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
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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