Baculoviruses encode a conserved sulfhydryl oxidase, P33, which is necessary for budded virus (BV) production and multinucleocapsid occlusion-derived virus (ODV) formation. Here, the structural and functional relationship of P33 was revealed by X-ray crystallography, site-directed mutagenesis, and functional analysis. Based on crystallographic characterization and structural analysis, a series of P33 mutants within three conserved regions, i.e., the active site, the dimer interface, and the R127-E183 salt bridge, were constructed. In vitro experiments showed that mutations within the active site and dimer interface severely impaired the sulfhydryl oxidase activity of P33, while the mutations in the salt bridge had a relatively minor influence. Recombinant viruses containing mutated P33 were constructed and assayed in vivo. Except for the active-site mutant AXXA, all other mutants produced infectious BVs, although certain mutants had a decreased BV production. The active-site mutant H114A, the dimer interface mutant H227D, and the salt bridge mutant R127A-E183A were further analyzed by electron microscopy and bioassays. The occlusion bodies (OBs) of mutants H114A and R127A-E183A had a ragged surface and contained mostly ODVs with a single nucleocapsid. The OBs of all three mutants contained lower numbers of ODVs and had a significantly reduced oral infectivity in comparison to control virus. Crystallographic analyses further revealed that all three regions may coordinate with one another to achieve optimal function of P33. Taken together, our data revealed that all the three conserved regions are involved in P33 activity and are crucial for virus morphogenesis and peroral infectivity.

IMPORTANCE Sulfhydryl oxidase catalyzes disulfide bond formation of substrate proteins. P33, a baculovirus-encoded sulfhydryl oxidase, is different from other cellular and viral sulfhydryl oxidases, bearing unique features in tertiary and quaternary structure organizations. In this study, we found that three conserved regions, i.e., the active site, dimer interface, and the R127-E183 salt bridge, play important roles in the enzymatic activity and function of P33. Previous observations showed that deletion of p33 results in a total loss of budded virus (BV) production and in morphological changes in occlusion-derived virus (ODV). Our study revealed that certain P33 mutants lead to occlusion bodies (OBs) with a ragged surface, decreased embedded ODVs, and reduced oral infectivity. Interestingly, some P33 mutants with impaired ODV/OB still retained BV productivity, indicating that the impacts on BV and on ODV/OB are two distinctly different functions of P33, which are likely to be performed via different substrate proteins.

杆状病毒编码一种保守的巯基氧化酶 P33，这是出芽病毒 (BV) 生产和多核衣壳闭塞衍生病毒 (ODV) 形成所必需的。在这里，通过 X 射线晶体学、定点诱变和功能分析揭示了 P33 的结构和功能关系。基于晶体学表征和结构分析，构建了三个保守区域（即活性位点、二聚体界面和R127-E183 盐桥）内的一系列P33 突变体。体外实验表明，活性位点和二聚体界面内的突变严重损害了 P33 的巯基氧化酶活性，而盐桥中的突变影响相对较小。构建并在体内检测含有突变 P33 的重组病毒. 除活性位点突变体 AXXA 外，所有其他突变体均产生感染性 BV，尽管某些突变体的 BV 产量降低。通过电子显微镜和生物测定进一步分析了活性位点突变体 H114A、二聚体界面突变体 H227D 和盐桥突变体 R127A-E183A。突变体 H114A 和 R127A-E183A 的包涵体 (OB) 表面参差不齐，主要包含具有单个核衣壳的 ODV。与对照病毒相比，所有三种突变体的 OB 都含有较少数量的 ODV，并且口腔感染性显着降低。晶体学分析进一步表明，所有三个区域可能相互协调以实现 P33 的最佳功能。综合起来，

重要性巯基氧化酶催化底物蛋白的二硫键形成。P33 是一种杆状病毒编码的巯基氧化酶，不同于其他细胞和病毒的巯基氧化酶，在三级和四级结构组织中具有独特的特征。在这项研究中，我们发现三个保守区域，即活性位点、二聚体界面和R127-E183 盐桥，在P33 的酶活性和功能中起重要作用。先前的观察表明，删除p33导致芽生病毒 (BV) 生产的完全丧失和闭塞衍生病毒 (ODV) 的形态变化。我们的研究表明，某些 P33 突变体会​​导致表面粗糙的包涵体 (OB)、嵌入的 ODV 减少以及口腔传染性降低。有趣的是，一些 ODV/OB 受损的 P33 突变体仍保留 BV 生产力，表明对 BV 和 ODV/OB 的影响是 P33 的两种截然不同的功能，这可能是通过不同的底物蛋白进行的。