The African Swine Fever Virus Virion Membrane Protein pE248R Is Required for Virus Infectivity and an Early Postentry Event

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...

Section: Discussionmentioning

confidence: 99%

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

Passarelli

2010

“…These proteins are associated with the viral outer membranes and have important roles in the virion assembly process. Examples include the L1R protein in poxviruses (36) and pE248R in ASFV (37). Baculoviruses are morphologically and compositionally distinct from NCLDVs, such that clear counterparts to the NCLDV structural proteins are not evident in baculoviruses (21).…”

Section: Discussionmentioning

confidence: 99%

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

Hakim

Mandelbaum

Fass

2011

Genomes of nucleocytoplasmic large DNA viruses (NCLDVs) encode enzymes that catalyze the formation of disulfide bonds between cysteine amino acid residues in proteins, a function essential for the proper assembly and propagation of NCLDV virions. Recently, a catalyst of disulfide formation was identified in baculoviruses, a group of large double-stranded DNA viruses considered phylogenetically distinct from NCLDVs. The NCLDV and baculovirus disulfide catalysts are flavin adenine dinucleotide (FAD)-binding sulfhydryl oxidases related to the cellular Erv enzyme family, but the baculovirus enzyme, the product of the Ac92 gene in Autographa californica multiple nucleopolyhedrovirus (AcMNPV), is highly divergent at the amino acid sequence level. The crystal structure of the Ac92 protein presented here shows a configuration of the active-site cysteine residues and bound cofactor similar to that observed in other Erv sulfhydryl oxidases. However, Ac92 has a complex quaternary structural arrangement not previously seen in cellular or viral enzymes of this family. This novel assembly comprises a dimer of pseudodimers with a striking 40-degree kink in the interface helix between subunits. The diversification of the Erv sulfhydryl oxidase enzymes in large double-stranded DNA viruses exemplifies the extreme degree to which these viruses can push the boundaries of protein family folds.In contrast to proteins that traverse the secretory pathway, cytosolic and nuclear proteins in mesophilic organisms rarely evolve to contain structural disulfide bonds. Some exceptions to this generalization are structural proteins encoded by nucleocytoplasmic large DNA viruses (NCLDVs), which do contain disulfides despite folding in an environment that typically is reducing (16). To promote disulfide formation in the cytosol or nucleus, NCLDVs (such as poxviruses, mimivirus, African swine fever virus [ASFV], iridoviruses, phycodnaviruses, and others) encode catalysts of disulfide formation (38, 40) similarly to cellular enzymes of the Erv (for essential for respiration and viability) family. The cellular Erv enzymes promote oxidative folding in the mitochondrial intermembrane space (30) and certain other biological settings (14, 41). 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).The sulfhydryl oxidase encoded by the Ac92 gene in the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) contains amino acid sequence features shared by other cellular and viral sulfhydryl oxidases (28). In particular, a CXXC motif at the amino terminus of a predicted helix corresponds to the active-site disulfide. A motif consisting of a tryptophan, three histidine, and two asparagine amino acid...

“…Importantly, core delivery depends on viral protein pE248R, an N-myristoylated transmembrane protein located at the inner membrane (18). With use of a conditional lethal mutant, it has been shown that virus assembly and egress occur normally in the absence of pE248R expression.…”

Section: Membrane Fusion and Core Deliverymentioning

confidence: 99%

“…With use of a conditional lethal mutant, it has been shown that virus assembly and egress occur normally in the absence of pE248R expression. However, the pE248R-defective virus progeny is noninfectious due to a postentry blockage (18). When analyzed at the electron microscope level, incoming pE248R-defective particles become disrupted as normal virions but fail to penetrate into the cytosol and accumulate in lysosomes (12).…”

Section: Membrane Fusion and Core Deliverymentioning

confidence: 99%

African Swine Fever Virus Gets Undressed: New Insights on the Entry Pathway

Andrés

2017

African swine fever virus (ASFV) is a large, multienveloped DNA virus composed of a genome-containing core successively wrapped by an inner lipid envelope, an icosahedral protein capsid, and an outer lipid envelope. In keeping with this structural complexity, recent studies have revealed an intricate entry program. This Gem highlights how ASFV uses two alternative pathways, macropinocytosis and clathrin-mediated endocytosis, to enter into the host macrophage and how the endocytosed particles undergo a stepwise, low pH-driven disassembly leading to inner envelope fusion and core delivery in the cytoplasm.