Bowzard J B, Bennett R P, Krishna N K, Ernst S M, Rein A, Wills J W
Department of Microbiology and Immunology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033, USA.
J Virol. 1998 Nov;72(11):9034-44. doi: 10.1128/JVI.72.11.9034-9044.1998.
The Gag proteins of Rous sarcoma virus (RSV) and human immunodeficiency virus (HIV) contain small interaction (I) domains within their nucleocapsid (NC) sequences. These overlap the zinc finger motifs and function to provide the proper density to viral particles. There are two zinc fingers and at least two I domains within these Gag proteins. To more thoroughly characterize the important sequence features and properties of I domains, we analyzed Gag proteins that contain one or no zinc finger motifs. Chimeric proteins containing the amino-terminal half of RSV Gag and various portions of the carboxy terminus of murine leukemia virus (MLV) (containing one zinc finger) Gag had only one I domain, whereas similar chimeras with human foamy virus (HFV) (containing no zinc fingers) Gag had at least two. Mutational analysis of the MLV NC sequence and inspection of I domain sequences within the zinc-fingerless C terminus of HFV Gag suggested that clusters of basic residues, but not the zinc finger motif residues themselves, are required for the formation of particles of proper density. In support of this, a simple string of strongly basic residues was found to be able to substitute for the RSV I domains. We also explored the possibility that differences in I domains (e.g., their number) account for differences in the ability of Gag proteins to be rescued into particles when they are unable to bind to membranes. Previously published experiments have shown that such membrane-binding mutants of RSV and HIV (two I domains) can be rescued but that those of MLV (one I domain) cannot. Complementation rescue experiments with RSV-MLV chimeras now map this difference to the NC sequence of MLV. Importantly, the same RSV-MLV chimeras could be rescued by complementation when the block to budding was after, rather than before, transport to the membrane. These results suggest that MLV Gag molecules begin to interact at a much later time after synthesis than those of RSV and HIV.
劳氏肉瘤病毒（RSV）和人类免疫缺陷病毒（HIV）的Gag蛋白在其核衣壳（NC）序列中包含小相互作用（I）结构域。这些结构域与锌指基序重叠，其功能是为病毒颗粒提供合适的密度。这些Gag蛋白中有两个锌指和至少两个I结构域。为了更全面地描述I结构域的重要序列特征和特性，我们分析了含有一个锌指基序或不含锌指基序的Gag蛋白。含有RSV Gag氨基末端一半和鼠白血病病毒（MLV）（含有一个锌指）Gag羧基末端不同部分的嵌合蛋白只有一个I结构域，而与人类泡沫病毒（HFV）（不含锌指）Gag的类似嵌合体至少有两个I结构域。对MLV NC序列的突变分析以及对HFV Gag无锌指C末端内I结构域序列的检查表明，形成合适密度颗粒需要碱性残基簇，而不是锌指基序残基本身。支持这一点的是，发现一串简单的强碱性残基能够替代RSV的I结构域。我们还探讨了I结构域差异（例如其数量）是否解释了Gag蛋白在无法结合膜时被拯救到颗粒中的能力差异。先前发表的实验表明，RSV和HIV（两个I结构域）的此类膜结合突变体可以被拯救，但MLV（一个I结构域）的则不能。现在，用RSV-MLV嵌合体进行的互补拯救实验将这种差异定位到MLV的NC序列。重要的是，当出芽受阻发生在运输到膜之后而非之前时，相同的RSV-MLV嵌合体可以通过互补被拯救。这些结果表明，MLV Gag分子在合成后开始相互作用的时间比RSV和HIV的要晚得多。
