Transgene Sequence Codon Optimization and Composition Determines Replication Competence of Self-Amplifying RNA.

In trans RNA replication of a defective self-amplifying RNA lowers RNA replication but enhances protein translation.

GC content mismatch of transgene destabilizes RNA virus genomes

ABSTRACT GC content—the proportion of guanine and cytosine nucleotides—varies widely among organisms and viruses. Although GC content is recognized to have biological significance, its functional role in viruses remains poorly understood. Here, we examined the impact of GC-content bias using three positive-sense single-stranded RNA viruses with distinct GC profiles: severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), Japanese encephalitis virus (JEV), and hepatitis C virus (HCV). Variants of the NanoLuc (Nluc) reporter gene, engineered with synonymous mutations to alter GC content, were inserted into each viral genome. Serial passaging experiments revealed strong effects on genome stability and viral fitness. In SARS-CoV-2, which has low GC content, the introduction of a GC-rich Nluc gene disrupted genome stability, leading to frequent deletions of Nluc. In both SARS-CoV-2 and HCV, Nluc with mismatched GC levels accumulated substitutions that optimized GC content toward that of the viral genome, predominantly at the third codon position. tRNA-seq analysis revealed a shift in the host tRNA pool toward a more GC-rich composition during HCV infection, consistent with this substitution bias. In contrast, JEV (intermediate GC content) maintained the Nluc variants across serial passages, suggesting reduced selective pressure. Taken together, these findings demonstrate that mismatches between viral genome GC content and inserted sequences profoundly affect genetic stability, suggesting evolutionary constraints that may shape RNA virus composition more broadly. This study provides mechanistic insight into how GC content influences viral genome maintenance and offers a framework for designing genetically stable recombinant reporter viruses. IMPORTANCE GC content is a fundamental genomic feature that influences gene expression, genome architecture, and adaptation. Although its role in cellular genomes has been extensively studied, the functional significance of GC content in viral genomes remains poorly understood. In this study, we show that incompatibility in GC content between a viral genome and an inserted transgene destabilizes RNA secondary structures, compromising viral genome integrity, and is accompanied by changes in the host tRNA pool consistent with this compositional bias. These findings identify GC content as a previously underappreciated determinant of virus–host compatibility and viral fitness. Importantly, our results provide mechanistic insight into how the GC content of a viral genome can be matched to its host cellular environment. By demonstrating that GC content matching is critical for the stability of recombinant viruses, this study provides a basis for the rational design of reporter viruses, vaccine platforms, and antiviral screening tools. GC content is a fundamental genomic feature that influences gene expression, genome architecture, and adaptation. Although its role in cellular genomes has been extensively studied, the functional significance of GC content in viral genomes remains poorly understood. In this study, we show that incompatibility in GC content between a viral genome and an inserted transgene destabilizes RNA secondary structures, compromising viral genome integrity, and is accompanied by changes in the host tRNA pool consistent with this compositional bias. These findings identify GC content as a previously underappreciated determinant of virus–host compatibility and viral fitness. Importantly, our results provide mechanistic insight into how the GC content of a viral genome can be matched to its host cellular environment. By demonstrating that GC content matching is critical for the stability of recombinant viruses, this study provides a basis for the rational design of reporter viruses, vaccine platforms, and antiviral screening tools.

IRES Mutation Confers a Replicative Advantage to SVA by Enhancing Translation Efficiency

The internal ribosome entry site (IRES) is a cis‐acting element found in certain RNA viruses. In virus‐infected cells, the Senecavirus A (SVA) IRES can directly recruit the small ribosomal subunit to an internal initiation codon on the mRNA, enabling translation initiation independently of both the 5′ cap structure of the viral genome and the host cell eukaryotic initiation factor 4F (eIF4F). As a small RNA virus, SVA frequently accumulates genetic mutations during transmission. This study aimed to characterize the mutational and evolutionary patterns of the SVA genome during its transmission within host cells exhibiting enhanced innate immunity. SVA was serially passaged for 80 generations in preactivated 3D4/21 cells that had been established in an antiviral innate immune state. During serial passaging, two stable single‐nucleotide mutations were consistently identified in the IRES region of the viral genomic RNA from passages 60–80: a uridine (U) insertion at genomic position 109, and a guanine (G)‐to‐adenine (A) substitution at position 265. Furthermore, the rescued SVA mutants harboring these mutations exhibited significantly higher replication titers than the parental strain. Notably, our results indicated that the mutant virus was unable to evade host antiviral innate immunity, whereas it significantly enhanced the translational activity of the SVA IRES. Collectively, these findings provide a foundation for understanding how single nucleotide polymorphisms (SNPs) in the 5′ untranslated region (UTR) region influence viral IRES activity and the replication capacity of recombinant viruses.

Functional atlas of vertebrate viral RNA elements that stabilize RNA and enhance translation.

Viruses encode diverse regulatory elements, but their breadth and mechanisms remain poorly defined. To address this gap, we performed massively parallel reporter assays spanning ∼200,000 genomic segments from 297 vertebrate-infecting viral genera. We identified numerous viral elements that enhance RNA stability and translation through TENT4-mediated mixed tailing, distributed across 19 genera and grouped into six distinct subclasses, indicating extensive convergent evolution. We also found diverse TENT4-independent elements acting through alternative pathways. One such element, Pt1 from Potamipivirus, stabilizes linear mRNA to levels comparable to circular RNA, suggesting its potential for RNA therapeutics. Pt1 directly recruits canonical poly(A) polymerases (PAPγ/α)-previously thought to function exclusively in transcription-coupled nuclear pre-mRNA processing-to drive cytoplasmic polyadenylation. Together, these findings chart the rich landscape of viral regulation, extend the scope of poly(A)-tail biology, and establish the virome as a valuable source for uncovering host RNA regulatory mechanisms.

Engineering of T7 DNA-dependent RNA polymerase with activity at elevated temperature

Bacteriophage T7 RNA polymerase (T7 RNAP) is a key enzyme for in vitro transcription (IVT) and plays a central role in the production of synthetic mRNA for research and therapeutic applications. However, IVT frequently generates double-stranded RNA (dsRNA) as an undesired by-product, which can trigger innate immune responses and compromise mRNA quality. Increasing reaction temperatures reduces dsRNA formation, however, the wild-type T7 RNA polymerase exhibits limited stability under such conditions. Therefore, polymerase variants with enhanced thermotolerance enable more robust transcription at elevated temperatures while minimizing dsRNA generation. To address this limitation, we aimed at obtaining T7 RNA polymerase variants with increased thermotolerance using the Protein Repair One Stop Shop (PROSS) web server. Four crystal structures of T7 RNA polymerase, comprising a promoter complex, an initiation complex, and two elongation complexes were used as input for independent PROSS runs. Mutations shared across all four designs for each PROSS index were then combined to generate multi-structure PROSS Combined Designs (PCDs). In the subset evaluated experimentally, PCD9 retained full-length transcription activity at temperatures up to 48 °C, whereas wild-type T7 RNA polymerase showed strong loss of activity under the same buffer conditions. At 48 °C, PCD9 supported the synthesis of kilobase-length scale transcripts and produced no detectable dsRNA signal in a dot blot assay. In contrast, the wild-type enzyme generated strong dsRNA signals at 37 °C and failed to produce detectable RNA at 48 °C. Additional PROSS variants derived exclusively from the elongation complex structure were inactive at both 37 °C and 48 °C. Together, these results show that multi-structure PROSS design can yield a thermotolerant T7 RNA polymerase with improved performance at elevated temperature and reduced dsRNA byproduct formation. The findings also suggest that restricting stability design to a single structural state may not fully capture the requirements of a highly dynamic enzyme.

Expanding Reverse Genetics of Positive-Strand RNA Viruses: Optimised Rescue Platforms and Construction of a Novel Fluorescent Reporter Nidovirus

Reverse genetics systems are crucial for facilitating the precise manipulation of viruses across a wide spectrum of translational and fundamental research pipelines. Here, we compared Circular polymerase extension reaction (CPER), Gibson assembly, and infectious subgenomic amplicons (ISA) for bacteria-free recovery of a positive sense RNA virus. Through optimisation of CPER, we demonstrated accelerated virus recovery and enhanced viral yields. We further investigated strategies to improve rescue efficiency across diverse positive-sense RNA virus families through incorporation of alternative promoters and non-coding elements. To evaluate the performance of the Aedes aegypti polyubiquitin promoter (AePUb) in tandem with a hammerhead ribozyme (HH Rbz) and a polymerase pause site for virus recovery in insect cells, we constructed a new fluorescent reporter genome using a 20 kb insect-specific mesonivirus. In vitro recovery by CPER of the mesonivirus was achievable in 1 day when using AePUb with HH Rbz, in comparison to a four-day recovery when using the minimal OpIE2-CA promoter. These elements were additionally assessed for rescue of the orthoflaviviruses, Binjari virus (BinJV) and dengue virus 2 (DENV-2), in insect cells (using AePUb); or in mammalian cells (using the CMV promoter) and for launch of DENV2 and SARS-CoV-2. Both BinJV and DENV-2 demonstrated improved rescue with the AePUb promoter and HH Rbz. However, the addition of the HH Rbz and the polymerase pause site to the CMV linker fragment showed no substantial differences to the standard CMV promoter systems for both DENV-2 and SARS-CoV-2, highlighting the context-specific benefits of their implementation. In summary, we demonstrated that a potent constitutive promoter system and a hammerhead ribozyme enhance the efficiency of positive-sense RNA virus rescue using CPER. Importance Reverse genetics systems are often limited by plasmid instability and variable efficiency of promoters across diverse cell lines. Extensive comparative approaches have yielded improvements across a variety of systems, however, there has been a paucity of publications that empirically compare novel advancements to established approaches. Here, we formalised and compared a series of reverse genetics advancements in the form of bacteria-free assembly methods, host promoters, pause sites, and ribozymes. These streamlined approaches expedite the existing methodologies and provide fundamental improvements to the field of synthetic virology. The advancements herein may support applications requiring efficient recovery of low fitness mutants and diverse mutational libraries and barcoded virus populations.