ABSTRACT
Enveloped viruses utilize surface glycoproteins to bind and fuse with a target cell membrane. The zoonotic Hendra virus (HeV), a member of the family Paramyxoviridae, utilizes the attachment protein (G) and the fusion protein (F) to perform these critical functions. Upon triggering, the trimeric F protein undergoes a large, irreversible conformation change to drive membrane fusion. Previously, we have shown that the transmembrane (TM) domain of the F protein, separate from the rest of the protein, is present in a monomer-trimer equilibrium. This TM-TM association contributes to the stability of the prefusion form of the protein, supporting a role for TM-TM interactions in the control of F protein conformational changes. To determine the impact of disrupting TM-TM interactions, constructs expressing the HeV F TM with limited flanking sequences were synthesized. Coexpression of these constructs with HeV F resulted in dramatic reductions in the stability of F protein expression and fusion activity. In contrast, no effects were observed when the HeV F TM constructs were coexpressed with the nonhomologous parainfluenza virus 5 (PIV5) fusion protein, indicating a requirement for specific interactions. To further examine this, a TM peptide homologous to the PIV5 F TM domain was synthesized. Addition of the peptide prior to infection inhibited infection with PIV5 but did not significantly affect infection with human metapneumovirus, a related virus. These results indicate that targeted disruption of TM-TM interactions significantly impact viral fusion protein stability and function, presenting these interactions as a novel target for antiviral development.
IMPORTANCE Enveloped viruses require virus-cell membrane fusion to release the viral genome and replicate. The viral fusion protein triggers from the pre- to the postfusion conformation, an essentially irreversible change, to drive membrane fusion. We found that small proteins containing the TM and a limited flanking region homologous to the fusion protein of the zoonotic Hendra virus reduced protein expression and fusion activity. The introduction of exogenous TM peptides may displace a TM domain, disrupting native TM-TM interactions and globally destabilizing the fusion protein. Supporting this hypothesis, we showed that a sequence-specific transmembrane peptide dramatically reduced viral infection in another enveloped virus model, suggesting a broader inhibitory mechanism. Viral fusion protein TM-TM interactions are important for protein function, and disruption of these interactions dramatically reduces protein stability.
INTRODUCTION
The importance of transmembrane (TM) domains in protein oligomerization has been shown for several cellular proteins. For example, the amyloid precursor protein (APP) TM must dimerize for the generation of amyloid β, which is linked to Alzheimer’s disease (1). The TM domain of neuropilin 1 (NRP1), a coreceptor for vascular endothelial growth factor receptor 2, contains a GXXXG motif that is required for dimerization and, ultimately, downstream signaling (2). It has been shown that targeting the NRP1 TM domain with synthetic peptides could inhibit glioma tumor growth in vivo (3). Together, these studies exemplify the potential of targeting the transmembrane domain to disrupt protein function and/or trafficking, which could lead to potential therapeutic targets. Moreover, the importance of TM-TM interactions in protein function is apparent from these studies. In the case of viral systems, replacement of the viral fusion protein TM domain with other fusion protein TM domains or lipid anchors has been shown to alter fusion protein function in examples, including influenza virus, Newcastle disease virus, vesicular stomatitis virus, parainfluenza virus 5 (PIV5), and measles virus (4–9). Additionally, mutation of motifs within the TM domain known to promote protein oligomerization, such as GXXXG motifs, affects fusion protein function. For example, mutation of the GXXXG motif of the Hendra virus (HeV) fusion (F) protein TM domain resulted in a reduction of active fusion protein at the cell surface, possibly as a result of reduced protein stability or changes in protein trafficking (10, 11). Mutation of the GXXXG motif of HIV gp41 also affected intracellular trafficking of the HIV Env protein (12, 13).
The life cycle of an enveloped virus requires fusion of the viral envelope with a target cell membrane. There are several points in the early stages of the paramyxovirus fusion process that can be targeted for disruption: the receptor binding step mediated by the attachment protein, the interaction between the attachment protein and the fusion protein that facilitates triggering of F, and the overall refolding of the fusion protein necessary for membrane fusion (14). Class I fusion proteins, including those of the family Paramyxoviridae, are folded as trimers (15), and it has been shown that the TM domain is important for proper protein folding and function (5, 6, 9, 12, 16–18). To drive membrane fusion, the fusion protein must undergo dramatic conformational rearrangements from the metastable prefusion conformation to the postfusion conformation. The triggering process of the fusion protein is an essentially irreversible process, so spatiotemporal control of the triggering and refolding events is crucial. While there is little sequence homology among class I fusion proteins, the steps critical for membrane fusion appear to be conserved (15).
Previously, we have shown that the HeV F protein TM domain associates in a monomer-trimer equilibrium in isolation and contributes to overall F protein stability. More specifically, we have shown that HeV F TM-TM association is sequence specific, with a leucine/isoleucine (L/I) zipper motif significantly contributing to the interaction (10, 18). The HeV F protein is synthesized as a trimer with a domain structure composed of two heptad repeat domains (HRA and HRB), a highly hydrophobic fusion peptide (FP), a TM domain, and a cytoplasmic tail (CT) (Fig. 1A) (19, 20). Since the TM domain self-associates and has been shown to be important for F protein trafficking and function (21), we hypothesized that introduction of exogenous TM proteins homologous to the native F protein would competitively disrupt the TM-TM interactions in the native F protein, resulting in premature triggering or protein misfolding. To test this, exogenous constructs containing the Hendra virus F TM and limited flanking sequences were coexpressed with the full-length F protein. We demonstrate that the homologous TM protein constructs interacted with F and reduced the expression and fusion activity of the full-length protein. Furthermore, we show that the effects seen upon coexpression of the TM proteins are sequence specific. Since HeV is a biosafety level 4 (BSL4) pathogen, PIV5, another paramyxovirus, was used to test whether F protein function could be disrupted in viral particles. A synthetic TM peptide homologous to the PIV5 F TM domain successfully inhibited viral infection in cells, and the effect was specific, as treatment of human metapneumovirus (HMPV) with the PIV5 F TM peptide did not significantly affect infectivity. Together, these results emphasize the importance of the TM domain in fusion protein function and present fusion protein TM-TM interactions as a potential antiviral target.
HeV F TM constructs colocalize and interact with wild-type HeV F. (A) Domain structure of the HeV F protein containing a fusion peptide (FP), two heptad repeat regions (HRA and HRB), a transmembrane domain (TM), and a C tail (CT). A general schematic of TM protein design is shown, with amino acid sites indicated. Each construct includes a signal peptide, the full TM and CT, an HA tag, and a linker region that has varying lengths of the HRB domain. (B) Immunofluorescence of coexpression of HeV F (green) and each of the TM constructs (red). The insets show ×4 magnifications of the boxed areas. (C) Coimmunoprecipitation of HeV F with the TM constructs. Cells were starved for 45 min, radiolabeled for 30 min, and chased for 1.5 h prior to lysis and pulldown with an anti-HA antibody.
RESULTS
HeV F TM proteins interact with the full-length HeV F.To test whether addition of homologous TM domains affects F protein folding, trafficking, or function, exogenous constructs containing the TM were designed based on the HeV F protein sequence. Three constructs were synthesized to contain a signal peptide, a variable-length linker, the full-length TM domain and C tail, and a hemagglutinin (HA) tag (Fig. 1A). A signal peptide was included to target the TM constructs to the endoplasmic reticulum (ER) during translation. The linker represented varying lengths of the HRB domain upstream of the TM domain added to assist with protein solubility, as it was unclear how stable the TM domain would be by itself. These different constructs are designated short linker (SL), long linker (LL), and HRB TM (HRB) (Fig. 1A) here. The C tail was included to maintain proper protein trafficking (22, 23), as well as for antibody detection, and the HA tag was added for antibody detection purposes. During HeV F synthesis, the inactive form of the protein (F0) is trafficked through the ER to the plasma membrane, endocytosed, cleaved to its active form (F1) by cathepsin L, and trafficked back to the plasma membrane, where it can ultimately promote membrane fusion. To determine if the signal peptide directed the TM proteins to the secretory pathway, immunofluorescence assays were performed to determine the coexpression of HeV F with each of the TM constructs. Wild-type (WT) HeV F (green) and each of the TM proteins (red) colocalized in similar regions throughout the cell (Fig. 1B). A strong signal appeared near the nucleus at the expected location of the ER, which was to be expected with the addition of the signal peptide on the TM proteins. These constructs were then coexpressed with the full-length HeV F protein in Vero cells at a DNA transfection ratio of 2:1 (F/TM). Cells were radiolabeled for 30 minutes and chased for 1.5 h; then, pulldown with an anti-HA antibody was used to examine if the full-length HeV F protein would coimmunoprecipitate (co-IP) with the HA-tagged TM proteins. As a control, a plasmid encoding an HA-tagged HeV F protein was cotransfected with the mock control, and then, F-HA was immunoprecipitated with the anti-HA antibody (Fig. 1C). When the TM constructs were cotransfected with WT F, the uncleaved form, F0, was pulled down with the SL and LL TM constructs. Interestingly, the HRB TM construct did not appear to pull down F. These results demonstrated that the TM proteins colocalize and that the SL and LL constructs interact with the full-length F protein.
Exogenous TM proteins affect HeV F expression and protein stability.To facilitate fusion, the HeV F protein must be at the surface and in its cleaved (F1+F2) form. Previous work has shown that disruption of the HeV TM interactions significantly lowered the amount of F protein expressed at the cell surface (18). To determine whether the F protein was trafficked to the cell surface in the presence of the exogenous TM proteins, the proteins were analyzed with a radiolabel surface biotinylation assay. When HeV F was coexpressed with an empty vector and cells were radiolabeled for 3 h, the F protein was observed as two bands, F0 (uncleaved) and F1 (cleaved), in both total expression and the surface population. Upon coexpression of each of the TM constructs, total and surface expression levels of HeV F were reduced 20 to 30% compared to the HeV F expressed with the empty vector (Fig. 2A and B). Additional transfection ratios were tested using a radiolabel immunoprecipitation (IP), but the 2:1 HeV F/TM construct ratio resulted in the greatest change in F protein expression levels and was the transfection ratio used for the rest of the experiments, unless otherwise noted (Fig. 2D). Though the presence of the exogenous TM constructs dramatically reduced the levels of HeV F detected in cells, a small population of cleaved, potentially fusogenically active F was detected at the surface. A pulse-chase experiment was performed to determine whether the TM proteins affected initial F protein synthesis or stability over time (Fig. 2C). When coexpressed with mock vector at the early time points, F was detected in its uncleaved form, F0. By 8 h, the majority of F was present in the cleaved form (F1+F2; only F1 was visible on the gel), consistent with the previously reported time necessary for cathepsin L cleavage (24). Coexpression of HeV F with the SL TM protein resulted in a dramatic decrease in expression starting at early time points. However, the overall expression of F was not affected at the earlier time points for LL or HRB TM protein coexpression, indicating that the presence of these proteins does not affect initial protein synthesis. The coexpression of the LL TM protein instead affected the overall stability of HeV F over time, as shown by the reduced amount of either form of F detected at the 6-h and 8-h time points. The HRB TM protein appeared to have a less dramatic effect but still showed a reduced amount of either form of F at 8 h, indicating that some protein destabilization might be occurring. In the presence of each of the TM proteins at the earlier time points (0 to 2 h), a band appeared between the F0 and F1 bands, which might have been a degradation product or the result of a change in glycosylation. Coexpression with any of the three TM proteins still allowed some level of cleavage (F1+F2; only F1 was visible on the gel) by the 2-h time point, which is when the most cleaved product is detected for the mock coexpression. Differences in protein expression between Fig. 2A and C can be accounted for by differences in metabolic labeling and chase times; Fig. 2A represents a 3-h label and no chase, while Fig. 2C represents a 30-min label and the indicated chase times. These findings suggest that the presence of the exogenous TM proteins, especially LL and SL, destabilized the full-length F protein but did not affect the ability of synthesized protein to be cleaved to its fusogenically active form.
Coexpression of HeV F with the TM proteins reduces expression, protein stability, and stable oligomerization. (A) Surface biotinylation was used to analyze the total and surface populations of HeV F cotransfected with mock empty vector or each of the TM constructs. Proteins were radiolabeled for 3 h. (B) Relative HeV F protein expression was quantified using band densitometry (ImageQuant); experiments were performed in triplicate, with the standard deviations shown. (C) Pulse-chase radiolabel IP was used to determine the effects the TM constructs had on WT HeV F over time. Proteins were radiolabeled for 30 min, with the chase times noted above each gel in hours. (D) Various transfection ratios of the HeV F-TM constructs were tested in a radiolabel IP using the SL construct. (E) The effects of exogenous TM construct coexpression on HeV F protein oligomerization were analyzed by nonreducing SDS-PAGE.
Since HeV F associates as a homotrimer, an assay to detect higher-molecular-weight complexes was performed to determine if destabilization of the protein in the presence of exogenous TM proteins was due to destabilization of F trimerization. Cells transfected with F alone or F plus the TM constructs were radiolabeled for 45 min, and chase medium was added for 30 min. The cell lysates were then immunoprecipitated with anti-HeV F antibody to the C tail; heated at various temperatures to destabilize oligomeric interactions, as indicated; and separated on a gel under nonreducing conditions (Fig. 2E). When HeV F protein was expressed alone, a majority of the protein migrated as a monomer; however, heat-stable trimers and dimers are also detectable. These results are consistent with previous reports on other paramyxoviruses that have shown that uncleaved forms of the fusion protein can migrate as stable trimers, even up to 100°C (25). However, when any of the TM proteins were present, a large amount of the protein migrated in the monomeric form. These results suggest that the presence of the TM proteins may destabilize HeV F protein trimeric associations at early time points in protein synthesis. Interestingly, this destabilization of protein oligomerization appears to occur even before protein expression levels are affected in the presence of the LL and HRB constructs (compare LL and HRB in Fig. 2E to the 1- to 2-h time point in Fig. 2C).
Exogenous TM proteins reduce HeV F fusion activity.A syncytium fusion assay was used to determine whether the TM proteins affected F protein function. The F protein and its homotypic attachment protein, G, were coexpressed in Vero cells with each of the TM constructs at a G/F/TM transfection ratio of 3:1:1. After 24 h, the cells were imaged to visualize syncytium formation. HeV F and G coexpressed with the empty vector resulted in syncytium formation, as indicated by the white arrows in Fig. 3A. When the TM constructs were coexpressed with HeV and G, syncytium formation was ablated. The reduced overall expression levels of HeV F might explain a reduction in syncytium formation. However, a small population of fusogenically active HeV F was still detected in the surface population in the presence of the TM proteins (Fig. 2A), so reduced levels of fusion activity, rather than a complete loss, would be expected. Previous work demonstrated that reduction of HeV F expression to 20 to 30% of the wild-type level resulted in fusion levels at 30 to 40% of the wild-type level (26). Interestingly, coexpression of the TM constructs reduced HeV F expression to these levels, but fusion activity was completely ablated. The lack of fusion seen may be the result of premature triggering or misfolding of the fusion protein present at the plasma membrane. Alternatively, the presence of the TM proteins could prevent the fusion protein from being able to fully trigger or affect further conformational changes needed to drive fusion.
Coexpression of HeV F with the TM proteins reduces fusion activity. (A) To determine the fusion activity of HeV F in the presence of the TM constructs, cells were imaged for the presence of syncytia (indicated by arrows) at 24 h after transfection of HeV F and HeV G with each of the TM constructs. (B) Schematic of the reporter gene fusion assay experimental setup, showing experimental condition 1, with the TM constructs in the same cells as HeV F and G, and experimental condition 2, in which the TM constructs were in the target (BSR T7) cells. (C and D) Results from the luciferase reporter gene assay for condition 1 (C) and condition 2 (D). The results are representative of three independent experiments, each performed in duplicate. Significance, compared to the WT, was determined by Student's t test. *, P < 0.05; **, P < 0.001.
To further investigate the fusion activity of F in the presence of the TM constructs, a luciferase reporter gene assay was performed. HeV F and G, a luciferase plasmid under the control of a T7 promoter (labeled T7-Luciferase plasmid in Fig. 3B), and each of the TM constructs was coexpressed in Vero cells. At 18 to 24 h posttransfection, BSR cells that constitutively expressed the T7 polymerase were overlaid onto the Vero cells at a ratio of 1:1 and allowed to incubate for 3 h (Fig. 3B, Experimental Condition #1). After 3 h, the cells were lysed and incubated with luciferin, and then the luminescence was measured as an output of the fusion. The relative luminescence compared to HeV F and G without the TM constructs indicated that fusion was significantly reduced with each construct (Fig. 3C). The presence of the SL and LL constructs reduced the fusion to background levels, suggesting again that the SL and LL constructs interfere with the fusion activity of HeV F beyond just reducing protein surface expression. Fusion in the presence of the HRB construct, however, reduced fusion to levels consistent with that expected with the cleaved population of F present at the cell surface (Fig. 1B). However, when these results are considered along with the lack of syncytium formation (Fig. 3A) and the destabilization of protein oligomerization (Fig. 2E) seen in the presence of the HRB construct, it may suggest that this construct has an effect on F protein fusion pore expansion or other cellular rearrangements needed for syncytium formation.
Upon triggering, HeV F extends into a hairpin structure that inserts the fusion peptide into the target membrane. Since interactions between viral fusion protein TMs and the fusion peptide have previously been shown (27), a luciferase gene reporter assay was again employed to investigate the effects the exogenous TM peptides had when located in the target cell membrane (Fig. 3B, Experimental Condition #2). The relative luminescence compared to WT HeV demonstrated that when the SL and HRB TM proteins were present in the target cell membrane there was a 20 to 30% reduction in fusion (Fig. 3D). The LL TM protein did not produce a significant decrease in fusion. This reduction may be due to disruption of proper refolding of HeV F or destabilization of the fusion peptides caused by the exogenous TM proteins associated with the fusion peptide. Though an effect was seen under these conditions, it is apparent that the TM peptide effects are most significant when the peptides are coexpressed with HeV F.
Effects of exogenous TM proteins are sequence specific.The previous results demonstrated that HeV F protein levels were reduced upon coexpression of homologous TM constructs. To determine whether the HeV F TM proteins were specifically targeting the HeV F protein, analogous experiments were performed with another class I fusion protein of the paramyxovirus family, PIV5 F. The predicted TM domain sequences of HeV F and PIV5 F exhibit approximately 40% homology, as calculated with the ExPASy SIM alignment tool (Fig. 4A). Coexpression of the exogenous HeV F TM constructs with PIV5 F in Vero cells demonstrated no significant change in PIV5 F protein expression levels. The TM proteins also did not co-IP the PIV5 F protein in its F0 or F1 form (Fig. 4B and C). Immunofluorescence of PIV5 F with the HeV F TM constructs was also performed. While, the HRB TM protein did not appear to colocalize with PIV5 F in immunofluorescence staining, the SL and LL TM proteins were expressed in cellular compartments similar to those for PIV5 F (Fig. 4E), and the PIV5 F protein was able to drive membrane fusion and promote syncytium formation in the presence of all three exogenous TM constructs, as indicated by the arrows in Fig. 4D. Together, these data indicate that the TM constructs designed to target the HeV F TM domain do not produce the same effects on PIV5 F expression or fusion activity, suggesting that the effect is sequence specific.
PIV5 F expression and fusion activity are not affected by HeV F TM proteins. (A) TM sequences of HeV and PIV5 F proteins, predicted with TMHMM Server v2.0. (B) PIV5 F expression when coexpressed with each of the HeV TM proteins, determined by radiolabel immunoprecipitation with pulldown by anti-PIV5 F or anti-HA. Protein expression levels were determined by band density. (C) Relative protein expression levels of PIV5 F in the presence of the HeV F TM constructs compared to that of PIV5 F+mock were determined by band density. Experiments were performed in triplicate, with standard deviations shown. (D) Syncytium formation assay revealed that coexpression of TM constructs did not inhibit PIV5 F fusion activity. Syncytia are indicated by arrows. (E) Immunofluorescence analysis of the coexpression of PIV5 F (green) with HeV TM constructs (red).
TM peptides reduced viral infection.The results presented above utilized transient-transfection experiments to test whether the TM domain can be targeted to affect fusion protein expression and function. To determine if the F protein TM domain could be targeted in the context of a viral particle, an infection assay was performed. As Hendra virus is a BSL4 pathogen, TM peptide effects were examined in two other closely related enveloped viruses, PIV5 and HMPV, which both utilize class I fusion proteins to mediate membrane fusion. The predicted TM domains for the two viruses, however, exhibit only approximately 26% sequence homology. Recombinant viruses contained a green fluorescent protein (GFP) gene for either virus to allow visualization of infection. A peptide was designed based on the sequence of the PIV5 F TM domain, not including the C tail or HRB domain, and the highly hydrophobic peptide was resuspended in dimethyl sulfoxide (DMSO). HMPV or PIV5 was incubated with varying concentrations of peptide for 30 min and then added to Vero cells to allow infection (multiplicity of infection [MOI] = 1 PFU/cell). After 24 h, the cells were imaged and prepared for flow cytometry to count GFP-expressing cells (Fig. 5A). When the viruses were mock treated with DMSO (0 μM peptide), the infections for both were widespread. Treatment with TM peptide reduced the number of GFP-expressing cells for PIV5 (Fig. 5B). Addition of 10 μM peptide resulted in approximately 75% reduction in PIV5 infection, as determined by flow cytometry (P < 0.0001; Student’s t test). The HMPV samples treated with peptide did not exhibit a significant reduction in GFP-expressing cells, suggesting that the effect of the TM peptide utilized was specific to PIV5 F. Together, these results demonstrate that the TM domain can be targeted to disrupt F protein function and viral infection in a sequence-specific manner.
PIV5 F TM peptide inhibits PIV5 infection selectively. (A) GFP-expressing PIV5 or HMPV was incubated with TM peptide designed to target the PIV5 F TM domain; then, cells were infected with the treated virus. (B) GFP-expressing cells were imaged 24 h later and counted for quantification via flow cytometry. Experiments were performed in triplicate. Student's t test was used to determine significance; *, P < 0.0001.
DISCUSSION
Viral fusion proteins drive the fusion of viral and cellular membranes, a key early step in the entry of enveloped viruses. We have previously shown that the TM domains of several paramyxovirus fusion proteins associate in isolation and that disruption of TM-TM association by mutagenesis of a key L/I zipper motif resulted in a HeV F protein that triggered prematurely (11, 18). Based on the previous studies, we examined the ability of paramyxovirus TM-TM interactions to be targeted with exogenous proteins containing the TM domain to cause disruption in overall protein structure and function (Fig. 6). With the creation of three constructs containing the HeV F TM domain, we confirmed that coexpression of the native HeV F protein with any of the three constructs resulted in reduction of HeV F protein expression (Fig. 2A), protein stability (Fig. 2C), and disruption of the trimeric interactions of HeV F (Fig. 2E). Interestingly, the successful cleavage and presence of HeV F on the cell surface (Fig. 2A), as well as the cleavage patterns seen in the pulse-chase experiment (Fig. 2C), suggests that the exogenous TM-containing proteins did not alter proper trafficking of the F protein that successfully exited the ER. Additionally, examination of protein stability and oligomerization suggests that either the destabilization of trimeric interactions and/or premature triggering could cause early protein degradation, as it has been previously shown that prematurely triggered protein is quickly degraded (28).
Model for TM peptide interaction with fusion protein. The introduction of homologous TM protein may cause the fusion protein to misfold or prematurely trigger, as a result of the exogenous TM protein interacting with the native fusion protein TM domain.
The fusion process by which paramyxoviruses enter cells involves a series of steps, including attachment protein-mediated receptor binding; a triggering event of the fusion protein facilitated by the attachment protein; a dramatic, irreversible conformational change to merge the cellular and viral membranes to form a fusion pore; and fusion pore expansion (29). Not only were protein expression, stability, and stable oligomerization of native HeV F disrupted in the presence of the TM constructs, the coexpression of the TM constructs also dramatically reduced the fusion activity of HeV F (Fig. 3). The presence of the SL and LL constructs reduced fusion to background levels in both syncytia (Fig. 3A) and reporter gene fusion assays (Fig. 3C), suggesting the presence of these constructs may disrupt key steps in the membrane fusion process, such as protein triggering or protein conformational changes. Interestingly, the presence of the HRB construct appeared to completely abolish syncytium formation (Fig. 3A), similar to the other constructs; however, in reporter gene fusion experiments, HeV F was still able to facilitate about 20 to 30% of WT levels of fusion (Fig. 3C). It has been previously shown that there is a correlation between cell surface expression and fusogenic activity (26), and while the reporter gene assay results in the presence of the HRB construct would fit with that correlation, the lack of syncytium formation would not. This discrepancy may be due to the presence of HRB constructs disrupting late stages of fusion, such as fusion pore expansion or further cellular rearrangements, as reporter gene fusion assays require only fusion pore opening and expansion while syncytium formation requires additional dramatic cellular rearrangements. A previous study has shown that several enveloped viruses demonstrated luciferase fusion activity while they did not show syncytium formation (30), suggesting that downstream cellular rearrangements may be blocked even when fusion pore formation and expansion have occurred. Additionally, the reporter gene assay results with the exogenous TM constructs inserted into the target membrane (Fig. 3B, Experimental Condition #2) provided evidence that the nature of TM construct disruption was not due simply to global membrane alterations. Interestingly, a small but significant decrease was seen with the addition of the SL and HRB constructs in the target membrane (Fig. 3D). Since it has been previously shown that the TM domain and the fusion peptide of HeV F interact (27), the presence of these constructs may interfere with the ability of the fusion peptide to induce the membrane disorder needed for cell-cell fusion. Alternatively, the HRB construct may interfere with the formation of the six-helix-bundle formation, a critical step in paramyxovirus membrane fusion (19), as it has been shown for the viral family that peptides mimicking the HRB region can interact with the HRA in the hairpin intermediate to block viral fusion (31).
While the experiments presented in this paper support a model in which disruptions to TM domain interactions cause lower protein expression and function, the constructs created do contain other portions of the F protein (various lengths of the HRB, C tail, and signal peptides and an HA tag). Based on the cellular experiments completed (Fig. 2 and 3), more dramatic reductions in protein expression and fusion activity correlated with the presence of shorter lengths of the HRB region (the SL construct elicited the largest effect, followed by the LL construct). This suggests that these effects are not due to the HRB region. While the cellular experiments cannot rule out the possibility that the HA tag, C tail, or signal peptides present are not causing the examined effects, the peptides tested in the PIV5 infection system (Fig. 5) contained only the TM domain and still promoted a reduction in viral infection. This demonstrates that the exogenous TM domain alone can create effects similar to those seen in the cellular experiments. In addition to recent work completed in suppression of hyperactive immune cells (32), inhibition or suppression of tumor growth in several cancers (33–35), and inhibition of oncogenic activation induced by Epstein-Barr virus (36), these experiments further support the notion that the effects seen are specific to destabilizing TM interactions.
The effects on protein stabilization and fusion activity in the presence of the HRB construct were less dramatic than when the SL or LL construct was present. We have previously shown that including the full-length HRB destabilizes HeV F TM-TM interactions (10), supporting the idea that the lower effectiveness of these constructs could be due to the full-length HRB creating weakened TM interactions between the exogenous construct and the TM of the native F protein. This may impair the ability of the HRB construct to interact with the native F protein, as shown by the failure to co-IP (Fig. 1C). Alternatively, the full-length HRB on the exogenous TM construct could interact with HRA of the native protein (31), reducing the ability to associate with the native F protein TM, or the HRB on the exogenous TM construct could be self-trimerizing, causing the exogenous TM proteins to traffic as a trimeric unit. In the infection assay, which served as a proof-of-concept tool, a peptide containing only the TM domain could specifically affect viral infection. Our cellular experiments presented here demonstrate that small portions of the protein ectodomain (the HRB region) can be added without significantly affecting the reduction of viral-protein-mediated fusion. The inclusion of these small portions of this region may help with the peptide solubility of the highly hydrophobic TM peptides.
F proteins of paramyxoviruses and other enveloped viruses have specific sequence requirements for their TM domains, and simply substituting the TM domain of one paramyxovirus F protein into another, related F protein can abolish membrane fusion of that F protein without affecting overall cell surface expression (5). The specificity of the effect of exogenous TM protein was determined by coexpressing PIV5 F with the HeV F TM-containing proteins in cellular experiments and coexpressing HMPV F with PIV5 F TM peptides in the infection system. Neither PIV5 F (Fig. 4) nor HMPV F (Fig. 5) demonstrated a significant effect on expression or function in the presence of a nonhomologous exogenous TM construct or peptide. Interestingly, these PIV5 F and HMPV F proteins are predicted to have a heptad repeat leucine/isoleucine zipper motif in the TM domain similar to that of HeV F (18), so while this is an important oligomerization motif in TM-TM interactions for these proteins, there appear to be sequence-specific requirements for these interactions happening beyond this motif. These findings support the premise that the TM-TM interactions are sequence-specific interactions and not simply the result of proximity to certain interaction motifs or hydrophobicity.
The results from the PIV5 infection system, taken together with the results from the HeV F cellular experiments, highlight the critical nature of fusion protein TM-TM interactions for maintenance of viral infectivity and demonstrate a potential for small-molecule inhibitors that disrupt TM-TM interactions or modulators of lipid environment to serve as antiviral candidates. A study with paramyxoviruses found that a broad-spectrum small-molecule drug (JL103) could prevent membrane fusion by altering lipid architecture (37). Changes in the lipid environment caused by small-molecule inhibitors may be able to alter fusion protein TM-TM interactions and therefore disrupt fusion protein function. A recent study with Epstein-Barr virus showed that a small molecule targeting the TM domain could disrupt homotrimerization of latent membrane protein 1, a step that is critical for oncogenic activation of the virus (36). Other studies have shown that targeting antiviral peptides to the cell membrane with cholesterol enhanced efficacy (38). The inclusion of some portion of the TM domain may be able to further enhance specificity, in addition to physically anchoring the peptide. Beyond viral proteins, the concept of disrupting TM-TM interactions may prove to be a viable therapeutic option. The tyrosine kinase receptor ErbB2 was found to be overexpressed in high-grade inflammatory breast cancer. ErbB2 requires dimerization in order to trigger downstream signaling cascades that include the MAPK pathway. TM peptides that disrupt ErbB2 TM dimerization were found to reduce tumor cell growth and metastasis (39). Other studies have demonstrated the potential use of small molecules that disrupt TM domain oligomerization to suppress tumor growth in glioma and metastasis by targeting the TM of plexin A1 (34) and to inhibit tumor growth in melanoma by TM interactions in p75NTR (33). The results presented may further help in development or improvement of antivirals and other TM-targeting therapeutics. More importantly, our results show the important role of TM-TM interactions in the stability of the viral fusion protein on a global level. Utilizing TM-TM interactions to disrupt fusion protein function may also extend beyond paramyxoviruses, as we have recently shown that TM-TM interactions occur for fusion proteins of the families Coronaviridae, Rhabdoviridae, Orthomyxoviridae, and Filoviridae (40). These studies further broaden our understanding of the hydrophobic TM domain and demonstrate their functional role beyond serving as a hydrophobic membrane anchor.
MATERIALS AND METHODS
Cell lines and culture.Vero cells and BSR T7/5 cells (generously provided by Karl-Klaus Conzelman, Pettenkofer Institute) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen) supplemented with 10% fetal bovine serum (FBS). BSR T7/5 cells were selected for the expression of the T7 polymerase by the addition of G418 sulfate (Gibco/Invitrogen) to the DMEM every third passage.
Plasmids, viruses, and antibodies.Plasmids containing Hendra virus F or G were generously provided by Lin-Fa Wang (Australian Animal Health Laboratory) and were transiently expressed in Vero cells, as described previously (22). The Hendra virus F TM constructs were designed to include the predicted HeV F signal peptide, varying lengths of the HRB domain, the full-length TM domain, the C tail, and an HA tag. The Hendra virus F TM constructs were synthesized with GenScript and provided in the pUC57 vector. Each construct was subcloned into the eukaryotic expression vector pCAGGS, as described previously for Hendra virus F (10). Anti-peptide antibodies to residues 527 to 539 of the Hendra virus F cytoplasmic tail were used to pull down F and the TM proteins. Additionally, monoclonal antibody (MAb) 5G7, provided by Christopher Broder (Uniformed Services University [USUHS]), was used for immunoprecipitation to detect Hendra virus F. Coimmunoprecipitation studies were performed with a mouse anti-HA antibody (12CA5; Roche). For immunofluorescence assays, a rabbit anti-HA antibody (Abcam; ab9110) was used at 1:300 dilution to detect the TM proteins. Secondary antibodies used for immunofluorescence assays were goat anti-mouse–fluorescein isothiocyanate (FITC) and goat anti-mouse–tetramethyl rhodamine isocyanate (TRITC) (Jackson Immuno Research). The recombinant HMPV (rgHMPV) was kindly provided by Peter L. Collins and Ursula J. Buchholz (NIAID, Bethesda, MD). The recombinant PIV5 (rgPIV5) was kindly provided by Robert Lamb (Howard Hughes Medical Institute).
Surface biotinylation.Vero cells (80 to 90% confluence) in 60-mm dishes were transiently transfected using Lipofectamine and Plus reagent (4 μg of DNA) (Invitrogen) according to the manufacturer’s protocol. For coexpression experiments, the ratio of DNA transfected was 2:1 HeV F/TM, unless otherwise noted. Eighteen to 24 h posttransfection, cells were washed with phosphate-buffered saline (PBS) and starved for 45 min in DMEM deficient in cysteine and methionine. The cells were labeled for 3 h with DMEM deficient in cysteine and methionine and containing Tran35S label (100 μCi/ml; MP Biomedicals), biotinylated with 1 mg/ml EZ-Link Sulfo-NHS biotin (Pierce) diluted in PBS, pH 8.0, at 4°C for 35 min, followed by 15 min at room temperature. The cells were then lysed in RIPA lysis buffer, and the supernatants were cleared by centrifugation at 135,000 × g for 15 min at 4°C; 4 μl of an anti-peptide Hendra virus F antibody was added to the supernatants and incubated at 4°C for 3 h with rocking and then incubated with 30 μl of protein A-Sepharose beads (GE Healthcare). The immunoprecipitated proteins were then washed two times with each of the following, in order: radioimmunoprecipitation assay (RIPA) buffer containing 0.3 M NaCl, RIPA buffer containing 0.15 M NaCl, and SDS wash II buffer (150 mM NaCl, 50 mM Tris-HCL [pH 7.4], 2.5 mM EDTA). Samples were then boiled away from the beads in 10% SDS. Ten percent of the protein was removed as the “total” sample, and the remainder (“surface”) was diluted in biotinylation dilution buffer and incubated with immobilized streptavidin beads (Pierce) at 4°C for 1 h. Samples were again washed as described above. The samples were analyzed by SDS-15% PAGE, exposed to a phosphor screen, and visualized using the Typhoon imaging system (GE Healthcare), as previously described (11). Band densitometry using ImageQuant 5.2 was performed for each experiment to quantitate the amount of F expressed, which was reported as percent expression, the sum of F0 and F1, normalized to the F-plus-mock control.
Time course immunoprecipitation.Hendra virus F protein was coexpressed with the TM constructs in subconfluent Vero cells using Lipofectamine and Plus reagent (Invitrogen) as previously described. The next day, the cells were washed with PBS and starved for 45 min at 37°C in cysteine-methionine-deficient DMEM. The cells were then labeled for 30 min with Tran35S metabolic label (100 μCi/ml; MP Biomedicals). At different time points, the cells were washed three times with PBS and lysed with RIPA lysis buffer. Immunoprecipitation with the anti-peptide Hendra virus F antibody and protein A-sepharose beads and analysis were performed as described for surface biotinylation.
Higher-molecular-weight immunoprecipitation and native gel electrophoresis.Hendra virus F protein was coexpressed with the TM constructs in subconfluent Vero cells using Lipofectamine and Plus reagent (Invitrogen) as previously described. The next day, the cells were washed with PBS and starved for 45 min at 37°C in cysteine-methionine-deficient DMEM. The cells were then labeled for 30 min with Tran35S metabolic label (100 μCi/ml; MP Biomedicals) and then washed three times with PBS and allowed to incubate at 37°C for the indicated chase times. The cells were washed three times with PBS and lysed with RIPA lysis buffer. The lysates were incubated with anti-peptide antibodies to residues 527 to 539 of the Hendra virus F cytoplasmic tail for 3 h and subsequently with protein A-Sepharose beads for 30 min, as described for surface biotinylation. After the washing steps were completed, 30 μl of 2× loading buffer without dithiothreitol (DTT) was added to each of the samples. The samples were then boiled away from the beads at 60°C, 80°C, or 100°C, as indicated, for 10 min and analyzed on a 3.5% acrylamide gel under nonreducing conditions, and the gel was imaged on the same system described for surface biotinylation.
Syncytium assay.Subconfluent Vero cells in 6-well plates were transiently transfected with Hendra virus F, Hendra virus G, and TM protein at a ratio of 1:3:1 using Lipofectamine and Plus reagent (Invitrogen) according to the manufacturer’s protocol. Syncytium formation was observed 24 to 48 h posttransfection. Images were taken using a Nikon digital camera mounted atop a Nikon TS100 microscope with a 10× objective.
Luciferase reporter gene assay.Vero cells in 6-well plates were transiently transfected with Hendra virus G, Hendra virus F, T7 luciferase under the control of the T7 promoter, and the TM constructs at a ratio of 3:1:3:1 (2 μg total DNA) using Lipofectamine and Plus reagent according to the manufacturer’s protocol. Eighteen hours posttransfection, the Vero cells were washed once with PBS and overlaid with BSR cells (approximately 1:1) stably expressing the T7 polymerase for 3 h at 37°C. The cells were lysed and assayed for luciferase activity using the Luciferase assay system from Promega (Madison, WI) according to the manufacturer’s instructions. An Lmax luminometer (Molecular Devices, Sunnyvale, CA) was used to measure light emission; a 2-s delay and a 5-s read time were used. Values were normalized to those of samples containing wild-type Hendra virus F and G, with the wild type set at 100%, after subtraction of the values for Hendra virus G alone.
Peptide inhibition assay.Recombinant GFP-expressing HMPV or PIV5 was pretreated for 30 min at room temperature with TM peptide corresponding to the TM domain sequence of the PIV5 F protein. The peptide was synthesized by LifeTein (Somerset, NJ) with the following sequence: 485VLSIIAICLGCLGLILIILLSVVVWKLL512 (accession no. P04849). The peptide was solubilized in sterile DMSO. Virus was diluted in Opti-MEM for infection at an MOI of 1 PFU/cell. Vero cells (70 to 90% confluence) were washed twice with PBS and incubated with 500 μl of peptide-treated virus for 4 h at 37°C. After the incubation, the infection medium was removed, and the cells were washed twice with PBS and left overnight with DMEM plus FBS. The following day, the cells were imaged for GFP-positive cells with a Nikon Axiovert-100 microscope. For flow cytometry analysis, the cells were lifted with 100 μl of trypsin-EDTA and fixed with an equal volume of PBS-2% paraformaldehyde-50 mM EDTA. For each sample, 50,000 cells were counted for flow analysis. Analysis was performed on the LSR II flow cytometer by the UK Flow Cytometry Core.
Immunofluorescence.Cells grown in 6-well plates containing coverslips were cotransfected with HeV F and the TM constructs. After 24 h, the cells were washed in PBS and fixed in 4% paraformaldehyde for 15 min at room temperature. The cells were then permeabilized in 1% Triton X-100 for 15 min at 4°C, followed by blocking in 1% normal goat serum. The cells were incubated with the corresponding primary antibody overnight at 4°C. The following day, the cells were washed with 0.05% Tween-PBS, secondary antibodies were added, and the cells were incubated at 4°C for 1 h. The coverslips were then mounted on glass slides using Vectashield mounting medium (Vectorlabs, Burlingame, CA). Photographs were taken using a Nikon 1A confocal microscope and analyzed with NIS Elements software. All the images were processed in Adobe Photoshop, with equivalent adjustments made to all panels.
ACKNOWLEDGMENTS
We thank the Dutch laboratory for input on the manuscript.
This work was supported by NIAID grant R01AI051517 and NIH grant 2P20 RR02017 to R.E.D. and an NRSA grant (F31 AI120653) to S.R.W.
FOOTNOTES
- Received 21 May 2019.
- Accepted 12 June 2019.
- Accepted manuscript posted online 19 June 2019.
- Copyright © 2019 American Society for Microbiology.
