Disruption of the baculovirus core gene ac78 results in decreased production of multiple nucleocapsid-enveloped occlusion-derived virions and the failure of primary infection in vivo

The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac78 gene is one of the baculovirus core genes. Recent studies showed that ac78 is essential for budded virion (BV) production and the embedding of occlusion-derived virion (ODV) into occlusion body during the AcMNPV life cycle. Here, we report that an ac78-knockout AcMNPV (vAc78KO) constructed in this study had different phenotypes than those described in the previous studies. A few infectious BVs were detected using titer assays, immunoblot analyses and plaque assays, indicating that ac78 is not essential for BV formation. Electron microscopy confirmed that the ac78 deletion did not affect nucleocapsid assembly and ODV formation. However, the numbers of multiple nucleocapsid-enveloped ODVs and ODV-embedded occlusion bodies were significantly decreased. Subsequently, the highly conserved amino acid residues 2-25 and 64-88 of Ac78, which are homologous to an oxidoreductase and cytochrome c oxidase, respectively, were demonstrated to play a crucial role in the morphogenesis of multiple nucleocapsid-enveloped ODV. Immunoblot analysis found that Ac78 was an ODV envelope-associated protein. Consistently, amino acid residues 56-93 of Ac78 were identified as an inner nuclear membrane sorting motif, which may direct the localization of Ac78 to the ODV envelope. In vivo infectivity assays showed that the occlusion bodies of vAc78KO Were unable to establish primary infection in the midgut of Trichoplusia ni larvae. Taken together, our results suggest that ac78 plays an important role in BV production and proper multiple nucleocapsid-enveloped ODV formation, as well as AcMNPV primary infection in vivo. (C) 2014 Elsevier B.V. All rights reserved.

苜蓿银纹夜蛾核型多角体病毒（AcMNPV）的ac78基因是杆状病毒的核心基因之一。近期研究表明，在AcMNPV的生命周期中，ac78对于出芽型病毒粒子（BV）的产生以及将包埋型病毒粒子（ODV）嵌入包涵体是必不可少的。在此，我们报道本研究中构建的ac78基因敲除的AcMNPV（vAc78KO）具有与先前研究中所描述的不同的表型。通过滴度测定、免疫印迹分析和空斑试验检测到少量有感染性的BV，这表明ac78对于BV的形成并非必不可少。电子显微镜观察证实ac78的缺失不影响核衣壳组装和ODV的形成。然而，多核衣壳包裹的ODV以及嵌入ODV的包涵体的数量显著减少。随后，证明了Ac78中高度保守的氨基酸残基2 - 25和64 - 88分别与一种氧化还原酶和细胞色素c氧化酶同源，它们在多核衣壳包裹的ODV的形态发生中起关键作用。免疫印迹分析发现Ac78是一种与ODV包膜相关的蛋白质。一致地，Ac78的氨基酸残基56 - 93被鉴定为一种内核膜分选基序，它可能引导Ac78定位到ODV包膜。体内感染性试验表明，vAc78KO的包涵体无法在粉纹夜蛾幼虫的中肠建立初次感染。综上所述，我们的结果表明ac78在BV产生、正确的多核衣壳包裹的ODV形成以及AcMNPV体内初次感染中起重要作用。（C）2014爱思唯尔公司。保留所有权利。