- Open Access
Evidence for the interaction of the human metapneumovirus G and F proteins during virus-like particle formation
© Loo et al.; licensee BioMed Central Ltd. 2013
- Received: 26 June 2013
- Accepted: 26 August 2013
- Published: 25 September 2013
Human metapneumovirus (HMPV) is now a major cause of lower respiratory infection in children. Although primary isolation of HMPV has been achieved in several different cell lines, the low level of virus replication and the subsequent recovery of low levels of infectious HMPV have hampered biochemical studies on the virus. These experimental methodologies usually require higher levels of biological material that can be achieved following HMPV infection. In this study we demonstrate that expression of the HMPV F, G and M proteins in mammalian cells leads to HMPV virus-like particles (VLP) formation. This experimental strategy will serve as a model system to allow the process of HMPV virus assembly to be examined.
The HMPV F, G and M proteins were expressed in mammalian cell lines. Protein cross-linking studies, sucrose gradient centrifugation and in situ imaging was used to examine interactions between the virus proteins. VLP formation was examined using sucrose density gradient centrifugation and electron microscopy analysis.
Analysis of cells co-expressing the F, G and M proteins demonstrated that these proteins interacted. Furthermore, in cells co-expression the three HMPV proteins the formation VLPs was observed. Image analysis revealed the VLPs had a similar morphology to the filamentous virus morphology that we observed on HMPV-infected cells. The capacity of each protein to initiate VLP formation was examined using a VLP formation assay. Individual expression of each virus protein showed that the G protein was able to form VLPs in the absence of the other virus proteins. Furthermore, co-expression of the G protein with either the M or F proteins facilitated their incorporation into the VLP fraction.
Co-expression of the F, G and M proteins leads to the formation of VLPs, and that incorporation of the F and M proteins into VLPs is facilitated by their interaction with the G protein. Our data suggests that the G protein plays a central role in VLP formation, and further suggests that the G protein may also play a role in the recruitment of the F and M proteins to sites of virus particle formation during HMPV infection.
- Human metapneumovirus
- Virus-like particle
- Recombinant expression
- HMPV F protein
- HMPV G protein
- Protein interactions
Human metapneumovirus (HMPV) is a new member of Paramyxoviridae that was first identified in children with respiratory diseases in Netherlands . The clinical symptoms that are caused by HMPV infections in children are similar to those observed with respiratory syncytial virus (RSV) infection; ranging from mild symptoms to pneumonia. HMPV is now a globally recognised cause of lower respiratory infection in children [2, 3]). Genetic analysis identified two major genogroups A and B [4–6]. HMPV expresses two major integral membrane proteins that play a role in virus entry. The attachment (G) protein plays a role in virus attachment and is expressed as a single polypeptide chain, which subsequently undergoes extensive N- and O-linked glycosylation . The fusion (F) protein mediates fusion of the virus and host-cell membranes, and is initially synthesised as a single polypeptide chain (F0) that undergoes proteolytic cleavage to generate the mature and active form of the protein, consisting of F1 and F2 protein subunits . The virus also expresses a third membrane-associated protein called the matrix (M) protein, which is analogous to the M protein of RSV and is a major determinant of virus morphology .
Primary isolation of HMPV has been achieved in several different cell lines [4, 10, 11], however tissue culture adapted isolates can require up to 21 days incubation before cytopathic effects are visualised [7, 11–13]. This low level of virus replication and the subsequent recovery of low levels of infectious HMPV in standard cell culture have hampered biochemical studies on the virus. These experimental methodologies usually require higher levels of biological material than can be achieved following HMPV infection.
Virus-like particle (VLP) formation following the co-expression of specific virus structural proteins has been demonstrated in several paramyxoviruses [14–18]. These studies have allowed the identification of essential virus proteins that are required for virus particle assembly. Although a central role for the M protein in VLP formation has been reported for human parainfluenza type 1 virus  and Newcastle disease virus , the expression of the M protein alone was insufficient for VLP production in simian virus type 5  and avian pneumovirus type C . The use of recombinant HMPV protein expression to drive the formation of similar HMPV VLPs can potentially overcome the problems associated with the poor cultivation of HMPV in tissue culture. In addition, by direct cloning of the virus genes from clinical material the expression of gene sequences that have not been subjected to extensive tissue culture adaptation can be examined. This therefore affords a relatively simple experimental system with which to examine HMPV morphogenesis. In this study we have examined the capacity of the HMPV F, G and M proteins to form VLPs in mammalian cells, and to further examine the minimal virus protein requirements that lead to VLP formation.
Expression of the HMPV F, M and G proteins in mammalian cells
In pCAGGS/G-FLAG transfected cells the G protein migrated as two relatively broad Endo-H-resistant protein bands of approximately 60 kDa (G60) and 120 kDa (G120) (Figure 1A). The size of G60 was consistent with monomeric G protein, while the GA120 kDa protein species was consistent with an oligomeric form of G protein. Several studies have demonstrated the propensity of the HMPV G protein to form oligomers when analysed by immunoblotting. Both G60 and G120 were Endo-H resistant, however following PNGaseF treatment the G protein was reduced to a faint protein smear. This indicated removal of the N-linked glycans, and the residual protein smear was presumably due to heterogeneity of the remaining attached O-linked glycans . The glycosylation analysis was consistent with the presence of mature forms of the surface-expressed F and G proteins. The presence of unusually high levels G protein oligomers in SDS PAGE analysis has been reported previously in avian metapneumovirus [22, 23]. Due to its location beneath the plasma membrane, as expected we failed to detect the presence of biotinylated M protein. Immunoblotting of lysates prepared from pCAGGS/M transfected cells with MAbM3F8 (anti-M) revealed the presence of a 28 kDa protein, the expected size of the HMPV M protein (Figure 1C).
Lysates prepared from pCAGGS, pCAGGS/G-FLAG or pCAGGS/F-cmyc transfected cells were immunoprecipitated using either anti-cmyc or anti-FLAG and the immunoprecipitates then immunoblotted with either anti-cmyc (Figure 1D) or anti-FLAG (Figure 1E). Only pCAGGS/F-cmyc transfected cells immunoprecipitated and immunoblotted with anti-cmyc showed the presence of F proteins species corresponding in size to the monomeric and oligomeric F protein (Figure 1D). In a similar analysis G120 was only detected in pCAGGS/G-FLAG transfected cells immunoprecipitated and then immunoblotted with anti-FLAG (Figure 1E). This demonstrated the specificity of the immunoprecipitation assay.
Interaction between the HMPV F and G proteins
The F and G proteins are both integral membranes and we hypothesized that the presence of the lipid membrane may be required for stabilization of the protein complex in situ. In this scenario the removal the lipid membrane by detergent extraction prior to immunoprecipitation could lead to destabilization of the protein complex. We therefore used Dithiobis [succinimidylpropionate] (DSP) to stabilise the protein complex by in situ cross-linking prior to detergent extraction of the virus proteins as described previously [25, 26]. Although DSP cross-linked complexes were predicted to be beyond the resolution of SDS-PAGE analysis (>250 kDa), following immunoprecipitation the individual components of the stabilised protein complexes could be released by removal of the covalent cross-links by reductive cleavage using β-mercaptoethanol-treatment.
293T cells were co-transfected with pCAGGS/F-cmyc and pCAGGS/G-FLAG, and the cell monolayers treated with increasing DSP concentrations. Cell lysates were prepared and immunoprecipitated with anti-cmyc, and the presence of the G protein and F protein in the immunoprecipitation assays detected by immunoblotting with anti-FLAG (Figure 2E) and anti-cmyc (Figure 2G) respectively. Immunoblotting with anti-FLAG revealed the appearance of increasing amounts of a 170 kDa FLAG-tagged protein (the expected mass of GA170 protein) with increasing DSP concentrations (Figure 2E). This protein was not present in lysates prepared from non-treated cells. Probing with anti-cmyc revealed similar levels of the monomeric (F65) and oligomeric (F145) F protein species in both non-treated and DSP-treated cell monolayers (Figure 2G).
Similarly lysates immunoprecipitated with anti-FLAG and immunoblotted using anti-cmyc revealed increasing amounts of F145 with increasing DSP concentration, together with a cmyc-tagged protein species >300 kDa (Figure 2F) whose molecular mass was consistent with higher oligomeric forms of the F protein. Probing with anti-FLAG revealed monomeric (G70) and oligomeric forms (G170) of the G protein in both non-treated and DSP-treated monolayers (Figure 2H).
These data suggested that DSP was able to stabilise a protein complex that formed between the F and G proteins. The sedimentation characteristic of cross-linked F and G protein complex was examined using sucrose gradient centrifugation. Cells were co-transfected with pCAGGS/F-cmyc and pCAGGS/G-FLAG and treated with 0.1 mM DSP. A detergent extract was prepared and then applied to a continuous 5-30% (w/v) sucrose gradient. After centrifugation the gradient was fractionated, and the individual fractions immunoprecipitated with anti-cmyc and immunoblotted with anti-FLAG (Figure 2I) or immunoprecipitated with anti-FLAG and immunoblotted with anti-cmyc (Figure 2J). This assay revealed that the co-precipitating F and G proteins were mainly located within fractions 6 to 9, consistent with the co-migration of the DSP stabilized F and G protein complex.
The HMPV F and G protein complex interacts with the M protein
Co-expression of the F, G and M proteins lead to the formation of virus-like particles
The G protein facilitates VLP formation
A similar analysis was performed on FG-transfected, GM-transfected, and FM-transfected cells. In FG-transfected cells the presence of the F and G proteins was detected in the cell extract confirming expression (Figure 8D). However, in FG-transfected cells F65 together with G70 and G170 was detected at the 20-50% (w/v) sucrose interface. This indicated that co-expression of the F and G proteins facilitated incorporation of F65 into the VLP fraction. In GM-transfected cells the presence of the M and G proteins were detected in the cell extract confirming expression (Figure 8E). However, a significantly higher level of M protein was detected in the 20-50% (w/v) sucrose interface together with the two G protein species. Interestingly, although both proteins were present within the 20-50% (w/v) sucrose interface we noted no change in the electrophoretic migration of the G protein. In FM-transfected cells the presence of the F and M proteins were detected in the cell extracts (Figure 8F), however the levels of F and M protein in the sucrose interfaces were similar to those detected in cells singly expressing either the F protein (Figure 8B) or M protein (Figure 8C).
The HMPV F and G proteins have also been shown to bind heparan sulphate  and heparin  respectively. The HMPV G protein exhibits many similarities to the corresponding RSV G protein e.g. it is heavily modified by O-linked glycosylation and can bind glycosaminoglycans [7, 31, 32], which suggests a similar role for the HMPV G protein during HMPV infection. Evidence for the interaction between the RSV F and G proteins in the virus envelope [25, 33] has provided support for a single protein complex involving both proteins. In this present study we have also provided evidence for the existence of a similar protein complex involving the HMPV F and G proteins. By analogy with RSV, we can hypothesise that such a protein complex will form during HMPV replication.
Our study indicated that co-expression of the HMPV protein was able to form VLPs, and the imaging analysis indicated that the recombinant HMPV particles formed as filamentous structures in a similar manner to that in HMPV-infected cells. Similar virus filaments are formed during morphogenesis of the closely related RSV, suggesting some common features in the assembly process of these different viruses. In this regard we noted that the HMPV G protein facilitated VLP formation independently of other HMPV proteins. Our data also indicated that the interaction of the G protein and the M and F proteins facilitated the incorporation of the latter into these structures. Although the M protein has been proposed to play a role in RSV morphogenesis, recent data suggests that it does not play a role in the initiation of virus particle formation . Our data suggested that expression of the G protein in the absence of other virus proteins is sufficient to form these structures, and its interaction with other virus (and possibly other unknown cell factors) may lead to their active recruitment into VLPs. In particular, our data suggested the presence of the F protein in these structures is largely dependent on its association with the G protein.
It is likely that there are significant differences in the homeostasis in virus-infected and transfected cells which can influence virus processes. Although the VLPs and virus particles are similar in appearance it is also likely that there will be subtle differences between the virus architecture in VLPs and mature virus particles that formed on infected cells . However, given these caveats, we noted an overall similar morphology in the HMPV VLPs and HMPV particles. Given the technical difficulties in growing HMPV in mammalian tissue culture cells (in particular low passage clinical isolates), the generation of HMPV VLPs in tissue culture cells may form the basis of an experimental system to examine aspects of the HMPV maturation process, in particular using protein sequences derived from non-tissue culture adapted viruses). These VLPs may also be a potential source of particulate virus antigen that can form the basis of a vaccine candidate, and future studies will examine these possibilities.
Cloning of the HMPV F, G and M genes from nasopharyngeal washings
Nasopharyngeal washing were collected from children admitted to KK Women’s and Children’s Hospital for respiratory infection as described previously . The F, G and M genes were amplified from the HMPV A2 positive Nasopharyngeal washings (SIN06-NTU271) using the primers F271pCAGGf F271pCAGGmycr, G271pCAGGf G271pCAGGflagr, M84pCAGGf and M84pCAGGr (Additional file 2: Table S1). The PCR products were then inserted into the vector pCAGGS .
The anti-FLAG and anti-cmyc were purchased from Sigma Aldrich and Cell Signalling respectively. M3F8 (anti-M) was prepared from bacterially expressed HMPV M protein. The HMPV G (AT1) and F proteins (Mab58) were obtained from Geoff Toms .
The HMPV A2 stain NCL03-4/174 was used in the LLC-MK2 cell line in DMEM (BSA, 0.5 μgml TPCK-trypsin) at 37°C for 7 days.
Expression of F, G and M proteins in (HEK) 293T cells
Cells were transfected using Lipofectamine 2000 reagent (Invitrogen, USA) following the manufacturer’s instructions. The media was changed after 4 hr and the cells were incubated at 37°C for 48 hr before further analysis.
The proteins were separated by SDS–PAGE, transferred onto PVDF membranes (Immobilon-P, Milipore, USA) as described previously. The tagged proteins were detected with rabbit anti-FLAG antibodies (Sigma-Aldrich, USA), mouse anti-cmyc antibodies (Cell Signaling Technology, USA) or mouse anti-M antibodies (Nanyang Technological University) as appropriate. Protein bands were visualised using the ECL system (GE Healthcare, USA). Molecular masses were estimated using Kaleidoscope markers (Biorad, USA).
Cell extracts were prepared at 4°C using RIP buffer (1% NP-40, 0.1% SDS, 150 mM NaCl, 1 mM EDTA, 2 mM PMSF, 2 mM lysine, 20 mM Tris–HCl, pH7.5) and clarified by centrifugation (13,000 g, 10 min 4°C) and immunoprecipitated as described previously  using appropriate antibodies. The immunoprecipitation assays were separated using SDS-PAGE.
Cells were surface-labelled using EZ-Link Sulfo-NHS-LC-LC-Biotin (Pierce Biotechnology, USA) as described previously . Briefly, cell monolayers were incubated in 0.5 mg/ml solution of EZ-Link Sulfo-NHS-LC-LC-Biotin (Pierce Biotechnology, USA) in PBS pH 8 for 1 hr at room temperature. The lysates were immunoprecipitated with either anti-cymc or anti-FLAG as appropriate.
In situ cross-linking was performed using Dithiobis[succinimidylpropionate] (DSP) (Pierce Biotechnology, USA) as described previously . Briefly, the cell monolayers were treated with between 0.0 and 1.0 mM DSP (100 mM stock solution in DMSO) in PBS pH 8.0, and the cells were incubated at room temperature for 1 hour. The monolayers were then washed with PBS pH 8.0 containing 2 mM lysine prior to detergent extraction with RIP buffer.
Transfected cells were lysed at 100°C for 10 min in denaturation buffer (0 · 5% SDS, 40 mM DTT). The samples were then made up to a final concentration of either 50 mM sodium phosphate + 1% NP-40 (pH7 5) or 50 mM sodium citrate (pH5 5) and incubated at 37°C overnight with 1000 U PNGase F (New England Biolabs, USA) or 1000 U Endo_H (New England Biolabs, USA) respectively.
Density gradient centrifugation analysis of crosslinked protein complexes
All steps were performed at 4°C. Cell monolayers were extracted using RIP buffer, and the resulting lysate clarified by centrifugation (13,000 g, 10 min). The clarified lysate was layered onto a 5–30% sucrose (w/v) gradient prepared in TEN buffer (1 mM EDTA, 100 mM NaCl, 10 mM Tris-Cl pH 8 + 0.1% Triton X-100). The gradient was centrifuged for 18 hr at 150,000 g and 4°C (Hitachi CP90WX preparative ultracentrifuge; Hitachi Co Ltd, Japan).
Isolation of VLPs
Cell suspension was subjected to freeze-thaw by ethanol-dry ice and a 37°C water bath. After 3 rounds of freeze-thaw, the cell suspension was clarified (2,500 g for 10 min) and loaded onto a sucrose cushion (10% w/v sucrose in TEN buffer), and centrifuged at 200,000 g for 1 hr at 4°C (Hitachi CP90WX ultracentrifuge). The resulting pellet was resuspended in 200 μl of TEN buffer and loaded onto a discontinuous sucrose gradient (20%, 50% and 60% sucrose (w/v) in TEN buffer). The material was harvested from each sucrose interface was harvested for further analysis. For the continuous centrifugation analysis the sucrose pellet harvested from the sucrose cushion was resuspended in TEN buffer and applied to a continuous gradient (10% to 60% sucrose (w/v) in TEN buffer) and centrifuged at 200,000 g for 18 hr at 4°C. The gradient was harvested by removing 1ml fractions, which were then analysed further.
Immunofluorescence and confocal microscopy
Cells transfected overnight on 10 mm glass coverslips were fixed with methanol:acetone (1:1) for 15 min at 4°C. The cells were labelled using anti-cmyc, anti-FLAG or anti-M antibodies and the appropriate secondary antibodies (conjugated to either FITC or Alexa Fluor 555) as described previously . The cells were visualized using a Zeiss Axioplan 2 confocal microscope using appropriate machine settings
Scanning electron microscopy (SEM)
Cells were processed as described previously . Briefly, transfected cells were fixed using 0.1% glutaraldehyde and labeled using anti-FLAG and anti-rabbit IgG (1/100 dilution) conjugated with colloidal gold (10 nm) (Sigma-Aldrich, USA) for 4 hr at room temperature. The cells were then post-fixed in 2.5% glutaraldehyde and 1% OsO4 prior to critical point drying. The cells were carbon coated and viewed using either a Jeol 5600 or a Jeol FE-SEM7000 using appropriate machine settings.
We acknowledge the National Medical Research Council, Singapore, for research funding (NMRC/0956/2005). We thank Geoff Toms for the HMPV A2 stain NCL03-4/174 and the AT1 (anti-G) and Mab58 antibodies. L.H. Loo was a recipient of a NMRC-Lee foundation PhD scholarship.
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