From the Program in Cellular Biotechnology,
Institute of Biotechnology, Viikki Biocenter, P.O. Box 56, University
of Helsinki, FIN-00014 Helsinki, and the
Department of
Biochemistry and Pharmacy, Åbo Academi and University,
FIN-20520 Turku, Finland
Received for publication, July 11, 2000, and in revised form, December 1, 2000
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ABSTRACT |
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nsP3 is one of the four RNA replicase subunits
encoded by alphaviruses. The specific essential functions of nsP3
remain unknown, but it is known to be phosphorylated on serine and
threonine residues. Here we have completed mapping of the individual
phosphorylation sites on Semliki Forest virus nsP3 (482 amino acids) by
point mutational analysis of threonine residues. This showed that
threonines 344 and 345 represented the major threonine phosphorylation
sites in nsP3. Experiments with deletion variants suggested that nsP3 itself had no kinase activity; instead, it was likely to be
phosphorylated by multiple cellular kinases. Phosphorylation was not
necessary for the peripheral membrane association of nsP3, which was
mediated by the N-terminal region preceding the phosphorylation sites. Two deletion variants of nsP3 with either reduced or undetectable phosphorylation were studied in the context of virus infection. Cells
infected with mutant viruses produced close to wild type levels of
infectious virions; however, the rate of viral RNA synthesis was
significantly reduced in the mutants. A virus totally defective in nsP3
phosphorylation and exhibiting a decreased rate of RNA synthesis also
exhibited greatly reduced pathogenicity in mice.
Phosphorylation and dephosphorylation have been recognized as
major processes by which protein function is regulated. A wide range of
proteins display phosphorylation state-dependent activity, including proteins involved in signal transduction, transcription, and
the cell cycle. In the field of RNA virus replication, the phosphoprotein P of negative-strand RNA viruses with unsegmented genomes such as rhabdoviruses (order Mononegavirales) has a central function in regulating viral mRNA transcription and possibly RNA replication (1).
The alphaviruses are a globally distributed group of enveloped
positive-strand RNA animal viruses capable of causing fatal encephalitis; representative members include Sindbis virus
(SIN)1 and Semliki Forest
virus (SFV). The RNA synthesis of alphaviruses occurs in the cytoplasm,
where the 5' two-thirds of the genomic RNA (total length, ~11.5
kilobases) is translated into a large polyprotein of ~2500 amino
acids (aa). This polyprotein, termed P1234, is autoproteolytically
cleaved to yield the four subunits of the viral
RNA-dependent RNA polymerase, the nonstructural proteins nsP1-nsP4 (reviewed in Refs. 2 and 3). Polyprotein processing intermediates have distinct essential functions during the early phase
of RNA replication, the synthesis of negative strands (4-6). Later in
infection, the negative strands are used as stable templates for
synthesis of progeny-positive strands, and for synthesis of subgenomic
mRNAs coding for the structural proteins of the virus.
nsP1 is an enzyme responsible for methylation and capping of viral
mRNAs (7, 8). In addition, it mediates membrane association of the
RNA replication complex (9) and its targeting onto the cytoplasmic
surface of endosomes and lysosomes (10). nsP2 is an RNA helicase (11),
RNA triphosphatase (12), and an autoprotease responsible for the
cleavage of the nonstructural polyprotein (3). nsP4 is the catalytic
subunit of this RNA-dependent RNA polymerase (13). Each of
these three polypeptides has conserved amino acid sequence motifs, when
compared with cellular and viral proteins of similar function: nsP4
with RNA-dependent polymerases, nsP2 with nucleic acid
helicases and papain-like cysteine proteases, and nsP1 with
methyltransferases (14, 15).
In contrast, the functions of nsP3 are not well defined, although the
protein is essential for RNA replication (16). Studies of
temperature-sensitive and linker insertion mutants of SIN nsP3 have
implicated it in negative-strand RNA synthesis, and possibly also in
the synthesis of subgenomic mRNA (6, 17). The amino acid sequence
of SFV nsP3 (482 aa) can be divided into three almost equal sections.
The first third forms a small domain conserved in alphaviruses, rubella
virus, hepatitis E virus, and coronaviruses (14). Recently, through
genome sequencing, it has become apparent that this domain is widely,
although not universally distributed in bacteria, archae, and
eukaryotes. It usually exists on its own as a small open reading frame,
but a more divergent version can be found attached to unusual histone
variants, the macrohistones H2A (18). For the moment, this
unanticipated conservation imparts little insight, since none of the
nsP3-related proteins have been functionally characterized. The middle
third of nsP3 is only conserved between alphaviruses, whereas the last
third (after Tyr324; Fig. 1) is hypervariable, showing no
discernible conservation. Even the size of this C-terminal "tail"
varies in different alphaviruses: SFV nsP3 has 158 aa and SIN nsP3 232 aa (3). The tail is rich in acidic residues, as well as in serine,
threonine, and proline, and devoid of predicted secondary structure.
nsP3 is the only alphavirus nonstructural protein modified by
phosphorylation (19, 20). Phosphoamino acid analysis of SFV nsP3 showed
that serine and threonine residues are phosphorylated, approximately in
2:1 ratio, whereas no phosphotyrosine could be found (19). The major
phosphorylation sites of SFV nsP3 were determined by mass spectrometric
analysis in conjugation with on-target alkaline phosphatase digestion,
as well as two-dimensional peptide mapping and Edman sequencing (21).
In SFV nsP3 the serines 320, 327, 332, and 335 and from 7 to 12 residues in peptide Gly338-Lys415 can be
phosphorylated. SIN nsP3 is even more heavily phosphorylated on serine
and threonine, leading to formation of several species of different
electrophoretic mobility (20). Deletions in the nonconserved tail
region of SIN nsP3 considerably reduce its phosphorylation, suggesting
that the modification may primarily occur in this part of the protein
(22). nsP3 is capable of associating with cellular membranes when
expressed alone (23).
Here we have constructed truncated and point-mutated derivatives of SFV
nsP3 and studied their phosphorylation. This enabled the introduction
of phosphorylation-defective nsP3 variants to the SFV genome, to better
understand the role of phosphorylation of nsP3 in the alphavirus life
cycle. The resultant mutant viruses were characterized in cell culture
with respect to growth and RNA synthesis. Finally, a virus encoding
nonphosphorylated nsP3 was used in studies of neurovirulence in mice.
Plasmids and Plasmid Construction--
Deletions and point
mutations were made in SFV nsp3 present in plasmid pTSF3
under the T7 promoter (24). The C-terminal nsp3 deletion
mutant
Primers 1-8 were constructed as follows: primer 1, 5'-CTCAAGCTTACGTAGATGCGGCATACTTCCGCGG-3'; primer 2, 5'-CTCAAGCTTATGCACCCGCGCGGCCTAGTCG-3'; primer 3, 5'-ATCACCATGGCACCATCCTACAGAGTTAAG-3'; primer 4, 5'-TCGTAGTCCAAGTCAAACCCTCGTAACGACCGATC-3'; primer 5, 5'-TGGACTACGAGCCAATGGCTCCCATAGTAGTGACGG-3'; primer 6, 5'-CAGTGTACGAGCCAATGGCTCCCATAGTAGTGACGG-3'; primer 7, 5'-TCGTACACTGAAGGTACCGTCGGGACGAACAGG-3'; and primer 8, 5'-ATCCATATGACCATGGACCCGACGGTACCTTCAGTGG-3'.
Cells and Viruses--
HeLa cell monolayers were grown in
Dulbecco's modified minimal essential medium supplemented with 10%
heat-inactivated fetal bovine serum and 100 units/ml streptomycin and
penicillin. The modified recombinant vaccinia virus Ankara was kindly
provided by Dr. Moss (National Institutes of Health, Bethesda, MD), and stock was grown in baby hamster kidney (BHK) cells as described (26).
Propagation of the SFV strain and cultivation of BHK and Vero cells
have been described previously (27).
Protein Expression in HeLa Cells--
HeLa cells (80-90%
confluent) were infected with modified vaccinia Ankara using 20-50
plaque-forming units (pfu)/cell. After a 45-min adsorption period, the
cells were washed and transfected in OptiMEM (Life Technologies, Inc.)
with pTSF3 or its derivatives, using Lipofectin (Life Technologies,
Inc.). 7 µg of DNA in 12 µl of Lipofectin, and 10 µg in 50 µl
were used for 60- and 100-mm plates, respectively. After 3-4 h the
transfection mixture was replaced with normal growth medium containing
serum, and the incubation was continued for 1-3 h.
For in vivo labeling, transfected cells were washed twice
with Dulbecco's medium containing 0.2% bovine serum albumin at 2 h after transfection and a methionine/cysteine-free or a phosphate-free medium was added. Cells were labeled at 3 h after transfection with either 150 µCi of [35S]methionine/cysteine (>1000
Ci/mmol, Redivue PRO-mix, Amersham Pharmacia Biotech) or 200-500 µCi
of carrier-free [32P]orthophosphate (Amersham Pharmacia
Biotech) per 100-mm plate. After 3-5 h of labeling, cells were washed
carefully, harvested, and lysed in 1% SDS. After shearing the DNA with
a syringe and a 27-gauge needle, proteins were denatured by boiling and
nsP3 was immunoprecipitated as described below, followed by SDS-PAGE analysis. The radioactive proteins were detected using a Fuji Bas 1500 Bioimaging Analyzer instrument.
Immunoprecipitation, Immunoblotting, and
Immunofluorescence--
HeLa cells were lysed in phosphate-buffered
saline (PBS) containing 1% SDS, 10 mM sodium fluoride
(Merck), and protease inhibitor mixture (CompleteTM; Roche Molecular
Biochemicals). After boiling the samples for 2 min, immunoprecipitation
was carried out with a polyclonal anti-nsP3 rabbit antiserum as
described previously (19). For immunoblotting, the samples, resolved by
SDS-PAGE in 10% gels, were transferred to nitrocellulose membrane
(Hybond ECL or Hybond-C-extra; Amersham Pharmacia Biotech) as described previously (7). Polyclonal anti-nsP3 rabbit antiserum was used in 1 to
10,000 dilution, polyclonal anti-nsP3 guinea pig antiserum in 1 to
10,000 dilution, and anti-phosphothreonine (Zymed
Laboratories Inc.) in 1 to 1,000 dilution. The secondary
antibodies were swine-anti-rabbit IgG (Dako) and donkey-anti-guinea pig
IgG (Jackson Immunoresearch Laboratories) conjugated to horseradish
peroxidase. The proteins were visualized using an enhanced
chemiluminescence detection system (Amersham Pharmacia Biotech).
Indirect immunofluorescence microscopy was carried out for transfected
cells at 4 h after transfection using Bio-Rad MRC1024 confocal
microscope. The cells were fixed with 4% paraformaldehyde in cytosolic
buffer (10 mM MES, pH 6.0, 138 mM KCl, 3 mM MgCl2, 2 mM EGTA, and 11%
sucrose) for 15 min and permeabilized with 0.1% Triton X-100 for 1 min. The cells were labeled with anti-nsP3 antiserum (1:500), followed by donkey rhodamine red X-conjugated anti-rabbit IgG (1:300, Jackson Immunoresearch Laboratories) as a secondary antibody.
Comparison of Virus Growth in BHK, Vero, and NIH
Cells--
Derivative of the infectious cDNA (pSP6-SFV4)
containing nsp3 deletion nsp3 Viral RNA Synthesis--
BHK cells (2 × 106
cells/35-mm Petri dish) were infected with 50 pfu/cell wild-type SFV,
SFV nsP3 Mouse Pathogenicity--
Two mouse groups, both consisting of 5 4-to-6 week-old female mice, were infected intraperitoneally with the
wild type SFV4 and phosphorylation-deficient mutant SFV nsP3 Phosphorylation of Wild Type nsP3 and Deletion Mutant
N-nsP3--
To study phosphorylation of SFV nsP3, we synthesized the
protein in relatively large amounts in eukaryotic cells by
transfection. HeLa cells were infected with the attenuated vaccinia
virus Ankara, which encodes bacteriophage T7 RNA polymerase (26),
followed by transfection with a plasmid encoding nsP3 under the T7
promoter. The cultures were labeled with
[32P]orthophosphate from 3 to 6 h after
transfection. In preliminary experiments, phosphoamino acid analysis
had revealed that the wild type nsP3 was phosphorylated on serine and
threonine, as described for the protein synthesized during SFV
infection (data not shown; Ref. 19). Next, we expressed the wild type
nsP3 and N-nsP3 (nsP3
The major phosphorylation sites of nsP3 have been determined by mass
spectrometric analysis in conjugation with on-target alkaline
phosphatase digestion as well as two-dimensional peptide mapping and
Edman sequencing (21). At least serines 320, 327, 332, and 335 and from
7 to 12 residues in peptide Gly338-Lys415 are
potential phosphorylation sites (Fig. 1B). However, the mass spectrometric analysis as well as two dimensional peptide mapping and
Edman sequencing failed to identify phosphorylated residues in peptide
Gly338-Lys415. Therefore, to complete the
mapping of SFV nsP3 phosphorylation sites, and to confirm the results
of mass spectrometric analysis, we performed point mutational studies
and constructed additional deletions.
Phosphorylation of Threonines--
To further study the
phosphorylated threonine residues of SFV nsP3, we made eight different
constructs mutating to alanine all the 11 threonines present in the
C-terminal region of nsP3 (Fig. 1B), individually or in
pairs. All constructs were expressed in HeLa cells as above. Western
blotting with anti-nsP3 antiserum showed that the relative amounts of
mutant nsP3 proteins were similar to that of the wild type nsP3 (Fig.
2B, lower panel). Probing a parallel
blot with anti-phosphothreonine antiserum revealed that a double mutant
T344A/T345A reacted only very weakly, whereas other mutants retained
approximately wild type levels of threonine phosphorylation (Fig.
2B, upper panel). When
Thr344 and Thr345 were mutated separately to
alanine and the resulting derivatives expressed in HeLa cells, repeated
experiments indicated that both threonines were needed for efficient
phosphorylation of this site (Fig. 2B, lower
panel, lanes 10 and 11).
Mass spectrometric analysis has revealed that tryptic peptide
Gly338-Lys415 carried from 7 to 12 phosphates
(21). Thus, in proteins carrying 12 phosphates, threonines 350 and 354 were also phosphorylated (assuming that phosphorylation of
Thr378 could be excluded based on phosphorylation studies
of deletion mutant nsP3 Phosphorylation of C-terminal Peptide (aa 312-482)--
To study
whether full-length nsP3 was needed either to provide appropriate
conformation and/or kinase activity to phosphorylate the
serines/threonines in region 320-368, we made a deletion construct where the N-terminal first two thirds of nsP3 were removed (C-nsP3, nsP3 Effects of Point Mutations and Deletions on the Phosphorylation of
nsP3--
To study the phosphorylation of individual amino acids, we
made various point mutations in nsP3 and N-nsP3, expressed the mutated
derivatives in HeLa cells by transfection, and labeled the proteins
with [32P]orthophosphate. The expression level was
determined by Western blotting with anti-nsP3 antiserum (Fig.
4B), and the ability of the
mutant proteins to incorporate [32P]orthophosphate was
compared with that of wild type nsP3 (Fig. 4A). The total
phosphorylation level of the T344A/T345A threonine double-mutant was
~40% less than that of the wild type nsP3, as quantified with a
phosphorimager (Fig. 4, lanes 3 and
9). When either of the threonines 344 or 345 was singly
mutated, a similar decrease in phosphorylation was observed (Fig. 4,
lanes 2 and 3). Mutation of
Ser320, which in mass spectrometric analysis seemed to be
one of the major serine phosphorylation sites, also decreased the
phosphorylation degree of nsP3 by ~50% (Fig. 4, lane
4). However, when mutations T344A/T345A and S320A were
combined, no further decrease in phosphorylation was observed (Fig. 4,
lane 5). These results suggest that the different
phosphorylation sites may influence each other in a rather complex
manner, and therefore phosphorylation of individual sites cannot be
simply quantified based on point mutational studies. The
phosphorylation level of N-nsP3 was about 1% as compared with the wild
type (Fig. 4, lane 7), as already seen in Fig.
2A. Mutation of Ser317 and Ser320
did not greatly affect the phosphorylation level of N-nsP3 (Fig. 4,
lane 8), suggesting that it is phosphorylated on
the remaining known site Ser327 (Fig. 1B). A
small internal deletion of 26 amino acids, nsP3
Next, we wanted to generate a nsP3 derivative, in which all the known
phosphorylation sites of nsP3 would be eliminated. In this construct 50 aa were deleted, removing the entire phosphorylated region (nsP3 Effect of Phosphorylation on Membrane Association of nsP3--
In
SFV-infected BHK cells, nsP3 is associated with modified endosomes and
lysosomes (29). When nsP3 is expressed alone in mammalian cells by
transfection, it can also be found in vesicular and vacuolar structures
in the cytoplasm (23). It has been suggested previously that there
could be a connection between phosphorylation and membrane association
of nsP3 (19). To further examine this issue, we studied the
localization of transfected nsP3 derivatives by indirect
immunofluorescence. Both the wild type nsP3 (Fig. 5A) and the nonphosphorylated
derivative nsP3
To study the membrane association of nsP3 biochemically, extracts of
cells expressing the wild type nsP3 or the truncated derivative N-nsP3
were floated in a discontinuous sucrose gradient. Approximately one
third of both nsP3 and N-nsP3 floated with membranes in low salt buffer
(Fig. 5, E and F). Since N-nsP3 is minimally phosphorylated, phosphorylation does not contribute to the membrane association of nsP3. Instead, the biochemical determinants of membrane
association are located within the conserved N-terminal region of nsP3.
The association of nsP3 and N-nsP3 with membranes was peripheral in
nature, since both proteins were efficiently solubilized by 0.5 M NaCl (Fig. 5, G and H).
nsP3 Deletion Mutants in SFV Infection--
To study the effect of
nsP3 phosphorylation in the natural context of SFV infection, we
introduced the segments coding for the 26- and 50-aa deletion mutants
of nsP3 into the infectious cDNA clone of SFV. In vitro
transcribed full-length viral RNAs were used to transfect BHK cells,
which led to visually observed cytopathic effect, indicating that the
mutant constructs generated infectious viruses. As a control we
transcribed wild type icDNA of SFV4. Cell supernatants were used to
grow second-generation virus stocks, which were plaque-titrated. The
titers of the viruses were 7 × 108 pfu/ml for SFV
nsP3
The efficiencies of virus replication for the wild type SFV and the
mutant viruses were compared in one-step growth experiments in BHK,
Vero, and NIH cells using 50 pfu/cell for infection. Plaque assays of
samples collected at different time points from cells infected with
mutant viruses SFV nsP3 Reduced Pathogenecity of nsP3 Phosphorylation-defective Mutant
Virus--
To study the effects of nsP3 phosphorylation in an animal
system, mice were injected intraperitoneally with 107 pfu
of wild type or nsP3 phosphorylation-defective virus, SFV nsP3 In this study, we have completed the identification of the main
phosphorylation sites of SFV nsP3 by showing that the major threonines
phosphorylated are Thr344 and/or Thr345 (Fig.
2). Several serine residues between amino acids 320 and 368 were also
phosphorylated, possibly in a heterogeneous manner. Thus, all the
identified phosphorylation sites were concentrated in a small, highly
phosphorylated region (Fig. 1B). Mutation of one or more
serines/threonines affected the phosphorylation of other residues,
since the effects of point mutations were not additive (Fig. 4).
Specifically, mutation of either Thr344 and
Thr345 or Ser320 reduced the overall
phosphorylation level of nsP3 by 40-50%, but combination of these two
mutations gave no additional reduction. The phosphorylated region was
located in the beginning of the nonconserved C-terminal "tail"
region of nsP3 (Fig. 1), extending slightly into the conserved domain,
as Ser320 could be phosphorylated. However, this serine is
not conserved among alphaviruses, and thus there appear to be no
conserved phosphorylation sites shared by nsP3s from different
alphaviruses. Nevertheless, the general feature of extensive
phosphorylation of the tail seems to be shared with SIN nsP3 (22).
Among alphaviruses, the nsP3 variable region differs in length and in
composition (3). General similarities in the nonconserved regions of
alphavirus nsP3s include the presence of proline-rich and acidic
regions, which, however, show variation in length and distribution. In
the SFV nsP3 nonconserved region, there are two acidic domains: aa
340-406 (15 Asp+Glu in 67 aa, 22%), and aa 453-473 (8 Asp+Glu in 21 aa, 38%) separated by a proline-rich stretch between aa 408-439 (11 prolines in 32 aa, 34%). Most of the phosphorylated sites of SFV nsP3
were in the first acidic region (Fig. 1B), making it even
more negatively charged. It remains to be determined whether
phosphorylation is concentrated in a small subregion of nsP3 also in
other alphaviruses. Both acidic and proline-rich domains have been
suggested to be consensus motifs for transcriptional activation domains
(30, 31).
The C-terminal region of nsP3 (C-nsP3; Fig. 1A) was capable
of becoming phosphorylated when expressed alone, although at reduced level as compared with the full-length protein (Fig. 3). This reduced
phosphorylation may reflect a different conformation leading to
differential kinase recognition. Coexpression with the wild type nsP3
did not increase the phosphorylation of the C-terminal peptide,
indicating that the N-terminal portion is unlikely to contribute any
kinase activity itself. The N-terminal domain is related to a large
protein family with unknown function, but unrelated to known kinases
(18). An alternative explanation for the reduced phosphorylation of
C-nsP3 is offered by the fact that it was localized in the cytoplasm,
whereas the wild type nsP3 was associated with vesicular structures.
Thus, C-nsP3 might not be equally accessible to membrane-associated
kinases, which may play a role in phosphorylation of the wild type nsP3
since membrane-associated nsP3 is more heavily phosphorylated than
soluble nsP3 (19). The phosphorylation of nsP3 was, however, not
required for its peripheral membrane association.
Casein kinase II (CK II) has been suggested to be the host enzyme
phosphorylating SIN nsP3 (20), based on an experiment in which nsP3 was
immunoprecipitated from SIN-infected cells and phosphorylated in
vitro by a kinase present in the precipitate. The properties of
this kinase resembled those of CK II, as assessed by heparin
inhibition, polyamine activation, and use of both ATP and GTP as
substrate (20). However, the SIN nsP3 studied in this experiment was
most likely already phosphorylated within the cells. This may have
favored the binding of, and phosphorylation by CK II, which recognizes
serines/threonines followed by acidic residues, including
phosphorylated residues (32). Thus, CK II is one of the kinases
involved in nsP3 phosphorylation, but there are likely to be others. In
SFV nsP3, phosphorylation sites at Ser327 and
Ser367 are the best potential sites for CKII (preferred
recognition sequence (S/T)-X-X-(D/E)), but
phosphorylation of other sites may become possible, if other sites are
already phosphorylated by additional kinases (see sequence in Fig.
1B). Among SFV nsP3 phosphorylation sites,
Ser320, Ser332, and Ser335 are
potential protein kinase C sites (recognition sequence
(S/T)-X-(R/K); Ref. 32). SFV nsP3 Ser320 is a
consensus site also for p34cdc2 kinase. Among
negative-strand RNA viruses, the phosphoprotein P may also be a
substrate for multiple kinases; examples include phosphorylation of
canine distemper and measles virus P proteins by CK II and protein
kinase C (33, 34), rabies virus P protein by a unique cellular protein
kinase and protein kinase C (35), vesicular stomatis virus P protein by
CKII and an L protein-associated kinase (36, 37), and Sendai virus P
protein by protein kinase C and a proline-directed protein kinase (38,
39).
Mutant viruses coding for truncated forms of SFV nsP3 replicated to
high titers in BHK, Vero, and NIH cells (Fig. 7A). nsP3 mutant When used to infect mice, the SFV nsP3
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
329-482 (N-nsp3, Fig. 1) was generated by PCR
utilizing downstream primer 1 (see list of primers below), and upstream
primer 2. The PCR product was cloned into
NcoI/HindIII-digested pTSF3. The internal
nsp3 deletion
343-368 was made
using PCR with primer pairs 2 and 3, and 4 and 5. The PCR products were purified (Qiagen PCR purification kit) and used as a template in a
second PCR with primers 2 and 4. The internal deletion construct nsp3
319-368 was made using primers 6 and 7 together with primers 2 and 4 as described for
nsp3
343-368. The C-terminal construct nsp3
1-311 (C-nsp3) was made using
primers 8 and 2. Selected serine and threonine residues were mutated to
alanine by using the unique site elimination mutagenesis kit (Amersham
Pharmacia Biotech) according to the manufacturer's instructions. All
deletions and point mutations were verified by DNA sequencing. The
deletions nsp3
319-368
(nsp3
50) was cloned into the infectious
cDNA (pSP6-SFV4; Ref. 25) via an intermediate clone
containing the SacI/BglII fragment of the
infectious cDNA clone in vector pSP73 (Promega).
50 was
transcribed in vitro, and the obtained RNA was used to
transfect BHK cells. The cells were incubated overnight (37 °C), and
the virus was collected, centrifuged at low speed to remove cell
debris, and used to infect fresh BHK cells (100 µl of supernatant to
150 cm2 cells) to obtain a second virus passage. After
overnight incubation the virus was collected and centrifuged at low
speed, and 5% of glycerol was added to the virus stock. Virus titers
were determined by plaque assay (27). Monolayers of BHK, NIH, or Vero
were infected using 50 pfu/cell. At 1 h after infection, the media
were changed and the incubation was continued at 37 °C. Samples were
taken from the media at 2, 4, 6, 8, 10, and 12 h postinfection
(p.i.), and the amount of the virus was determined by plaque formation and hemagglutination assays (27).
26, SFV nsP3
50, or mock-infected. At 1 h p.i., the
virus was removed and the medium including actinomycin D (2 µg/ml)
was added. Duplicate dishes of each set were pulse-labeled with 50 µCi/dish for 30 min in the presence of actinomycin D (2 µg/ml) at
1.5, 2.5, 3.5, 4.5, and 5.5 h p.i. At the end of pulse period,
monolayer was washed twice with cold PBS and cells were solubilized
into buffer containing 0.5% SDS, 10 mM Tris-HCl, pH 7.5, 100 mM NaCl at 65 °C. After shearing the DNA with a
27-gauge needle, the proteins were denatured by boiling. Part of the
sample (5%) was used for determining the total protein content
(Protein Assay, Bio-Rad), while proteinase K (200 µg/ml, Roche
Diagnostics GmbH) and 4 mM CaCl2 was added to
the rest of the sample and the samples were incubated at 37 °C for
30 min. The viral RNA in double sets of 200-, 300-, and 400-µl
aliquots was precipitated with 10% trichloroacetic acid. After a
30-min incubation on ice, the precipitates were collected onto glass
fiber filters (Milllipore AP40), which were washed twice with 5%
trichloroacetic acid and dried at 100 °C for 20 min. The
incorporated [3H]uridine was quantified by liquid
scintillation counting.
50. The
amount of virus used, determined by plaque assay, was 107
pfu in PBS per mouse. Two groups of 10 mice were also infected, with
the same dose, for immunohistochemical studies. Two mice were
sacrificed daily from 3 days to 7 days after infection. The blood
samples were centrifuged at low speed at 4 °C and diluted 1:10 in
PBS, and the virus titers were determined as described earlier (27).
After perfusing the mice with cold PBS, the brains were divided
sagittally. Half of the brain was homogenized in PBS, and supernatant
was used for virus titration. For preparation of cryosections, one half
of the brain was further divided corollary in two pieces, which were
covered with Tissue Tek optimal cooling compound (Miles, IN) and frozen
in an isopentane/dry ice bath. The samples were stored in
70 °C
until cryosections of 10 µm were prepared on silanized glass. The
slices were air-dried, fixed in
20 °C acetone for 10 min, dried,
and stored at
20 °C until double-stained with a polyclonal
SFVA7(74)-specific antibody (28) using the avidin-biotin-peroxidase
detection method (Vectastain ABC kit, Vector Laboratories) and
hematoxylin staining for nuclei.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
329-482) from which the nonconserved
C-terminal region had been deleted (Fig.
1). The transfected and
32P-labeled cells were harvested, and the lysates were
subjected to immunoprecipitation with a specific anti-nsP3 antiserum,
followed by SDS-PAGE analysis. Both the wild type nsP3 and the
truncated derivative N-nsP3 were detected in Western blotting by
anti-nsP3 antibodies (Fig. 2A,
lanes 3 and 4). N-nsP3 was poorly
labeled with [32P]phosphate (lanes
1 and 2), representing only about 1%
32P activity as compared with the wild type. In Western
blots, an anti-phosphothreonine antiserum detected the wild type nsP3
but failed to recognize N-nsP3 (Fig. 2A, lanes
5 and 6). Based on these experiments, the
conserved region of nsP3 (aa 1-328) did not contain phosphorylated
threonine residues, and it was poorly phosphorylated on serine
residues.
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Fig. 1.
A, scheme of the SFV nsP3 derivatives
used in this work. The conserved and nonconserved regions are marked at
the top. The nomenclature of the derivatives, as well as the
deleted residues, are indicated on the left. The amino acid
residues included in the constructs, are shown on the right.
Internally deleted regions are denoted by thin
lines. B, amino acid sequence of the C-terminal
part of SFV nsP3. Residues mutated during the course of this work are
shown in boldface, and potentially phosphorylated residues
(21) are marked with asterisks above the residues.
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Fig. 2.
Threonine phosphorylation of nsP3.
A, the full-length nsP3 or the N-terminal portion N-nsP3
were expressed in HeLa cells by transfection, and the cells were
labeled with [32P]orthophosphate. Cell extracts either
were immunoprecipitated with anti-nsP3 antibodies and analyzed by
SDS-PAGE and autoradiography to detect labeling or were analyzed by
Western blotting with anti-nsP3 or anti-phosphothreonine antibodies, as
indicated at the top. Molecular mass markers (in kDa) are
shown on the left. B, the indicated point mutated
derivatives of nsP3 were expressed in HeLa cells. Cell extracts were
analyzed by Western blotting with anti-phosphothreonine
(upper panel) and anti-nsP3 (lower
panel) antibodies.
50, see below). However, double
mutation of T350A/T354A did not show decreased reaction with
anti-phosphothreonine antiserum (Fig. 2), indicating that the major
phosphothreonines in SFV nsP3 were Thr344 and
Thr345. Mutating either threonine alone appeared to
decrease phosphorylation to a degree almost similar to the double
mutation, and either or both of them could be phosphorylated in the
wild type nsP3. It is noteworthy that the phosphorylated threonine
residues were in close vicinity of the phosphorylated serines,
indicating that a small subregion of nsP3 contained large majority of
the phosphorylated residues (Fig. 1B).
1-311; Fig. 1A). Anti-nsP3 antiserum was able to
recognize this C-terminal peptide expressed in HeLa cells by
transfection (Fig. 3, lane
2). This peptide could be labeled with
[32P]orthophosphate (Fig. 3, lane
4). According to metabolic labeling with
[35S]methionine and [32P]orthophosphate the
degree of phosphorylation of C-nsP3 was about 30% of that of the
full-length protein. This difference may be due to altered conformation
of the peptide. Alternatively, the subcellular localization of C-nsP3
may be different from that of the full-length protein, which might make
it differentially accessible to kinases. To study the possibility of
nsP3 autophosphorylation, the C-terminal peptide was cotransfected with
the wild type nsP3. No increase in the phosphorylation level of C-nsP3
was detected (Fig. 3, lane 5). Since it is
unlikely that the variable C-terminal region with an unusual amino
composition would have kinase activity, our results suggest that the
phosphorylation of C-nsP3 and the wild type nsP3 is catalyzed by
cellular kinases.
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Fig. 3.
Phosphorylation of nsP3 and C-nsP3. nsP3
and its C-terminal domain C-nsP3 were expressed in HeLa cells. The
cells were labeled with either [32P]orthophosphate or
[35S]methionine/cysteine as indicated at the
top. Cell extracts were immunoprecipitated with anti-nsP3
antibodies and analyzed by SDS-PAGE and autoradiography. Molecular mass
markers (in kDa) are shown on the left.
26 (nsP3
343-368;
Fig. 1A), reduced nsP3 phosphorylation by 90% (Fig. 4,
lane 6).
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Fig. 4.
Phosphorylation of nsP3 derivatives. The
nsP3 derivatives indicated at the top were expressed in HeLa
cells. The cells were labeled with [32P]orthophosphate,
and cell extracts were analyzed by immunoprecipitation with anti-nsP3
rabbit antiserum and autoradiography to detect labeling (A)
or by Western blotting with anti-nsP3 guinea pig antiserum to verify
the level of expression (B). The numbers
below panel A indicate the amount of
phosphorus labeling as normalized with respect to the wild type
nsP3.
50;
Fig. 1A). The construct was expressed in transfected HeLa
cells at levels similar to the wild type nsP3, and did not incorporate
detectable amounts of [32P]orthophosphate (see below).
50 (Fig. 5B) were associated with
vesicular structures in HeLa cells. Therefore, phosphorylation of nsP3
is not needed for its ability to associate with these structures. In
contrast, the phosphorylated C-terminal peptide C-nsP3 showed a diffuse
cytoplasmic distribution (Fig. 5C), indicating that it does
not contain determinants for membrane association.
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Fig. 5.
Membrane association and localization of nsP3
and its derivatives. A-D, HeLa cells expressing wild
type nsP3 (A), nsP3 50 (B), C-nsP3
(C), or mock-transfected HeLa cells (D) were
fixed at 4 h after transfection and subjected to immunofluorescent
labeling with anti-nsP3 antiserum. E-H, HeLa cells
expressing the wild type nsP3 or the truncated derivative N-nsP3 were
broken by Dounce homogenization (8), and the extracts were analyzed in
discontinuous sucrose gradients consisting of 67%, 60%, 50%, and 5%
(w/w) layers in 100 mM NaCl, 50 mM Tris, pH 7.5 (E and F), or in the same buffer containing 500 mM NaCl (G and H). The samples were
adjusted to 60% (w/w) sucrose and loaded on top of the 67% layer.
During centrifugation (Beckman SW50.1 rotor at 35,000 rpm for 16 h), membrane-associated material floated to the interface of 50% and
5% layers (9).
26, and 3 × 109 pfu/ml for SFV4 and SFV
nsP3
50. BHK cells were infected with these viruses using 50 pfu/cell, and the cells were labeled with [35S]methionine/cysteine and
[32P]orthophosphate. nsP3 was immunoprecipitated from the
cell lysates using anti-nsP3 antiserum. The mutant viruses produced
nsP3 of the expected size (Fig. 6,
lanes 1-3) but comparing to the wild-type SFV
nsP3 only 10% of [32P]orthophosphate was incorporated to
mutated nsP3 produced in SFV nsP3
26 infection (Fig. 6,
lanes 5 and 6). Moreover, no
detectable amounts of [32P]orthophosphate was
incorporated to the mutated nsP3 in SFV nsP3
50 infection (Fig. 6,
lane 4), showing that the mutant viruses
possessed the expected nsP3 phosphorylation phenotypes.
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Fig. 6.
Phosphorylation of nsP3 and its derivatives
in SFV-infected cells. BHK cells were infected with viruses
encoding wild type nsP3 or its mutated derivative nsP3 26 or
nsP3
50, as indicated at the top. The cells were labeled
with [35S]methionine/cysteine (lanes
1-3) or [32P]orthophosphate (lanes
4-6). Cell extracts were collected, immunoprecipitated with
anti-nsP3 antibody, and analyzed by SDS-PAGE and fluorography.
Molecular mass markers (in kDa) are shown on the left.
26 and SFV nsP3
50 showed titers similar to
wild type SFV in BHK and Vero cells (Fig.
7A). In NIH cells the mutated
virus titers were 1 order of magnitude lower at 2-12 h p.i. However,
after 23 h p.i., the titers of the mutated viruses were similar to
the wild-type SFV (Fig. 7A). This indicates that nsP3
phosphorylation did not greatly increase infectious SFV production in
these cell types. However, since a small difference in growth rate
could be seen at 4 h p.i. also in BHK and Vero cells, we
investigated the level of virus-specific RNA synthesized in BHK cells
in early infection. For this, BHK were infected with wild type and
mutated viruses using 50 pfu/cell and the viral RNA was metabolically
labeled for 30 min at different time points. As shown in Fig.
7B, the virus-specific RNA levels of both deletion mutants
were significantly reduced at early stages of infection.
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Fig. 7.
Comparison of virus growth in BHK, Vero, and
NIH cells and synthesis of viral RNA in BHK cells. A,
for growth curves, cells were infected with wild-type SFV, SFV
nsP3 26, and SFV nsP3
50 using 50 pfu/cell at 37 °C. Samples
from media were taken at 2, 4, 6, 8, 10, and 12 h p.i., and
titrated on BHK cells. B, for analysis of viral RNA
synthesis, BHK cells were infected with the indicated virus using 50 pfu/cell and labeled with [3H]uridine for 30 min in the
presence of actinomycin D. The RNA was precipitated with 10%
trichloroacetic acid and quantified by liquid scintillation. The values
shown represent the average of three parallel samples; bars
indicate S.D.
50. As
described above, this phosphorylation-deficient mutant was able to
propagate in BHK, Vero, and NIH cells to titers as high as the wild
type SFV. However, this mutant virus was avirulent for mice, since none
of the 5 animals infected with SFV nsP3
50 was killed or had clinical
symptoms during the 3 weeks follow-up period. All five mice infected
with the same dose of the wild type SFV (107 pfu) died
within 6 days. Histochemical analysis revealed that 4 days after
infection wild type SFV was widely spread within the brain (both in
cerebellum and cerebrum) of the mice having severe paralysis (Fig.
8, A, B, and
E). The virus titers of the wild type SFV in the brain of
the mice after 5 and 6 days p.i. were 3 × 106 pfu/g
of brain and 5 × 106 pfu/g of brain, respectively. In
contrast, when mice were infected with SFV nsP3
50 mutant virus, no
staining of virus structural proteins could been seen in the central
nervous system of 6 individual mice. In the brains of 2 mice, isolated
viral foci were observed in cerebellum (Fig. 8, C and
D) and cerebrum (Fig. 8F). The focal replication
of SFV nsP3
50 was associated with perivascular areas as shown for
cerebellum (Fig. 8D). The titer of SFV nsP3
50 5 days
after infection in the brain of 1 mouse was 3 × 103
pfu/g of brain, and virus titers in the brains of the other 7 mice were
under the detection limit between days 4 and 7 after infection. Thus,
the nsP3 phosphorylation-defective mutant virus had greatly reduced
neurovirulence for adult mice.
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Fig. 8.
Immunoperoxidase staining of brains from mice
infected with SFV4 (A, B, and
E) and SFV nsP3 50
(C, D, and F) 5 days
after intraperitoneal infection of 107 pfu/mouse.
Staining with hematoxylin and polyclonal anti-SFV antibodies was
followed by avidin-biotin peroxidase staining. A, general
infection of neuronal cells in cerebellum. B, a larger
magnification of cerebellar region of infected Purkinje cells with
antigen-positive dendritic region (arrows). C, a
cluster of SFV nsP3
50 infected cerebellar cells in perivascular
region shown enlarged in D). E, multiple infected
foci in SFV4 infected cerebrum. F, isolated focus in
cerebrum of mutant virus-infected mouse. Bars, 500 µm in
A and C; 100 µm in B and
D-F.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
26 had 10% level of phosphorylation remaining as compared with wild type, whereas mutant
50 was not detectably phosphorylated. These results indicate that the mutated nsP3 proteins were able to
carry out all their essential functions needed for RNA replication and
for high rates of virus production. Therefore, these internal deletions
in a region that was predicted to be devoid of secondary structure did
not cause gross misfolding of the protein.
50 mutant virus was avirulent
even with a dose of 107 pfu/mouse. The rate of viral RNA
synthesis of this mutant was significantly lower than that of the wild
type SFV resulting the decreased virulence in mice. However, it cannot
be simply stated that the decreased RNA synthesis rate is caused solely
by the lack of phosphorylation of nsP3, since deletion of amino acids around the phosphorylation sites may have made a contribution to this
phenomenon. Recent study with an avirulent SFV mutant A7(74) (40) has
revealed the importance of nsP3 in the neuropathogenicity of SFV (28).
Phosphorylation of nsP3, as well as the entire heterogenous C-terminal
tail, which has been subject to rapid alteration during alphavirus
evolution, may serve to fine-tune the replication of alphaviruses in
different cell types. Further studies should reveal the role of the
phosphorylated C-terminal region of nsP3 in specific interactions with
components of the neuronal cells leading to neurovirulence of Semliki
Forest virus.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Petra Nygårdas (Åbo Academi and Turku Immunology Center, Turku, Finland) for performing the immunohistochemical assays. We also thank Airi Sinkko and Tarja Välimäki for excellent technical assistance and Dr. Marja Makarow for critical reading of this manuscript.
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FOOTNOTES |
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* This work was supported in part by Academy of Finland Grant 8397 and by grants from the Technology Development Center and Helsinki University Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Present address: Pfizer Global Research and Development, Ramsgate Rd., Sandwich CT13 9NJ, United Kingdom.
§ To whom correspondence should be addressed: Inst. of Biotechnology, P.O. Box 56, Viikinkaari 9, University of Helsinki, Helsinki FIN-00014, Finland. Tel.: 358-9-191-59650; Fax: 358-9-191-59560; E-mail: helena.vihinen@helsinki.fi.
** Biocentrum Helsinki fellow.
Published, JBC Papers in Press, December 4, 2000, DOI 10.1074/jbc.M006077200
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ABBREVIATIONS |
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The abbreviations used are: SIN, Sindbis virus; SFV, Semliki Forest virus; nsP, nonstructural protein; BHK, baby hamster kidney; pfu, plaque-forming unit; PBS, phosphate-buffered saline; p.i., postinfection; CK II, casein kinase II; PAGE, polyacrylamide gel electrophoresis; aa, amino acid(s); MES, 4-morpholineethanesulfonic acid.
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