khan R, Giedroc D P
Department of Biochemistry and Biophysics, Texas A&M University, College Station 77843-2128.
J Biol Chem. 1994 Sep 9;269(36):22538-46.
The nucleocapsid protein (NC) of all animal retroviruses is the major structural protein of the core ribonucleoprotein complex, bound to genomic RNA in mature virions. In a previous report, we showed that recombinant NC protein from HIV-1, a 71-amino-acid protein (NC71), is apparently able to form two types of protein-nucleic acid complexes under low [NaCl], pH 8.3 and 25 degrees C. These appeared to differ in occluded apparent site size, napp, forming n = 8 and n = 14 complexes on poly(A) (Dib-Hajj, F., Khan, R., and Giedroc, D. P. (1993) Protein Sci. 2, 331-243) under conditions of high and low protein-nucleotide ratios, respectively. Here we show that both NC71-poly(A) complexes strongly scatter light under these solution conditions. Examination of the wavelength dependence of the light scattering at lambda < or = 320 nm indicates that each complex is characterized by a different scattering coefficient. Optical density measurements suggest that upon formation of the saturated n = 8 complex, additional polynucleotide is not incorporated into the complex over a period of hours, i.e. the n = 14 complex is not formed via redistribution of the n = 8 complex under low salt conditions, 25 degrees C. In contrast, the n = 14 complex readily incorporates additional protein until that sufficient to form the n = 8 complex is present. The n = 14 complex efficiently precipitates poly(A) and shows spectral characteristics expected for an extensively charge-neutralized nucleic acid complex. At [NC71] in excess of that required to form the n = 8 complex, this n = 14 complex is best described as a kinetic intermediate on the pathway to the n = 8 complex, which forms over a period of hours under low salt conditions, 25 degrees C. This slow kinetics of binding provides a possible explanation for the finding that the previously observed moderate cooperativity of Zn2 NC71 binding to poly(A) (omega = 200) at pH 8.3 and 0.29 M NaCl (Khan, R., and Giedroc, D. P. (1992) J. Biol. Chem. 267, 6689-6695) is shown here to represent a nonequilibrium phenomenon, apparently converting to a low or no cooperativity complex over a period of hours. Proteolytic removal of the COOH-terminal 14 amino acids from NC71, forming a 57-amino-acid protein (denoted NC57), removes this apparent binding site size heterogeneity of NC71 on poly(A). At 20 mM NaCl, NC57 binds with n = 6-7 nucleotides, in a manner which is independent of the protein-poly(A) nucleotide ratio. The implications of these findings on processing of the gag precursor which leads to mature NC in HIV-1 virions is discussed.
所有动物逆转录病毒的核衣壳蛋白（NC）是核心核糖核蛋白复合体的主要结构蛋白，在成熟病毒粒子中与基因组RNA结合。在之前的一份报告中，我们表明来自HIV-1的重组NC蛋白，一种71个氨基酸的蛋白（NC71），在低[NaCl]、pH 8.3和25℃条件下显然能够形成两种类型的蛋白质-核酸复合体。这些复合体在封闭的表观位点大小（napp）上似乎有所不同，在高和低蛋白质-核苷酸比率条件下，分别在聚（A）上形成n = 8和n = 14的复合体（迪布-哈吉，F.，汗，R.，和吉德罗克，D. P.（1993年）《蛋白质科学》2，331 - 243）。在这里我们表明，在这些溶液条件下，两种NC71-聚（A）复合体都强烈散射光。对λ≤320 nm处光散射的波长依赖性的研究表明，每种复合体都具有不同的散射系数。光密度测量表明，在形成饱和的n = 8复合体后，在数小时内没有额外的多核苷酸掺入该复合体，即n = 14复合体不是在低盐条件（25℃）下通过n = 8复合体的重新分布形成的。相反，n = 14复合体很容易掺入额外的蛋白质，直到存在足以形成n = 8复合体的蛋白质。n = 14复合体有效地沉淀聚（A），并显示出广泛电荷中和的核酸复合体所预期的光谱特征。当[NC71]超过形成n = 8复合体所需的量时，这种n = 14复合体最好被描述为在低盐条件（25℃）下数小时内形成n = 8复合体的途径上的动力学中间体。这种缓慢的结合动力学为以下发现提供了一种可能的解释：先前观察到的在pH 8.3和0.29 M NaCl条件下Zn2 NC71与聚（A）结合的适度协同性（ω = 200）（汗，R.，和吉德罗克，D. P.（1992年）《生物化学杂志》267，6689 - 6695）在这里被证明代表一种非平衡现象，显然在数小时内转变为低协同性或无协同性的复合体。从NC71上蛋白水解去除COOH末端的14个氨基酸后，形成一种57个氨基酸的蛋白（称为NC57），消除了NC71在聚（A）上这种明显的结合位点大小异质性。在20 mM NaCl条件下，NC57以与蛋白质-聚（A）核苷酸比率无关的方式与n = 6 - 7个核苷酸结合。讨论了这些发现对导致HIV-1病毒粒子中成熟NC的gag前体加工的影响。
