A Vaccinia Virus Recombinant Transcribing an Alphavirus
Replicon and Expressing Alphavirus Structural Proteins
Leads to Packaging of Alphavirus Infectious Single Cycle
Juana M. Sa ´nchez-Puig, Marı ´a M. Lorenzo, Rafael Blasco*
Departamento de Biotecnologı ´a, Instituto Nacional de Investigacio ´n y Tecnologı ´a Agraria y Alimentaria (I.N.I.A.), Madrid, Spain
Poxviruses and Alphaviruses constitute two promising viral vectors that have been used extensively as expression systems,
or as vehicles for vaccine purposes. Poxviruses, like vaccinia virus (VV) are well-established vaccine vectors having large
insertion capacity, excellent stability, and ease of administration. In turn, replicons derived from Alphaviruses like Semliki
Forest virus (SFV) are potent protein expression and immunization vectors but stocks are difficult to produce and maintain.
In an attempt to demonstrate the use of a Poxvirus as a means for the delivery of small vaccine vectors, we have
constructed and characterized VV/SFV hybrid vectors. A SFV replicon cDNA was inserted in the VV genome and placed
under the control of a VV early promoter. The replicon, transcribed from the VV genome as an early transcript, was
functional, and thus capable of initiating its own replication and transcription. Further, we constructed a VV recombinant
additionally expressing the SFV structural proteins under the control of a vaccinia synthetic early/late promoter. Infection
with this recombinant produced concurrent transcription of the replicon and expression of SFV structural proteins, and led
to the generation of replicon-containing SFV particles that were released to the medium and were able to infect additional
cells. This combined VV/SFV system in a single virus allows the use of VV as a SFV delivery vehicle in vivo. The combination
of two vectors, and the possibility of generating in vivo single-cycle, replicon containing alphavirus particles, may open new
strategies in vaccine development or in the design of oncolytic viruses.
Citation: Sa ´nchez-Puig JM, Lorenzo MM, Blasco R (2013) A Vaccinia Virus Recombinant Transcribing an Alphavirus Replicon and Expressing Alphavirus Structural
Proteins Leads to Packaging of Alphavirus Infectious Single Cycle Particles. PLoS ONE 8(10): e75574. doi:10.1371/journal.pone.0075574
Editor: Volker Thiel, Kantonal Hospital St. Gallen, Switzerland
Received June 30, 2013; Accepted August 15, 2013; Published October 9, 2013
Copyright: ? 2013 Sa ´nchez-Puig et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by contracts QLK2-CT2002-01867 and CT2006-037536 from the European Commission, and grants BFU2005-05124 and
BIO2008-03713 from Plan Nacional de Investigacio ´n Cientı ´fica, Desarrollo e Innovacio ´n Tecnolo ´gica and by grant S2009/TIC-1476 from Comunidad Auto ´noma de
Madrid. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The work described is part of the following patent application: Recombinant DNA viruses comprising a DNA that is complementary to an
RNA virus replicon, and applications thereof. Application Number, EP20040742039 20040702; Publication Number, 1642982(A1). This does not alter the authors’
adherence to all the PLOS ONE policies on sharing data and materials, as detailed in PLOS ONE guide for authors.
* E-mail: email@example.com
Virus-based expression systems have been derived from
members of diverse virus families, including widely different
RNA and DNA viruses. Among those, Vaccinia virus (VV), the
representative member of the Poxviridae, constitutes an exten-
sively used protein expression and vaccine vector. In addition to
many beneficial characteristics for vaccine use, a major advantage
of VV vectors is their large DNA genome that provides
considerable insertion capacity, thus allowing the expression of
large and/or multiple genes. In contrast, Alphavirus-based vectors
are expression systems which are smaller in size and insertion
capacity, but constitute attractive vaccine candidates shown to
induce strong immune responses. For reviews on Alphavirus
vectors see [1,2,3,4].
Alphaviruses are members of the family Togaviridae, whose
genome is a positive-sense single-stranded RNA molecule of
approximately 12 kb. After infection, the 59 two-thirds of the
incoming genome is translated, producing the viral replicase
nonstructural proteins (nsP1–4). Next, the replicase synthesizes
negative-sense copies of the genome, which serve as templates for
both progeny genomes and for transcription of an mRNA from
the internal subgenomic promoter .
Self-amplifying Alphavirus replicons are derived from the viral
genome by replacing the genomic region coding for the viral
structural proteins by a foreign gene . Therefore, such replicons
consist of a single RNA molecule which, when transfected into
cells, is translated into the viral replicase, which amplifies the
replicon and transcribes a subgenomic RNA encompassing the
foreign gene. To facilitate introduction in cells, replicon RNA
molecules have been packaged by Alphavirus structural proteins
provided by a helper replicon in trans. By packaging the replicon
RNA into SFV particles, single-cycle virus particles are produced,
that can be subsequently used to introduce the replicon into new
cells. Over the last years, considerable experience has been
accumulated in the use of alphavirus-based systems for immuni-
Since the design of Alphavirus replicons, a number of strategies
have been used to introduce the replicon into cells. Original
systems relied on transfection of replicons transcribed in vitro using
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T7 or SP6 polymerases. Subsequently, other methods for
intracellular delivery of Alphavirus replicons have been developed,
including transcription from transfected plasmid DNA  [8,9] or
expression from baculovirus .
In this work we have sought to use vaccinia virus as a vehicle
capable of delivering and packaging an alphavirus replicon within
cells. Using this strategy, a Vaccinia virus/SFV combined vector
can potentially be used as a single immunizing agent.
Coinfection of Cells with Vaccinia and SFV Particles
To ascertain whether VV and SFV replication cycles are
compatible, i.e., can take place concurrently in the same cells, we
carried out coinfections of cells with vaccinia virus and SFV
replicons. In a first experiment, we used a vaccinia virus b–
Glucuronidase recombinant and single-cycle SFV particles har-
boring the b–Galactosidase gene, and gene expression mediated
by each system was measured. To compensate for kinetic
differences between the two systems, SFV infections were started
at different times after VV infection. After 48 hours, expression of
b–Galactosidase and b–Glucuronidase were quantitated by
measuring enzymatic activity with the specific substrates ONPG
and PNG, respectively (Fig. 1A). Simultaneous infection with the
two viruses resulted in significant amounts of b–Galactosidase and
b–Glucuronidase, indicating that replication and gene expression
of the two viruses were compatible. Notably, SFV-directed
expression of b–Galactosidase was unaffected or enhanced in cells
coinfected with vaccinia virus, provided that SFV infection was
initiated within the first two hours after vaccinia infection. Also,
virus production from coinfected cultures was within the normal
ranges for both SFV and VV (Fig. 1B). Those results did not reveal
any interference in the replication cycle of the two viruses, and
indicate that the complete replication cycle of both viruses can
SFV Replicons can be Transcribed in Vaccinia Virus-
In the SFV-based expression system originally described by
Liljestrom and Garoff [6,11] a capped and polyadenilated replicon
RNA is generated from a linearized plasmid by in vitro transcrip-
tion using SP6 RNA polymerase. However, generating a
functional replicon from the VV genome would require transcrip-
tion to take place in the cytoplasm of VV-infected cells. To
examine if SFV replicons can be produced by SP6 RNA
polymerase in VV-infected cells, plasmid pSFV-LacZ, in which
the SFV replicon is placed downstream of the SP6 promoter, was
transfected in cells previously infected with a vaccinia virus
recombinant expressing SP6 RNA polymerase. 36 hours after
transfection, b–Galactosidase activity was detected in the cell
cultures (Fig. 2A). Interestingly, transfection of both linearized or
closed circular plasmid resulted in b–Galactosidase expressing
cells. In contrast, no b–Galactosidase was detected in parallel
cultures transfected with pSFV-LacZ and infected with WR virus.
The number of b–Galactosidase positive cells was higher after
transfection of the uncut plasmid, probably revealing the higher
efficiency of transfection, its amplification and/or the higher
stability of the circular plasmid relative to the linearized plasmid
inside cells. Those results indicate that functional SFV replicons
can be generated in transfected cells by SP6 transcription. In
addition, the fact that circular plasmids induced b–Galactosidase
expression suggests that a functional 39 end of the replicon RNA
was generated inside cells after SP6 polymerase transcription.
SFV Replicons can be Packaged by SFV Structural
Proteins Provided in trans by Vaccinia Virus
A VV recombinant expressing the SFV structural proteins was
constructed by inserting the complete set of SFV structural genes
in the VV genome, downstream of the F13L gene and under the
control of a synthetic vaccinia early/late promoter. To avoid any
homology between the sequence inserted in vaccinia and the
replicon, the bare coding sequence of the SFV structural proteins
was used to insert in the VV genome. Therefore, the ensuing
transcript would lack 59 and 39 non-coding sequences normally
present in genomic (plus strand) SFV RNA or in SFV subgenomic
RNA. The VV recombinant isolated was termed V-Helper and
was shown to express SFV structural proteins by immunoblotting
(Fig. 2C). To test the ability of the proteins expressed by V-Helper
to package SFV replicon RNA, BHK-21 cells were transfected
with an in vitro transcribed SFV-GFP replicon RNA, and then
infected with V-Helper. The titer of single-cycle SFPs in cell
supernatants after 36 hours was estimated by infecting fresh BHK
cell monolayers. Titers were in the range of 105to 106SFPs/ml,
and were similar to those obtained when co-transfecting in vitro
transcribed replicon and SFV-Helper RNAs (Fig. 2B). An
additional proof of the packaging ability of SFV structural proteins
expressed by V-Helper was obtained by passaging single-cycle
SFPs on monolayers of V-Helper- infected cells, which typically
produced titers in the range of 106SFP/ml. Those results indicate
that SFV replicons can be packaged into infective particles within
vaccinia virus infected cells using packaging proteins provided in
trans by a vaccinia-virus recombinant.
SFV Replicons can be Generated as Early VV Transcripts
The compatibility of vaccinia virus and SFV replication opened
the possibility to generate SFV replicons as VV early transcripts.
With this objective, we inserted the complete SFV replicon cDNA
into the VV thymidine kinase locus, placing the 59 end of the
replicon immediately downstream of the normal TK promoter. To
facilitate complete transcription of the replicon, we mutagenized a
vaccinia early transcription termination sequence (TTTTTnT)
that was present in the non-structural region of the replicon
Insertion in the VV genome was performed in a two-step
process (Fig. 3). First, about two-thirds of the SFV genome,
encompassing the genes coding for non-structural proteins,
together with a dsRed gene cassette was inserted into the TK
locus of vaccinia WR. The resulting virus, termed W-RednsTK,
was subsequently used for the second step, in which the 39 end of
the replicon cDNA was inserted. The latter portion of the replicon
included a GFP gene placed downstream of the SFV sub-genomic
To test the requirement of SFV sequences in the 39 end of the
replicon, we constructed viruses that incorporated the 70, 170 or
340 nucleotides immediately adjacent to the terminal Poly-A
sequence. In addition, a 70 nt-long poly-A sequence was either
included or not in each of the constructs. During isolation, virus
harboring replicons devoid of the Poly-A sequence failed to
produce GFP fluorescence. In contrast, viruses that included Poly-
A sequence at the 39 end of the replicon formed plaques displaying
green fluorescence. Notably, GFP fluorescence was similar in cells
infected with viruses that included 340, 170 or 70 nucleotides of 39
terminus sequence. Therefore, the virus which contained 70
nucleotides and the cDNA Poly-A sequence, termed W-SFR, was
selected for further study.
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Construction of a Virus Containing the SFV Replicon and
the Genes Coding for SFV Structural Proteins
In the original SFV-derived system, the RNA replicon is
encapsidated and released from cells expressing SFV structural
proteins. This is usually accomplished by transfecting an in vitro
transcribed helper replicon RNA devoid of the SFV packaging
signal. We tested if expression of SFV structural genes from VV
recombinant V-Helper would result in packaging of the replicon in
W-SFR infected cells. Coinfection of cells with W-SFR and V-
Helper resulted in production of SFPs in the medium of infected
cells (not shown), indicating that W-SFR-derived replicon RNA
was functional for packaging, and further confirmed that replicon
packaging could take place in vaccinia-infected cells.
To construct a recombinant virus that would transcribe the
replicon and also provide the structural proteins, we assembled the
complete replicon in the TK locus as above, but starting from the
V-Helper virus containing the SFV structural genes downstream
of the F13L gene (Fig. 3). Using the same two-step process
described above, virus W-H-SFR was isolated taking advantage of
the GFP fluorescence produced by the virus during the isolation
Plaque Formation and SFP Production by W-H-SFR
In the standard VV plaquing assay, virus plaques are usually
allowed to develop in cell monolayers maintained under liquid
medium. Under those conditions, the size and shape of the virus
plaques are good indicators of cell-to-cell virus transmission and
extracellular virus release. Some VV strains that are well
transmitted locally but release low numbers of infectious virus to
the culture medium give rise to round, well defined plaques. In
contrast, VV strains which release more extracellular virus
typically produce plaques with a comet shape, indicative of
secondary infections caused by virus released from the primary
plaque. When W-SFR and W-H-SFR were subjected to a plaque
assay on BSC-1 cells, a clear difference between the two viruses
was noted. W-SFR plaques were of the normal round phenotype,
similar to plaques formed by the parental WR virus. In contrast,
W-H-SFR produced a comet-shaped plaque phenotype reminis-
Figure 1. Coinfection of VV and SFV. A) BSC-1 cells were infected with VV expressing b–Glucuronidase and coinfected at different times with
SFPs containing a SFV replicon with the b–Galactosidase gene. Both infections were carried out at a moi of 5 pfu/cell. 48 hours after the first infection,
cells were lysed, and the b-galactosidase and b-glucuronidase activities in extracts corresponding to 103cells were assayed. B) BSC cells were infected
with VV and then infected with SFV at different times post-vaccinia infection. At 48 hours, titers of Vaccinia virus were obtained by plaquing lysed
cells (grey bars), and SFV titers were determined from culture media (black bars).
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cent of vaccinia viruses producing more extracellular virus like
IHD-J (Fig. 3).
We hypothesized that cells infected by W-H-SFR within the
primary plaque were releasing SFPs encapsidating the replicon-
GFP gene that would infect distant cells within the monolayer.
The comet tail would therefore be the result of cythophatic effect
caused by those secondary infections. That this was the case was
corroborated by the observation that when anti-SFV antibody was
included in the culture medium during the plaque assay, comet
tails in W-H-SFR plaques were not formed, but instead round
plaques developed (Fig. 4).
In addition to the appearance of comet tails, if infective SFV
particles containing the replicon-GFP RNA were being released, a
singular distribution of VV and SFP –infected cells within the virus
plaque would be expected. To explore this idea, the distribution of
cells infected with VV within the virus plaques was visualized by
immunofluorescence, and compared with those of cells with
replicon-mediated expression of GFP (Fig. 5). In W-SFR plaques,
VV staining and the GFP signal appeared within the virus plaque,
consistent with the replicon being produced in W-SFR infected
cells. Even in the small tails found in some plaques, GFP
fluorescence was coincident with VV-positive cells, indicating
release of some vaccinia extracellular particles. Significantly,
immunofluorescence staining of W-H-SFR plaques revealed a
singular distribution (Fig. 5). Within the plaque, the coincidence of
VV and GFP positive cells was lower than in the case of W-SFR
plaques, being GFP-positive cells more abundant in the outer
region of the plaques. Also, while vaccinia staining was present
almost exclusively in the primary plaque, GFP-expressing cells
were detected both in the area of the primary plaques and in the
comet tails. Notably, GFP expressing cells in the comet tail area
were not stained by anti-VV antibody, suggesting that they were
produced by infecting SFPs (Fig. 5).
Additionally, a similar plaque assay was carried out in BHK-21
cells, which are highly susceptible for SFV (Fig. 6). Compared with
BSC-1 cells, comet tails were much larger in BHK-21 cells, and
displayed brighter GFP fluorescence, consistent with the high
susceptibility of BHK-21 cells to replicon-mediated expression.
Remarkably, the sizes of the primary plaques formed by W-H-
SFR were smaller than those formed by W-SFR, probably
revealing competition of SFPs with vaccinia virus for cells within
the plaque area.
The above results indicate that infection with W-H-SFR, a VV
recombinant expressing the SFV replicon and SFV structural
proteins, is able to generate infectious SFV particles that, in a
second round of infection, promote the expression of a foreign
Electron Microscopy of W-H-SFR Infected Cells
Infectious material in the medium of W-H-SFR was filterable
through 100 nm filters that completely removed VV infectivity,
suggesting that replicon molecules were encapsidated in small
Figure 2. Transcription and packaging of the SFV replicon in VV-infected cells. A) BSC-1 cells were infected with vaccinia virus expressing
SP6 RNA polymerase (VV-Sp6) and subsequently transfected with plasmid pSFV-LacZ. At 48 hours, cells were either stained for b-galactosidase by
addition of X-Gal to the cultures or lysed to assay b-galactosidase activity. Transf.: Number of b-galactosidase positive cell in a 24-well plate well. b-Gal
T: b-galactosidase in 105cells (pg). b-Gal/cel: ratio of b-galactosidase activity per cell. B) Packaging of replicon RNA by SFV structural proteins
expressed from V-Helper. BHK-21 cells were transfected with plasmid pSFV-GFP, or with in vitro transcribed RNA from pSFV-GFP linearized with SpeI,
and mock infected or infected with V-Helper at a moi of 5 pfu/cell. Column SFPs/ml shows the titers of SFPs in the culture media at 48 h post-
infection. C) Western blot analysis of cell extracts infected with the viruses indicated at the top. The positions of SFV mature structural proteins p62,
E1 and C are indicated.
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particles like those of SFV. To directly visualize the particles being
formed, cells infected by W-SFR or W-H-SFR were analyzed by
electron microscopy. In addition of the well known stages of VV
morphogenesis, typical alphavirus particles, approximately 60 nm
in size, were evident (Fig. 7 A–E). Those Alphavirus-like particles
were commonly found in groups or attached to the plasma
membrane of cells. Consistently with the morphogenetic pathways
for SFV, budding events at the plasma membrane could also be
identified. As expected, the appearance of SFV-like particles was
dependent on expression of SFV structural proteins, since it was
not detected in W-SFR infected cells (Fig. 7-F). In addition, the
presence of extracellular SFV particles was not dependent on the
exit of VV, since it occurred normally in W-H-SFR infected cells
treated with IMCBH, an inhibitor of VV release (not shown).
Production of SFPs in the Presence of VV Inhibitors
We studied the effect of VV inhibitors in the production of SFPs
from W-H-SFR infected cells (Fig. 8). HeLa cells infected with W-
H-SFR produced increasing amounts of SFPs up to 36 hours post-
infection. As expected, SFP production was only slightly affected
by Rifampicin or IMCBH, two specific inhibitors of VV
morphogenesis and release that do not inhibit viral gene
expression. In contrast, a marked decrease was noted by treatment
with AraC, an inhibitor of DNA replication that blocks VV
intermediate and late gene expression. Since AraC should not
affect RNA replication, we consider it likely that the effect of AraC
is the consequence of the lower expression of SFV structural
proteins, which is dependent, in W-H-SFR, on a VV early/late
promoter. As the inhibition of VV DNA replication abolishes late
gene expression, it is expected that SFV structural proteins will be
limiting at late times, in conditions where replicon amplification
can continue unabated.
Absence of Viable SFV
One important consideration in the design of alphavirus vectors
is the possibility of generating viable viruses by RNA recombina-
tion, a prospect imposing important biosafety limitations to the
system. In the established replicon/helper system, replication-
proficient viruses were detectable in the stocks in ratios to SFPs of
1023to 1026. This frequency has been lowered by using
several strategies, including the mutation of the spike glycoprotein,
the use of two different Helper RNAs, promoterless helpers or the
inclusion of miRNA targets in the helper RNA [12,13,14,15].
The system described here should hamper the production of
replicating SFV, since the structural proteins are produced via a
VV RNA with no homology to the replicon and lacking 59 and 39
Figure 3. Construction of VV containing a SFV replicon. Upper panel: Schematic representation of the virus genome, indicating the F13L and
the TK loci. Viruses W-RednsTK and W-H-RednsTK were obtained by recombination of VV WR and VV-Helper, respectively, with plasmid pRednsTK.
Viruses W-SFR and W-H-SFR were obtained by recombination of W-RednsTK and W-H-RednsTK, respectively, with plasmid pMix-f70An. Boxes labeled
TKL and TKR denote the left and right recombination flanks or the TK gene. Boxes ns1–4* correspond to the genomic region coding the non-
structural SFV proteins with an early VV transcription termination signal mutated. Black circle: VV synthetic early/late promoter. dsRed: red fluorescent
protein gene. Helper: SFV genes for structural proteins, inserted downstream of the F13L gene under the control of a VV synthetic early/late
promoter. rsGFP: green fluorescent protein gene. f: sequence from the 39end of the SFV replicon, including 70 nucleotides adjacent to the PolyA and
a 70 nt PolyA sequence. In the lower right, plaques formed by W-SFR or W-H-SFR on monolayers of BSC-1 cells for 48 hours. Below, sequence of the
VV TK promoter and the predicted 59 end of the SFV replicon (arrow).
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non-translated sequences of the subgenomic RNA. We tested if
replication competent SFV were present in SFP stocks generated
from W-H-SFR. To this aim, 107SFPs were used to infect BHK-
21 cell monolayers, and after 48 hours, the supernatant was
harvested and used to infect fresh BHK-21 cell monolayers, in an
attempt to detect any replicating SFV. Consistently, in three
independent experiments, we failed to detect replication-compe-
tent SFVs in our SFP stocks, suggesting a significant improvement
over the replicon/helper RNA system.
The strategy outlined in this work demonstrates that a single
vaccinia virus can serve as a vehicle to produce Alphavirus
particles in infected cells. Due to the technical complexity and
considerable difficulties to produce Alphavirus replicons in a cost-
effective manner, it may be desirable to generate and package a
self-amplifying replicon in vivo. This strategy not only obviates
recombinant SFP production problems, but also provides a
potential means to greatly enhance the efficacy of Alphavirus or
Poxvirus -based vaccines.
The concept of combining two viral vectors may be used to
exploit the benefits of each of the individual vectors involved. So
far, a number of different combinations have been described,
including Adeno-Retrovirus [16,17,18,19,20] and SFV – Retro-
virus [21,22,23]. Many of these systems require the coinfection of
several trans-complementing virus recombinants or trans-comple-
menting cell lines, since the virus recombinants used do not
accommodate all the genetic information required. Poxviruses
constitute an attractive alternative to those since, in addition to
their well-known capabilities as vaccines and reliable expression
systems, they allow the insertion of large and multiple DNA
sequences. Taking advantage of these features, VV-retrovirus
hybrid vectors have been developed previously, and shown to be
capable of producing transduction-competent retroviral particles
Vaccinia virus offers several advantages to act as a ‘‘shuttle’’
vector for an RNA virus vector. Most important, the coding
capacity of the vector and the well characterized transcriptional
control of vaccinia genes make it possible to express both the
replicon and the genes coding for the structural proteins from
different locations in the genome. In the past, a vaccinia virus
recombinant expressing a Venezuelan Equine Encephalitis repli-
con from a T7 promoter, in which the replicon RNA could be
transcribed after coinfection with a second recombinant expressing
the T7 RNA polymerase, has been described . There are two
main differences in our system with respect to the previous one.
First, here the transcription of both the replicon and the genes
coding for the packaging protein are dependent on VV promoters,
and therefore expression of the T7 RNA polymerase is not
Figure 4. Plaque formation and SFP production by W-H-SFR.
Plaque phenotype of the viruses indicated was determined in a
standard vaccinia plaque assay on BSC-1 monolayers. Virus plaques
were allowed to develop for 48 hours and stained with crystal violet
solution. In the cultures treated with anti-SFV antibody, culture medium
was replaced at 2 hours post-infection with medium containing
polyclonal antiserum to SFV proteins (a-SFV).
Figure 5. Distribution of cells infected with VV and/or
expressing the SFV replicon in BSC-1 cells. BSC-1 cell monolayers
were infected with dilutions of W-SFR or W-H-SFR for 48 h, fixed and
stained with anti-B5 antibody. Fluorescence within and around virus
plaques was visualized in an inverted fluorescence microscope. Merged
images result from the combination of monochrome images in red
(anti-B5 antibody) and green (direct expression of GFP). Images
covering whole plaques and tails were assembled from multiple
individual images stitched together as described in the Materials and
Methods section. White boxes specify the areas inside (plaque) or
outside (tail) the virus plaques that are shown enlarged in the smaller
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required. Second, the packaging proteins are expressed from the
same VV recombinant, and therefore no coinfections are required
to trigger the replicon and the production of suicide SFPs. Those
facts are crucial for the use of this, or similar viruses, as
immunizing agents in vivo, with the aim of using VV as a shuttle
to generate suicide SFPs inside the injected tissues.
One potential improvement of the system described here over
existing SFV expression systems is the low generation of
replication competent SFV. To prevent recombination leading
to replicative SFV, we designed the constructs to avoid the
occurrence of any repeated sequences between the replicon and
the gene coding for the SFV structural proteins. We anticipated
that generating viable SFV would be an extremely rare event,
since the VV mRNA coding for SFV structural genes in our
recombinant lacks a SFV subgenomic promoter, as well as any
non-coding sequences at the 39 end. Thus, reconstruction of a
viable SFV genome would need two precise recombination events
in regions with no homology. There may be additional reasons for
the absence of SFV in our preparations. For instance, if the
mRNA for the SFV structural proteins behaves like a normal VV
transcript and does not interact with the SFV replicase, physical
interaction with the replicon RNA may not be favored, and they
might be compartmentalized in different subcellular locations as a
consequence of the different transcription/replication strategy for
the two viral systems.
Another feature related to the safety of the vector is that the
system could be applied to replication-defective Poxvirus vectors
(like MVA) that do not expand within the tissues in mammalian
hosts. In that situation, even with limited spread of the poxvirus,
the combined vector might maintain a higher level of expression
due to the second cycle of expression in the SFP-infected cells.
This ‘‘expanded expression’’ effect is likely to be dependent on the
susceptibility of cells to the SFV, as exemplified by plaque assays in
different cell lines (compare the GFP expression in Figs. 5 and 6).
One interesting question is how this double expression might
affect the immunological response to the relevant antigen. Up to
date, a number of approaches have been used to enhance the
response to the foreign antigen in a context of multiple vector-
specific antigens. For instance non-replicating or growth-restricted
Figure 6. Distribution of cells infected with VV and/or
expressing the SFV replicon in BHK-21 cells. BHK-21 monolayers
were infected with dilutions of VV-rsGFP, W-SFR or W-H-SFR. At 48 h.p.i,
cell monolayers were fixed and stained with polyclonal antiserum to VV
proteins. Merged images result from the combination of monochrome
images in red (anti-VV polyclonal serum) and green (direct expression of
GFP). The larger panel was assembled using overlapping photographs
stitched together as described in the Materials and Methods section.
Figure 7. Electron microscopy of W-H-SFR infected cells. Vaccinia virus (particles around 200–250 nm) and smaller, SFV-like particles (SFPs,
spherical, approximately 50 nm diameter) were present in W-H-SFR infected cells (Panels A-E). Note that SFPs tend to aggregate in clusters (central
panels). A budding image at the plasma membrane can be seen in panel C. An image of the control of W-SFR infected cells (panel F) is shown.
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vaccines such as plasmid DNA or VV MVA are often used in
combination with other heterologous vectors delivering a common
antigen in prime-boost regimens. It is tempting to speculate that
the two vector system described here, that directs expression of the
antigen in two different cellular contexts (the VV-infected cell and
the SFP-infected cell) might have consequences with respect to the
immune responses to the antigen. Further, this system could be
used in combination with additional immunizing agents to further
increase the responses.
Materials and Methods
Cells and Viruses
BSC-1 cells were grown in Eagle’s minimal essential medium
(EMEM) supplemented with 0.1 mg/ml penicillin, 0.1 mg/ml
streptomycin, 2 mM L-glutamine (Bio Whittaker) and 5% fetal
bovine serum (FBS). BHK-21 cells (ATCCCCL10) were grown in
BHK-21 Glasgow minimal essential medium (Glasgow-MEM,
GibcoBRL) containing 5% FBS, 3 g/ml tryptose phosphate broth,
0.01 M HEPES and supplemented with antibiotics and glutamine.
Virus infections were carried out in medium containing 2% FBS.
Plaque assays and crystal violet staining was carried out as
described  .
Vaccinia virus expressing b-Glucuronidase (V-Gus) was isolated
by insertion of the b-Glucuronidase gene downstream of the F13L
gene under the control of a synthetic early/late vaccinia promoter.
For this, plasmid pRB21-bGus  was transfected into cells
infected with virus vRB12 , and recombinant viruses were
subsequently isolated by plaque selection . Vaccinia virus
expressing Sp6 DNA polymerase vSIMBE/L  was kindly
made available by B. Moss.
Generation of Semliki Forest Virus Suicide Particles
Semliki Forest Particles (SFPs) were generated in cell cultures by
electroporation of plasmids containing the SFV replicon down-
stream of the SP6 promoter together with pSFV-Helper1 helper
plasmid . In vitro RNA synthesis, transfections and SFV
infections were performed as described [11,36].
Construction of Plasmid pSFV-GFP
The GFPst gene was obtained by PCR from the virus W-
GFPs65T   with oligonucleotides pRB21–59BamHI 59-
cloned into the plasmid pSFV1 (a generous gift of Henrik Garoff,
Karolinska Institutet) between sites BamHI/SmaI. SFPs for
expression of LacZ or GFP were obtained by transfection of
in vitro transcribed RNA from plasmids pSFV3-lacZ ( a
generous gift of Henrik Garoff) or pSFV-GFP.
Construction of V-Helper
Plasmid pRB-Helper was constructed by inserting the genes for
SFV structural proteins (C-p62-6K-E1) downstream of a synthetic
VV early/late promoter in expression plasmid pRB21 . SFV
sequences were obtained by digestion of pSFV-Helper1 and
cloned in pRB21 in two steps. First, an EcoRI-HindIII fragment
(pSFV-Helper1 coordinates 2653–5281) was ligated into the
Figure 8. Particle production in the presence of vaccinia virus inhibitors. Hela cells were infected at a m.o.i. of 5 pfu per cell with
recombinant virus W-H-SFR and after adsorption, medium was replaced with fresh normal medium (brown rhombs) or medium containing 100 mg/ml
AraC (red squares), 10 mg/ml IMCBH (black triangles) or 100 mg/ml Rifampicin (blue squares). Samples of the culture medium were collected every 12
hours, clarified, filtered and stored at 4uC until last point was collected. To determine the titer of SFPs, fresh BHK cells were infected with serial
dilutions of the filtered supernatant, and GFP-positive cells were counted 24 hours later in the fluorescence microscope. Lower panels: Vaccinia
virus Extracellular virus (EV) or cell associated (IV) production. Hela cell monolayers were infected at a multiplicity of 5 pfu per cell with W-H-SFR
recombinant virus, and after 1 hour medium was replaced with medium containing AraC (100 mg/ml), rifampicin (100 mg/ml) or IMCBH (10 mg/ml).
Virus was harvested at 24 hpi and Vaccinia virus titers in the culture medium (EV) (left panel) and cell lysates (IV) (right panel) were obtained by
plaquing in BSC-1 cell monolayers and are represented relative to the control with no inhibitor.
Vaccinia/Alphavirus Combined Vector
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EcoRI/HindIII sites in pRB-21. Subsequently, an EcoRI
fragment (pSFV-Helper1 coordinates 1298–2653) was cloned into
the resulting single EcoRI site.
Vaccinia virus recombinant VV-Helper was isolated by a
plaque size selection method . Plasmid pRB-Helper was
transfected into cells infected with v-RB12 virus, and a virus with
large plaque phenotype was isolated through successive rounds of
Generation of Recombinant Viruses W-SFR and W-H-SFR
Recombinant viruses with the SFV replicon inserted in the VV
thymidine kinase (TK) locus were isolated in two steps. First,
intermediate viruses were isolated by recombination of the viral
genome with plasmid pRednsTK, which contains two-thirds of the
SFV replicon cDNA and a dsRed2 cassette flanked by recombi-
nation flanks for insertion into the TK locus (see Fig. 3). In a
second step, the foreign gene was inserted in place of the dsRed2
Plasmid pRednsTK was isolated as follows. Recombination
flanks for the TK locus were amplified from vaccinia WR DNA.
SFV sequence was assembled precisely at the transcriptional start
nucleotide of the VV TK promoter, by recombinant PCR using
two fragments amplified with oligonucleotides.
GACT-3 (VV left TK flank, amplified on WR virus DNA) and
p TK 1F 59-GAATAAAGTGAACAATAATTAATTCTCGAC-
andSFV 700 XhoI
CAGTTTGTG-39. This PCR fragment spans the 59 end of the
SFV cDNA, which included the BsiWI site at coordinate 514 of
pSFV-1 used later to clone additional SFV sequences.
Right VV TK flank was amplified with oligonucleotides TKRL
5-TTGGGTGAGGATCCCGAGATAGAAATAA-39 and TKRR
59-TTTTTGATGCATCGTAGATATTCCTCATC-39. An in-
termediate plasmid containing the TK flanks and the 59 end of the
SFV genome was termed pRedTK.
SFV sequences were obtained as follows: First, a TTTTTGT
sequence, (which constitutes a VV early transcription termination
signal) present in the ns4 gene of the SFV replicon was mutated to
TTTCTGT in plasmid pSFV-1. Subsequently, a 6887 bp
fragment containing the most of the sequence of the non-structural
genes of the SFV replicon was obtained from this plasmid by
digestion with BsiWI and BamHI (coordinates 514 to 7404 in
pSFV-1) and was inserted into the corresponding sites in plasmid
pRedTK, generating plasmid pnsTK. Then, a fragment contain-
ing dsRed gene under a synthetic early/late promoter was digested
with BamHI and BglII from pRBdsRed2  and inserted in
BamHI site of plasmid pnsTK. The resulting plasmid, pRednsTK,
contains a dsRed cassette and most of the SFV replicon flanked by
WR TK flanking sequences.
Plasmid pMix-f70An, designed for insertion of foreign genes in
the sub-genomic region of the SFV replicon, was derived from
pRednsTK by substituting a SphI-HindIII fragment containing
the nsP1–4 genes, the dsRed2 cassette and the TK left flank by a
fragment encompassing a portion of nsP4 gene and the GFP
marker gene placed downstream of the SFV subgenomic
promoter. This fragment was amplified by PCR from pSFV-
rsGFP using oligonucleotides ns-4 f 59-CGGTCGGCATGCAAC-
GAGATGTCA-39 and SFV GFP Hind R 59-GGGATGTAA-
TAAGCTTAATTACCCGG-39. Then, a fragment spanning
140 nt of the replicon 39 sequence (including a 70 nucleotide-
long polyA tail) was amplified by PCR from pSFV-1 using
oligonucleotides F70Xma 59-GGCAATACCCGGGAGCTTA-
CATAAG-39 and FStuRPoliA 59-GCGCGTAGGCCTATT-
CATTAATGCA-39 and inserted in the Xma-I and Stu-I sites in
pMix using the corresponding sites included in the oligonucleo-
tides. The resultant plasmid, pMix-f70An contains a portion of the
nsP4 gene as the left recombination flank, the GFP gene under the
SFV subgenomic promoter, 70 nucleotides of 39 sequence, plus a
70 nt polyA sequence of the SFV replicon and the right flank of
Plasmid pRednsTK was used to generate VV recombinants
containing the ns1–4 genomic region of the SFV replicon. Thus,
pRednsTK was transfected into cells infected with WR virus or
VV-Helper to generate recombinant viruses W- RednsTK or W-
H-RednsTK (Figure 3). These recombinant viruses were subse-
quently used for insertion of the 39 end of the replicon.
The final combined vectors W-SFR and W-H-SFR were
isolated by recombination of pMix-f into the W-RednsTK or
W-H-RednsTK recombinant viruses, respectively (See scheme in
Fig. 3). Recombinant viruses were isolated by plaque isolation, by
identifying plaques under the fluorescence microscope by GFP
expression or absence of dsRed2 expression.
Quantitation of b–Galactosidase and b–Glucuronidase
BHK-21 cells grown in 12-well plates were mock infected,
infected with WR or with vaccinia virus recombinant vSIMBE/L
which express Sp6 RNA polymerase at a moi of 5 PFU/cell. One
hour later, the cells were transfected with 2 mg of pSFV-LacZ
plasmid using Lipofectin, following manufacturer’s protocols. At
5 h posttransfection medium was replaced with fresh medium. At
36 h the cells were lysed in 300 ml of lysis buffer (1% NP40,
50 mM Tris-HCl pH 7.6, 150 mM NaCl, 2 mM EDTA, 1 mg/ml
PMSF), for 10 min at 4uC. b–Galactosidase or b–Glucuronidase
in the cell extracts was measured using ONPG (Sigma N1127) or
4-NPG (Sigma N1627) as substrates, respectively. After a 20 min
incubation at 37 uC the enzymatic reaction was followed by color
development at 414 nm. 100 ml of diluted lisate was tested
addition 100 ml of BufferZ-ONPG2X .
Monolayers of Hela cells in p100 culture plates were infected
with W-H-SFR virus at a moi of 5 pfu/cell. At 24 hpi, the culture
medium was removed and the cells were fixed by adding a solution
of 2% glutaraldehyde, 1% tannic acid, and 0.4 M HEPES
(pH 7.2) directly to the monolayer. After 2 hours of fixation at
room temperature, cells were scraped and pelleted by centrifuga-
tion at 2000 rpm for 5 min. Cells were resuspended in 1 ml of
HEPES buffer and were included in Epon 812 resin as described
. Ultrathin sections were obtained with a Ultracut microtome,
which were deposited on copper grids coated with a film of
coloidon/carbon. Finally, the preparations were contrasted with
lead citrate 0.2% under nitrogen and examined by electron
To generate stocks of single-cycle Semliki Forest virus particles
(SFPs), BHK 21 cells were infected with W-H-SFV at a moi of
5 pfu/cell. At 24 h.p.i, media were collected, clarified (centrifuged
5 minutes at 2000 rpm) and filtered through a 0.1 mm filter to
remove vaccinia virus. Filtered media were aliquoted and kept as
SFP stocks. In order to measure the titer of SFPs, cell culture
supernatants were titrated on fresh monolayers of BHK-21 or
BSC-1 cells grown to 70–80% confluence. Different concentra-
tions of the filtered medium were used to infect cells seeded in 6-
well plates. After a 2 hour adsorption period, the cells were
incubated for at least 24 hours. Finally, the titer of SFV particles
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PLOS ONE | www.plosone.org9 October 2013 | Volume 8 | Issue 10 | e75574
was estimated by counting cells displaying GFP fluorescence under Download full-text
a Nikon Eclipse TE2000-E inverted fluorescence microscope.
To visualize large virus plaques in single images, a number of
overlapping microscopy images were acquired from individual
virus plaques. These images were stitched together using a Grid/
Collection Stitching plug-in for ImageJ .
We thank Henrik Garoff for the generous gift of pSFV-1 and pSFV-Helper
1, and Dr. Bernard Moss for vSIMBE/L.
Conceived and designed the experiments: JMS MML RB. Performed the
experiments: JMS MML. Analyzed the data: JMS MML RB. Wrote the
paper: JMS MML RB.
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