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Journal of Virology, December 1998, p. 10265-10269, Vol. 72, No. 12
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Alphavirus Replicase Protein NSP1 Induces
Filopodia and Rearrangement of Actin Filaments
Pirjo
Laakkonen,
Petri
Auvinen,
Pekka
Kujala, and
Leevi
Kääriäinen*
Institute of Biotechnology, Viikki Biocenter,
University of Helsinki, Helsinki, Finland
Received 18 May 1998/Accepted 25 August 1998
 |
ABSTRACT |
Expression of the NSP1 protein of Semliki Forest virus and Sindbis
virus in cultured cells induced filopodia-like extensions containing
NSP1 but not F actin. The actin stress fibers disappeared, whereas
vimentin, keratin, and tubulin networks remained intact. The effects of
NSP1 were dependent on its palmitoylation but not on its enzymatic
activities and were also observed in virus-infected cells.
| |
TEXT |
The RNA replication of alphaviruses
such as Semliki Forest virus (SFV) and Sindbis virus (SIN) is directed
by virus-specific nonstructural proteins (NSP1 to -4) and takes place
in the cytoplasm of infected cells on modified endosomes and lysosomes
(5, 17). The functions of the individual NSPs have been
studied extensively (27). NSP1 (537 and 540 amino acids in
SFV and SIN, respectively) is an mRNA capping enzyme with
guanine-7-methyltransferase (MT) (11, 15) and
guanylyltransferase (GT) activities (1, 2). Our recent study
showed that SFV NSP1 was tightly associated with membranes due to the
palmitoylation of cysteine residues 418 to 420. Interestingly,
acylation also affected the subcellular distribution of NSP1, since
only the palmitoyolated NSP1 was associated with the surface extensions
of HeLa cells (10), which expressed NSP1 from the plasmid
pTSF1 (18) by the aid of vaccinia virus vector vTF7-3
(6, 26).
Induction of filopodia by expression of SFV and SIN NSP1.
To
study this phenomenon further, we expressed the acylated and unacylated
forms of NSP1 of SIN. The gene coding for NSP1 was obtained from the
infectious cDNA clone pToto1101 (19) by PCR, sequenced, and
placed under the T7 promoter in pGEM3 to yield pTSIN1. HeLa cells were
first infected with vTF7-3 for 45 min, followed by transfection with
plasmid pTSF1 or pTSIN1 with the Lipofectin reagent (GIBCO, BRL)
(10, 18) to express NSP1 of SFV or SIN, respectively.
Hydroxyurea (10 mM) (Sigma) was applied to the cells simultaneously
with Lipofectin to inhibit vaccinia virus DNA replication (3,
30). The cells were examined at 3 h posttransfection by
using phase-contrast optics. Mock-infected (not shown) and
vTF7-3-infected (Fig. 1A) cells served as
controls. The cells expressing SFV (Fig. 1B) or SIN (Fig. 1C) NSP1
showed induction of a large number of filopodia-like structures, which hereafter are designated filopodia. These extensions often had a thick
root and were branched at their distal parts to thin microspikes or
filopodia. Similar structures were also observed in SFV- and SIN-infected cells, as demonstrated for SFV by scanning electron microscopy, which was stabilized as described elsewhere (24, 25) (Fig. 1D).
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FIG. 1.
Phase-contrast images of HeLa cells after 3 h of
incubation in the transfection medium. Shown are cells infected with
vaccinia virus vTF7-3 for 45 min followed by incubation in transfection
medium (A) and cells infected first with vTF7-3 for 45 min followed by
transfection with plasmid pTSF1 encoding SFV NSP1 (B) or with pSIN1
encoding SIN NSP1 (C). Also shown is a scanning electron micrograph of
an SFV-infected BHK cell at 4 h postinfection (D). Bars, 10 µm.
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|
Tunneling microscopy revealed that the diameter of the filopodia was
fairly uniform (about 50 nm [Fig.
2A])
and that many
of them were branched (Fig.
2A and insert). Whole-mount
immunotransmission
electron microscopy of saponin (0.5%)-permeabilized
cells carried
out as described elsewhere (
24,
25) revealed
that NSP1 was
present along the whole length of the filopodia (Fig.
2B).
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FIG. 2.
Filopodia contain NSP1 along their whole length. HeLa
cells infected with vTF7-3 and transfected with pTSF1 as described in
the legend to Fig. 1 were processed at 3 h posttransfection for
tunneling microscopy (A) or for whole-mount immunotransmission electron
microscopy (B). For immunotransmission electron microscopy, treatment
with anti-NSP1 was followed by the addition of protein A-gold
particles. Arrowheads, retraction fibers; asterisks, filopodia.
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Reorganization of F actin in cells expressing NSP1.
Since the
formation of filopodia in normal cells is driven by the extension of F
actin filaments, we labeled pTSF1-transfected cells with fluorescent
phalloidin to visualize F actin. In mock-infected cells (Fig.
3A), phalloidin decorated the actin
stress fibers, whereas no stress fibers were detectable in
NSP1-expressing cells (Fig. 3C). Instead, F actin was brightly stained
in the cell periphery and in the more thickly branched roots of the
cellular extensions (Fig. 3C). However, double staining with anti-NSP1
and phalloidin revealed that the narrow filopodia-like branches did not
contain detectable F actin filaments (compare Fig. 3B and C). Filopodia were also induced in BHK and PC12 cells and in the human fibrosarcoma cell line HT1080, all expressing SFV NSP1 (not shown).
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FIG. 3.
Expression of NSP1 causes destruction of actin stress
fibers. (A) Oregon-green-conjugated phalloidin staining of stress
fibers in a control HeLa cell. (B and C) Lack of stress fibers in a
HeLa cell expressing NSP1 3 h after transfection with pTSF1;
staining with anti-NSP1 antibodies (B) and Oregon-green phalloidin
staining of the same cell (C). Note that stress fibers are present in a
neighboring cell, which does not express NSP1. Bars, 10 µm.
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|
We have previously identified critical amino acid residues in SFV NSP1,
which are required for both MT and GT activities (e.g.,
D64 and D90),
only for GT activity (H38) (
2) or for palmitoylation
(C418-420) (
10). Moreover, removal of amino acid residues
121
to 148 destroyed both the MT and the GT activities and inhibited
acylation and membrane association of this truncated NSP1 derivative
(
10). To study the effects of these mutations on the
induction
of filopodia and disappearance of stress fibers, the mutated
NSP1
derivatives were expressed with the vaccinia virus vTF7-3 system
(
6). Table
1 shows that
neither MT nor GT activity was required
for the induction of filopodia
and disassembly of actin stress
fibers. However, the acylation of NSP1
of both SFV and SIN was
necessary to cause these alterations.
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TABLE 1.
Correlation of actin redistribution, palmitoylation, and
enzymatic activities of NSP1 in HeLa cells expressing wild-type or
mutant SFV or SIN NSP1 proteins
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|
NSP1-induced extensions differ from normal filopodia.
Cytochalasin D (CD) inhibits the polymerization of actin filaments,
resulting in the disappearance of actin stress fibers and in the
appearance of actin aggregates in the cytoplasm (Fig. 4B). When NSP1-expressing cells were
treated with CD (0.1 µg/ml), numerous filopodia labeled with
anti-nsP1 antiserum were observed around the cell periphery (Fig. 4A),
indicating that F actin was not needed for their formation. The thicker
surface extensions, which sometimes resembled neuronal growth cones
(e.g., Fig. 3C), were never seen in CD-treated cells, suggesting that F
actin had participated in their formation. In addition, no evidence for the presence of tubulin, vimentin, or keratin filaments in the filopodia of NSP1-expressing HeLa cells was obtained by staining with
the respective antibodies (not shown). Addition of 0.1 or 0.5 mM
nocodazole (12) or 50 µM vinblastine from 1 to 3 h
posttransfection to disrupt microtubules did not inhibit the formation
of filopodia, indicating that the integrity of microtubular and
intermediate filament networks was not necessary for their formation
(not shown).
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FIG. 4.
CD (0.1 µg/ml) was added at 1 h posttransfection
to HeLa cells infected with vTF7-3 and transfected with pTSF1 1 h
after transfection. (A and B) HeLa cell stained with anti-NSP1 (A) and
phalloidin (B) of the same cell. (C through F)
Double-immunofluorescence staining of HeLa cells 3 h after
transfection with pTSF1 stained with anti-NSP1 (C and E), anti-ezrin
antibodies (D), and anti-CD44 antibodies (F). Bars, 10 µm.
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|
Paxillin and vinculin, which are typical components of the focal
adhesions for F actin bundles (
4), were distributed
similarly
at the cell periphery both in mock- and pTSF1-transfected
cells,
suggesting that focal adhesions were not affected by NSP1 (not
shown). Nor was
1-integrin, which normally mediates the contact
of
filopodia with the external matrix at the focal contact points
(
31), found in the filopodia (not shown). Instead, both the
transmembrane protein CD44 (
8) and the intracellularly
located
peripheral plasma membrane protein ezrin (
29)
colocalized with
NSP1 in the filopodial extensions (Fig.
4C to
F).
According to the present study, alphavirus NSP1 causes disassembly of
the actin cytoskeleton when it is expressed in several
different cell
lines. In addition, it induces numerous surface
extensions which stain
positively with anti-NSP1 antibodies and
morphologically resemble
filopodia but lack, e.g., F actin. Only
the acylated, tightly
membrane-associated form of NSP1 causes
these effects. The same
phenomena take place in virus-infected
cells, indicating that they are
natural responses for alphavirus
infection. Expression vectors which
lack the genes for structural
proteins have been developed both for SFV
and SIN (
13,
23).
Since these vectors express the RNA
replicase proteins, including
NSP1, they also cause induction of
filopodia and disruption of
stress fibers, a fact which has to be taken
into account when
analyzing the effects of the expressed foreign
proteins.
Analysis of interactions of viral proteins with host proteins has
expanded our knowledge of many central cellular processes,
such as DNA
replication, transcriptional control, regulation of
translation,
nuclear transport, signal transduction, and apoptotic
cell death, as
well as folding, modification, transport, and targeting
of exocytic
membrane proteins (
9). The present results for
alphavirus
NSP1 offer the possibility of studying its intervention
with the
delicately controlled and complex regulation of the actin
cytoskeleton
(
7,
14,
16,
20-22,
28,
32). Understanding
of the mechanism
of action of NSP1 should reveal new aspects in
this regulatory cascade,
which controls the shape and movements
of the
cell.
| |
ACKNOWLEDGMENTS |
We thank Marja Makarow for critical reading of the manuscript and
Tarja Välimäki and Airi Sinkko for technical assistance. We
thank Sirpa Jalkanen, Eero Lehtonen, Ismo Virtanen, and Jari Ylänne for antibodies. Charles M. Rice is acknowledged for the full-length SIN infectious cDNA clone pToto1101 and Tero Ahola for the
SIN NSP1 constructs.
This work has been supported by Academy of Finland, Biocentrum
Helsinki, and the Helsinki University Foundation.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Institute of
Biotechnology, P.O. Box 56, Viikinkaari 9, 00014 University of
Helsinki, Helsinki, Finland. Phone: 358-9-70859400. Fax:
358-9-70859560. E-mail: leevi.kaariainen{at}helsinki.fi.
| |
REFERENCES |
| 1.
|
Ahola, T., and L. Kääriäinen.
1995.
Reaction in alphavirus mRNA capping: formation of a covalent complex of nonstructural protein nsP1 with 7-methyl-GMP.
Proc. Natl. Acad. Sci. USA
92:507-511[Abstract/Free Full Text].
|
| 2.
|
Ahola, T.,
P. Laakkonen,
H. Vihinen, and L. Kääriäinen.
1997.
Critical residues of Semliki Forest virus RNA capping enzyme involved in methyltransferase and guanylyltransferase-like activities.
J. Virol.
71:392-397[Abstract].
|
| 3.
|
Bucci, C.,
R. G. Parton,
I. H. Mather,
H. G. Stunnenberg,
K. Simons,
B. Hoflack, and M. Zerial.
1992.
The small GTPase rab5 functions as a regulatory factor in the early endocytic pathway.
Cell
70:715-728[Medline].
|
| 4.
|
Burridge, K.,
M. Chrzanowska-Wodnicka, and C. Zhong.
1997.
Focal adhesion assembly.
Trends Cell Biol.
7:342-347.
[Medline] |
| 5.
|
Froshauer, S.,
J. Kartenbeck, and A. Helenius.
1988.
Alphavirus RNA replicase is located on the cytoplasmic surface of endosomes and lysosomes.
J. Cell Biol.
107:2075-2086[Abstract/Free Full Text].
|
| 6.
|
Fuerst, T. R.,
E. G. Niles,
F. W. Studier, and B. Moss.
1986.
Eukaryotic transient expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase.
Proc. Natl. Acad. Sci. USA
83:8122-8126[Abstract/Free Full Text].
|
| 7.
|
Hall, A.
1998.
Rho GTPases and the actin cytoskeleton.
Science
279:509-514[Abstract/Free Full Text].
|
| 8.
|
Jalkanen, S. T.,
R. F. Bargatze,
L. R. Herron, and E. C. Butcher.
1986.
A lymphoid cell surface glycoprotein involved in endothelial cell recognition and lymphocyte homing in man.
Eur. J. Immunol.
16:1195-1202[Medline].
|
| 9.
|
Knipe, D. M.
1996.
Virus-host cell interactions, p. 273-299.
In
B. N. Fields, D. M. Knipe, P. M. Howley, et al. (ed.), Virology. Lippincott-Raven, Philadelphia, Pa.
|
| 10.
|
Laakkonen, P.,
T. Ahola, and L. Kääriäinen.
1996.
The effects of palmitoylation on membrane association of Semliki Forest virus RNA capping enzyme.
J. Biol. Chem.
271:28567-28571[Abstract/Free Full Text].
|
| 11.
|
Laakkonen, P.,
M. Hyvönen,
J. Peränen, and L. Kääriäinen.
1994.
Expression of Semliki Forest virus nsP1-specific methyltransferase in insect cells and in Escherichia coli.
J. Virol.
68:7418-7425[Abstract/Free Full Text].
|
| 12.
|
Liao, G.,
T. Nagasaki, and G. G. Gundersen.
1995.
Low concentrations of nocodazole interfere with fibroblast locomotion without significantly affecting microtubule level: implications for the role of dynamic microtubules in cell locomotion.
J. Cell Sci.
108:3473-3483[Abstract].
|
| 13.
|
Liljeström, P.,
S. Lusa,
D. Huylebroeck, and H. Garoff.
1991.
In vitro mutagenesis of a full-length cDNA clone of Semliki Forest virus: the small 6,000-molecular-weight membrane protein modulates virus release.
J. Virol.
65:4107-4113[Abstract/Free Full Text].
|
| 14.
|
Machesky, L. M., and A. Hall.
1996.
Rho: a connection between membrane receptor signalling and the cytoskeleton.
Trends Cell Biol.
6:304-310.
[Medline] |
| 15.
|
Mi, S., and V. Stollar.
1991.
Expression of Sindbis virus NSP1 and methyltransferase activity in Escherichia coli.
Virology
184:423-427[Medline].
|
| 16.
|
Nobes, C. D., and A. Hall.
1995.
Rho, Rac, and Cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin fibers, lamellipodia, and filopodia.
Cell
81:53-62[Medline].
|
| 17.
|
Peränen, J., and L. Kääriäinen.
1991.
Biogenesis of type I cytopathic vacuoles in Semliki Forest virus-infected BHK cells.
J. Virol.
65:1623-1627[Abstract/Free Full Text].
|
| 18.
|
Peränen, J.,
P. Laakkonen,
M. Hyvönen, and L. Kääriäinen.
1995.
The alphavirus replicase protein nsP1 is membrane-associated and has affinity to endocytic organelles.
Virology
208:610-620[Medline].
|
| 19.
|
Rice, C. M.,
R. Levis,
J. H. Strauss, and H. V. Huang.
1987.
Production of infectious RNA transcripts from Sindbis virus cDNA clones: mapping of lethal mutations, rescue of a temperature-sensitive marker, and in vitro mutagenesis to generate defined mutants.
J. Virol.
61:3809-3819[Abstract/Free Full Text].
|
| 20.
|
Ridley, A. J., and A. Hall.
1994.
Signal transduction pathways regulating Rho-mediated stress fibre formation: requirement for a tyrosine kinase.
EMBO J.
13:2600-2610[Medline].
|
| 21.
|
Ridley, A. J.,
H. F. Paterson,
C. L. Johnston,
D. Diekmann, and A. Hall.
1992.
The small GTP-binding protein rac regulates growth factor-induced membrane ruffling.
Cell
70:401-410[Medline].
|
| 22.
|
Schafer, D. A., and J. A. Cooper.
1995.
Control of actin assembly at filament ends.
Annu. Rev. Cell Dev. Biol.
11:497-518[Medline].
|
| 23.
|
Schlesinger, S.
1993.
Alphaviruses vectors for the expression of heterologous genes.
Trends Biotechnol.
11:18-22[Medline].
|
| 24.
|
Singer, R. H.,
G. L. Langevin, and J. B. Lawrence.
1989.
Ultrastructural visualization of cytoskeletal mRNAs and their associated proteins using double-label in situ hybridization.
J. Cell Biol.
108:2342-2353.
|
| 25.
|
Sormunen, R.
1997.
 -Spectin in detergent-extracted whole-mount cytoskeletons of chicken embryo heart fibroblasts.
Histochem. J.
25:678-686.
|
| 26.
|
Stenmark, H.,
C. Bucci, and M. Zerial.
1995.
Expression of Rab GTPases using recombinant vaccinia viruses.
Methods Enzymol.
257:155-164[Medline].
|
| 27.
|
Strauss, J. H., and E. G. Strauss.
1994.
The alphaviruses: gene expression, replication, and evolution.
Microbiol. Rev.
58:491-562[Abstract/Free Full Text].
|
| 28.
|
Tsukita, S.,
K. Oishi,
N. Sato,
J. Sagara, and A. Kawai.
1994.
ERM family members as molecular linkers between the cell surface glycoprotein CD44 and actin-based cytoskeletons.
J. Cell Biol.
126:391-401[Abstract/Free Full Text].
|
| 29.
|
Vaheri, A.,
O. Carpén,
L. Heiska,
T. S. Helander,
J. Jääskeläinen,
P. Majander-Nordenswan,
M. Sainio,
T. Timonen, and O. Turunen.
1997.
The ezrin protein family: membrane-cytoskeleton interactions and disease associations.
Curr. Opin. Cell Biol.
9:659-666[Medline].
|
| 30.
|
Vos, J. C., and H. G. Stunnenberg.
1988.
Derepression of a novel class of vaccinia virus genes upon DNA replication.
EMBO J.
7:3487-3492[Medline].
|
| 31.
|
Ylänne, J.,
D. A. Cheresh, and I. Virtanen.
1990.
Localization of beta 1, beta 3, alpha 5, alpha v, and alpha lib subunits of the integrin family in spreading human erythroleukemia cell.
Blood
76:570-577[Abstract/Free Full Text].
|
| 32.
|
Zigmond, S. H.
1996.
Signal transduction and actin filament organization.
Curr. Opin. Cell Biol.
8:66-73[Medline].
|
Journal of Virology, December 1998, p. 10265-10269, Vol. 72, No. 12
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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