Effect of Foot-and-Mouth Disease Virus 2B Viroporin on Expression and Extraction of Mammalian Cell Culture Produced Foot-and-Mouth Disease Virus-like Particles

To improve the production of foot-and-mouth disease (FMD) molecular vaccines, we sought to understand the effects of the FMD virus (FMDV) 2B viroporin in an experimental, plasmid-based, virus-like particle (VLP) vaccine. Inclusion of the FMDV viroporin 2B into the human Adenovirus 5 vectored FMD vaccine enhanced transgene expression despite independent 2B expression negatively affecting cell viability. Evaluating both wildtype 2B and mutants with disrupted viroporin activity, we confirmed that viroporin activity is detrimental to overall transgene expression when expressed independently. However, the incorporation of 2B into an FMD molecular vaccine construct containing a wildtype FMDV 3C protease, a viral encoded protease responsible for processing structural proteins, resulted in enhancement of transgene expression, validating previous observations. This benefit to transgene expression was negated when using the FMDV 3CL127P mutant, which has reduced processing of host cellular proteins, a reversion resulting from 2B viroporin activity. Inclusion of 2B into VLP production constructs also adversely impacted antigen extraction, a possible side effect of 2B-dependent rearrangement of cellular membranes. These results demonstrate that inclusion of 2B enhanced transgene expression when a wildtype 3C protease is present but was detrimental to transgene expression with the 3CL127P mutant. This has implications for future molecular FMD vaccine constructs, which may utilize mutant FMDV 3C proteases.


Introduction
Foot-and-mouth disease virus (FMDV), the causative agent of foot-and-mouth disease (FMD), is a member of the Aphthovirus genus in the Picornavirus family. Like other picornaviruses, the FMDV genome encodes a single polypeptide containing structural proteins, comprising the capsid, and non-structural proteins, responsible for viral replication.
Intact capsid structures are required to generate protective immunity from FMD. Molecular FMD vaccines require expression of the P1 polypeptide and the non-structural 3C protease, which processes the P1 into VP0, VP3, and VP1 prior to assembly of viruslike particles (VLPs); peptide VP0 is further processed into VP2 and VP4 by an unknown mechanism upon capsid assembly [1]. A drawback of 3C protease expression is its ability to process multiple host cellular proteins, resulting in detrimental effects on cells and reduced transgene expression [2]. To avoid the deleterious effects of 3C expression, vaccine constructs have incorporated methods to reduce either 3C expression or activity [2][3][4][5][6]. Notable among these is the 3C L127P mutant (3C L127P ), which reduces the ability of 3C to process multiple host proteins, enhancing transgene expression [2].
The FMDV 2B protein is a membrane-bound viroporin with both N-and C-terminal domains extending into the cytosol [7][8][9]. Expression of 2B alters cellular Ca 2+ concentrations inducing autophagy of host cells [7]. It is unique in containing two distinct pore domains, each able to mediate pore formation, and disrupted by mutations of residues 62 to 65 (2B ∆A ) or 128 to 130 (2B ∆B ) [10]. The 2B protein is not known to have a direct role in either processing or assembly of FMDV capsids. However, incorporation into human Adenovirus 5 (Ad5)-vectored FMD vaccines improves transgene expression, potency, and efficacy [11,12]. This enhancement would appear counterintuitive, as independent expression of 2B results in cellular toxicity related to its viroporin function [7,13].
Recently a FMDV VLP vaccine platform utilizing mammalian cell culture transiently transfected with plasmids expressing only the P1 and 3C protease was capable of inducing protection from disease in swine [14]. This platform utilized the 3C L127P protease to enhance transgene expression over wildtype 3C (3C wt ), [2], followed by extraction utilizing a lysis buffer [14]. VLPs produced utilizing this methodology were able to confer protection from challenge with FMDV O1 Manisa in all swine following the administration of two doses [14].
In this report we investigated the effects of 2B on transgene expression independently and upon incorporation into the FMDV VLP producing constructs in mammalian cell culture. Results demonstrate a complicated relationship between 2B expression, viroporin activity, and transgene expression dependent upon 3C activity. We also found the inclusion of 2B altered the ability to extract capsid antigens using established methodologies.
For independent expression, the O1 Manisa 2B sequence was synthesized into the pJJP plasmid without a luciferase reporter. The sequence of O1 Manisa VP1 was also synthesized into the pJJP plasmid for use as a control. The construct A24P1-L127P was constructed by synthesis of the P1-2A from FMDV A24 Cruzeiro (GI: AY593768) and replacement of the O1 Manisa P1-2A sequence in pJJP P1-L127P.

Transfection of Cell Cultures with Single Plasmids
HEK293-T (ATCC, CRL-11268) and CHO-K1 (ATCC, CCL-61) cell cultures were transfected with single plasmids utilizing Lipofectamine 2000, as per manufacturers suggestions (Thermo Fisher Scientific, Waltham, MA, USA). Transfected cells were maintained at 37 • C and 5% CO 2 for 24 h before media was harvested for luciferase assays.

Transfection of Cell Cultures with Two Plasmids
For transfection of two plasmids simultaneously, HEK293-T cells were cultured in six-well plates and simultaneously transfected with 2 µg of each plasmid utilizing Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA) as per manufacturers suggestions. Transfected cells were maintained at 37 • C and 5% CO 2 for 24 h after which media was harvested for use in luciferase assays and cells lysed with M-PER (Thermo Fisher Scientific, Waltham, MA, USA) for antigen detection.

Luciferase Assay
Luciferase assays were performed as previously described [15]. In brief, cell culture supernatant from transiently transfected cell cultures were harvested 24 h post-transfection and clarified through centrifugation at 500× g for 3 min. Clarified media was diluted to a 1:4 dilution to prevent signal over-saturation and evaluated for luciferase activity utilizing a 96-well BioSystems Veritas luminometer (Turner Biosystems, Sunnyvale, CA, USA) with 100 µL of diluted sample in each well. Readings were taken following injection of 100 µL of 50 µg water-soluble coelentrazine (Nanolight Technology, Pinetop, AZ, USA) using an integration time of 0.5 s both before and after injection of substrate.

Electron Microscopy
The formation of arrays comprised of FMDV VLPs produced using this system had been previously established using immuno-electron microscopy [16]. For TEM processing, cells were fixed in 2.5% glutaraldehyde, 2% paraformaldehyde in 0.1 M sodium cacodylate buffer, postfixed with 1% osmium tetroxide, stained en bloc with 2% uranyl acetate, dehydrated through a graded series of ethanol then propylene oxide, and embedded in Spurr's resin (Electron Microscopy Sciences, Hatfield, PA, USA). Ultrathin (80 nm) sections were cut on a Leica UC6 microtome, post-stained with uranyl acetate and lead citrate, and imaged on a Hitachi 7600 TEM with a 2k × 2k AMT camera at 80 kV.
In brief, cells were removed by flushing with cell culture media and transferred to 15 mL conical tubes. Cells were pelleted by centrifugation at 500× g for 5 min, and the supernatant discarded. Cells were washed by resuspension in 1 mL of dPBS and pelleted by centrifugation at 500× g for 5 min. Cell pellets were resuspended in lysis buffer, either LB9, LB9A, LB9D, or RIPA, and incubated for 10 min at room temperature on a shaker to lyse cells. Following incubation, cellular debris was removed by centrifugation at 500× g for 5 min, and supernatant was retained for western blotting. For cells lysed with RIPA, 1 µL of DNase I (Sigma-Aldrich, St. Louis, MO, USA) was added, and the mixture was incubated at 37 • C for 1 h.

Expression of 2B Had a Negative Effect on Transgene Expression
We utilized the GLuc-∆1D2A reporter system, Figure 1A, to evaluate transgene expression by 2B viroporin activity using either single trans-membrane domain alanine mutants, 2B ∆A or 2B ∆B , or a mutation of both trans-membrane domains, 2B ∆A/B . In both HEK293-T and CHO-K1 transfected cells, all three mutants enhanced transgene expression over 2B, and mutant 2B ∆B produced the greatest enhancement, Figure 1B.

Expression of 2B Had a Negative Effect on Transgene Expression
We utilized the GLuc-Δ1D2A reporter system, Figure 1A, to evaluate transgene expression by 2B viroporin activity using either single trans-membrane domain alanine mutants, 2B ΔA or 2B ΔB , or a mutation of both trans-membrane domains, 2B ΔA/B . In both HEK293-T and CHO-K1 transfected cells, all three mutants enhanced transgene expression over 2B, and mutant 2B ΔB produced the greatest enhancement, Figure 1B.

Enhancement of Transgene Expression by 2B Was Dependent upon 3C Activity
Plasmid vectors expressing P1-2A, 3C, and the Δ1D2A-GLucΔ1M reporter, Figure  2A, were previously constructed to evaluate VLP assembly and transgene expression [15]. For this study we incorporated a 2B containing sequence mirroring that of previous Ad5 vectors, Figure 2B. The incorporated sequence contained 2B and additional sequences derived from FMDV non-structural proteins 2C, 3A, and 3B, Figure 2C.

Enhancement of Transgene Expression by 2B Was Dependent upon 3C Activity
Plasmid vectors expressing P1-2A, 3C, and the ∆1D2A-GLuc∆1M reporter, Figure 2A, were previously constructed to evaluate VLP assembly and transgene expression [15]. For this study we incorporated a 2B containing sequence mirroring that of previous Ad5 vectors, Figure 2B. The incorporated sequence contained 2B and additional sequences derived from FMDV non-structural proteins 2C, 3A, and 3B, Figure 2C. Incorporation of 2B into VLP constructs expressing 3C wt enhanced transgene expression in both HEK293-T and CHO-K1 cell lines, Figure 3A. In contrast, incorporation of 2B into VLP constructs containing the 3C L127P mutant decreased transgene expression, Figure  3B. This decrease was partially abated by mutation of 2B viroporin domains, Figure 3B. To evaluate if 2B reduction of transgene expression with 3C L127P was the result of an altered construct design we expressed 2B independently of P1-L127P using a two-plasmid system. In this system, only the P1-L127P plasmid contained a luciferase reporter. While expression of P1-L127P was within the standard deviation of that observed when VP1 was expressed from a second plasmid, a substantial decrease in expression was observed when 2B was expressed, Figure 3C, suggesting that the reduced transgene expression caused by 2B expression with 3C L127P was independent of construct design. This 2B-dependent decrease was replicated with a plasmid encoding the P1 polypeptide from FMDV serotype A strain Cruzeiro, A24P1-L127P, demonstrating independence from the FMDV O1 Manisa P1 polypeptide, Figure 3D. Incorporation of 2B into VLP constructs expressing 3C wt enhanced transgene expression in both HEK293-T and CHO-K1 cell lines, Figure 3A. In contrast, incorporation of 2B into VLP constructs containing the 3C L127P mutant decreased transgene expression, Figure 3B. This decrease was partially abated by mutation of 2B viroporin domains, Figure 3B. To evaluate if 2B reduction of transgene expression with 3C L127P was the result of an altered construct design we expressed 2B independently of P1-L127P using a two-plasmid system. In this system, only the P1-L127P plasmid contained a luciferase reporter. While expression of P1-L127P was within the standard deviation of that observed when VP1 was expressed from a second plasmid, a substantial decrease in expression was observed when 2B was expressed, Figure 3C, suggesting that the reduced transgene expression caused by 2B expression with 3C L127P was independent of construct design. This 2B-dependent decrease was replicated with a plasmid encoding the P1 polypeptide from FMDV serotype A strain Cruzeiro, A24P1-L127P, demonstrating independence from the FMDV O1 Manisa P1 polypeptide, Figure 3D. . This decrease in transgene expression was partially negated in constructs containing mutated viroporin domains. (C) Simultaneous transfection of two plasmids resulted in a decrease in luciferase activity with a 3C L127P construct in the presence of 2B, demonstrating that reduction of transgene expression was independent of construct design. (D) This decrease was replicated with a P1 corresponding to the A24 serotype. * denotes a significant difference, p < 0.001 as determined by Student's T-test, between expression of constructs with and without wildtype 2B or 2B viroporin mutants.

Incorporation of 2B in Plasmids Did Not Affect the Presence of VLP Arrays in Transfected Cell Culture
Previous utilization of the P1-L127P plasmid demonstrated the formation of VLP arrays within transfected cells [2,15,16]. Studies using the Ad5 vaccine did not observe arrays in infected cells but did demonstrate an increase in cytoplasmic vesicles following 2B incorporation [11]. In this study, VLP arrays were observed independent of 2B inclusion, Figure 4. In cells transfected with the P1-2B-L127P construct many VLP arrays, but not all, were fully encircled by membrane vesicles, Figure 4. No arrays or similar structures were This decrease was replicated with a P1 corresponding to the A24 serotype. * denotes a significant difference, p < 0.001 as determined by Student's T-test, between expression of constructs with and without wildtype 2B or 2B viroporin mutants.

Incorporation of 2B in Plasmids Did Not Affect the Presence of VLP Arrays in Transfected Cell Culture
Previous utilization of the P1-L127P plasmid demonstrated the formation of VLP arrays within transfected cells [2,15,16]. Studies using the Ad5 vaccine did not observe arrays in infected cells but did demonstrate an increase in cytoplasmic vesicles following 2B incorporation [11]. In this study, VLP arrays were observed independent of 2B inclusion, Figure 4. In cells transfected with the P1-2B-L127P construct many VLP arrays, but not all, were fully encircled by membrane vesicles, Figure 4. No arrays or similar structures were observed in cells transfected with constructs containing a 3C C163A protease mutant [2,15], which is unable to process the P1 (data not shown).
observed in cells transfected with constructs containing a 3C C163A protease mutant [2,15], which is unable to process the P1 (data not shown).

Inclusion of 2B Inhibits Antigen Extraction
Antigen from transfected cell cultures were extracted utilizing LB9 and evaluated by western blotting. Antigens were detected with multiple monoclonal antibodies using the P1-L127P extract but not detected with P1-2B-L127P extract, Figure 5A. This effect was replicated utilizing a two-plasmid transfection system, Figure 5B.

Inclusion of 2B Inhibits Antigen Extraction
Antigen from transfected cell cultures were extracted utilizing LB9 and evaluated by western blotting. Antigens were detected with multiple monoclonal antibodies using the P1-L127P extract but not detected with P1-2B-L127P extract, Figure 5A. This effect was replicated utilizing a two-plasmid transfection system, Figure 5B.
Lysis of cells with buffers containing either more TritonX-100, LB9A, or SDS, LB9D and RIPA, did not fully counteract the observed effect, although use of RIPA buffer for extraction revealed possible low molecular weight degradation products, Figure 6. These products were detected with two different VP0/2 antibodies, F1412SA and 1F1E4. Interestingly no impairment of GLuc detection by western blot was observed, Figures 5A and 6. Lysis of cells with buffers containing either more TritonX-100, LB9A, or SDS, LB9D and RIPA, did not fully counteract the observed effect, although use of RIPA buffer for extraction revealed possible low molecular weight degradation products, Figure 6. These products were detected with two different VP0/2 antibodies, F1412SA and 1F1E4. Interestingly no impairment of GLuc detection by western blot was observed, Figures 5A and 6.

Discussion
Expression of either FMDV 2B viroporin or 3C protease in mammalian cells is detrimental to cellular activity and transgene expression [2,7]. In our studies, the negative effect of 2B on transgene expression was related to its viroporin activity, and mutations that

Discussion
Expression of either FMDV 2B viroporin or 3C protease in mammalian cells is detrimental to cellular activity and transgene expression [2,7]. In our studies, the negative effect of 2B on transgene expression was related to its viroporin activity, and mutations that removed or reduced this activity resulted in enhancement of transgene expression, Figure 1B. Published results documenting that inclusion of 2B enhances FMD vaccine expression, potency, and efficacy were performed using the Ad5 vector system expressing a 3C wt protease [11,12]. We also found that when 2B was incorporated into constructs utilizing 3C wt , transgene expression was enhanced, Figure 3A, in agreement with Ad5 reports [12]. Our results agree with both reports of 2B expression having harmful effects on cells, and its inclusion enhancing FMD vaccine expression. This increase could result in improved Ad5 vaccine potency and efficacy, independent of other potential alterations caused by the presence of 2B.
Expression of FMDV 3C wt is deleterious to both host cells and transgene expression due to its ability to process host cellular proteins, however this impact can be negated by utilizing the 3C L127P mutant [2]. Multiple FMD molecular vaccine platforms incorporate strategies to mitigate 3C activity for enhancement of expression, such as IRES [3,4], HIV frameshift [6], or 3C mutagenesis [4][5][6]15]. However, it is unknown how incorporation of 2B would influence transgene expression for each strategy. Therefore, the relationship between 2B, 3C, and transgene expression reported here suggest multiple possibilities and demonstrate the critical need to evaluate the effects of including 2B in each circumstance.
A possible reason for the beneficial effects of 2B with 3C wt is the design of the constructs. The 2B-containing sequence utilized here and previously [11,12], contains non-2B-related sequences, Figure 2C, including junctions processed by 3C. Incorporation of these potential 3C processing sites may play a role in the enhancement seen with 3C wt constructs by providing additional substrate for the protease. In the Ad5 vectored vaccines, the addition of 2B with 94 truncated residues along with 3B removed these potential 3C processing sites and did not enhance transgene expression [12]. While a reduction in expression was observed with P1-2B-L127P relative to P1-L127P, expression was still greater than that observed with P1-2B-3C wt . Inclusion of both 3C L127P and 2B in an Ad5 vectored FMD vaccine resulted in full protection in cattle [2], demonstrating that 3C L127P and 2B remain compatible with inducing protection from clinical disease.
Inclusion of 2B is not necessary for the construction of virus like particles [2,6,15,16], and the work presented here demonstrated the presence of VLP arrays in transfected cells without 2B, Figure 4. VLPs produced using P1-L127P constructs elicit protection from clinical FMD in both cattle and swine [14]. The same extraction methodology applied to P1-2B-L127P-expressing cells resulted in undetectable antigens in western blots with multiple antibodies, Figure 5A. This effect was duplicated using a two-plasmid expression system with P1-L127P, Figure 5B, demonstrating its independence of the P1-2B-L127P construct and dependent upon 2B expression.
Significant membrane rearrangements are reported following 2B expression [7,11]. While not a universal effect, multiple VLP arrays observed in P1-2B-L127P-transfected cells were encapsulated within membrane vesicles, Figure 4. It is unknown if this was due to an interaction of VLPs, or precursors, with 2B or whether 2B-induced membrane rearrangement created opportunities for the occurrence.
Interestingly, antigen could not be readily extracted from P1-2B-L127P-expressing cells using the LB9 lysis methodology, Figure 5. Low levels of LB9 extracted antigen were visible by using 1-Step™ TMB-Blotting Substrate Solution, Figure 6. To enhance lysis of transfected cells, lysis buffers containing alternative detergent formulations were utilized. While these buffers may enhance extraction to a minor degree, they were unable to produce major changes in visualization of the extracted antigen, Figure 6. These results demonstrated that the previous LB9-based methodology of VLP extraction was ineffective when 2B was expressed. This effect appears independent of viroporin activity, although it is possible that limited residual activity is retained in viroporin mutants.
The use of extraction buffers LB9D and RIPA, containing the detergent SDS, resulted in the presentation of some lower molecular weight bands around 14 kDa, possibly representative of degradation products, Figure 6. As 2B is known to induce autophagy in expressing cells, these bands may represent degradation products of VP0 or VP2 resulting from autophagy within the transfected cell.

Conclusions
Incorporation of 2B into FMDV VLP constructs had different effects on transgene expression dependent upon the 3C protease sequence incorporated. Utilizing a 3C wt construct resulted in inclusion of 2B enhancing transgene expression, while 3C L127P constructs had reduced transgene expression in the presence of 2B. The addition of 2B into 3C L127P constructs severely limited the ability to extract capsid antigens detectable by western blotting. It remains unknown if the VLPs produced by 2B-containing constructs are structurally or immunologically different and whether this can influence vaccine potency or efficacy. These findings demonstrate the need to define circumstances in which inclusion of 2B will be beneficial to FMD VLP vaccine production, potency, and efficacy.