Differential Contributions of Tacaribe Arenavirus Nucleoprotein N-Terminal and C-Terminal Residues to Nucleocapsid Functional Activity
Differential Contributions of Tacaribe Arenavirus Nucleoprotein N-Terminal and C-Terminal Residues to Nucleocapsid Functional Activity
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DOI：
10.1128/jvi.00321-14
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发表时间：
2014-06-01
影响因子：
5.4
通讯作者：
Lopez, Nora
中科院分区：
文献类型：
--
作者：
D'Antuono, Alejandra;Eugenia Loureiro, Maria;Lopez, Nora
沙粒病毒核蛋白（NP）是病毒核衣壳的主要蛋白组分，并且是由L聚合酶介导的病毒基因组复制所严格需要的。NP的同源寡聚化被认为在核衣壳组装中起重要作用，尽管核衣壳活性中NP-NP相互作用的潜在机制和相关性仍然知之甚少。在这里，我们评估新世界塔卡里贝病毒（TCRV）NP自我相互作用的核衣壳功能活性的贡献。我们表明，丙氨酸取代的N-末端残基预测可用于NP-NP相互作用强烈影响NP的自关联，通过免疫共沉淀测定，产生了急剧抑制TCRV小基因组RNA的转录和复制，并削弱NP与RNA的结合。突变和功能分析还表明，虽然NP自身相互作用的限制，在C-末端结构域的关键氨基酸是必不可少的RNA合成。此外，在这些C-末端残基的突变使得NP不能结合RNA在体内和体外，但没有影响与L聚合酶的相互作用。此外，虽然测试的所有寡聚化缺陷的变体表现出不变的能力，以维持NP-L相互作用，NP缺失突变体完全不能结合L，这表明，而NP自缔合是不稳定的，N-末端和C-末端结构域的完整性是需要结合的L聚合酶。总的来说，我们的研究结果表明，NP的N-末端结构域介导的自我相互作用可能在TCRV核衣壳组装和活性中发挥关键作用，NP的C-末端结构域与RNA结合有关。
The arenavirus nucleoprotein (NP) is the main protein component of viral nucleocapsids and is strictly required for viral genome replication mediated by the L polymerase. Homo-oligomerization of NP is presumed to play an important role in nucleocapsid assembly, albeit the underlying mechanism and the relevance of NP-NP interaction in nucleocapsid activity are still poorly understood. Here, we evaluate the contribution of the New World Tacaribe virus (TCRV) NP self-interaction to nucleocapsid functional activity. We show that alanine substitution of N-terminal residues predicted to be available for NP-NP interaction strongly affected NP self-association, as determined by coimmunoprecipitation assays, produced a drastic inhibition of transcription and replication of a TCRV minigenome RNA, and impaired NP binding to RNA. Mutagenesis and functional analysis also revealed that, while dispensable for NP self-interaction, key amino acids at the C-terminal domain were essential for RNA synthesis. Furthermore, mutations at these C-terminal residues rendered NP unable to bind RNA both in vivo and in vitro but had no effect on the interaction with the L polymerase. In addition, while all oligomerization-defective variants tested exhibited unaltered capacities to sustain NP-L interaction, NP deletion mutants were fully incompetent to bind L, suggesting that, whereas NP self-association is dispensable, the integrity of both the N-terminal and C-terminal domains is required for binding the L polymerase. Overall, our results suggest that NP self-interaction mediated by the N-terminal domain may play a critical role in TCRV nucleocapsid assembly and activity and that the C-terminal domain of NP is implicated in RNA binding.