1 Laboratory of Public Health, Department of Environmental Veterinary Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan
2 Laboratory of Anatomy, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan
3 Department of Pathology, Institute of Tropical Medicine, Nagasaki University, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan
Correspondence
Ikuo Takashima
takasima{at}vetmed.hokudai.ac.jp
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ABSTRACT |
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INTRODUCTION |
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It has been reported that the envelope proteins play an important role in the budding process of many viruses. Expression of the M and E envelope proteins of mouse hepatitis virus, without other viral proteins, led to the secretion of virus-like particles (VLPs), which were morphologically similar to native virions (de Haan et al., 1998; Vennema et al., 1996
). The hepatitis B virus (HBV) surface proteins can be secreted as subviral particles, but their morphology is quite different from HBV virions (Patzer et al., 1986
; Simon et al., 1988
). In the case of flaviviruses, slowly sedimenting haemagglutinin (sHA), which lacks infectivity, is secreted from virus-infected cells (Gritsun et al., 1989
; Heinz & Kunz, 1977
). sHA has viral envelope proteins but lacks nucleocapsid protein and viral RNA, and its particulate structure is similar to the infectious virion, except for lower density and smaller size. Expression of the prM and E protein of several flaviviruses without other viral proteins results in the secretion of VLPs, which are similar to sHA particles (Allison et al., 1995b
; Konishi et al., 1992
; Mason et al., 1991
).
The flavivirus envelope has two proteins: the major envelope glycoprotein E (molecular mass 52 kDa) and the small membrane protein M (molecular mass 78 kDa). Both proteins are synthesized as part of a polyprotein precursor and then co- and post-translationally cleaved into the individual proteins (Lindenbach & Rice, 2001). The M protein is cleaved first into an intermediate precursor called prM, before final processing.
E protein is a well-characterized viral protein in flavivirus. E protein mediates virus entry via receptor-mediated endocytosis and also carries the major antigenic epitopes leading to a protective immune response (Heinz & Mandl, 1993). The X-ray crystallographic resolution of the structure of the E ectodomain of TBE virus revealed that E protein forms head-to-tail homodimers that lie parallel to the viral envelope (Rey et al., 1995
). In low-pH condition, such as in endocytic vesicles, these homodimers dissociate and lead to the irreversible formation of homotrimers (Allison et al., 1995a
; Stiasny et al., 2001
, 2002
).
M is synthesized as precursor protein, prM (molecular mass 25 kDa) in ER, carrying one N-linked oligosaccharide. One of the roles of prM protein reported previously is a chaperone-like activity for the folding and maturation of E (Konishi & Mason, 1993; Lorenz et al., 2002
). Newly synthesized E and prM proteins associate to form heterodimers that are incorporated into immature virions (Wengler & Wengler, 1989
). This heterodimerization leads to the final native conformation of E and protects E from inactivation by acidification in the transport vesicles (Heinz & Allison, 2000
). Shortly before release from the cell, the immature particles are converted to the active form by cleavage of the pr-portion from prM by a cellular furin protease in trans-Golgi network and prM turns into M (Elshuber et al., 2003
; Stadler et al., 1997
).
Recent examination of the assembly and maturation of Kunjin virus revealed that the assembly of virions occurs within the lumen of the rough ER (Mackenzie & Westaway, 2001). Furthermore, the structure of immature flavivirus particles containing prM was analysed by cryoelectron microscopy (Zhang et al., 2003
). Sixty trimeric spikes were organized icosahedrally on the surface of the particles, in contrast to the smooth surface of mature virions reported previously (Kuhn et al., 2002
). In the spike structure, prM covers the fusion peptides of E in a manner similar to the organization of the glycoproteins in alphavirus spikes (Zhang et al., 2002
). In this way, various approaches have revealed the morphological assembly and maturation processes of virus particles, but the molecular mechanism of virus budding and secretion remains obscure.
In this study, we constructed plasmids expressing mutant prM and E proteins of TBE virus, and tested the effect of these mutations on the production of VLPs when expressed in mammalian cells. This allowed the identification of a single point mutation in prM that induced a reduction of secretion of VLPs. The mutation in prM did not affect the oxidative folding of the viral envelope proteins nor the chaperone-like activity of prM. The envelope proteins not secreted from the cells due to the prM mutation accumulated in the ER, and the transport of viral envelope proteins to the Golgi complex was also inhibited. By electron microscopy, tubular structures were observed in the lumen of the ER. When the point mutation in prM was introduced into the TBE virus genome, it severely reduced the ability of the mutant viral RNA to produce infectious particles. This data points out the critical role of prM protein in the virus budding process.
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METHODS |
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Antibodies.
For detection of TBE virus prM and E proteins, ELISA, immunoprecipitation and immunofluorescence experiments, mouse anti-E mAb 1H4 and 4H8, prepared in our laboratory, were used (Komoro et al., 2000). Rabbit polyclonal anti-prM and anti-E antibodies were prepared by immunization with recombinant prM and E proteins expressed in the pET43 system (Novagen). For the immunofluorescence colocalization studies, anti-calreticulin rabbit polyclonal antiserum (Affinity BioReagents) or anti-giantin rabbit polyclonal antiserum (Covance Research Products) was applied. FITC conjugated anti-mouse IgG antibodies and Texas red conjugated anti-rabbit IgG antibodies (Jackson Immunoresearch) were used as secondary antibodies in the immunofluorescence assays.
Plasmid construction.
The production of the recombinant plasmid pCAGprME expressing prM and full-length E, derived from the Oshima 5-10 strain of TBE virus (GenBank accession no. AB062063), was described previously (Yoshii et al., 2003). For the construction of the mutant plasmids, TBE viral RNA was extracted from virus-inoculated suckling mouse brain and RT-PCR was performed as described previously (Takashima et al., 1997
). Amplification of mutated DNA coding prM and E gene was carried out twice by error-prone PCR using XhoIMEf and ClaIrNS1 primers (Table 1
), with AmpliTaq DNA polymerase (Applied Biosystems), in the presence of a high concentration of Mg2+, at low annealing temperature. To generate the mutant expression plasmid, clone 55, the PCR products were then digested with XhoI and ClaI, and cloned into the pCAGGS/MCSR plasmid (Niwa et al., 1991
).
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For the construction of the TBE virus infectious cDNA clone containing the ProSer mutation at position 63 in prM, the site-directed mutation was substituted into pGEMT-CprME using the pr63Sf and pr63Sr primers described above. The reconstructed plasmid was then digested with SpeI and the fragment containing the mutation was replaced into Oshima IC-pt, as described previously (Hayasaka et al., 2004
). The new cDNA clone construct was designated Oshima IC-pr63S.
Transfection.
293T cells, grown to 6070 % confluence in six-well culture plates, were transfected with 2 µg each plasmid complexed to TransIT-LT1 reagent (PanVera) in Opti-MEM (Invitrogen). At 24 h post-transfection (or as otherwise stated), the cells and supernatant were harvested and used for further experiments.
ELISA.
Transfected cells were lysed with 1 % Triton X-100 in 10 mM Tris-buffered saline (TBS) and the supernatants were treated with 1 % Triton X-100. Triton X-100-solubilized samples were added to mAb 1H4-coated wells of 96-well microtitre ELISA plates, previously blocked with 3 % BSA. TBE virus E protein was detected by adding biotinylated MAb 4H8 and HRP-conjugated streptavidin (Sigma). The HRP activity was detected by adding o-phenylenediamine dihydrochroride (Sigma) in the presence of 0·03 % H2O2.
Immunoprecipitation.
293T cells were transfected with the wild-type or pr63S pCAGprME plasmid as described above. At 24 h post-transfection, the cells were lysed with Triton X-100 in 10 mM TBS, incubated on ice for 20 min and then centrifuged at 16 000 g for 20 min. The supernatant, which excluded the nuclear fraction, was precleared on Protein G-Sepharose beads (Amersham Pharmacia Biotech) for 2 h at 4 °C. Precleared lysates were combined with protein G-Sepharose beads with MAb 1H4 and precipitated by incubation for 2 h at 4 °C. Immune complexes were pelleted at 10 000 g for 10 s and washed four times with 1 % Triton X-100 in 10 mM TBS. Subsequently, the precipitated materials were solubilized by adding Laemmli buffer (Laemmli, 1970) and by heating to 95 °C for 2 min and then analysed by SDS-PAGE and Western blotting.
SDS-PAGE and Western blotting.
Transfected cells were lysed with Laemmli buffer under nonreducing or reducing (in the presence of 2-mercaptoethanol) conditions. Protein samples were electrophoresed through 8 and 15 % polyacrylamide-SDS gels. The protein bands were transferred onto PVDF membranes, then incubated with 1 % gelatin in 25 mM TBS containing 0·01 % Tween 20 (TBST) for 30 min at room temperature. After washing with TBST, the membranes were reacted with polyclonal anti-E or anti-prM rabbit IgG for 1 h, followed by alkaline phosphatase conjugated anti-rabbit IgG (Promega) for 30 min at room temperature. For the detection of glycosylation of envelope proteins, the membranes were treated with biotin conjugated concanavalin A (Honen Corporation) and then with alkaline phosphatase conjugated streptavidin (Sigma). Protein bands were visualized using the AP detection reagent kit (Novagen).
Immunofluorescence assay.
293T cells grown on eight-well chamber slides (Nalge Nunc International) were transfected with the wild-type or pr63S pCAGprME plasmids. At 8 h post-transfection, cells were rinsed with PBS and fixed with 4 % paraformaldehyde for 10 min, then permeabilized with 0·2 % Triton X-100 for 4 min at room temperature. After blocking with 2 % BSA for 30 min, the cells were incubated at room temperature for 1 h with mouse mAb 1H4 and antibodies that recognize marker proteins of various cellular organelles, at dilutions between 1 : 100 and 1 : 1000 in antibody-dilution buffer (PBS containing 0·1 % Triton X-100 and 2 mg BSA ml1). After extensive washing, the cells were incubated at room temperature for 1 h with fluorescence-label conjugated secondary antibodies, diluted 1 : 200. The cells were washed three times with PBS, followed by mounting of the coverslips on glass slides. Images were viewed and collected with an Olympus IX70 confocal microscope.
Electron microscopy.
293T cells were transfected with the wild-type or pr63S pCAGprME plasmids. At 24 h post-transfection, cells were harvested and centrifuged at 1000 g for 5 min. The pellets were fixed with 3 % (v/v) glutaraldehyde in 0·1 M phosphate buffer (pH 7·2) for 3 h and then rinsed three times with 0·1 M phosphate buffer. After post-fixation in a 1 % (w/v) osmium tetraoxide solution for 1·5 h, the pellets were dehydrated through a series of graded ethanols and embedded in Epon 812 via QY1 (Nishin EM). Ultrathin sections were cut, stained with uranyl acetate and lead citrate, and examined under a JEM 1210 transmission electron microscope (JEOL) at an acceleration voltage of 80 kV.
RNA transcription and transfection.
Oshima IC-pt or Oshima IC-pr63S were digested with SpeI and extracted using a QIAquick gel extraction kit (Qiagen). Infectious RNA was transcribed in vitro using mMESSAGE mMACHINE SP6 kits (Ambion) in 20 µl reaction volumes, with an additional 1 µl GTP solution. After the transcription at 37 °C for 2 h, template DNA was removed by DNase I digestion 37 °C for 15 min. RNA was precipitated with lithium chloride, washed with 70 % ethanol, resuspended in RNase-free water, and stored in aliquots at 80 °C.
Approximately 5x106 BHK cells in 0·5 ml cold PBS were electroporated with 10 µg RNA in 0·4 cm cuvettes using a GenePulser apparatus (Bio-Rad), pulsing twice at settings of 1·3 kV, 25 µF and maximum resistance. Transfected cells were equally divided into two T-25 flasks. After an overnight recovery, cell debris resulting from electroporation was washed away twice with PBS and fresh medium was added. At various times post-electroporation, aliquots of media were harvested as a source of recovered viruses. Infectious virus titre was assayed by the focus count assay, as described previously (Takashima et al., 1997).
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RESULTS |
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Complete sequencing of the pCAGprME mutant clone 55 allowed identification of 5 nt changes that induce 4 aa changes (Fig. 1). The mutant prM protein had 2 aa changes at positions 63 and 88 in the pr region, which was eventually removed by an intracellular furin protease to produce the mature M protein (Stadler et al., 1997
). The mutant E protein had two amino acid changes at positions 276 and 464. The Ile
Val substitution at position 276 is a conservative amino acid change and maps to the domain II of E proteins (Rey et al., 1995
). The Leu
Pro substitution at position 464 maps to the first transmembrane region of E protein, which constitutes a membrane anchor (Allison et al., 1999
; Mandl et al., 1989
; Rice, 1996
).
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Intracellular localization of recombinant TBE virus envelope proteins
To determine the intracellular distribution of the viral envelope proteins, 293T cells were transfected with pCAGprME wild-type or pr63S plasmids. The cells were fixed, permeabilized and double stained for TBE virus envelope proteins and cellular marker antigens. Anti-calreticulin (Michalak et al., 1992) was used as a marker for ER (Fig. 5b and e
), and anti-giantin (Linstedt & Hauri, 1993
) was used as a marker for the Golgi complex (Fig. 5h and k
). A mouse mAb anti-E (Fig. 5a, d, g and j
) was used to stain viral envelope proteins. In wild-type-transfected cells, the distribution of viral envelope protein overlapped almost completely with the ER marker (Fig. 5c
) and Golgi marker (Fig. 5i
), indicating that viral envelope protein was transported into the Golgi complex. While distribution of viral envelope protein in the ER was observed (Fig. 5f
), overlap of viral envelope proteins and Golgi marker was hardly seen in pr63S-transfected cells (Fig. 5l
). These data suggest that the mutation at position 63 in prM proteins causes the accumulation of viral envelope proteins in the ER.
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DISCUSSION |
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The interaction between prM and E proteins is important during the early events of virus particle maturation and secretion. It has been reported that E protein cannot attain full maturity when expressed alone, while prM protein is able to fold independently of other viral components (Lorenz et al., 2002). Furthermore, the secretion of E protein requires cosynthesis with prM, as demonstrated previously in many flaviviruses (Allison et al., 1995b
; Konishi & Mason, 1993
; Ocazionez Jimenez & Lopes da Fonseca, 2000
). Heterodimerization of prM and E leads to the final native conformation of E protein, which is an early process in virus maturation. Therefore, we first examined whether the reduced VLP secretion induced by the position 63 mutation in prM was related to the alteration of interaction between the prM and E proteins. The results of immunoprecipitation with anti-E monoclonal antibodies also indicate that heterodimerization between prM and E, as well as oxidative folding and glycosylation of viral envelope proteins, occurred normally in the presence of the position 63 mutation in prM (Fig. 4
). Furthermore, total production level of protein E, which had conformational structures, was not affected by the prM mutation (Fig. 3a
), indicating that the position-63 mutation in prM does not detrimentally affect the maturation of protein E. These data suggest that the reduction of VLP secretion induced by the prM mutation was due to a later step of virus particle budding and secretion, not to the prM and E interaction in the early events of virus budding.
It has been reported that flavivirus particles are assembled into the ER lumen (Mackenzie & Westaway, 2001). Thus, the mechanism of flavivirus secretion can be divided into two steps. Virus particle budding in ER membrane, followed by virus transport through the secretory pathway. To identify the influence of the prM position 63 mutation in virus secretion, the intracellular localization of the viral envelope proteins was examined. Envelope proteins expressed with the mutated prM were not transported to the Golgi complex, and accumulated in the ER (Fig. 5
). Electron microscopic analysis revealed that many tubular structures, which differed from spherical VLPs in shape, were observed in the ER lumen of cells transfected with a plasmid, with the mutation in prM (Fig. 6
). In the Lorenz et al. (2003)
study, it was reported that similar tubular structures were occasionally seen in cells expressing TBE virus prM and E, and that these structures were not observed in the Golgi complex. The tubular structures observed in cells expressing mutated prM and E in our study may be comparable to those noted in the Lorenz study, and they may not undergo secretion due to their abnormal budding. Another possibility is that the tubular structures are membrane components that detach from the ER lumen due to the damage to membrane structures caused by accumulation of viral envelope proteins. In any case, the position 63 mutation in prM clearly affects the budding process of the virus particle.
In many enveloped viruses, the cytoplasmic domain of the envelope proteins has been assigned an important role in virus assembly. For vesicular stomatitis virus, the cytoplasmic domain is important for incorporation of the glycoprotein (Owens & Rose, 1993; Whitt et al., 1989
). For alphaviruses, it has been shown that the cytoplasmic domain of the E2 glycoprotein has a critical role in virus budding (Kail et al., 1991
; Owen & Kuhn, 1997
; Zhao et al., 1994
). Deletion of the cytoplasmic tails of influenza virus haemagglutinin and neuraminidase (NA) leads to irregularly shaped virions, and deletion of the NA cytoplasmic domain reduces the incorporation of NA into virions (Jin et al., 1997
; Mitnaul et al., 1996
). But unlike these viruses, flavivirus prM and E proteins have cytoplasmic loops consisting of only a few amino acid residues between their two transmembrane segments. Thus, it is thought that the luminal domains, or the two transmembrane domains of prM and E, play more critical roles in the assembly of these viruses.
In a recent study by Zhang et al. (2003), the structure of prM-containing immature particles of dengue and yellow fever virus was analysed by cryoelectron microscopy and image reconstruction techniques. The surface of the immature particles was characterized by the presence of 60 fairly prominent projections or spikes, which differed from the smooth surface of mature virus (Kuhn et al., 2002
). In the spike structure, prM protein covered the fusion peptides of domain II of the E protein, similar to the case of alphaviruses, where the E2 glycoproteins protect the fusion peptides of the E1 glycoproteins within a trimeric spike (Zhang et al., 2002
). Thus, it is suggested that the position 63 mutation in prM may induce conformational changes in the domain exposed on the outer side of the viral envelope, which is important for the virus budding process.
The cellular membranes involved in membrane transport normally form vesicles on the cytoplasmic side, such as clathrin coated vesicles, and COP I and COP II vesicles (Schekman & Orci, 1996). It is possible that prM-E heterodimers, alone or with cellular factors in the ER lumen, assemble laterally and induce the membrane curvature into an isometric lattice, like the assembly of coat proteins in membrane transport vesicles (Keen et al., 1979
; Wieland & Harter, 1999
). The abnormal budding induced by the prM mutation may be due to a dysfunction in this process, caused by structural changes in prM or by loss of interaction with a cellular component. Alternatively, the prM mutation may be related to the pinching off of particles from the ER membrane, as is the case for dynamin in clathrin-coated vesicles, and it might be possible that VLPs could not be pinched off properly due to the prM mutation (McNiven, 1998
).
In summary, by analysis of a prM mutation that induces the reduction of VLP and virus particle secretion, we demonstrated a critical function for prM in the virus budding process. This mutation does not affect the heterodimerization between prM and E, and E proteins can reach the native conformation in spite of the prM mutation, suggesting the preservation of prM's chaperone-like role. Envelope proteins that are not secreted due to the prM mutation accumulate in the ER, indicating the failure of virus particle budding. Molecular approaches focused on the ectodomain of prM protein should enable further investigation of the mechanisms during the virus budding process.
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ACKNOWLEDGEMENTS |
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Received 6 April 2004;
accepted 28 June 2004.