高致病性沙粒病毒,Junín病毒(JUNV),表达其核蛋白(NP)的三个截短的替代亚型,即,NP53kD,NP47kD,和NP40kD。虽然NP47kD和NP40kD先前已被证明是半胱天冬酶裂解的产物,在这里,我们显示第三同工型NP53kD的表达是由于来自M80的替代框内翻译。根据这些信息,我们能够产生缺乏这些同工型的重组JUNV。这些突变体的感染表明,虽然所有三种同工型都有助于有效控制胱天蛋白酶的激活,NP40kD起主导作用。与全长NP(即,NP65kD),局限于包涵体,病毒RNA合成发生的地方,这些同种型中N-末端卷曲螺旋区部分的丢失导致弥漫性细胞质分布和病毒RNA合成中的功能丧失。尽管如此,NP53kD,NP47kD,和NP40kD都保留了强大的干扰素拮抗和3'-5'核酸外切酶活性。我们建议,这些NP同种型的定位改变使它们可以被激活的胱天蛋白酶更有效地靶向,以裂解为诱饵底物,并更好地降解在病毒感染期间在细胞质中积累的病毒双链(DS)RNA种类和/或与细胞溶质RNA传感器相互作用,从而限制dsRNA介导的先天免疫应答。一起来看,这项工作深入了解了JUNV在感染过程中利用细胞凋亡产生生物学上不同的NP库的机制,并有助于我们理解病毒感染过程中替代蛋白质亚型的表达和生物学相关性.IMPORTANCEA有限的编码能力意味着RNA病毒需要策略来使其蛋白质组多样化。高致病性沙粒病毒Junín病毒(JUNV)的核蛋白(NP)产生三种N末端截短的同工型:已知两种(NP47kD和NP40kD)是通过caspase裂解产生的,while,在这里,我们表明NP53kD是通过替代翻译起始产生的。缺乏单个NP同种型的重组JUNV表明,所有三种同种型都有助于抑制感染期间的caspase激活,但卵裂产生NP40kD的贡献最大。重要的是,所有三种同工型都保留了消化双链(ds)RNA和抑制干扰素启动子激活的能力,但具有弥漫性的细胞质分布。鉴于两种异常病毒dsRNA的细胞质定位,以及dsRNA传感器和先天免疫激活途径的许多其他细胞成分,我们认为,NP同工型的产生不仅有助于逃避细胞凋亡,而且还能强有力地控制抗病毒反应。
The highly pathogenic arenavirus, Junín virus (JUNV), expresses three truncated alternative isoforms of its nucleoprotein (NP), i.e., NP53kD, NP47kD, and NP40kD. While both NP47kD and NP40kD have been previously shown to be products of caspase cleavage, here, we show that expression of the third isoform NP53kD is due to alternative in-frame translation from M80. Based on this information, we were able to generate recombinant JUNVs lacking each of these isoforms. Infection with these mutants revealed that, while all three isoforms contribute to the efficient control of caspase activation, NP40kD plays the predominant role. In contrast to full-length NP (i.e., NP65kD), which is localized to inclusion bodies, where viral RNA synthesis takes place, the loss of portions of the N-terminal coiled-coil region in these isoforms leads to a diffuse cytoplasmic distribution and a loss of function in viral RNA synthesis. Nonetheless, NP53kD, NP47kD, and NP40kD all retain robust interferon antagonistic and 3\'-5\' exonuclease activities. We suggest that the altered localization of these NP isoforms allows them to be more efficiently targeted by activated caspases for cleavage as decoy substrates, and to be better positioned to degrade viral double-stranded (ds)RNA species that accumulate in the cytoplasm during virus infection and/or interact with cytosolic RNA sensors, thereby limiting dsRNA-mediated innate immune responses. Taken together, this work provides insight into the mechanism by which JUNV leverages apoptosis during infection to generate biologically distinct pools of NP and contributes to our understanding of the expression and biological relevance of alternative protein isoforms during virus infection.IMPORTANCEA limited coding capacity means that RNA viruses need strategies to diversify their proteome. The nucleoprotein (NP) of the highly pathogenic arenavirus Junín virus (JUNV) produces three N-terminally truncated isoforms: two (NP47kD and NP40kD) are known to be produced by caspase cleavage, while, here, we show that NP53kD is produced by alternative translation initiation. Recombinant JUNVs lacking individual NP isoforms revealed that all three isoforms contribute to inhibiting caspase activation during infection, but cleavage to generate NP40kD makes the biggest contribution. Importantly, all three isoforms retain their ability to digest double-stranded (ds)RNA and inhibit interferon promoter activation but have a diffuse cytoplasmic distribution. Given the cytoplasmic localization of both aberrant viral dsRNAs, as well as dsRNA sensors and many other cellular components of innate immune activation pathways, we suggest that the generation of NP isoforms not only contributes to evasion of apoptosis but also robust control of the antiviral response.