Viroids, small circular non-coding RNAs, act as infectious pathogens in higher plants, demonstrating high stability despite consisting solely of naked RNA. Their dependence of replication on host machinery poses the question of whether RNA modifications play a role in viroid biology. Here, we explore RNA modifications in the avocado sunblotch viroid (ASBVd) and the citrus exocortis viroid (CEVd), representative members of viroids replicating in chloroplasts and the nucleus, respectively, using LC - MS and Oxford Nanopore Technology (ONT) direct RNA sequencing. Although no modification was detected in ASBVd, CEVd contained approximately one m6A per RNA molecule. ONT sequencing predicted three m6A positions. Employing orthogonal SELECT method, we confirmed m6A in two positions A353 and A360, which are highly conserved among CEVd variants. These positions are located in the left terminal region of the CEVd rod-like structure where likely RNA Pol II and and TFIIIA-7ZF bind, thus suggesting potential biological role of methylation in viroid replication.

• Klíčová slova
• 6-methyladenosine, LC-MS, m6A SELECT, RNA modification, Viroid, direct RNA-seq,
• MeSH
• konformace nukleové kyseliny MeSH
• kruhová RNA * genetika   metabolismus   MeSH
• nemoci rostlin virologie   MeSH
• posttranskripční úpravy RNA * MeSH
• replikace viru MeSH
• RNA virová * genetika   metabolismus   chemie   MeSH
• sekvenční analýza RNA MeSH
• viroidy * genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• kruhová RNA * MeSH
• RNA virová * MeSH

The Picornavirales include viruses that infect vertebrates, insects, and plants. It was believed that they pack only their genomic mRNA in the particles; thus, we envisaged these viruses as excellent model systems for studies of mRNA modifications. We used LC-MS to analyze digested RNA isolated from particles of the sacbrood and deformed wing iflaviruses as well as of the echovirus 18 and rhinovirus 2 picornaviruses. Whereas in the picornavirus RNAs we detected only N6 -methyladenosine and 2'-O-methylated nucleosides, the iflavirus RNAs contained a wide range of methylated nucleosides, such as 1-methyladenosine (m1 A) and 5-methylcytidine (m5 C). Mapping of m1 A and m5 C through RNA sequencing of the SBV and DWV RNAs revealed the presence of tRNA molecules. Both modifications were detected only in tRNA. Further analysis revealed that tRNAs are present in form of 3' and 5' fragments and they are packed selectively. Moreover, these tRNAs are typically packed by other viruses.

• Klíčová slova
• LC-MS, Picornavirales, RNA methylation, human Echovirus 18, human rhinovirus type 2, tRNA fragments,
• MeSH
• messenger RNA MeSH
• nukleosidy * MeSH
• RNA transferová * genetika   MeSH
• RNA MeSH
• včely genetika   MeSH
• virion genetika   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• messenger RNA MeSH
• nukleosidy * MeSH
• RNA transferová * MeSH
• RNA MeSH

Viral proteases are indispensable for successful virion maturation, thus making them a prominent drug target. Their enzyme activity is tightly spatiotemporally regulated by expression in the precursor form with little or no activity, followed by activation via autoprocessing. These cleavage events are frequently triggered upon transportation to a specific compartment inside the host cell. Typically, precursor oligomerization or the presence of a co-factor is needed for activation. A detailed understanding of these mechanisms will allow ligands with non-canonical mechanisms of action to be designed, which would specifically modulate the initial irreversible steps of viral protease autoactivation. Binding sites exclusive to the precursor, including binding sites beyond the protease domain, can be exploited. Both inhibition and up-regulation of the proteolytic activity of viral proteases can be detrimental for the virus. All these possibilities are discussed using examples of medically relevant viruses including herpesviruses, adenoviruses, retroviruses, picornaviruses, caliciviruses, togaviruses, flaviviruses, and coronaviruses.

• Klíčová slova
• Human Immunodeficiency Virus (HIV), Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), activation, adenoviruses, autoprocessing, flaviviruses, herpesviruses, precursor, protease,
• MeSH
• antivirové látky farmakologie   MeSH
• Flavivirus účinky léků   metabolismus   MeSH
• Herpesviridae účinky léků   metabolismus   MeSH
• HIV-1 účinky léků   MeSH
• inhibitory virových proteáz farmakologie   MeSH
• lidé MeSH
• lidské adenoviry účinky léků   metabolismus   MeSH
• SARS-CoV-2 účinky léků   metabolismus   MeSH
• virové nemoci farmakoterapie   MeSH
• virové proteasy biosyntéza   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• antivirové látky MeSH
• inhibitory virových proteáz MeSH
• virové proteasy MeSH