Possible Hidden Physicochemical Properties and PTMs' role in Cross-Species Transmission

Viral zoonotic diseases are viral illnesses transmitted from other organisms to humans. Think of pandemics and epidemics such as the Antonine Plague, Influenza pandemics in the late 1800s and throughout the 1900s, SARS-CoV-1 and 2, Ebola, Yellow Fever, MERS, and even new ones like Nipah. None of these diseases that led to pandemics had killed fewer than one hundred thousand people, showing how unexpected and deadly these illnesses can be.

¾ of new diseases are zoonotic, and there are over 200 viral zoonoses worldwide. Around the world, scientists are working hard trying to predict future zoonoses. Therefore, the purpose of this project is to identify signature features in viral zoonoses to predict the likelihood of future emerging diseases when observing Cysteine residues for disulfide bonds that help with stability, N-linked glycosylation, which shields viruses from the host’s immune system, relative mutability for unique factors and hydropathy for conserved regions as well as exposed aqueous ones.

In the past, researchers have mainly observed phylogenetic characteristics and geological properties to identify patterns, but few have ever viewed the main part of the infection. Attachment. Attachment is the first of three parts to consider an infection successful. Attachment proteins bind to cell receptors, so they are highly selective for a virus’s ability to attach. They are also the main factor that alters when viruses infect a new host, enabling them to fit with the new receptor.

STEPS:

1) Virus collection: 50 zoonotic viruses were identified through data availability of protein sequence in Uniprot, with a maximum of 5 viruses per family to minimize phylogenetic bias. The following variables were recorded: genus, family, order, animal carriers, genome type and envelope's presence to later apply in analysis. Figure1.

2) Sequence collection: Attachment proteins' sequences were collected through Uniprot via FASTA sequence. Proteins are evidence at the protein level or reviewed (Swiss-Prot). Signal peptide and other proteins in the precursor were preferably removed through annotation.

Analysis is conducted at both the individual and family levels.

3) Post-Translational Modifications (PTMs) Analysis: The chosen modifications are widely presented in proteins, and have key identifiers for easy tracking. Cysteine content is measured to represent the highest possibility for disulfide bonds, which highly influence protein folding and stability, especially for viruses that have to deal with extracellular stress. N-linked glycosylation also influences protein folding and acts as a shield for epitopes against the host's immune system.

The amount of cysteine and N-linked glycosylation sites was calculated using an Excel formula before transferring to a percentage of the sequence length. The results are then analyzed for trends between viruses.

4) Physicochemical Analysis: The Kyte-Doolittle hydropathy index is chosen since it's constructed using observations of protein interactions, and Dayhoff's relative mutability is used to calculate mutations since attachment proteins are known to be highly mutative. In this project, the values on both scales are each used within different windows to calculate the average through EXPASY Protscale. Post-calculation numerical values were exported and analyzed for similar regions.

5) Clustal Omega MSA: MSA is chosen to show the regular alignment of sequences and was conducted using Clustal Omega. The result is analyzed for conservation, showing alignment density, which is used to determine the alignment's quality, using Jalview.

RESULTS

This study analyzed viral zoonotic attachment proteins from 50 viruses on cysteine residues, N-linked glycosylation sites, Dayhoff’s relative mutability values, and Kyte-Doolittle hydropathy scores as well as MSA alignment. Analyses were conducted at both the family and individual virus levels.

N-LINKED GLYCOSYLATION SITES

No strong correlation was observed among viruses in N-linked glycosylation rates according to the Pearson Correlation Coefficient (r² = 0.25) when grouped by family. Graph1 & 2. Standard deviation is relatively high at 3.48% compared to the average of 3.64%. Furthermore, Pearson Correlation Coefficient is low even when grouped by family, but when grouped arbitrarily, Pearson Correlation Coefficient decreased to r2= 0.067.

CYSTEINE LEVEL

Cysteine residue content showed a weak correlation overall (r² = 0.11). Graph1 & 3. There is a highly diversified range at 0.10-9.03%. compared to the average of 3.35%. All viruses that have an over 4% in Cysteine level all belong to Bunyavirales but are relatively low in N-linked glycosylation sites at 0.56-2.38%. Arenaviridae, although also belonging to the same order, have more N-linked glycosylation sites and lower cysteine levels.

Viruses generally exhibited either a significantly higher cysteine residue content or a higher N-linked glycosylation rate, with the exceptions of most non-enveloped viruses, Flaviviridae, VSIV, and CHPV, making up 76%. Graph4 & 5.

HYDROPATHY RATE

Kyte-Doolittle hydropathy analysis showed that sequences tend to remain relatively hydrophilic with few, thinly scattered hydrophobic peaks. Figure3. The most alignment detected is at positions 41 and 42, with a rounded value of -0.25 and 17 viruses aligned.

RELATIVE MUTABILITY

Analysis conducted with Dayhoff’s relative mutability shows minimal correlations and alignments. Most alignment observed was at positions 11, 40, 101, 122 and 216 with 13 viruses in alignment. Residues with the highest mutability scores appeared more frequently than residues with the lowest mutability scores by 241.61%.

MSA

Multiple sequence alignment completed with Jalview shows minimal alignment, with conserved regions ranging from 0-4 (scale: 0-11). Quality analysis shows moderate scores, which suggests coincidental alignments.

NON-ENVELOPED VIRUS

Non-enveloped viruses exhibited particularly low N-linked glycosylation level, ranging from 0.62% to 6.44%, with ARV reaching the higher end at 6.44%, while others remained below 3.5%. Their cysteine level remains low with 0.21-1.35% compared to the average of 3.35%. Graph6. The most hydropathy alignment was found on position 181, with a value of -0.25, with 5 out of 6 viruses in alignment. In relative mutability, it is shown that these viruses do not have many conserved regions at 2.53%.

CONCLUSIONS

Viral zoonoses do not exhibit significant patterns in the analyzed PTMs and physicochemical properties factors outside of their family, with no major alignments. A long range and high standard deviation further indicate that there are minimal similarities. This result is predicted, given that attachment proteins mutate rapidly.

Bunyavirales viruses show a high cysteine level, which could explain the numerous successful mutations to having a stable structure. My results show viruses possibly favouring either N-linked glycosylation or disulfide bonds, rarely both, implying that these viruses require different factors in protein folding and immune invasion.

Non-enveloped viruses are generally low in cysteine level and N-linked glycosylation sites since their proteins are made from ribosomes in the cytoplasm, minimizing their chance of acquiring these PTMs. Non-enveloped viruses generally focus on a flexible but tight capsid through oligomerization, and certain PTMs could affect oligomerization.

Attachment proteins are highly mutable, suggesting that these viruses don't share unique patterns, but rather mutatable residues to bind the host cell receptor. In conclusion, there were no significant identified results that PTMs and physicochemical properties play a direct role in cross-species transmission, indicating that these factors do not directly contribute to cross-species transmission, proving my first hypothesis null and do not contradict my second hypothesis.

Hydrophobic peaks could suggest transmembrane domains, which mainly help with stabilization but also later during assembly and budding. I hypothesized that the transmembrane region could play a key role for viruses that is usually affected by plasma membrane restrictions.

LIMITATIONS

15 protein sequences are part of a precursor, as well as having a possible signal peptide attached translating to a 12-30% error rate. This has a drastic impact on my project, given that some attachment proteins might be in non-alignment. There may be phylogenetic bias, given that 17 viruses were derived from the same order.

FUTURE IMPROVEMENTS

To improve this project, I would add more analysis factors, especially for non-enveloped viruses that don't often encode N-linked glycosylation or disulfide bonds. I also want to learn more about these viruses to be able to give more insights into my results.

A special thank you to Ms. Morris, Ms. Anderson and Ms. Magnon for their support. And Fraser Valley Regional Science Fair for selecting me to represent them at the Canada-Wide Science Fair.

The biggest thank you goes to my parents for everything they've done for me. Especially my mom, who is my biggest inspiration.

*Sequence accession is in the attachment*

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