Activity of Ac92 as an FAD-linked sulfhydryl oxidase has been previously determined (15
); however, the role of this activity during virus replication or morphogenesis is unknown. In order to determine the effects of this gene during viral replication, we constructed an ac92
-null virus, Ac92KO-PG, and a virus with mutations within a conserved motif necessary for sulfhydryl oxidase activity, Ac92HAM1-PG. Both viruses were characterized and compared to three other viruses, AcWT-PG, carrying unaltered ac92
at its native locus; Ac92Rep-PG, carrying ac92
at the polyhedrin
locus; and Ac92HARep-PG, carrying an epitope-tagged ac92
at the polyhedrin
locus. This approach allowed us to contrast the effects of Ac92 and its known enzymatic activity during virus replication and determine if ac92
has a function independent of sulfhydryl oxidation.
RT-PCR analysis and mapping of the 5′ end of the ac92 transcripts.
We determined the timing of ac92 transcription in an effort to determine when the gene was important during virus replication. RT-PCR was performed using total RNA extracted from AcMNPV-infected cells at different time points. ac92 transcripts were detected from 9 h p.i. to 72 h p.i. (Fig. , RT+). No signals were obtained when avian myeloblastosis virus (AMV) reverse transcriptase was not included (Fig. , RT−). Addition of the protein synthesis inhibitor cycloheximide and DNA synthesis inhibitor aphidicolin to infected cells abolished transcription, indicting that ac92 is a late gene, requiring DNA and prior protein synthesis (Fig. ).
FIG. 1. Transcription kinetics of ac92 and production of Ac92 in AcMNPV-infected cells. (A) RT-PCR analysis of ac92 transcripts. Total RNA was extracted from AcMNPV-infected cells at designated time points. AMV reverse transcriptase was added (RT+) or (more ...)
The ac92 transcriptional initiation site was determined by 5′ RACE analysis using total RNAs isolated from AcMNPV-infected cells at 24 h p.i. Five clones derived from the 5′ RACE products were sequenced, and transcripts were initiated at the first A of the typical baculovirus late promoter motif TAAG, located 184 nucleotides (nt) upstream of the ac92 translation initiation codon ATG (Fig. ). Together, RT-PCR and the 5′ RACE analyses indicate that ac92 is a late gene.
Synthesis of Ac92 during infection.
Synthesis of Ac92 from Ac92HARep-PG was analyzed by immunoblotting lysates from Ac92HARep-PG-infected cells by using HA antibody. An immunoreactive band of approximately 33 kDa, the expected mass, was first detected at 24 h p.i. and persisted at 96 h p.i. (Fig. ). The protein synthesis inhibitor cycloheximide and the DNA synthesis inhibitor aphidicolin were individually added to infected cells; no proteins were detected in the presence of cycloheximide as expected, and Ac92 was not detected in aphidicolin-treated cells (Fig. ). The requirement for DNA synthesis indicates that Ac92 is produced at late times.
Construction of ac92-knockout bacmid and confirmation of its construction.
Ac92KO was constructed by deleting most of ac92
but retaining 150 nt from the 5′ end and 61 nt from the 3′ end of the ac92
coding region to ensure expression of the neighboring genes, ac93
(Fig. ). The remaining 567-nt coding sequence (nt 78762 to nt 79328 of AcMNPV [1
]) was replaced with the Cm
cassette (Fig. ).
FIG. 2. Construction of recombinant bacmids. (A) Construction strategy of Ac92KO. A fragment (567 bp) of the ac92 ORF was deleted and replaced with the chloramphenicol resistance gene (Cm) between nt 78762 and nt 79328 of the AcMNPV genome (1). (B) PCR confirmation (more ...)
PCR was performed to confirm that ac92 had been deleted from the ac92 locus of AcBAC and that the Cm cassette was inserted (Fig. ). Primers Ac9251 and Ac9231 were used to confirm the deletion of 567 bp within the ac92 coding region and its replacement with the 1,038-bp Cm. Primer pairs Ac9251/Cm3 and Cm5/Ac9231 (Fig. ) were used to examine the recombination junctions upstream and downstream of ac92, respectively. Primers Cm5/Cm3 were used to confirm the insertion of the Cm cassette. The sizes of the PCR-amplified products are as expected (Fig. ).
Construction of Ac92KO-PG, Ac92Rep-PG, Ac92HARep-PG, and AcWT-PG AcBACs containing polyhedrin and egfp.
To determine if deletion of ac92 had any effects on occlusion morphogenesis and to facilitate examination of virus-infected cells, an ac92-knockout mutant, Ac92KO-PG, containing polyhedrin and egfp, was constructed by transposition of polyhedrin and egfp into the polyhedrin locus of Ac92KO (Fig. , row i). Two repair bacmids were made to rescue and confirm the phenotype resulting from the deletion of ac92, Ac92Rep-PG (Fig. , row ii) and Ac92HARep-PG, with an HA tag at the C terminus (Fig. , row iii). AcWT-PG, AcBAC with polyhedrin and egfp, was used as a positive control (Fig. ). All constructs were confirmed by PCR, expression of egfp, and the formation of occlusions (data not shown).
Analysis of Ac92KO-PG, Ac92Rep-PG, Ac92HARep-PG, and AcWT-PG replication in bacmid DNA-transfected cells.
To determine the effect of deleting ac92 on viral replication, cells were transfected with DNA from Ac92KO-PG, Ac92Rep-PG, Ac92HARep-PG, or AcWT-PG. Transfected cells were monitored by fluorescence microscopy, and no obvious differences in the numbers of fluorescent cells and fluorescence intensities were observed at 24 h posttransfection (p.t.) (Fig. , 24 h p.t.). eGFP fluorescence was observed in most AcWT-PG-, Ac92Rep-PG-, or Ac92HARep-PG-transfected cells by 72 h p.t. (Fig. , 72 h p.t.). In contrast, the number of cells transfected with Ac92KO-PG DNA did not increase in eGFP fluorescence significantly from 24 to 72 h p.t. (Fig. , 72 h p.t.). Inspection of cells by light microscopy showed no differences in occlusion body formation in any of the viruses up to 48 h p.t. (data not shown). However, many cells transfected with AcWT-PG, Ac92Rep-PG, or Ac92HARep-PG DNA contained occlusion bodies by 72 h p.t., while Ac92KO-PG DNA-transfected cells showed no increase in the number of cells that contained occlusion bodies (Fig. , 72 h p.t.). By 96 h p.t., most cells transfected with AcWT-PG, Ac92Rep-PG, or Ac92HARep-PG DNA showed eGFP fluorescence and contained occlusion bodies (data not shown); Ac92KO-PG DNA-transfected cells had no obvious differences, except for several small plaques (data not shown).
FIG. 3. Analysis of viral replication and BV production in cells. (A) The top panels show cells transfected with the indicated recombinant bacmids at 24 h p.t. The middle panels show the progression of virus infection at 72 h p.t. The bottom panels show occlusion (more ...)
One-step virus growth curve analyses were performed to assess the effect of deleting ac92 on virus replication. Cells were transfected with bacmid DNA, and at selected time points the BV titers were determined by TCID50 endpoint dilution assays. Cells transfected with AcWT-PG, Ac92Rep-PG, or Ac92HARep-PG revealed a steady increase in virus production (Fig. ). However, Ac92KO-PG-infected cells had single-cell infections as indicated by single eGFP-positive cells. Accounting for single eGFP-positive cells in titers yielded a very low titer starting at 24 h p.t., where the production of BV was 10 million times less than that in ac92-carrying viruses at 120 h p.t. (Fig. , panel ii). If single-cell infections were excluded, no detectable infectious BV was produced by Ac92KO-PG (Fig. , panel i). The supernatants from Ac92KO-PG DNA-transfected cells were collected and used for subsequent infections. At 96 h p.i., only 10 to 20 individual cells per 106 cells were eGFP positive. Supernatant from infected cells was collected and used to infect naive cells, and by 96 h p.i., no additional eGFP-positive cells were detected (data not shown). Therefore, deletion of ac92 is essential for budded virus production.
Plaque assays were performed to better assess virus spreading to neighboring cells. By 4 days p.t., large plaques were formed in AcWT-PG-, Ac92Rep-PG-, and Ac92HARep-PG DNA-transfected cells (Fig. ), while plaques from Ac92KO-PG DNA-transfected cells consisted of single cells (Fig. ), indicating a defect in virus production.
Quantitative analysis of viral DNA replication.
To assess the role of ac92
in viral DNA replication, we performed quantitative real-time PCR. AcGP64KO, lacking the envelope fusion protein, was used as a noninfectious control since the virus was reported to be unable to infect from cell to cell (16
). Cells transfected with Ac92KO-PG or AcGP64KO bacmid DNA were harvested at 0, 24, 48, and 72 h p.t. Total intracellular DNA was extracted, and virus-specific DNA was quantified by real-time PCR (Fig. ). We assumed that the efficiencies of viral and cellular DNA purification from viral DNA-transfected cells were equivalent. A standard calibration curve was generated, and a highly reproducible linear curve was obtained when the log concentration of purified AcMNPV genomic DNA was plotted against the cycle threshold (data not shown). Melting-curve analysis revealed that PCR products were specific (data not shown). There were no significant differences when comparing Ac92KO-PG and AcGP64KO at 0 and 24 h p.t.; however, viral DNA copy numbers were significantly higher from 0 to 24 h p.t. for each virus, clearly demonstrating that DNA replication was occurring in both viruses (Fig. ). However, AcGP64KO produced more viral DNA at 48 h p.t. (P
< 0.001) and 72 h p.t. (P
< 0.05) than did Ac92KO-PG. Although it is possible that Ac92KO-PG has slight defects in DNA synthesis, we have observed that AcGP64KO has a slight leaky spreading defect that would lead to additional rounds of infections and thus more DNA copies (W. Wu and A. L. Passarelli, unpublished results) and therefore favor the later scenario.
FIG. 4. Viral DNA replication. Cells were transfected with bacmid DNA in triplicate. At 0, 24, 48, and 72 h p.t., total intracellular DNA was purified, digested with DpnI, and analyzed by real-time PCR. Two-way analysis of variance was carried out with GraphPad (more ...) Deletion of ac92 does not affect IE-1 and VP39 synthesis.
To evaluate the effect of deleting ac92 on specific viral protein synthesis, proteins from AcWT-PG- or Ac92KO-PG DNA-transfected cells were harvested at designated time points and the early regulatory gene product IE-1 or the late major capsid gene product VP39 was detected with anti-IE-1 antibodies or anti-VP39 antiserum, respectively. IE-1 and VP39 were detected from 12 h p.t. or 18 h p.t., respectively, from AcWT-PG or Ac92KO-PG DNA-transfected cells (Fig. ). The results indicate that deletion of ac92 did not affect IE-1 or VP39 protein production and, possibly, does not affect genes in these temporal phases.
Viral protein synthesis. Cells were transfected with Ac92KO-PG (KO) or AcWT-PG (WT) bacmid DNA and harvested at the designated time points. Proteins in whole-cell lysates were immunoblotted and probed with anti-IE-1 or anti-VP39.
Ac92 is localized to both BV and ODV.
BV or ODV was prepared from the supernatant of Ac92HARep-PG-infected cells or whole larvae, respectively. Virions were purified and biochemically fractionated into nucleocapsid and envelope fractions (Fig. , lanes NC and E), and HA-tagged Ac92 was detected by immunoblotting using anti-HA antibody. Ac92 was detected in both BV and ODV and was associated with the envelope fraction of BV. In the ODV, Ac92 appeared on both nucleocapsid and envelope fractions; however, proteins in the envelope fraction are more prominent than those in the nucleocapsid fraction. At this time, we cannot rule out contamination of the ODV nucleocapsid fraction with proteins from envelopes. To confirm fractionation efficiency, the nucleocapsid protein VP39 and the BV envelope protein GP64 were also analyzed by immunoblotting, and both VP39 and GP64 were detected in the expected fractions. These results indicate that Ac92 is associated with both BV and ODV and localized to the envelope fractions.
FIG. 6. Localization of Ac92 in purified and fractionated virions. BV and ODV were purified and fractionated, and proteins were detected by immunoblotting. Blots were probed with anti-HA antibody to detect Ac92, with anti-GP64 antibody to detect the BV envelope (more ...) Transmission electron microscopy analyses of Ac92KO-PG-, Ac92HARep-PG-, and AcWT-PG DNA-transfected cells.
To determine whether deletion of ac92 affected virion morphogenesis, thin sections from bacmid DNA-transfected cells were visualized by electron microscopy. At 72 h p.t., cells transfected with AcWT-PG DNA showed typical characteristics of baculovirus infection, including the presence of the virogenic stroma (VS), an electron-dense net-shaped baculovirus-induced structure, containing numerous rod-shaped nucleocapsids (Fig. , panel i), and virus-induced intranuclear microvesicles and bundles of enveloped nucleocapsids aligning within the ring zone (Fig. , panel ii) and the mature multiply enveloped nucleocapsid within the ring zone (Fig. , panel ii, arrows, and Fig. , panel iii) or embedded in polyhedra (Fig. , panel iv). Cells transfected with Ac92HARep-PG showed characteristics similar to those of AcWT-PG DNA-transfected cells (data not shown).
FIG. 7. Transmission electron microscopy analysis of cells transfected with AcWT-PG, Ac92KO-PG, or Ac92HAM1-PG DNA. (A) Cells transfected with AcWT-PG DNA and harvested at 72 h p.t. (i) Rod-shaped nucleocapsids in the virogenic stroma (VS). (ii) Intranuclear (more ...)
In Ac92KO-PG DNA-transfected cells, the VS and rod-shaped nucleocapsids were also observed in the nucleus (Fig. , panel i). Intranuclear microvesicles appeared within the ring zone (Fig. , panels ii and iv). However, ODVs with multiply enveloped nucleocapsids were not detected in the ring zone; instead, ODVs with singly enveloped nucleocapsids accumulated along with the microvesicles (Fig. , panels ii and iv). The shape and the size of polyhedra formed after transfection of cells with Ac92KO-PG DNA were similar to those of polyhedra formed by AcWT-PG or Ac92HARep-PG DNA-transfected cells; however, some did not contain ODVs (Fig. , panel vi), at least in the cross sections viewed, and some contained ODVs with singly enveloped nucleocapsids (Fig. , panel v). Together, these observations indicate that deletion of ac92 did not affect the formation of the VS, nucleocapsids, or polyhedra. However, it affected the formation of ODVs with multiply enveloped nucleocapsids. To our knowledge, ac92 is the only gene affecting this step in virion assembly.
Construction and characterization of the Ac92 C155XXC158 to A155XXA158 point mutation virus Ac92HAM1-PG. ac92
encodes an FAD-linked sulfhydryl oxidase, a member of the ERV1/ALR family of sulfhydryl oxidases (15
). The presence of a C-X-X-C active-site motif, where X is a variable amino acid, is the hallmark of proteins that are involved in forming or breaking disulfide bonds (29
). To assess whether the C-X-X-C motif in Ac92 is essential for sulfhydryl oxidase activity and to determine the effects of this activity on virus morphogenesis, Ac92HAM1-PG, in which the predicted cysteine amino acids 155 and 158 of Ac92 were mutated to alanines and an HA tag was inserted at the C terminus of ac92
, was constructed. The ac92
in Ac92HAM1-PG was driven by the ac92
native promoter, and the virus carried polyhedrin
at the polyhedrin
locus (Fig. , row iv).
Cells were transfected with Ac92HARep-PG or Ac92HAM1- PG DNA and harvested at 24 and 48 h p.t., and proteins were detected by immunoblotting using anti-HA antibody. A specific immunoreactive band of approximately 33 kDa, corresponding to epitope-tagged Ac92, was detected in transfected cells (Fig. ). The signal was stronger in Ac92HARep-PG DNA-transfected cells at 48 h p.t., probably due to virus spread to and protein production from adjacent cells, while there was no virus spread from Ac92HAM1-PG-transfected cells (data not shown and Fig. ).
Expression of Ac92 mutant in Ac92HAM1-PG-transfected cells. Cells were transfected with Ac92HAM1-PG or Ac92HARep-PG bacmid DNA and harvested at 24 and 48 h p.t. Total cellular proteins were immunoblotted using anti-HA antibody.
Replication of Ac92HAM1-PG in insect cells was similar to that of Ac92KO-PG (Fig. ), including infection of single cells. In addition, plaques from Ac92HAM1-PG DNA-transfected cells consisted of single cells, reminiscent of those formed by Ac92KO-PG (Fig. ). Electron microscopy analysis of Ac92HAM1-PG DNA-transfected cells also showed results similar to those for Ac92KO-PG DNA-transfected cells, where singly enveloped nucleocapsids accumulated with microvesicles in the ring zone (Fig. , panel vii). Thus, altering the C-X-X-C motif affected both BV production and ODV envelopment of multiple nucleocapsids, suggesting that sulfhydryl oxidase activity affected these processes.
Sulfhydryl oxidase activity of Ac92 (C155XXC158) and C-X-X-C (A155XXA158) mutant proteins.
Ac92 (Ac92HA) and Ac92HA cysteine mutant protein (Ac92HAM1) were expressed in E. coli
and affinity purified (data not shown). The purified proteins were assayed for sulfhydryl oxidase activity by using DTT as a substrate. Reactions were initiated by adding purified proteins to the substrate mixture, and aliquots were withdrawn at different times thereafter. The thiol content was measured in collected aliquots by DTNB reactivity (15
). Ac92HA was able to oxidize DTT in a time-dependent manner and with similar oxidation efficiencies from 0 to 120 min, while Ac92HAM1 was not able to oxidize the substrate (Fig. ). Thus, mutation of the C-X-X-C motif in Ac92 hinders sulfhydryl oxidase activity.
FIG. 9. Sulfhydryl oxidase activity of Ac92 (Ac92HA) and mutant (Ac92HAM1) proteins. Hexahistidyl-tagged proteins were expressed in E. coli and purified by nickel agarose. Sulfhydryl oxidase assays were carried out using 0.5 mM DTT as substrate, without recombinant (more ...)