Li Zhuorui, Zhang Nan, Zhang Tao, Wang Zhiying, Li Jiang, Wang Manli, Hu Zhihong, Wang Xi
Key Laboratory of Virology and Biosafety, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China.
University of Chinese Academy of Sciences, Beijing, China.
J Virol. 2024 Jun 13;98(6):e0023524. doi: 10.1128/jvi.00235-24. Epub 2024 May 22.
Baculoviruses enter insect midgut epithelial cells via a set of occlusion-derived virion (ODV) envelope proteins called infectivity factors (PIFs). P74 of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV), which was the first identified PIF, is cleaved by an endogenous proteinase embedded within the occlusion body during infection, but the target site(s) and function of the cleavage have not yet been ascertained. Here, based on bioinformatics analyses, we report that cleavage was predicted at an arginine and lysine-rich region in the middle of P74. A series of recombinant viruses with site-directed mutants in this region of P74 were generated. R325 or R334 was identified as primary cleavage site. In addition, we showed that P74 is also cleaved by brush border membrane vesicles (BBMV) of the host insect at R325 or R334, instead of R195, R196, and R199, as previously reported. Simultaneous mutations in R195, R196, and R199 lead to instability of P74 during ODV release. Bioassays showed that mutations at both R325 and R334 significantly affected oral infectivity. Taken together, our data show that both R325 and R334 of AcMNPV P74 are the primary cleavage site for both occlusion body endogenous proteinase and BBMV proteinase during ODV release and are critical for oral infection.
Cleavage of viral envelope proteins is usually an important trigger for viral entry into host cells. Baculoviruses are insect-specific viruses that infect host insects via the oral route. P74, a infectivity factor of baculoviruses, is cleaved during viral entry. However, the function and precise cleavage sites of P74 remain unknown. In this study, we found that R325 or R334 between the N- and C-conserved domains of P74 was the primary cleavage site by proteinase either from the occlusion body or host midgut. The biological significance of cleavage seems to be the release of the potential fusion peptide at the N-terminus of the cleaved C-terminal P74. Our results shed light on the cleavage model of P74 and imply its role in membrane fusion in baculovirus infection.
杆状病毒通过一组称为感染因子（PIFs）的源自包涵体的病毒粒子（ODV）包膜蛋白进入昆虫中肠上皮细胞。苜蓿银纹夜蛾多粒包埋核型多角体病毒（AcMNPV）的P74是首个被鉴定出的PIF，在感染期间被包埋在包涵体内的一种内源性蛋白酶切割，但切割的靶位点和功能尚未确定。在此，基于生物信息学分析，我们报告预测P74中部富含精氨酸和赖氨酸的区域会发生切割。构建了一系列在P74该区域带有定点突变的重组病毒。确定R325或R334为主要切割位点。此外，我们发现P74在R325或R334处也会被宿主昆虫的刷状缘膜泡（BBMV）切割，而不是如先前报道的在R195、R196和R199处。R195、R196和R199同时发生突变会导致P74在ODV释放过程中不稳定。生物测定表明，R325和R334处的突变均显著影响经口感染性。综上所述，我们的数据表明，AcMNPV P74的R325和R334都是ODV释放过程中包涵体内源性蛋白酶和BBMV蛋白酶的主要切割位点，对经口感染至关重要。
病毒包膜蛋白的切割通常是病毒进入宿主细胞的重要触发因素。杆状病毒是昆虫特异性病毒，通过口感染宿主昆虫。P74是杆状病毒的一种感染因子，在病毒进入过程中会被切割。然而，P74的功能及精确切割位点仍不清楚。在本研究中，我们发现P74的N端和C端保守结构域之间的R325或R334是来自包涵体或宿主中肠的蛋白酶的主要切割位点。切割的生物学意义似乎是在切割后的C端P74的N端释放潜在的融合肽。我们的结果揭示了P74的切割模型，并暗示了其在杆状病毒感染中的膜融合作用。
