The Autographa californica M Nucleopolyhedrovirus ac79 Gene
Encodes an Early Gene Product with Structural Similarities to UvrC
and Intron-Encoded Endonucleases That Is Required for Efficient
Budded Virus Production
Wenbi Wu and A. Lorena Passarelli
Molecular, Cellular, and Developmental Biology Program, Division of Biology, Kansas State University, Manhattan, Kansas, USA
The Autographa californica M nucleopolyhedrovirus (AcMNPV) orf79 (ac79) gene is a conserved gene in baculoviruses and
knockout bacmid was generated through homologous recombination in Escherichia coli. Titration assays showed that budded
viruses include four genera: Alphabaculovirus, Betabaculovirus,
Gammabaculovirus, and Deltabaculovirus (13). Alphabaculovi-
ruses and betabaculoviruses infect larvae of Lepidoptera; while
gammabaculoviruses and deltabaculoviruses, infect larvae of
Hymenoptera and Diptera, respectively (13).
During alphabaculovirus replication, two types of virions are
(ODV). The BV is produced as the nucleocapsid buds out from
the plasma membrane of the infected cell. BVs are essential for
establishing systemic infection in an infected insect by transmit-
ting infection from cell to cell. The ODV is formed in the nucleus
of the infected cell prior to being embedded within a crystalline
infection from larvae to larvae when a dead host releases ODV-
containing polyhedra into the environment and polyhedra are
ingested by another host.
The Autographa californica M nucleopolyhedrovirus (AcMNPV)
open reading frame (ORF) orf79 (ac79) has not been studied in
detail, and our knowledge of the function of ac79 stems from
genomic or proteomic analyses. The genomic sequence of ac79
ular mass of 12.2 kDa (2). Homologs of ac79 are found in baculo-
viruses of the genera Alphabaculovirus and Betabaculovirus, asco-
viruses, iridoviruses, and bacteria (23). A previous proteomic
concluded that Ac79 was associated with ODVs (5). It was sug-
gested that Ac79 is a member of the DNA repair UvrC endonu-
aculoviruses have circular double-stranded DNA genomes of
between 80 and 180 kbp (12) and infect arthropods. Baculo-
clease superfamily with similarities to intron-encoded endonu-
cleases, since it is predicted to contain two tyrosines spaced by
about 10 amino acids, the hallmark sequence RX3[YH], and a key
glutamate downstream of this sequence (1).
In this study, we characterized ac79 by mapping the transcrip-
the role of Ac79 during AcMNPV replication, we generated an
ac79-knockout virus, Ac79KO-PG, through homologous recom-
bination in Escherichia coli. The deletion of ac79 resulted in a de-
crease in BV production but did not affect viral DNA replication
or late and very late protein accumulation. Ac79KO-PG was able
to produce BV, but more virions than the control virus were not
infectious. Electron micrographs did not reveal virion structure
defects in the absence of ac79. Elongated tubular structures con-
taining the major capsid protein VP39 were produced in ac79-
knockout virus-infected cells. These tubular structures were not
observed for viruses carrying ac79 mutations in the UvrC/intron-
encoded endonuclease conserved residues.
Received 8 September 2011 Accepted 1 March 2012
Published ahead of print 14 March 2012
Address correspondence to A. Lorena Passarelli, firstname.lastname@example.org.
This article is contribution 11-401-J from the Kansas Agricultural Experiment
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
jvi.asm.org 0022-538X/12/$12.00 Journal of Virologyp. 5614–5625
MATERIALS AND METHODS
Viruses, cell lines, and bacterial strains. Bacmid bMON14272 (Invitro-
gen), here referred to as AcBAC, containing an AcMNPV genome, was
propagated in E. coli BJ5183 cells as described previously (3). The virus
AcWT-PG was constructed by introducing the polyhedrin gene and the
enhanced green fluorescent protein gene (egfp) at the polyhedrin locus of
AcBAC (32). DH10B cells with helper plasmid pMON7124, encoding a
transposase, were purchased from Invitrogen. The Sf9 insect cell line,
27°C in TC-100 medium (Invitrogen) supplemented with 10% fetal bo-
vine serum, penicillin G (60 ?g/ml), streptomycin sulfate (200 ?g/ml),
and amphotericin B (0.5 ?g/ml).
sites for ac79, RNA was extracted from AcWT-PG-infected Sf9 cells and
collected at 6, 12, and 24 h postinfection (p.i.) by use of TRIzol reagent
measuring the optical density at 260 nm. Rapid amplification of 5= cDNA
ends (5= RACE) was performed by using a 5= RACE kit, version 2.0, ac-
cording to the handbook provided by the manufacturer (Invitrogen).
ac79-specific primer Ac79SP1 (5=-GGTTTGGCTTTGATGAGACGC-3=)
was used to synthesize first-strand cDNA. A nested ac79-specific primer,
Ac79SP2 (5=-GCTGTGATACACGAGCCGTA-3=), and the provided
strand cDNA as a template. PCR products were gel purified with the Gel
Extraction kit (Qiagen) and cloned into pCRII (Invitrogen) prior to de-
riving the nucleotide sequence. A total of five clones per time point were
sequenced in each of two independent experiments.
Time course analysis of Ac79 expression. A monolayer of Sf9 cells
(1 ? 106cells) was infected at a multiplicity of infection (MOI) of 5 with
locus (see below). The protein synthesis inhibitor cycloheximide and the
DNA synthesis inhibitor aphidicolin were added, and cell samples were
collected as previously described (32). Proteins were analyzed by sodium
dodecyl sulfate (SDS)–15% polyacrylamide gel electrophoresis (PAGE)
followed by immunoblotting or stored at ?20°C until further use.
Cell fractionation into cytoplasmic and nuclear fractions. Sf9 cells
(1 ? 106cells) were infected with Ac79HARep-PG at an MOI of 5 and
collected at 6, 12, 24, 48, and 72 h p.i. To fractionate the cells into cyto-
plasmic and nuclear fractions, the cell pellet was resuspended in 50 ?l of
dithiothreitol, 0.5% [vol/vol] NP-40) and kept on ice for 5 min. The cells
were disrupted and nuclei were released by using a prechilled Dounce
homogenizer (10 strokes with a tight pestle). Cells were centrifuged at
1,000 ? g for 5 min, and the supernatant was retained as the cytoplas-
mic fraction. Pelleted nuclei were washed five times with buffer A and
resuspended in 50 ?l of buffer A. Fractions were stored at ?20°C until
Immunoblotting. Immunoblotting was performed as previously de-
scribed (32). The primary antibodies used in these study were (i) mouse
monoclonal anti- hemagglutinin (HA) antibody (Covance), to detect
clonal anti-GP64 antibody (eBioscience), to detect the viral fusion envelope
protein; (iv) mouse monoclonal anti-IE-1 antibody (a gift from Linda Gua-
dotsugata MNPV (OpMNPV) polyhedrin antiserum (a gift from George
Generation of the ac79-knockout bacmid. The ac79-knockout bac-
mid was generated through homologous recombination in E. coli as pre-
the chloramphenicol resistance gene (Cm). A 1,178-bp PCR fragment,
containing a 1,038-bp Cm cassette and 70 bp of ac79 flanking regions at
each end, was amplified by using pCMR (29) as the template and primers
TTCGAATAAA-3=) and Ac79D2 (5=-TTATGCAACAAAAGTGGTTTG
AAGATTAAACCAGCAATAGACAAA-3=). Purified PCR fragments (1
?g) were electroporated into electrocompetent BJ5183 cells, and the re-
combinant ac79-knockout bacmid (Ac79KO) was selected, as previously
described (32), and confirmed by PCR amplification of inserted and
replacement of ac79 by Cm in the ac79 locus of AcBAC. Primers Cm5
(5=-CTTCGAATAAATACCTGTGA-3=) and Cm3 (5=-AACCAGCAATA
GACATAAGC-3=) were used to detect the correct insertion of the Cm
region of recombination, were used to confirm the expected deleted region
and the insertion of Cm at the ac79 locus. Primer pairs Ac7951/Cm3 and
Construction of the ac79-knockout and repair viruses and AcBAC
carrying the egfp and polyhedrin genes. To facilitate the examination of
virus-infected cells and to determine if the deletion of ac79 had any effect
into the polyhedrin locus of AcBAC by site-specific transposition, as
previously described (32). To this end, several donor plasmids were
constructed. Two primers, Ac7952(5=-GAGCTCATCTGCGTCTGCCA
ACATAT-3= [the SacI restriction site is underlined]) and Ac7933 (5=-TC
derlined]), were designed to amplify an 816-bp fragment containing the
native ac79 promoter (300 bp upstream of the ac79 ATG) and the ac79
ORF (Ac79POA), using AcBAC DNA as a template. This PCR product
was ligated into pCRII, and the resulting plasmid, pCRII-Ac79POA, was
confirmed by nucleotide sequencing. pCRII-Ac79POA was digested with
SacI and XbaI, and the Ac79POA fragment was inserted into pFB1-PH-
ACGTCGTATGGGTACAACTTATTTGCTAACAGGA-3= [the XbaI re-
striction site is underlined]) were used to amplify a 654-bp fragment that
contained the ac79 native promoter and the ac79 ORF with an HA tag
prior to the stop codon (Ac79POHA). The PCR product was ligated into
pCRII and confirmed by nucleotide sequencing. The resulting plasmid,
pCRII-Ac79POHA, was digested with SacI and XbaI to obtain the
Ac79POHA fragment and inserted into pFB-PG-pA (32), generating the
taining the helper plasmid pMON7124 and Ac79KO were transformed
with donor plasmid pFB1-PH-GFP, pFB-PG-Ac79POA, or pFB-PG-
Ac79POHA to generate the ac79-knockout virus Ac79KO-PG and two
repair viruses, Ac79Rep-PG with ac79 and Ac79HARep-PG with an HA-
genes, was described previously (32). All bacmids were cured of helper
plasmids as described previously (32). Bacmid DNA was extracted and
Construction of bacmids Ac79Y24AG26A-PG, Ac79R34K-PG, and
Ac79E72D-PG. To determine whether conserved amino acids in Ac79
were important for function, we changed the nucleotide sequences of
tyrosine and glycine at positions 24 and 26, respectively, to make
Ac79Y24AG26A-PG; arginine at position 34 to encode lysine and con-
struct Ac79R34K-PG; or glutamic acid at position 72 to encode aspar-
tic acid and construct Ac79E72D-PG. Three primer pairs were used
to construct donor plasmids pFB-PG-Ac79Y24AG26A, pFB-PG-
Ac79R34K, and pFB-PG-Ac79E72D for transposition by using a
QuikChange XL site-directed mutagenesis kit (Agilent Technologies)
and pFB-PG-Ac79POHA as the template. Primers Ac79 GIYmut f (5=-
CCAATTTTCCATTGTCTTG-3=) were designed to introduce mutations
into Ac79Y24AG26A-PG, primers Ac79 RtoKmut f (5=-CACGGGCATC
Efficient Infectious BV Production Requires Ac79
May 2012 Volume 86 Number 10 jvi.asm.org 5615
Ac79 RtoKmut r (5=-GTTCGAATGCTGTTTTATCTTTCTGTTAAGAT
TGCTCGTGATGCCCGTG-3=) were designed to introduce a mutation
into Ac79R34K-PG, and primers Ac79 EtoDmut f (5=-GCCGCCCGCAT
GGATTACAATCTTAAGCGTAAATGC-3=) and Ac79 EtoDmut r (5=-G
CATTTACGCTTAAGATTGTAATCCATGCGGGCGGC-3=) were de-
signed to introduce a mutation into Ac79E72D-PG. Electrocompetent
DH10B cells containing helper plasmid pMON7124 and Ac79KO were
transformed with donor plasmid pFB-PG-Ac79Y24AG26A, pFB-PG-
Ac79R34K, or pFB-PG-Ac79E72D to generate Ac79Y24AG26A-PG,
Ac79R34K-PG, or Ac79E72D-PG, as described above.
Viral growth curve analyses and plaque assays. Sf9 cells (1.0 ? 106
cells/35-mm-diameter dish) were transfected with 1 ?g of bacmid DNA
using Grace’s unsupplemented medium (Invitrogen) and Lipofectin (6),
The supernatants of transfected or infected cells were collected at various
time points to determine titers by 50% tissue culture infective dose
(TCID50) endpoint dilution assays (22) using Sf9 cells. Time zero was
defined as the time when the DNA-Lipofectin mixture or virus was re-
placed with fresh medium.
Sf9 cells were infected with AcWT-PG or Ac79KO-PG at an MOI of
0.01, 0.005, or 0.001, and plaque assays were performed as previously
fluorescence microscope using an eyepiece micrometer. The results were
analyzed by using GraphPad Prism, version 5.01 (GraphPad Software,
Comparison of AcWT-PG and Ac79KO-PG BV genome copy num-
bers and structural proteins. To compare the numbers of BV genome
copies of Ac79KO-PG and AcWT-PG, the same number of infectious BV
units (based on TCID50, 5 ? 107PFU) of each virus was concentrated by
centrifugation, and genomic DNA was purified as previously described
5 ?l of DNA was used as the template for quantitative PCR (Q-PCR) as
previously described (32).
The same number of infectious Ac79KO-PG or AcWT-PG units (5 ?
anti-GP64 antibody or anti-VP39 antiserum.
Viral DNA replication analysis. Sf9 cells (1.0 ? 106cells/35-mm-
diameter dish) were infected in triplicate with Ac79KO-PG or
AcWT-PG at an MOI of 5 and collected at different time points. Total
DNA was prepared, and Q-PCR was used to determine viral DNA
replication in virus-infected cells as previously described (32). The
results were analyzed by using GraphPad Prism, version 5.01 (Graph-
Pad Software, Inc.).
effect of ac79 on viral protein expression, Sf9 cells were infected with
A79KO-PG or AcWT-PG at an MOI of 5. At different time points, cells
were collected, and samples were prepared for immunoblotting as
described above. VP39 antiserum and OpMNPV polyhedrin antise-
rum, which cross-reacts with AcMNPV polyhedrin, were used as pri-
mary antibodies to immunodetect representative late and very late
Purification of BV and ODV. Sf9 cells were infected with
and the supernatant were separated by centrifugation. BV was concen-
third-instar Trichoplusia ni larvae were infected by the contamination of
an artificial diet with Ac79HARep-PG polyhedra, which were prepared
from Ac79HARep-PG-infected Sf9 cells. ODVs were purified as previ-
ously described (30).
Purification of BV and negative staining for transmission electron
microscopy. Sf9 cells were infected with Ac79KO-PG or AcWT-PG at
an MOI of 0.01. The supernatant of infected cells containing BV was
collected at 120 h p.i. The purification of BV was carried out as previ-
ously described (22). For negative staining, an electron microscope copper
grid was placed over 10 ?l of purified BV to absorb virions. Grid-bound
virions were stained with 10 ?l of a 2% uranyl acetate solution in water.
Stained virions were observed by using a Philips CM 100 transmission elec-
Electron microscopy and immunoelectron microscopy. Sf9 cells
(1.0 ? 106cells/35-mm-diameter dish) were infected with the indicated
virus at an MOI of 5. Infected cells were collected at 72 h p.i., fixed,
dehydrated, embedded, sectioned, and stained as described previously
(17). Samples were viewed as described above.
For immunoelectron microscopy, cells were embedded in LR White
resin (Ted Pella, Inc.), according to the technical notes provided by the
manufacturer. VP39 in Ac79KO-PG-infected cells was detected with rab-
bit polyclonal VP39 antisera, and HA-tagged Ac79 was detected with
The Ac79SP2 primer used for 5= RACE analysis is underlined. The asterisks and triangles indicate products from 5= RACE corresponding to the RACE products
in panel A.
Wu and Passarelli
jvi.asm.org Journal of Virology
anti-HA antibody in Ac79HARep-PG-infected cells. These primary anti-
bodies and goat anti-rabbit immunoglobulin G 10-nm gold-conjugated
ing the primary antibody were used as background labeling controls. Im-
ages were obtained with a Philips CM 100 transmission electron micro-
scope at an accelerating voltage of 100 kV.
Mapping of the ac79 transcription start sites. The ac79 tran-
scription initiation sites were determined by 5= RACE analyses
using total RNAs isolated at 6, 12, and 24 h p.i. from AcWT-
PG-infected cells. 5= RACE products (Fig. 1A) obtained from
RNAs harvested at each time point were cloned, sequenced,
and mapped to four transcription start sites (Fig. 1B, asterisks).
At 6 and 12 h p.i., we detected 5= RACE products that mapped
to 55, 166, 167, and 168 nucleotides (nt) upstream of the pre-
dicted Ac79 translational start site (Fig. 1B). These sites were
not reminiscent of early or late start sites (i.e., TATA box, ini-
tiator sequence, or TAAG). The larger 5= RACE product ob-
cells were harvested, and total cellular proteins were resolved by SDS-PAGE and detected with anti-HA antibody. mi, mock-infected cells. The numbers
to the left indicate the molecular masses of protein standards. The arrow indicates the expected migration for Ac79. (B) Cells were infected with
Ac79HARep-PG in the presence (?) or absence (?) of the DNA synthesis inhibitor aphidicolin or the protein synthesis inhibitor cycloheximide. Total
cellular proteins were immunoblotted using anti-HA antibody to detect Ac79 or anti-IE-1 antibody to detect IE-1. (C) Cells were infected with
Ac79HARep-PG at an MOI of 5, and cytoplasm and nuclear fractions were prepared at the indicated times p.i. Fractionated proteins were immunoblotted
using anti-HA, anti-GP64, or anti-IE1 antibody to detect Ac79, GP64, or IE-1, respectively. (D) BV and ODV were purified, and proteins were detected
by immunoblotting with anti-HA antibody. Ac92HARep-PG BV was used as a positive control. Anti-IE-1 antibody was used to detect the BV- and
ODV-associated IE-1. (E) Images of a cross section of Ac79HARep-PG-infected cells prepared at 72 h p.i., showing Ac79 by using an immunogold-
conjugated anti-HA antibody. Bars, 500 nm.
Efficient Infectious BV Production Requires Ac79
May 2012 Volume 86 Number 10 jvi.asm.org 5617
served at 24 h p.i. (Fig. 1A, triangle) maps to 18 nt upstream of
the gp41 translation initiation codon and corresponds to the
gp41 transcription start site (Fig. 1B). gp41 is present upstream
of ac79 and is transcribed in the same direction.
Synthesis and subcellular localization of Ac79. Sf9 cells were
infected with Ac79HARep-PG, a bacmid carrying an HA-tagged
immunoreactive band of approximately 16 kDa was detected at 6
h p.i., and detection continued through 96 h p.i. (Fig. 2A). The
hibitor cycloheximide were individually added to cells to assess if
Ac79 synthesis required prior DNA replication. The synthesis of
Ac79 was reduced but was detected in aphidicolin-treated cells;
however, as expected, it was nearly abolished in the presence of
cycloheximide (Fig. 2B), suggesting that late genes or DNA repli-
cation was not necessary for Ac79 synthesis. As a positive control,
the same membrane was stripped from antibodies and reprobed
with anti-IE-1 antibody to detect an early viral protein (Fig. 2B).
To determine the subcellular localization of Ac79 during infec-
tion, Sf9 cells were infected with Ac79HARep-PG and collected at
plasmic and nuclear fractions, and proteins were detected following
at late and very late phases (Fig. 2C). To assess the fractionation effi-
We also examined whether Ac79 was associated with the BV
or ODV. BV or ODV was prepared from the supernatant of
Ac79HARep-PG-infected cells or whole larvae, respectively, and
HA-tagged Ac79 was detected by immunoblotting with anti-HA
to detect an HA-tagged protein. Ac79 was associated with neither
BV nor ODV (Fig. 2D). To verify the purification and loading of
protein. In addition, Ac79 did not specifically localize within
Ac79HARep-PG-infected cells (Fig. 2E).
of its construction by PCR. Ac79KO was constructed by deleting
the central portion of ac79 and retaining 115 nt from the 5= end
and 95 nt from the 3= end of the ac79 coding region to ensure the
expression of the neighboring genes ac78 and gp41 (ac80) (Fig.
AcMNPV ) was replaced with the Cm cassette (Fig. 3A).
The deletion of ac79 from the ac79 locus of AcBAC and the
insertion of the Cm cassette were confirmed by PCR (Fig. 3B).
Primers Ac7951 and Ac7931 (Fig. 3A) were used to confirm the
deletion of 105 bp within the ac79 coding region and its replace-
ment with the 1,038-bp Cm cassette. Primers pairs Ac7951/Cm3
and Cm5/Ac7931 (Fig. 3A) were used to confirm the recombina-
tion junctions upstream and downstream of ac79, respectively.
Primer pair Cm5/Cm3 was also used to confirm the insertion of
were as predicted following successful recombination (Fig. 3B).
FIG 3 Construction of recombinant bacmids. (A) Strategy for construction of Ac79KO. An ac79 fragment (105 nt) was deleted and replaced with the
chloramphenicol resistance gene (Cm) between nt 65385 and nt 65498 of the AcMNPV genome (2). (B) PCR confirmation of Ac79KO. The table below
the polyhedrin (polh) and enhanced green fluorescent protein (egfp) genes inserted into the polyhedrin locus by Tn7-mediated transposition. (D) Schematic
diagram of Ac79KO-PG, Ac79Rep-PG, and Ac79HARep-PG, showing polh and egfp inserted into the polyhedrin locus after Tn7-mediated transposition.
Wu and Passarelli
jvi.asm.orgJournal of Virology
facilitate an examination of the effects of the deletion of ac79 on
virus infection, an ac79-knockout mutant, Ac79KO-PG, contain-
ing the polyhedrin gene under polyhedrin promoter control and
Ac79KO (Fig. 3Di). Two repair bacmids were constructed to res-
cue and confirm the phenotype resulting from the deletion of
ac79, Ac79Rep-PG, carrying the ac79, polyhedrin, and egfp genes
(B to D) BV growth curves determined by TCID50endpoint dilution assays. Titers were determined from virus present in the supernatants of cells transfected
with the indicated recombinant bacmids (B) or from cells infected at an MOI of 0.01 (C) or 5 (D). (E) Plaque diameters in AcWT-PG- or Ac79KO-PG-infected
cells. An arbitrary plaque diameter of 10 is equivalent to 1 mm. A total of 54 plaques were measured in AcWT-PG-infected cells, and 66 were measured in
Ac79KO-PG-infected cells. Plaque size differences were analyzed by using the Mann-Whitney test with Graph Pad Prism software. Bars indicate the medians of
samples. ***, significant difference (P ? 0.001).
Efficient Infectious BV Production Requires Ac79
May 2012 Volume 86 Number 10jvi.asm.org 5619
(Fig. 3Dii), and Ac79HARep-PG, carrying the polyhedrin gene,
egfp, and ac79 with an HA tag at the C terminus (Fig. 3Diii).
AcWT-PG, AcBAC carrying the polyhedrin and egfp genes, was
described previously (32) and used as a positive control (Fig. 3C).
the formation of occlusions (data not shown).
AcWT-PG replication in viral DNA-transfected and virus-in-
fected cells. Sf9 cells were transfected with DNA from Ac79KO-
PG, Ac79Rep-PG, Ac79HARep-PG, or AcWT-PG, and the ex-
pression of egfp was monitored by fluorescence microscopy. No
h posttransfection (p.t.) among the viruses (Fig. 4A). An inspec-
tion of cells by light microscopy showed no differences in the
approximate number of occlusion bodies formed and the timing
of occlusion body formation at 48 h p.t. (data not shown) and 72
h p.t. (Fig. 4A).
of the deletion of ac79 on virus replication. Cells were transfected
with bacmid DNA, and at selected time points, BV titers were
in virus production was observed with each virus. However, the
virus titer of Ac79KO-PG was lower than the titers of the other
viruses, with obvious differences starting at 48 h p.i. (Fig. 4B). At
72 h p.t., the titer of Ac79KO-PG was 25-fold lower than that of
Ac79HARep-PG. To confirm this defect, cells were infected with
were determined by TCID50endpoint dilution assays at selected
time points. The titer of Ac79KO-PG was again lower than those
of any of the other viruses from about 48 to 96 h p.t. at both
multiplicities of infection (Fig. 4C and D), and the onset of BV
example, Ac79KO-PG-infected cells at two different MOIs pro-
FIG 5 Infectious and noninfectious BV production mixtures and BV struc-
ber analysis by Q-PCR. Viral DNA was purified from the same units of infec-
tious AcWT-PG and Ac79KO-PG BV and subjected to Q-PCR. Column
heights indicate the averages of data from three repeats, and the error bars
represent the standard deviations. (B) The same units of infectious AcWT-PG
(WT) and Ac79KO-PG (KO) BV were purified, and virion proteins were de-
tected by immunoblotting with anti-GP64 antibody or anti-VP39 antiserum.
microscopy. Bars, 100 nm.
FIG 6 Viral DNA replication. Cells were infected with AcWT-PG or
purified and analyzed by Q-PCR. The error bars represent the standard devi-
ations from three independent experiments.
FIG 7 Viral protein synthesis. Cells were infected with AcWT-PG (WT) or
Proteins were analyzed by immunoblotting using anti-VP39 or anti-polyhe-
Wu and Passarelli
jvi.asm.orgJournal of Virology
duced 10.5-fold-lower titers at 72 h p.i. (Fig. 4C) or 9.15-fold-
lower titers at 48 h p.i. (Fig. 4D) than Ac79HARep-PG. Together,
these data show a reduction rather than a delay in BV production
in the absence of ac79.
To confirm the defect of BV production in the ac79-knockout
virus and, thus, virus spread, the diameters of randomly selected
plaques were measured at 72 h p.i. Plaques from AcWT-PG-in-
infected cells (P ? 0.001) (Fig. 4E), supporting the defect of
Ac79KO-PG in BV production.
Production of infectious BV from AcWT-PG- and Ac79KO-
from cells infected with Ac79KO-PG compared to those from
AcWT-PG-infected cells could be attributed to a reduction in the
amount of infectious BV. To determine if Ac79KO-PG produced
less infectious BV than AcWT-PG, we first used Q-PCR to deter-
mine the relative number of viral DNA copies produced during
infection. To this end, viral DNA was purified from 5 ? 107PFU
and used as a template for Q-PCR. Using the same units of infec-
tious BV, more viral genomic DNA was amplified in Ac79KO-PG
noblotting was performed using the same amount of infectious
Ac79KO-PG- or AcWT-PG-purified BVs, and blots were probed
was much stronger with proteins from Ac79KO-PG than that with
proteins from AcWT-PG. This suggested that more noninfectious
To evaluate if the lack of ac79 affected the structure of BV,
BV was purified and prepared for visualization by transmission
electron microscopy. The rod-shaped structures and sizes of
Quantitative analysis of viral DNA replication. To assess
Q-PCR was performed. Cells were infected with AcWT-PG or
Ac79KO-PG at an MOI of 5 and collected at selected time points.
Total DNA was purified and used as a template, amplifying gp41
by Q-PCR. DNA replication dynamics between the viruses
showed no significant differences (Fig. 6), confirming that ac79
did not affect viral DNA replication.
Deletion of ac79 does not affect production of VP39 and
polyhedrin. To explore the effects of ac79 on late and very late
FIG 8 Transmission electron microscopy analysis of cells infected with AcWT-PG or Ac79KO-PG and visualized at 72 h p.i. (A) AcWT-PG-infected cell. (B)
Portion of a cell infected with Ac79KO-PG. (C and D) Images of the nuclei of Ac79KO-PG-infected cells showing elongated tubular structures bound to
gold-conjugated VP39 antisera. Bars, 500 nm.
Efficient Infectious BV Production Requires Ac79
May 2012 Volume 86 Number 10jvi.asm.org 5621
FIG 9 Alignment of Ac79 homologs and characterization of Ac79Y24AG26A-PG, Ac79R34K-PG, and Ac79E72D-PG. (A) Alignment of Ac79 homologs
in the Uri motif. The numbers before and after each sequence indicate the first and last amino acids of that sequence, respectively. The amino acids deleted in
Wu and Passarelli
jvi.asm.org Journal of Virology
hedrin from a virus lacking ac79 were compared to those from a
virus carrying ac79. VP39 and polyhedrin were detected from 18
and 24 h p.i., respectively, in either Ac79KO-PG- or AcWT-PG-
did not affect the relative accumulation of VP39 and polyhedrin.
Transmission electron microscopy analyses of cells infected
Thin sections from virus-infected cells collected at 72 h p.i. were
ical characteristics of baculovirus replication, that is, the appear-
ance of rod-shaped nucleocapsids in the electron-dense virogenic
stroma and in bundle formations in the ring zone, microvesicles,
and polyhedra containing embedded ODVs in the ring zone (Fig.
8A). Cells infected with Ac79Rep-PG or Ac79HARep-PG showed
characteristics similar to those of AcWT-PG-infected cells (data
not shown). In general, typical infection characteristics were also
observed for Ac79KO-PG-infected cells (Fig. 8B); however, in
contrast to cells infected with AcWT-PG, Ac79KO-PG-infected
cells contained clusters of elongated tubular structures in the ring
zone (Fig. 8B, arrows). These tubular structures appeared trans-
lucent, suggesting that they lacked DNA. To confirm whether
these structures were capsid assemblages, VP39 antiserum and
gold-conjugated antibodies were applied prior to transmission
electron microscopy visualization. The tubular structures in
Ac79KO-PG-infected cells reacted specifically with VP39 antise-
of noninfectious BV was related to these tubular structures.
Construction and characterization of Ac79Y24AG26A-PG,
Ac79R34K-PG, and Ac79E72D-PG in bacmid DNA-transfected
cells. Iterative database searches revealed significant similarities
among motifs present in UvrC, intron-encoded endonucleases,
and other proteins, including Ac79 (1). This motif consists of
and is referred to as the Uri motif (UvrC and intron-encoded
endonucleases). The tyrosines are present in the GIY-YIG super-
part of the catalytic domain (25). The arginine and glutamic acid
corresponding to amino acids 34 and 72, respectively, in Ac79 are
important for intron-encoded endonuclease catalysis (9, 15). In-
tron-encoded endonucleases and UvrC endonucleases were sug-
gested previously to have the same lineage (10).
The alignment of baculovirus Ac79 orthologs shows that the
GIY-YIG sequence present in intron-encoded endonucleases is
not completely conserved in baculovirus Ac79 orthologs, which
lack the first glycine (Fig. 9A). In Ac79KO-PG, the conserved glu-
tamic acid was deleted, but the rest of the Uri motif was not (Fig.
9A, top line following the alignment). We constructed three re-
combinant bacmids, Ac79Y24AG26A-PG, Ac79R34K-PG, and
Ac79E72D-PG, with mutations in the Uri motif of Ac79 (Fig. 9A,
bottom). These mutations include two residues in the GIY-YIG-
corresponding sequence, arginine 34 and a conserved glutamic
acid at amino acid 72. These viruses expressed the altered Ac79
proteins following the transfection of bacmid DNA into Sf9 cells
(Fig. 9B). To assess whether the mutations affected virus replica-
tion, virus growth curve analyses were performed. Cells were
transfected with bacmid DNA, and BV titers were determined by
increase in virus production was observed with each virus.
However, the virus titer of Ac79E72D-PG was lower through-
out the time course than that of other viruses (Fig. 9C), similar
to the reduction observed for Ac79KO-PG-infected cells.
Ac79Y24AG26A-PG and Ac79R34K-PG were comparable to
the control virus, Ac79HARep-PG (Fig. 9C).
Transmission electron microscopy analysis was also per-
formed to determine whether the mutations affected the virus
DNA-transfected cells showed similar ultrastructural characteris-
tics of baculovirus replication (Fig. 9D). No elongated tubular
structures were found in the ring zone of the transfected cells.
baculovirus genes has increased with the development of bacmid
technology, facilitating the generation of viruses null for genes of
DNA-transfected cells. We sought to characterize ac79 since it is
conserved in many baculoviruses, and orthologs are found in
other viruses and bacteria. ac79 is not an essential gene for virus
replication, and viruses lacking ac79 were able to produce infec-
tious BVs (Fig. 4) and ODVs that were infectious in insects (data
not shown). However, it is required for efficient levels of infec-
10-fold decrease in levels of infectious BV. Since ac79 is not con-
served in all baculovirus genomes, its function may be partially
an advantage to virus replication in some hosts.
of DNA replication, its timing of expression, and its transcription
initiation being at a site other than a late or very late consensus
start site. ac79 does not appear to affect late or very late protein
sion at any phase was affected. Ac79 was detected from 6 to 96 h
p.i. (Fig. 2A). It localized to the nucleus and cytoplasm (Fig. 2C)
but accumulated in the nucleus as the time course of infection
progressed, suggesting a nuclear-specific role during virus repli-
cation. The appearance of tubular structures containing capsid
a nuclear role.
Viruses lacking ac79 produced reduced levels (about 10-fold)
late gene expression appeared to be unaffected, suggesting the
Ac79KO-PG are indicated on the top line of the bottom panel. Ac79Y24AG26A-PG, Ac79R34K-PG, and Ac79E72D-PG are shown below, with altered amino
2, Ac79Y24AG26A-PG; lane 3, Ac79R34K-PG; lane 4, Ac79E72D-PG. (C) BV growth curves determined by TCID50endpoint dilution assays. Titers were
determined from virus present in the supernatants of cells transfected with the indicated recombinant bacmid DNA. (D) Transmission electron microscopy
analysis of cells transfected with Ac79Y24AG26A-PG, Ac79R34K-PG, or Ac79E72D-PG. Bars, 500 nm.
Efficient Infectious BV Production Requires Ac79
May 2012 Volume 86 Number 10 jvi.asm.org 5623
completion of the replication cycle. In addition, the timing of the
onset of infectious BV production was similar for all viruses. To-
gether, these observations suggest that Ac79 may have a role in
sid assembly or virion maturation, the efficient transport of nu-
cleocapsids from the nucleus to the cytoplasm, or virus budding
from the cell.
Previous studies showed that the deletion of the AcMNPV
gp64, ac17, exon0, ac66, me53, or pp31 gene results in a reduction
in the level of BV production but that viral DNA replication re-
process of BV production may differ. Defects in BV production
could be due to defective virus egress; impaired nucleocapsid
within the cell, packaging, or virus assembly. The lack of ac79 did
not appear to affect the egress of BV from the cell or the transport
of nucleocapsids from the nucleus to the cytoplasm, since infec-
tious and noninfectious virions were released (Fig. 4 and 5). In
addition, consistent with the finding that Ac79 was not a struc-
the lack of ac79 did not affect the gross morphology of budded
virions released from the cells (Fig. 5C). This is supported by pre-
vious studies that did not identify Ac79 as a component of BV by
proteomic methods (28).
structures along the inner nuclear membrane. Interestingly, pre-
vious studies that described mutations in the ac53, 38K, vlf-1, and
alkaline nuclease genes also described similar tubular sheaths (18,
transport, nucleic acid resolution, or packaging defects. In addi-
tion, capsid protein-containing tubular structures have been ob-
served following treatment with cytochalasin D, a microfilament
elongation inhibitor, even though viral DNA synthesis was not
affected (27). The interference of cytochalasin D with capsid as-
sembly indicated that microfilaments were involved in this nu-
clear process (27). Given that defects in different genes result in
similar phenotypes, it makes it difficult to determine if ac79 func-
tions in any of these processes or has another function.
A previous study suggested that Ac79 may be related to bacte-
rial DNA repair UvrC excision endonucleases and intron-en-
coded endonucleases, based on the presence of the Uri motif (1).
To explore this further, we compared the Ac79 peptide sequence
to sequences in protein structure databases using HHpred v 2.0
(4). The results showed significant predicted structural similari-
ties between Ac79 and UvrC, bacteriophage T4 endonuclease II,
and the I-TevI intron-encoded endonucleases with the GIY-YIG
domain (data not shown), indicating structural parallels between
similarities, along with the presence of key residues important for
nuclease function, suggest that Ac79 is related to these endonu-
To test the importance of residues conserved between Ac79
and GIY-YIG-containing endonucleases, we constructed viruses
with mutations in the conserved GIY-YIG-corresponding motif
(Ac79 amino acids Y24 and G26) or in residues predicted to par-
ticipate in endonucleolytic catalysis (Ac79 amino acids R34 and
E72). None of the mutants showed tubular capsid-like structures
similar to those observed in Ac79KO-PG-infected cells. It is pos-
sible that the elongated structures were caused by the presence of
the undeleted N-terminal fragment of Ac79 (amino acids 1 to 38)
in Ac79KO-PG, which contained the conserved tyrosines and the
RX3H sequence, which may have hindered the activity of cellular
or viral proteins necessary for proper nucleocapsid formation.
However, this N-terminal peptide does not have a dominant neg-
ative function, since it is also present in the amino acid point
mutants. Among the viruses with point mutations in Ac79, only
the virus with the conservative E72D change showed reduced BV
production. This glutamic acid was also deleted in Ac79KO-PG,
Curiously, we did not observe reduced BV production when Y24
and G26 or R34 were mutated. It is possible that these mutations
were repaired by recombination events, with the N-terminal 38
amino acids remaining at the ac79 locus, even though our exper-
iments were carried out with transfected bacmid DNA to mini-
mize homologous recombination events during reiterative virus
replication cycles. Further work is needed to determine the re-
quirement of Y24, G26, and R34 in infectious BV production. It
appears that the tubular capsid protein-containing structures ob-
in infectious BV production. Although the E72D mutation sug-
gests that endonucleolytic activity is important for infectious BV
production, additional experiments will be required to further
define this role.
Mutations in baculovirus DNA replication and processing
genes result in altered capsid protein-containing structures, sug-
gesting that viral DNA affects the nucleocapsid architecture (23).
We tested whether there is an interaction between Ac79 and
VLF-1, which is involved in viral genome processing, hypothesiz-
ing that Ac79 may provide the endonuclease activity needed dur-
interaction by coimmunoprecipitation (our unpublished data).
Although Ac79KO-PG produces elongated capsid protein-con-
taining structures, it also produces normal nucleocapsids in both
Ac79 functions similarly to UvrC, cleaving phosphodiester bonds
to reutilize nucleotides for its DNA synthesis, or whether it func-
tions in creating double-strand breaks characteristic of a homing
determine if Ac79 has endonucleolytic activity and to further de-
fine its role during BV production.
This research was supported by U.S. Department of Agriculture award
We thank Rollie Clem for valuable discussions.
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Efficient Infectious BV Production Requires Ac79
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