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Published Ahead of Print 26 June 2013.
2013, 87(17):9928. DOI: 10.1128/JVI.01259-13. J. Virol.
Peter Tijssen
Perreault, Hiroko Shike, Jozsef Szelei, Max Bergoin and
Hanh T. Pham, Françoise-Xavière Jousset, Jonathan
Densovirus
Expression Strategy of Aedes albopictus
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Expression Strategy of Aedes albopictus Densovirus
Hanh T. Pham,
a
Françoise-Xavière Jousset,
b
Jonathan Perreault,
a
Hiroko Shike,
c
Jozsef Szelei,
a
Max Bergoin,
a
Peter Tijssen
a
INRS-Institut Armand-Frappier, Laval, Québec, Canada
a
; Laboratoire de Pathologie Comparée, Université Montpellier II, Montpellier, France
b
; Division of Clinical Pathology,
Milton S. Hershey Medical Center, Pennsylvania State University, Hershey, Pennsylvania, USA
c
The transcription map of the Aedes albopictus densovirus (AalDNV) brevidensovirus was identified by Northern blotting, rapid
amplification of cDNA ends (RACE) analysis, and RNase protection assays. AalDNV produced mRNAs of 3,359 (NS1), 3,345
(NS2), and 1,246 (VP) nucleotides. The two overlapping P7/7.4 NS promoters employed closely located alternate transcription
initiation sites, positioned at either side of the NS1 initiation codon. All NS mRNAs coterminated with VP mRNA. All promot-
ers, explored using luciferase assays, were functional in insect and human cell lines.
I
nvertebrate parvoviruses (densoviruses [DNVs]) are subdivided
into four genera: Densovirus, Pefudensovirus, Iteravirus, and
Brevidensovirus (1–3). Brevidensoviruses have a 4.1-kb single-
stranded DNA (ssDNA) genome with three open reading frames
(ORFs) on the same strand (about 790 [NS1], 360 [NS2], and 350
[VP] amino acids). The genome of Aedes albopictus densovirus
(AalDNV) (GenBank accession no. NC_004285) has terminal, T-
shaped hairpins (4). Brevidensoviruses were isolated from medi-
cally important mosquito vectors, such as Aedes albopictus
(AalDNV) (4), Aedes aegypti (AaeDNV) (5), and Anopheles gam-
biae (AgDNV) (6). AalDNV was isolated from Aedes albopictus
C6/36 cells (7) but is infectious for Aedes aegypti larvae (8, 9).
AaeDNV expression has been studied to some extent (10, 11).
ORF
NS1
of AaeDNV was reported to have a 57-amino-acid N-ter-
minal extension compared to that of AalDNV (4, 5). -Galactosi-
dase fusion proteins with the three ORFs were enzymatically ac-
tive, except for NS1 (12) unless NS1--gal was constructed
downstream of the corresponding AalDNV AUG
NS1
(11). Primer
extension demonstrated that AaeDNV VP transcription started at
nucleotide (nt) 2402 (10). Here, AalDNV transcription was ana-
lyzed by Northern hybridization, 5= and 3= rapid amplification of
cDNA ends (RACE), amplicon mapping, and RNase protection
assays (RPAs). AalDNV promoter activities were also determined.
AalDNV transcripts were obtained after transfection of
pCR2.1-AalDNV (containing the AalDNV genome of 4,176 nt [4]
between its EcoRI sites), using Lipofectamine (Invitrogen proto-
col), in permissive C6/36 cells (7) grown in RPMI medium sup-
plemented with 5% fetal bovine serum (FBS). After 48 h, RNA was
isolated using the Stratagene Absolutely RNA miniprep kit.
Northern blotting.
32
P-labeled RNA probes for Northern
blots targeted overlapping domains of the NS1 and NS2 ORFs or
VP ORF. PCR-amplified products (primers in Table 1) were tran-
scribed in vitro with [␣-
32
P]UTP and T7 RNA polymerase (NEB)
(13). Northern blots using 10 g of total RNA (13, 14) revealed
transcript sizes of 3.4 and 1.3 kb with the VP-specific probe and a
3.4-kb transcript with the NS-specific probe (Fig. 1A).
RACE experiments. The FirstChoice RLM RACE kit (Invitro-
gen) was used to identify the 5= starts and 3= ends of the polyadenyl-
ated transcripts. The 5= adapter primer (IP) and the 3=-anchored
primer (AP) (in the kit) were used in PCR with AalDNV-specific
primers (Table 1), as shown in Fig. 1B. Amplicons obtained with
A626R/IP (⬃325 bp), A497R/IP (⬃200 bp), A2740R/IP (⬃350 bp),
A2540R/IP (⬃150 bp), A3162F/AP (⬃600 bp), and A3461F (⬃300
bp) (data not shown) were sequenced. Sequencing revealed that NS1
transcription started at nt 329 (AGTA), 6 nt upstream of AUG
NS1
,
and that VP transcription started at nt 2441 (CAGTCG), 158 nt up-
stream of AUG
VP
(Fig. 1B) (sequence data not shown). Sequencing of
the 300- and 600-bp amplicons showed a transcription termination
position at 3680, 18 nt downstream of the polyadenylation signal at
position 3662 (Fig. 1C) (sequence data not shown).
As is common for other densoviruses, NS2 could be translated
from nt 411 by leaky scanning (13) on the NS1 transcript. The
short untranscribed region of NS1 (UTR
NS1
), in spite of a consen-
sus AnnAUGG sequence for NS1 initiation, could favor it (13). A
canonical initiator Inr
NS2
sequence (CAGT) is located at nt 342.
After TfiI digestion (NEB) at nt 338 of the A831R/IP PCR product,
followed by a nested PCR (A626R/IP or A497R/IP) for specific
amplification of putative NS2 transcripts, bands of 330 and 200 nt
were produced (data not shown). Their sequencing revealed that
NS2 transcription started at nt 343 (sequence data not shown).
Thus, NS1 and NS2 AalDNV transcription starts were separated
by 14 nt on either side of AUG
NS1
.
Amplicon mapping. In order to confirm that NS and VP tran-
scripts all coterminated at position 3680, primer A2380F (up-
stream of the VP transcript) and two reverse primers, A3650R
(upstream) and A3744R (downstream), of the VP transcript end
were designed for reverse transcription (RT)-PCR amplification
(Table 1). Only a band of about 1,300 nt was obtained using the
A2380F/A3650R set of primers for NS transcripts (Fig. 1C).
RNase protection assays. RPAs were employed to confirm
NS1, NS2, and VP transcription starts. RPA probes for NS and VP
transcripts were prepared by PCR (Table 1; Fig. 2A), and in vitro
transcription as described for Northern blot probes. For positive
controls, a sense RNA was generated by in vitro transcription and
RPA was performed in parallel: for NS, two positive-control RNAs
spanning from nt 329 to 439 (predicted for NS1) and nt 343 to 439
(predicted for NS2) were used, and for VP one from nt 2441 to
2542 was used. The probes and control RNA were purified from
polyacrylamide gel and used in the RPA III kit (Invitrogen). Size
markers were generated according to the 5= RACE results. The NS
Received 9 May 2013 Accepted 18 June 2013
Published ahead of print 26 June 2013
Address correspondence to Peter Tijssen, peter.tijssen@iaf.inrs.ca.
Copyright © 2013, American Society for Microbiology. All Rights Reserved.
doi:10.1128/JVI.01259-13
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probe protected the expected 96 and 110 nt of the NS transcript 5=
ends (Fig. 2B), confirming NS1 and NS2 mRNA start positions.
Similarly, the VP probe confirmed the VP transcript 5= extremity
by protecting 101 nt (Fig. 2B). A protected band at 219 nt, slightly
smaller than the entire probe (236 nt, including 17 nt of unspecific
sequence), showed the coexistence of NS transcripts.
Promoter activity. NS (P7/7.4) and VP (P60) promoter re-
gions were amplified by PCR (primers in Table 1) and cloned into
TABLE 1 Primers used for Northern blot probes, RACE, amplicon mapping, and RPAs
Primer name
a
Sequence Purpose
A639F GCTCCAGAGCCTCTGAACAGCTTG NS probe, Northern blot
A1345R (⫹T7 sequence) TAATACGACTCACTATAGGGGTTCTGACTCTTGTGCTGTTTC
NS probe, Northern blot
A3106F CTAGAAACAGTTGCAGCAACCGGAC VP probe, Northern blot
A3509R (⫹T7 sequence) TAATACGACTCACTATAGGCGTACTTGATATCTGAATTTCATG
VP probe, Northern blot
A3372F AACTACAACATATGCCACGTCAG 3= RACE
A3461F ACAAGTTCCAGACGAAACAGG 3= RACE
A497R GTTCGTAATTGTTGGCATTCCT 5= RACE of NS
A626R GTGGGTAGATGTTATCAACGG 5= RACE of NS
A831R CTTGCCTGTGACCCGTTATTATCC 5= RACE of NS
A2540R GTGCGTTGTCTTCTTCTTCTATC 5= RACE of VP
A2740R GACCAAACATTACGGAAATGG 5= RACE of VP
A3126F CGGACCATTAGCACAACAAAC 3= RACE
A2380F GAGTATACAACACAGAGAAG Amplicon mapping
A3650R TCATAA GGCATACATGCTAC Amplicon mapping
A3744R TCTGTCGTGGACATTATCAG Amplicon mapping
A272F (⫹UTS) GCGATGAATGAACACTGAATCCACCACCACATGATCC
RPA NS probe
A329F (⫹T7 sequence) TAATACGACTCACTATAGGAGTAGTATGGAATCAG RPA-positive control for NS1
A343F (⫹T7 sequence) TAATACGACTCACTATAGGGTCTGCAGTGAACATTCG
RPA-positive control for NS2
A439R TCTCCTCCTGGATTTACACTG RPA-positive control for NS1 and NS2
A439R (⫹T7 sequence) TAATACGACTCACTATAGGTCTCCTCCTGGATTTACACTG
RPA NS probe
A2323F (⫹UTS) GCGATGAATGAACACTGGCATATGAACGAAACCTCAC
RPA VP probe
A2441F (⫹T7 sequence) TAATACGACTCACTATAGGAGTCGGCCACCAGGTCTTGTAG
RPA-positive control for VP
A2542R ATGTGCGTTGTCTTCTTCTTC RPA-positive control for VP
A2542R (⫹T7 sequence) TAATACGACTCACTATAGGATGTGCGTTGTCTTCTTCTTC RPA VP probe
A148F TCCAATTGGAACACACGGAC P7/7.4 promoter for AalDNV
A333R CTACTGACTCTCCCTTC P7/7.4 promoter for AalDNV
A2431F CAAACTCATCAGTCGGCCAC P60 promoter for AalDNV
A2597R CCTCTGCTTCTTCTTTTGC P60 promoter for AalDNV
a
In the primer names, “UTS” represents the unspecific target sequence and “R” and “F” represent sense and antisense, respectively. The numbers in the primer names indicate the
5= end of the primer sequence in AalDNV. The underlined sequences represent the UTS or T7 sequences.
FIG 1 (A) Northern blot analysis of AalDNV transcription 48 h after transfection with vectors with virus-specific inserts (lanes 1) or without inserts (lanes 2).
The VP probe also detected bands corresponding to the size of NS transcript indicating that VP and NS transcripts may coterminate. (B) Strategy of 5= and 3=
RACE. Above the ORFs are the specific primers that were designed, and below are the ORFs and IP and AP primers from the Invitrogen kit (indicated with
arrows). We took advantage of the TfiI restriction site between the putative Inr of NS1 and NS2 to distinguish between these transcripts. (After digestion, only
amplicons from the second Inr would be obtained.) (C) Confirmation of termini of NS transcripts of AalDNV. The diagrams represent transcript map results by
5= and 3= RACE. Forward primers that do not recognize VP transcripts and reverse primers, both upstream and downstream of the VP transcript end (3= RACE),
were used in a PCR (indicated in the diagrams). As expected, only the inboard reverse primer (A3650R in lane 1) gave a product. M, markers; lane 2,
A2380F/A3744R.
Expression of the AalDNV Brevidensovirus
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EcoRV in pGL4.20, upstream of the luciferase gene (Promega;
GenBank accession no. DQ188840) to estimate their functional-
ity. Those with the reverse promoter orientation, shown by se-
quencing, served as controls. The Mythimna loreyi (MlDNV) NS
promoter (an insect virus replicating in LD652 cells) (15) and the
simian virus 40 (SV40) promoter were also cloned into pGL4.20
vectors to serve as positive controls. Transfection of LD652 using
DOTAP (Roche) and HeLa cells using Lipofectamine (Invitrogen)
in 24-well plates was performed according to the suppliers’ in-
structions. Cells were harvested 40 h posttransfection and resus-
pended in 100 l of Bright-Glo lysis buffer (Promega), and rela-
tive luciferase activity was determined according to Promega’s
instructions. The AalDNV P7/7.4 and P60 promoters were func-
tional both in insect cells and in human cells, in contrast to con-
structs lacking a promoter or with promoters in the reverse orien-
tation, whereas the SV40 promoter preferred HeLa cells and the
MlDNV promoter preferred insect cells (Fig. 2C).
(i) Promoter 7/7.4 elements. The RACE experiments and
RPA (Fig. 2) revealed the location of Inr1 and Inr2 at either side
of the NS1 protein initiation codon (Fig. 3C). Consequently,
the NS1 transcript could potentially be translated into both
NS1 and NS2 proteins, whereas the NS2 transcript could only
code for the NS2 protein. Different constructs, leaving Inr1 and
its upstream elements intact (for NS1 transcription) but with
mutations in the NS2 promoter and NS1/2 translation ele-
ments, were made with a luciferase reporter gene (Fig. 3B and
C). Constructs containing the intact luciferase AUG (F1 to F7)
served as a positive control for transcription, whereas those
lacking the luciferase AUG (F1⫺ to F7⫺) showed the impact of
the various mutations.
P7/7.4 consisted of three segments (Fig. 3B). The KpnI-SacI
segment I contained upstream promoter elements, the SacI-PstI
segment II included both TATA boxes, ATG
NS1
, and both Inrs
and, finally, the PstI-HindIII segment III (about 20 nt for the
NS1 ORF and about 70 nt for the NS2 ORF) contained the
downstream promoter elements (DPE) and ATG
NS2
. These
segments were connected in pBluescript (PCR with primers
containing appropriate restriction sites and pCR2.1-AalDNV
template) and were transferred as a whole (or after mutation)
to the pGL4.20 luciferase reporter. The diagram in Fig. 3C
delineates the different constructs and knockouts (using the
Transformer kit according to Clontech’s instructions) (Fig.
3C). Certain ATGs were mutated to TTG, Inr (CATG) was
mutated to GCCG, and the TATA box of NS2 was mutated to
GCTCGAG. In addition, alternates of F1 to F7 that lacked the
luciferase initiation codon (F1⫺ to F7⫺) as well as two constructs
from which the NS2 core promoter was mutated (F2* and F5⫺*)
were obtained. C6/36 and 293T (as for HeLa) cells were trans-
fected, and luciferase activity was determined. Except for F2* and
F5⫺*, all constructs were expected to yield both NS1 and NS2
mRNAs. The observed luciferase activity in C6/36 and 293T cells
(Fig. 3E) corresponded well with the expected luciferase activities,
summarized in Fig. 3D, from (i) fusion proteins with NS1 (F1 and
F1⫺) and NS2 (F5 and F5⫺), (ii) directly from the luciferase
FIG 2 (A) Diagram of AalDNV expression and location of probes. (B) RNase protection assay of starts of NS and VP transcripts of AalDNV. Lanes: 1, NS probe
with a specific length of 184 nt and short nonspecific extra terminal sequences; 2, positive control for NS1 with specific size obtained with RACE experiments; 3,
positive control for NS2 with specific size obtained with RACE experiments; 4, total RNA from C6/36; 5, total RNA from AalDNV-infected C6/36 (15 g); 6, total
RNA from AalDNV-infected C6/36 (40 g); 7, VP probe; 8, positive control for VP; 9, total RNA from C6/36; 10, total RNA from AalDNV-infected C6/36 (15
g); 11, total RNA from AalDNV-infected C6/36 (40 g). The band at 101 nt confirmed the VP start, and the band at 219 nt represented protection of the VP
probe on the NS transcript. (C) Luciferase activity 40 h after transfection with different promoter constructs as fold increase over background (transfected pGL4
without insert). Inverse orientation of the promoters did not increase activity significantly over background. SV40 (not shown) and the insect virus MlDNV NS
promoter (Ml-NSP) had a strong preference for cells from vertebrates and invertebrates, respectively. Surprisingly, AalDNV had significant activity in both types
of cells.
Pham et al.
9930 jvi.asm.org Journal of Virology
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initiation codon (F1 to F7), or (iii) after leaky scanning (for F1, F2,
and F5 to F7). Therefore, individual elements of both NS1 and
NS2 promoters contributed to mRNA expression.
Nevertheless, NS2 could be generated on the NS1 transcript by
leaky scanning. Blocking NS2 transcription by mutating its core
promoter in F5⫺* yielded an ⬃90% drop in luciferase activity in
C6/36 cells compared to F5⫺. The remaining activity could be
ascribed to a fusion protein from ATG
NS2
on the NS1 transcript
since the introduction of an NS2 stop codon in frame after the NS2
initiation codon in F6⫺ or knocking out ATG
NS2
in F7⫺ abol
-
ished luciferase activity. Consequently, for F2* the remaining lu-
ciferase activity was due mainly to leaky scanning on the NS1
transcript and initiation from ATG
luc
. In conclusion, although
NS1 and NS2 mRNAs have their own promoter elements in p7/
7.4, NS1 mRNA contributes significantly through leaky scanning
to NS2 expression.
In conclusion, Northern blotting, RACE, amplicon mapping,
and RPA results were all in agreement. AalDNV used one pro-
moter region with closely overlapping elements to start transcrip-
tion of NS1 and NS2 at positions that are just 14 nt apart at either
side of ATG
NS1
. No clear TATA-like motif sequences were found
upstream of the initiator sequence CAGT of the VP of AalDNV
and AaeDNV, suggesting that these promoters were under the
control of DPE (16, 17). This regulatory circuit is likely to be one
FIG 3 Analysis of AalDNV P7/7.4 promoter elements using a luciferase reporter gene. (A) Diagram of ORFs in AalDNV, where nt 77 to 423 contain typical
sequences of core promoter elements for NS1 and NS2. (B) Three segments (I to III) were connected via pBluescript in pGL4 and could be individually mutated
and swapped. (C) In the constructs, filled boxes represent elements thought to direct NS1 mRNA expression and open boxes those thought to direct NS2 mRNA
expression, filled triangles represent transcript starts that were established, crosses represent knockouts, and gray boxes represent replaced sequences. Addition-
ally, mutants were made for all constructs in which the initiation codon of luciferase was knocked out (F1⫺ to F7⫺), and in 2 constructs TATA2 and Inr2 (for
NS2 transcripts) were knocked out (indicated by *). (D) Expected activities using the luciferase reporter gene in the pGL4 vector. (“l.s.” represents luciferase
activity if leaky scanning occurs.) (E) The observed luciferase activity matched the expected activity, except for the very low F7⫺ activity (none expected). These
results indicated that the NS initiation codons, and hence the two sets of promoter elements for transcription, were individually important for expression but
nevertheless leaky scanning on the NS1 transcript could also contribute to the expression of NS2.
Expression of the AalDNV Brevidensovirus
September 2013 Volume 87 Number 17 jvi.asm.org 9931
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means by which insect virus networks can transmit transcriptional
signals, such as those from DPE-specific and TATA-specific en-
hancers, via distinct pathways (18), to regulate NS and VP expres-
sion.
ACKNOWLEDGMENTS
This work was supported by a grant from the Natural Sciences and Engi-
neering Research Council of Canada to P.T. and scholarships from the
Agence Universitaire de la Francophonie and the Fondation Armand-
Frappier to H.T.P.
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