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J&us Research, 24 (1992) 65-75
0 1992 Elsevier Science Publishers B.V. All rights reserved 0168-1702/92/$05.00
65
VIRUS 00769
Nuclear transport of influenza virus polymerase
PA protein *
Amelia Nieto 2, Susana de la Luna 1,2, Juan Bhcena 3, Agustin Portela 3,
Juan Valchrcel ‘s2,#,
JosC A. Melero 3 and Juan Ortin 1,2
’ Centro National de Biotecnologia (CSIC), Uniuersidad Aut&oma, Cantoblanco, Madrid, Spain,
’ Centro de Biologia Molecular (CSIC-UAM), Universidad Autdnoma, Cantoblanco, Madrid, Spain
and 3 Servicio de Biologia Molecular, Centro National de Microbiologia, Instituto Carlos III,
Majadahonda, 28220 Madrid, Spain
(Received 21 October 1991; revision received and accepted 13 January 1992)
Summary
The subcellular distribution of influenza polymerase PA subunit has been
studied using a SV40-recombinant virus (SVPA76), which allows the expression
and accumulation of this protein in COS-1 cells. In contrast to the complete
nuclear localization observed for the PA subunit several hours after influenza virus
infection, when COS-1 cells were infected with the SVPA76 recombinant, the PA
protein accumulated either in the nucleus, in the cytoplasm or was distributed
throughout the cell. When cells were infected with the SVPA76 recombinant and
superinfected with influenza virus, a clear increase in the proportion of cells
showing nuclear localization of the PA protein was observed, suggesting that some
truns-factor may be required to allow complete nuclear accumulation of the
protein. Double infections using SVPA76 recombinant and either SVPBl or SVNS
recombinant viruses showed a complete correlation between expression of poly-
merase PBl subunit or NSl protein and nuclear localization of polymerase PA
subunit. However, no such correlation was observed in the double infections of
SVPA76 and SVNP recombinants. These results suggest that polymerase PBl
Correspondence to: J. Ortin, Centro National de Biotecnologia (CSIC), Cantoblanco, 28049 Madrid,
Spain.
* Presented in part at the Eighth International Conference on Negative Strand Viruses, Charleston,
SC, U.S.A., 15-20 September 1991.
# Present address: Program in Molecular Medicine, University of Massachussets Medical Center, 373
Plantation street Worcester, MA 01605, U.S.A.
subunit and the non-structural proteins could be involved in the nuclear targeting
or nuclear retention of influenza polymerase PA protein.
Influenza virus polymerase; PA subunit
Introduction
Influenza virus genome consists of eight single stranded RNA segments of
negative polarity that encode ten proteins (Lamb, 1989). Their transcription and
replication is carried out by complexes that include at least the three polymerase
subunits (PBl, PB2 and PA) and the nucleoprotein (NP) (Braam et al., 1983;
Horisberger, 1980; Huang et al., 1990). Both transcription and replication take
place in the nucleus of the infected cells (Herz et al., 1981; Jackson et al., 1982;
Lopez Turiso et al., 1990; Shapiro and Krug, 1988) where soluble complexes of the
three polymerase subunits can be detected (Detjen et al., 1987).
Influenza virus nucleoprotein is transported into the nucleus when expressed
from cloned DNA (Lin and Lai, 1983; Portela et al., 1985) and the sequences
responsible for the nuclear accumulation have been identified (Davey et al., 198.5).
Likewise, all three polymerase subunits move into the nucleus during influenza
virus infection (Akkina et al., 1987; Jones et al., 1986), as well as when expressed
from vaccinia virus recombinants (Smith et al., 1987), but their kinetics of nuclear
accumulation may not be identical (Akkina et al., 1987). The sequences responsible
for the nuclear transport of PBl and PB2 subunits have been shown to consist of
two separate elements (Mukaigawa and Nayak, 1991; Nath and Nayak, 1990), as is
also the case for NSl protein (Greenspan et al., 1988).
In this report we have analyzed the localization of the PA subunit of influenza
virus polymerase when expressed from a SV40 recombinant. Unexpectedly, a large
proportion of the cells expressing PA protein showed a cytoplasmic localization.
However, coexpression of PBl or non-structural proteins, but not NP, correlated
to a complete nuclear localization of PA protein.
Materials and Methods
Biological materials
Plasmid pBSL-4, used as intermediate vector, contains the SV40 genome late
region, from the Hind111 site (5171) to the BclI site (2770) cloned between the
Hind111 and BamHI sites of Bluescript plasmid (Stratagene). All plasmids were
maintained in Escherichia co/i DH-5.
The MDCK and CV-1 cell lines were obtained from the American Type Culture
Collection. The COS-1 cell line (Gluzman, 1981) was obtained from Y. Gluzman.
Influenza viruses A/Victoria/3/75 WIG) and A/PR/8/34 (PRS) were grown in
MDCK cells as reported previously (Ortin et al., 1980). Recombinant vaccinia virus
VA-C expressing A/PR/8/34 PA protein (Smith et al., 1987) was obtained from
G. Smith.
Monoclonal antibodies specific for the PA subunit were prepared using as
antigen the full-length protein expressed in E. coli. Their complete description will
be presented elsewhere (Barcena et al., in preparation). Rabbit anti-NSl sera were
provided by P. Palese and J. Young. Rabbit anti-NP serum was prepared by
immunization with purified viral RNPs. Peptide MPBl/l (positions 70-81 of the
VIC virus PBl sequence; de la Luna et al., 1989) was synthesized on a lysine core
as described (Tam, 1988), using the FMOC chemistry. Anti-peptide serum was
prepared by intradermal immunization of rabbits with purified peptide mixed with
CFA, followed at monthly intervals by two intramuscular injections of IFA-peptide
mixture.
Molecular cloning and gene expression
DNA manipulations including restriction enzyme digestions, DNA ligations and
E. coli transformations were done by standard procedures (Sambrook et al., 1989).
SV40 recombinant viruses were obtained by transfection of the corresponding
SVX DNAs into COS-1 cells using the DEAE-dextran method (Lai and Nathans,
1974). Culture supernatants of the transfected cells were used to produce high titre
virus stocks by infection of COS-1 cells.
Protein labelling was carried out at 60 hpi or 5 hpi in cells infected with SV40
recombinants or influenza virus, respectively. Cell monolayers were washed and
incubated for 1 h with methionine-free DMEM medium. [35S]Met was then added
and incubation was continued for 1 h. After washing in PBS, total cell extracts
were prepared in loading buffer and analyzed by polyacrylamide gel electrophore-
sis as described (Studier, 1972).
Immunqfluorescence
Influenza virus infections were done at a m.o.i. of 10 pfu per cell either in
MDCK or COS-1 cells; CV-1 cells were infected with VA-C virus at a m.o.i. of 30
pfu per cell; SV40 recombinant virus infections were done in COS-1 cells using a
m.o.i. of l-5 pfu per cell. Monolayers of mock infected or virus infected cells were
fixed at different times after infection with methanol at -20°C for 20 min and
stored in phosphate buffer saline at 4°C until use. Fixed cells were incubated with
PA-specific monoclonal antibodies (culture supernatants) and, in double staining
experiments, with either rabbit anti-MPBl/l peptide serum (dilution 1: 1000)
rabbit anti-NSl protein serum (dilution 1: 200) or rabbit anti-NP serum (dilution
1: 200) for 1 h at room temperature. The cells were then washed with PBS and
incubated with Texas red-labelled sheep anti-mouse immunoglobulin antibodies
(dilution 1: 200). In double staining experiments, fluorescein-conjugated donkey
anti-rabbit immunoglobulin antibodies and the nuclear Hoechst dye (0.5 pg/ml)
were also used. Dilutions of sera were done in PBS-2% BSA. After further
68
washing in PBS, preparations were mounted in Mowiol and photographed in a
Zeiss fluorescence microscope. Pictures of each preparation were taken with
identical exposure times.
Results and Discussion
Expression of polymerase subunits in COS-I cells
The cDNAs corresponding to WC influenza virus RNA segments 1, 2, 3 and 5
were synthesized and cloned into pUC18 plasmid as described previously (de la
Luna et al., 1989) The appropriate inserts were subcloned into the pBSL interme-
diate vector to obtain the pSEX-plasmid series. After removing the bacteria-de-
rived sequences from these recombinants by XbaI digestion (Fig. 11, the sv40
recombinant DNAs were circularized and transfected into COS-1 cells. sv40
Xba I IPsp 718
PBl *
PA *
NP x
- 94
- 68
- 45
- 29
Fig. 1. Expression of influenza virus genes in COS-1 cells. Plasmids of the pSEX serie were constructed
by transferring segment 1, 2, 3 and 5 cDNAs from the corresponding pUC1R recombinants (pUX serie),
to plasmid pBSL, Bluescript-derived DNA was eliminated by XbaI digestion. After recircularization,
the SVX DNAs were transfected into COS-1 cells to amplify the SV40 recombinants. Mock-infected,
influenza virus-infected or SV40 recombinant-infected COS-1 cells were labelled with [“‘Slmethionine.
total cell extracts were prepared and analyzed by polyacrylamide gel electrophoresis as described under
Materials and Methods. Left panel: (black bar), SV40 DNA, (line) Bluescript DNA; (white bar).
Influenza virus cDNA. Right panel: MOCK, VIC, SVPBl, SVPA and SVNP indicate the elec-
trophoretic patterns obtained with extracts from uninfected cells or cells infected by influenza virus
(Victoria strain), SVPBl, SVPA, and SVNP recombinant viruses, respectively. Numbers to the right (in
kDa) indicate the position of molecular weight markers.
69
pseudovirus stocks were collected and amplified by serial infections in COS-1 cells.
Segment 8 was cloned into vector pBSV9 by oriented cDNA synthesis and
circularization, as described (Portela et al., 1985).
The expression of the cloned genes was studied by pulse labelling SV40
recombinant-virus infected cells. The electrophoretic analyses of the labelled
protein extracts obtained are shown in Fig. 1 and indicate the presence of bands
with apparent molecular weights identical to those obtained in COS-1 cells
infected with VIC virus. The identification of the PBl, PA and NP proteins
expressed as bona fide influenza virus gene products was confirmed by their
immunologic detection with specific antibodies (see below).
Subcellular distribution of polymerase PA subunit in influenza or SV40-recombinant
virus infected cells
Cultures of COS-1 cells were infected with VIC virus or with SVPA76 recombi-
nant. At several times after infection the cells were fixed and processed for
indirect immunofluorescence using PA subunit-specific monoclonal antibodies.
The results obtained are presented in Figs. 2 and 3. Early after influenza virus
infection PA protein could be detected in the cytoplasm of the cells, when other
nuclear influenza virus proteins as PBl, NP or NSl were already localized in the
nuclei (Fig. 2). This observation is in agreement with previous results (Akkina et
al., 1987) and suggests that the transport of the PA subunit to the nucleus may be
regulated during infection. Six hours post-infection, the PA protein accumulated in
the nucleus and the immunofluorescence pattern indicates that, even at 10 hpi, it
did not leave this cellular compartment. This behaviour contrasts with that
observed for other nuclear proteins like NP and NSl that appeared distributed all
over the cell at those very late times post-infection.
When PA protein was expressed from the SVPA76 recombinant, some of the
cells showed nuclear localization, while in others the PA subunit accumulated in
the cytoplasm or was distributed throughout the cell (Fig. 3). The percentage of
cells with cytoplasmic PA protein localization ranged between 20 and 60% in
different infections. These data suggest that PA protein contains a nuclear trans-
port signal, but when expressed as a single influenza virus specific protein, its
accumulation in the nucleus is inefficient. The differences in the distribution
patterns observed in influenza virus and SVPA76 recombinant infected cells
suggest that, for an efficient accumulation of PA protein in the nucleus to occur,
some other viral function(s) are required.
The results obtained with the SVPA76 recombinant are in contradistinction to
those published earlier (Smith et al., 1987), using a vaccinia-PA (VA-C) recombi-
nant. In fact, using VA-C recombinant, we have confirmed that the PA subunit
could be found preferentially in the nuclei of the infected cells, although cytoplas-
mic fluorescence was also apparent. The same pattern of subcellular distribution
could be observed with PA76 clone when expressed from a pGEM vector using the
vaccinia virus-T7 expression system (Fuerst et al., 1987) (data not shown). This
would suggest that the vaccinia expression system acts as a helper for the PA
70
Oh
4h
6h
8h
10h
Fig. 2. Localization by indirect immunofluorescence of influenza virus-specific proteins in COS-I
infected cells. Cells were fixed at various times post-infection and stained with either PA-specific
monoclonal antibodies (pa), anti-MPBl/l peptide serum (pbl), anti-NSl protein serum (nsl) or
anti-NP serum (np) as described under Materials and Methods. Numbers to the left indicate the time
after influenza virus infection in hours.
protein nuclear accumulation. The alternative possibility, i.e., that the SV40
expression system would be defective in nuclear transport is unlikely, since PBl,
NP and NSl proteins accumulate into the nucleus when expressed from the
71
SVPA76 SVPA76 + PR8
Fig. 3. Localization of PA protein in cells infected with SVPA76 recombinant: effect of influenza virus
superinfection. COS-1 cell cultures were infected with the PR8 strain of influenza virus for 12 h (PRS)
or with SVPA76 recombinant for 48 h (SVPA76). Some of the SVPA76 recombinant-infected cultures
were then superinfected with the PR8 strain of influenza virus for 12 more hours (SVPA76 + PR8).
After these incubation times the cells were fixed and the PA protein was localized by indirect
immunofluorescence using a PA-specific monoclonal antibody that poorly recognizes the PA protein of
PR8 strain, as described under Materials and Methods.
corresponding SV40 recombinants (see below). The mechanisms responsible for
the differences observed when the PA protein was expressed by either a SV40 or a
vaccinia virus system are not clear, but the strong cellular shutoff induced by
vaccinia virus may be relevant in this context. Thus, the activity of nuclear
proteases may decrease as a consequence of this shut-off and hence the concentra-
tion of nuclear PA protein may be higher than in SV40 recombinant infected cells.
It could be argued that the molecular clone that we have expressed in SVPA76
recombinant is a fortuitous mutant, defective in nuclear transport. However, this is
unlikely since two independent molecular clones (PA76 and PA1051 had identical
nucleotide sequence. Moreover, the PA protein expressed from SVPA76 recombi-
nant was fully functional, since this recombinant was able to induce the synthesis
of CAT enzyme from a PBZCAT chimeric vRNA when it was transfected into
cells coinfected with the corresponding PBl, PB2 and NP SV40 recombinants
(data not shown).
Effect of influenza virus infection on the localization of the PA subunit expressed
from the SWA76 recombinant
In order to study if any viral trans factors could be responsible for the efficient
nuclear localization of the PA subunit observed in influenza virus infected cells,
COS-1 cells were infected with the SVPA76 recombinant and 48 h postinfection
the cells were superinfected with the PR8 strain of influenza virus. Twelve hours
thereafter, the cultures were fixed and the localization of the VIC virus PA subunit
was carried out by immunofluorescence using a monoclonal antibody which poorly
recognized the PR8 strain PA protein. The results are shown in Fig. 3. In the cells
infected with PR8 influenza virus, the pattern of PA protein subcellular localiza-
tion observed (studied with a cross-reactive monoclonal antibody) was identical to
SVPA76 + SVPBl
SVPA76 + SVNP
Fig. 4. Effect of specific influenza virus proteins in the subcellular localization of the PA polymerase
subunit. Cultures of COS-1 cells were double-infected with SVPA76 recombinant and other SV40
recombinants expressing either PBl (SVPA76 + SVPBl), the non-structural proteins (SVPA76 + SVNS)
or nucleoprotein (SVPA76 + SVNP). Forty eight hours post-infection the cells were fixed and influenza
virus proteins were localized by indirect immunofluorescence using PA-specific monoclonal antibodies
(pa) and either anti-MPBl/l peptide serum (pbl), anti-NSl protein serum (nsl) or anti-NP serum (np).
In all cases, nuclei were visualized by staining with the Hoechst nuclear dye (h), as described under
Materials and Methods, Arrows in the right panels show cells with cytoplasmic staining of PA protein,
whereas asterisks indicate nuclear staining.
73
that obtained in cells infected with the VIC strain (data not shown), i.e., the
antigen is found in the nuclei of the cells. When the cells were infected with the
SVPA76 recombinant and superinfected with PR8 virus, a dramatic increase in the
nuclear localization of VIC virus PA protein was observed (Fig. 3). These results
strongly suggest that some influenza specific or induced protein is involved in the
nuclear targeting or in the nuclear retention of polymerase PA protein.
Tram effect of specific influenza virus gene products in the subcellular distribution of
the polymerase PA subunit
The evidence presented above, supporting the notion that an influenza virus
induced protein is involved in the nuclear accumulation of the polymerase PA
subunit, prompted us to carry out double infection experiments in which COS-1
cells were coinfected with the SVPA76 recombinant and other SV40-recombinants
able to express PBl, NP or the non-structural proteins. The results of double
infections with SVPA76 plus SVPBl or SVPA76 plus SVNS recombinants showed
an absolute correlation between PBl or NSl protein expression and nuclear
localization of polymerase PA subunit (Fig. 41, as well as an increase in the
proportion of cells showing nuclear accumulation of the PA subunit. This effect
was not as complete as in the case of influenza virus-SVPA76 doubly infected cells,
due to the lower multiplicity of infection attainable with SV40 recombinants. In
contrast, no correlation was observed when cells were double infected with
SVPA76 and SVNP recombinants (Fig. 4).
Conclusions
The results presented above indicate that the PA protein can be transported to
the nucleus of the cell, even when expressed in the absence of other influenza virus
proteins, in accordance with previous results (Smith et al., 1987). However, the
nuclear accumulation observed was not complete when a SV40 recombinant was
used, unless other viral proteins were present (Fig. 3). These results suggest that,
although the PA protein may move into the nucleus via active transport mediated
by a nuclear targeting signal, other viral factors may play a role in its accumulation
in this cellular compartment, in agreement to its delayed localization in the
nucleus in the influenza virus infection cycle (Fig. 2) (Akkina et al., 1987). Several
mechanisms may be conceived for such a helper function: (i> physical association to
a viral or virus-induced protein that is efficiently transported to the nucleus; (ii>
virus-mediated post-translational modification of the PA protein to increase its
transport; (iii) intranuclear binding that would increase PA protein retention in the
nucleus; and (iv) metabolic stabilization of intranuclear PA subunit, i.e. binding to
a virus-specific or modification by a virus-specific protein that would lead to an
increased half-life of the PA subunit in the nucleus. It is difficult at present to
74
decide which of these mechanisms is really operating, specially since the involve-
ment of PBl subunit and any of the non-structural proteins (Fig. 4) suggests that
more than one of them may play a role in the process.
The involvement of PBl protein is not unexpected, in view of its presence in a
complex with PB2 and PA in the nucleus (Detjen et al., 1987) and therefore a
retention and/or stabilization mechanism would seem reasonable. In fact, prelimi-
nary results suggest a similar involvement of PB2 subunit (data not shown). The
mechanism by which the RNA segment 8 genes mediate PA protein nuclear
accumulation is far less obvious. It should be first pointed out that, although NSl
protein has been used as a marker for segment 8 expression, we can not be certain
at present whether NSl or NS2 protein is involved. With this caveat in mind, it is
tempting to speculate that the phenotype of ts mutants in the NSl cistron,
compatible with a lack of vRNA synthesis (Shimizu et al., 19821, may be in relation
with the genetic interaction with the PA gene. In this context, it is also worth
mentioning the intriguing phenotype shown by a mutant in the NS2 cistron in
which a specific accumulation of PA defective vRNA is observed (Odagiri and
Tobita, 19901. Taken together, these results suggest a role for the non-structural
protein(s) in modulating the PA protein function in influenza virus-infected cells.
Acknowledgements
We are grateful to Dr. C.R. Howard for training us in the peptide synthesis
techniques. We thank A. Beloso, J. Rebelles and F. Ocafia for their excellent
technical assistance and G. Smith, J. Young and P. Palese for providing the
VAC-C recombinant and anti-NSl sera, respectively. J.B. was a predoctoral fellow
of the Plan de Formation de Personal Investigador. This work was supported by
Comision Interministerial de Ciencia y Tecnologia, grants BI088-1091 and BI089-
0545, and by an institutional grant of Fundacion Areces to the CBM.
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