Liu Yang, Nikolaitchik Olga A, Rahman Sheikh Abdul, Chen Jianbo, Pathak Vinay K, Hu Wei-Shau
Viral Recombination Section, HIV Dynamics and Replication Program, National Cancer Institute, Frederick, MD 21702, USA.
Viral Mutation Section, HIV Dynamics and Replication Program, National Cancer Institute, Frederick, MD 21702, USA.
J Mol Biol. 2017 Aug 4;429(16):2542-2555. doi: 10.1016/j.jmb.2017.06.018. Epub 2017 Jun 30.
Genome packaging is an essential step to generate infectious HIV-1 virions and is mediated by interactions between the viral protein Gag and cis-acting elements in the full-length RNA. The sequence necessary and sufficient to allow RNA genome packaging into an HIV-1 particle has not been defined. Here, we used two distinct reporter systems to determine the HIV-1 sequence required for heterologous, non-viral RNAs to be packaged into viral particles. Although the 5' untranslated region (UTR) of the HIV-1 RNA is known to be important for RNA packaging, we found that its ability to mediate packaging relies heavily on the context of the downstream sequences. Insertion of the 5' UTR and the first 32-nt of gag into two different reporter RNAs is not sufficient to mediate the packaging of these RNA into HIV-1 particles. However, adding the 5' half of the gag gene to the 5' UTR strongly facilitates the packaging of two reporter RNAs; such RNAs can be packaged at >50% of the efficiencies of an HIV-1 near full-length vector. To further examine the role of the gag sequence in RNA packaging, we replaced the 5' gag sequence in the HIV-1 genome with two codon-optimized gag sequences and found that such substitutions only resulted in a moderate decrease of RNA packaging efficiencies. Taken together, these results indicated that both HIV-1 5' UTR and the 5' gag sequence are required for efficient packaging of non-viral RNA into HIV-1 particles, although the gag sequence likely plays an indirect role in genome packaging.
基因组包装是产生具有感染性的HIV-1病毒粒子的关键步骤，由病毒蛋白Gag与全长RNA中的顺式作用元件之间的相互作用介导。允许RNA基因组包装到HIV-1颗粒中的必要且充分的序列尚未明确。在此，我们使用了两种不同的报告系统来确定将异源非病毒RNA包装到病毒颗粒中所需的HIV-1序列。尽管已知HIV-1 RNA的5'非翻译区（UTR）对RNA包装很重要，但我们发现其介导包装的能力在很大程度上依赖于下游序列的背景。将5'UTR和gag的前32个核苷酸插入两种不同的报告RNA中不足以介导这些RNA包装到HIV-1颗粒中。然而，将gag基因的5'半部分添加到5'UTR可强烈促进两种报告RNA的包装；此类RNA的包装效率可达到HIV-1近全长载体效率的50%以上。为了进一步研究gag序列在RNA包装中的作用，我们用两个密码子优化的gag序列替换了HIV-1基因组中的5'gag序列，发现这种替换仅导致RNA包装效率适度下降。综上所述，这些结果表明，HIV-1 5'UTR和5'gag序列对于将非病毒RNA有效包装到HIV-1颗粒中都是必需的，尽管gag序列可能在基因组包装中起间接作用。
