Kemler Iris, Barraza Roman, Poeschla Eric M
Molecular Medicine Program, Departments of Immunology and Medicine, Mayo Clinic, Rochester, Minnesota 55905, USA.
J Virol. 2002 Dec;76(23):11889-903. doi: 10.1128/jvi.76.23.11889-11903.2002.
Encapsidation of retroviral RNA involves specific interactions between viral proteins and cis-acting genomic RNA sequences. Human immunodeficiency virus type 1 (HIV-1) RNA encapsidation determinants appear to be more complex and dispersed than those of murine retroviruses. Feline lentiviral (feline immunodeficiency virus [FIV]) encapsidation has not been studied. To gain comparative insight into lentiviral encapsidation and to optimize FIV-based vectors, we used RNase protection assays of cellular and virion RNAs to determine packaging efficiencies of FIV deletion mutants, and we studied replicative phenotypes of mutant viruses. Unlike the case for other mammalian retroviruses, the sequences between the major splice donor (MSD) and the start codon of gag contribute negligibly to FIV encapsidation. Moreover, molecular clones having deletions in this region were replication competent. In contrast, sequences upstream of the MSD were important for encapsidation, and deletion of the U5 element markedly reduced genomic RNA packaging. The contribution of gag sequences to packaging was systematically investigated with subgenomic FIV vectors containing variable portions of the gag open reading frame, with all virion proteins supplied in trans. When no gag sequence was present, packaging was abolished and marker gene transduction was absent. Inclusion of the first 144 nucleotides (nt) of gag increased vector encapsidation to detectable levels, while inclusion of the first 311 nt increased it to nearly wild-type levels and resulted in high-titer FIV vectors. However, the identified proximal gag sequence is necessary but not sufficient, since viral mRNAs that contain all coding regions, with or without as much as 119 nt of adjacent upstream 5' leader, were excluded from encapsidation. The results identify a mechanism whereby FIV can encapsidate its genomic mRNA in preference to subgenomic mRNAs.
逆转录病毒RNA的衣壳化涉及病毒蛋白与顺式作用基因组RNA序列之间的特异性相互作用。1型人类免疫缺陷病毒（HIV-1）RNA的衣壳化决定因素似乎比鼠类逆转录病毒更为复杂和分散。猫慢病毒（猫免疫缺陷病毒 [FIV]）的衣壳化尚未得到研究。为了深入比较慢病毒的衣壳化情况并优化基于FIV的载体，我们利用细胞RNA和病毒粒子RNA的核糖核酸酶保护试验来确定FIV缺失突变体的包装效率，并研究了突变病毒的复制表型。与其他哺乳动物逆转录病毒不同，主要剪接供体（MSD）与gag起始密码子之间的序列对FIV衣壳化的贡献可忽略不计。此外，该区域存在缺失的分子克隆具有复制能力。相比之下，MSD上游的序列对衣壳化很重要，U5元件的缺失显著降低了基因组RNA的包装。我们使用含有gag开放阅读框可变部分的亚基因组FIV载体，在反式提供所有病毒粒子蛋白的情况下，系统地研究了gag序列对包装的贡献。当不存在gag序列时，包装被消除且标记基因转导缺失。包含gag的前144个核苷酸（nt）可将载体包装提高到可检测水平，而包含前311 nt则可将其提高到接近野生型水平，并产生高滴度的FIV载体。然而，已确定的近端gag序列是必要的但不是充分的，因为包含所有编码区的病毒mRNA，无论是否含有多达119 nt的相邻上游5'前导序列，都被排除在衣壳化之外。这些结果确定了一种机制，通过该机制FIV能够优先将其基因组mRNA包装成衣壳，而不是亚基因组mRNA。
