Vaccinia virus A6 is essential for virion membrane biogenesis and localization of virion membrane proteins to sites of virion assembly.
ABSTRACT Poxvirus acquires its primary envelope through a process that is distinct from those of other enveloped viruses. The molecular mechanism of this process is poorly understood, but several poxvirus proteins essential for the process have been identified in studies of vaccinia virus (VACV), the prototypical poxvirus. Previously, we identified VACV A6 as an essential factor for virion morphogenesis by studying a temperature-sensitive mutant with a lesion in A6. Here, we further studied A6 by constructing and characterizing an inducible virus (iA6) that could more stringently repress A6 expression. When A6 expression was induced by the inducer isopropyl-β-D-thiogalactoside (IPTG), iA6 replicated normally, and membrane proteins of mature virions (MVs) predominantly localized in viral factories where virions were assembled. However, when A6 expression was repressed, electron microscopy of infected cells showed the accumulation of large viroplasm inclusions containing virion core proteins but no viral membranes. Immunofluorescence and cell fractionation studies showed that the major MV membrane proteins A13, A14, D8, and H3 did not localize to viral factories but instead accumulated in the secretory compartments, including the endoplasmic reticulum. Overall, our results show that A6 is an additional VACV protein that participates in an early step of virion membrane biogenesis. Furthermore, A6 is required for MV membrane protein localization to sites of virion assembly, suggesting that MV membrane proteins or precursors of MV membranes are trafficked to sites of virion assembly through an active, virus-mediated process that requires A6.
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ABSTRACT: Essential vaccinia virus genes are often studied with conditional-lethal inducible mutants. Here, we constructed a deletion mutant of the essential H7R gene (ΔH7) with a H7-expressing cell line. Compared to an inducible H7 mutant, ΔH7 showed defect at an earlier step of virion membrane biogenesis, before the development of short crescent-shaped precursors of viral envelope. Our studies refine the role of H7 in virion membrane biogenesis and highlight the values of analyzing deletion mutants.Journal of Virology 05/2013; · 4.65 Impact Factor
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ABSTRACT: Vaccinia virus (VACV) has achieved unprecedented success as a live viral vaccine for smallpox which mitigated eradication of the disease. Vaccinia virus has a complex virion morphology and recent advances have been made to answer some of the key outstanding questions, in particular, the origin and biogenesis of the virion membrane, the transformation from immature virion (IV) to mature virus (MV), and the role of several novel genes, which were previously uncharacterized, but have now been shown to be essential for VACV virion formation. This new knowledge will undoubtedly contribute to the rational design of safe, immunogenic vaccine candidates, or effective antivirals in the future. This review endeavors to provide an update on our current knowledge of the VACV maturation processes with a specific focus on the initiation of VACV replication through to the formation of mature virions.Viruses 10/2014; 6(10):3787-3808. · 3.28 Impact Factor
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ABSTRACT: Crescents, consisting of a single lipoprotein membrane with an external protein scaffold, comprise initial structural elements of poxvirus morphogenesis. Crescents enlarge to form spherical immature virions, which enclose viroplasm consisting of proteins destined to form the core of mature virions. Previous studies suggested that the L2 protein participates in recruitment of endoplasmic reticulum-derived membranes to form immature virions within assembly sites of cytoplasmic factories. Here we showed that L2 interacts with the previously uncharacterized 42-amino acid A30.5 protein. An open reading frame similar in size to the one encoding A30.5 was at the same genome location in representatives of all chordopoxvirus genera. A30.5 has a putative transmembrane domain and co-localized with markers of the endoplasmic reticulum and with L2. By constructing a complementing cell line expressing A30.5, we isolated a deletion mutant virus that exhibited a defect in morphogenesis in normal cells. Large, electron dense, cytoplasmic inclusions and clusters of scaffold protein-coated membranes that resembled crescents and immature virions devoid of viroplasm were seen in place of normal structures. Crescent-shaped membranes were continuous with the endoplasmic reticulum membrane and oriented with the convex scaffold protein-coated side facing the lumen, while clusters of completed spherical immature virion-like forms were trapped within the expanded lumen. Immature virion-like structures were more abundant in infected RK-13 cells than in BS-C-1 or HeLa cells, in which cytoplasmic inclusions were decorated with scaffold protein-coated membrane arcs. We suggest that the outer surface of the poxvirus virion is derived from the luminal side of the ER membrane.Journal of Virology 09/2013; · 4.65 Impact Factor
Vaccinia Virus A6 Is Essential for Virion Membrane Biogenesis and
Localization of Virion Membrane Proteins to Sites of Virion Assembly
Xiangzhi Meng, Addie Embry, Lloyd Rose, Bo Yan, Chungui Xu, and Yan Xiang
Department of Microbiology and Immunology, The University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA
family of large, complex viruses that replicate entirely in the cyto-
plasm (29). The best-characterized family member is vaccinia vi-
rus (VACV), which encodes more than 200 proteins in a 190-kb
intermediate steps that are discernible by electron microscopy
cytoplasmic DNA staining in fluorescence microscopy and by a
The viral structures that appear first in the factories are electron-
dense viroplasms containing viral core proteins. Crescent-shaped
membranes consisting of a single lipid bilayer stabilized with a
lattice of VACV D13 protein (11, 41) then develop at the periph-
eries of viroplasms. The crescent membranes engulf part of the
viroplasm and circularize to form the spherical immature virions
(IV). The viral genome is encapsidated in IV before the IV mem-
brane completely closes off, forming IV with an electron-dense
nucleoid (IVN). Concomitant with proteolytic processing of sev-
eral major virion core proteins, including A10 (17, 36), IVNs ma-
ture into the brick-shaped intracellular mature virions (MV),
which are the majority of infectious particles produced during
Many details, as well as underlying mechanisms, of the virion
morphogenesis process remain enigmatic. A longstanding ques-
tion has been the origin and biogenesis of the crescent-shaped
membranes that ultimately become the primary envelope of
VACV. The apparent lack of continuity of crescent membranes
with cellular membranes initially prompted a model in which the
crescent membranes are synthesized de novo (5). More recent
models, however, suggest that the crescent membranes are ac-
he majority of enveloped viruses obtain their lipid envelope
ment between the endoplasmic reticulum and Golgi apparatus
(ERGIC) (33, 40) or the endoplasmic reticulum (ER) (13). Con-
the MV envelope are synthesized on the ER (13, 37), and a path-
way exists for the trafficking of MV membrane proteins from the
ER to IV (13). However, it is unclear whether precursors of MV
membranes or MV membrane proteins need to be actively traf-
(16) and no specific signal is required for MV membrane protein
A9 to be incorporated into IV (14).
conditional-lethal mutants as essential for viral membrane bio-
genesis. F10 (42, 43), A11 (32), H7 (38), and L2 (21) are required
at an early stage, as repression of their individual expression re-
required at a later stage, as repression of A14 or A17 expression
tures at the boundaries of large viroplasm inclusions. A defect in
ature-sensitive (ts) mutants with a lesion in G5 (3) or H5 (7).
However, G5 does not directly participate in crescent membrane
biogenesis, as normal IV formation was observed in a G5R dele-
tion mutant (39). H5 may also affect membrane formation indi-
Received 7 February 2012 Accepted 28 February 2012
Published ahead of print 7 March 2012
Address correspondence to Yan Xiang, email@example.com.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
0022-538X/12/$12.00 Journal of Virologyp. 5603–5613jvi.asm.org
rectly, as it plays a role in the transcription of viral genes and
replication of the viral genome (6, 19).
VACV A6 is a minor component of the virion core (1) and is
A6 plays an essential role in virion morphogenesis by studying a
temperature-sensitive A6 mutant (24). Because the temperature-
sensitive mutant synthesizes a small amount of A6 at a nonper-
more stringently represses A6 expression. The characterization of
the inducible A6 mutant showed that A6 is required for an early
step in viral membrane biogenesis. Furthermore, we found that
A6 is required for localization of several major MV membrane
or precursors of MV membranes are trafficked to viral factories
through a virus-mediated process.
MATERIALS AND METHODS
medium with Earle’s salts supplemented with 10% fetal bovine serum
(FBS). Baby hamster kidney (BHK) cells and HeLa cells were maintained
monoclonal antibodies (MAbs) against V5 (Sigma-Aldrich), PDI (Enzo
Life Sciences), ERGIC53 (Enzo Life Sciences), and Golgin 97(Invitrogen)
purchased from the vendors. Murine MAbs against VACV proteins A10
(26) (BG3 clone), A13 (48) (11F7), A14 (26) (FE11), D8 (26) (BD6), H3
previously. The murine MAb against I1 was derived from a mouse in-
fected with VACV, similar to that described previously (48). Polyclonal
mouse sera against L1 or F9 were obtained from mice immunized with
recombinant L1 fused with glutathione S-transferase (GST) or F9 fused
with maltose-binding protein (MBP), respectively.
Plasmids and viruses. The plasmid pVote-A6L-V5 was constructed
epitope tag from pLJ2 (24), followed by insertion into NcoI and BamHI
sites of pVote1 (46). vYB21, a VACV with the inducible A6L-V5 gene at
through homologous recombination of pVote-A6L-V5 with vT7LacOI
(provided by Bernard Moss ). vYB21 was isolated after three rounds
of plaque purification under selection conditions for guanine phosphori-
bosyl transferase (GPT) according to a standard protocol (8).
pA5-GFP-A7, the transfer plasmid for replacing the endogenous A6L
gene with a green fluorescent protein (GFP) gene, was constructed as
follows. Approximately 500-bp left and right flanking sequences of A6L
were PCR amplified from genomic DNA of WR virus with primer pairs
5=-CGGGATCCTTAGTTGTTTAATTTATTTGTGC-3= plus 5=-CCCGA
TAAGCTTTACGAACTACATCTGATATTATT-3= and 5=-CGGGAGCT
CCGCTGTTCAAAGTCTTATCAAATTCA-3=, respectively. The PCR
products were then sequentially cloned into pYW31 (25) by using the
restriction enzyme sites underlined in the primer sequences (BamHI,
HindIII, SacI, and NotI) to flank the open reading frame (ORF) of GFP
under the control of the P11 late promoter. Isopropyl-?-D-thiogalacto-
side (IPTG)-inducible, conditional-lethal A6L virus (iA6)-GFP was con-
structed through homologous recombination of pA5-GFP-A7 with
vYB21 and was isolated after three rounds of plaque purification of GFP-
expressing virus in the presence of 500 ?M IPTG.
pA5-GUS-A7, the transfer plasmid for replacing the endogenous A6L
gene with ?-glucuronidase (GUS), was constructed by replacing the GFP
cassette between BamHI and NotI sites in pA5-GFP-A7 with a cassette of
GUS between BamHI and NotI sites of pBSgptgus (15). iA6-GUS was
constructed through homologous recombination of pA5-GUS-A7 with
vYB21 and was isolated after three rounds of plaque purification of GUS-
expressing viruses in the presence of 500 ?M IPTG, according to a stan-
in the presence of 100 ?M IPTG.
Virus yield determination. BS-C-1 cells in a 12-well tissue culture
of infection (MOI) of 5 PFU per cell. After 1 h of adsorption at room
temperature, the inoculum was removed and the cells were washed with
DMEM plus 1% FBS. The infected cells were incubated with medium in
the presence or absence of various concentrations of IPTG. At various
times after infection, cells were harvested and viral titers in the cell lysates
were determined by plaque assay in the presence of 100 ?M IPTG.
Western blot analysis. Western blot analysis was performed as previ-
ously described (25). Briefly, the samples were solubilized in sodium do-
decyl sulfate (SDS) sample buffer, resolved by SDS-polyacrylamide gel
electrophoresis (PAGE), transferred to nitrocellulose membranes, and
blocked with Tris-buffered saline supplemented with 5% nonfat dried
luminescence reagent (Pierce).
Fluorescence microscopy. BHK or HeLa cells grown on coverslips
were infected at 0.5 PFU/cell in the presence or absence of 100 ?M IPTG.
for 20 min, permeabilized with 0.5% saponin (Sigma-Aldrich) for 5 min,
1 h and an appropriate secondary antibody for an additional hour. The
DNA was stained with DAPI (4=,6-diamidino-2-phenylindole) (Invitro-
gen). The coverslips were imaged with an Olympus FV-1000 laser scan-
ning confocal system.
Subcellular fractionation. Subcellular fractionation was performed
similarly to the method described by Earley et al. (9). HeLa cells grown in
a 150-mm dish were infected with the virus at an MOI of 5 PFU per cell.
After 2 h of adsorption at room temperature, the inoculum was replaced
with fresh medium with or without 100 ?M IPTG. At 8 h p.i., the cells
were harvested by scraping them into medium, collected by centrifuga-
(0.25 M sucrose, 1 mM EDTA, 10 mM HEPES, pH 7.4) containing 0.2
(Roche Diagnostics), and broken by passage through a 27-gauge syringe
needle 10 times. The nuclei and unbroken cells were removed by centrif-
ugation at 2,400 ? g for 5 min. The postnuclear supernatant was loaded
onto a 10.5-ml preformed 5 to 25% continuous iodixanol gradient (Axis
Shield) and centrifuged at 200,000 ? g for 2.5 h at 4°C. After centrifuga-
in each fraction was precipitated with trichloroacetic acid and washed
buffer, resolved by SDS-PAGE, and processed for Western blotting as
Electron microscopy. For transmission electron microscopy, BS-C-1
cells in 60-mm-diameter dishes were infected with 1 PFU/cell of
for transmission electron microscopy by the Electron Microscopy Core
Construction of iA6. We used the vT7LacOI and pVote system
developed by Ward and coworkers (46) to construct two recom-
binant viruses that expressed A6 only in the presence of inducer
IPTG. The recombinant viruses were constructed in two steps.
First, an intermediate recombinant virus that contains both an
inducible A6L gene and the endogenous A6L gene was con-
structed. Then, the endogenous A6L gene of the intermediate vi-
rus was replaced with either Green fluorescent protein (GFP) or
?-glucuronidase (GUS) by homologous recombination in the
Meng et al.
jvi.asm.orgJournal of Virology
in an IPTG dose-dependent manner, as determined by Western
blotting with an antibody against the V5 epitope tag appended to
greater than 100 ?M, in addition to the full-length A6 protein, a
smaller protein of approximately 25 kDa was also detected by the
sample preparation. In the absence of IPTG, the inducible viruses
did not express a detectable level of A6 (Fig. 1A), produce any
D). The yield of inducible viruses at 24 h p.i. depended on the
?M IPTG (Fig. 1B). In the presence of 100 ?M IPTG, the growth
kinetics of the inducible viruses was similar to that of the parental
vT7LacOI in one-step growth experiments, achieving maximum
amplification in titers (?2 log) at 24 h p.i. (Fig. 1D). The replica-
specifically to repression of A6, as it could be rescued by transfec-
tion of a plasmid containing A6L under the control of its natural
FIG 1 Characterization of IPTG-inducible A6 VACVs. (A) IPTG-dependent
an MOI of 5 PFU/cell in the presence of various concentrations of IPTG. The
cells were harvested at 8 h p.i. and analyzed by Western blotting (WB) with a
MAb against the V5 epitope appended to the C terminus of A6. (B) Plaque
morphology of A6L inducible viruses. BS-C-1 cells were infected with
IPTG for 48 h. The cells were fixed and stained with crystal violet. (C) IPTG-
dependent replication by iA6-GFP. BS-C-1 cells were infected with vT7lacOI
or iA6-GFP at an MOI of 5 PFU/cell in the presence of various concentrations
of IPTG. After 24 h, the cells were harvested, and the viral yields were deter-
mined by plaque assay in the presence of 100 ?M IPTG. (D) One-step growth
curves of iA6-GFP and iA6-Gus. BS-C-1 cells were infected at an MOI of 5
PFU/cell with vT7lacOI, iA6-GFP, or iA6-Gus. The infection by iA6-GFP and
iA6-Gus was performed either in the presence (?IPTG) or the absence of 100
virus titers were determined by plaque assay in the presence of 100 ?M IPTG.
FIG 2 iA6-GFP is defective in virion morphogenesis in the absence of IPTG.
BS-C-1 cells were infected with iA6-GFP at an MOI of 1 PFU/cell in the pres-
prepared for transmission electron microscopy. Panels B, D, and F are higher
magnifications of the boxed areas in panels A, C, and E, respectively. Note the
short membrane arcs (arrow) at the peripheries of viroplasm inclusions in
panels E and F but not in panels C and D. N, nucleus; MV, mature virion; C,
crescent; IV, immature virion; V, viroplasm.
Vaccinia Virus Virion Membrane Formation
May 2012 Volume 86 Number 10 jvi.asm.org 5605
promoter (data not shown). Metabolic labeling of infected cells
with [35S]methionine and [35S]cysteine showed no gross differ-
ence in viral protein synthesis in the presence or absence of IPTG
(data not shown), consistent with previous observations with the
temperature-sensitive A6L mutant (24).
A6 is required for crescent membrane formation. We then
studied the effect of A6 repression on virion morphogenesis with
transmission electron microscopy. In cells infected with iA6-GFP
in the presence of IPTG, the entire spectrum of normal morpho-
logical forms of VACV, including crescents, IV, MVs, and
wrapped virions, was observed at 24 h postinfection (Fig. 2A and
B). In contrast, no crescents or IV were seen in cells infected with
electron-dense viroplasm accumulated in the cells as inclusions
that measured up to 2 to 3 ?m in diameter. The majority of the
2C and D), but some inclusions had short membrane arcs at their
observed when H7 or L2 expression was repressed (21, 38).
of MV virion proteins. Recently, it was reported that a subset of
and includes F9 and L1. We thus assessed the effect of A6 repres-
blots were then performed with antibodies against various VACV
proteins. F9 and L1 proteins were detected in IPTG-treated cells,
but they were undetectable in cells not supplemented with IPTG
(Fig. 3E and F), indicating that, similar to L2, A11, and A17, A6 is
required for the stability of L1 and F9. Based on densitometry,
almost half of the A10 proteins (46%) were proteolytically pro-
cessed in IPTG-treated cells, but 85% of the A10 proteins re-
mained in the precursor form when A6 was repressed (Fig. 3G),
with proteolytic processing of virion core proteins. Two forms of
infected cells (Fig. 3B); the latter was previously shown to result
from N-glycosylation (28). There was a significant increase in the
also consistent with a previous finding that A14 glycosylation was
increased when virion membrane biogenesis was blocked due to
the lack of a functional F10, H5, A17 (28), or A11 (32). There was
no obvious change in protein level or posttranslational modifica-
was repressed (Fig. 3A, C, and D).
brane proteins. Since MV membrane formation was blocked
when A6 was repressed, we were curious about the localization of
MV membrane proteins in infected cells. We infected BHK cells
processed the cells for immunofluorescence microscopy by using
various MAbs against VACV proteins. In the presence of IPTG,
A13, A14, D8, and H3 predominantly localized to viral-DNA-
containing factories (labeled “F” in Fig. 4), which were stained
with DAPI. Weak staining of these proteins was sometimes ob-
served outside the factories. GFP, which was under the control of
the P11 late promoter, was diffused throughout the cell. In the
absence of IPTG, however, inclusions of GFP, with diameters
4). Similar GFP inclusions were previously observed when H7
with this idea, the GFP inclusions were also stained with antibod-
to associate with viroplasm. Most interestingly, most of the A13,
A14, D8, and H3 proteins localized to cytoplasmic areas outside
the viral factories (Fig. 4A to D). A13 also accumulated in a peri-
Fig. 4A). Similar changes in the localization of MV membrane
proteins were also observed at 16 and 24 h postinfection and in
HeLa cells (data not shown). In contrast to altered localization of
tein of extracellular enveloped virions (EV), and I1, a DNA bind-
ing protein (18), did not change. In the presence or absence of
apparatus (data not shown).
MV membrane proteins localized to cellular secretory com-
partments when A6 was repressed. To find out whether MV
membrane proteins localized to specific cellular organelles when
for 8 h in the presence or absence of IPTG and costained the cells
with antibodies against viral proteins and markers for cellular or-
ganelles. iA6-GUS was used here instead of iA6-GFP because the
former did not express GFP and thus was more convenient for
double labeling in immunofluorescence analysis. In the presence
FIG 3 Effect of A6 on stability and posttranslational processing of MV virion proteins. BS-C-1 cells were infected with iA6-GFP at an MOI of 5 PFU/cell in the
antibodies against A13, A14, D8, H3, F9, L1, A10, and cellular heat shock protein 70 (HSP70).
Meng et al.
jvi.asm.orgJournal of Virology
for ERGIC (Fig. 5). In the absence of IPTG, A13 and A14 colocal-
ized extensively with PDI. The area where A13 accumulated was
In an additional confirmatory experiment, D8 and H3, as well as
A13 and A14, were found to colocalize extensively with calnexin,
an integral membrane protein of the ER, when A6 expression was
repressed (Fig. 6).
Altogether, the immunofluorescence results in Fig. 4 to 6
partments outside the viral factories.
MV membrane proteins cosedimented with secretory com-
partments when A6 was repressed. To confirm the immunoflu-
examined the subcellular localization of VACV proteins by frac-
tionating the cells and assessing the distribution of viral proteins
in the cell fractions. HeLa cells were infected with iA6-GFP in the
fractionated with a continuous iodixanol gradient. Proteins from
gradient fractions were precipitated and analyzed by Western
the gradient (fractions 12 to 15 in Fig. 7), consistent with their
presence in MV virions, which are heavier than most cellular or-
A14 were also present in lighter fractions that also contained PDI
(fractions 6 to 11). Since only unglycosylated A14 is incorporated
into the virions (27) while glycosylation of A14 indicates its traf-
ficking through the ER, the separation of glycosylated A14 from
the gradient was able to separate the ER from the virions. A6 was
consistent with its localization, not to viral factories, but to the
cytoplasm in immunofluorescence (24). WR148 and full-length
A10 were also present at the top of the gradient (fractions 1 to 4).
In the absence of IPTG, WR148 and full-length A10 remained at
D8, and the mature form of A10 were found in the denser part of
the gradient, where virions sedimented (fractions 12 to 15). A14,
FIG 4 Effect of A6 on intracellular localization of VACV proteins. BHK cells were infected with iA6-GFP at an MOI of 0.5 PFU/cell in the presence or absence
and goat anti-mouse IgG coupled to Cy3. The cells were imaged with an Olympus FV-1000 laser scanning confocal system. F, viral DNA factories. The arrow
points to a perinuclear area where A13 accumulated.
Vaccinia Virus Virion Membrane Formation
May 2012 Volume 86 Number 10jvi.asm.org 5607
jvi.asm.orgJournal of Virology
which was mostly glycosylated, cosedimented closely with PDI
(fractions 6 to 12). A13 and D8 were present in lighter fractions
that contained the Golgi marker Golgin 97, as well as in fractions
that contained PDI. There was a very small amount of A6 at the
that when A6 expression was repressed, major MV membrane
proteins were predominantly associated with membrane struc-
tures that were similar in density to the ER.
that A6 was essential for VACV virion morphogenesis (24). The
A6 mutation in the ts mutant reduced A6 protein stability in a
temperature-dependent manner. As a result, cells infected by the
ts mutant at the higher nonpermissive temperature showed re-
low level of A6 protein at 42°C might allow the formation of a
small number of IV (24). Therefore, to further define the role of
A6 in virion morphogenesis, we constructed an IPTG-inducible
A6L mutant that showed more stringent repression of A6 expres-
sion at normal temperature. In the absence of the inducer IPTG,
the A6L inducible mutant did not express any detectable amount
of A6, and there was no growth in the viral titer over 48 h of
generate an IPTG-inducible A6 mutant by utilizing a modified
pVote transfer vector. In our previous attempts, A6L was
cloned in pVot (10), which was derived from pVote by remov-
ing the encephalomyocarditis virus (EMCV) internal ribosome
entry site (IRES) sequence and was used previously to make an
inducible A22R virus (10). Our intention in using pVot was to
generate a virus that would repress A6 expression more strin-
gently than a pVote-derived virus, as the former would not
allow any cap-independent translation of A6 from potential
read-through transcripts derived from promoters upstream of
the inducible T7 promoter. We were able to use a pVot-derived
transfer vector to generate an intermediate virus with both the
endogenous and the inducible A6L gene. However, we could
not delete the endogenous A6L gene from the intermediate
virus under IPTG-inducible conditions for unknown reasons.
In retrospect, we think that the EMCV IRES sequence might be
important for inducible expression of essential proteins that
are needed at higher levels for viral replication.
Characterization of the A6L inducible mutant showed that A6
belongs to a class of viral proteins that play an essential role in an
EM. When A6 expression was repressed, large, dense viroplasm
inclusions accumulated in the cells with no sign of a crescent
membrane (Fig. 2), similar to the phenotypes associated with the
lack of F10, A11, H7, or L2 (21, 27, 32, 38, 42). Furthermore, A6
repression caused a dramatic increase in the glycosylation of MV
membrane protein A14 and a great reduction in the levels of MV
membrane proteins F9 and L1 (Fig. 3). Both of these phenotypes
are associated with VACV mutants that are defective in virion
membrane biogenesis. A14 is one of the few VACV MV mem-
brane proteins that has an N-glycosylation site, but A14 is glyco-
sylated at a low level during normal viral replication (28). A14
glycosylation was increased, however, when virion membrane
biogenesis was blocked, presumably because A14 was retained or
diverted to the ER in the absence of viral membranes. When A6
expression was repressed, almost all A14 protein was glycosylated
(Fig. 3 and 7). This level of A14 glycosylation appeared to be as
much as, if not more than, that in the absence of a functional A11
(32), A17, F10, or H5 (28). Some VACV MV membrane proteins
that are part of the entry-fusion complex (EFC) became unstable
when viral membrane formation was blocked by repression of
L2, A11, H7, or A17 (22, 38). Similarly, EFC proteins F9 and L1
were undetectable in the absence of A6 (Fig. 3). Thus, based on
similarity in EM morphology and the fate of several MV mem-
brane proteins under nonpermissive conditions, A6 can be
classified together with F10, A11, H7, and L2 in a class of viral
proteins that play essential roles in an early step of virion mem-
brane biogenesis. Although the molecular functions of this
group of VACV proteins are largely unknown, it is critical to
identify all the viral proteins that participate in the virion
membrane biogenesis process in order to gain a molecular un-
derstanding of the unique mechanism used by the poxviruses
to acquire their primary envelope.
Similar to most proteins that function in early steps of virion
membrane formation, A6 does not appear to be a structural pro-
tein of MV. Mass spectrometry analysis previously identified A6,
and an A6 ortholog in myxoma virus, as a minor virion compo-
nent (1, 31, 49). Western blot analysis further demonstrated that
not specifically localize to viral factories but instead localized
throughout the cytoplasm (24). Indeed, cell fractionation with a
lighter cytoplasmic fractions and only a small amount of A6 was
present in the heavier virion fractions (Fig. 7). It is likely that A6
mainly functions in the cytoplasm and is only packaged into viri-
ons nonspecifically. Similarly, L2 appears to be nonspecifically
incorporated into virions (22), while H7 and A11 are not pack-
in membrane biogenesis is probably not a structural one, as it
A6 in that it localizes to the cytoplasm during infection (38). In
contrast, L2 is a membrane protein that localizes to the ER (22),
tion of F10 is unclear, as one report suggested that it localizes to
to Fig. 4. The cells were stained with both a primary antibody against a viral protein (either anti-A13 [11F7; IgG2a] or anti-A14 [FE11; IgG2b]) and a primary
antibody against a cellular organelle marker (either anti-PDI [IgG1] or anti-ERGIC53 [IgG1]). This was followed by staining with secondary antibodies against
point to perinuclear areas where both A13 and ERGIC53 accumulated.
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May 2012 Volume 86 Number 10jvi.asm.org 5609
jvi.asm.orgJournal of Virology
viral factories (42) while another report suggested that it localizes
to the ER and ERGIC (30).
has not been previously reported for VACV mutants that are de-
fective in virion membrane biogenesis. During normal VACV in-
fection, MV membrane proteins predominantly localize to viral
tion of MV membrane proteins in the absence of A11, H7, or L2
was previously studied by immunofluorescence analysis of MV
In contrast, when A6L expression was repressed, there was a pro-
found defect in localization of MV membrane proteins to viral
factories. This defect was demonstrated with four different MV
membrane proteins that were inserted into membranes through
(37), while H3 and presumably D8 were synthesized on free ribo-
somes and posttranslationally inserted into membranes (4). The
the cells but dramatically affected their intracellular localization.
Immunofluorescence analysis showed that they no longer local-
ized to viral factories but instead colocalized extensively with ER
markers PDI and calnexin outside the factories (Fig. 4, 5, and 6).
marker for ERGIC. Furthermore, cell fractionation studies
showed that, in the absence of A6, only a small amount of these
MV proteins sedimented with virions, while the majority of the
were similar to that of the ER (Fig. 7).
EM analysis of VACV-infected cells previously showed that
some MV membrane proteins were associated with ER or ERGIC
immunofluorescence microscopy showed that MV membrane
proteins predominantly localized to viral factories and rarely co-
localized with any cellular organelle markers (40). Similarly, de-
the A14 proteins are unglycosylated, and minor glycosylated A14
proteins are not packaged in the virions (27). It is thus unprece-
membrane proteins are present in secretory compartments out-
side viral factories and almost all A14 proteins are glycosylated.
This is unlikely to be the result of MV membrane proteins being
diverted to the ER located outside the factories from their normal
A6, MV membrane proteins are synthesized outside the factories
and fail to be recruited to the factories. This further suggests that
ER components are not passively incorporated into viral factories
but are actively recruited to viral factories through a virus-medi-
ated process that requires at least A6. Thus, when A6 is absent, no
proteins are inappropriately synthesized in the ER outside the
viral factories. Alternatively, MV membrane proteins could be
cifically recruited, along with the underlying membranes, to fac-
tories through an active, virus-mediated process that requires A6.
likely to be quite different from intracellular trafficking of mem-
brane vesicles from the ER to the Golgi apparatus, since blocking
FIG 6 Colocalization of MV membrane proteins with calnexin when A6 expression was repressed. BHK cells were infected with iA6-GUS and processed for
immunofluorescence microscopy as described in the legend to Fig. 5. The cells were stained with both a primary mouse antibody against a viral protein and the
of 100 ?M IPTG. At 8 h p.i., the cells were harvested, and the postnuclear supernatant was loaded onto a 5 to 25% continuous iodixanol gradient, centrifuged,
and fractionated. Trichloroacetic acid-precipitated proteins were analyzed by SDS-PAGE, followed by Western blotting with antibodies against Golgin 97, PDI,
A13, A14, D8, A10, WR148, and V5. The direction of the density gradient is indicated above the lanes.
Vaccinia Virus Virion Membrane Formation
May 2012 Volume 86 Number 10jvi.asm.org 5611
ER-to-Golgi vesicle transport with brefeldin A or a dominant-
negative Sar-1 GTPase failed to inhibit the formation of IV (12,
45). Determining the molecular mechanism by which A6 func-
tions may provide an intriguing story of how viruses manipulate
intracellular membrane trafficking.
This work was supported by a grant from NIAID to Y. Xiang (AI079217).
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