Download Hutational analysis of the influenza virus A/Victoria/3/75 PA protein

Journal of General Virology (1996), 77, 1745-1749.
Printedin Great Britain
SHORT
COMMUNICATION
Hutational analysis of the influenza virus A/Victoria/3/75 PA
protein: studies of interaction with PB1 protein and
identification of a dominant negative mutant
Thomas Zercher,t Susana de la Luna, Juan J. Sanz-Ezquerro, Amelia Nieto and Juan Ort(n
Centro Nacional de Biotecnologia (CSIC), Universidad Autonoma, Cantoblanco, 28049 Madrid, Spain
The RNA polymerase activity and PB1 binding of
influenza virus PA mutants were studied using an in
vivo-reconstituted polymerase assay and a two
hybrid system. Deletions covering the whole PA
protein abolished polymerase activity, but the
deletion of the 154 N-terminal amino acids allowed
PB1 binding, indicating that the PA protein N
terminus is not absolutely required for this interaction. Further internal or C-terminal deletions
abolished PB1 interaction, suggesting that most of
the protein is involved in this association. As a novel
finding we showed that a single amino acid insertion
mutant, PAI672, was responsible for a temperature-sensitive phenotype. Hutant PAS509, which
had a serine insertion at position 509, bound to
PBI like wild-type PA but did not show any
polymerase activity. Over-expression of PAS509
interfered with the polymerase activity of wild-type
PA, identifying PAS509 as a dominant negative
mutant.
The influenza virus RNA polymerase is composed of three
polymerase proteins, PB1, PB2 and PA, and catalyses two
distinct types of RNA synthesis: (i) synthesis of mRNA
(transcription) and (ii) amplification of the vRNA (replication).
For mRNA synthesis, 5'-capped, host cell-derived RNA
fragments are used as primers and polyadenylation occurs at a
signal located 17-22 nucleotides before the 5' end of the
template. Replication occurs without primer and the vRNA
template is copied to a full-length positive-stranded RNA
(cRNA), which serves as template for vRNA synthesis. It has
been shown that free nucleoprotein (NP) might be a control
element for anti-termination, but little is known about the
Authorfor correspondence:Juan Ortfn.
Fax + 3 4 1 585 4506. e-mail [email protected]
1 Presentaddress:National Institute for Medical Research,The
Ridgeway, Mill Hill, London NW7 1AA, UK
0001-3930 © 1996 SGN
mechanism of the transcription-replication switch and the
detailed role of the components of the RNA polymerase (Krug
et al., 1989). Comparative sequence analysis and experimental
data suggest that PBI is the polymerase itself (Biswas &
Nayak, 1994; Poch et al., I990). PB2 binds CAP1 structures
and is probably responsible for the binding of the vRNA
promoter (Fodor ef al., 1993; Ulmanen et al., 1981) and the
endonucleolytic cleavage of the host cell primers (Licheng et
aI., I995). The function of PA is unknown. It is essential for the
activity of in aiao-reconstituted polymerase (reviewed in Mena
et al., 1995) and genetic evidence suggests a role for PA in
replication rather than in transcription (reviewed in Mahy,
1983). PA protein induces a general proteolysis of coexpressed proteins (Sanz-Ezquerro et al., 1995), but it is not
clear if this property is essential for polymerase activity. The
region of PA responsible for the induction of proteolysis maps
to the N-terminal third of PA, as determined by mutational
analysis (Sanz-Ezquerro et al., 1996). The expression of the P
proteins in Xenopus oocytes showed that complex formation
occurs in the absence of virus RNA. The co-immunoprecipitation of pairs of P proteins indicated that PB2 and PA can
interact independently with PB1, yet cannot form a complex
directly with each other (Digard ef al., 1989).
In this report we describe the use of two in aivoreconstitution assays to analyse the RNA polymerase activity
and the PBI binding of a large set of PA mutants. Polymerase
deficient, but interacting mutants have been characterized as
dominant negative and therefore identify regions of the
molecule relevant for its biochemical activity. Fig. 1 shows the
most relevant mutants used in this study. C-temlinal, Nterminal and internal deletions covered the entire PA protein
sequence. In addition, three point mutants in the N terminus
(positions 151, 154 and 162) and three insertion mutants in the
C-terminal third of PA (PAI550, PAI672 and PASS09) were
analysed. Mutant PASS09 contained a serine insertion after
position 509 just downstream of a conserved sequence with
homology to a nucleotide-binding motif (position 502-509).
The construction of all mutants has been described previously
(Sanz-Ezquerro et aI., 1996). The subcellular localization and
expression of the individual mutants used in this study have
been shown previously (Sanz-Ezquerro et al., 1996).
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74!
iiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiii iiii iii iiii iiiiiiiiiiiiiii
100
200
300
400
500
600
.
___1___,
Wt PA and
700 PA mutant
Association
with PB1
Polymerase
activity
wt (716)
100
100
C (683)
ND
ND
St (626)
ND
ND
Bg (509)
ND
ND
u (407)
ND
ND
Sc (342)
ND
ND
H (24~3
ND
ND
S (186)
ND
ND
ii
K (al-85)
ND
ND
66 + 23
ND
BS (A509-626)
ND
ND
BA (A407-511)
ND
ND
SB (A341-407)
ND
ND
HS (A246-342)
ND
ND
SH (A186-247)
ND
ND
1 672
102 _+26
ND/tS
1 550
57 + 15
20 -+0/ts
1 509
123 + 52
ND
ND
ND
D (A1-154)
i
154 (E-~G)
Internal deletion
Point mutation
Single amino acid insertion
m
~"/']~/vP16 fusion
NDNot detected
ts Temperature-sensitive
ii
Fig. I. PA mutants: PBI association and polymerase activities. The last PA-encoded amino acid of each C-terminal deletion
mutant is indicated. Black boxes indicate a frameshift and a short stretch of unspecific sequence until the next stop codon is
reached. For the N-terminal and internal mutants the size and location of the deletion is indicated. The insertion mutants are
named with the inserted amino acid and the position upstream of the insertion. The PBI-PA interaction was measured using
the two hybrid system as described in the text, The PBI association of the interacting PA mutants is indicated as the
percentage luciferase activity of that achieved by the wt VPI co-PAfusion protein. The interaction assay of the non-interacting
mutants was repeated at least twice and the values for the interacting mutants represent the average of six independent
experiments. The polymerase activity of the individual PA mutants was determined by reconstitution of the RNA polymerase in
vivo as described. The activity of mutant 1550 is indicated as the percentage CAT activity of that achieved by the wt PA protein,
The polymerase assay was repeated two to three times with each mutant,
Firstly, we tested whether the P A mutants were able to
reconstitute in vivo an active polymerase complex. The
expression of the three P proteins and NP using the vaccinia
virus-T7 R N A polymerase expression system allowed the
expression of an influenza virus-like v R N A containing the
74(
CAT gene flanked by the non<oding regions of the NS gene
(Piccone et aL 1993) as previously described (Mena e/ aI.,
1994). In addition to the mutants shown in Fig. 1, three
mutants with smaller deletions in the C-terminal half of PA
(PAA463-511, PAzX407-51I and PAA407-425) and one
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(a)
expression of wt PA allowed CAT expression (data not
shown).
We considered it of interest to examine if any of the
"~-dAC-CM mutations introduced a temperature-sensitive (ts) phenotype.
Thus, the RNA polymerase activity was reconstituted at 3 7 °C
or 33 °C as described (Mena eta]., 1994). Mutant PAI54 and
-,.~--Ac-CM
Q
Q
I
O e
all deletion mutants remained inactive at 33 °C and mutants
PA151 and PAI62 showed only slightly increased activity at
III
OII
II
ell
"~-CM
33 °C compared to 37 °C (data not shown). Nevertheless,
interesting results were obtained with the insertion mutants
(b) wt
I550 I672 $509 wt
I550 I672 $509
(Fig. 2). Mutant PAI672, completely inactive at 37 °C, reached
100 BG 20 BG BG 1100 BG 53 4.4 BG Exp. 1
3"4% of wt activity at 33 °C, a level significantly higher than
100 BG 20 BG BG ]100 BG 37 2.3 BG Exp.2
background (0"3%). Mutant PAI550 was also about twofold
I
100 BG 20+0 BG BG 1100 BG 45+11 3.4+1.5BG Avg
more active at the permissive temperature. Similar insertion
mutants
with a ts phenotype for RNA polymerase activity
Fig. 2. CAT activity and its competition by PA mutants. CAT activity of
inseRion mutants at permissive and non-permissive temperatures. The
have been observed for the PB2 gene (Perales eta]., 1996).
influenza virus transcription-replication system was reconstituted as
Interestingly, a ts mutant (ts263) of fowl plague virus carries a
described (IVlena et at., t994) and duplicates of transfected COS-1
missense
mutation (Ala to Val change) at position 67i of PA
cultures were incubated at 37 °C (restrictive temperature) or 33 °C
(permissive temperature). Total protein extracts were prepared and
(Herget & Scholtissek, I993). Insertion mutants, in contrast to
assayed for CAT activity. (a) Thin layer chromatography assay illustrating
missense mutations that are in general found to be responsible
CAT activities (de la Luna et el., 1993). The CAT activities for the wt
for ts mutations, could be genetically quite stable since
control and mutant 1550 were not in the range of this assay. The positions
of chloramphenicol (CiVl), 3-acetyl-chloramphenicol (Ac-ClM) and 1,3reversion would require the deletion of exactly three nucleoacetyl-chloramphenicol (dAc-Cb4) are indicated. (b) Quantitative analysis
tides. Nevertheless mutations in PA or other virus genes might
of CAT activities usin 9 a previously described phase extraction assay (de
suppress the ts phenotype (Mandler et a]., 1991). Influenza
la Luna et al., 1993) in two separate experiments, with the average. The
background (BG) in this assay was lower than 0.3°2'0 of the wt activity. The
viruses with ts insertion mutations could be rescued using
activities are shown as a percentage of the activity of vet PA. Transfected
described procedures (for review see Garc{a-Sastre & Palest,
PA plasmids: wt, pGEM3PAwt;-, pGEN13; 1550, pGEM3PA1550; 1672,
1993) to generate recombinants with reduced pathogenicity
pGEM3PAI672; $509, pGEM3PAS509.
and enhanced genetic stability.
For the mutants that were negative for CAT activity it
could not be distinguished if the active site of PA, complex
additional deletion mutant (PAASA) in the N-terminal half
formation or just the general folding of PA was affected by the
(A186-280) of PA were analysed (data not shown). The
mutation. Thus, we further studied the PA-PB1 association of
polymerase activities of the PA mutants are summarized in Fig.
the mutants using a GAL4/VP16-based two hybrid system
1. None of these deletion mutants was active in the polymerase
developed for the transfection of animal cells (Sadowski et al.,
assay. This result is not surprising since these drastic mutations
1988 and references therein). Ptasmids were constructed
might not only affect the structure, the intraceIlular localization
expressing the GAL4 DNA-binding domain fused to the N
or the active site of PA but also complex formation and thus
terminus of PB1, and the VP16 activation domain fused to the
indirectly the activity of the other polymerase subunits. The
N terminus of PA. For the construction of GAL4 fusions the
point mutant PA154, the only cytoplasmic mutant beside
multiple cloning site (MCS) of pSG424 (Sadowski & Patshne,
PAASA and PAASH, was inactive, whereas the nuclear mutants
1989) was replaced by the MCS of pTM1 (Elroy-Stein eta].,
(PA151, PAI62) with mutations in the same region (Sanz1989). PCR fragments of PB1 and PA cDNAs of strain
Ezquerro et al., 1996) remained active (data not shown). Of the
A/Victoria/75 with a SmaI restriction site upstream of the
three insertion mutants only PAI550 showed activity, albeit to
ATG and a XbaI site downstream of the stop codon were
a reduced level (20%). The expression of the individual PA
transferred to the modified pSG424 using the introduced Sinai
mutants was analysed by Western blot using a part of the same
and XbaI sites. The VP16-PAwt, -PAAD and -PAAK fusion
cell extract that was analysed for CAT activity (data not
shown), confirming the results previously obtained (Sanzplasmids were constructed by transferring the PA genes of
pSG424PA, pGEM3PAAD or pGEM3PAAK into the EcoRIEzquerro et al., 1996). All mutant proteins were of the expected
XbaI site of pAASVVP16 (Aso et aL, 1992) whose polylinker
size and the majority of the mutants was expressed to similar
had been previously replaced by the MCS of pGEM3
or higher levels (PAS, PAASA, PAASH, PA154) than the wild(EcoRI-HindIII). All other pVPPA mutant plasmids were
type (wt) control (data not shown). Only the mutants PASc
constructed by replacing the EcoNI-XbaI fragment of pVPPA
and PAH were reproducibly accumulated at levels lower than
with the corresponding fragment from the pGEM3PA mutant
wt PA and other mutants. However, the low expression levels
plasmid. The expression of the VPPA fusion proteins was
of these mutants cannot be the reason for the negative result in
confirmed by Western blot analysis using GAL4-VP16- and
polymerase activity, since much lower, almost undetectable
37 °C
33 °C
!
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174;
10000.
1000
i
~- 100
o
1C
1
no
competitor
100 %
2
wt PA
3
PAS509
4
PAABA
47 %
0.4 %
36 %
Fig. 3. Competition of the reconstituted transcription replication system by
over-expression of PA mutants. The influenza virus
transcription-replication system was reconstituted at 37 °C as described in
the text. COS-1 cells infected with T7 RNA polymerase-expressing
vaccinia virus were transfected with 5 ng of pGEM3PAwt, 1O0 ng of
pGEM3PB1 and pGEM3PB2, 2 pg of pGEM3NP and an additional 1OO ng
of different PA protein-expressing plasmids or control plasmids, as
indicated: column 1, pGEM3; column 2, pGEM3PAwt; column 3,
pGEIv]3PAS509; column 4, pGEfvl3PAABA. CAT activities were measured
using a phase extraction assay (de la Luna et el., 1993).
PA-specific antisera (data not shown). All VPPA mutant
proteins were of the expected size and accumulated to similar
levels as wt VPPA, except for the two shortest mutants
expressing C-terminal portions of PA (VPPAH, VPPAS),
which were five- to 10-fold less abundant than wt VPPA. The
reporter plasmid pGL-G5 (constructed and provided by P.
Staheli, Department of Virology, University of Freiburg,
Germany) contains five GAL4 DNA-binding sites upstream of
an EIB promoter and the luciferase gene.
The association of PA and PB1 in vivo was measured by
transfection of subconfluent monolayers of COS-I cells (p35
dishes) with 1 pg of pGL-GS, 2"5 IJg of pVPPA (or mutant
pVPPA) and 250 or 500 ng of pSG424PBI, using cationic
liposomes for transfection. Cells were harvested 48 h posttransfection and assayed for luciferase activity (Brasier et al.,
1989). The transactivation mediated by this association of
VPPA and GAL4-PB1 resulted in a high level of Iuciferase
expression, reaching about I0% of that achieved with
GAL4-VP16 by transfection of I p,g of pSGVPA490 (Sadowski
& Patshne, 1989; data not shown). When either pSG424PB1 or
pVPPA were transfected together with plasmids expressing
either the unfused VP16 activation domain or the unfused
GAL4 binding domain, only basal level of luciferase activity
(1-3 % of wt activity) could be detected. Thus, neither VPPA
nor GAL4-PB1 had transactivation ability by itself.
The interaction of the PA mutants with PB I is shown in Fig.
1. Deletion of the 154 N-terminal amino acids (mutant
'4~
VPPAaD) barely affected the association with PB1, indicating
that the N terminus is not absolutely required for such
interaction. Since none of the 12 C-terminal and internal PA
deletion mutants showed binding to PB1 it might be concluded
that the entire C-terminal three quarters of PA are involved in
this association. PBI may be bound at several points and/or by
a highly structure-dependent element formed by discontinuous
regions of PA. The structure is indeed important since a
deletion of the 154 N-terminal amino acids did not affect
binding to PB1, but a deletion of only 85 amino acids (mutant
VPPAaK) or the amino acid substitution at position 154
(mutant VPPA154) were inhibitory. Thus, the sequence located
between position 85 and 154 does not contribute to interaction
but can inhibit the binding mediated by other sequences. The
binding capacity of other mutants might be inhibited similarly.
The insertion mutants VPPASS09 and VPPAI672 bound to
PB1 with similar affinity to VPPAwt, and VPPAI550 showed
about 50% of the binding activity of VPPAwt.
In the experiments presented so far, we identified three
mutants (PAAD, PASS09 and PAI672) which were negative in
polymerase activity, but interacted with PB1 to a similar extent
as wt PA. We were interested to know whether these mutants
could enter the polymerase complex and thus inhibit its
activity in a dominant negative manner. Therefore, we
performed in vivo competition experiments at 37 °C (Fig. 3). A
low amount of wt pGEM3PA (5 ng per dish) was transfected
together with i00 ng of pGEMdPB2 and pGEM3PB1 for the
reconstitution of the polymerase complex. In spite of this, the
system was saturated with PA protein, since transfection of an
additional i00 ng of pGEMdPAwt did not increase CAT
activity (Fig. 3). The decrease in CAT activity observed after
over-expression of wt PA or the non-interacting mutant
PAABA (Fig. 3) can be explained by the proteolytic activity
induced by PA protein, which reduced the concentration of the
co-expressed proteins (PB1, PB2, NP and CAT; Sanz-Ezquerro
et al., 1995). The over-expression of PASS09 (Fig. 3) reduced
CAT expression more than 100-fold compared to competition
with wt PA or PAhBA proteins, indicating that PASS09
competes for complex formation. It has been shown that the
proteolytic activities induced by PAS509, wt PA and PAd~BA
are identical (Sanz-Ezquerro et al., 1996) and hence cannot be
responsible for this result. Thus, mutation $509 did not alter
the structure but might have affected an active site of PA. In
this mutant a serine is inserted just downstream of a conserved
putative nucleotide-binding motif (Gs0~FIIKGs07Rs0sSs09),
which closely resembles the classic GXXXXGKT/S nucleotidebinding motif, and suggests that the function of PA requires
the binding of nucleotides. Despite their strong interaction
with PB1 in the two hybrid system, PAAD and PAI672 could
not compete efficiently under these conditions (data not
shown), indicating that their binding properties are not
complete in terms of complex formation.
In conclusion, two in vivo-reconstituted systems were used
to screen a series of PA mutants for their polymerase activity
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and complex formation capacities. The inability to identify a
small region of PA able to interact with PB1 suggests that
several distant positions of its amino acid sequence might fold
together to form conformation-dependent interaction domains.
Several mutations in the C-terminal third of the protein
affected its polymerase activity without dramatically altering
the PBl-binding properties. In particular, the insertion of a
serine downstream of a putative nucleotide-binding domain
generated a dominant negative mutant, identifying this region
as a potential active site of PA. Further experiments will show
if this dominant negative mutant is able to interfere with RNA
replication in influenza virus-infected cells,
We thank B. Moss, P. Palese and P. St/iheli for providing biological
materials. The plasmids used for the two-hybrid system were derived
from the laboratory of S. M. Weissman. The technical assistance of J.
Fern~indezand M. Pastor is gratefully acknowledged. T. Z. was supported
by a fellowship of the Ministerio de Education y Cienciaand from a longterm fellowship of the Human Frontier Science Program (HFSP). This
work was supported by Comisi6n Interministerial de Ciencia y
Tecnologfa (grant BIO92-1044), Comunidad Aut6noma de Madrid
(grant A0063), Programa Sectorial de Promocfon General del Conocimiento (grant PB-1542) and EU HCM Program (grant ERBCHRXCT
949453).
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Received 6 February 1996; Accepted 23 April 1996
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'4!