The results indicate that −1PRF and spike protein maturation in alphaviruses is tuned by the dynamic remodeling of the ER translocon, which could allow polyprotein biogenesis to adapt to different stages of viral replication and to distinct host or vector environments.
Abstract
Like other enveloped RNA viruses, alphaviruses synthesize and assemble their envelope glycoproteins at the endoplasmic reticulum (ER) membrane. There, protein-conducting channels called translocons provide nascent proteins access to the membrane and allow them to fold into their correct shapes. We previously showed that a hydrophobic segment in the Sindbis virus structural polyprotein forms cotranslational interactions with the translocon that enhance −1 programmed ribosomal frameshifting (−1PRF), a recoding event that regulates polyprotein biogenesis. Recent discoveries concerning translocon remodeling suggest this segment, which corresponds to the second transmembrane domain of the E2 protein, could serve as a signal that recruits the multipass translocon (MPT). Here, we show that knocking out certain components of the MPT increases −1PRF efficiency. These differences in recoding coincide with changes in the membrane topology of the nascent polyprotein and in its downstream proteolytic processing in a manner that ultimately reduces viral fitness. Together, our results indicate that −1PRF and spike protein maturation in alphaviruses is tuned by the dynamic remodeling of the ER translocon. Such coupling could allow polyprotein biogenesis to adapt to different stages of viral replication and to distinct host or vector environments.
ABSTRACT Programmed −1 ribosomal frameshifting (−1 PRF) is a translational recoding mechanism used by many RNA viruses to regulate the expression of viral replication proteins. In coronaviruses, including SARS-CoV-2, −1 PRF is controlled by a conserved frameshift stimulation element containing a three-stemmed RNA pseudoknot located downstream of a slippery sequence. Studies have shown that conformational dynamics, mechanical stability, and structural variability of the pseudoknot influence ribosome pausing and frameshifting efficiency, identifying viral RNA structures as potential antiviral targets. This review outlines the structural organization, mechanistic basis, and conformational dynamics of viral frameshifting pseudoknots, with emphasis on the SARS-CoV-2 frameshift stimulation element. Advances in cryo-electron microscopy, single-molecule biophysics, molecular dynamics simulations, and computational modeling have identified multiple pseudoknot conformations involved in translational recoding and ribosome–RNA interactions. RNA-targeted therapeutic approaches used to suppress or modulate −1 PRF are also discussed, including small-molecule RNA binders, antisense oligonucleotides, peptide nucleic acids, and ribonuclease-targeting chimeras. These approaches act on distinct aspects of RNA structure, conformational flexibility, and stability to inhibit viral translation or promote selective RNA degradation. Major challenges include selective targeting of highly dynamic RNA structures, optimization of intracellular delivery, and minimizing off-target effects. Integration of structural biology, computational modeling, and RNA-targeted therapeutic strategies may support the development of next-generation antivirals targeting conserved viral RNA regulatory elements.
Neha Jeena, I. Khan· Antimicrobial Agents and Che...· 0 citations
The orthoflavivirus capsid (C) protein is a multifunctional protein that plays essential roles throughout the viral life cycle. Besides viral RNA encapsidation for nucleocapsid assembly, it associates with lipid droplets, interacts with host proteins, and translocates to the nucleus, although its nuclear functions are still poorly understood. How these diverse activities are coordinated remains an open question. Post-translational modifications (PTMs), which are key regulators of protein function, have emerged as critical modulators of the infection cycle in many RNA viruses. However, little is known about the occurrence and functional significance of PTMs in orthoflavivirus C proteins. Here, we review the current evidence on PTMs in orthoflavivirus C proteins and integrate insights from studies of other RNA viruses to propose mechanisms by which PTMs may regulate C protein function. To complement this review, we performed a comparative in silico analysis of predicted PTM sites in the C proteins of dengue, Zika, West Nile, and Japanese encephalitis viruses. By integrating PTM predictions with experimentally validated modification sites, residue conservation, and structural mapping, we identified conserved regulatory hotspots that represent promising targets for future experimental validation. Together, these findings highlight PTMs as an underexplored regulatory mechanism in orthoflavivirus capsid biology and provide a framework for future mechanistic investigations.
N. C. Mebus-Antunes, Dayane Henriques, Andrea T. Da Poian· Molecules· 0 citations
Severe acute respiratory syndrome coronavirus 2 assembles at the ER–Golgi intermediate compartment (ERGIC), yet the molecular basis of nucleocapsid (N) protein interactions with host membranes remains unclear. Using in vitro reconstituted lipid membranes and viral RNA– N complexes, we show that full-length N binds phosphatidylinositol (PI)- and phosphatidylserine (PS)-containing membranes and induces lipid clustering, an effect amplified by viral RNA and ERGIC-like membrane lipid composition. The isolated N-terminal domain lacks this activity, whereas the C-terminal domain retains membrane-associated multimerization. Although, lipid bilayers promote co-clustering of N and PI lipids, facilitating ribonucleoprotein (RNP) assembly, even on simple membranes. Importantly, ERGIC- mimicking membranes enhances this co-clustering further stabilizing RNPs of dimensions matching the viral core. In cells, viral RNA enhances N clustering without altering particle production. These findings reveal cooperative interactions between N, viral RNA, and ERGIC lipids as key drivers of lipid-dependent viral core formation, providing a mechanistic framework for the early steps of viral assembly.
Shovon Swarnakar, J. Mishra, Virgile Rat et al.· bioRxiv· 0 citations
Translation of key viral replicative proteins in coronaviruses requires a programmed –1 ribosomal frameshifting (–1 PRF) event controlled by the viral frameshift-stimulatory element (FSE). Although previous studies have analyzed host factor dependencies of coronaviruses, how host cellular factors alter –1 PRF efficiency and affect viral replication remains poorly understood. Here, using RNA pull-down combined with LC-MS/MS analysis, we identified heterogeneous nuclear ribonucleoprotein C (hnRNPC) as a major interacting protein of FSE RNA. Coronavirus infection triggers hnRNPC mRNA decay, alters hnRNPC protein levels, and induces its cytoplasmic relocalization, where it appears to bind directly to FSE RNA through residues Asn7 and Asn83. This binding is associated with increased –1 PRF efficiency and may facilitate coronavirus replication. Deletion mapping analysis shows that hnRNPC preferentially binds U-rich regions of the FSE RNA. Finally, we demonstrated that the small molecule Elbasvir directly binds hnRNPC, disrupting the interaction between hnRNPC and FSE RNA and inhibiting coronavirus replication by decreasing –1 PRF efficiency. Collectively, our study identifies hnRNPC as a key host cofactor for coronaviruses and provides a novel target for broad-spectrum antiviral drug development.
Jingchen Xu, Hongying Li, Jianrui Li et al.· PLoS Pathogens· 0 citations
Endocytosis is central to cellular trafficking and signaling across eukaryotes, yet whether and how plant viruses actively reprogram this pathway remains unclear. Here, we show that the geminiviral betasatellite–encoded βC1 reprograms the host VPS9a-Rab5 endocytic module to promote viral infection. βC1 associates with the Rab5 GTPases ARA6 and ARA7 as well as their guanine nucleotide exchange factor VPS9a, stabilizing the VPS9a-Rab5 complex and enhancing nucleotide exchange. This catalytic potentiation sustains Rab5 activation and drives endosome proliferation, which, in turn, stabilizes βC1 to support efficient viral replication. Genetic disruption of Rab5 or VPS9a compromises endocytosis, reduces βC1 accumulation, and restricts infection by multiple geminiviruses. Together, these findings define the VPS9a-Rab5 module as a central proviral hub linking host membrane dynamics to effector stability and viral DNA amplification, revealing an unanticipated strategy in which a viral effector amplifies a cellular regulatory catalyst to reprogram a fundamental cellular pathway for virus infection.
Tuxunaili Aizitili, Asigul Ismayil, Aiping Cao et al.· Science Advances· 1 citation
It is proposed that MraW modification of 16S rRNA enhances translation efficiency in general, and that specific transcripts have evolved structural features that fine-tune protein levels that may be prevalent in bacteria which exhibit uncoupled transcription and translation.
Zachory M. Park, Christina R. Savage, Amanda R. Decker-Farrell et al.· Cell Reports· 0 citations
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