A SARS-CoV-2 packaging signal is identified within the nsp12 coding region and shows that the nucleocapsid protein mediates selective genome packaging through its C-terminal domain, establishing the mechanistic basis for SARS-CoV-2 genome packaging and offer a potential antiviral target.
Abstract
Selective genome packaging is a critical step for RNA viruses, which must distinguish genomic RNA from other abundant transcripts. For SARS-CoV-2, the cis-acting packaging signal is thought to be recognized by the nucleocapsid (N) protein, but its identity and mechanistic basis for selective recognition remain undefined. Here we identify the packaging signal within the nsp12 polymerase-coding region. CLIP-seq maps N-bound sites and, together with virus-like particle assays, pinpoints a conserved segment with strong packaging activity. An orthogonal defective-interfering RNA approach confirms its role in genome selection. We further delineate two critical subregions, α and β, that engage the N C-terminal domain. Synonymous mutations in either subregion selectively disrupt packaging and reduce viral fitness. Notably, the α subregion encompasses the ribosomal frameshifting element, revealing its dual role in viral translation and assembly. These findings establish the mechanistic basis for SARS-CoV-2 genome packaging and offer a potential antiviral target. Here, Park et al. identify a SARS-CoV-2 packaging signal within the nsp12 coding region and show that the nucleocapsid protein mediates selective genome packaging through its C-terminal domain. Synonymous mutations disrupt packaging and reduce viral fitness without affecting genome replication.
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
Coronaviruses (CoVs) produce dsRNA during genome replication and mRNA synthesis. Upon sensing dsRNA, host cells activate numerous antiviral pathways. CoVs encode multiple proteins that antagonize antiviral responses. The conserved CoV nsp15 contains an endoribonuclease (EndoU) that cleaves viral ssRNA to limit dsRNA accumulation. Similarly, the viral protein, NS6, encoded by open reading frame (ORF)6 is expressed by severe acute respiratory syndrome (SARS)-CoV and SARS-CoV-2 and has been shown to block STAT translocation, but its absence has minimal impact on IFN-stimulated genes (ISGs). A viral genome has limited coding capacity, and ORFs encoding proteins without a significant function are typically mutated or deleted over time. Therefore, we infer that ORF6 plays an essential role in immune evasion, perhaps dependent upon other viral proteins.
A recombinant SARS-CoV-2 encoding an inactive EndoU (nsp15mut) and an interrupted ORF6 (ORF6stop) was constructed. Viral kinetics and innate immune induction were assessed in both Calu-3 cells and primary nasal air-liquid interface (ALI) cultures using RT-qPCR and Western blot analysis.
Replication of SARS-CoV-2 nsp15mut/ORF6stop in nasal ALI cultures was attenuated at both 33 °C and 37 °C compared to WT SARS-CoV-2. A significant increase in type I and III IFN transcripts was also observed in nasal ALI cultures infected with nsp15mut/ORF6stop compared to WT and each single mutant. Robust induction of various ISGs were also observed via Western blot analysis, with a faster induction observed at 37 °C.
The synergistic relationship between nsp15 and ORF6 is essential for efficient SARS-CoV-2 replication. We hypothesize that since nsp15 functions by reducing dsRNA upstream of ORF6, nsp15 acts as a more potent antagonist leaving little activity for ORF6 to account for. However, when both EndoU activity and NS6 are absent, a robust induction of IFN and ISGs are induced to levels higher than that of the nsp15mut alone.
This work was supported by National Institutes of Health grants R01 AI140442 (SRW), R01AI169537 (SRW&NAC), RO1A1AI161175 (LM-S); Department of Veterans Affairs Merit Review 1-I01-BX005432-01 (NAC&SRW); the Penn Center for Research on Emerging Viruses (SRW
Viral Immunology (VIR)
Nicole R. Bracci, Clayton J. Otter, Nicholas A. Parenti et al.· Journal of Immunology· 0 citations
Structural and biochemical studies have elucidated the architecture of the nsp14 N7-methyltransferase domain, revealing an S-adenosyl-L-methionine (SAM)-dependent fold with a defined cofactor-binding site and an adjacent cap-binding pocket that orients the RNA substrate for methyl transfer.
Yifan Zhao, Rhea Guo, Yang Yang et al.· Microorganisms· 0 citations
It is suggested that the ribosome-associated Ski2,3,8 proteins block 60S subunit joining on polyA− mRNAs, suggesting that yeast viruses will continue to be a fertile area for study of viral pathogenesis and host anti-viral systems.
Abstract SARS-CoV-2 manipulates host gene expression through multiple mechanisms, including disruption of RNA processing. Here, we identify a novel function of the viral non-structural protein 14 (NSP14) in inducing N7-methylguanosine (m7G) modification in the internal sequences of host mRNA. We demonstrate that NSP14 catalyzes the conversion of GTP to m7GTP, which is subsequently incorporated into mRNA by RNA polymerase II, resulting in widespread internal m7G modification. This activity is dependent on NSP14’s N7-methyltransferase (N7-MTase) domain, and the NSP10–NSP14 interaction increases cellular m7G levels primarily by increasing NSP14 protein abundance. NSP14-induced m7G modification is conserved across alpha-, beta-, and gamma-coronaviruses. Mechanistically, we show that this RNA modification is associated with altered splicing, particularly in genes regulating genome stability, RNA metabolism, and nuclear processes. Importantly, using SARS-CoV-2 infection models, we show that viral replication is associated with increased cellular m7G signal, supporting the relevance of this pathway during infection. Inhibition of NSP14 N7-MTase or RNA polymerase II reduces SARS-CoV-2 replication, consistent with a model in which NSP14-induced m7G modification may contribute to viral replication. Our findings reveal a previously unrecognized epitranscriptomic mechanism and suggest that NSP14-induced m7G modification may contribute to the remodeling of host gene expression during coronavirus infection.
