1 CNRS UPR9051, Hôpital Saint-Louis, Conventionné par
l'Université Paris 7, 1 avenue Claude Vellefaux, 75475 Paris Cedex 10,
France
2 Laboratoire des Lyssavirus, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris
Cedex 15, France
* Author for correspondence (e-mail: alisaib{at}infobiogen.fr)
Accepted 14 April 2003
![]() |
Summary |
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Key words: Retrovirus, Cytoskeleton, Trafficking, Spumavirus
![]() |
Introduction |
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Spumaviruses, also known as foamy viruses (FVs), are complex animal
retroviruses that encode structural (Gag, Env), enzymatic (Pol) and regulatory
products (Tas, Bet). Although surface Env glycoproteins are needed to enter
target cells, interaction and subsequent self-multimerization of Gag molecules
lead to capsid formation (Eastman and
Linial, 2001; Tobaly-Tapiero
et al., 2001
). Several functional regions were identified along
the scaffold Gag protein. There are three glycine(G)/arginine(R)-rich
sequences, the so-called GR boxes, in the C-terminus that are implicated in
viral nucleic acid binding and harbor a nuclear localization sequence (NLS)
(Schliephake and Rethwilm,
1994
; Yu et al.,
1996
), a 18 amino acid motif (amino acids 43-60) resembling the
cytoplasmic targeting and retention signal (CTRS) of type D retroviruses that
allows cytoplasmic targeting of Gag
(Eastman and Linial, 2001
) and,
finally, a coiled-coil domain (amino acids 130-160) that is necessary for
Gag-Gag interaction (Tobaly-Tapiero et
al., 2001
). The last two motifs are required for capsid assembly.
Although compelling studies have demonstrated the requirement for Gag in the
late steps of infection (i.e. packaging of genomic RNA or virus egress),
little is known about its role during the early steps of virus cycle.
We have previously shown that incoming human foamy virus (HFV), the
prototype of FVs, targets the microtubule organizing center (MTOC) prior to
nuclear translocation. Centrosomal targeting of incoming viral proteins and
subsequent viral replication were inhibited by a treatment with nocodazole,
demonstrating the involvement of the MT network in FV replication
(Saib et al., 1997). However,
the precise status of the viral material at the MTOC and the mechanism of
transport towards this organelle were unknown. Here, we show that incoming HFV
reaches the MTOC as naked and structured capsids. Remarkably, the Gag protein
by itself harbors the determinant to target the MTOC, requiring a domain of 30
amino acids predicted to fold into a coiled coil. Transport of Gag to the MTOC
necessitates the retrograde, MT-dependent, dynein/dynactin motor because it is
prevented by overexpression of the central coiled-coil domain (CC1) of
p150Glued, the dynactin sidearm subunit. Finally, a direct
interaction between HFV Gag and the cytoplasmic light chain of the dynein LC8
was described, identifying a molecular basis of retroviral transport in
infected cells during the early steps of infection.
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Materials and Methods |
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Cell culture and transfection
Hamster BHK21 and simian Cos6 cells were maintained in DMEM (Gibco)
supplemented with 5% fetal calf serum, 100 µg ml1
penicillin, 50 µg ml1 streptomycin, 2 mM glutamine and
240 mM Hepes. For transfection assays, 2x105 Cos6 cells were
transfected with 2 µg recombinant plasmid DNA with the Lipofectin reagent
(Gibco BRL) as specified by the manufacturer. 20 hours after transfection,
cells were fixed and permeabilized with methanol at 20°C for 5
minutes. HFV Gag was detected by rabbit polyclonal anti-Gag antibodies (1/400
dilution). Monoclonal antibodies (mAbs) against vimentin (clone V18, used at
1/100 dilution; Sigma) and antibodies directed against a centriolar component
(provided by M. Bornens, Curie Institute; clone CTR910, used at 1/100
dilution) were used in some experiments. Fluorescein (FITC) or Texas red
(TR)-coupled antibodies were used as second fluorescent conjugates (Biosys,
dilution 1/800 in PBS-Tween 0.1%). Cells nuclei were stained with DAPI.
For confocal analysis, cells were mounted in Mowiol (Calbiochem) and examined using a BioRad MRC-1024 confocal imaging system (BioRad Microscience) and an inverted Diaphot 300 Nikon microscope. For fluorescein, a krypton/argon laser (Ion Laser Technology) with a 488 nm filter was used. For TR staining, an ion laser with a 568 nm filter was used. Images of FITC, TR and DAPI were pseudocolored in green, red and blue, respectively.
Virus titers were determined using indicator FV-activated GFP expression
(FAG) cells, which harbor the GFP ORF under the control of the HFV long
terminal repeat (LTR), as previously described
(Tobaly-Tapiero et al., 2001).
Thus, the titers were measured as fluorescent cell-forming-units (FCFU) per
ml.
Protein analysis
24 hours after transfection with pSG-Gag575,
pSG-Gag575L171G or pLC8Flag, or co-transfection of these
vectors, 293T cells were labeled with [35S]methionine-cysteine (200
µCi ml1, 1.175 Ci mmol1 specific
activity, Dupont NEN) for 3 hours. Cells were rinsed with cold PBS and then
lysed with 10 mM Tris-HCl (pH 7.5), 50 mM NaCl, 1 mM CaCl2, 3 mM
MgCl2 and anti-protease cocktail (Sigma), followed by
centrifugation at 20,000 g for 5 minutes at 4°C.
Cytoplasmic proteins were directly analysed by western blot using rabbit
anti-Gag antibodies following denaturation in Laemmli buffer or
immunoprecipitated with anti-Flag M2 mAb (Sigma) in CHAPS buffer (10 mM Tris
pH 7.5, 150 mM NaCl, 1% CHAPS, 1 mM PMSF). For that purpose, pre-cleared cell
extracts were incubated overnight at 4°C with the M2 anti-Flag mAb in
CHAPS buffer, absorbed in protein-A/Sepharose. Immune complexes were
centrifuged and washed four times in CHAPS buffer and analysed by western blot
using anti-Gag antibodies (1/100). For western-blot analyses, proteins were
resolved using SDS 10% polyacrylamide gel electrophoresis.
Culture supernatants of Cos6 cells transfected with pHFV13, pHFVGagL171G or a plasmid encoding only the Gag and Pol proteins (pCgp9; kindly provided by A. Rethwilm) were first cleared from cellular debris and then centrifuged through a 20% sucrose cushion in a solution containing 100 mM NaCl, 10 mM Tris-HCl (pH 7.4), and 1 mM EDTA at 80,000 g for 3 hours in a SW41 rotor at 4°C. The resulting pellets (containing the extracellular viruses) and cellular viral protein extracts from the parental cells were visualized by immunoblotting with rabbit anti-Gag antibodies (1/100) and peroxidase-conjugated antibodies, and revealed by enhanced chemiluminescence (Amersham). GFP-Gag fusions were analyzed by western blot using a polyclonal anti-GFP antibody (sc-8334, TEBU, 1/100). Prediction of the coiled-coil motifs was performed with the COILS program (http://www.ch.embnet.org/software/COILS_form.html).
Electron microscopy
Monolayers were fixed in situ with 1.6% glutaraldehyde (Taab Laboratory
Equipment, Reading, UK) in 0.1 M Sörensen phosphate buffer, pH 7.3-7.4
for 1 hour at 4°C. Cells were scraped from their plastic substratum and
centrifuged. The resulting pellets were successively post-fixed with 2%
aqueous osmium tetroxide for 1 hour at room temperature, dehydrated in ethanol
and embedded in Epon. Ultrathin sections were collected on 200-mesh copper
grids coated with Formvar and carbon, and stained with uranyl acetate and lead
citrate prior to observation with a Philips 400 transmission electron
microscope, at 80 kV, at 2800-36,000 x magnification.
![]() |
Results |
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Because Gag is the main component of capsids, we next assessed whether Gag by itself, in the absence of the viral genome and other viral proteins, could still reach the centrosome. For that purpose, a full-length Gag expression vector (pSG-Gag) was transfected into Cos6 cells and Gag localization was followed by confocal microscopy using polyclonal anti-Gag antibodies. 20 hours after transfection, Gag was mainly detected as a unique cytoplasmic dot, close to the nucleus (Fig. 2, left). Double labeling using a mAb (clone CTR910) directed against a centriolar component confirmed that this staining corresponds to the accumulation of Gag molecules around the centrosome (Fig. 2, left). Expression of other structural (Pol and Env) or regulatory (Tas and Bet) viral proteins never leads to a centrosomal staining (data not shown).
|
It has been recently shown that cytoplasm-replicating large DNA viruses
such as poxviruses and African swine fever virus (ASFV), assemble in
aggresomes (Heath et al.,
2001), a structure located close to the MTOC and generated in
response to high levels of misfolded proteins
(Johnston et al., 1998
;
Kopito, 2000
). Aggresome
formation induces a striking redistribution of the intermediate filament
protein vimentin into a characteristic halo-like cage around aggregates of
proteins (Kopito, 2000
). To
determine whether accumulation of Gag around the MTOC corresponded to the
formation of an aggresome owing to a high level of Gag expression in
transfected cells, Cos6 cells transfected with pSG-Gag were labeled with both
anti-Gag and anti-vimentin antibodies. As shown by fluorescence microscopy
(Fig. 2, right), the vimentin
network is not affected in Gag-expressing cells, demonstrating that
accumulation of Gag around the MTOC does not lead to the formation of an
aggresome.
Mapping of the minimal Gag sequence involved in MTOC targeting
Several functional domains have been already mapped along the HFV Gag
protein. To determine whether these domains (the C-terminal GR boxes and the
two recently characterized N-terminal regions responsible for Gag
self-assembly) are involved in MTOC targeting, the subcellular localization of
a full-length GFP-Gag fusion protein and of deletion mutants was investigated
(Fig. 3A). Cos6 cells were
transfected with these constructs and the GFP fluorescence was directly
observed 20 hours after transfection, following cell fixation. Although GFP
alone diffusely localizes in both the cytoplasm and the nucleus of transfected
cells, and never accumulates around the MTOC (data not shown), fusion of GFP
to the full-length Gag targets the fusion protein to the centrosome
(Fig. 3C). Serial deletions of
the C-terminus of Gag, up to residue 270 (mutant M1), do not impair MTOC
targeting of the corresponding GFP-Gag fusions. Deletion of amino acids
180-200 (mutant M3) or 200-250 (mutant M4) has no effect on MTOC targeting,
whereas deletion of amino acids 165-180 (mutant M5) totally abolishes
centrosomal targeting. Finally, GFP-Gag150-250 (mutant M2) is present around
this organelle. Taken together, these results suggest that the domain
implicated in MTOC targeting encompasses amino acids 150-180. This conclusion
is, however, clouded by the fact that the M6 mutant encoding for GFP-Gag1-201
is no longer detected at the centrosome. To assess whether the absence of MTOC
targeting of mutant M6 is due to inadequate positioning of the putative
implicated domain regarding the entire fusion protein, the first 200 amino
acids of Gag were fused to the N-terminus of GFP and transfected in Cos6
cells. In that case, the fusion protein Gag1-200-GFP (mutant M7) was detected
at the centrosome, suggesting that the domain involved in MTOC targeting must
be flanked with minimal amino acid sequences at both sides to be functional.
Taken together, these results indicate that proper centrosomal targeting does
not require the presence of the GR boxes or the domains involved in capsid
assembly, but necessitates the sequence between amino acids 150 and 180. All
constructions were controlled by sequence analysis and protein expression was
verified by western blot (Fig.
3B).
|
It is noteworthy that the N-terminus of HFV Gag harbors three putative
coiled-coil motifs as revealed by the COILS program
(Tobaly-Tapiero et al., 2001).
We have previously shown that the coiled-coil motif located between amino
acids 130 and 160 is crucial for Gag-Gag interaction
(Tobaly-Tapiero et al., 2001
).
Interestingly, the second coiled-coil encompassing amino acids 160-180
strikingly matches the domain required in MTOC targeting identified here. To
assess whether this motif is involved in centriolar targeting, we have
generated a GFP-Gag fusion harboring a mutation at position 171 exchanging a
crucial leucine into a glycine, disrupting the coiled-coil motif (mutant M8).
As shown in Fig. 3C, this
mutant is no longer able to reach the centrosome in transfected Cos6
cells.
Dynein-dynactin is required for centrosomal targeting of Gag
Retrograde movements along the MTs (minus-end-directed MT transport)
require the dynein motor complex, which is assisted by the 20S dynactin
cofactor (Karki and Holzbaur,
1999). The motor activity can be dislodged from the cargo by
overexpression of p50/dynamitin, a component of the dynactin complex,
disrupting their association (Burkhardt et
al., 1997
). This approach is widely used to demonstrate the
involvement of the dynein-dynactin complex in the movement of viruses
(Alonso et al., 2001
;
Dohner et al., 2002
;
Sodeik et al., 1997
;
Suomalainen et al., 1999
).
However, whereas dynamitin overexpression was reported to have no other effect
on the cytoskeleton in HeLa cells
(Burkhardt et al., 1997
), it
might affect MT organization and centrosome integrity in Cos cells
(Quintyne et al., 1999
).
Interestingly, it has been shown that overexpression of the central
coiled-coil domain (CC1) of p150Glued, the dynactin
sidearm subunit, has a more specific inhibitory effect on dynein-based
transport than does p50/dynamitin
(Quintyne et al., 1999
). Thus,
in our assays, HeLa cells and a CC1-expressing vector were used instead of
Cos6 cells and p50/dynamitin. As shown in
Fig. 4A, overexpression of
DsRed-CC1 did not affect the MT network, confirming previous studies. However,
the presence of CC1 prevented MTOC targeting of Gag in co-transfected HeLa
cells, whereas Gag was still associated with this organelle in cells
transfected with empty DS-Red vector. These observations demonstrate that
targeting of Gag to the MTOC requires the dynein-dynactin retrograde motor. In
all these experiments, we only analysed cells that contained evenly
distributed recombinant proteins, avoiding those (10% of the transfected
cells) that harbored large protein aggregates already reported when using
DsRed expressing vectors.
|
Finally, GFP-Gag transfected Cos6 cells were analysed by immunofluorescence following or not a treatment with nocodazole, 20 hours after transfection. As shown in Fig. 4B, Gag remains diffuse and punctate throughout the cytoplasm in treated cells, and is never detected at the MTOC under these settings. This observation suggests that the MT network is required to target the centrosome but also to stabilize Gag at the pericentrosomal region.
HFV Gag directly interacts with the cytoplasmic light chain of
dynein
Cytoplasmic dynein is a large multisubunit motor complex that is composed
of heavy, intermediate and light chains
(Karki and Holzbaur, 1999).
For lyssaviruses, a specific interaction has been demonstrated between the
dynein light chain 8 (LC8) and the P phosphoprotein, a component of the viral
ribonucleoprotein complex, providing a molecular basis for the axonal
transport of these neurotropic viruses
(Jacob et al., 2000
;
Raux et al., 2000
). To assess
whether HFV Gag could interact with LC8, we first examined their subcellular
distribution in co-transfected cells. Confocal microscopy revealed that
between 87% and 98% of Gag localized with GFP-LC8 at the MTOC, depending on
the experiment analysed (Fig.
5A), consistent with a possible interaction between the molecules,
whereas GFP-LC8 was distributed throughout the cytoplasm when expressed alone
(Fig. 5A)
(Alonso et al., 2001
;
Jacob et al., 2000
).
|
To investigate this interaction further, co-immunoprecipitation experiments
were performed following cell transfection. For this purpose, 293T cells were
transfected with a vector encoding Gag, GagL171G or LC8Flag, or
with a combination of these vectors. A plasmid encoding the first 575 amino
acids of Gag was used rather than the full-length molecule because the
full-length molecule is hardly immunoprecipitated by anti-Gag antibodies
(Tobaly-Tapiero et al., 2001).
Direct analysis of protein extracts from transfected cells using rabbit
anti-Gag antibodies revealed the presence of Gag575 and the L171G
mutant by western blot (Fig.
6B). By contrast, LC8Flag was detected by
immunoprecipitation with anti-Flag antibodies in the corresponding transfected
cells following metabolic labeling. These immune complexes were subsequently
analysed by western blot using anti-Gag antibodies, revealing the presence of
Gag575 in cells co-transfected with pLC8Flag and
pSG-Gag575, whereas no signal was detected in cells expressing
Gag575L171G and LC8Flag
(Fig. 6B). Similarly, following
the same procedure, an interaction with LC8 was detected when using the M2
mutant encoding GFP-Gag150-250, whereas no interaction was observed in cells
co-transfected with a vector encoding GFP-Gag deleted from amino acids 144-277
(mutant M9), which lacks the domain required to target the MTOC.
|
These results demonstrate a direct interaction between the cytoplasmic light chain of dynein LC8 and the structural HFV Gag protein.
Effect of the GagL171G mutation on the viral cycle
Finally, to assess the biological role of the coiled-coil motif between
amino acids 150 and 180 of HFV Gag in the virus cycle, a full-length viral
clone harboring the L171G helix-breaking point mutation was constructed, and
its behavior in tissue culture was compared with that of the parental
infectious proviral clone. Cos6 were transfected with the wild-type pHFV13,
pHFVGagL171G harboring the L171G mutation in the Gag ORF, pSG-Gag or pCgp9 (a
vector encoding for only HFV Gag and Pol viral proteins); 72 hours later,
viral protein expression in transfected cells and virus production in culture
supernatants were evaluated. As shown in Fig. 7, in cell extracts both
proviral clones express similar levels of Gag molecules, which are detected as
a doublet of 71 kDa and 68 kDa by polyclonal anti-Gag antibodies,
demonstrating that the L171G point mutation has no effect on Gag expression,
stability and maturation. Indeed, the 71 kDa band corresponds to the
full-length Gag precursor (p71), as visualized in pSG-Gag transfected cells,
whereas the 68 kDa product represents its main cleavage product (p68),
mirroring the presence of an active viral protease
(Giron et al., 1997). To
assess whether the Gag mutation affected virus egress, supernatants from
transfected cells were ultracentrifuged on a sucrose gradient and the
resulting pellets, which contained extracellular virions were analysed by
western blot using polyclonal anti-Gag antibodies. In that case, no major
difference was observed between pHFV13 and pHFVGagL171G, demonstrating that
virus egress was not impaired by this helix-breaking point mutation in Gag. No
viral particles were produced from neither pSG-Gag- or pCgp9-transfected cells
as expected because, in contrast to other retroviruses, FV particle formation
requires the presence of the homologous envelope
(Pietschmann et al.,
1999
).
Finally, virus production in culture supernatants was evaluated following
infection of an indicator cell line (the FAG cells), which harbor the GFP ORF
under the control of the HFV LTR as already described
(Tobaly-Tapiero et al., 2001).
Direct fluorescence observed 48 hours after infection reveals a significant
virus production from pHFV13 transfected cells (9x104 FCFU
ml1), whereas the Gag mutation dramatically reduced
infectivity of the corresponding provirus but did not totally abolished it
(6.2x103 FCFU ml1). Values are averages of
three independent experiments.
Altogether, our data demonstrate that the L171G mutation in the Gag ORF has no effect on virus egress but drastically reduces infectivity of the corresponding proviral clone.
![]() |
Discussion |
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By EM, we show here that incoming HFV proteins concentrate in the
pericentrosomal region as free and structured capsids close to MTs. More viral
capsids surround the MTOC than expected from the multiplicity of infection
used. Indeed, most extracellular retroviral particles produced during an
infection harbor a defective genome and are therefore unable to transactivate
the viral LTR of GFP-indicator cells
(Tobaly-Tapiero et al., 2001).
In the case of HIV-1, it has been reported that infectivity to particle ratio
is as low as 1 in 60,000 (Kimpton and
Emerman, 1992
; Piatak et al.,
1993
), whereas a more recent study softened this conclusion by
showing a ratio up to 1 infectious particle in 10
(Andreadis et al., 2000
).
Furthermore, we demonstrate that the determinants required to reach the
pericentrosomal area are harbored by the scaffold component of viral capsids,
the structural Gag protein, because this protein alone targets the centrosome
in transfected cells in the absence of other viral partners.
Because the vimentin network is not affected in Gag-transfected cells,
accumulation of Gag around the MTOC does not trigger the formation of an
aggresome. However, similar to the formation of this inclusion body
(Garcia-Mata et al., 1999),
Gag transport towards the MTOC involves the retrograde dynein-dynactin motor,
because it is prevented by overproduction of CC1, the central coiled-coil
domain of p150Glued, reported to disrupt the function of
this retrograde motor. Other viruses have been shown to use the MT network for
their intracellular transport. For instance, adenovirus type 2 (Ad2) and HSV,
two nuclear replicative DNA viruses entering the cell through
receptor-mediated endocytosis and direct membrane fusion respectively, are
transported as naked capsids towards the nucleus via the MT network
(Sodeik et al., 1997
;
Soll, 1997
). For ASFV and
rabies viruses, two other viruses that use the MT network to move within the
infected cell, an interaction with LC8 forms a molecular basis of this
trafficking (Alonso et al.,
2001
; Jacob et al.,
2000
; Raux et al.,
2000
). The interaction between LC8 and HFV Gag described here
could play such a role in the case of FVs.
LC8 was first identified as an integral component of the Chlamydomonas
reinhardtii outer dynein arm (Pfister
et al., 1982; Piperno and
Luck, 1979
). Since then, this evolutionarily conserved molecule
has been shown to interact with many cellular complexes, such as the nitric
oxide synthase (Jaffrey and Snyder,
1996
) and myosin V (Goode et
al., 2000
; Naisbitt et al.,
2000
), an actin-based motor mainly located at the plasma membrane
that shuttles between the cell periphery and the MTOC along the MT network
(Espreafico et al., 1998
;
Tsakraklides et al., 1999
).
LC8 is encoded by at least two genes in humans
(Naisbitt et al., 2000
),
leading to the synthesis of dynein light chain 2 (DLC2) and dynein light chain
1 (DLC1), the isoform used in our work. Interestingly, myosin V has been shown
to interact with DLC2 but not with DLC1
(Naisbitt et al., 2000
;
Puthalakath et al., 2001
).
Thus, migration of HFV capsids towards the centrosome on the microtubule
network probably involves an interaction with dynein LC8 (DLC1) rather than
myosin V LC8 (DLC2). In that sense, HFV efficiently targets the centrosomal
region and replicates in myosin-V-deficient melanoma S91-6 cells (E.
Espreafico and A. Saïb, unpublished). However, we cannot exclude the
possibility that HFV Gag also interacts with DLC2 as a component of myosin V,
favoring its trafficking on actin beneath the plasma membrane devoid of
microtubules. Therefore, interaction between incoming FV capsids and the
multifunctional LC8 could provide a bridge to shuttle between an actin-based
motor beneath the plasma membrane and the MT network within the cytoplasm.
Remarkably, a similar MT- and dynein-dependent trafficking towards the
centrosome was recently described for incoming HIV-1, although the viral and
cellular protagonists involved in this retrograde transport were not
determined (McDonald et al.,
2002
). Nevertheless, these observations suggest that distinct
classes of retroviruses might tether the dynein-dynactin complex motor on the
MT network during the early stage of infection to reach the nucleus, opening
new perspectives in the development of anti-retroviral drugs.
Analysis of the subcellular distribution of Gag deletion mutants reveals
that the GR boxes as well as the determinants involved in capsid assembly (the
Gag-Gag interaction domain and the CTRS) are not required to target the
centrosome, whereas amino acids 150-180 are necessary to reach this organelle.
This region does not harbor any obvious similarities with the two previously
described LC8-interacting motifs, (K/R)XTQT and GIQVD
(Lo et al., 2001;
Rodriguez-Crespo et al.,
2001
), but a coiled-coil motif conserved in all primate foamy
viruses (Tobaly-Tapiero et al.,
2001
) seems to be involved in centriolar targeting of Gag. In that
sense, a single point mutation (L171G) in the Gag ORF, leading to the
disruption of the coiled-coil motif, abolishes MTOC targeting of the
corresponding GFP-Gag fusion and renders a full-length viral clone
non-infectious. Indeed, whereas Gag assembly and virus release are not
affected by this helix-breaking point mutation, productive infection of naive
cells is dramatically reduced. Because this mutation was shown to prevent
Gag/LC8 interaction, tethering of the dynein retrograde motor following its
entry into the cytoplasm might be impaired, a hypothesis that we are currently
exploring. The L171G point mutation does not totally abolish infectivity,
suggesting that other trafficking pathways might be used by FVs to reach the
center of the cell. In that sense, intracellular trafficking involving the
actin cytoskeleton might account for this observation, an alternative route
already put forward for HIV-1 (McDonald et
al., 2002
) and previously suggested for FVs because Gag was also
shown to interact with actin (Giron et
al., 1997
).
HFV Gag is not the unique partner of LC8 lacking the two characterized
LC8-interacting motifs. Interestingly, all these proteins harbor an
-helical coiled-coil in the domain identified as the LC8-binding
region. For example, the LC8-interacting domain of myosin V is predicted to
fold into an amphiphilic helix (Naisbitt
et al., 2000
) and, similarly, the signal response domain of
I
B
, which was shown to interact with LC8
(Crepieux et al., 1997
), folds
into a coiled coil as revealed by the COILS program (data not shown). Such a
hypothesis might also apply to the neuronal form of nitric oxide synthase
(nNOS) (Benashski et al.,
1997
). Given that LC8 harbors a similar amphiphilic helix
triggering its multimerization (Benashski
et al., 1997
), interactions between LC8 and myosin V,
I
B
, nNOS and HFV Gag might occur via such specific protein motif
interface, constituting a new mode of contact with LC8.
One remaining issue is how the viral genome, wrapped by Gag molecules, is
imported into the nucleus. Interestingly, the dynein-dynactin complex has
recently been shown to associate with the nuclear envelope, facilitating the
remodeling of this physical frontier
(Busson et al., 1998;
Salina et al., 2002
).
Therefore, interaction of Gag with the dynein-dynactin complex could not only
drive incoming viral capsids towards the centrosome but also be involved in
their subsequent targeting to the nuclear envelope. Yet, HFV capsids were
never detected within the nucleus, nor close to nuclear pores, even later
during the replication cycle [here and Fischer et al.
(Fischer et al., 1998
)],
whereas unassembled Gag proteins and the viral genome are detected in the
nucleus early after infection (Saib et
al., 1997
; Schliephake and
Rethwilm, 1994
). Therefore, in contrast to Ad2 or HSV, those
capsids dock to the nuclear pore triggering nuclear translocation of the viral
genome (Greber et al., 1997
;
Ojala et al., 2000
;
Trotman et al., 2001
), nuclear
import of HFV Gag and genome must be accompanied by disassembly or significant
deformation of the core particle at the MTOC.
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Acknowledgments |
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