Three Conserved Regions in Baculovirus Sulfhydryl Oxidase P33 Are Critical for Enzymatic Activity and Function

Abstract

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. 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 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. 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 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.

Baculovirus Per Os Infectivity Factor Complex: Components and Assembly

“…Antibodies. The rabbit polyclonal antibodies (pAbs) anti-AcPIF0, anti-AcPIF1, anti-AcPIF2, anti-AcPIF5, anti-AcODV-E66, anti-AcODV-E25, anti-AcGP41, anti-GAPDH, anti-lamin B, anti-AcGP64, anti-AcVP80, anti-AcP33, anti-Ac-polyhedrin, anti-AcVP39, anti-HaPIF0, anti-HaPIF1, anti-HaPIF2, and anti-HaPIF3 were produced in our lab as reported previously (36)(37)(38)(39). Other antibodies are reported here for the first time; these include the rabbit pAbs anti-AcPIF8 (amino acids [aa] 466 to 847), anti-AcEC43, anti-Ac49K, anti-AcVP80, anti-Ac66, anti-Ac81 (aa 15 to 164), anti-HaPIF5 (aa 1 to 318), and anti-HaPIF8 (aa 180 to 816) as well as the mouse pAbs anti-AcPIF1 (aa 24 to 530) and anti-AcODV-E18 (aa 20 to 62).…”

Systematic analysis of nuclear localization of Autographa californica multiple nucleopolyhedrovirus proteins

“…(1) P33 is one of the core genes in baculoviruses and is also present in nudiviruses and hytrosaviruses. It contains sulphhydryl oxidase activity, and is essential for BV formation and ODV morphogenesis [30]. TLP is a conserved gene in all lepidopteran baculoviruses (alphaand betabaculoviruses) [7].…”

Autographa californica Multiple Nucleopolyhedrovirusorf13is Required for Efficient Nuclear Egress of Nucleocapsids

“…OBs were purified from larvae by differential centrifugation according to the method described by Gross et al (50) and observed by TEM (Hitachi Co., Ltd., Tokyo, Japan) and SEM (HITACHII SU-8010; Tokyo, Japan) as described previously (51). To observe ODVs embedded within OBs, 10 μL of OB suspension (10 OBs/mL) were loaded onto a copper grid for min.…”