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Previous Article | Next Article 
Journal of Virology, January 2000, p. 547-551, Vol. 74, No. 1
0022-538X/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Plasmid-Driven Formation of Influenza
Virus-Like Particles
Gabriele
Neumann,1
Tokiko
Watanabe,1,2 and
Yoshihiro
Kawaoka1,*
Department of Pathobiological Sciences,
School of Veterinary Medicine, University of Wisconsin
Madison,
Madison, Wisconsin 53706,1 and
Laboratory of Microbiology, Department of Disease Control,
Graduate School of Veterinary Medicine, Hokkaido University, Sapporo
060-0818, Japan2
Received 24 June 1999/Accepted 16 September 1999
 |
ABSTRACT |
We established a plasmid-based system for generating infectious
influenza virus-like particles entirely from cloned cDNAs. Human
embryonic kidney cells (293T) were transfected with plasmids encoding
the influenza A virus structural proteins and with a plasmid encoding
an influenza virus-like viral RNA (vRNA) which contained an antisense
copy of the cDNA for green fluorescence protein (GFP) flanked by an RNA
polymerase I promoter and terminator. Intracellular transcription of
the latter construct by RNA polymerase I generated GFP vRNA that was
packaged into influenza virus-like particles. This system, which
produced more than 104 infectious particles per ml of
supernatant, would be useful in studies of influenza virus replication
and particle formation. It might also benefit efforts in vaccine
production and in the development of improved gene therapy vectors.
| |
TEXT |
Influenza A viruses possess a genome
of eight single-stranded negative-sense viral RNAs (vRNAs) that encode
a total of 10 proteins. The influenza virus life cycle begins with
binding of the hemagglutinin (HA) to sialic acid-containing receptors
on the surface of the host cell (reviewed in reference
6), followed by receptor-mediated endocytosis. The
low pH in late endosomes triggers a conformational shift in the HA,
resulting in fusion of the viral and endosomal membranes and the
consequent release of the matrix protein (M1) and ribonucleoprotein
complexes (RNPs) into the cytosol of infected cells. RNPs consist of
the nucleoprotein (NP), which encapsidates vRNA, and the viral
polymerase complex, which is formed by the PA, PB1, and PB2 proteins.
RNPs are transported into the nucleus, where transcription and
replication take place. Newly synthesized RNPs are then exported from
the nucleus and transported to the cellular membrane, where progeny
virus particles are assembled. The neuraminidase (NA) protein plays a
crucial role late in infection by removing sialic acid from
sialyloligosaccharides, thus releasing newly assembled virions from the
cell surface and preventing the self-aggregation of virus particles.
Although virus assembly involves protein-protein and protein-vRNA
interactions, the nature of these interactions is largely unknown.
A vaccinia virus-based system for the generation of influenza
virus-like particles (VLPs) has been established (2, 7). In
this system, an influenza virus-like vRNA carrying a reporter gene is
transcribed in vitro and transfected into eukaryotic cells. All 10 influenza virus proteins are expressed from plasmids under the control
of a T7 RNA polymerase promoter. When the transfected cells are
infected with a recombinant vaccinia virus that expresses T7 RNA
polymerase, they produce influenza VLPs containing the vRNA of an
artificial reporter gene (7). However, vaccinia virus
expresses more than 80 proteins, any of which could affect the
influenza virus life cycle. We therefore sought to establish an
efficient plasmid-driven system for the generation of infectious influenza VLPs containing a virus-like RNA segment.
Expression of the influenza virus proteins PB2, PB1, PA, and NP
leads to replication and transcription of an artificial vRNA.
To
generate influenza VLPs, we used the RNA polymerase I system for the
intracellular synthesis of influenza virus RNAs (Fig. 1) (12). In this system, a
cDNA carrying a reporter gene in antisense orientation is flanked by
the 5' and 3' noncoding regions of an influenza virus RNA (Fig.
2). This cassette is inserted between an
RNA polymerase I promoter and terminator. Transfection of such
constructs into eukaryotic cells leads to transcription of the reporter
gene by cellular RNA polymerase I, thereby generating influenza
virus-like RNAs (12). Upon influenza virus infection, the
artificial vRNAs are replicated and transcribed by the viral polymerase
complex, resulting in the expression of the reporter gene
(12).
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FIG. 1.
Schematic diagram of VLP generation strategy. Individual
protein expression plasmids and a plasmid containing the RNA polymerase
I promoter, a cDNA encoding the GFP reporter gene, and the RNA
polymerase I terminator are transfected into 293T cells. Intracellular
transcription by RNA polymerase I yields GFP vRNA of negative polarity,
as indicated by inverted letters. Supernatants containing VLPs are
harvested, mixed with influenza helper virus, and inoculated into MDCK
cells.
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FIG. 2.
The pPolI-GFP plasmid for generating influenza
virus-like RNA encoding the GFP protein. The GFP gene (derived from
pEGFP-N1 [Clontech, Palo Alto, Calif.]) was inserted in the antisense
orientation into pHL1844 (E. Hoffmann, Ph.D. thesis, Justus-Liebig
University, Giessen, Germany), which contains the 5' and 3' noncoding
regions of influenza A virus segment 5 (NP-NTRs). The gene was flanked
by multiple cloning sites (MCS1 and MCS2) and by the human RNA
polymerase I promoter and the mouse RNA polymerase I terminator.
Asterisks indicate mutations introduced to upregulate promoter activity
(10a).
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|
We first tested whether expression of the PB2, PB1, PA, and NP proteins
leads to expression of the reporter gene carried by the RNA polymerase
I-derived transcript. Plasmids (1 µg each) expressing the NP protein
of A/WSN/33 (H1N1) virus under the control of the chicken 
-actin
promoter (pCAGGS-WSN-NP0/14) (10), the polymerase proteins
of A/PR/8/34 virus under the control of the cytomegalovirus promoter
[pcDNA762(PB2), pcDNA774(PB1), and pcDNA787(PA)] (14), and
an RNA polymerase I reporter gene construct (pPolI-GFP) (Fig. 2) were
transfected into human embryonic kidney (293T) cells as described
previously (11). Forty-eight hours later, 30 to 40% of the
cells were expressing green fluorescence protein (GFP) (Fig.
3). In contrast, GFP expression could not
be detected in transfected cells lacking the polymerase or NP proteins.
These results indicate that NP and the three influenza virus polymerase proteins had formed a functional complex that replicated and
transcribed the RNA polymerase I-derived GFP vRNA.
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FIG. 3.
The PA, PB1, PB2, and NP proteins of influenza A virus
encapsidate GFP vRNA produced by RNA polymerase I, leading to GFP
expression. 293T cells were transfected with plasmids expressing the
PB2, PB1, PA, and NP proteins (A) or with all plasmids except the one
expressing the NP protein (B), together with the RNA polymerase I-GFP
gene plasmid for intracellular synthesis of reporter gene vRNA. Cells
were fixed 48 h after transfection, and GFP expression was
determined with a fluorescence microscope. Magnification, ×100.
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Optimal vRNA transcription and replication.
To determine the
amounts of plasmid DNA required for optimal reporter GFP expression, we
modulated the expression of the polymerase proteins and NP. Previous
studies had indicated that large amounts of PA reduce the extent of
reporter gene expression in transcription-replication systems
(7). We therefore reduced in a stepwise manner the expression of PA from the plasmid, identifying 0.1 µg of pcDNA787(PA) as the template amount yielding the strongest expression of GFP (data
not shown). With NP, the major structural component of RNP complexes,
large amounts of protein expression plasmid may be required. However,
larger amounts of the plasmid did not appreciably affect the number of
GFP-positive 293T cells (data not shown). In addition, various amounts
of the PB2 and PB1 protein expression plasmids (ranging from 1.0 to
0.03 µg) did not affect GFP expression in 293T cells (data not
shown). Hence, in all subsequent experiments, we used 0.1 µg of
pcDNA787(PA) and 1.0 µg of pcDNA774(PB1), pcDNA762(PB2), and
pCAGGS-WSN-NP0/14.
Formation of influenza VLPs from cloned cDNAs.
Previous
studies with the vaccinia virus T7 RNA polymerase system showed that
the formation of influenza VLPs requires nine influenza virus proteins:
PB2, PB1, PA, HA, NA, NP, M1, M2, and NS2 (7). The NS1
protein, in contrast, is dispensable for particle formation
(7). To establish an efficient plasmid-driven system for VLP
generation, we generated cDNAs encoding the HA, NA, M1, M2, and NS2
genes and cloned them into the eukaryotic expression vector pCAGGS/MCS
(controlled by the chicken -actin promoter) (13),
resulting in pEWSN-HA, pCAGGS-WNA15, pCAGGS-WSN-M1-2/1, pEP24c, and
pCA-NS2, respectively. Expression of each protein was confirmed by
Western blot analysis (data not shown).
To generate VLPs, we first transfected 106 293T cells with
1.0 µg of each protein expression plasmid [with the exception of pcDNA787(PA), for which we used 0.1 µg] and with 1 µg of the
reporter gene construct pPolI-GFP. Culture supernatants were harvested 48 h after transfection and mixed with A/WSN/33 virus to provide the influenza virus proteins required for replication and transcription of GFP vRNA. The mixture was then inoculated into MDCK cells (Fig. 1).
Ten hours after incubation, we detected GFP-positive MDCK cells,
corresponding to ~450 particles/ml of supernatant (Table 1). Thus, plasmid-driven expression of
all influenza virus structural proteins resulted in the formation of
infectious influenza VLPs containing GFP vRNA that could be delivered
into subsequent cells.
Optimal assembly of influenza virus.
VLP formation was also
studied in cells expressing different amounts of the RNA polymerase I
reporter gene construct, as well as HA, NA, M1, M2, and NS2 plasmid
DNAs. In experiments with pPolI-GFP, 1.0 µg of the plasmid DNA was
highly efficient in generating VLPs, whereas the efficiency was
significantly reduced for 2.0 or 3.0 µg (data not shown). Because the
NS2 and M2 proteins are expressed in small amounts late in infection,
we reasoned that relatively small amounts of the expression plasmids
would be needed for optimal VLP formation. Reduction of the M2
expression construct from 1.0 to 0.1 µg resulted in more than a
10-fold increase in the number of GFP-positive MDCK cells (Table 1);
further reduction to 0.03 µg did not increase the number of VLPs. For
NS2, smaller amounts of plasmid (0.1 µg) were associated with less
efficient formation of VLPs (Table 1).
The M1 protein is the major structural component of the virion. Thus,
high levels of M1 expression are likely required for efficient
formation of VLPs. This prediction was tested in experiments comparing
VLP formation in cells transfected with 1.0 or 2.0 µg of M1 plasmid
DNA. As shown in Table 1, larger amounts of plasmid resulted in more
than a 10-fold increase in the number of GFP-positive MDCK cells.
Comparison of two different amounts (1 and 2 µg) of plasmids
expressing the HA and NA proteins did not reveal any appreciable
differences in VLP formation (data not shown), leading to selection of
1 µg of each plasmid (pEWSN-HA and pCAGGS-WNA15) for use in
subsequent experiments. Overall, these studies resulted in a >100-fold
increase in the efficiency of VLP formation, ultimately leading to the
production of more than 104 infectious influenza virus
particles per ml of supernatant (Fig. 4).
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FIG. 4.
Generation of infectious influenza VLPs. 293T cells were
transfected with nine plasmids, each expressing a different viral
structural protein (A), or with eight plasmids omitting the construct
for NP (B), together with the RNA polymerase I-GFP gene plasmid.
Forty-eight hours after transfection, supernatants were collected,
mixed with A/WSN/33 helper virus, and inoculated into MDCK cells. Cells
were fixed at 10 h after infection, and GFP expression in
VLP-infected MDCK cells was determined with a fluorescence microscope.
Magnification, ×100.
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Authenticity of VLPs produced entirely from plasmids.
To
verify that VLPs initiate infection in the same manner as authentic
influenza viruses, we attempted to neutralize the VLPs with antibody to
the A/WSN/33 HA. VLP-containing supernatants derived from
plasmid-transfected 293T cells were incubated with a pool of
anti-A/WSN/33 HA monoclonal antibodies or with a monoclonal antibody to
the G protein of vesicular stomatitis virus (VSV) (negative control)
for 1 h at room temperature. A/PR/8/34 helper virus, which is not
neutralized by the pool of anti-A/WSN/33 HA monoclonal antibodies, was
added to the mixture and inoculated into MDCK cells. Only the A/WSN/33
HA-specific monoclonal antibody neutralized the VLPs (data not shown),
indicating that the HA mediates the attachment and entry of VLPs into cells.
Next, we identified the minimal set of proteins required for the
formation of VLPs. Others have established that the three influenza
virus polymerases and the NP are essential for the replication and
transcription of vRNA (3). Therefore, we included each of
these four proteins in our assay but consecutively omitted HA, NA, M1,
M2, or NS2. Exclusion of either of the plasmids mentioned above did not
affect the replication-transcription of GFP vRNA in transfected 293T
cells (data not shown). Supernatants derived from transfected 293T
cells that lacked the HA, NA, M1, or NS2 protein did not promote GFP
expression in infected MDCK cells, indicating the absence of infectious
VLPs. Infectious VLPs were detected with the omission of M2 but the
number was low (>500-fold reduction compared to the full set of
structural proteins). Thus, all influenza virus structural proteins are
required for the efficient formation of infectious VLPs, in accord with
data from studies of the vaccinia virus-based system (7).
VSV glycoprotein can replace the HA and NA proteins in the
production of VLPs.
To test the utility of our plasmid-based
system for virus assembly studies, we replaced the influenza virus HA
and NA proteins with the VSV-G protein, which functions in receptor
binding and fusion. When 293T cells were transfected with pPolI-GFP,
with optimal amounts of the PB2, PB1, PA, NP, M1, M2, and NS2
expression constructs, and with 1 µg of the VSV-G construct
(pCAGGS-VSV-G), substitution of the VSV-G protein for influenza virus
glycoproteins did not adversely affect VLP formation (data not shown).
In fact, we reproducibly found slightly higher numbers (~1.2-fold) of
GFP-positive cells when VSV-G, rather than the HA and NA, served as the
viral glycoprotein. Thus, the VSV-G protein can be efficiently
incorporated into virions and can function as well as the HA and NA in
virus release and entry.
An efficient system for generating infectious influenza virus particles
would be an asset in research with this virus and potentially in the
production of vaccines and vectors for gene therapy. Moreover, this VLP
system leads to the generation of influenza A viruses entirely from
cloned cDNA (11). The VLP production strategy described here
is highly efficient, both in the initial transfection of cells and in
the yield of VLPs (>104 infectious particles/ml of
supernatant). Moreover, it is driven entirely by plasmids expressing
influenza virus proteins (i.e., in the absence of any other viral
proteins), which greatly simplifies the interpretation of results.
Another major advantage is the capability of studying the effects of
lethal mutations in virion formation, the packaging of RNP complexes,
the budding of virus replication, and the binding and fusion processes.
In addition, we expect that our system would operate equally well with
other viruses, e.g., paramyxoviruses and rhabdoviruses.
We demonstrated that the influenza virus HA and NA proteins can be
functionally replaced by the VSV-G protein. Influenza viruses failed to
incorporate VSV-G protein when provided by recombinant simian virus 40 (9). However, assays had not been available to replace the
influenza virus glycoproteins by foreign glycoproteins. Therefore, the
question of whether interactions of HA and/or NA with other viral
proteins are essential for virus formation remained open. Although a
novel virus has been generated upon expression of VSV-G protein as well
as a Semliki Forest virus replicon in the same cell, the infectious
titer of this new virus is at least 105-fold lower than
that of normal VSV (15). Thus, the Semliki Forest virus
replicon is not efficiently incorporated by VSV-G. By contrast, the
infectious titers of VLPs with VSV-G, instead of HA and NA, were even
higher than those of authentic influenza VLPs. Therefore, our finding
suggests that neither HA nor NA is essential for the formation of VLPs.
However, we cannot rule out a role for these glycoproteins in
interactions with other viral proteins, which would affect the
structure of virions and the efficiency of virion formation, as
suggested by the elongated shapes of viruses expressing tailless HAs,
NAs, or both (1, 4, 5, 8).
We emphasize the potential value of our plasmid-based system for
therapeutic gene delivery. One can now generate VLPs that contain a
vRNA encoding the proteins required for transcription and replication
(i.e., the NP and the polymerases), as well as a vRNA encoding the
protein of interest. These particles are infectious and would deliver a
designated gene into target cells, where it would replicate and be
transcribed. Because these particles do not contain a full complement
of viral genes, they cannot produce infectious progeny viruses. This
feature, together with the lack of integration of the viral genome into
host chromosomes, would ensure the biological safety of gene delivery
in human and nonhuman subjects. Finally, the availability of 15 HA and
9 NA subtypes and their variants would allow one to administer VLPs
repeatedly, thereby overcoming immunoresistance to vector-generated
proteins, which is one of the major obstacles faced with repeated use
of other viral vectors, such as adenoviruses. We suggest that the greatest benefit of our plasmid-driven system would be realized in
situations requiring only short-term expression of foreign proteins, as
in cancer treatment.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Pathobiological Sciences, School of Veterinary Medicine, University of WisconsinMadison, 2015 Linden Dr. West, Madison, WI 53706. Phone: (608) 265-4925. Fax: (608) 265-5622. E-mail:
kawaokay{at}svm.vetmed.wisc.edu.
| |
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Journal of Virology, January 2000, p. 547-551, Vol. 74, No. 1
0022-538X/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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