@phdthesis{fdi:010089166,
  title = {{T}he interplay of {RNA} {N}7-and 2'{O}-methylation in viral replication},
  author = {{E}l {K}azzi, {P}.{B}.},
  editor = {},
  language = {{ENG}},
  abstract = {{I}n recent years, epitranscriptomic modifications have been detected in numerous viral {RNA}, but the physiological function of most of them remains barely understood. {A}mong these modifications, {RNA} methylations are an important modification induced by specific viral or cellular {RNA} methyltransferases. {T}he first part of this manuscript focuses on {RNA} 2'{O}-methylation. {T}his post-transcriptional modification prevents viral {RNA} detection by {RIG}-like receptors ({RIG}-{I} and {MDA}5) that regulate type-1 interferon expression and is therefore considered a 'self' marker. {I}n this work, we address the possibility that {RNA} 2'{O}-methylation may interplay with the antiviral action of an interferon-induced restriction factor, namely {ISG}20. {B}riefly, we show that {ISG}20 exonuclease activity is strongly impaired when it encounters a 2'{O}-methylation mark within the {RNA}. {ISG}20 stops two nucleotides upstream ({N}-2) and at the methylated residue ({N}0). {S}tructure-function analyses revealed that {ISG}20 {R}53 and {D}90 residues play a key role in the {RNA} hydrolysis impairment, which results from a steric clash of these residues with the 2'{O}-methylated nucleotide. {W}e next extrapolated our observations to {HIV}-1 which is naturally 2'{O}-methylated by the host {FTSJ}3. {B}y comparing the sensitivity to {ISG}20 of {HIV}-1 {RNA}s extracted from hypo-methylated viruses (produced in {FTSJ}3-{KO} cells) and from normally methylated viruses, we showed that internal 2'{O}-methylation protects the {HIV}-1 genome from {ISG}20 degradation. {W}e confirmed this observation in infected cells and showed that ectopically expressed {ISG}20 drastically reduces the replication of hypomethylated {VSV}-{G} pseudotyped {HIV}-1 virus, as a consequence of impaired reverse transcription. {A}ltogether, our results shed light on a new pro-viral role of viral {RNA} 2'{O}-methylation. {I}ndeed, we demonstrated that {HIV}-1 2'{O}-methylation promotes viral replication by limiting {ISG}20-mediated restriction. {I}n the second part of the manuscript, we characterized the {C}o{V} nonstructural protein 14 (nsp14) which is a bifunctional protein harboring an {N}-terminal 3'-to-5' {E}xo{N} domain and a {C}-terminal {N}7-{MT}ase domain that is presumably involved in viral m{RNA} capping. {W}e integrate structural, biochemical, and virological data to identify 4 conserved regions essential for nsp14's enzymatic activities and virus viability. {W}e identified several residues involved in the formation of the {N}7-{MT}ase catalytic pocket, which presents a fold distinct from the {R}ossmann fold observed in most known {MT}ases and we assess their importance for in vitro enzymatic activity and for virus replication. {O}ur results identify the {N}7-{MT}ase as a critical enzyme for betacoronavirus replication and define key residues of its catalytic pocket that can be targeted to design inhibitors with a potential pan-coronaviral activity spectrum.},
  keywords = {},
  address = {{M}arseille},
  publisher = {{U}niversit{\'e} {A}ix-{M}arseille ; {IRD}},
  pages = {166  multigr.},
  year = {2022},
  URL = {https://www.documentation.ird.fr/hor/fdi:010089166},
}