Since the emergence of SARS-CoV-2, mutations in all subunits of the RNA-dependent RNA polymerase (RdRp) of the virus have been repeatedly reported. Although RdRp represents a primary target for antiviral drugs, experimental studies exploring the phenotypic effect of these mutations have been limited. This study focuses on the phenotypic effects of substitutions in the three RdRp subunits: nsp7, nsp8, and nsp12, selected based on their occurrence rate and potential impact. We employed nano-differential scanning fluorimetry and microscale thermophoresis to examine the impact of these mutations on protein stability and RdRp complex assembly. We observed diverse impacts; notably, a single mutation in nsp8 significantly increased its stability as evidenced by a 13°C increase in melting temperature, whereas certain mutations in nsp7 and nsp8 reduced their binding affinity to nsp12 during RdRp complex formation. Using a fluorometric enzymatic assay, we assessed the overall effect on RNA polymerase activity. We found that most of the examined mutations altered the polymerase activity, often as a direct result of changes in stability or affinity to the other components of the RdRp complex. Intriguingly, a combination of nsp8 A21V and nsp12 P323L mutations resulted in a 50% increase in polymerase activity. To our knowledge, this is the first biochemical study to demonstrate the impact of amino acid mutations across all components constituting the RdRp complex in emerging SARS-CoV-2 subvariants.

• Klíčová slova
• RNA‐dependent RNA polymerase (RdRp), SARS‐CoV‐2, mutations, phenotypic effect,
• MeSH
• COVID-19 virologie   MeSH
• koronavirová RNA-replikasa * genetika   metabolismus   chemie   MeSH
• lidé MeSH
• mutace * MeSH
• RNA-dependentní RNA-polymerasa genetika   chemie   metabolismus   MeSH
• SARS-CoV-2 * genetika   enzymologie   MeSH
• stabilita proteinů MeSH
• vazba proteinů MeSH
• virové nestrukturální proteiny * genetika   chemie   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• koronavirová RNA-replikasa * MeSH
• NS8 protein, SARS-CoV-2 MeSH Prohlížeč
• NSP12 protein, SARS-CoV-2 MeSH Prohlížeč
• NSP7 protein, SARS-CoV-2 MeSH Prohlížeč
• RNA-dependentní RNA-polymerasa MeSH
• virové nestrukturální proteiny * MeSH

Fungi harbor a vast diversity of mobile genetic elements (MGEs). Recently, novel fungal MGEs, tentatively referred to as 'ambiviruses,' were described. 'Ambiviruses' have single-stranded RNA genomes of about 4-5 kb in length that contain at least two open reading frames (ORFs) in non-overlapping ambisense orientation. Both ORFs are conserved among all currently known 'ambiviruses,' and one of them encodes a distinct viral RNA-directed RNA polymerase (RdRP), the hallmark gene of ribovirian kingdom Orthornavirae. However, 'ambivirus' genomes are circular and predicted to replicate via a rolling-circle mechanism. Their genomes are also predicted to form rod-like structures and contain ribozymes in various combinations in both sense and antisense orientations-features reminiscent of viroids, virusoids, ribozyvirian kolmiovirids, and yet-unclassified MGEs (such as 'epsilonviruses,' 'zetaviruses,' and some 'obelisks'). As a first step toward the formal classification of 'ambiviruses,' the International Committee on Taxonomy of Viruses (ICTV) recently approved the establishment of a novel ribovirian phylum, Ambiviricota, to accommodate an initial set of 20 members with well-annotated genome sequences.

• Klíčová slova
• Ambiviricota, Ascomycota, Basidiomycota, ICTV, International Committee on Taxonomy of Viruses, Riboviria, ambiviricot, ambivirus, deltavirus, fungi, fungus, hairpin, hammerhead, phylum, ribozyme, twister, viroid, virus classification, virus nomenclature, virus taxonomy,
• MeSH
• fylogeneze MeSH
• genom virový * MeSH
• houby genetika   virologie   MeSH
• mykoviry genetika   klasifikace   izolace a purifikace   MeSH
• otevřené čtecí rámce * MeSH
• RNA virová genetika   MeSH
• RNA-dependentní RNA-polymerasa genetika   MeSH
• RNA-viry genetika   klasifikace   MeSH
• viroidy * genetika   klasifikace   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• RNA virová MeSH
• RNA-dependentní RNA-polymerasa MeSH

In April 2023, following the annual International Committee on Taxonomy of Viruses (ICTV) ratification vote on newly proposed taxa, the phylum Negarnaviricota was amended and emended. The phylum was expanded by one new family, 14 new genera, and 140 new species. Two genera and 538 species were renamed. One species was moved, and four were abolished. This article presents the updated taxonomy of Negarnaviricota as now accepted by the ICTV.

• Klíčová slova
• Aliusviridae, Arenaviridae, Articulavirales, Artoviridae, Aspiviridae, Bornaviridae, Bunyavirales, Crepuscuviridae, Discoviridae, Filoviridae, Fimoviridae, Goujianvirales, Hantaviridae, ICTV, International Committee on Taxonomy of Viruses, Jingchuvirales, Lispiviridae, Mononegavirales, Muvirales, Mymonaviridae, Myriaviridae, Nairoviridae, Natareviridae, Negarnaviricota, Nyamiviridae, Orthomyxoviridae, Orthornavirae, Paramyxoviridae, Peribunyaviridae, Phasmaviridae, Phenuiviridae, Pneumoviridae, Rhabdoviridae, Riboviria, Serpentovirales, Sunviridae, Tenuivirus, Tosoviridae, Tospoviridae, Tulasviridae, articulaviral, bunyaviral, bunyavirus, goujianviral, megaclassification, megataxonomy, mononegaviral, muviral, negarnaviricot, serpentoviral, virus classification, virus nomenclature, virus taxonomy,
• MeSH
• negativní RNA-viry * MeSH
• RNA-dependentní RNA-polymerasa genetika   MeSH
• RNA-viry * genetika   MeSH
• Publikační typ
• dopisy MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, N.I.H., Intramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• RNA-dependentní RNA-polymerasa MeSH

Here, we report the discovery and complete genome sequence of a novel virus, designated as "Phytophthora heveae alphaendornavirus 1" (PhAEV1), from a single isolate of the plant pathogenic oomycete Phytophthora heveae (kingdom Stramenipila) isolated from a tropical evergreen lowland rainforest in northern Vietnam. PhAEV1 was detected by both cellulose affinity chromatography of dsRNA and high-throughput sequencing of total RNA, and its presence and sequence were confirmed by RT-PCR and Sanger sequencing. The PhAEV1 genome, 12,820 nucleotides (nt) in length, was predicted to encode a single large polyprotein with the catalytic core domain of viral (superfamily 1) RNA helicase (HEL, amino acid [aa] positions 1,287-1,531), glycosyltransferase (GT, aa positions ca. 2,800-3,125), and RNA-directed RNA polymerase (RdRp, aa positions 3,875-4,112). PhAEV1 is the most similar to Phytophthora cactorum alphaendornavirus 3, sharing 39.4% and 39.1% nt and aa sequence identity, respectively. In addition to the first 5'-terminal AUG codon, three additional in-frame methionine codons were found in close proximity (nt 14-16, 96-98, and 176-178), suggesting potential additional translation initiation sites. Conserved RdRp motifs (A-E) similar to those detected in related endornaviruses were identified in PhAEV1, as well as in several previously described alphaendornaviruses from other Phytophthora species in which these motifs had not been identified previously. Phylogenetic analysis showed that PhAEV1 clusters with members of the genus Alphaendornavirus in the family Endornaviridae and is basal to two other alphaendornaviruses described from another oomycete, Phytophthora cactorum. PhAEV1 is the first virus reported in P. heveae.

• MeSH
• fylogeneze MeSH
• genom virový MeSH
• otevřené čtecí rámce MeSH
• Phytophthora * genetika   MeSH
• RNA virová genetika   MeSH
• RNA-dependentní RNA-polymerasa genetika   MeSH
• RNA-viry * genetika   MeSH
• sekvence aminokyselin MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• RNA virová MeSH
• RNA-dependentní RNA-polymerasa MeSH

We have identified seven putative guanine quadruplexes (G4) in the RNA genome of tick-borne encephalitis virus (TBEV), a flavivirus causing thousands of human infections and numerous deaths every year. The formation of G4s was confirmed by biophysical methods on synthetic oligonucleotides derived from the predicted TBEV sequences. TBEV-5, located at the NS4b/NS5 boundary and conserved among all known flaviviruses, was tested along with its mutated variants for interactions with a panel of known G4 ligands, for the ability to affect RNA synthesis by the flaviviral RNA-dependent RNA polymerase (RdRp) and for effects on TBEV replication fitness in cells. G4-stabilizing TBEV-5 mutations strongly inhibited RdRp RNA synthesis and exhibited substantially reduced replication fitness, different plaque morphology and increased sensitivity to G4-binding ligands in cell-based systems. In contrast, strongly destabilizing TBEV-5 G4 mutations caused rapid reversion to the wild-type genotype. Our results suggest that there is a threshold of stability for G4 sequences in the TBEV genome, with any deviation resulting in either dramatic changes in viral phenotype or a rapid return to this optimal level of G4 stability. The data indicate that G4s are critical elements for efficient TBEV replication and are suitable targets to tackle TBEV infection.

• MeSH
• antivirové látky * farmakologie   terapeutické užití   MeSH
• G-kvadruplexy * MeSH
• klíšťová encefalitida farmakoterapie   genetika   MeSH
• lidé MeSH
• ligandy MeSH
• RNA virová genetika   MeSH
• RNA-dependentní RNA-polymerasa genetika   MeSH
• viry klíšťové encefalitidy * účinky léků   genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antivirové látky * MeSH
• ligandy MeSH
• RNA virová MeSH
• RNA-dependentní RNA-polymerasa MeSH

Leishmania spp. are important pathogens causing a vector-borne disease with a broad range of clinical manifestations from self-healing ulcers to the life-threatening visceral forms. Presence of Leishmania RNA virus (LRV) confers survival advantage to these parasites by suppressing anti-leishmanial immunity in the vertebrate host. The two viral species, LRV1 and LRV2 infect species of the subgenera Viannia and Leishmania, respectively. In this work we investigated co-phylogenetic patterns of leishmaniae and their viruses on a small scale (LRV2 in L. major) and demonstrated their predominant coevolution, occasionally broken by intraspecific host switches. Our analysis of the two viral genes, encoding the capsid and RNA-dependent RNA polymerase (RDRP), revealed them to be under the pressure of purifying selection, which was considerably stronger for the former gene across the whole tree. The selective pressure also differs between the LRV clades and correlates with the frequency of interspecific host switches. In addition, using experimental (capsid) and predicted (RDRP) models we demonstrated that the evolutionary variability across the structure is strikingly different in these two viral proteins.

• Klíčová slova
• Leishmaniavirus, coevolution, phylogenomics,
• MeSH
• Leishmania virologie   MeSH
• leishmanióza virologie   MeSH
• lidé MeSH
• RNA virová analýza   MeSH
• RNA-dependentní RNA-polymerasa genetika   MeSH
• RNA-viry genetika   MeSH
• virové plášťové proteiny genetika   MeSH
• virové proteiny genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• RNA virová MeSH
• RNA-dependentní RNA-polymerasa MeSH
• virové plášťové proteiny MeSH
• virové proteiny MeSH

The genome sequence of a mitovirus found in an isolate of Diaporthe rudis, one of the causal agents of Phomopsis dieback on grapevines, was determined by two high-throughput sequencing approaches, small RNA and total RNA sequencing. The genome of this mitovirus is 2,455 nt in length and includes a single large open reading frame (ORF) encoding an RNA-dependent RNA polymerase (RdRp). A BLASTx comparison of the full-length genome sequence showed the highest similarity (54.15%) with that of Colletotrichum falcatum mitovirus 1 (CfMV1). Our results reveal a new member of the genus Mitovirus first detected in D. rudis (Fr.) Nitschke, with the proposed name "Diaporthe rudis mitovirus 1" (DrMV1).

• MeSH
• délka genomu MeSH
• exprese genu MeSH
• fylogeneze * MeSH
• genom virový * MeSH
• mykoviry klasifikace   genetika   izolace a purifikace   MeSH
• nemoci rostlin mikrobiologie   MeSH
• otevřené čtecí rámce MeSH
• RNA-dependentní RNA-polymerasa genetika   MeSH
• Saccharomycetales virologie   MeSH
• sekvenování celého genomu MeSH
• virové proteiny genetika   MeSH
• Vitis mikrobiologie   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• RNA-dependentní RNA-polymerasa MeSH
• virové proteiny MeSH

Tick-borne encephalitis virus (TBEV) is a medically important representative of the Flaviviridae family. The TBEV genome encodes a single polyprotein, which is co/post-translationally cleaved into three structural and seven non-structural proteins. Of the non-structural proteins, NS5, contains an RNA-dependent RNA polymerase (RdRp) domain that is highly conserved and is responsible for the genome replication. Screening for potential antivirals was done using a hybrid receptor and ligand-based pharmacophore search likely targeting the RdRp domain. For the identification of pharmacophores, a mixture of small probe molecules and nucleotide triphosphates were used. The ligand/receptor interaction screenings of structures from the ZINC database resulted in five compounds. Zinc 3677 and 7151 exhibited lower cytotoxicity and were tested for their antiviral effect against TBEV in vitro. Zinc 3677 inhibited TBEV at micromolar concentrations. The results indicate that Zinc 3677 represents a good target for structure-activity optimizations leading potentially to a discovery of effective TBEV antivirals.

• Klíčová slova
• Molecular, Non-structural proteins, Simulation, TBEV, Viral inhibition,
• MeSH
• antivirové látky farmakologie   MeSH
• inhibitory enzymů farmakologie   MeSH
• klíšťata virologie   MeSH
• klíšťová encefalitida virologie   MeSH
• lidé MeSH
• replikace viru účinky léků   MeSH
• RNA-dependentní RNA-polymerasa antagonisté a inhibitory   genetika   metabolismus   MeSH
• virové proteiny antagonisté a inhibitory   genetika   metabolismus   MeSH
• viry klíšťové encefalitidy účinky léků   enzymologie   genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antivirové látky MeSH
• inhibitory enzymů MeSH
• RNA-dependentní RNA-polymerasa MeSH
• virové proteiny MeSH

RNA-dependent RNA polymerase 3Dpol is a key enzyme for the replication of picornaviruses. The viral genome is translated into a single polyprotein that is subsequently proteolytically processed into matured products. The 3Dpol enzyme arises from a stable 3CD precursor that has high proteolytic activity but no polymerase activity. Upon cleavage of the precursor the newly established N-terminus of 3Dpol is liberated and inserts itself into a pocket on the surface of the 3Dpol enzyme. The essential residue for this mechanism is the very first glycine that is conserved among almost all picornaviruses. However, kobuviruses and siciniviruses have a serine residue instead. Intrigued by this anomaly we sought to solve the crystal structure of these 3Dpol enzymes. The structures revealed a unique fold of the 3Dpol N-termini but the very first serine residues were inserted into a charged pocket in a similar manner as the glycine residue in other picornaviruses. These structures revealed a common underlying mechanism of 3Dpol activation that lies in activation of the α10 helix containing a key catalytical residue Asp238 that forms a hydrogen bond with the 2' hydroxyl group of the incoming NTP nucleotide.

• Klíčová slova
• Crystal structure, Kobuvirus, Picornavirus, Polymerase, RNA,
• MeSH
• HeLa buňky MeSH
• Kobuvirus enzymologie   MeSH
• krystalografie rentgenová MeSH
• lidé MeSH
• mutageneze cílená MeSH
• Picornaviridae enzymologie   MeSH
• průtoková cytometrie MeSH
• RNA-dependentní RNA-polymerasa chemie   genetika   metabolismus   MeSH
• virové proteiny chemie   genetika   metabolismus   MeSH
• vodíková vazba MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• RNA-dependentní RNA-polymerasa MeSH
• virové proteiny MeSH

The family Partitiviridae consists of dsRNA viruses with genome separated into two segments and encoding replicase and capsid protein only. We examined the nucleotide diversity expressed as the ratio dN/dS of nonsynonymous and synonymous substitutions, which has been calculated for 12 representative viruses of all five genera of partitiviruses. We can state that strong purifying selection works on both the RdRp and CP genes and propose that putative positive selection occurs also on the RdRp genes in two viruses. Among the 95 evaluated viruses, wherein both segments had been sequenced, 8 viruses in betapartitiviruses and 9 in alphapartitiviruses were identified as reassortment candidates because they differ extremely in their CP identity even as they are related in terms of RdRp. Furthermore, there are indications that reassortants are present among isolates of different viruses.

• Klíčová slova
• Capsid protein, Negative/positive selection, RNA polymerase, Reassortment, Symbiosis,
• MeSH
• dvouvláknová RNA genetika   MeSH
• genetická variace MeSH
• molekulární evoluce * MeSH
• mutace MeSH
• RNA virová genetika   MeSH
• RNA-dependentní RNA-polymerasa genetika   MeSH
• RNA-viry genetika   MeSH
• virové plášťové proteiny genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• dvouvláknová RNA MeSH
• RNA virová MeSH
• RNA-dependentní RNA-polymerasa MeSH
• virové plášťové proteiny MeSH