Functional Characterization of Autographa Californica Multiple Nucleopolyhedrovirus Gp16 (Ac130)

Virology 464-465 (2014) 341–352

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Virology

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Functional characterization of Autographa californica multiple nucleopolyhedrovirus gp16 (ac130)

Ming Yang, Cui Huang, Duo-Duo Qian, Lu-Lin Li n

Hubei Key Laboratory of Genetic Regulation and Integrative Biology, College of Life Sciences, Central China Normal University, Wuhan 430079, China article info abstract

Article history: To investigate the function of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) gp16, Received 2 January 2014 multiple gp16-knockout and repair mutants were constructed and characterized. No obvious difference Returned to author for revisions in productivity of budded virus, DNA synthesis, late gene expression and morphogenesis was observed 7 May 2014 between gp16-knockout and repair viruses, but gp16 deletion resulted in six hours of lengthening in ST50 Accepted 18 July 2014 to the third instar Spodoptera exigua larvae in bioassays. GP16 was fractionated mainly in the light Available online 9 August 2014 membrane fraction, by subcellular fractionation. A GP16-EGFP fusion protein was predominantly Keywords: localized close around the nuclear membrane in infected cells, being coincident with formation of the Autographa californica multiple vesicles associated with the nuclear membrane, which hosted nucleocapsids released from the nucleus. nucleopolyhedrovirus These data suggest that gp16 is not required for viral replication, but may be involved in membrane gp16 trafﬁcking associated with the envelopment/de-envelopment of budded viruses when they cross over ac130 gp16 knockout the nuclear membrane and pass through cytoplasm. & 2014 Elsevier Inc. All rights reserved.

Introduction insect, ODV virions are released in the midgut of the insect to initiate a new infection cycle (Rohrmann, 2013), so that, function The Baculoviridae is a family of arthropod-specific, rod-shaped, to spread infection between host insects. enveloped viruses with large circular, supercoiled, double- During infection, the viral genes are transcribed by host RNA stranded DNA genomes. Autographa californica multiple nucleopo- polymerase II or a viral RNA polymerase. Prior studies suggested lyhedrovirus (AcMNPV) is the type species of the alphabaculo- that the gene expression of baculoviruses could be divided into viruses and the most studied baculovirus (King et al., 2012). early and late phases, which could be further subdivided into Baculoviruses produce two types of virions during infection. The immediate early, delayed early, late and very late phases. Early nucleocapsids of progeny virus are assembled in the nuclei of gene expression was shown to be dependent on host RNA poly- infected cells. Some of them exit the nucleus and acquire an merase and late expression to reply on the viral RNA polymerase envelope by budding through the cytoplasma membrane, produ- (Grula et al., 1981; Guarino et al., 1998; Hoopes and Rohrmann, cing budded viruses (BVs). In the process to bud out of the nucleus, 1991). A recent study on the transcriptoem of AcMNPV in a the nucleocapsids obtain an envelope from the nuclear membrane. Trichoplusia ni cell line showed that the viral genes could be This envelope is lost during transit through the cytoplasm classified into four clusters, named G1 to G4, based on the (MacKinnon et al., 1974; Knudson and Harrap, 1976; Granados temporal abundance and the pattern of changes in transcript and Lawler, 1981). BVs released from infected cells attach to other accumulation through the infection cycle. G1 and G2 genes are susceptible cells to initiate secondary infections (Granados and mainly transcribed by host cell RNA polymerase II and many are Lawler, 1981; Keddie et al., 1989). The nucleocapsids remaining in associated with the regulation of DNA replication and transcrip- the nucleus are enveloped by viral induced membranes within the tion. The majorities of G3 and G4 genes are transcribed by the viral nucleoplasm and occluded in a protein crystal matrix, named RNA polymerase and mostly encode structural proteins or proteins occlusion body (OB) or polyhedra. The virions embedded in OBs involving the processing of newly synthesized viral genomes and are called occlusion-derived viruses (ODVs). OBs are released into the assembly of the new virions or occlusion bodies (Chen et al., the environment upon lysis of the infected cells and disruption of 2013). the body of host insect. When OBs are ingested by another host Although many baculovirus genes have been characterized, there is little information on ac130, a gene found in the genomes of all Group I and most Group II alphabaculoviruses sequenced to n fi Corresponding author. Tel.: þ86 027 87878981. date. It is located within a cluster of ve late expressed orfs E-mail address: [email protected] (L.-L. Li). (orf129-133), which are oriented in the same direction on the http://dx.doi.org/10.1016/j.virol.2014.07.030 0042-6822/& 2014 Elsevier Inc. All rights reserved. 342 M. Yang et al. / Virology 464-465 (2014) 341–352 genome of AcMNPV (Gombart et al., 1989; Oellig et al., 1987; Ayres in cells transfected with the three individual bacmids. By 96 hpt, et al., 1994). In Orgyia pseudotsugata MNPV (OpMNPV), the product most cells in each culture were fluorescent (Fig. 2A). No obvious of the homologous gene of ac130 was identified as a 16 kD N- difference was observed among the wt, gp16 knockout and gp16 glycosylated protein and was called GP16 (Gross et al., 1993). The repair bacmids, showing that all the three bacmids replicated in OpMNPV GP16 was shown to be associated with lamellar like the transfected cells. To detect BV production in transfected cells, structures peripheral to the nuclear membrane and with envel- supernatants from the transfected cultures above were collected at opes of viruses budded into the cytoplasm through the nuclear 96 hpt, titered, and used to infect Sf9 cells at an MOI of 5. membrane but not detected in BVs and ODVs. It was proposed to Fluorescence was first observed in the infected cells at 18 hpi be involved in facilitating the movement of nucleocapsids through (data not shown). By 96 hpi, almost all cells in the plates inoculated the nuclear membrane. If it is true, GP16 may be required for virus with the three different supernatants were filled with fluorescence replication. However, a deletion of the ortholog of ac130 (bm107) (Fig. 2A), indicating that all these three viruses could generate was found to be viable, in Bombyx mori NPV (BmNPV) (Katsuma et infectious BVs. Another set of gp16 knockout (vAcgp16ko-PHs), gp16 al., 2012). The purposes of this study was to verify whether ac132 knockout repaired (vAcgp16ko-rep-PH), and wild-type control (vAcPHs) (gp16) was essential for replication and infectivity of AcMNPV, and bacmids were also used for transfection–infection of Sf9 cells. These to investigate if GP16 was associated with egress of nuclecapsids bacmids contain the polh gene as a marker to determine effects of the from the nucleus. deletion of gp16 on virus replication. As a result, OBs were observed To understand the role of gp16 in the AcMNPV replication cycle, in cells transfected with vAcPHs and vAcgp16ko-rep-PH,aswellasthe we constructed and characterized AcMNPV mutants lacking gp16. cells transfected with vAcgp16ko-PHs, under phase contrast microscopy. Effects of gp16 knockout on BV reproduction, morphogenesis, DNA When Sf9 cells were inoculated with the supernatants from the replication and late gene expression in infected cells and infectiv- transfected cell cultures, OBs were observed as early as 24 hpi, in all ity to host larvae were evaluated. In addition, the subcellular cases. By 96 hpi, most cells in the infected cultures were filled with location of the GP16 in cells infected by wild type AcMNPV was OBs (Fig. 2B). These phenotypes suggested that the gp16 was not characterized. required for production of BV and formation of OB. To determine the replication kinetics of the recombinant viruses, virus growth curve analyses were performed. For this Results experiment, Sf9 cells were infected with BVs at an MOI of 5. At selected time points, the BV titers were determined by a gfp Generation of gp16 knockout and repair AcMNPV bacmids TCID50 end-point dilution assay. Sf9 cells infected with wt (vAc or vAcPHs), gp16 knockout (vAcgp16ko-gfp or vAcgp16 ko-PHs)orgp16 To investigate the function of gp16 during the viral infection repair (vAcgp16 ko-rep-gfp or vAcgp16 ko-rep-PH) viruses revealed a cycle, a knockout of gp16 in the AcMNPV bacmid, bMON14272, steady increase in virus production, and the slope of the growth was constructed using a λ-Red homologous recombination system curves of these three viruses had no significant difference (Datsenko and Wanner, 2000). The orf of gp16 (nt 110524 to (Fig. 2C). However, the growth curves of the OB-negative and 0 110844) is 321 nt. The 5 237 nt of it was deleted and replaced with the OB-positive viruses separated into two distinctive groups. 0 the cat gene (Fig. 1A and B). The remaining 84 nt at 3 end was The titers of OB-negative viruses were higher by a factor of about retained to avoid affecting transcription of an adjacent down- 10 than the titers of the OB-positive viruses. These results were stream gene. The deletion was confirmed by PCR screening (data consistent with the phenotype of AcMNPV fp25K mutants, which not shown) and was further verified by western-blot of the exhibited reduced polyhedrin synthesis and increased release of extracts of the cells infected separately by wild type AcMNPV infectious BV (Harrison and Summers, 1995). and vAcgp16ko, using antiserum against OpMNPV GP16. A peptide of about 16 kD was detected in extracts of the cells infected by Deletion of gp16 does not affect virus DNA replication AcMNPV but was not present in extracts of the cells infected by vAcgp16ko (Fig. 1C). To facilitate detection of viruses in cells, the To determine if gp16 had any impact on viral DNA replication, polyhedrin (polh) gene and the reporter gene egfp was separately the levels of viral DNA replication in the cells infected with inserted into vAcgp16ko and the wt bacmid through Tn7-mediated vAcgp16ko-PHs were measured over a 96 h time-course and compared transposition at the polh locus (Fig. 1 A and B). vAcgfp and vAcPHs or with the ones in the cells infected with vAcPHs or vAcgp16ko-rep-PH. vAcPH were used as wild-type bacmids in experiments. The infected Sf9 cells were collected at designated time-points, and To ensure that the traits of the gp16 knockout were due to the the total DNA was extracted and analyzed by qPCR. DNA synthesis removal of the gp16 gene, two kinds of gp16 repair bacmids named began increasing at 12 hpi and continued until 72 hpi, and the vAcgp16ko-rep-gfp and vAcgp16ko-rep-PH were constructed. vAcgp16ko-rep-gfp levels of DNA detected for the three viruses were similar through- has the reporter gene egfp fused with the GP16 coding sequence and out the whole processes (Fig. 3). These results indicated that the inserted at the polh locus; vAcgp16ko-rep-PH has a copy of polh and a gp16 total level of DNA replication in individual infected cells was not with their native promoters inserted at the polh locus in opposite affected by deletion of gp16. orientation (Fig. 1A and B). The transposition events occurring in the construction of the recombinant bacmids were confirmed by EGFP expression or formation of occlusionbodiesinSf9cellstransfected Effects of gp16 deletion on expression of VP39 and polyhedrin with the bacmids (Fig. 2AandB). Western blot assays were performed with antibodies against Propagation of the gp16 knockout bacmids in Sf9 cells the major nucleocapsid protein VP39 and polyhedrin to determine if late and very late gene expression was affected by the gp16. To examine the effects of the deletion of the gp16 gene on virus Expression of VP39 was similar for vAcgp16ko-PHs and vAcgp16ko-rep-PH. replication, the bacmids with the gp16 gene deleted (vAcgp16ko-gfp), A peptide of 39 kD was detected with polyclonal antibodies against repaired by insertion of gp16 at the polh locus (vAcgp16ko-rep-gfp), or the VP39 in the infected cells by 18 hpi, after which levels increased the wild-type control (vAcgfp) were transfected into Sf9 cells. The until 72 hpi. Polyhedrin could be detected in the infected cells for transfected cells were monitored by fluorescence microscopy. both vAcgp16ko-PHs and vAcgp16ko-rep-PH by 24 hpi, and levels increased Fluorescence was first observed, at 24 h post transfection (hpt), steadily until 96 hpi. The levels of polyhedrin for vAcgp16ko-PHs M. Yang et al. / Virology 464-465 (2014) 341–352 343

A Ac129 gp16 Ac131 237bp 84bp gp16PU gp16PD

locus bMON14272 gp16 polh locus

vAcgfp gentamicin Pgp16 egfp SV40

vAcPH(s) gentamicin polh SV40 B Ac129 cat Ac131 1027bp 84bp

catPF catPR

locus bMON14272 gp16 polh locus

gp16ko-gfp vAc gentamicin Pgp16 egfp SV40

vAcgp16ko-PH(s) gentamicin polh SV40

vAcgp16ko-rep-gfp gentamicin Pgp16 gp16-egfp SV40

vAcgp16ko-rep-PH gentamicin SV40 polh gp16 SV40

Fig. 1. Construction of gp16 knockout, repair and wt AcMNPV bacmids. (A) Maps of the gp16 locus showing primers used for analysis and how the polh locus was modiﬁed to produce three ‘wt’ AcMNPV bacmids. In vAcgfp, a copy of the egfp orf under control of AcMNPV gp16 promoter was insert into the polh locus; vAcPH(s) represents vAcPHs and vAcPH, in which a copy of polh with native promoter (92 nt for vAcPHs and 223 nt for vAcPH) was re-insert into the polh locus. (B) Maps showing the modiﬁcation of the gp16 and polh loci in gp16 knockout and repair AcMNPV bacmids. At the gp16 locus, a 237 bp sequence of the gp16 orf was replaced with the cat. In the polh locus, a copy of egfp under the control of the gp16 promoter (vAcgp16ko-gfp), or a copy of polh with its native promoter (vAcgp16ko-PH(s)), or a copy of gp16 fused with egfp and under control of the gp16 promoter (vAcgp16ko-rep-gfp) or a copy of gp16 with native promoter and a copy of polh with native promoter in opposite orientation was insert by transposition (vAcgp16ko-rep-PH). vAcgp16ko-PH(s) represents vAcgp16ko-PHs (with a 92-nt polh promoter) and vAcgp16ko-PH (with a 223-nt polh promoter). (C) Western blot analysis for the presence or absence of GP16 protein in extracts of the cells infected by vAcgp16ko (ko) or wild type AcMNPV (wt). M, protein marker SM0671 (Fermentas).

looked a little lower than that for vAcgp16ko-rep-PH at all time points 223 nt). In additional western blot assays, the levels of polyhedrin (Fig. 4). The difference in the polyhedrin expression was found to be detected in infected cells for vAcgp16ko-PH,whichcontainedapolh due to the different lengths of the polh promoters contained in promoter same as the one in vAcgp16ko-rep-PH, were similar to the these two recombinant viruses. The polh promoter in vAcgp16ko-PHs polyhedrin levels for vAcgp16ko-rep-PH and higher than those for was much shorter than the one in vAcgp16ko-rep-PH (92 nt versus vAcgp16ko-PHs (data not shown). 344 M. Yang et al. / Virology 464-465 (2014) 341–352

1.E+09

1.E+08

1.E+07

1.E+06 vAcPHvAc

vAcgp16ko-PHvAc /ml 1.E+05 50 vAcgp16ko-rep-PHvAc 1.E+04 TCID vAcgfpvAc 1.E+03 vAcgp16ko-gfpvAc

1.E+02 vAcgp16ko-rep-gfpvAc

1.E+01

1.E+00 024487296120 Hours post infection

Fig. 2. Analysis of viral replication in Sf9 cells. (A) Fluoresence microscopy of Sf9 cells transfected/infected with vAcgfp, vAcgp16ko-gfp and vAcgp16ko-rep-gfp at 24 and 96 hpt and 96 hpi. (B) Phase contrast microscopy of Sf9 cells infected with vAcPHs, vAcgp16ko-PHs and vAcgp16ko-rep-PH at 24 and 96 hpi. (C) Virus growth curves of gp16 knockout, repair and wt viruses in Sf9 cells. Sf9 cells were infected with each virus at an MOI of 5. The supernatants were harvested at the designated time points, and virus titers were determined by TCID50 end-point dilution assays. Each data point represents the average titer of three independent infections. Error bars indicate standard deviations. M. Yang et al. / Virology 464-465 (2014) 341–352 345

released from the nucleus into vesicles which were associated with the outer surface of the nuclear membrane (Fig. 5B). Similar vesicles containing enveloped nucleocapsids were also observed in the cells infected by vAcgp16ko-PHs (Fig. 5E). Some bundles of nucleocapsids in a single envelope appeared in the vesicles. Similar phenotypes were also observed in insect cells infected by Spodoptera frugiperda NPV or Trichoplusia ni,NPV, in early studies (MacKinnon et al., 1974; Knudson and Harrap, 1976). Fig. 5C and F, respectively, shows enveloped nucleocapsids embedding into the developing OBs in the nuclei of the cells infected by vAcgp16ko-rep-PH and vAcgp16ko-PHs at 72 hpi. Fig. 5GshowsanOBwithenveloped nucleocapsids embedded, in a vAcgp16ko-PHs infected cell. The OB has cracks, and its surface is rough. The OBs purified from the larvae infected by vAcgp16ko-PHs or vAcgp16ko-rep-PH were further examined by SEM. vAcgp16ko-rep-PH OBs had a smooth surface (Fig. 6A). In comparison, the OBs gp16ko-PHs Fig. 3. Quantitative-PCR analysis of viral DNA replication in Sf9 cells infected by wt, produced by vAc were cubic. Most of them were smaller gp16 knockout or gp16 knockout-repaired AcMNPV recombinants. Total DNA was and had pitted surfaces (Fig. 6B). This was similar to the pheno- purified from the cells infected with vAcPHs, vAcgp16ko-PHs, or vAcgp16ko-rep-PH,at types resulted from deletion of pep and/or p10 in OpMNPV (Gross 0, 12, 24, 48, 72 and 96 hpi, digested with Dpn I to eliminate input bacmid DNA, and et al., 1994). Since the polh promoter in vAcgp16ko-PHs, which was the gp41 sequence was analyzed by real-time PCR. The values displayed represent gp16ko-rep-PH the averages from infections performed in triplicate with error bars indicating from pFastBac I, was much shorter than the one in vAc , gp16ko-PH standard deviations. vAc containing a polh promoter same as the one in vAcgp16ko-rep-PH was constructed and the OBs were analyzed by SEM. As shown in Fig. 6C, the appearance of the vAcgp16ko-PH OBs was basically the same as that of the vAcgp16ko-rep-PH OBs. This implied that the deletion of gp16 did not have significant impact on formation of OBs. The deficient OBs of vAcgp16ko-PHs obviously resulted from the short polh promoter contained, which likely induced inefficient expression of polyhedrin, in comparison with the polh promoter contained in vAcgp16ko-rep-PH, as shown in Fig. 4.

Localization of GP16 in AcMNPV-infected insect cells

To determine the subcellular localization of GP16 during infection, Sf9 cells infected with AcMNPV were fractionated into cytoplasmic and nuclear fractions, and proteins were detected by immunoblotting. As a result, GP16 was only fractionated in the cytoplasmic fraction (Fig. 7A). To assess the fractionation effi- ciency, the same membrane was re-probed with GP64 or PP31 antibodies. As expected, GP64 was detected only in cytoplasmic fraction, while PP31was detected mainly in nuclear fraction (Fig. 7A) (Volkman et al., 1984; Guarino et al., 1992). When the cytoplasmic fraction of the cells at 48 hpi was further fractionated Fig. 4. Western blot analysis of VP39 and polyhedrin synthesis in Sf9 cells infected into the heavy membranes, light membranes and cytosol fractions, with vAcgp16ko-PHs or vAcgp16ko-rep-PH. Sf9 cells were infected at an MOI of 5, and GP16 was detected mainly in the light membrane fraction and less harvested at the designated times post-infection, and cell lysates were prepared for in the cytosol fraction, while GP64 was presented in both of the western blot analysis. Each protein was analyzed with polyclonal antibodies against heavy and light membrane fractions (Fig. 7A). the protein. An anti-actin antibody was used as a loading control. Subcellular localization of GP16 was also monitored directly by fluorescence microscopy. Sf9 cells infected with vAcgp16ko-rep-gfp,in Electron microscopy analysis of Sf9 cells infected by gp16-knochout which gp16 with native promoter and fused with egfp was inserted and repair viruses into the polh locus of gp16 knockout bacmid vAcgp16ko (Fig. 1B), were examined by confocal laser scanning microscope. GFP- TEM analysis was performed with thin sections of the cells specific fluorescence was first observed in infected Sf9 cells at infected with vAcPHs, vAcgp16ko-PHs or vAcgp16ko-rep-PH at 36, 48 and 18 hpi (not shown). In the cells infected 24 hpi, fluorescence was 72 hpi, to determine if gp16 had any effect on virus morphogen- found to be associated with particles distributing close around the esis. The typical characteristics of a baculovirus infection, such as nucleus. A few fluorescent particles sticking into the nucleus were virogenic stroma inundated with rod-shaped nucleocapsids, and observed in the cells infected 24 or 48 hpi. At 72 hpi, concentra- nucleocapsids budding through cytoplasmic membrane, were tions were observed at the outer side of the nuclear membrane observed in all the cells infected with vAcPHs (data not present), (Fig. 7B). Hoechst-staining of the infected cells showed that green vAcgp16ko-PHs or vAcgp16ko-rep-PH (Fig. 5A and D). In Fig. 5D, which fluorescence was mostly observed close around the blue area shows a cell infected by vAcgp16ko-PHs at 36 hpi, it can be seen that (nucleus) stained by Hoechst (Fig. 7C). Since GP16 was detected three nucleocapsids were budding out of the nuclear membrane. only in the cytoplasmic fraction, it unlikely bound the nuclear At another site in the same cell, a single enveloped nucleocapsid membrane directly. The fluorescent particles sticking into the was almost out off the nucleus. Around the nucleocapsid was an nuclei might result from invagination of the nuclear membrane. empty space, which might be a developing vesicle. In the cells In the cells infected with vAcgfp, fluorescence distributed through- infected by vAcgp16ko-rep-PH, some enveloped nucleocapsids were out the cytoplasm and the nucleus (Fig. 7B). 346 M. Yang et al. / Virology 464-465 (2014) 341–352

NM VC NM

VN NM NM

Fig. 5. TEM of Sf9 cells infected with vAcgp16ko-rep-PH (A, B and C) or vAcgp16ko-PHs (D, E, F and G), at 36 hpi. (A and D) or 72 hpi. (B, C, E, F, and G). NM: nuclear membrane; NC: nucleocapsid; VC: vesicle. VN: virion; VS: virogenic stroma. Scale bar¼500 nm. M. Yang et al. / Virology 464-465 (2014) 341–352 347

Fig. 6. SEM of the OBs of vAcgp16ko-rep-PH (A), vAcgp16ko-PHs (B) and vAcgp16ko-PH (C).

The effect of gp16 deletion on infectivity of AcMNPV for Spodoptera expression of polh by interacting with the polh coding sequence or exigua larvae polyhedrin protein. Both AcMNPV and OpMNPV are group I NPVs. OpMNPV GP16 The infectivity of vAcPH,vAcgp16ko-PH and vAcgp16ko-rep-PH was was found to be associated with aggregates of lamellar tested for newly molted third-instar S. exigua larvae by feeding the membrane-like structures and nucleocapsids budding through larvae with viral OBs and determining LD50 and ST50 in bioassays. nuclear membrane in infected L. dispar cells, and was proposed As shown in Table 2, there was no significant difference in LD50 to be involved in facilitating the movement of nucleocapsids between the gp16 knockout mutant and the wt or gp16 repair through nuclear membrane, or altering the nuclear membrane to virus. However, the ST50 of gp16 knockout mutant was 6 h longer facilitate the movement of protein into nuclear membrane (Gross than that of the wt and the gp16 repair virus in two of the three et al., 1993). In this study, AcMNPV GP16 was detected mainly in trials (Table 3). In another trial, the ST50 of gp16 knockout mutant the light membrane fraction in subcellular fractionation of the was 6 h and 12 h longer than that of the wt and the gp16 repair infected cells. Generally, the light membrane fraction includes virus, respectively, but the values seemed not accurate since the plasma membranes, golgi membranes, microsomes, endoplasmic difference between the wt and the gp16 repair virus was statisti- reticulum and ribosomes. Golgi apparatus has lamellar cally not significant. membrane-like structures and is involved in vesicular transport in cells. Since golgi apparatus is the site for glycocylation of proteins, locating of GP16, as a glycoprotein, on golgi apparatus is logical. It is not clear if GP16 was presented in other membranes Discussion in the light membrane fraction, in addition to golgi and cytosol. In this study, the GP16-EGFP fusion protein was found located In this study, AcMNPV mutants lacking gp16 were constructed close around the nuclear membrane in the cells infected by and used for transfection/infection of Sf9 cells, to determine if gp16 vAcgp16ko-rep-gfp. This was coincident with formation of the vesicles was required for viral replication. As a result, the mutants lacking which were associated the nuclear membrane and hosted the gp16, with or without polh, could replicate in Sf9 cells and had nucleocapsids released from the nucleus (Fig. 5). Relation of GP16 similar BV productivities as the wild type and gp16-knockout to the nucleocapsid-containing vesicles remains to be determined. repair viruses, indicating that gp16 was not essential for virus In herpes simplex virus (HSV), egress of the virions from cells replication. The deficiency in vAcgp16ko-PHs OBs shown by electron involves extensive modification of cellular membranes and sequen- microscopy was obviously caused by less than optimal polyhedrin tial envelopment and de-envelopment steps. Capsids in the nucleus expression driven by the 92-nt polh promoter from the transfer undergo primary envelopment at the inner nuclear membrane, and vector pFastBac I, suggesting that a longer length of polh promoter then enveloped virus particles undergo de-envelopment by fusing was required to achieve optimal polh gene expression. The polh with the outer nuclear membrane. Capsids delivered into the promoter of AcMNPV was previously characterized with reporter cytoplasm then undergo secondary envelopment, involving trans- genes (Matsuura et al., 1987; Possee and Howard, 1987; Rankin Golgi network membranes. HSV glycoproteins play important role in et al., 1988). It was shown that deletion of the 240 nucleotides these processes. HSV glycoproteins gB and gH/gL are involved in de- upstream from the 95 of the polh or making insertions in the site envelopment. gB, gD and gE/gI function to promote secondary did not affect the ability of the polh promoter to express a reporter envelopment. These HSV glycoproteins act in a redundant fashion gene, and a sequence 69 nucleotides upstream to the normal in these two steps (Johnson et al., 2011; Farnsworth et al., 2007, position of the polh translation initiation codon was sufficient for 2003). The processes of nuclear egress of BVs of baculoviruses are not maximum promoter activity (Possee and Howard, 1987; Gearing clear yet. As shown in Fig. 5, some nucleocapsids budded through the and Possee, 1990). The result from our study implies that the nuclear membrane and were wrapped into vesicles in the infected sequence upstream from the 92 site may have subtle effects on cells. How these enveloped nucleocapsids were released from the 348 M. Yang et al. / Virology 464-465 (2014) 341–352

GFP Hoechst Merge 24 hpi 72 hpi

Fig. 7. Subcellular localization of the GP16. (A) Cells were infected with AcMNPV at an MOI of 5, and cytoplasmic, heavy membrane (HM), light membrane (LM), cytosol (CL), and nuclear fractions were prepared at the indicated time points post-infection. Fractionated proteins were immunoblotted using individual polyclonal antibodies speciﬁcto GP16, GP64 and PP31 to detect correspondent proteins. (B) Sf9 cells infected with vAcgp16ko-rep-gfp or vAcgfp were observed for ﬂuorescence by confocal laser scanning microscopy at 24, 48 and 72 hpi. (C) Sf9 cells infected with vAcgp16ko-rep-gfp or vAcgfp were sampled at 48 and 72 hpi, stained with Hoechst33258 to mark nuclei (blue), and subjected to confocal microscopy. Scale bar¼10 μm. M. Yang et al. / Virology 464-465 (2014) 341–352 349

Table 1 Oligonucleotides used to generate knockout and repair bacmid constructs.

Name Sequencea

gp16koPF 50-CCTGAATAAATAATAATAAGGGTTTTGTACGATTTCAACACTTCGAATAAATACCTGTG-30 gp16koPR 50-ATAACACGTTAATTTTTTCGTCGAGATTGGATATTTTTTTAACCAGCAATAGACATAAG-30 UP486 50-ATGTCACCTTTTCTTAGCAACGGC-30 DN498 50-GGTCTGCAATGAGGAGAACGTG-30 catPF 50-CGAATAAATACCTGTGACGGAAGAT-30 catPR 50-GAAAACGGTGTAACAAGGGTGAAC-30 polhPF1 50-GCTGTCGACATGCCGGATTATTC-30 polhPR 50-TAGCTGCAGTTAATACGCCGGACC-30 polhPF2 50- TCGGTCGACAACAGTTCACCTCCC-30 0 0 Pgp16PF1 5 -TAGGGATCCTCATAGCAACGATGCGGCC-3 gp16PR1 50-GGGCTGCAGTTATTGAACGTTTAG-30 0 0 Pgp16PF2 5 -TAGGTCGACTCATAGCAACGATGCGGCC-3 SV40PR 50-GCGTACGTAGCAGTGATCAGATCCAGAC-30 gfpPF 50-ATTGTCGACATGGTGAGCAAGGGCG-30 gfpPR 50-GCCCTGCAGTTACTTGTACAGCTCGTCC-30 0 0 Pgp16PF3 5 -GACTACGTATCATAGCAACGATGCGGCCG-3 0 0 Pgp16PR1 5 -GGCGTCGACTGTTGAAATCGTACAAAACCCTTAT-3 gp16PR2 50-GGCGTCGACTTGAACGTTTAGCAC-30 M13F 50-GTTTTCCCAGTCACGAC-30 M13R 50-CAGGAAACAGCTATGAC-30

a Sequences of restriction sites are underlined.

Table 2 Dose-mortality of vAcPH, vAcgp16ko-PH and vAcgp16ko-rep-PH for third-instar Spodoptera exigua larvae.

3 Virus LD50 (95% CL) ( 10 OB/larva) Relative median potency (95% CL)

vAcPH vAcgp16ko-PH

Test 1 vAcPH 1.515 (0.802–2.794) —— vAcgp16ko-PH 1.354 (0.713–2.496) 0.894 (0.366–2.135) — vAcgp16ko-rep-PH 1.354 (0.711–2.500) 0.893 (0.365–2.137) 1.000 (0.413–2.418)

Test 2 vAcPH 1.474 (0.834–2.553) —— vAcgp16ko-PH 1.530 (0.867–2.646) 1.038 (0.474–2.290) — vAcgp16ko-rep-PH 1.402 (0.793–2.423) 0.951 (0.431–2.083) 0.916 (0.414–1.998)

Test 3 vAcPH 1.300 (0.677–2.409) —— vAcgp16ko-PH 1.426 (0.750–2.633) 1.098 (0.456–2.696) — vAcgp16ko-rep-PH 1.241 (0.647–2.291) 0.955 (0.391–2.316) 0.870 (0.353–2.085) vesicles and de-enveloped before reached the plasma membrane and In conclusion, the experimental results from this study demon- budded through remains to be determined. Apart from GP16, there strate that AcMNPV gp16 is not essential for virus replication, but the have been, at least, five additional glycoproteins identified in GP16 protein may be involved in formation or transport of the baculoviruses, including GP64, F, vFGF, GP41 and GP37. GP64 and F nucleocapsid-containing vesicles around the nuclear membrane and protein are low pH activated envelope fusion proteins and are associated with trafficking of BVs through nuclear membrane and required for entry of BVs into cells (IJkeletal.,2000;Pearsonetal., cytoplasm. 2000; Monsma et al., 1996). GP64 is also required for efficient viral budding (Oomens and Blissard, 1999). AcMNPV GP41, an O-glycosylated tegument protein located between virion envelope Materials and methods and capsids, is required for egress of nucleocapsids from the nucleus in the pathway of budded virus synthesis (Olszewski and Miller, Virus, cell line and insects 1997). GP37 has a similar subcellular localization in infected cells as GP16. GP37 of Mamestra brassicae MNPV distributed in a double The Sf9 cell line, a clonal isolate of the parent cell line IPLB- semicircle or ring in the cytoplasm adjacent to infected nuclei Sf21-AE from the fall armyworm Spodoptera frugiperda (Vaughn (Phanis et al., 1999). The GP37 of AcMNPV is associated with BV et al., 1977) were cultured at 26 1C in Grace's medium (Invitrogen and is also nonessential for replication (Cheng et al., 2001). Since Life Technologies) containing 10% fetal bovine serum, penicillin deletion of gp16 did not reduce BV production, in this study, it is (100 μg/ml) and streptomycin (100 μg/ml). The AcMNPV bacmid probably not essential for movement of nucleocapsids through bMON14272 derived from the AcMNPV strain E2 was maintained nuclear membrane. However, GP16 may be involved in envelopment in DH10B cells as described previously (Luckow et al., 1993). and/or de-envelopment of BVs when they cross over the nuclear membrane. It may be redundant with another viral protein, such as Construction of gp16 knockout AcMNPV bacmid GP37. Considering that deletion of gp16 did not have obvious effects on BV production and OB formation in Sf9 cells, it did result in time AcMNPV bacmid bMON14272 was used to generate the gp16 lengthening to kill S. exigua larvae, GP16 may have effects on viral KO viruses through recombination in Escherichia coli using the replication or virus-host interaction in host species or cell type λ-Red system, as previously described (Datsenko and Wanner, specificstyles. 2000). The DNA primers used in the experiments are listed in 350 M. Yang et al. / Virology 464-465 (2014) 341–352

Table 3 Time-mortality of vAcPH, vAcgp16ko-PH and vAcgp16ko-rep-PH for third-instar Spodoptera exigua larvae.

Virus ST50 7SEM (95% CI) (h) Log rank(Mantel–Cox)

vAcPH vAcgp16ko-PH

χ2 p χ2 p

Test 1 vAcPH 108 71.89 (104.29–111.71) ———— vAcgp16ko-PH 120 71.76 (116.56–123.44) 14.653 0.000 —— vAcgp16ko-rep-PH 114 71.51 (111.05–116.96) 0.609 0.435 15.587 0.000

Test 2 vAcPH 108 71.73 (104.61–111.39) ———— vAcgp16ko-PH 114 71.80 (110.48–117.52) 8.104 0.004 —— vAcgp16ko-rep-PH 108 72.22 (103.65–112.35) 0.059 0.808 5.775 0.016

Test 3 vAcPH 108 71.48 (105.10–110.90) ———— vAcgp16ko-PH 114 71.95 (110.18–117.82) 4.099. 0.043 —— vAcgp16ko-rep-PH 108 70.974 (106.09–109.91) 0.408 0.523 6.527 0.011

Table 1. Briefly, the chloramphenicol acetyltransferase gene (cat) gp16 promoter was synthesized using primers Pgp16PF3 and cassette was PCR-amplified from a pKS-cat template plasmid by Pgp16PR1 and inserted between SnaB I and Sal I sites of pFB-gfp PCR with primers gp16koPF and gp16koPR, which contained a 40 to obtain pFB-Pgp16-gfp, in which the egfp was under control of the nt homologous arm corresponding to the sequence (nt 110484 to gp16 promoter. pFB-Pgp16-gfp was electroporated into the DH10B 110523) immediately upstream of the start codon ATG and the containing vAcgp16ko and pMON7124 and the DH10B containing 30-end (nt 110761 to 110800) of AcMNPV gp16 (nt 110524 to bMON14272 and pMON7124 to generate vAcgp16ko-gfp and vAcgfp, 110844) respectively. The PCR product was digested with Dpn I to respectively (Fig. 1B). A 514-bp fragment containing gp16 with its completely remove any remaining template plasmids before it was native promoter and without the stop codon was amplified using electro-transformed into arabinose-preinduced DH10B cells har- primers Pgp16PF3 and gp16PR2, digested with SnaB I and Sal I and boring bMON14272 and plasmid pKD46 encoding λ-Red recombi- inserted into pFB-gfp to obtain pFB-gp16-gfp, in which egfp was nase. The colonies resistant to kanamycin and chloramphenicol fused with gp16 in frame at the 30-end. pFB-gp16-gfp was electro- were selected. The resultant bacmid was named vAcgp16ko (Fig. 1B). porated into the DH10B containing vAcgp16ko and pMON7124 to Four sets of primers, UP486/DN498, UP486/catPR, catPF/DN498 produce vAcgp16ko-rep-gfp(Fig. 1B). and catPF/catPR, were used in PCR to confirm the proper replace- All the transfer vectors above were sequenced to confirm the ment of gp16 with cat cassette in it. construction. All of the bacmid constructs were confirmed by PCR with primer set M13F/R (Table 1). Construction of gp16 knockout, repair, and wt AcMNPV bacmids containing egfp or polh Titration of BV

A fragment containing AcMNPV polh orf was PCR-amplified using Sf9 cells were transfected with the appropriate bacmids (2.0 μg/ primers polhPF1 and polhPR, digested with Sal I and Pst I, and ligated well) or infected with infectious cell culture supernatants. The cell with pFastBac1 (Invitrogen) cut with the same enzymes to obtain culture supernatants were collected at various time points post pFB-PHs. pFB-PHs was electroporated into E. coli DH10B containing infection, and the budded virus titers were determined using a bMON14272 and a help plasmid pMON7124 (Luckow et al., 1993), TCID50 end-point dilution assay (O’Reilly et al., 1992). Virus infection which encodes a transposase, to generate vAcPHs (Fig. 1A). pFB-PHs was determined by cytopathology (appearance of OBs in cells) or by was electroporated into E. coli DH10B containing vAcgp16ko and monitoring EGFP expression by fluorescence microscopy. pMON7124 to generate vAcgp16ko-PHs (Fig. 1B). A fragment containing AcMNPV polh with its native promoter Quantitative real-time PCR was PCR-amplified using primers polhPF2 and polhPR, and inserted between Sal I and Pst I sites of pFastBac1 to make pFB- Viral DNA replication was assayed by real-time PCR, as pre-

PH0. A 514-bp fragment containing gp16 with its native promoter viously described (Vanarsdall et al. 2004). Briefly, Sf9 cells seeded 6 was PCR-amplified using primers Pgp16PF1 and gp16PR1 and was in 35 mm plates (1.0 10 cells/plate) were infected with inserted between BamH I and Pst I sites of pFastBac1 to obtain vAcgp16ko-PHs, vAcgp16ko-rep-PH, or vAcgp64ko at an MOI of 0.1 and pFB-gp16. Using pFB-gp16 as a template, a new fragment contain- harvested at designated time points post infection. The DNA was ing gp16 with promoter and the SV40 polyA signal was amplified extracted, digested with Dpn I and combined with the SYBR-Green using primers Pgp16PF2 and SV40PR, digested with Sal I and SnaB I I Real-Time PCR Master Mix Kit (Toyobo) and the qPCR primers and inserted into pFB-PH0 to generate pFB-gp16-PH. pFB-gp16-PH Q-65972F and Q-66072R (Vanarsdall et al., 2004). The samples was electroporated into E. coli DH10B containing vAcgp16ko and were analyzed in a Bio-Rad CFX96 qPCR cycler. The results were pMON7124 to generate gp16-repaired bacmid vAcgp16ko-rep-PH analyzed using CFX Manager 2.1 (Bio-Rad) software.

(Fig. 1B). pFB-PH0 was cut with SnaB I and Sal I to remove the redundant PPH, end-blunt with T4 DNA polymerase, then, Western blot analysis and antisera re-circularized to make pFB-PH. pFB-PH was electroporated into E. coli DH10B containing bMON14272 and pMON7124 to generate The protein samples were separated by SDS-12% polyacryla- vAcPH (Fig. 1A), and electroporated into E. coli DH10B containing mide gel electrophoresis (PAGE) and transferred to BioTace PVDF vAcgp16ko and pMON7124 to generate vAcgp16ko-PH (Fig. 1B). membrane (PALL Life Science) with a liquid transfer apparatuses. A fragment containing egfp orf was PCR-amplified using pri- The blots were probed with individual specific polyclonal antisera. mers gfpPF and gfpPR, digested with Sal I and Pst I and inserted The OpMNPV GP16-specific polyclonal antiserum was provided by into pFastBac1 to obtain pFB-gfp. A fragment (193 bp) containing Dr. George Rohrmann (Gross et al., 1993). GP64-specific rabbit M. Yang et al. / Virology 464-465 (2014) 341–352 351 polyclonal antiserum (1:3000) was provided by Dr. Zhihong Hu. 3000 rpm for 5 min. The cell pellets were embedded, sectioned, and The AcMNPV VP39-specific rabbit polyclonal antiserum (1:10,000), stained as described previously (van Lent et al., 1990), then examined and the PP31-specific mouse polyclonal antiserum (1:10,000) with a FEI Tecnai G2 20 TWIN transmission electron microscope at an were separately arose with His-tagged AcMNPV VP39 and PP31, accelerating voltage of 200 kV. which were expressed in E. coli. IRDye-800CW conjugated goat For Scanning electron microscopy (SEM), third instar larvae of anti-rabbit (or -mouse) antibody (1:10,000) (LI-COR) was used as S. exigua were feed with artificial diet contaminated with viral OBs the secondary antibody. Fluorescence was detected by LI-COR and incubated at 26 1C. OBs were purified from larval cadavers Odyssey. SDS-PAGE and immunohybridizations to western blots with the method described by Gross et al. (1994). Briefly, three were performed in accordance with standard protocols and larval cadavers were smashed in 0.5 ml of 2% Triton-100 and manufacturer's instruction (Sambrook and Russell, 2001). incubated at 37 1C for 1 h. Then, 0.25 ml of 2% sodium deoxycholate was added and the sample was incubated further for 1 h. The Subcelular fractionation suspended material was transferred to a new tube and centrifuged at 9000g, for 15 min, the supernatant removed, and the pellet Cytoplasmic and nuclear fractionation was performed as pre- suspended in 1.0 ml of TNS solution. The sample was centrifuged viously described (Wu and Passarelli, 2012). Briefly, Sf9 cells for 30 s and the pellet was resuspended in 200 μl TNS, layered on seeded in 35 mm plates (1 106 /plate) were infected with 4.5 ml of 50% sucrose in TNS and centrifuged at 10,000 rpm (in infectious supernatant of AcMNPV at an MOI of 5 and harvested MLS-50 rotor) for 30 min. The sucrose solution was removed and at 0, 24, 48 and 72 h post-infection (hp i). The cells were washed the pellet was resuspended in 1 ml TNS. Following washing twice with 1 ml of PBS, dislodged and centrifuged at 3000 rpm for 5 min. with TNS, the OB pellet was dried by lyophilization. For SEM, OBs The pellet was resuspended in 50 μl of ice-cold buffer A (10 mM were spread on metal stubs, sputter-coated with gold and

Tris [pH 7.9], 10 mM NaCl, 5 mM MgCl2, 1 mM dithiothreitol, 0.5% observed with a JFOL6700F scanning electron microscope at an [vol/vol] NP-40) and incubated on ice for 5 min. The cells were accelerating voltage of 10 kV. homogenized with a prechilled Dounce homogenizer (10 strokes with a type B pestle) and centrifuged at 1000g for 5 min. The supernatant was retained as the cytoplasmic fraction. The pelleted Insects and bioassays nuclei were resuspended in 50 μl of buffer A, following washing five times with the same buffer. Fractions were stored at 20 1Cpriorto Larvae of S. exigua were reared on an artificial diet at 26 1C. For western-blot assays. Heavy membrane and light membrane fractiona- occluded virus production, Fifth-instar S. exigua larvae were 7 tion were done as bellow. The cell pellet (2 10 cells) was resus- injected with 10 μl/insect infectious supernatant from cell culture pended in 4 ml of hypotonic lysis buffer (2 mM Tris–HCl, pH 7.4, 0.02% transfected with bacmids containing the complete polyhedrin 6 Triton-X 100, 0.1% mM MgCl2 and 1 complete protease inhibitor gene, at a concentration of 2 10 TCID50/ml. Bioassays on the cocktail tablet [Roche Molecular Biochemicals]) and incubated on ice infectivity of AcMNPV mutants were performed as previously for 1 h. The cells were homogenized with 30 strokes in a Dounce described (Liu et al., 2011). To determine the median lethal dose homogenizer using a type A pestle. The lysate was centrifuged twice at (LD50), OBs purified from larval cadavers were suspended in 400 g for 10 min at 4 1C to remove nuclei and intact cells. The double-distilled water. 10 μl of the samples containing 0 (control), supernatant was centrifuged twice at 10,000 g for 10 min at 4 1C. 2 102,1 103,2 103,1 104 and 2 104 OBs, was respectively The heavy membrane pellet (mitochondria, lysosomes, peroxisomes) applied onto a small piece of artificial diet in a well of a twelve- was resuspended in 120 μl of complete solubilization/extraction buffer well plate. Newly molted third-instar S. exigua larvae were allowed (20 mM sodium phosphate buffer, pH 7.5, 500 mM NaCl, 0.1% SDS, 1% to feed individually until all the diet was consumed. Those larvae Nonidet P-40, 0.5% sodium deoxycholate, and 0.02% sodium azide, and completely consuming virus inocula within 12 h were included in 1 complete protease inhibitor cocktail tablet). The supernatant was the assay and were transferred to an artificial diet, with fresh diet centrifuged at 200,000g for 90 min at 4 1C. The light membrane pellet added as needed for the duration of the bioassay. Mortality was (plasma membranes, golgi membranes, microsomes, ribosomes) was recorded daily after infection until larvae died or pupated. 36 resuspended in 40 μl of solubilization/extraction buffer. The fractions larvae per dosage were used in the infection experiments and the 1 were stored at 80 Cpriortowestern-blotassays. experiments were repeated in triplicate. The LD50 values were determined by the probit analysis calculated compared with a Confocal microscopy relative median potency method. To determine median survival

time (ST50), newly molted third-instar S. exigua larvae were Sf9 cells seeded in a 35 mm plate were infected with AcMNPV starved for 6 h and then inoculated with a solution containing mutants containing egfp, at an MOI of 5, respectively. At 18, 24, 48 4% sucrose (w/v), 0.01% blue food coloring, and OBs (3 107 and 72 hpi, cells were visualized with a ZEISS, LSM710 NLO OBs/ml). Inoculated larvae were maintained individually in 12- confocal laser scanning microscope for fluorescence using a well plates provided with artificial diet; Mortality was recorded wavelength of 488 nm laser line for EGFP. To stain nuclei, Sf9 cells every 6 h after infection until larvae died or pupated. 36 larvae per seeded on the surface of cover slips placed in 35 mm plates were virus were used in the infection experiments and the experiments infected with AcMNPV mutants. At 48 and 72 hpi, the cells were were repeated three times. The ST50 values were calculated with fixed, stained with Hoechst33258 and visualized with confocal the Kaplan–Meier estimator and compared by the log-rank test. laser scanning microscope using a wavelength of 352 nm for Hoechst33258. All images were digitally recorded and merged by the use of ZEISS software. Acknowledgments Electron microscopy We thank Professor George F Rohrmann for suggestion, critical For transmission electron microscopy (TEM), 1 106 Sf9 cells per reviewing the manuscript and providing the GP16-specific anti- plate (35 mm) were infected with vAcgp16ko-PHs or vAcgp16ko-rep-PH,at serum. Thank Professor Jian-Fang Gui, Jun Zhang and Xiao-Chun an MOI of 5. At 36, 48 and 72 hpi, cells were fixed, dehydrated, then Luo for assisting in Confocal microscopy and images. Thank dislodged with a rubber policeman and precipitated by centrifuge at Professor Xiu-Liang Sun, Dr. Yin Zhou for assisting in bioassays. 352 M. Yang et al. / Virology 464-465 (2014) 341–352

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