Department of Zoology, University of British Columbia, 6270 University Boulevard, Vancouver, BC, Canada V6T 1Z4
Correspondence
Nelly Panté
pante{at}zoology.ubc.ca
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ABSTRACT |
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INTRODUCTION |
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Viruses have evolved various strategies to gain entry to the nucleus. Most viruses enter through the NPCs, using specific nuclear localization sequences (NLSs) in viral proteins that bind cellular nuclear transport receptors (importins or karyopherins). The viral proteinimportin complex then docks at the cytoplasmic face of the NPC and is actively transported into the nucleus; in contrast, many retroviruses enter the nucleus only during mitosis, when the membrane barrier is temporarily absent (reviewed by Smith & Helenius, 2004; Whittaker et al., 2000
).
At approximately 26 nm in diameter, parvoviruses are among the smallest known viruses (Llamas-Saiz et al., 1997; Tsao et al., 1991
). They are non-enveloped, with a single-stranded DNA genome of
5 kb, and replicate in the nucleus of actively dividing cells during S phase (Muzyczka & Berns, 2001
). Thus, transport into the host nucleus is necessary for productive infection. Entry of parvoviruses into the host cell has been studied, as have the early stages of virus trafficking (reviewed by Vihinen-Ranta et al., 2004
). Parvoviruses enter cells by receptor-mediated endocytosis. Several cell surface receptors have been identified for different parvoviruses and the uptake process appears to involve clathrin-coated vesicles (Bartlett et al., 2000
; Parker & Parrish, 2000
). Despite these important advances in the parvovirus cellular entry pathway, very little is known about nuclear import of parvoviruses.
Immunofluorescence experiments either with fluorescently labelled virus or with antibodies against capsid proteins have shown that the capsid enters the nucleus (Bartlett et al., 2000; Seisenberger et al., 2001
; Vihinen-Ranta et al., 2000
). However, these studies lack the resolution necessary to visualize whether the virus particles entering the nucleus are partially disassembled. For example, it is possible that the capsid disassembles in the cytosol and that the fluorescent signal seen in the nucleus comes from fluorescent-labelled capsid proteins. Nevertheless, it has been largely assumed that the intact parvovirus enters the nucleus through the NPC. In contrast, a single study has suggested that for adeno-associated virus (AAV), nuclear entry occurs independently of the NPC (Hansen et al., 2001
). This suggestion is based on experiments in which AAV was incubated with purified nuclei in the absence of factors necessary for NPC-mediated import and in the presence of wheat germ agglutinin (WGA), a lectin that is known to bind to the NPC and inhibit nuclear import (Dabauvalle et al., 1988
; Finlay et al., 1987
). WGA did not inhibit nuclear import of AAV, suggesting that the NPC is not necessary for virus entry. However, an alternative mechanism has not been defined.
Despite limited knowledge of the mechanisms of parvovirus nuclear import, some work has been done on nuclear import of parvoviral capsid proteins. Parvoviral capsids consist of 60 copies of three proteins, VP1 (83 kDa), VP2 (64 kDa) and VP3 (60 kDa) (Muzyczka & Berns, 2001). VP2 is the major structural protein composing
90 % of the capsid. VP1 and VP2 are expressed from overlapping reading frames and thus the two proteins are identical in amino acid sequence except for the first 142 residues of VP1 (Weichert et al., 1998
). Several potential NLSs are located in the N-terminal sequence of VP1 and one of these is able to mediate the nuclear import of a heterologous protein (Vihinen-Ranta et al., 1997
). Thus, it has been suggested that VP1 may mediate nuclear import of the parvovirus. However, the N-terminal sequence of VP1 is enclosed within the capsid (Cotmore et al., 1999
) and therefore it is not clear how it would mediate nuclear import of the virus. Similarly, a sequence directing nuclear import of VP2 has been identified (Lombardo et al., 2000
; Pillet et al., 2003
). Because newly synthesized capsid proteins must reach the nucleus to permit assembly of new virus particles, the NLSs on VP1 and VP2 may be involved only in the nuclear import of newly synthesized capsid proteins and not in the nuclear import of intact capsids. Thus, additional work is required to determine whether these sequences serve as signals directing not only nuclear import of the capsid proteins, but nuclear import of the intact capsids.
To investigate the mechanism of parvovirus nuclear transport, the parvovirus Minute virus of mice (MVM) was microinjected into the cytoplasm of Xenopus oocytes and its nuclear uptake was followed by electron microscopy (EM). Our data indicate that MVM causes small breaks in the NE that are able to support nuclear import of protein in oocytes where the NPCs were blocked with WGA. A mechanism of parvovirus nuclear entry is proposed that involves disruption of the NE and import through the resulting breaks.
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METHODS |
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Mature (stage VI) oocytes were surgically removed from narcotized Xenopus laevis as described by Panté (2006). Oocytes were washed three times with modified Barth's saline buffer [MBS: 88 mM NaCl, 1 mM KCl, 0·82 mM MgSO4, 0·33 mM Ca(NO3)2, 0·41 mM CaCl2, 10 mM HEPES, pH 7·5] and defolliculated by treatment with collagenase (5 mg ml1; Sigma) in calcium-free MBS for 1 h. Defolliculated oocytes were washed three times with MBS and stored in MBS.
Oocyte microinjection.
Micropipettes (Microcaps; Drummond) were heated and pulled using a micropipette puller (Inject-Matic). Microinjection of Xenopus oocytes was performed as described by Panté (2006) using an oocyte microinjector (Inject-Matic). Oocytes were injected with about 100 nl purified MVM in the cytoplasm at the transitional zone between the animal and vegetal hemispheres. As control experiments, oocytes were mock-injected with 100 nl PBS.
For time-dependence experiments, oocytes were microinjected with purified MVM at a cellular concentration of 9·8x104 p.f.u. ml1 and then incubated at room temperature in MBS for 30 min, 1 h or 2 h. For concentration-dependence experiments, oocytes were microinjected with purified MVM at a cellular concentration of 9·8x101, 9·8x103 or 9·8x104 p.f.u. ml1 and then incubated at room temperature in MBS for 1 h.
For inhibition of nuclear import through the NPC, oocytes were cytoplasmically microinjected with 50 nl WGA (20 mg ml1; Sigma) and incubated at room temperature for 6 h. After this incubation period, oocytes were microinjected with purified MVM at a cellular concentration of 9·8x104 p.f.u. ml1 and then incubated at room temperature in MBS for 2 h. Inhibition of nuclear import by WGA was confirmed in control oocytes that were microinjected with WGA, incubated as above and then microinjected with an import-competent substrate instead of MVM. This substrate was BSA cross-linked with synthetic Simian virus 40 large T antigen NLS peptide (CGGGPKKKRKVED) and conjugated to colloidal gold (BSA-NLSg). The BSA-NLS was custom made by Sigma Genosys. Both colloidal gold particles (diameter 8 nm) and NLS-BSAg were prepared as described by Panté (2006)
.
Preparation of injected oocytes for EM.
After microinjection and incubation at room temperature for the indicated time, oocytes were prepared for embedding and thin-section EM following the protocol of Panté (2006). Briefly, oocytes were first fixed overnight at 4 °C with 2 % glutaraldehyde in MBS. The following day, oocytes were washed with MBS and their animal hemispheres were dissected and fixed with 2 % glutaraldehyde in low-salt buffer (LSB: 1 mM KCl, 0·5 mM MgCl2, 10 mM HEPES, pH 7·5) for 1 h at room temperature. Dissected oocytes were washed with LSB, embedded in 2 % low-melting agarose and post-fixed with 1 % OsO4. Fixed oocytes were sequentially dehydrated in ethanol and embedded in Epon 812 (Fluka) as described by Panté (2006)
.
Thin sections (50 nm) through the NE were cut on a Leica Ultracut Ultramicrotome (Leica Microsystems) using a diamond knife (Diatome). Sections were placed on phalloidin/carbon-coated copper EM grids and stained with 2 % uranyl acetate for 30 min and 2 % lead citrate for 5 min. Micrographs were digitally recorded with a Hitachi-7600 transmission electron microscope.
Organelle isolation and assay.
Rat liver nuclei were isolated as previously described (Gerace et al., 1978). Approximately 8x106 rat liver nuclei were incubated for 2 h at room temperature in LSB with MVM at a ratio of 1 p.f.u. per nucleus. After incubation, nuclei were centrifuged at 16 000 g for 5 min. The resulting pellet was fixed and prepared for thin-section EM as described above.
Mitochondria were purified from HeLa cells using a kit from Sigma. Briefly, cells were homogenized in extraction buffer (10 mM HEPES, 200 mM mannitol, 70 mM sucrose, 1 mM EGTA, pH 7·5) by 25 strokes in a 3 ml homogenizer, to achieve 80 % lysis. Homogenization was followed by low- (600 g) and high-speed (11 000 g) centrifugation at 4 °C. The pellet from the high-speed centrifugation contained mitochondria, which were then stored in storage buffer (10 mM HEPES, 250 mM sucrose, 1 mM ATP, 0·08 mM ADP, 5 mM sodium succinate, 2 mM K2HPO4, 1 mM DTT, pH 7·5).
Mitochondria from approximately 7·2x106 cells were incubated for 2 h at room temperature in storage buffer with MVM at a ratio of 1 p.f.u. per cell. After incubation, mitochondria were pelleted and prepared for EM as described above.
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RESULTS |
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In contrast to oocytes microinjected with other import substrates that have shown the substrate crossing the NPC (Görlich et al., 1996; Panté & Aebi, 1996
; Panté & Kann, 2002
; Rabe et al., 2003
; Rollenhagen et al., 2003
), MVM was not seen in transit through NPCs. Instead, it was observed that at 1 h post-injection (p.i.), the virus caused small breaks (
100200 nm) in the NE (Fig. 1
). Damage was predominantly to the ONM and was often near the NPCs (Fig. 1b
; breaks marked with *). MVM particles, identified by their round shape, diameter and dense appearance, appeared to be associated with the ONM and close to where the ONM was disrupted (Fig. 1b
, breaks marked with **, and 1d, arrows). Occasionally, virus particles were observed between the ONM and INM, in close proximity to breaks in the ONM (Fig. 1c
, arrows). Unusual membrane structures associated with the NE were also observed; for example, vesicles between the ONM and INM (Fig. 1e
and Fig. 3d
, arrows).
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NE damage induced by MVM is concentration-dependent
In addition to being time-dependent, damage to the NE was also concentration-dependent (Fig. 3). Oocytes were injected with MVM at cellular concentrations of 9·8x101, 9·8x103 or 9·8x104 p.f.u. ml1, incubated at room temperature for 1 h and prepared for EM. As illustrated in Fig. 3
, it was found that damage increased with virus concentration. Membrane damage was quantified as described above for time-dependence experiments: the mean break length, frequency of breaks, and proportion of NE damaged in mock injections and oocytes injected with different concentrations of MVM are represented in Fig. 3(e and f)
. Because of the 1 h incubation period, damage was usually restricted to the ONM for the different virus concentrations. As shown in the bar graph in Fig. 3(e)
, the length and frequency of breaks increased with the intracellular virus concentration. Although the mean break length was similar in oocytes injected with 9·8x101 and 9·8x103 p.f.u. ml1, the frequency of breaks increased in oocytes injected with 9·8x103 p.f.u. ml1. Similarly, the proportion of NE damaged increased with virus concentration.
NE damage induced by MVM is independent of the NPC
To determine whether the NPC is necessary for nuclear import of MVM, MVM was microinjected into oocytes after blocking the NPCs with WGA. Oocytes were pre-injected with WGA (20 mg ml1), incubated for 6 h, microinjected with MVM, incubated for 2 h and prepared for thin-section EM. Import-competent NLS-BSAg was microinjected into the cytoplasm of control oocytes to confirm inhibition of nuclear import by WGA (Fig. 4a). When BSA-NLSg was microinjected into Xenopus oocytes in the absence of WGA, gold particles were observed passing through the NPC and in the nucleus (data not shown). However, in oocytes pre-injected with WGA, BSA-NLSg was observed near the NPCs in range of the NPC filaments (Fig. 4a
, arrows), but was not observed passing through the NPCs or in the nucleus. These observations are in agreement with similar results reported by Panté & Aebi (1996)
and effectively demonstrate that the NPCs were blocked by WGA.
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MVM does not induce damage in all intracellular membranes
Next, the question of whether MVM affects other intracellular membranes in addition to the NE was investigated. In Xenopus oocytes there are many mitochondria in the cytoplasm, which are structurally well preserved for EM. Damage to mitochondrial membranes in oocytes microinjected with MVM was not observed; in fact, perfectly intact mitochondrial membranes were observed directly next to NE exhibiting severe damage (Fig. 6). However, to further investigate this question, the effects of MVM on purified rat liver nuclei and on mitochondria purified from HeLa cells were examined.
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DISCUSSION |
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Traditionally, crossing the NPC has been considered the only way for nuclear import substrates to enter the nucleus. Evidence has shown that several viruses that replicate in the nucleus also use the NPC to gain access to the nucleus (reviewed by Izaurralde et al., 1999; Smith & Helenius, 2004
; Whittaker et al., 2000
). However, our data show that MVM is an exception. Two possible ways to enter an intact nucleus from the cytoplasm without crossing the NPC can be envisioned. The first is by passing through the ONM and INM using a budding mechanism similar to pinocytosis. Such a pathway has been observed in nuclear export of herpesviruses (Granzow et al., 2001
), but has not been observed as a means of nuclear import of any virus or any other import substrate. In the case of herpesviruses, after assembly in the nucleus, the capsids bud through the INM and acquire an envelope. In this process, the INM is left intact and enveloped viruses are seen in the perinuclear space. This does not seem to be the case for parvoviruses, since clear breaks in the ONM of MVM-injected oocytes were found and enveloped MVM particles were not observed in the perinuclear space.
The second possible mechanism for nuclear import that does not involve the NPC is disruption of the NE and entering the nucleus through the breaks. This is the mechanism that we favour for MVM. Ruptures of the NE to allow nuclear import have also been reported for the human immunodeficiency virus (HIV) protein R (Vpr) (de Noronha et al., 2001). Vpr can induce transient herniations in the NE that burst, creating breaks in the NE (de Noronha et al., 2001
); it is thought that the pre-integration complex of HIV is then imported into the nucleus through these breaks (Segura-Totten & Wilson, 2001
). How Vpr induces herniations is not well understood, but it is thought that after Vpr enters the nucleus through the NPC it weakens the NE at a few sites (Segura-Totten & Wilson, 2001
). Thus, it is likely that NE damage starts at the INM and not at the ONM as for MVM.
Our results have revealed interesting characteristics of the distinct mechanism used by MVM to enter the nucleus. The NE damage induced by MVM seems to be independent of the NPC, since breaks are still observed in vivo in oocytes microinjected with MVM in the presence of WGA. This is consistent with in vitro studies showing that WGA does not inhibit nuclear uptake of AAV in purified nuclei (Hansen et al., 2001). That BSA-NLSg gained entry to the nucleus in oocytes where the NPCs were blocked when MVM was co-injected demonstrates that NE breaks induced by MVM facilitate nuclear import in an NPC-independent manner. Our data reinforce the conclusion that parvoviruses do not require the NPC for nuclear import, but rather use a mode of virus entry to the nucleus that is different to that used by any other group of viruses known to date. An NPC-mediated mechanism of nuclear import for parvoviruses cannot be ruled out, but if this exists it is as one of multiple entry pathways. Since virus particles were never observed crossing the NPC in microinjected oocytes throughout the course of our EM work, we believe that entry through disruptions in the NE is the main nuclear import route of MVM.
In addition to being independent of the NPC, damage to the NE is also observed when MVM is incubated with purified rat liver nuclei, suggesting that cytosolic factors are not necessary for MVM to cause breaks in the NE. Moreover, MVM does not affect mitochondrial membranes, indicating that this mechanism is specific to certain intracellular membranes, including the NE and perhaps other similar membranes. The basis of this specificity remains to be determined.
The unconventional MVM nuclear import route could be explained if MVM capsid proteins have membrane-lytic properties. Membrane-lytic peptides have been discovered in insects, amphibians and mammals, and may lyse prokaryotic or eukaryotic membranes (reviewed by Shai, 1999). Although the mechanism is not completely understood, it is thought that membrane-lytic peptides work either by inserting themselves into the membrane and creating hydrophilic pores or by interacting with phosphate head groups to disrupt membrane curvature, resulting in membrane lysis (Shai, 1999
). It has also been shown that Vpr has membrane-lytic properties and can effectively permeabilize membranes (Coeytaux et al., 2003
). It is possible that both MVM and Vpr cause NE breaks by directly lysing the NE, thereby facilitating nuclear entry. Thus, membrane-lytic peptides may play a previously unrecognized role in the nuclear import of diverse groups of viruses, including DNA and retroviruses.
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ACKNOWLEDGEMENTS |
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Received 12 February 2005;
accepted 31 August 2005.
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