Baculovirus FP25K Localization: Role of the Coiled-Coil Domain

Abstract

Two types of viruses are produced during the baculovirus life cycle: budded virus (BV) and occlusion-derived virus (ODV). A particular baculovirus protein, FP25K, is involved in the switch from BV to ODV production. Previously, FP25K from the model alphabaculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) was shown to traffic ODV envelope proteins. However, FP25K localization and the domains involved are inconclusive. Here we used a quantitative approach to study FP25K subcellular localization during infection using an AcMNPV bacmid virus that produces a functional AcMNPV FP25K-green fluorescent protein (GFP) fusion protein. During cell infection, FP25K-GFP localized primarily to the cytoplasm, particularly amorphous structures, with a small fraction being localized in the nucleus. To investigate the sequences involved in FP25K localization, an alignment of baculovirus FP25K sequences revealed that the N-terminal putative coiled-coil domain is present in all alphabaculoviruses but absent in betabaculoviruses. Structural prediction indicated a strong relatedness of AcMNPV FP25K to long interspersed element 1 (LINE-1) open reading frame 1 protein (ORF1p), which contains an N-terminal coiled-coil domain responsible for cytoplasmic retention. Point mutations and deletions of this domain lead to a change in AcMNPV FP25K localization from cytoplasmic to nuclear. The coiled-coil and C-terminal deletion viruses increased BV production. Furthermore, a betabaculovirus FP25K protein lacking this N-terminal coiled-coil domain localized predominantly to the nucleus and exhibited increased BV production. These data suggest that the acquisition of this N-terminal coiled-coil domain in FP25K is important for the evolution of alphabaculoviruses. Moreover, with the divergence of preocclusion nuclear membrane breakdown in betabaculoviruses and membrane integrity in alphabaculoviruses, this domain represents an alphabaculovirus adaptation for nuclear trafficking of occlusion-associated proteins. IMPORTANCEBaculovirus infection produces two forms of viruses: BV and ODV. Manufacturing of ODV involves trafficking of envelope proteins to the inner nuclear membrane, mediated partly through the FP25K protein. Since FP25K is present in alpha-, beta-, and gammabaculoviruses, it is uncertain if this trafficking function is conserved. In this study, we looked at alpha-and betabaculovirus FP25K trafficking by its localization. Alphabaculovirus FP25K localized primarily to the cytoplasm, whereas betabaculovirus FP25K localized to the nucleus. We found that an N-terminal coiled-coil domain present in all alphabaculovirus FP25K proteins, but absent in betabaculovirus FP25K, was critical for alphabaculovirus FP25K cytoplasmic localization. We believe that this represents an evolutionary process that partly led to the gain of function of this N-terminal coiled-coil domain in alphabaculovirus FP25K to aid in nuclear trafficking of occlusion-associated proteins. Due to betabaculovirus breakdown of the nuclear membrane be...

Identification and characterization of coiled-coil motifs across Autographa californica multiple nucleopolyhedrovirus genome

“…Among them, some (Ac13, BV/ODV-E26, IE2, GP64, F protein, Exon0, and Ac51) are reported to contain CC motifs previously ( Biswas et al., 2018 ; Qiu et al., 2019 ; Rohrmann, 2019a ; Singh et al., 1999 ). However, FP25K, which was also predicted to contain a CC motif ( Garretson et al., 2016 ), was not identified in this study, as its scores in two prediction software could not reach the cutoff score. 12.3% of AcMNPV proteins were predicted to contain CC structures ( Figure 1 A), comparable with the average 10% in eukaryotes ( Liu and Rost, 2001 ), instead of 4–5% in prokaryotes and archaea ( Liu and Rost, 2001 ), suggesting that large eukaryotic DNA viruses may be more similar to eukaryotes in the evolution of CC structures.…”

A novel method for construction of baculovirus bacmids

“…Given the advantages of bacmids in gene deletion and modi cation, more bacmid systems are urgently needed, especially for those baculoviruses with species-speci c genes and features, as the authors in the article called for the construction of a betabaculovirus bacmid system [30]. Owing to its ability to increase the proportions of recombinants by means of both puri cation and linearization, the novel method presented here serves as a highly competitive strategy for generating bacmids for baculoviruses that are capable of homologous recombination in cultured cells and provides a potential solution to bacmid generation for baculoviruses that do not produce high titers of viruses.…”

Improvement of protein production in baculovirus expression vector system by removing a total of 10 kb of nonessential fragments from Autographa californica multiple nucleopolyhedrovirus genome

“…In AcMNPV and BmNPV, it has been shown that individual deletion of Ac55 ( Bm44 ), Ac56 ( Bm45 ), Ac57 ( Bm46 ), Ac58/59 ( Bm47 ) and Ac60 ( Bm48 ) does not affect the virus reproduction ( Ono et al, 2012 ; Chen T. et al, 2021 ). For Ac61 , its gene product FP25K is involved in nuclear trafficking of occlusion-associated proteins ( Garretson et al, 2016 ). Studies have demonstrated that the gene knockout does not reduce BV production in BmNPV and AcMNPV ( Ono et al, 2012 ; Garretson et al, 2016 ; Yu et al, 2023 ), but results in a ‘few polyhedra phenotype’ which are influenced by the host insect cells ( Cheng et al, 2012 ).…”

“…For Ac61 , its gene product FP25K is involved in nuclear trafficking of occlusion-associated proteins ( Garretson et al, 2016 ). Studies have demonstrated that the gene knockout does not reduce BV production in BmNPV and AcMNPV ( Ono et al, 2012 ; Garretson et al, 2016 ; Yu et al, 2023 ), but results in a ‘few polyhedra phenotype’ which are influenced by the host insect cells ( Cheng et al, 2012 ). As virus mutants with single gene deletion in Ac55-61 are normal for BV production, it is still unclear why vAcΔ55-61 has reduced BV productivity.…”