The ha72 Core Gene of Baculovirus Is Essential for Budded Virus Production and Occlusion-Derived Virus Embedding, and Amino Acid K22 Plays an Important Role in Its Function

Abstract

b ha72 of Helicoverpa armigera nucleopolyhedrovirus (a homologue of ac78) was identified as a conserved late baculovirus gene and characterized. HA72 localizes in the intranuclear ring zone. By generating mutants, we showed that HA72 is essential for budded virus (BD) production and occlusion-derived virus (ODV) embedding. HA72 also interacted with P33, a baculoviral sulfhydryl oxidase. A point mutation of amino acid 22 from lysine to glutamic acid curtailed BV production and precluded ODV occlusion as well as interaction with P33. Baculoviruses are pathogens that infect insects from the orders Lepidoptera, Diptera, and Hymenoptera (1). To date, 60 baculovirus genomes have been fully sequenced, and all of them contain the 37 core genes (2) that play critical roles in baculovirus infection. ac78 is a newly identified baculovirus core gene (2). A previous study showed that deletion of the homologue of ac78 (bm64) in Bombyx mori nucleopolyhedrovirus (BmNPV) prevented the production of infectious budded virus (BV) (3). Its homologue in Helicoverpa armigera NPV (HA72) was shown to be a component of the occlusion-derived virus (ODV) envelope (4). However, its role in the viral life cycle is still unclear, and the present study was undertaken to provide initial biochemical, microscopic, and genetic characterizations of HA72.HA72 contains an IPLKL motif and a putative fumarate reductase flavoprotein C-terminal motif. Bioinformatics analysis showed that HA72 has two recognized motifs ( Fig. 1A): an IPLKL motif at the N terminus, required for a peroxisome-targeting signal in plants (5), and a putative fumarate reductase flavoprotein (FRF) C-terminal motif that has been implicated in electron transfer in the oxidation-reduction process (6, 7). The IPLKL motif is highly conserved in all baculoviral HA72 homologues, while the FRF-C is conserved at a low amino acid identity in all of the baculoviruses ( Fig. 1A and B).Time course analyses of transcription and expression suggest that ha72 is a late gene. Reverse transcription-PCR (RT-PCR) and Western blotting were performed to assess the temporal transcription and expression of ha72. The data summarized in Fig. 1C show that transcription was first detected at 18 hours postinfection (hpi). A rabbit polyconal antibody against the full length of HA72 was used for Western blotting, and a specific immunoreactive band was initially detected at 36 hpi, indicating that ha72 is a late gene. The gene-specific primer ha72up-R (Table 1) was used for 5=-random amplification of cDNA ends (5=-RACE) and, consistent with the above data, sequencing of the 5=-RACE products showed that expression of the ha72 transcript was initiated from the first A of a late gene transcriptional start site motif, TTAAG at 87 nucleotides upstream of the predicted start codon. For the 3=-RACE analysis, an oligo(dT) 15 three-site adaptor primer was used for generating cDNA, and subsequent PCR amplification was performed by using a three-site adaptor primer and an ha72-specific forward primer (Table 1). The 3=-RACE...

Bombyx mori nucleopolyhedrovirus (BmNPV) Bm64 is required for BV production and per os infection

“…A recent study demonstrated that ac78 was required for nucleocapsid egress from the nucleus [ 12 ]. Many proteins are localized to the ring zone of infected nuclei, such as Ac76 [ 29 ], P33 [ 13 ], Ac93 [ 19 ], and E25 [ 30 ]. The ring zone is very important for nucleocapsid envelopment and egress from the nucleus.…”

“…HA72 was demonstrated to be required for BV production and ODV embedding. Moreover, the IPLKL motif at the N terminus was shown to play an important role in its function [ 13 ]. More recently, Li et al found that Ac78 was not essential for BV production and ODV formation [ 11 ], which was a contradictory result.…”

The Trichoplusia ni single nucleopolyhedrovirus tn79 gene encodes a functional sulfhydryl oxidase enzyme that is able to support the replication of Autographa californica multiple nucleopolyhedrovirus lacking the sulfhydryl oxidase ac92 gene

“…It is not clear if abnormal nucleocapsids cannot be embedded because defective or missing structural proteins affect nucleocapsid morphology, are necessary to interact with other factors to mediate the embedding process, or provide nucleocapsid stability. Ac92 has been reported to interact with the human and insect p53 (Prikhod’ko et al, 1999; Wu et al, 2013) and HA72 (Ac78 ortholog), a Heliothis armiguera NPV factor (HearNPV) with a putative fumarate reductase flavoprotein motif (Huang et al, 2014). However, whether these interactions have functional significance has not been demonstrated.…”

“…Deletion of ha72 , the ac78 ortholog, and ac92 result in BV production and occlusion defects (Huang et al, 2014; Nie et al, 2011; Tao et al, 2013a; Wu and Passarelli, 2010). In addition, P33 (HearNPV Ac92 ortholog) and HA72 localize to the intranuclear ring zone of infected cells (Huang et al, 2014; Nie et al, 2011).…”

“…In addition, P33 (HearNPV Ac92 ortholog) and HA72 localize to the intranuclear ring zone of infected cells (Huang et al, 2014; Nie et al, 2011). Thus, it is possible that Ac92 (P33) and HA72 (and possibly Ac78) work in the same redox pathway.…”