The Trichoplusia Ni Single Nucleopolyhedrovirus Tn79 Gene

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Virology 460-461 (2014) 207–216

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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

Stian A. Clem, Wenbi Wu, A. Lorena Passarelli n

Molecular, Cellular, and Developmental Biology Program, Division of Biology, Kansas State University, Manhattan, KS 66506-4901, USA

article info abstract

Article history: The Autographa californica multiple nucleopolyhedrovirus ac92 is a conserved baculovirus gene with Received 11 March 2014 homology to flavin adenine dinucleotide-linked sulfhydryl oxidases. Its product, Ac92, is a functional Returned to author for revisions sulfhydryl oxidase. Deletion of ac92 results in almost negligible levels of budded virus (BV) production, 31 March 2014 defects in occlusion-derived virus (ODV) co-envelopment and their inefficient incorporation into Accepted 6 May 2014 occlusion bodies. To determine the role of sulfhydryl oxidation in the production of BV, envelopment Available online 13 June 2014 of nucleocapsids, and nucleocapsid incorporation into occlusion bodies, the Trichoplusia ni single Keywords: nucleopolyhedrovirus ortholog, tn79, was substituted for ac92. Tn79 was found to be an active sulfhydryl Baculovirus oxidase that substituted for Ac92, resulting in the production of infectious BV, albeit about 10-fold less Sulfhydryl oxidase than an ac92-containing virus. Tn79 rescued defects in ODV morphogenesis caused by a lack of ac92. ac92 Active Tn79 sulfhydryl oxidase activity is required for efficient BV production, ODV envelopment, and tn79 their subsequent incorporation into occlusion bodies in the absence of ac92. & 2014 Elsevier Inc. All rights reserved.

Introduction Besides baculoviruses, these enzymes are also encoded by nucleo- cytoplasmic large DNA viruses (NCLDV) (Hakim and Fass, 2010), Cellular sulfhydryl oxidases catalyze intra- and inter-protein large DNA-containing viruses that infect eukaryotes and have disulfide bridges using free sulfhydryl groups in cysteine amino cytoplasmic and nuclear replication phases (Koonin and Yutin, acids. During sulfhydryl oxidation, electrons are lost from cysteine 2012). The sulfhydryl oxidase and/or interacting redox pathway thiol groups and transferred to the acceptor molecule, molecular proteins of the vaccinia virus (Poxviridae)(Senkevich et al., 2002) oxygen. A metal or a flavin serves as an intermediate in the and African swine fever virus (Asfaviridae)(Hakim and Fass, 2009; transfer of electrons. Disulfide bridges are important for protein Rodriguez et al., 2006) have been characterized. In addition, the stability, folding, and the catalytic activity of some enzymes. structure of a sulfhydryl oxidase from the NCLDV mimivirus has Disulfide bonds are present in the extracellular domains of been derived (Hakim et al., 2012). The sulfhydryl oxidases of the secreted and membrane proteins, and these bonds are formed in baculoviruses Autographa californica M nucleopolyhedrovirus intracellular organelle compartments. In the cytoplasm, disulfide (AcMNPV), Ac92 (P33), and Bombyx mori NPV, Bm75, have also reductases maintain proteins in a reduced state. Sulfhydryl been characterized structurally (Hakim et al., 2011; Hou et al., oxidases have been classified into four families, ERV/ALR (essential 2012) and functionally (Hou et al., 2012; Nie et al., 2011; Wu and for respiration and vegetative growth/augmenter of liver regen- Passarelli, 2010). eration), QSOX (quiescin sulfhydryl oxidase), Ero (endoplasmic Ac92 was shown to be a flavin adenine dinucleotide (FAD)- reticulum oxidase), and secreted fungal sulfhydryl oxidases. Sulf- binding sulfhydryl oxidase related to the EVR/ALR family of hydryl oxidases have been identified in prokaryotes, eukaryotes, sulfhydryl oxidases (Long et al., 2009). It has conserved motifs and certain viruses of eukaryotes [reviewed in (Faccio et al., 2011)]. found in cellular sulfhydryl oxidases, including a dicysteine motif

All baculovirus genomes sequenced to date encode a sulfhydryl in the sequence CX2C (where X is any amino acid), which is oxidase homolog (Garavaglia et al., 2012; Herniou et al., 2003). essential for function (Wu and Passarelli, 2010), and a ﬂavin binding domain comprised of eight key amino acids (Long et al., 2009). In the absence of ac92 or in the presence of a mutation n Corresponding author. Tel.: þ1 785 532 3195. in the dicysteine motif, budded virus (BV) production was E-mail address: [email protected] (A. Lorena Passarelli). nearly abolished (Nie et al., 2011; Wu and Passarelli, 2010) and

http://dx.doi.org/10.1016/j.virol.2014.05.006 0042-6822/& 2014 Elsevier Inc. All rights reserved. 208 S.A. Clem et al. / Virology 460-461 (2014) 207–216 nucleocapsids in the nucleus, which are normally enveloped in we cloned the TnSNPV ac92 ortholog, tn79, and tested its func- bundles, were enveloped as single nucleocapsids (Wu and tionality and ability to rescue an ac92 mutant bacmid. Passarelli, 2010). However, viral genome biosynthesis and gene expression at different phases appeared unaffected (Nie et al., 2011; Wu and Passarelli, 2010). Conservation of baculovirus sulfhydryl oxidase genes During AcMNPV replication, two virus forms are produced. The BV is synthesized at late times post-infection (p.i.) of permissive Members of the Evr-like sulfhydryl oxidase family contain a cells as it buds through the cell membrane, becoming available to CX C active center sequence motif within the sequence G-X -W- infect neighboring cells. The occlusion-derived virus (ODV) is 2 3 X -H-X -F/Y-X -P-C-X -C-X -H-N-X -N (where X is any amino produced at very late times p.i. in the nucleus of infected cells. It 3 5 23 2 N 2 acid and the subscripted number or letter indicate the number of is thought to acquire its membrane from the inner nuclear membrane residues between amino acids or a variable number of amino or intranuclear microvesicles (Hong et al., 1997) where several acids, respectively). This sequence is in close proximity to the FAD nucleocapsids stack together and are then co-enveloped. This pheno- site, where the FAD cofactor is tethered (Fass, 2008). Ac92 and type led to the name “multiple” (M) for AcMNPV and other NPVs, in Tn79 contain this motif with invariant dicysteines (Fig. 1). contrast to “single” (S)NPVs,wheresinglenucleocapsidsareenvel- An alignment of sulfhydryl oxidase genes from all baculoviruses oped. Enveloped nucleocapsids are then embedded into an occlusion sequenced to date shows that all orthologs have the CX Cconserved body, which is mainly composed of the protein polyhedrin. During 2 motif and most of the sequence encompassing the motif. The amino infection of the insect host, the embedded ODV is protected in the acids in boldface in the sequence above and an additional histidine environment and available to infect new hosts that consume the nexttothedicysteinemotifarepresentinthebaculovirussulfhydryl occlusionbodybyfeeding. oxidase predicted sequences (Supplementary figure and (Long et al., Since the absence of sulfhydryl oxidase activity in AcMNPV 2009)). The importance of this additional histidine is not known. Only resulted in the formation of single nucleocapsids enveloped in the one other cysteine upstream of the dicysteine motif is present in most nucleus of cells, we hypothesized that rescuing the ac92 mutation baculovirus sulfhydryl oxidases (Supplementary figure), including with a sulfhydryl oxidase ortholog from an SNPV would result in Ac92. Members of the genera Deltabaculovirus and Gammabaculovirus an SNPV-like phenotype. To this end, we replaced ac92 with the and six other baculovirus sulfhydryl oxidases, including Tn79, lack this sulfhydryl oxidase from Trichoplusia ni SNPV, tn79. An AcMNPV additional cysteine (Supplementary figure). In contrast, additional recombinant bacmid carrying tn79 instead of ac92 was able to dicysteines involved in electron transfer are present upstream or produce infectious BV, although less efficiently than a bacmid downstream of the CX CinotherEvr-likeenzymes(Fass, 2008). Since containing ac92, and ODV were co-enveloped, indicating that 2 the significance of other non-active center cysteines is not clear, we sulfhydryl oxidase by itself did not determine the envelopment cannot make general inferences at this time, but a number of other phenotype of ODV nucleocapsids of the MNPV or SNPV type. viral sulfhydryl oxidases do not encode these additional dicysteines (Fass, 2008). To address evolutionary relationships among sulfhydryl oxi- Results dases of MNPV, SNPV, and granuloviruses, we derived a non- rooted phylogenetic tree (Fig. 2). The homologs from the four Viruses with a deletion in ac92 or cysteine point mutations in baculovirus genera group accordingly, consistent with sulfhydryl the active sulfhydryl oxidase motif resulted in two obvious oxidases being present in the baculovirus lineage prior to the phenotypes in Sf9 cells. The first phenotype was almost no divergence of these groups. detectable production of infectious BV (Nie et al., 2011; Wu and Passarelli, 2010). The second phenotype exhibited occluded and pre-occluded virions that contained single nucleocapsids, remi- niscent of the SNPV phenotype (Wu and Passarelli, 2010). Since Construction of an AcMNPV-based bacmid encoding TnSNPV tn79 these processes occur at late times, the associated phenotypes in ac92-deficient viruses are consistent with the expression of ac92 at To determine if the sulfhydryl oxidase from an SNPV, specifi- late times and the normal onset and levels of viral DNA synthesis cally, the TnSNPV tn79, was able to substitute for that of an MNPV, observed with these mutants (Nie et al., 2011; Wu and Passarelli, we introduced an HA-tagged tn79 driven by the ac92 promoter 2010). Given the phenotypic switch from an MNPV to an SNPV-like into a previously characterized AcMNPV bacmid lacking ac92, phenotype in the absence of ac92, we considered the possibility Ac92KO (Wu and Passarelli, 2010), to construct Ac92KO-Tn79HA- that the sulfhydryl oxidase enzymes of MNPV and SNPV, either PG (Fig. 3A). Insertion of tn79 into the correct site was confirmed directly or through their interaction with other viral or host by PCR (results not shown). Two similar bacmid constructs in an proteins, may confer nucleocapsid envelopment differences. Thus, Ac92KO backbone were used as controls, Ac92HARep-PG and

Fig. 1. Comparison of the AcMNPV Ac92 and the TnSNPV Tn79 predicted protein sequences. Alignment of Ac92 and Tn79 was made using ClustalX and edited in Genedoc.

The conserved baculovirus amino acid motif W-X3-H-X37 41-C-X2-C-H-X5572-H-N-X2-N (Long et al., 2009), which contains a CX2C motif at the active site of sulfhydryl oxidases, is shown under the alignment. The numbers above and at the end are amino acid numbers. The asterisks above the sequences were placed every twenty amino acids. S.A. Clem et al. / Virology 460-461 (2014) 207–216 209

86 AcMNPV 86 RoMNPV 80 PlxyMNPV 99 BmNPV MaviNPV 31 ThorNPV EppoNPV 46 87 CfMNPV-DEF 99 AgNPV HycuNPV OpMNPV 100 69 ChroNPV 47 99 ChocNPV 99 CfMNPV PhcyNPV 100 ApNPV 98 100 LyxyMNPV LdMNPV 100 AdorNPV AdhoNPV 24 100 LeseNPV SlNPV Alphabaculovirus 35 76 HaNPV-G4 73 HzSNPV 100 HaSNPV 100 TnSNPV ChchSNPV 34 28 100 ChchNPV 32 HespNPV OlNPV 61 EupsNPV 93 ApciNPV 31 37 EoNPV 50 43 ClbiNPV 79 HaMNPV 99 MabrMNPV 100 MacoNPV-B MacoNPV-A 94 AgseNPV 100 AgipMNPV 64 SfMNPV 95 SlNPV-II 99 SeMNPV 43 XecnGV 100 HaGV 20 PsunGV SlGV AdorGV 99 PhopGV 99 28 CpGV Betabaculovirus 26 CrleGV 14 25 ChocGV ClanGV 37 AgseGV PlxyGV 8 34 PrGV 28 EpapGV NeseNPV Gammabaculovirus 100 NeabNPV 100 NeleNPV CuniNPV Deltabaculovirus

0.1

Fig. 2. Phylogenetic analysis of Ac92 homologs. Sixty-one predicted baculovirus protein sequences with homology to Ac92 were aligned with ClustalX and a non-rooted phylogenetic tree was derived using MEGA 5.0.2 with 1000 bootstrap replicates. Numbers at the nodes indicate bootstrap scores for the NJ analysis. Virus abbreviations are as follows: AcMNPV: Autographa californica multiple nucleopolyhedrovirus (MNPV); AdhoNPV: Adoxophyes honmai NPV; AdorGV: Adoxophyes orana granulovirus (GV); AdorNPV: Adoxophyes orana NPV; AgipMNPV: Agrotis ipsilon MNPV; AgNPV: Anticarsia gemmatalis NPV; AgseGV: Agrotis segetum GV; AgseNPV: Agrotis segetum NPV; AnbiGV: Andraca bipunctata GV; ApciNPV: Apocheima cinerarium NPV; ApNPV: Antheraea pernyi NPV; MabrMNPV: Mamestra brassicae MNPV; BmNPV: Bombyx mori NPV; CfMNPV: Choristoneura fumiferana MNPV; CfMNPV-DEF: Choristoneura fumiferana DEF MNPV; ChchNPV: Chrysodeixis chalcites NPV; ChchSNPV: Chrysodeixis chalcites single NPV; ChocGV: Choristoneura occidentalis GV; ChocNPV: Choristoneura occidentalis NPV; ChroNPV: Choristoneura rosaceana NPV; ClanGV: Clostera anachoreta GV; ClbiNPV: Clanis bilineata NPV; CpGV: Cydia pomonella GV; CrleGV: Cryptophlebia leucotreta GV; CuniNPV: Culex nigripalpus NPV; EoNPV: Eotropis obliqua NPV; EpapGV: Epinotia aporema GV; EppoNPV: Epiphyas postvittana NPV; EupsNPV: Euproctis pseudoconspersa NPV; HaGV: Helicoverpa armigera GV; HaMNPV: Helicoverpa armigera MNPV; HaNPV- G4: Helicoverpa armigera NPV G4; HaSNPV: Helicoverpa armigera NPV NNg1; HespNPV: Hemileuca sp. NPV; HycuNPV: Hyphantria cunea NPV; HzSNPV: Helicoverpa zea single NPV; LdMNPV: Lymantria dispar MNPV; LeseNPV: Leucania separata NPV; LyxyMNPV: Lymantria xylina MNPV; MabrMNPV: Mamestra brassicae MNPV; MacoNPV-A: Mamestra conﬁgurata NPV-A; MacoNPV-B: Mamestra conﬁgurata NPV-B; MaviMNPV: Maruca vitrata MNPV; NeabNPV: Neodiprion abietis NPV; NeleNPV: Neodiprion lecontei NPV; NeseNPV: Neodiprion sertifer NPV; OlNPV: Orgyia leucostigma NPV; OpMNPV: Orgyia pseudotsugata MNPV; PhcyNPV: Philosamia cynthia ricini NPV; PhopGV: Phthorimaea operculella GV; PlxyGV: Plutella xylostella GV; PlxyMNPV: Plutella xylostella MNPV; PrGV: Pieris rapae GV; PsunGV: Pseudaletia unipuncta GV; RoMNPV: Rachiplusia ou MNPV; SeMNPV: Spodoptera exigua MNPV; SfMNPV: Spodoptera frugiperda MNPV; SlGV: Spodoptera litura GV; SlNPV: Spodoptera litura NPV; SlNPV-II: Spodoptera litura NPV II; ThorNPV: Thysanoplusia orichalcea NPV; TnSNPV: Trichoplusia ni single NPV (noted with an asterisk); XecnGV: Xestia c-nigrum GV.

Ac92HAM1-PG, where an HA-tagged wild-type ac92 or a mutant Replication of Ac92KO-Tn79HA-PG in Sf9 cells of ac92 carrying catalytic site dicysteine mutations, respectively, were expressed under ac92 promoter control after insertion into Bacmid DNA transfections revealed that Ac92KO-Tn79HA-PG the polyhedrin (polh) locus (Wu and Passarelli, 2010). All bacmids was able to produce infectious BV (data not shown). BV produced also carried polh driven by its promoter and enhanced green from Ac92KO-Tn79HA-PG or Ac92HARep-PG DNA transfections ﬂuorescent protein gene (egfp) under control of the AcMNPV ie-1 was titered and used to infect Sf9 cells at a low or high multiplicity promoter, and both were inserted at the polh locus (Fig. 3A). of infection (MOI; 0.01 and 5 PFU/cell, respectively), and cells 210 S.A. Clem et al. / Virology 460-461 (2014) 207–216

Cm Ac92KO-Tn79HA-PG Ac92HARep-PG MOI = 0.01MOI = 5 MOI = 0.01 MOI = 5 ac92 locus Ac92KO polh locus

Tn7R Tn7L 24 h p.i. Gentamicin

polh ie-1 Ac92KO-PG polh egfp polh ac92 ie-1 Ac92HARep-PG polh ac92HA egfp

polh ac92 ie-1 Ac92HAM1-PG polh ac92HAM1 egfp 96 h p.i. polh ac92 ie-1 Ac92KO-Tn79HA-PG polh tn79HA egfp

Fig. 3. Construction of recombinant bacmids and their replication in Sf9 cells. (A) The construction of Ac92KO-PG, Ac92HARep-PG, Ac92HAM1-PG, and Ac92KO-Tn79HA-PG is diagramed. C-terminally HA-tagged viral genes, polyhedrin (polh), and the enhanced green fluorescent protein gene (egfp) were inserted by Tn7-mediated transposition in the polh locus. Promoters and direction of transcription driving each gene is noted by the square arrows. (B) Infection of Sf9 cells with Ac92KO-Tn79HA-PG and Ac92HARep- PG at two different MOI as indicated at the top were visualized at the times p.i. shown on the left. The bottom row panels are bright fields corresponding to the visual fields in middle panels and showing occlusion body formation in infected cells. expressing egfp were monitored every 24 h p.i. Increasing num- Ac92HARep-PG. Tn368 cells were infected at an MOI of 5 PFU/cell and bers of eGFP-positive cells in both infections at 96 h p.i. indicated BV aliquots were collected throughout a time course of infection. that infectious BV was being produced (Fig. 3B). In addition, Similar to infections in Sf9 cells, BV production by Ac92KO-Tn79HA-PG infected cells produced occlusion bodies as shown at 96 h p.i., in Tn368 cells was reduced (Fig. 4D). indicating that the infection cycle proceeded into the very late stages of replication. Production of non-infectious virions in the absence of ac92 or tn79

Ac92KO-Tn79HA-PG infectious BV production kinetics in Sf9 When cells were transfected with Ac92KO-PG DNA, no infec- and Tn368 cells tious BV was detected (Nie et al., 2011; Wu and Passarelli, 2010), but it was not clear from those data whether non-infectious Although detection of increasing numbers of eGFP-positive virions were able to bud from the infected cells. Similarly, cells suggested production of infectious BV by Ac92KO-Tn79HA- Ac92KO-Tn79HA-PG produced reduced levels of infectious BV PG, we quantified their production throughout a time course of (Fig. 4), but simply performing growth curve experiments cannot infection. Ac92HARep-PG and Ac92KO-Tn79HA-PG were used to determine whether more non-infectious virions are budding from infect Sf9 cells at an MOI of 5 or 0.01 PFU/cell in case the the plasma membrane than from cells infected with Ac92HARep- phenotype was subtle and required several rounds of infection PG. Thus, we compared the total production of BV in Ac92HARep- to be more obvious. Aliquots were collected from the supernatant PG; Ac92KO-PG, a dicysteine mutant; Ac92HAM1-PG; and of infected cells from 0 to 96 h p.i. and titered in Sf9 cells. At both Ac92KO-Tn79HA-PG by detecting the presence of viral structural MOI, cells infected with the virus carrying tn79 exhibited about proteins and DNA in the supernatant of infected cell cultures. 10-fold lower infectious BV production starting after 24 h p.i. Cells were transfected with Ac92KO-PG, Ac92HAM1-PG, or (Fig. 4A and B). BV production remained less than that of Ac92HARep-PG DNA, aliquots from the supernatant were collected Ac92HARep-PG levels through 96 h p.i., indicating that there was between 48 and 96 h post-transfection (p.t.), and the BV structural not a delay in infectious BV production but a defect in producing proteins VP39 (major capsid protein) and GP64 (membrane fusion maximal levels. The production of Tn79 was lower than that of protein) were detected by immunoblotting. We detected GP64 and Ac92 (Fig. 4C), even though they are expressed from the same VP39 in all viral DNA-transfected cell supernatants (Fig. 5A), promoter. It is not known if protein or transcript stability is the indicating that the mutant viruses Ac92KO-PG and Ac92HAM1- same. This reduction may have resulted in reduced BV production PG, which lack a functional ac92, produced budded virions that in Ac92KO-Tn79HA-PG compared to Ac92HARep-PG. The timing of were not infectious. There was no significant contamination of BV production defects in Ac92KO-Tn79HA-PG-infected cells was viral proteins from ruptured cells in the supernatant, since VP39 consistent with the expression of Tn79 at late times p.i. (Fig. 4C) and GP64 are produced at high levels by 12 h p.i. (Blissard and and possibly a requirement for sulfhydryl oxidation during pro- Rohrmann, 1989; Jarvis and Garcia, 1994; Thiem and Miller, 1989), cesses that take place at late times. and some samples had low or undetectable levels of proteins at Since TnSNPV was originally isolated from the cabbage looper T. ni, 48 h p.t. (Fig. 5A). Detection of proteins was lower in samples from we considered that Tn79 may interact with a host-specificfactorfor both mutants than from Ac92HARep-PG, suggesting that less total optimal function; thus, we hypothesized that expression of Tn79 in BV (infectious and non-infectious) was produced from these Tn368 cells, which are derived from T. ni,wouldprovideabetter mutant viruses. Transfections with Ac92HARep-PG showed an context for activity and produce similar levels of infectious BV as increase in accumulated proteins at 48 and 96 h p.t., consistent S.A. Clem et al. / Virology 460-461 (2014) 207–216 211

Sf9 (MOI = 5) 9

7 mi 24 48 24 48 h p.i. 6 anti-IE-1 IE-1 (pfu/ml) 5 10 Ac92HARep-PG Ac92HA

log 4 anti-HA Ac92KO-Tn79HA-PG Tn79HA 3

2 0 24 48 72 96 Time (h p.i.)

Sf9 (MOI = 0.01) Tn368 (MOI = 5) 9 9 8 8 7 7 6 6 5 5 (pfu/ml) (pfu/ml) 4 10 4 10 3 Ac92HARep-PG log 3 Ac92HARep-PG log 2 Ac92KO-Tn79HA-PG Ac92KO-Tn79HA-PG 2 1 1 0 0 24 48 72 96 0 24 48 72 96 Tiime (h p.i.) Time (h p.i.)

Fig. 4. Viral replication kinetics. Sf9 cells (A and B) or Tn368 cells (D) were infected with Ac92HARep-PG or Ac92KO-Tn79HA-PG at an MOI of 5 (A and D) or 0.01 (B) and samples were titered at the indicated time points post infection by TCID50 endpoint dilution assay. The bars show standard errors of the mean from three independent experiments. (C) Expression of Tn79HA and Ac92HA (arrows) from Sf9 cells infected with Ac92KO-Tn79HA-PG or Ac92KO-Tn79HA-PG, respectively, was conﬁrmed by immunoblotting using anti-HA antibody. The upper portion of the blot was cut and processed to determine expression of the viral protein IE-1 as a control.

Ac92KO-PG Ac92HAM1-PG Ac92HARep-PG P = 0.8969 48 72 96 48 72 96 48 72 96 h p.t. 8

anti-GP64

anti-VP39

2 8 Genome copy number/PFU

48 h p.t. 6 0

72 h p.t.

4 96 h p.t. Ac92HARep-PG Log genome copy number Ac92KO-Tn79HA-PG 2 - + + + + + + + + + + DNaseI mi + AcWT-PG Ac92KO-PG Ac92HAM1-PG Ac92HARep-PG

Fig. 5. Production of non-infectious budded virions from Ac92KO-PG, Ac92HAM1-PG, Ac92HARep-PG, and Ac92KO-Tn79HA-PG. (A) At the indicated time points, supernatants containing budded virions were collected, passed through a 0.8 mm filter, and concentrated by centrifugation at 20,000g to pellet virus particles. Protein lysates from purified virions were analyzed by immunoblotting using anti-GP64 and anti-VP39 antibodies. (B) Q-PCR was performed to assess whether Ac92KO-PG and Ac92HAM1- PG produced non-infectious budded virus. Concentrated virus particles were treated with DNase I (þ) prior to DNA extraction to remove transfected or lysed cell DNA. DNA extracted from virus particles was used as a template for Q-PCR. AcWT-PG bacmid DNA was spiked in the supernatant of mock-infected cells (mi) and treated with DNase I before DNA extraction to control for DNA digestion. (C) Comparison of genome copy numbers per infectious unit between Ac92HARep-PG and Ac92KO-Tn79HA-PG by Q-PCR. Viral DNA was purified from the same amount of infectious budded virus and used as a template for Q-PCR. 212 S.A. Clem et al. / Virology 460-461 (2014) 207–216

70 20 40

25 DTT + buffer 10 DTT + Tn79 + buffer 15 Tn79 + buffer Thiol groups [nmol]

0 0 20 40 60 anti-HA Tn79HA Time (min)

Fig. 6. Sulfhydryl oxidase activity of HA-tagged Tn79. (A) His-tagged Tn79HA protein was expressed in E. coli, partially purified by affinity chromatography, resolved using SDS-PAGE, and stained with Coomassie blue (top panel). Expression of HA-tagged Tn79 was confirmed by immunoblotting using anti-HA antibody (bottom panel). (B) Sulfhydryl oxidase assays were carried out using purified Tn79HA and DTT as a substrate. with this virus being able to produce infectious BV that could was a functional sulfhydryl oxidase; a defect in enzymatic activity may infect additional cells (Fig. 5A). It is not clear why GP64 accumula- have manifested as a defect in BV production. We expressed an tion in Ac92HARep-PG-DNA-transfected cells was lower than that HA- and 6X histidine-tagged tn79 in Escherichia coli and partially of the mutant viruses at 48 h p.t., but this result was reproducible purified it by affinity chromatography (Fig. 6A). Recombinant Tn79 in independent experiments. In contrast, the levels of VP39 were was able to oxidize the thiol groups in dithiothreitol (DTT) (Fig. 6B), higher in Ac92HARep-PG at the same time point, making it indicating that it is an active sulfhydryl oxidase. We did not determine difficult to draw additional conclusions. More importantly, this specific activities of Tn79 and Ac92, since the enzymes were not experiment indicates that viruses with a defective ac92 produced purified to homogeneity; thus we cannot compare their activities. BV that was mostly non-infectious. Q-PCR was used to measure the amount of BV DNA from Effects of Tn79 on nucleocapsid envelopment in the nucleus supernatants of Ac92KO-PG-, Ac92HAM1-PG-, or Ac92HARep-PG- of infected cells DNA-transfected cells. DNase I treatment prior to BV DNA pur- ification was able to digest most of AcWT-PG DNA spiked into At very late times p.t. with Ac92KO-PG DNA, virions in the mock-infected samples (Fig. 5B, miþAcWT-PG lanes), indicating nucleus of cells contain single nucleocapsids and are poorly that viral DNA detected by Q-PCR from the transfected samples embedded in polyhedra (Wu and Passarelli, 2010). We concluded was from BV and not from transfected viral DNA or intracellular that sulfhydryl oxidation of cellular or viral proteins by Ac92 was viral DNA. BV genomes detected by Q-PCR increased with time in important for co-envelopment of nucleocapsids. Tn79 is encoded all samples, but it was more significant in samples from by a virus that produces ODV containing single nucleocapsids; Ac92HARep-PG-DNA-transfected cells, presumably due to the thus, we asked whether Tn79 would be able to substitute for Ac92 ability of Ac92HARep-PG to produce infectious BV that infect and produce co-enveloped nucleocapsids, or whether the resulting additional cells (Fig. 5B). Alternatively, we cannot rule out that ODV would have the SNPV-like phenotype. Examination of this increase in virion genome copies may also reflect a higher Ac92KO-Tn79HA-PG-transfected cells by transmission electron production of BV. Nevertheless, we concluded that Ac92KO-PG microscopy revealed the presence of co-enveloped nucleocapsids produced BV (Fig. 5A and B), but these were not infectious as in the nuclei (Fig. 7A) and embedded in polyhedra (Fig. 7B). previously reported (Nie et al., 2011; Wu and Passarelli, 2010). Consequently, it appears that sulfhydryl oxidase is not the sole Given that the ac92 mutant viruses produced non-infectious determinant for a specific nucleocapsid envelopment phenotype. BV, we considered that the decrease in infectious Ac92KO- We note that if Ac92KO-Tn79HA-PG would have produced single Tn79HA-PG BV production, as compared to that of Ac92HARep- nucleocapsid virions, we still could not conclude if tn79 was PG (Fig. 4), may also be due to the synthesis of an increased responsible for this phenotype, or just not able to fully comple- number of non-infectious particles. Thus, we performed Q-PCR on ment the ac92 mutant phenotype, since Ac92KO-PG also produces DNA extracted from the same amount of infectious Ac92KO- single nucleocapsid virions. However, the MNPV phenotype indi- 8 Tn79HA-PG and Ac92HARep-PG (1 10 PFU each) and calculated cates that expression of a functional sulfhydryl oxidase, even one the number of viral genome copies per PFU (Fig. 5C). In most from an SNPV, is sufficient to result in the MNPV phenotype experiments, we found that Ac92KO-Tn79HA-PG produced slightly during AcMNPV infection. We did not detect any obvious differ- more viral genome copies than Ac92HARep-PG per infectious unit, ences between Ac92KO-Tn79HA-PG and Ac92HARep-PG virions. but this difference was not statistically significant and does not explain the ten-fold difference in infectious BV production (Fig. 4). We concluded that the Ac92KO-Tn79HA-PG had a defect in BV Discussion production. The distinction between the SNPV and MNPV phenotypes provided Sulfhydryl oxidation activity of Tn79 a visual and simple method to classify NPVs in the past; however, with the advent of improved techniques for nucleotide sequencing and The sulfhydryl oxidase Tn79 only partially complemented the BV genome analysis, the availability of baculovirus genomes has exploded production defect in the ac92 deletion virus. We wondered if Tn79 and more accurate evolutionary relationships amongst sequenced S.A. Clem et al. / Virology 460-461 (2014) 207–216 213

Fig. 7. Transmission electron microscopy of Ac92KO-Tn79HA-PG-infected Sf9 cells at 48 h p.i. Co-enveloped nucleocapsids were observed in the ring zone of infected cells (A) and embedded in mature polyhedra (B). Bar: 500 nm. viruses have been revealed. The SNPV and MNPV distinctions are no emerging in which defects in ODV morphogenesis lead to defi- longer considered taxonomically relevant (Rohrmann, 2011), but ciencies in the process of embedding. Several of these implicated questions regarding what determines envelopment or co- genes (e.g., ac23, ac65, ac76, ac92 ac109, ac142, or their orthologs) envelopment of nucleocapsids destined to become ODV and their have been found to encode structural proteins (Braunagel et al., subsequent embedding into occlusionbodiesremainunanswered.The 2003; Deng et al., 2007; Fang et al., 2009; Hou et al., 2013; Hu SNPV/MNPV distinction is further complicated by observations that et al., 2010; Nie et al., 2011; Perera et al., 2007; Wang et al., 2010; viruses produce a mixture of single and multiple nucleocapsids per Wu and Passarelli, 2010), suggesting that abnormal nucleocapsid envelope (Ackermann and Smirnoff, 1983; Falcon and Hess, 1985; Feng structure can affect envelopment and/or embedding. It is not clear if etal.,2012;Rohrmann,2011;Yuetal.,2009), although one is usually abnormal nucleocapsids cannot beembeddedbecausedefectiveor predominant. Since sulfhydryl oxidation is not prevalent in the missing structural proteins affect nucleocapsid morphology, are reducing environment of nuclei, deleting ac92 should abolish sulfhy- necessary to interact with other factors to mediate the embedding dryl oxidation of proteins. Thus, since lack of sulfhydryl oxidation process, or provide nucleocapsid stability. Ac92 has been reported to results in a switch from multiple to single nucleocapsids per envelope, interact with the human and insect p53 (Prikhod'ko et al., 1999; Wu et we explored the function of the SNPV sulfhydryl oxidase, Tn79. We al., 2013) and HA72 (Ac78 ortholog), a Heliothis armiguera NPV factor caution that although the phenotype of ac92 mutants resembles that (HearNPV) with a putative fumarate reductase flavoprotein motif of SNPV, these mutant viruses cannot be considered bona fide SNPV, (Huang et al., 2014). However, whether these interactions have since there are also defects in occlusion and BV infectivity (Wu and functional significance has not been demonstrated. Passarelli, 2010). Deletion of ha72, the ac78 ortholog, and ac92 result in BV An analysis of available ac92 baculovirus homologs indicated that production and occlusion defects (Huang et al., 2014; Nie et al., thedicysteinemotifattheactivesitewasconserved(Supplementary 2011; Tao et al., 2013a; Wu and Passarelli, 2010). In addition, P33 figure). This motif is within the hallmark sequence of the Erv family of (HearNPV Ac92 ortholog) and HA72 localize to the intranuclear sulfhydryl oxidases, G-X3-W-X3-H-X5-F/Y-X23-P-C-X2-C-XN-H-N-X2-N, ring zone of infected cells (Huang et al., 2014; Nie et al., 2011). where the glycine allows space for the FAD isoalloxazine ring, a Thus, it is possible that Ac92 (P33) and HA72 (and possibly Ac78) tricyclic heteronuclear organic ring characteristic of flavins, to be work in the same redox pathway. positioned (Fass, 2008); however, this glycine is not conserved in The stabilization of capsids by disulfide bonds has been known baculovirus sulfhydryl oxidases (Long et al., 2009 and Supplementary to occur in several viruses for many years (Hare and Chan, 1968; figure). Two histidines downstream and upstream of the dicysteine Huang et al., 1972; Zweig et al., 1979). Treatment with reducing motif are also conserved and may participate in binding to the adenine agents resulted in dissociated and unstable virus particles. Studies of the FAD cofactor. These conserved sequence elements and the concluding that nucleocapsid disulfide bridges confer stability to ability of the Ac92 and Tn79 enzymes to oxidize thiol groups in DTT capsids has been more recently carried out in herpes simplex virus (Fig. 6 and Long et al., 2009; Wu and Passarelli, 2010)confirms their (Szczepaniak et al., 2011). In baculoviruses, it is possible that classification as sulfhydryl oxidases. disulfide bonds are necessary to provide structural stability to Mutations in MNPV genes, including ac92, have resulted in a VP39 or other nucleocapsid proteins in BV and/or ODV. higher number of single nucleocapsids enveloped and/or defects We found that the TnSNPV tn79 encodes a functional sulfhydryl in their ability to be embedded into polyhedra. This includes oxidase. The ac92 and tn79 predicted protein sequences are 51% mutations in the viral DNA polymerase gene (ac65) that confer identical. tn79 was able to complement ac92 defects, at least partially, drug resistance to aphidicolin or abacavir (Feng et al., 2012), the including BV and co-enveloped nucleocapsid production and subse- envelope fusion protein ac23 (Yu et al., 2009), the Spodoptera quent embedding into occlusion bodies. We do not know if the frugiperda MNPV sf9 (Simón et al., 2008), and ac43 (Tao et al., activity of Tn79 was not sufficient to fully complement BV production 2013b). Additionally, defects in ODV nucleocapsid envelopment in the absence of ac92 or if interactions with AcMNPV orthologs or have led to a block in occlusion (Alfonso et al., 2012; Hu et al., specific cellular factors were not as efficient, leading to partial 2010; Lehiy et al., 2013; McCarthy et al., 2008; Tian et al., 2009; complementation. Since Ac92KO-Tn79HA-PG produced about 10- Yuan et al., 2008) and defects in polyhedra morphology (Tao et al., fold less BV than Ac92HARep-PG, we have referred to this comple- 2013b). Although relatively few examples exist, a pattern is mentation as partial; however, we note that Ac92KO-Tn79HA-PG 214 S.A. Clem et al. / Virology 460-461 (2014) 207–216 produced up to 10,000-fold more infectious BV than viruses lacking Tn7951 (50-GTCGACATGATACCGTTGACACCACT-30, with SalI site ac92. Ac92KO-Tn79HA-PG-infected Tn368 cells also showed a defect underlined) and Tn7931 (50-TCTAGATTAGGCGTAATCTGGGACGTCG- in BV production. Even though Tn368 cells were derived from T. ni, TATGGGTATAGCTGTAACAATTTTTTGTATTC-30,withXbaIsiteunder- the host of TnSNPV, they may not be representative of all T. ni cells. lined). The PCR-amplified product containing tn79 was gel-purified The Mamestra configurata NPV ac92 ortholog, maco96,was and ligated into pCRII to generate T-tn79HA. The nucleotide substituted for ac92 (Nie et al., 2011). BV production in the sequence of the cloned HA-tagged tn79 was confirmed. The Tn79HA presence of maco96 was lower than that of a virus carrying ac92 fragment was digested from T-Tn79HA with SalI/XbaI, gel-purified, but higher than a virus lacking ac92, similar to the results and ligated to pFBPG-ac92P-pA, which was also digested with SalI/ presented with tn79 in this study. DNA synthesis accumulation XbaI to generate a final plasmid called pFBPG-ac92P-tn79HA. of the virus with maco96 was not as high as that of a virus with Electrocompetent E. coli DH10B cells containing the helper ac92. Also, Maco96 distribution in the nucleus was different to that plasmid pMON7124 and the Ac92KO bacmid were transformed of the parental virus (Nie et al., 2011). It was concluded from these with the donor plasmid pFBPG-ac92P-tn79HA to generate an experiments that distribution of Maco96 may affect full comple- Ac92KO-Tn79HA-PG recombinant bacmid. Bacmid DNA was iso- mentation in ac92-deficient viruses. We did not address the lated and transposition was confirmed by PCR. The verified localization of Tn79, but it is possible that altered localization Ac92KO-Tn79HA-PG recombinant bacmid was electroporated into within the cell affects its ability to interact with other factors to DH10B cells and screened for tetracycline sensitivity to ensure that function more efficiently. We are currently determining viral and the isolated bacmid was free of helper plasmid. Bacmid DNA was host substrates for viral sulfhydryl oxidases. Ac92KO-Tn79HA-PG extracted and purified with QIAGEN Large-Construct Kit and had a similar particle-to-PFU ratio to Ac92HARep-PG (Fig. 5C). quantified by optical density. Although it is possible that the defect in BV production (Fig. 4) may be due to a defect in viral DNA synthesis, this is not likely Transfections with bacmid DNA, virus infections, and viral growth since Ac92KO-PG has no apparent defects in viral DNA replication curve analyses (Nie et al., 2011; Wu and Passarelli, 2010). The small defect in Ac92KO-Tn79HA-PG replication may be in nucleocapsid assembly, Bacmid DNA was mixed with a non-commercial liposome nucleocapsid transport from the nucleus to the cytoplasm, or reagent and added to Sf9 cells as previously described (Crouch budding. Since tn79 was able to partially complement for ac92,it and Passarelli, 2002). Cells were incubated with the DNA/liposome provided a tool to define steps in which sulfhydryl oxidation was mixture at 27 1C for 5 h and then washed twice with TC-100 media. important during virus replication. Altogether, this heterologous TC-100 media containing 10% fetal bovine serum was then added to gene exchange was able to provide insight into the functions of transfected cells and incubation was continued at 27 1C. Super- sulfhydryl oxidation on the production of two virion types, BV and natant containing virus was collected at 96 h p.t. and the virus titer the morphology of virions destined to become ODV. It also adds to was determined by TCID50 endpoint dilution assays in Sf9 cells. our knowledge of the determinants for co-envelopment of multi- To generate viral growth curves, Sf9 or Tn368 cells were ple nucleocapsids produced in the nucleus of cells prior to their infected with the indicated virus at high (5 PFU/cell) or low localization into occlusion bodies. (0.01 PFU/cell) MOI as indicated. Virus MOI calculations were determined by titrations in either Sf9 or Tn368 cells, depending on the corresponding cell line being used for the experiment. Materials and methods Supernatants from the infected cells were collected at various time

points, and virus titers were determined by TCID50 endpoint Construction of recombinant virus dilution assays (O'Reilly et al., 1994).

The recombinant bacmids AcWT-PG, Ac92KO-PG, Ac92HARep- Immunoblotting PG, and Ac92HAM1-PG have been previously described (Wu and Passarelli, 2010). Briefly, Ac92KO-PG has a deletion in ac92, while Proteinlysatesweremixedwithequalvolumesof2Xprotein reintroduction of ac92 or a cysteine mutant of ac92 at the polh loading buffer (0.25 M Tris–HCl, pH 6.8, 4% SDS, 20% glycerol, 10% locus into an Ac92KO backbone resulted in Ac92HARep-PG and 2-mercaptoethanol and 0.02% bromophenol blue) and incubated at Ac92HAM1-PG, respectively. AcWT-PG does not have alterations at 100 1C for 5 min before resolving by SDS-PAGE. In some cases, the cell the ac92 locus. AcWT-PG, Ac92KO-PG, Ac92HARep-PG, and supernatants containing virions were filtered through a 0.8 μm filter Ac92HAM1-PG express egfp and polh. Ac92KO-Tn79HA-PG was to remove cell debris. Proteins were then transferred to a PVDF constructed to express tn79 under the ac92 promoter in an ac92- membrane (MILLIPORE) and probed with mouse monoclonal anti- knockout background. Previously, the ac92 promoter fragment HA antibody (Covance) or anti-GP64 (eBioscience), followed by was able to express ac92 in Ac92HARep-PG and complement ac92 incubation with horse radish peroxidase-conjugated secondary anti- defects in Ac92KO-PG (Wu and Passarelli, 2010). bodies (Sigma). Anti-VP39 was kindly provided by Dr. Kai Yang (Sun The ac92 promoter was amplified by PCR using two primers, Yat-sen University, China) and anti-IE-1 by Dr. Linda Guarino (Texas Ac92511 (50-GAATTCAATGCACATATGTCTTATAC-30, with EcoRI site A&M). Blots were developed using the SuperSignal West Pico Chemi- underlined) and Ac92311 (50-GTCGACGGCGACTAGCAAAACGATC- luminescent substrate (Pierce) and exposed to X-ray films. Immuno- GT-30, with SalI site underlined), and AcBAC DNA (Invitrogen) as a blots were performed from three independent infections and a template. PCR products were purified using the Qiagen Gel representative one is shown. Extraction Kit and ligated into pCRII (Invitrogen). The resulting plasmid, T-ac92P, was sequenced to verify that no errors were Genomic copy numbers in Ac92KO-PG, Ac92HAM1-PG, Ac92HARep- introduced by the polymerase. T-ac92P was digested with EcoRI/ PG and Ac92KO-Tn79HA-PG by Q-PCR SalI to cut out and gel-purify the ac92 promoter. The promoter was ligated to pFBPG-pA (Wu and Passarelli, 2010) and digested with To determine whether Ac92KO-PG produced non-infectious BV, the same enzymes to generate a donor plasmid, pFBPG-ac92P-pA. Sf9 cells (2 106 in 60-mm dishes) were transfected with 2 μg TnSNPV genomic DNA, kindly provided by David Theilmann Ac92KO-PG, Ac92HAM1-PG, or Ac92HARep-PG. Supernatants con- (Agriculture and Agri-Food Canada), was used as a template to taining BV were collected at 48, 72 and 96 h p.t., and BV were amplify tn79 with an HA epitope tag at the C terminus using primers purified and concentrated in 100 μl of sterile distilled water as S.A. Clem et al. / Virology 460-461 (2014) 207–216 215 described (O'Reilly et al., 1994). Prior to DNA extraction, super- This work was supported in part by the National Institutes of natants were treated with 2 units of DNase I (New England Health (NIH) Award 5RO1AI091972-3 to A.L.P. Biolabs) at 37 1C for 10 min to remove any remaining transfected This is contribution number 14-203-J from the Kansas Agricul- DNA and DNA from lysed cells. DNase I was inactivated by adding tural Experiment Station. EDTA and heating, according to the specifications of the manu- facturer. DNA from BV was then extracted as previously described (O'Reilly et al., 1994). AcWT-PG (Wu and Passarelli, 2010) bacmid Appendix A. Supporting information DNA (2 μg) was spiked into the supernatant of mock-infected Sf9 cells and treated with DNaseI before DNA extraction in order to Supplementary data associated with this article can be found in control for digestion of DNA and assess the levels of residual DNA. the online version at http://dx.doi.org/10.1016/j.virol.2014.05.006. DNA from BV was used as a template for Q-PCR. To compare the number of BV genome copies between Ac92HA- Rep-PG and Ac92KO-Tn79HA-PG, 1 108 PFU of infectious BV (based References on TCID50) of each virus was concentrated by centrifugation from four independent virus stocks and genomic DNA was purified from each Ackermann, H.W., Smirnoff, W.A., 1983. A morphological investigation of 23 stock as previously described (O'Reilly et al., 1994). Purified genomic baculoviruses. J. Invertebr. Pathol. 41, 269–280. Alfonso, V., Maroniche, G.A., Reca, S.R., Lopez, M.G., del Vas, M., Taboga, O., 2012. DNA was used as a template for Q-PCR as previously described (Wu AcMNPV core gene ac109 is required for budded virion transport to the nucleus and Passarelli, 2010)andrunintriplicate. and for occlusion of viral progeny. PLoS One 7, e46146. Blissard, G.W., Rohrmann, G.F., 1989. Location sequence transcriptional mapping fi and temporal expression of the gp64 envelope glycoprotein gene of the Orgyia Protein puri cation and sulfhydryl oxidase activity assays pseudotsugata multicapsid nuclear polyhedrosis virus. Virology 170, 537–555. Boyle, D.L., Takemoto, L., 1998. Finger-like projections of plasma membrane in the The E. coli expression vector pET28a-Tn79HA was constructed most senescent fiber cells of human lenses. Curr. Eye Res. 17, 1118–1123. Braunagel, S.C., Russell, W.K., Rosas-Acosta, G., Russell, D.H., Summers, M.D., 2003. to express a His-tagged Tn79 protein. Two primers, NdeI-Tn79F Determination of the protein composition of the occlusion-derived virus of 0 0 (5 -CATATGATACCGTTGACACCACT-3 , with NdeI restriction site Autographa californica nucleopolyhedrovirus. Proc. Natl. Acad. Sci. USA 100, underlined) and HindIII-HA-3 (50-AAGCTTTTAGGCGTAATCTGG- 9797–9802. 0 Crouch, E.A., Passarelli, A.L., 2002. Genetic requirements for homologous recombi- GACGT-3 , with HindIII restriction site underlined), were used to nation in Autographa californica nucleopolyhedrovirus. J. Virol. 76, 9323–9334. PCR-amplify Tn79HA from pFBPG-ac92P-tn79HA. The PCR product Deng, F., Wang, R., Fang, M., Jiang, Y., Xu, X., Wang, H., Chen, X., Arif, B.M., Guo, L., was gel-purified and ligated to pCRII, and the resulting plasmid, Wang, H., Hu, Z., 2007. Proteomics analysis of Helicoverpa armigera single fi T-Tn79HA-NH, was sequenced. 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