Biology subjects

Fominykh, K.

Publications and source records attributed to Fominykh, K..

5 recordsLinked to original sources

bioRxiv2026

The dynamics and strategy of RNA replication in astroviruses

Astroviruses are positive-sense single-stranded RNA viruses that cause significant disease across avian and mammalian hosts, yet their replication mechanisms remain poorly understood. The replication of astrovirus RNA occurs via a double-stranded RNA intermediate that is used as a template for the synthesis of new positive-sense RNA, which is covalently linked to the virus-encoded protein VPg. These viruses also produce a capsid-encoding subgenomic (sg) RNA that is 3'-coterminal with the genomic RNA. The mechanisms by which the astrovirus sgRNA is produced and regulated during infection have not yet been characterized. Using high throughput sequencing of RNA from cells infected with each of five different astrovirus strains, we demonstrate that the presence of a (-)sgRNA is a conserved feature of infection, supporting a premature termination model of subgenomic RNA production. A pronounced pile-up in the mapping positions of the 3' ends of negative-sense RNA reads marks the precise 3' terminus of the (-)sgRNA. We investigate the relative abundance and dynamics of positive and negative RNA species during virus replication and virion packaging, and perform a mutational analysis of conserved residues in the genomic and subgenomic 5' termini. Together, this work elucidates the dynamics of genomic and subgenomic RNA synthesis during astrovirus infection. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=70 SRC="FIGDIR/small/700307v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@1d71111org.highwire.dtl.DTLVardef@a1a024org.highwire.dtl.DTLVardef@118d126org.highwire.dtl.DTLVardef@4b3c4e_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2025

Viral protease-mediated polyprotein processing in human astroviruses

Positive-sense RNA viruses often encode large polyproteins that are proteolytically processed by viral and host proteases into functional replication proteins. Astroviruses infect intestinal and neuronal cells across diverse human and animal hosts and also rely on polyprotein cleavage for replication. In this study, we mapped the cleavage sites of the nonstructural polyproteins of classical human astrovirus 1 (HAstV1) and neurotropic astrovirus strain MLB2 using complementary N-terminomics of infected cells and analyses of untagged overexpressed polyproteins. Notably, we identified two adjacent cleavage sites at the N-terminus of HAstV1 and MLB2 proteases, as well as a similar dual cleavage site at the C-terminus of the MLB2 protease. We also demonstrated processing of the hypervariable region and VPg in both astrovirus strains. This allowed us to define the boundaries of individual protein products and identify conserved and divergent processing features between classical and non-classical astroviruses. Additionally, we characterized several polyprotein precursors and evaluated the replication properties of cleavage-deficient mutant replicons, revealing the critical role of polyprotein processing for functional replication complex formation. Understanding the dynamics of polyprotein processing is essential for interpreting the stages of viral infection and identifying new drug targets and antiviral strategies.

microbiology↗

bioRxiv2025

Structural and mechanistic insights into translation initiation on the enterovirus Type 1 IRES

Enteroviruses are a diverse group of pathogens that cause over one billion human infections annually. Upon cell entry, translation of the viral genome is directed by an internal ribosome entry site (IRES) within the 5' untranslated region. Despite early identification of the Type 1 poliovirus IRES, the structural and mechanistic basis for its activity remains poorly understood due to its size, flexibility and dependence on multiple host cell factors. Here, we reconstitute human translation initiation on a model poliovirus IRES and examine 48S complexes by cryo-electron microscopy. Our structures reveal how IRES domain IVc contacts ribosomal proteins uS19 and uS13, whilst a conserved GNRA tetraloop engages with the initiator tRNA during start-codon recognition. Disruption of these interfaces impairs IRES-dependent translation and viral replication. Together, our results provide new structural and mechanistic insights into initiation on the Type 1 IRES and reveal conserved RNA-RNA interactions critical for enterovirus translation.

molecular biology↗

bioRxiv2025

Phosphorylation enables allosteric control of a viral condensate

In many viruses, intrinsically disordered proteins (IDPs) drive the formation of replicative organelles essential for viral production. In species A rotaviruses, the disordered protein NSP5 forms condensates in cells via liquid-liquid phase separation (LLPS). Yet the sequence diversity of NSP5 raises the question of whether condensate formation is conserved across all strains and if distinct variants employ alternative mechanisms for nucleating phase separation. Using a machine learning approach, we demonstrate that NSP5 variants differ significantly in their propensity to phase-separate. We engineered a variant incorporating amino acid signatures from strains with low LLPS tendency, which failed to phase separate in vitro yet supported the formation of replicative condensates in recombinant viruses in cells. Low-tendency LLPS strains require phosphorylation of NSP5 to nucleate phase separation, whereas high-tendency strains do not, suggesting distinct nucleation mechanisms. Furthermore, hydrogen-deuterium exchange mass spectrometry revealed a phosphorylation-driven allosteric switch between binding sites on the high-propensity variant. These findings establish that phosphorylation plays a context-dependent role in the formation of replicative organelles across diverse rotaviruses.

biophysics↗

bioRxiv2025

Flexibility and modulation of translation initiation in enterovirus genomes

Enteroviruses comprise a large group of mammalian pathogens that often utilize two open reading frames (ORFs) to encode their proteins: the upstream protein (UP) and the main polyprotein. In some enteroviruses, in addition to the canonical upstream AUG (uAUG), there is another AUG that may represent an alternative upstream initiation site. An analysis of enterovirus sequences containing additional upstream AUGs identified several clusters, including strains of pathogenic Enterovirus alphacoxsackie and E. coxsackiepol. Using ribosome profiling on coxsackievirus CVA-13 (E. coxsackiepol), we demonstrate that both upstream AUG codons can be used for translation initiation in infected cells. Moreover, we confirm translation from both upstream AUGs using a reporter system. Mutating the additional upstream AUG in the context of CVA-13 did not result in phenotypic changes in immortalized cell lines. However, the wild-type virus outcompeted this mutant in human intestinal organoids and differentiated neuronal systems, representing an advantage in physiologically relevant infection sites. Mutation of the stop codon of the shorter upstream ORF led to dysregulated translation of the other ORFs in the reporter system, suggesting a potential role for the additional uORF in modulating the expression level of the other ORFs. These findings demonstrate the remarkable plasticity of enterovirus IRES-mediated initiation and the competitive advantage of double-upstream-AUG-containing viruses in terminally differentiated intestinal organoids and neuronal systems.

microbiology↗