Autographa californica Multiple Nucleopolyhedrovirus Ac92 (ORF92, P33) Is Required for Budded Virus Production and Multiply Enveloped Occlusion-Derived Virus Formation

Abstract

The Autographa californica multiple nucleopolyhedrovirus orf92 (p33), ac92, is one of 31 genes carried in all sequenced baculovirus genomes, thus suggesting an essential function. Ac92 has homology to the family of flavin adenine dinucleotide-linked sulfhydryl oxidases and is related to the ERV/ALR family of sulfhydryl oxidases. The role of ac92 during virus replication is unknown. Ac92 was associated with the envelope of both budded and occlusion-derived virus (ODV). To investigate the role of Ac92 during virus replication, an ac92-knockout bacmid was generated through homologous recombination in Escherichia coli. Titration and plaque assays showed no virus spread in ac92-knockout bacmid DNA-transfected insect cells. Deletion of ac92 did not affect viral DNA replication. However, ac92-knockout bacmid DNAtransfected cells lacked multiply enveloped occlusion-derived nucleocapsids; instead, singly enveloped nucleocapsids were detected. To gain insight into the requirement for sulfhydryl oxidation during virus replication, a virus was constructed in which the Ac92 C 155 XXC 158 amino acids, important for sulfhydryl oxidase activity, were mutated to A 155 XXA 158 . The mutant virus exhibited a phenotype similar to that of the knockout virus, suggesting that the C-X-X-C motif was essential for sulfhydryl oxidase activity and responsible for the altered ODV phenotype.Baculoviruses infect insects and are characterized by a circular double-stranded DNA genome ranging from 80 to 180 kbp (10), which is packaged within a rod-shaped capsid and surrounded by an envelope. Two types of virions are produced during the baculovirus replication cycle: the budded virus (BV) and the occlusion-derived virus (ODV). Although the nucleocapsids are similar in the two, the envelopes differ in composition, reflecting their different functions during infection (4). The virions have distinct tissue tropism: ODVs infect midgut epithelial cells up to 10,000-fold more efficiently than do BVs, whereas BVs infect cultured cells 1,000-fold more efficiently than do ODVs (24).Alphabaculovirus and Betabaculovirus are two genera of Baculoviridae, based primarily on occlusion body morphology and phylogeny (11). Alphabaculoviruses have been divided into single nucleopolyhedrovirus (SNPV) and multiple nucleopolyhedrovirus (MNPV) occlusions. The M or S refers to whether the ODVs contain multiply or singly enveloped nucleocapsids, respectively (33). The MNPVs have been isolated only from Lepidoptera (23). It has been proposed that the M phenotype has an advantage over the S phenotype. Although the establishment of primary infections may be the same, the presence of multiple virions in MNPVs accelerated the onset of systemic infections (32, 33).The Autographa californica MNPV (AcMNPV) open reading frame 92 (orf92) (ac92, p33) is one of 31 core genes present in all sequenced baculovirus genomes (24). Previous studies showed that Ac92 and its homologs, Culex nigripalpus NPV orf14 and Helicoverpa armigera SNPV orf80, are associated with the ODV (3, 8, 18). Pre...

Selection and Characterization of Autographa californica Multiple Nucleopolyhedrovirus DNA Polymerase Mutations

“…However, to our surprise, and in contrast to WT ODVs, containing mostly multiple nucleocapsids with only a few single nucleocapsids, very few or no multiple-nucleocapsid ODVs were observed in occlusion bodies from the two dnapol mutants. Although the mechanisms of nucleocapsid envelopment in ODVs probably differ, the lack of multiple-nucleocapsid ODVs in the ABC rep double point mutant was similar to that of the nonviable Ac92 and viable Ac23 knockout viruses (53,54). Moreover, the drug-resistant viruses generated polyhedra which were larger than those generated by the WT rep virus.…”

Structure of a Baculovirus Sulfhydryl Oxidase, a Highly Divergent Member of the Erv Flavoenzyme Family

“…Therefore, it is difficult to propose putative sulfhydryl oxidase substrates in baculoviruses on the basis of information derived from NCLDVs. However, despite their distant relationship, common to both baculoviruses and NCLDVs is a severe drop in infectious virion production upon the elimination of sulfhydryl oxidase activity (25,31,39,48), suggesting a fundamental role for disulfide formation in both cases. The molecular defects that lead to a dramatic drop in the number of infectious baculovirus particles produced in the absence of Ac92 have not yet been reported.…”

“…Recently, an enzyme capable of catalyzing disulfide formation was identified in baculoviruses (28), a group of large double-stranded DNA viruses not classified as NCLDVs (21). The baculovirus sulfhydryl oxidase enzyme, one of a core group of conserved baculovirus gene products, is essential for virion assembly and thus for virus propagation (31,48), similarly to its counterparts in NCLDVs (25,39).…”

Baculovirus nucleocapsid aggregation (MNPV vs SNPV): an evolutionary strategy, or a product of replication conditions?

“…Also a knockout (KO) of ac43 appeared to increase the number of SNPVs in occlusion bodies, and co-infection of the ac43 KO virus with a virus encoding ac43 restored the predominance of the MNPV morphotype [24]. It has also been shown that deletion of ac92, a core gene encoding a sulfhydryl oxidase, results in virions that resemble the SNPV morphotype [25]. However, they are not infectious, indicating that they have a structural defect in one or more viral or possibly cellular proteins required for the production of viable virions.…”