1 Istituto Pasteur-Fondazione Cenci Bolognetti, Dipartimento di Biotecnologie Cellulari ed Ematologia, Sezione di Genetica Molecolare, Università di Roma La Sapienza, Viale Regina Elena 324, 00161 Rome, Italy
2 e Dipartimento di Medicina Sperimentale e Patologia, Sezione di Virologia, Università di Roma La Sapienza, Viale Regina Elena 324, 00161 Rome, Italy
Correspondence
Marie-Isabelle Garcia
garcia{at}bce.uniroma1.it
Paolo Amati
amati{at}bce.uniroma1.it
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ABSTRACT |
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M. Caruso and M. Cavaldesi contributed equally to this work.
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INTRODUCTION |
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A major role of SA moieties in virus infection has been documented extensively in murine polyomavirus (MPyV), a double-stranded DNA tumour virus (Eckhart, 1990). Both MPyV binding and infectivity are greatly reduced when the total amount of SA residues available on host cell membrane is: (i) decreased by treatment with neuraminidase or use of N-glycosylation inhibitors (Chen & Benjamin, 1997
; Herrmann et al., 1997
; Caruso et al., 2003
); (ii) metabolically modified by the use of synthetic SA precursor analogues carrying elongated N-acyl side chains (Herrmann et al., 1997
); or (iii) blocked by lectins used as competitors (Chen & Benjamin, 1997
). Indeed, all MPyV strains recognize N-linked glycoproteins with oligosaccharides terminating in
(2,3)-linked SA as receptors, through the major coat protein VP1. In addition, MPyV strains carrying a Gly in position 91 of VP1 are also able to bind branched oligosaccharides that carry a second
(2,6)-linked SA (Fried et al., 1981
; Cahan et al., 1983
; Chen & Benjamin, 1997
). The crystal structure of the major coat protein VP1 in complex with sialyllactose has been solved at 1·9 Å resolution (Stehle et al., 1994
; Stehle & Harrison, 1996
, 1997
). As a result of these studies, the sialylated receptor has been predicted to bind to pre-formed pockets on the viral capsid surface. Pockets 1 and 2 accommodate the terminal SA and the penultimate
(2,3)-linked galactose, respectively, and pocket 3 accommodates a second
(2,6)-branched SA (Stehle & Harrison, 1996
, 1997
). Five residues (Y72, R77, G78, N93 and H298), mapped within the outfacing BC and HI loops of VP1, appear to be important for SA interactions, since they establish hydrogen bonds, salt bridges or hydrophobic interactions with the oligosaccharide (Stehle et al., 1994
; Stehle & Harrison, 1996
, 1997
). On the basis of these structural data, Bauer et al. (1999)
generated MPyV PTA mutant strains carrying single substitutions in each of these VP1 residues to abrogate such bonds. Transfection of mouse fibroblast cells with the mutated genomes, in contrast to transfection with the wt genome, did not result in any visible cytopathic effect even at 3 weeks post-transfection (p.t.), suggesting that each of these VP1 residues was crucial for MPyV infectivity; however, the reason for the non-infectivity of such mutants was not investigated (Bauer et al., 1999
). In particular, the precise effects of these mutations on the stability of the virus particles potentially formed after transfection and on their cell binding and entry as well as viral gene expression properties were not analysed.
In the past, several attempts to isolate and identify candidate MPyV receptor molecules have been unsuccessful (Griffith & Consigli, 1986; Marriott et al., 1987a
, b
; Bauer et al., 1999
). Recently, we demonstrated that the
4
1 integrin acts as a cell receptor for MPyV in fibroblast cells, probably by recognizing as a ligand the consensus LDV motif present in the DE loop of VP1. The interaction of MPyV with
4
1 integrin appears to be important for MPyV infectivity at a post-attachment level, since function-blocking antibodies directed against the ligand-binding site of the integrin block MPyV infectivity without affecting MPyV cell binding (Caruso et al., 2003
).
The aim of the present work was to study further the function of SA residues as MPyV cell receptors and the potential relationship between SA-containing receptors and 4
1 integrin. For this purpose, two isogenic VP1 virus mutants of the MPyV A2 large plaque strain, designated MPyV R77Q and H298Q, were constructed and their infectious properties were analysed in murine fibroblast cells. Binding properties of the corresponding wt or mutant virus-like particles (VLPs) were also studied by FACS analysis in cells overexpressing or not expressing the
4
1 integrin in both SA-positive and SA-deficient backgrounds.
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METHODS |
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To generate VP1 pseudocapsids, a 1·6 kb EcoRVXbaI fragment (nt 41102524) from the wt, R77Q or H298Q genomes of MPyV was introduced into the linearized StuIXbaI pFASTBAC1 cloning vector (Life Technologies). The cloned VP1 genes were then transferred by transposition to the Bacmid vector, as recommended by the manufacturer.
Cells.
Mouse fibroblast 3T6 and Swiss 3T3 cells were grown in DMEM supplemented with 10 % FCS (Gibco) in a 5 % CO2 atmosphere at 37 °C. Mock- or 4-transfected BALB/c 3T3 cells were grown in DMEM supplemented with 10 % FCS in the presence of 1·3 µg puromycin ml-1, as described previously (Caruso et al., 2003
). Lec-2 cells, defective in SA expression, and the parental cell line Pro-5 (expressing SA), both originating from a Chinese ovary hamster (CHO) cell line, were grown as monolayers, as described previously (Stanley et al., 1975
; Stanley & Siminovitch, 1977
). Pro-5 and Lec-2 cells were transfected with the empty or recombinant pRK5 vector carrying the entire cDNA of the murine
4 integrin subunit (Rietzler et al., 1998
) and the plasmid pBabe-Puro containing the puromycin-resistance gene as a selection marker (Morgenstern & Land, 1990
) using the Lipofectamine Plus reagent (Invitrogen). At 2 days after transfection, cells were split 1 : 5 in DMEM supplemented with 5 µg puromycin ml-1. Surviving colonies were pooled and amplified.
Insect Sf9 cells were grown as monolayers in SF900II medium (Gibco) supplemented with 10 % de-complemented FCS (Gibco) at 27 °C.
Viruses and VPLs.
To produce MPyV particles, fibroblast 3T6 cells were transfected with the re-ligated wt or mutant genomes, as described previously (Garcia et al., 2000), using the DEAE/dextran procedure (McCutchan & Pagano, 1968
). Cells were lysed 72 h p.t. by repeated freeze-thawing and centrifuging for 15 min at 8000 g. The resulting supernatant was collected and virus particles were then concentrated by centrifugation through a 20 % sucrose cushion in buffer B [150 mM NaCl, 10 mM Tris/HCl (pH 7·4) and 0·01 mM CaCl2]. For infection experiments, titration of non-infectious virus mutants MPyV R77Q and H298Q was performed by comparing their concentration in the supercoiled form I DNA molecules with that of wt MPyV particles titrated previously by plaque assay. For lipofection experiments, virus particles were purified by both CsCl and sucrose gradients, as described previously (Caruso et al., 2003
), and Lipofectamine Plus reagent was used according to the manufacturer's instructions.
To produce recombinant baculovirus particles, Sf9 cells were transfected with recombinant BacmidVP1 plasmids using CellFectin (Gibco). Initial transfection lysates were used to subsequently infect cells in order to increase baculovirus titres. Production and purification of VLPs were by CsCl and sucrose gradients, as described previously (Forstovà et al., 1995).
For cell-binding assays, VLPs or wheat germ agglutinin (WGA; Sigma) were biotinylated as follows: 15 µg protein was incubated with 1 mM biotin (Pierce) in a 50 mM Na2CO3 buffer (pH 8·5) for 30 min at room temperature and then dialysed extensively against buffer B. The efficiency of biotinylation was tested by Western blotting, as described previously (Caruso et al., 2003).
Negative staining.
Virus, viruslipofectamine complexes or VLP preparations were adsorbed to 400 mesh Formar carbon-coated copper grids and stained with 1 % uranyl acetate, pH 4·5. The preparations were observed under a Philips CM10 electron microscope operating at 80 kV.
Haemagglutination assays.
A 5 % suspension of sheep red blood cells (Sclavo Diagnostics) was washed three times in PBS just before use and adjusted to a final concentration of 0·4 % in PBS. Cells were added (100 µl) to 100 µl serially diluted VLPs in 96-well, round-bottomed culture dishes (Falcon). Haemagglutination was read after an incubation time of at least 4 h at 4 °C.
Virus replication and gene expression analysis.
For virus replication assays, total cellular DNA was obtained by cell lysis in 10 mM Tris/HCl (pH 8·0), 0·1 M EDTA, 0·5 % SDS and 200 µg proteinase K ml-1 and subsequent phenol/chloroform extraction. DNA was digested with EcoRI/MboI and restriction fragments were separated by electrophoresis in 1 % agarose gels and transferred to Gene Screen membranes (NEN Life Science Products), as recommended by the manufacturer. Filters were hybridized with the whole MPyV genome linearized by EcoRI, as described previously (Sambrook et al., 1989).
For viral gene expression assays, total cellular RNA was isolated from virus infected- or virus-lipofected cells by the method of Chomczynski & Sacchi (1987) and transferred to Gene Screen membranes. Filters were hybridized with the NdeIEcoRI (nt 15622738) and AseI (nt 29253835) fragments specific for early and late genes, respectively, as well as with the
-actin-specific probe. The
-actin-specific probe was generated by PCR on 3T3 cellular DNA extracts using the following primers: V94 (forward, 5'-atggatgacgatatcgctgcg-3') and V95 (reverse, 5'-atcttcatgaggtagtctgtcagg-3'). Autoradiography was carried out with Fuji photo films at -70 °C.
FACS analysis.
Cell monolayers were detached with PBS/5mM EDTA and washed with binding buffer BB (PBS, 0·1 mM CaCl2, 0·05 mM MgCl2 and 1 % BSA).
For integrin subunit cell surface expression, 4x105 cells were incubated with 0·5 µg monoclonal antibodies directed to the integrin subunits (CD49d, clone R1-2 for the 4 subunit, or CD29, clone HM
1-1 for the
1 subunit; BD Pharmigen) for 1 h on ice in buffer BB. Cells were washed twice in buffer BB and subsequently incubated with secondary R-phycoerythrin (PE)-conjugated anti-rat or anti-hamster antibodies (ICN) for 30 min on ice.
For cell-binding assays, 4x105 cells were incubated with biotin-labelled VLPs or WGA in buffer BB for 1 h on ice. Cells were washed twice in buffer BB and incubated with R-PE- or FITC-conjugated streptavidin (BD Pharmigen) for 30 min on ice.
After three final washes in buffer BB, cells were fixed in 4 % paraformaldehyde and their fluorescence intensity analysed on a FACS Calibur flow cytometer (Becton Dickinson) using the CELL QUEST software.
Western blotting.
Cells were lysed for 1 h at 4 °C in 50 mM Tris/HCl (pH 7·4), 1 % Triton X-100, 150 mM NaCl, 2mM CaCl2 and protease inhibitors (1 mM PMSF, 10 µg leupeptin ml-1 and 10 µg aprotinin ml-1). After centrifugation at 13 000 r.p.m. for 10 min at 4 °C, the protein content of each sample was quantified using the Bio-Rad Protein Assay reagent. A total of 300 µg protein was treated with or without 100 mU Clostridium perfringens neuraminidase (Sigma) for 3 h at 37 °C. Equal quantities of lysates were then run on 6 % SDS-polyacrylamide gels and transferred onto nitrocellulose filters (Schleicher & Schuell). Western blot analysis was carried out using anti-4 antibodies (C-20) or anti-
1 antibodies (M-106) (Santa Cruz Biotechnology) after blocking non-specific reactivity with 2 % non-fat dried-milk in TBS/0·05 % Tween 20. Bands were detected with horseradish peroxidase (HRP)-conjugated anti-goat secondary antiserum (Santa Cruz Biotechnology) or anti-rabbit secondary antiserum (Bio-Rad), followed by the enhanced chemiluminescence reaction (Pierce).
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RESULTS |
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Next, the infectious properties of the mutant viruses produced after transfection were tested by infecting a permissive fibroblast Swiss 3T3 cell line, with equal quantities of mutant or wt virions (m.o.i. of 10). The presence of early transcripts was analysed by Northern blotting. As shown in Fig. 1(D), early transcripts were detected in wt MPyV-infected cells after 18 h post-infection (p.i.), with an increase in signal at 24 h p.i. In contrast, no signal was ever observed in MPyV R77Q- or H298Q-infected cells, indicating that the mutant virus particles were not able to initiate early transcription, therefore confirming that MPyV R77Q and H298Q were not infectious.
Lipofection of mutant virions into fibroblast cells restores viral gene expression
To assess whether the lack of infectivity of the MPyV R77Q and H298Q mutants was due to a deficiency in cell entry, 1 or 2 µg purified mutant or wt virus particles (corresponding to 107 and 2x107 p.f.u., respectively) were delivered directly into the cytoplasm of Swiss 3T3 cells by lipofection. The equivalent m.o.i. was estimated to be of 10 and 20, respectively.
Total RNA was extracted after 24 h and viral gene expression was analysed by Northern blotting (Fig. 2). Early and late gene expression was enhanced in fibroblasts lipofected with wt virus particles as compared to cells inoculated with wt particles alone (without Lipofectamin Plus reagent). In addition, we found that lipofection of Swiss 3T3 cells with mutant virions could restore early and late gene expression efficiently. The positive signals obtained did not result from contaminating free DNA present in the virus preparation, as pre-treatment of viruses with DNase I prior to lipofection gave similar results (data not shown). Moreover, electron microscopy observation of viruslipofectamine complexes showed that the addition of lipofectamine to the virus did not affect either the morphology or the stability of virions (data not shown). Furthermore, we confirmed that the late messengers detected by Northern blot analysis in R77Q- or H298Q-lipofected cells corresponded to the mutant sequences (data not shown). This control was carried out as follows: total RNA from the samples was reverse-transcribed and the VP1-encoding region amplified specifically by PCR. Amplified fragments were then digested with BstNI or BsrGI to discriminate between the two mutants and wt sequences. Therefore, these data indicated that bypassing the entry step could restore mutant virus infectivity. Consequently, we concluded that mutant virus particles were altered at the cell entry stage.
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DISCUSSION |
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In accordance with data reported by Bauer et al. (1999), our MPyV strain A2 mutants MPyV R77Q and H298Q were unable to replicate in fibroblast cells. We showed further that MPyV mutant virus particles were formed and perfectly encapsidated, ruling out the possibility that the total loss of infectivity of MPyV mutants was due to structural alterations in the viral capsid. We also demonstrated that direct delivery of mutant viruses into the host cell cytoplasm allowed viral gene expression. The post-entry events, including uncoating, studied on adenovirus and liposomeadenovirus complexes (Fasbender et al., 1997
), have demonstrated that virus lipofection bypasses natural cell binding and uptake and that the subsequent steps necessary for efficient infection are adenovirus-dependent. The fact that lipofection of wt viruses enhances, rather than decreases, early gene expression with respect to natural infection may indicate that, as in adenoviruses, liposome-mediated MPyV cell entry does not specifically affect the process of virus uncoating and genome expression.
The hypothesis that the virus cycle of MPyV mutants is impaired at the cell entry level, as suggested by lipofection experiments, was confirmed by FACS analysis. Binding of mutant R77Q or H298Q VLPs appeared to be totally abrogated on both SA-expressing (BALB/c 3T3 and Pro-5) and SA-deficient (Lec-2) cells, whereas wt VLP binding occurred in a SA-positive background only. Therefore, abrogation of the VP1SA interaction (due to the VP1 R77Q and H298Q mutations or to the absence of SA residues on host cells) results in a total loss of virus cell binding. To our understanding, this represents the first biological demonstration that MPyV cell binding depends solely on SA-containing molecules, irrespective of cell type. In this regard, the critical role of SA in MPyV cell binding notably differs from that reported for other viruses. For example, binding of reoviruses to SA enhances virus infection by accelerating adsorption but does not represent an essential event for the virus life cycle (Barton et al., 2001b).
Despite the fact that cell binding for several viruses, such as reoviruses, influenza virus and rotaviruses, was identified initially as being SA-dependent, recent reports have indicated that cell binding/entry may also occur through recognition of other receptor molecules (Barton et al., 2001a, b
; Stray et al., 2000
; Arias et al., 2002
). For instance, rotavirus strain RRV uses SA as a primary cell receptor and subsequently interacts with the
2
1 integrin at a post-attachment step to infect host cells, while its neuraminidase-resistant (SA-independent) variant nar3 recognizes this integrin as its primary cell attachment site (Arias et al., 2002
). The identification of
4
1 integrin as one of the cell receptors involved at a post-attachment level for MPyV (Caruso et al., 2003
) prompted us to investigate whether a lack of SA-mediated cell binding could be rescued by cell surface overexpression of the
4
1 integrin receptor. FACS analysis showed that high expression levels of the post-attachment receptor could not restore either mutant VLP binding to SA-containing and SA-deficient cells or wt VLP binding to SA-deficient cells. These results indicate that, as with rotavirus strain RRV, SA residues and integrins are not alternative receptors for MPyV and confirm the two-step entry mechanism hypothesized previously, in which initial cell binding occurs strictly through VP1SA recognition; such an interaction may, in turn, trigger subsequent recognition of the
4
1 integrin ligand-binding site at post-attachment level (Caruso et al., 2003
).
SA residues used for MPyV cell binding are present on molecules the nature of which has not yet been identified. In the present work, we present evidence that the 4
1 integrin is one of these molecules. Indeed, expression of the sialylated
4
1 integrin in BALB/c 3T3 cells correlated with an increased cell surface SA content and enhanced the cell-binding ability of wt VLPs by twofold. We have reported recently that transfection of BALB/c 3T3 cells with the
4 subunit enhances MPyV infectivity and that function-blocking antibodies directed against the ligand-binding site of the
4
1 integrin reduce MPyV infectivity in
4-transfected BALB/c 3T3 cells without affecting virus cell binding (Caruso et al., 2003
). These data led us to propose a role for the
4
1 integrin, probably through its ligand-binding site, in MPyV infectivity at a post-attachment level. In a similar manner to MPyV particles, wt VLP cell binding is not reduced by pre-incubation of
4-transfected cells with anti-
4 antibodies (data not shown). Taken together with our present results, this suggests that the
4
1 integrin plays a dual function in MPyV infectivity, being involved both initially at a cell-attachment level as a SA-containing receptor and then at a post-attachment level through its ligand-binding site. These two different functions probably involve separate parts of the receptor molecule, as the potential N-glycosylation sites of the
4 and
1 subunit chains do not map to the ligand-binding site (Shih et al., 1993
; Irie et al., 1995
, 1997
). To date, the contribution of N-glycosylation to modulation of the
4
1 integrin function, in the presence of terminal SA residues, is not understood fully. Recent reports suggest that hyposialylation of integrins enhances binding of the integrin to fibronectin by exposing the active ligand-binding site to the ligand (Pretzlaff et al., 2000
; Semel et al., 2002
). Further studies will, therefore, be needed to answer the question of whether MPyV binding to the SA residues of
4
1 integrin induces modulation of the integrin activity by promoting exposure of the active binding site for interaction with the VP1 LDV motif.
The molecular mechanisms that determine MPyV tropism, pathogenicity and tumourigenicity are complex. They rely both on post-cell entry events (regulated by the enhancer and promoter elements of the viral genome) and on the ability of MPyV to recognize branched or straight SA chains and the tightness of the VP1SA interaction (Amati, 1985; Maione et al., 1985
; Caruso et al., 1990
; Freund et al., 1991a
, b
). We showed previously that
4
1 integrin enhances cellular permissivity to MPyV at a post-attachment level (Caruso et al., 2003
) and, in this study, we present evidence that this molecule is also used as a SA-containing cell attachment receptor. These observations suggest that the nature of the proteinaceous part of the SA-containing glycoprotein receptor is a major determinant of tissue tropism. In addition, we have proposed previously that more than one molecule is involved in this process (Caruso et al., 2003
). The characterization of other as yet unidentified MPyV cell receptors is therefore necessary to understand MPyV pathogenicity better. Moreover, according to this hypothesis, branched SA-containing molecules, also designated by Bauer et al. (1999)
as pseudoreceptors, could represent glycoproteins that allow MPyV cell attachment but not secondary post-attachment interactions with the protein moiety required for virus internalization. Further investigation in this direction is required to develop tissue-restricted MPyV VLP-based vectors for gene therapy (Forstovà et al., 1995
; Soeda et al., 1998
; Krauzewicz & Griffin, 2000
).
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ACKNOWLEDGEMENTS |
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Received 23 May 2003;
accepted 4 August 2003.