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Young Scientist Journal

Young Scientist Journal

The Impact of Synonymous Mutations on the Processing of the Foot-and Mouth Disease Virus Capsid Precursor Protein

ABSTRACT

RNA viruses produce mutations rapidly and can hijack cellular processes to facilitate their life cycle. A promising new strategy to mitigate threats of viral outbreaks incorporates synonymous mutations into viral genomes to attenuate viral efficiency. Here, the Foot-and-Mouth disease virus (FMDV) was utilized as a model system to observe the impacts of codon substitutions on cellular mechanisms. We have previously observed that cleavage of the P1 polyprotein by the viral protease 3Cpro results in more efficient production of FMDV structural proteins for wild type P1 than deoptimized P1. Therefore, we seek to identify how synonymous codon mutations affect cleavage of the P1 polyprotein by 3Cpro. Results confirmed expression of wild type and deoptimized P1 in HEK293T cells with and without 3Cpro cleavage. In the presence of 3Cpro, the structural protein VP1 was observed by Western blot for both wild type P1 and deoptimized P1 to confirm polyprotein cleavage. Silver stain analysis was also performed, and structural proteins VP0, VP1, and VP3 were present for both wild type and deoptimized P1. In addition, we will clarify findings by applying mass spectrometry analysis to protein band sequences. Conclusive results from this research study may present further understanding into the effect synonymous codon mutations have on FMDV attenuation.

INTRODUCTION.

Codon-Pair-Deoptimization (CPD) has enabled the ability of attenuation of viruses using Synonymous mutations. These modifications to viral genomes result in a significant reduction in viral fitness but still produce a host immune response resembling that of a virulent strain.  Highly efficient attenuation through the introduction of synonymous mutations into viral genomes was observed in a wide variety of RNA viruses including: Enterovirus C (poliovirus), Influenza A virus (IAV), Human immunodeficiency virus type 1 (HIV-1), Human respiratory syncytial virus [4,5] Indiana vesiculovirus [4,5]and Dengue virus [7,8]These studies provide a strong basis for the efficacy of the attenuation of viruses through the use of synonymous mutations.

The concept of codon pair bias involves maximizing the amount of underrepresented codon pair sequences for a given organism. Frequency of specific codon bases has a direct relationship with the relative abundance of tRNA in the active translation process. Altering codon bases to code for the same amino acid has caused slower co-translational speeds as the cytosolic tRNA pool is not enriched for that specific codon and slows translation elongation. It has been proposed that possible changes in frequency of dinucleotides cytosine and adenine constructs contribute to viral attenuation as the recoded viral genes elicit an innate immune response to viruses [Figure 1]. A recent study has discovered that a certain antiviral protein ZAP [1] factor is CpG-rich RNA targets them for degradation by the RNA exosome and has been considered very effective in inhibiting virus replication.

Figure 1. Schematic of Central Hypothesis. Faster translational speeds and effective cotranslational folding result from optimal codon usage. Non-optimal codon usage results in slowed translation elongation and altered proteostasis interactions. Figure made in BioRender.

Foot-and-mouth disease virus (FMDV).  

FMDV is characterized as a highly infectious disease that infects cloven-hooved animals including sheep, cattle, pigs, goats, and deer. The causative agent for this virus originates from the Aphthovirus genus of the Picornaviridae family. Its virion is a 140S particle consisting of a single-stranded RNA genome and 60 copies each of four structural proteins (VP1, VP2, VP3, and VP4) [1]. FMD poses a constraint on importation of major livestock and there are cases listed by the International Organization of Animal Health (OIE) as a reportable disease, and severe trading restrictions are imposed upon notification of an outbreak [2]. FMDV symptoms are characterized as fever, blisters, lameness, depression and reduced milk production. FMDV is a highly transmissible disease and has 60 subtypes (7 serotypes). The virus infects major livestock and decreases production of byproduct sold at the commercial level. For instance, the recent United Kingdom outbreak of 2001 afforded economic losses that surpassed $12 billion. Although it is contained in certain developed nations there were recent outbreaks in Europe, Taiwan, and Japan indicating that the virus is spreading rapidly [4,6].

MATERIALS AND METHODS.

The deoptimized P1 sequence from the FMDV A24 strain was previously generated using the SAVE method for deoptimization as described previously [2,5,6]. Wild type and deoptimized mammalian expression constructs with a C-terminal FLAG tag were designed and ordered from Twist Biosciences.

Cell Culture and Transfection.

HEK293T cells were cultured in Dulbeco’s Modified Eagle Medium (high glucose) with 10% fetal bovine serum,

1% penicillin/streptomycin, and 1% glutamine.  Cell cultures were maintained at 37 ºC and 5% CO2. Calcium phosphate method was used to transfect 10cm dishes with 3 µg GFP, WT P1, P1Deopt, and 1 µg 3Cpro.  Media was replaced after 16 hours, and cells were harvested 24 hours after media exchange by scraping on ice.

Co-immunoprecipitation.

 Co-immunoprecipitation was performed as previously described in Davies et. al [1] Cell pellets were lysed using RIPA buffer (50 mM Tris pH 7.5, 150 mM NaCl, 0.1% SDS, 1% Triton X-100, 0.5% deoxycholate) with “cOmplete” protease inhibitor (Roche). Lysis was performed for 30 minutes on ice and lysates were collected following a centrifugation step at 21,000x g for 10 minutes. Following lysis, protein concentrations were normalized using a BCA protein assay kit (Pierce). FLAG beads and Sepharose beads were obtained in 1 ml tubes and were resuspended and washed four times with RIPA. Normalized lysates were combined with 30 µL Sepharose 4B beads (Sigma) and incubated by rocking for 1 hour at 4 ºC. Following centrifugation at 400× g for 10 min, supernatant was transferred to 30 µL anti-DYKDDDDK resin (GenScript) and incubated while rocking overnight at 4 °C. The next day, FLAG beads were centrifuged at 400x g and washed with RIPA four times. Beads were dried using a 30G x ½ syringe needle to remove excess RIPA buffer. Protein bound to FLAG beads was eluted using 2% SDS in PBS for 5 minutes at 95 ºC.

Protein Detection by Western Blot and Silver Stain.

After Co-immunoprecipitation samples from lysates and elutions were loaded onto 10% and 12% acrylamide gels where the electrophoretic system ran the cassette for 1 hour at 180 volts [Figure 2]. The gel was then transferred to a membrane and blocked with 5% milk in TBST. Antibodies for FLAG tag (Sigma Aldrich), 2x-Strep tag (GenScript), and tubulin (BioRad) were used for detection of targeted proteins by Western blot.  Silver stain was performed using a commercially available kit (Thermo).  

In-gel digestion for LCMS/MS Analysis.

Following silver stain analysis of elution samples, bands of interest were excised from the gel using a scalpel. The gel was destained by following the manufacturer’s protocol (Thermo). Samples were reduced with 10 mM dithiothreitol (DTT) in 25 mM ammonium bicarbonate with 10% acetonitrile for 1 hour at 56 ºC. Samples were alkylated with 55 mM iodoacetamide in aqueous 25 mM ammonium bicarbonate in the dark at room temperature for 45 minutes. Gel pieces were vacuumed to complete dryness. One volume of trypsin solution (12.5 ng/µL trypsin in 25 mM aqueous ammonium bicarbonate) was added to the gel piece incubated for 30 minutes on ice. Excess trypsin solution was discarded, and the gel pieces were incubated overnight at 37 ºC. Water two times the volume of the gel piece was added to the digest and vortexed for 10 minutes, sonicated in a water bath for 5 minutes, and centrifuged. The supernatant was removed and a solution containing 45% water, 50% acetonitrile, and 5% formic acid was added to the tube containing the gel piece. Vortexing, sonication, and centrifugation was carried out as described above. The supernatant was removed and added to a fresh tube, and the previous step was repeated twice. The volume was reduced to ~10 µL using a vacuum centrifuge.

An Exploris480 mass spectrometer (Thermo) was used to perform LC-MS/MS analysis. Peptides were separated using a 21.5 cm fused silica microcapillary analytical column with a laser-pulled tip packed with Aqua C18, 3 µm, 100 Å resin (Phenomenex # 04A-4311). Peptides were loaded onto a trap column containing Aqua 5 µm C18 resin (Phenomenex # 04A-4299) using an autosampler. Samples were then analyzed using data-dependent acquisition mass spectrometry (DDA-MS) [Figure 2]. Peptide identification and protein quantifications were carried out using Proteome Discoverer (PD) 2.4 (Thermo).

Figure 2. Workflow for analysis of P1 cleavage in HEK293T cells. HEK293T cells are transfected with WT P1, P1Deopt, 3Cpro, WTP1+3Cpro, P1Deopt+3Cpro, and GFP. Following immunoprecipitation with anti-FLAG beads, samples were analyzed using Western blot and Silver Stain. In progress: Bands visualized by western blot are excised and digested for analysis by label free DIA-MS. Figure made in Bio Render.

 Data Analysis.

Western blot and silver stain images were obtained using a ChemiDoc MP Imaging System (BioRad), and images were analyzed using Image Lab software (BioRad). Mass spectrometry data analysis was performed using Proteome Discoverer (PD) 2.4 (Thermo) and Prism.

RESULTS.

In this study, FMDV was used as a model virus to study the effects of a synonymously deoptimized strain on co-translational speeds when using non-optimal codons. Analysis of protein detection through Western Blot showed that deoptimized P1 has less expression when compared to the wild type strain of the FMDV A24 strain. Expression through Western blot analysis allowed us to identify that the P1 polyprotein is present [Figure 3].

Figure 3. Validation of wild type P1 and deoptimized P1 (P1Deopt) expression in HEK293T cells. The synonymously mutated P1Deopt shows less expression when compared to WT P1. Tubulin was used as loading control.

Tubulin was used as a loading control to ensure all lanes were loaded evenly. Recent studies have determined that lesser expression of the P1-Deopt strain can be attributed to non-optimal codon usage in the synonymously mutated strain. Additionally, cleavage of the P1 polyprotein is confirmed through the presence of structural protein VP1 because the C-terminal FLAG tag is retained with this protein [Figure 3]. Western blots were further analyzed to quantify the production of VP1 following cleavage of P1 by 3Cpro . This is measured by observing the production of VP1 after following cleavage by the 3Cprotease by comparing the WTP/P1-Deopt ratios to for both the amount of VP1 and P1 polyprotein present. Validation of wild type P1 and deoptimized P1 (P1Deopt) expression in HEK293T cells. The synonymously mutated P1Deopt shows less expression when compared to WT P1. Tubulin was used as loading control. Approximately 7.5 fold more wild type P1 enriched compared to deoptimized P1, while there was only an approximately 1.5 fold difference in enrichment of the wild type and deoptimized polyproteins [Figure 4]. Therefore, it can be concluded that there is a difference in processing efficiency as a result of synonymous mutations. To confirm production of FMDV structural proteins VP0, VP3, and VP1, we performed a silver stain was performed to visualize all proteins present in  the elution samples. GFP and 3Cpro lanes served as negative controls to account for background signal in the gel. 3C pro only serves as a negative control because it does not contain a FLAG tag and should not be enriched. It was observed that the protease cleaved the polyprotein efficiently because of the presence of structural proteins VP0, VP3, and VP1. Full length P1 for both wild type and deoptimized P1 are not identified in samples that also had protease expression, indicating that both proteins are cleaved. Mass spectrometry analysis allowed for identification of the intact polyprotein and produced structural proteins. Full length WT P1 was identified with 63% sequence, and P1Deopt was identified with a sequence coverage of 66%. Full length P1 was found to have the highest number of identified peptides. Sequence of structural proteins was confirmed by Silver Stain of elution gel shows structural proteins VP0, VP1, and VP3. [Table 1] . Sequence coverage and peptide identifications for VP0 and VP1 in Both WT P1 and P1Deopt. No peptides were identified for VP3 likely due to issues with excising the bands from the gel [Table 1].

Figure 4. Quantification of Western blot following 3Cpro cleavage and FLAG IP.

 

Table 1. Summary of sequence coverage and peptide identifications from DDA-MS analysis of excised bands from silver stain. Sequence coverage readings are available for WT P1 and P1Deopt and its corresponding structural proteins VP0 and VP1 with the exception of VP3 as well as the number of peptides identified.
Protein Sequence Coverage (%) #Peptides
WT P1 63 34
P1Deopt 66 36
WT VP0 48 9
P1Deopt VP0 33 7
WT VP1 62 8
P1Deopt VP1 26 4
WT VP3 — —
P1Deopt VP3 — —

RNA viruses can adapt rapidly to new environments. Therefore, the need to develop effective therapeutics against these viruses is high. Variation in the genetic make-up FMDV previously showcased possible signs of attenuation. It’s known that there is a possible degeneracy in the genetic code and codons can possibly be replaced by less efficient non-optimal codons that correspond to less abundant tRNA intermediates. Genetically mutated strains of the FMDV virus capsid created utilizing the SAVE method has enabled research in identifying the mechanisms responsible for viral attenuation. In this study, we utilized quantitative analysis to characterize how synonymous mutations cause the 3Cpro to cleave the polyprotein less efficiently. FMDV virus constitutes a huge threat to the stability of a nation’s economy. Although FMDV is not fatal, the virus reduces the amount of milk produced and overall animal productivity. Researchers have pursued scientific endeavors apportioning time to discover ways to employ their research and develop therapeutic remedies in order to mitigate this threat. Previous studies have utilized synonymous mutations to attenuate certain viruses.

Attenuation by the use for synonymous mutations was used in the development in the vaccine that eradicated the poliovirus [2]. Additionally, it has been shown that the use of synonymous mutations result in attenuation of FMDV [1]. These findings support the theory that incorporating underrepresented codons in a genetic sequence can alter the efficiency of the virus. DNA plasmids for wild type P1 and deoptimized were expressed in HEK293T cells. Expression of WT P1 and P1Deopt was present, but there was less expression of the P1Deopt when compared to the WT P1 strain. The appearance of the fainter band suggests that the nonoptimal codon usage is can have an impact on translation elongation. After samples were bound to beads conjugated with anti-FLAG antibodies, the bound proteins were eluted and analyzed by Western blot. A FLAG co-immunoprecipitation was preformed to enrich P1 and structural proteins. The presence of structural proteins in both the Western blot and silver stain gel indicates that there was cleavage of the P1 protein. The structural proteins are visible when the protease cleaves the polyprotein into mature structural proteins in preparation of assembly of the virion capsid that encapsulates the viral genome. At the 25 kDa line there was presence of structural protein VP1-FLAG confirming cleavage of the polyprotein. Western Blot data was then quantified and slightly more polyprotein was pulled down for WT P1 as compared to deoptimized P1. However, there was a much greater amount of deoptimized VP1 produced than wild type VP1.

DISCUSSION.

We can conclude that synonymous mutations possibly have effects on the cleavage of the P1 polyprotein to produce viral structural proteins. Identification through mass spectrometry data has confirmed the sequence of each structural protein produced. There is high confidence that structural protein VP0 and VP1 are present but not VP1, which is possibly due to not fully excising the bands in preparing it for mass spectrometry. VP0 and VP1 were identified with high sequence coverage and identified peptides for both wild type and deoptimized P1. Though previous studies have investigated the cleavage efficiency of a ribosome on a synonymously mutated strain and identified sps interactions with other proteins involved in assembly of virus mechanisms. High profile interactions of sps in other studies has confirmed that the presence of the sps are possibly connected with facilitating viral replication. It’s not clear what cellular factors are responsible for differential processing, performing a Strep IP of 3Cpro and utilizing mass spectrometry will aid in having a deeper understanding in the host machineries involved with translation. Interestingly an IP on the 3Cpro allows for one-step affinity purification under less harsh conditions, in order to maintain the protease functionality creating an accurate read on assays and quantification. Possibly identifying specific proteins that may still be picked up after cleavage of maturated proteins will provide more insight and confirmation into all specific aiding proteins. Other studies have confirmed that cellular processes are driven by membrane bound signaling molecules and this could possibly elucidate to how a specific genetic code is translated [3]. Through advancement of genetic engineering researchers have repeatedly utilize viral clones derived from the actual virus and manipulate it’s genetic code to develop Live Attenuated Vaccines[4]. Although there may be some risks presented in inoculation with a mutated version of the virus. Aforementioned, research has suggested that there that it’s promotion of slower co-translational speed provides a window in which an immune response can combat the virus. Other studies have used this foundational knowledge and tested various ways in incorporating different mutation in the FMD viral genome. Segundo et. al discussed deletions in the FMDV strain that omitted the leader-codon region behaved like the WTP-P1 when used as a inactive vaccine. The results of their study showed that synonymous mutations caused too much attenuation of the virus. But a lack of the leader-codon suggested it being a safer alternative in FMDV vaccine development[5].

ACKNOWLEDGMENTS.

Special thanks to mentor Molly Sullivan for her advice, Valeria Garcia-Lopez, and the Plate Lab. Big thanks to Dr. Eeds and the School for Science and Math at Vanderbilt for their support and collaboration with the USDA.

REFERENCES.

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Posted by on Thursday, May 14, 2026 in May 2026.

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