Download Influenza virus naked RNA can be expressed upon transfection into

Journal of General Virology (1993), 74, 535-539.
535
Printed in Great Britain
Influenza virus naked RNA can be expressed upon transfection into cells
co-expressing the three subunits of the polymerase and the nucleoprotein
from simian virus 40 recombinant viruses
Susana de la L u n a , la Javier Martin,3t Agustin Portela 3 and Juan O r t i n ~a*
1Centro Nacional de Biotecnologia (CSIC), and 2Centro de Biologia Molecular ( CSIC-UAM), Universidad
Aut6noma, Cantoblanco, 28049 Madrid and 3Instituto Carlos III, Centro Nacional de Microbiologia, Inmunologia y
Virologia Sanitarias, Majadahonda, 28220 Madrid, Spain
The functionality of the influenza virus polymerase
subunits and the nucleoprotein expressed from simian
virus 40 (SV40) recombinants has been tested by their
ability to direct the in vivo expression of influenza viruslike RNAs. These RNAs, which contained either the
chloramphenicol acetyltransferase (CAT) or haemagglutinin (HA) genes, were synthesized and reconstituted
in vitro into viral ribonucleoproteins with a polymerase/
nucleoprotein mixture purified from influenza virusinfected cells. Only the coinfection with SV40
recombinant viruses expressing the three polymerase
subunits and the nucleoprotein allowed the expression
of the transfecting CAT or HA RNAs, confirming that
this set of viral genes is the minimal requirement for viral
gene expression. Unexpectedly, transfection of the
corresponding naked RNAs into SV40 recombinantinfected cells was as effective in directing the synthesis of
CAT or HA proteins as the standard reconstituted
ribonucleoprotein transfection. These results may be
important for the genetic analysis of trans-acting factors
involved in influenza virus transcription and replication
and may open the way to rescuing influenza viruses in
the absence of a helper virus.
Influenza virus RNA transcription and replication take
place in the nucleus of the infected cell (Herz et al., 1981 ;
Jackson et al., 1982) in ribonucleoprotein (RNP)
complexes consisting of at least the three polymerase
subunits (PB 1, PB2 and PA) and the nucleoprotein (NP)
in addition to positive or negative polarity RNA
segments (Beaton & Krug, 1986; Detjen et al., 1987;
Honda et al., 1988; van Wike et al., 1981). In spite of
the development of several in vitro transcription and
replication systems derived from infected cells (Beaton
& Krug, 1984; del Rio et al., 1985; Lopez-Turiso
et al., 1990; Shapiro & Krug, 1988; Takeuchi et al.,
1987), several questions remain unresolved relating to
the control of the transcription replication and
c R N A - v R N A synthesis switches, as well as the structure
of the transcription/replication complex(es).
In the last few years, an experimental approach has
been developed for the rescue of artificial influenza virus
RNAs into infectious virus (Enami & Palese, 1991;
Luytjes et al., 1989; Yamanaka et al., 1991). This
system involves the in vitro reconstitution of RNPs,
using synthetic RNA and a mixture of polymerase sub-
units and nucleoprotein purified from virions, and its
transfection into cells infected by a helper virus. Its use
has allowed the introduction of defined mutations into
viral RNA (Enami et al., 1990) and the analysis of the
structure of the viral promoter (Li & Palese, 1992; Seong
& Brownlee, 1992; Yamanaka et al., 1991). However,
the requirement for a helper virus infection has precluded
the study of trans-acting factors involved in the transcription and replication processes. An important breakthrough in that direction was the description of a
completely artificial system in which defined vaccinia
virus recombinants substituted for the helper virus
infection (Huang et al., 1990). Using this system, it was
concluded that the three polymerase subunits and the
nucleoprotein constitute the minimal requirements for
viral RNA transcription and replication (Huang et al.,
1990). In spite of previous evidence for the role of the
non-structural proteins NS1 and NS2 in viral RNA
synthesis (Koennecke et al., 1981; Odagiri & Tobita,
1990; Shimizu et al., 1982; Snyder et al., 1990) and their
location in the nucleus of the infected cells (Briedis et al.,
1981; Greenspan et al., 1985), no effect could be seen
when they were expressed in addition to the minimal
replication system (Huang et al., 1990).
In previous studies we have expressed a number of
~- Present address: National Institute for Medical Research, The
Ridgeway, Mill Hill, London NW7 1AA, U.K.
0001-1355 © 1993 SGM
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536
Short communication
(a)
m
O
SVPOLs
SVNP
Virus
O
+
+
+
SVPB1
SVPB2
+
+
SVPA
SVNP
+
-
+
-
+
+
+
+
-
_
_
+
+
+
+
+
+
+
+
+
+
+
(b)
Infection
Transfection
Experiment 1
Experiment 2
Mock
Mock
SVPOLs
SVPOLs + SVNP
A/Victoria/3/75
A/PR/8/34
+
+
+
+
+
ND
ND
ND
93 pmol/pg
7 pmol/~tg
-
ND
ND
ND
20 pmol/pg
4 pmol/pg
Fig. 1. Biological activity of the SV40 recombinants expressing the polymerase subunits and NP. Cultures of 3 × 105 COS-1 cells were
infected with the SV40 recombinants, with influenza virus or mock-infected. Independent controls were carried out to ensure that each
SV40 recombinant virus infected most of the cells in the culture. At 48 h post-infection, the cultures were transfected by the D E A E dextran/DMSO method with the T7 R N A polymerase transcript of plasmid pPB2CAT9. Transcription was carried out in the presence
of polymerase/NP mixture purified from P R / 8 influenza virus-infected cells. At 20 h after transfection, total cell protein extracts were
prepared and assayed for CAT activity. (a) Assays were carried out by the TLC procedure using 50 pg of protein, except for the sample
of cells infected by all four SV40 recombinants that had 10 pg, and an incubation time of 4 h. (b) Assays were carried out by the PE
method using 1 h incubation time and two doses of protein to ensure that the assay was not saturated. The values given indicate pmol
of [3H]acetate transferred to chloramphenicol per pg of protein lysate. SVPOLs indicates a mixture of SVPB1, SVPB2 and SVPA
recombinant viruses. ND, Activity not detectable.
influenza virus gene products by means of simian virus
40 (SV40) recombinants, including the three polymerase
subunits (PB1, PB2 and PA) (de la Luna, 1989; Nieto
et al., 1992) and the nucleoprotein (NP) (Portela et al.,
1985 b). In order to assay for the biological activity of the
proteins expressed from these recombinants we made
use of recombinant pPB2CAT9, which contains a
chloramphenicol acetyltransferase (CAT) gene in negative polarity, flanked by the terminal sequences of the
influenza virus RNA segment 1, under the control of the
T7 RNA polymerase promoter (M. Krystal, personal
communication). Cultures of COS-1 cells were infected
with recombinant viruses SVPB 1, SVPB2, SVPA, SVNP
or a combination of these and subsequently mocktransfected or transfected with the product of in vitro
transcription of plasmid pPB2CAT9 digested with HgaI
and filled in with Klenow polymerase. The transcription
was carried out in the presence of a mixture of NP and
the three subunits of the polymerase, purified from PR8
virus-infected cell extracts (Martin et al., 1992). As a
positive control, transfection was carried out on cultures
of influenza virus-infected cells. In standard assays,
approximately 300 ng of CAT-construct RNA, reconstituted into viral RNP, was transfected into 3 x 105
cells by either the DEAE-dextran/DMSO method
(Lopez-Turiso & Ortin, 1988; Luytjes et al., 1989) or with
lipofectin (BRL). Total protein extracts were prepared by
freezing and thawing at 20 to 24 h post-transfection. CAT
activity was at least twofold higher in iipofectin-mediated
than in DEAE-dextran/DMSO-mediated transfections.
The assay for CAT activity in the cell extracts was carried
out by two methods: (i) transfer of acetyl residues to
[l~C}chloramphenicol and separation of the acetylated
molecules by thin-layer chromatography (TLC assay)
(Gorman et at., 1982) and (ii) transfer of [aH]acetyl
moieties from [3H]acetyl-CoA to unlabelled chloramphenicol and separation of labelled products by extraction into the organic phase (PE assay) (Portela et al.,
1985a). The first method is somewhat more sensitive but
the second allows quantitative determinations, since it is
carried out in the presence of excess chloramphenicol.
In order to test whether the proteins expressed by the
SV40 recombinant viruses were competent in replication
and transcription of the CAT synthetic RNP in vivo,
COS-1 cell cultures were either mock-infected, singlyinfected, doubly-infected or infected with mixtures of
three or four SV40 recombinants. After CAT RNP
transfection, total cell extracts were prepared and CAT
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(a)
6 6
:z
: ?,i
/z '
SVPOLs
SVNP
Pol/NP mix
-
+
•
•
•
+
+
+
-
+
+
+
-
+
(b)
Expt no.
1
2
3
4
- PolfNP mix
352
198
278
232
pmol/gg
pmol/gg
pmol/gg
pmol/gg
+ Pol/NP mix
298
158
117
170
pmol/gg
pmol/gg
pmol/gg
pmol/gg
Fig. 2. Expression of naked influenza virus-like CAT RNA. Cultures of
COS-1 cells were infected with SV40 recombinants as described in Fig.
1. Infected cell cultures were transfected by the lipofectin method with
CAT RNA transcribed, as indicated in Fig. 1, in the presence or absence
of purified polymerase/NP mixture. At 20 h after transfection, total
cell protein extracts were prepared and asayed for CAT activity. (a)
Assays were carried out by the TLC procedure using 20 gg of protein
and an incubation time of 4 h. (b) The results of four independent
experiments in which the cultures were infected by all four SV40
recombinants and transfected with CAT RNA transcribed in the
presence or absence of polymerase/NP mixture are shown. The assays
were carried out by the PE method as in Fig. 1.
activity was determined by the TLC assay, using 5 to 50 gg
of total protein and 4 h incubation periods. As shown in
Fig 1 (a), CAT activity was reproducibly detectable only
in extracts of cells expressing the three subunits of the
influenza virus polymerase and the NP, at levels
comparable to those present in extracts from influenza
virus-infected cells. Neither single infections nor any
double or triple infection yielded any detectable CAT
activity.
The level of CAT activity was dependent on the time
after SV40 recombinant virus infection at which
transfection was carried out. Maximal activity was
obtained at 48 to 60 h post-infection, a time when the
level of SV40-expressed proteins was maximal (data not
shown). When CAT activity was quantitatively determined by the PE assay, it was clear that this activity is
approximately five to 15-fold higher in cells infected with
SV40 recombinants than in cells infected with helper
influenza virus (Fig. 1 b).
The results described above confirm that, as in the
other published expression system (Huang et al., 1990),
the three subunits of the viral polymerase and the NP are
sufficient for synthetic RNP expression. However, the
537
system described here behaves in a different way: whereas
the detection of CAT activity depends strictly on a prior
RNA incubation with purified polymerase/NP fraction,
both in a helper influenza virus infection and in the
vaccinia virus expression system (Huang et al., 1990;
Luytjes et al., 1989; Martin et al., 1992), the SV40
expression system is also very efficient in inducing CAT
activity by transfection of naked R N A (Fig. 2). In a
series of independent transfection experiments, the
activities obtained with naked RNA were 1.2- to 2.4-fold
higher than those observed in parallel standard RNP
transfections. The basis for the differences among the
various expression systems is not apparent. It is
conceivable that the former transfections demand a
more effective protection of the transfecting RNA than
the latter. Conversely, the higher concentration of
polymerase subunits provided by the SV40 expression
system (Nieto et al., 1992) and, more importantly, the
fact that they are free to complex the incoming RNA,
might lead to a more effective intracellular formation of
transcription complexes. In this respect, the SV40-based
system is similar to the one recently described by Kimura
et al. (1992) and, in addition, it offers an easier way of
expressing different combinations of proteins by means
of infection with the corresponding SV40 recombinants.
To check further the efficiency of the system in
directing influenza virus RNA expression, cell cultures
were infected with SVPB1, SVPB2, SVPA and SVNP
recombinant viruses and transfected with a negativepolarity RNA corresponding to segment 4 of WSN
influenza virus. This RNA was obtained by T3 RNA
polymerase transcription of plasmid pT3/WSN-HA
digested with Ksp632I and filled in with Klenow
polymerase (Enami & Palese, 1991), both in the presence
and in the absence of purified polymerase-NP mixture.
Sixteen hours after transfection, cell monolayers were
fixed and analysed by immunofluorescence. Immunostaining with a specific polyclonal antibody showed that
haemagglutinin (HA) was detected after transfection of
either naked HA RNA or reconstituted HA RNP into
cells infected with SV40 recombinants expressing the
three subunits of the polymerase and NP, but not in
mock-infected, in mock-transfected cells or in cells
expressing the three subunits of the polymerase (Fig. 3).
The expression of HA was observed in 10 to 20 % of the
transfected cells and its accumulation was comparable to
that observed during a normal influenza virus infection.
In conclusion, using influenza virus gene products
expressed by means of SV40 recombinants in COS-1
cells, we have confirmed that in this system the three
subunits of the polymerase and the nucleoprotein
constitute the minimal gene complement for the expression of a foreign, influenza virus-like, gene. Unexpectedly, however, the in vitro reconstitution of the
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538
Short communication
Mock
Mock/T
SVPOLs
o
Fig. 3. Expression of influenza virus HA naked RNA. Cultures of COS-1 cells were infected as described in Fig. 1. At 48 h postinfection, cells were mock-transfected or transfected by the lipofectin method with a T3 polymerase transcript of plasmid pT3/WSNHA prepared in the presence or absence of the purified polymerase/NP mixture. At 16 h after transfection, cultures were fixed with
methanol and stained with an anti-PR/8 virus rabbit antiserum and Texas red-tagged goat anti-rabbit IgG serum. SVPOLs indicates
a mixture of SVPB1, SVPB2 and SVPA viruses. Mock/T indicates transfection on a mock-infected culture.
transfected RNA into viral RNP was not required for its
expression. This result might be relevant from a practical
point of view, since it eliminates the necessity for
purification of the polymerase/NP mixture from either
virions or infected cells (Luytjes et al., 1989; Martin et al.,
1992). In addition, the possibility of expressing viral
genes after transfection of naked R N A eliminates the
problems that the incoming, transfecting viral proteins
might introduce in the interpretation of the phenotype of
viral gene mutants being assayed in the system. Furthermore, this possibility may open the way for a cleaner
procedure by which to rescue transfectant viruses, since
the purified polymerase/NP mixture, in spite of being
practically devoid of viral RNA, can produce infectious
virus when complemented in trans by SV40 recombinant
viruses expressing the polymerase and the NP (data not
shown).
We thank J. A. Melero for critically reading this manuscript and
A. Nieto for the characterization of the SVPA recombinant. Plasmids
pPB2CAT9 and pT3/WSN-HA and anti-PR/8 virus serum were
kindly provided by M. Krystal, P. Palese and J. J. Skehel, respectively.
The technical assistance of A. Beloso, J. Rebelles, F. Ocafia and V.
Blanco is gratefully acknowledged. This work was supported by
Comisidn Interministerial de Ciencia y Tecnologia (grants BIO88-0191
and BIO89-0545), by the EEC Science Programme (grant SC1"CT910688) and by an institutional grant of Fundacidn Areces to the
CBM.
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