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\begin{document}

\title{L452M in spike glycoprotein indicative of zoonotic links of SARS-CoV-2
XBC.1}



\author[1]{Krishna Khairnar}%
\author[1]{Shefali Rahangdale}%
\author[1]{Siddharth Singh Tomar}%
\author[1]{Ekant Tamboli}%
\author[1]{Sandhra Ravikumar}%
\author[1]{Lekha Salsekar}%
\affil[1]{CSIR-National Environmental Engineering Research Institute}%


\vspace{-1em}



  
  \date{May 21, 2024}


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\selectlanguage{english}
\begin{abstract}
Mutations at positions 452 and 486 of the spike glycoprotein receptor
binding domain (RBD) in the SARS-CoV-2 XBC.1 variant were analysed from
the viewpoint of change in hydrophobicity and amino acid charge. A
decrease in hydrophobicity due to mutations at positions 452 and 486 in
the spike glycoprotein was observed, which might affect the infectivity
of the XBC.1 variant. L452M and F486P improve the hACE2-RBD binding
affinity, which might negatively impact the efficacy of vaccines against
SARS-CoV-2, primarily based on spike glycoprotein. Notably, the mutation
L452M in XBC.1 also indicates its probable zoonotic links.%
\end{abstract}%



\sloppy


\textbf{L452M in spike glycoprotein indicative of zoonotic links of
SARS-CoV-2 XBC.1}

Authors: Shefali Rahangdale\textsuperscript{1}, Siddharth Singh
Tomar\textsuperscript{1,2}, Ekant Tamboli\textsuperscript{1}, Sandhra
Ravikumar\textsuperscript{1}, Lekha Salsekar\textsuperscript{1} and
Krishna Khairnar\textsuperscript{1,2*}

Author Affiliation: \textsuperscript{1}Environmental Virology Cell
(EVC), Council of Scientific and Industrial Research-National
Environmental Engineering Research Institute (CSIR-NEERI), Nagpur,
India. \&

\textsuperscript{2} Academy of Scientific and Innovative Research
(AcSIR), Ghaziabad, UP, India.

*Corresponding author: Correspondence to Krishna Khairnar e-mail:
k\_khairnar@neeri.res.in

\textbf{ABSTRACT}

Mutations at positions 452 and 486 of the spike glycoprotein receptor
binding domain (RBD) in the SARS-CoV-2 XBC.1 variant were analysed from
the viewpoint of change in hydrophobicity and amino acid charge. A
decrease in hydrophobicity due to mutations at positions 452 and 486 in
the spike glycoprotein was observed, which might affect the infectivity
of the XBC.1 variant. L452M and F486P improve the hACE2-RBD binding
affinity, which might negatively impact the efficacy of vaccines against
SARS-CoV-2, primarily based on spike glycoprotein. Notably, the mutation
L452M in XBC.1 also indicates its probable zoonotic links.

\textbf{BACKGROUND}

XBC.1 variant of SARS-CoV-2 is showing an increasing trend in countries
located in North America, Europe, Asia and
Australia.\textsuperscript{1}As per European Centre for Disease
Prevention and Control, XBC.1 is designated as a variant under
monitoring.\textsuperscript{2} L452M and F486P are two important
mutations in the XBC.1 spike protein within the receptor binding domain
(RBD). It has been found that L452M and F486P enhance the infectivity;
this may also impact the potency of SARS-CoV-2 vaccines which are based
on spike protein.\textsuperscript{3,4} Since the RBD-Human Angiotensin
Converting Enzyme 2 (hACE2) interaction enables viral entry into the
human cells, SARS-CoV-2 infectivity may be affected by the substitution
of amino acids in the spike protein RBD region.\textsuperscript{4,5}

\textbf{SPIKE GLYCOPROTEIN MUTATIONS AT POSITIONS 452 \& 486}

Position 452 is located in the receptor binding motif in the RBD, the
amino acid substitution L452M will increase the size of the side chain
of the spike protein RBD region.\textsuperscript{6} Position 486 is
pivotal in binding RBD to hACE2.\textsuperscript{7} Positions 452 and
486 of the SARS-CoV-2 sublineages of omicron have been reported to be
mutation prone; the mutational analysis of XBC.1 identified a unique
mutation, L452M when compared with seventeen SARS-CoV-2 sublineages of
omicron. In particular, BA.5, CH.1.1, BA.4.6, BA.4, BQ.1.1, BQ.1, BF.7
and XAY.2 were found to have L452R mutation (Figure).

Additionally, a rare mutation, F486P was found in the variants XBB.1.5,
XAY.2, and XBC.1. This mutation is caused by two nucleotide
polymorphisms in a single codon in comparison to the wild-type Wuhan
strain. CH.1.1, XBB, BA.2.75.2, XBB.1 and XBB.2 have F486S mutation; and
BQ.1.1, BA.4.6, BQ.1, BF.7, BA.5, and BA.4 have F486V mutation.

\textbf{AMINO ACID HYDROPHOBICITY AFFECTS RBD-hACE2 BINDING AFFINITY}

In the spike glycoprotein RBD positions at 452 and 486, the amino acids
in the wild-type Wuhan-Hu-01 are leucine and phenylalanine, which are
substituted by less hydrophobic amino acids methionine and proline in
XBC.1, respectively. These mutations in XBC.1, indicate, that a
selection pressure might be working towards acquiring the amino acids
that are relatively less hydrophobic in spike glycoprotein. Kyte and
Doolittle hydrophobicity index is considered for understanding the
relative hydrophobicity.\textsuperscript{8}

Hydrophobic interactions have a major role in stabilising the SARS-CoV-2
spike protein.\textsuperscript{9} When a neutral and hydrophobic amino
acid such as phenylalanine is substituted by a neutral and lesser
hydrophobic amino acid such as proline at the position 486, this
particular substitution might increase the RBD-hACE2 binding affinity.
In addition, another substitution at the 452 position where a neutral
and hydrophobic amino acid such as leucine is replaced by a neutral and
lesser hydrophobic amino acid such as methionine may alter the RBD-hACE2
binding affinity.\textsuperscript{10} Methionine substitution in XBC.1
spike glycoprotein at position 452 is predicted to increase the
RBD-hACE2 binding affinity.\textsuperscript{11} A general trend of
increasing RBD-hACE2 binding affinity is observed when a neutral and
less hydrophobic amino acid is incorporated. In contrast, there is a
reduction in the binding affinity of RBD-hACE2 when a negatively charged
and less hydrophobic amino acid is incorporated.

\textbf{ZOONOTIC ORIGIN} \textbf{OF} \textbf{L452M IN XBC.1}

The mutation L452M has been noticed in SARS-CoV-2 sequences collected
from minks; and has been linked to zoonotic transmissions between humans
and minks. However, the zoonotic transmissions were observed only when
the L452M mutation co-occurred with F486L.\textsuperscript{12} The
binding affinity studies show that the co-occurring mutations L452M and
F486L reduced the binding affinity between human ACE2 and RBD but
resulted in increased binding between the mink ACE2 receptor and RBD of
the spike glycoprotein.\textsuperscript{13} In the case of XBC.1, the
mutation L452M co-occurs with F486P, which might affect the RBD-hACE2
binding affinity. The cumulative effect of L452M and F486P on RBD-hACE2
binding affinity warrants further studies.

\textbf{CONFLICT OF INTERESTS}

The authors declare no conflicts of interest.

\textbf{AUTHOR CONTRIBUTIONS}

Shefali Rahangdale, Siddharth Singh Tomar, Ekant Tamboli, Sandhra
Ravikumar, and Lekha Salsekar contributed to literature survey, analysis
and interpretation, draft preparation. Dr. Krishna Khairnar: Conception,
Manuscript editing, and revising.

\textbf{ACKNOWLEDGMENTS}

This work was supported and funded by CSIR-NEERI, Nagpur.

\textbf{REFERENCES}

\begin{enumerate}
\tightlist
\item
  SARS-CoV-2 data explorer. (n.d.). outbreak.info. Retrieved January 11,
  2023, from \emph{https://outbreak.info}
\item
  SARS-CoV-2 variants of concern as of 9 January 2023. (n.d.). European
  Centre for Disease Prevention and
  Control.\emph{https://www.ecdc.europa.eu/en/covid-19/variants-concern}
\item
  Tan, T. S., Toyoda, M., Ode, H., Barabona, G., Hamana, H., Kitamatsu,
  M., Kishi, H., Motozono, C., Iwatani, Y., \& Ueno, T. (2022).
  Dissecting Naturally Arising Amino Acid Substitutions at Position L452
  of SARS-CoV-2 Spike. Journal of virology, 96(20),
  e0116222.\emph{https://doi.org/10.1128/jvi.01162-22}
\item
  Jackson, C. B., Farzan, M., Chen, B., \& Choe, H. (2021). Mechanisms
  of SARS-CoV-2 entry into cells. Nature Reviews Molecular Cell Biology,
  23(1), 3--20.\emph{https://doi.org/10.1038/s41580-021-00418-x}
\item
  Ding, C., He, J., Zhang, X., Jiang, C., Sun, Y., Zhang, Y., Chen, Q.,
  He, H., Li, W., Xie, J., Liu, Z., \& Gao, Y. (2021). Crucial Mutations
  of Spike Protein on SARS-CoV-2 Evolved to Variant Strains Escaping
  Neutralization of Convalescent Plasmas and RBD-Specific Monoclonal
  Antibodies. Frontiers in Immunology,
  12.\emph{https://doi.org/10.3389/fimmu.2021.693775}
\item
  Huo, J., Dijokaite-Guraliuc, A., Nutalai, R., Das, R., Zhou, D.,
  Mentzer, A. J., Fry, E. E., Mongkolsapaya, J., Ren, J., Stuart, D. I.,
  \& Screaton, G. R. (2022). Humoral responses against SARS-CoV-2
  Omicron BA.2.11, BA.2.12.1 and BA.2.13 from vaccine and BA.1 serum.
  Cell Discovery, 8(1).\emph{https://doi.org/10.1038/s41421-022-00482-3}
\item
  Shang, J., Ye, G., Shi, K., Wan, Y., Luo, C., Aihara, H., Geng, Q.,
  Auerbach, A., \& Li, F. (2020). Structural basis of receptor
  recognition by SARS-CoV-2. Nature, 581(7807),
  221--224.\emph{https://doi.org/10.1038/s41586-020-2179-y}
\item
  Kyte, J., \& Doolittle, R. F. (1982). A simple method for displaying
  the hydropathic character of a protein. Journal of Molecular Biology,
  157(1), 105--132.\emph{https://doi.org/10.1016/0022-2836(82)90515-0}
\item
  Young, L., Jernigan, R., \& Covell, D. (1994). A role for surface
  hydrophobicity in protein-protein recognition. Protein Science, 3(5),
  717--729.\emph{https://doi.org/10.1002/pro.5560030501}
\item
  Su, C., He, J., Han, P., Bai, B., Li, D., Cao, J., Tian, M., Hu, Y.,
  Zheng, A., Niu, S., Chen, Q., Rong, X., Zhang, Y., Li, W., Qi, J.,
  Zhao, X., Yang, M., Wang, Q., \& Gao, G. F. (2022). Molecular Basis of
  Mink ACE2 Binding to SARS-CoV-2 and Its Mink-Derived Variants. Journal
  of virology, 96(17),
  e0081422.\emph{https://doi.org/10.1128/jvi.00814-22}
\item
  Starr, T. N., Greaney, A. J., Hilton, S. K., Ellis, D., Crawford, K.
  H., Dingens, A. S., Navarro, M. J., Bowen, J. E., Tortorici, M. A.,
  Walls, A. C., King, N. P., Veesler, D., \& Bloom, J. D. (2020). Deep
  Mutational Scanning of SARS-CoV-2 Receptor Binding Domain Reveals
  Constraints on Folding and ACE2 Binding. Cell, 182(5),
  1295-1310.e20.\emph{https://doi.org/10.1016/j.cell.2020.08.012}
\item
  Burkholz, S., Pokhrel, S., Kraemer, B. R., Mochly-Rosen, D., Carback,
  R. T., Hodge, T., Harris, P., Ciotlos, S., Wang, L., Herst, C., \&
  Rubsamen, R. (2021). Paired SARS-CoV-2 spike protein mutations
  observed during ongoing SARS-CoV-2 viral transfer from humans to minks
  and back to humans. Infection, Genetics, and Evolution, 93,
  104897.\emph{https://doi.org/10.1016/j.meegid.2021.104897}
\item
  Su, C., He, J., Han, P., Bai, B., Li, D., Cao, J., Tian, M., Hu, Y.,
  Zheng, A., Niu, S., Chen, Q., Rong, X., Zhang, Y., Li, W., Qi, J.,
  Zhao, X., Yang, M., Wang, Q., \& Gao, G. F. (2022b). Molecular Basis
  of Mink ACE2 Binding to SARS-CoV-2 and Its Mink-Derived Variants.
  Journal of Virology,
  96(17).\emph{https://doi.org/10.1128/jvi.00814-22}
\end{enumerate}

\textbf{FIGURE LEGEND:} (A) Mutational analysis of spike glycoprotein
RBD region of SARS-CoV-2 Omicon. Hydrophobicity of amino acids according
to the Kyte and Doolittle scale is represented as superscript. (B)
Representation of RBD residues interacting specifically with the hACE2
receptor across 18 omicron sublineages.

Unique mutations are highlighted in red and hACE2 positions interacting
with RBD are highlighted below the sequence in orange.\selectlanguage{english}
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