Analysis of the susceptibility of refractory hepatitis C virus resistant to nonstructural 5A inhibitors.

Resistance-associated substitutions (RASs) of hepatitis C virus (HCV) affect the efficacy of direct-acting antivirals (DAAs). In this study, we aimed to clarify the susceptibility of the coexistence of nonstructural (NS) 5A Q24K/L28M/R30Q (or R30E)/A92K RASs, which were observed in patients with DAAs re-treatment failure and to consider new therapeutic agents. We used a subgenomic replicon system in which HCV genotype 1B strain 1B-4 was electroporated into OR6c cells derived from HuH-7 cells (Wild-type [WT]). We converted WT genes to NS5A Q24K/L28M/R30Q/A92K or Q24/L28K/R30E/A92K. Compared with the WT, the Q24K/L28M/R30Q/A92K RASs was 36,000-fold resistant to daclatasvir, 440,000-fold resistant to ledipasvir, 6300-fold resistant to velpatasvir, 3100-fold resistant to elbasvir, and 1.8-fold resistant to pibrentasvir. Compared with the WT, the Q24K/L28M/R30E/A92K RASs was 640,000-fold resistant to daclatasvir and ledipasvir, 150,000-fold resistant to velpatasvir, 44,000-fold resistant to elbasvir, and 1500-fold resistant to pibrentasvir. The Q24K/L28M/R30E/A92K RASs was 816.3 times more resistant to pibrentasvir than the Q24K/L28M/R30Q/A92K RASs. Furthermore, a combination of pibrentasvir and sofosbuvir showed therapeutic efficacy against these RASs. Combination regimens may eradicate HCV with NS5A Q24K/L28M/R30E/A92K RASs.

Modular Assembly of Synthetic Secondary Chromosomes.

The development of novel DNA assembly methods in recent years has paved the way for the construction of synthetic replicons to be used for basic research and biotechnological applications. A learning-by-building approach can now answer questions about how chromosomes must be constructed to maintain genetic information. Here we describe an efficient pipeline for the design and assembly of synthetic, secondary chromosomes in Escherichia coli based on the popular modular cloning (MoClo) system.

Genetic determinants of host- and virus-derived insertions for hepatitis E virus replication.

Hepatitis E virus (HEV) is a long-neglected RNA virus and the major causative agent of acute viral hepatitis in humans. Recent data suggest that HEV has a very heterogeneous hypervariable region (HVR), which can tolerate major genomic rearrangements. In this study, we identify insertions of previously undescribed sequence snippets in serum samples of a ribavirin treatment failure patient. These insertions increase viral replication while not affecting sensitivity towards ribavirin in a subgenomic replicon assay. All insertions contain a predicted nuclear localization sequence and alanine scanning mutagenesis of lysine residues in the HVR influences viral replication. Sequential replacement of lysine residues additionally alters intracellular localization in a fluorescence dye-coupled construct. Furthermore, distinct sequence patterns outside the HVR are identified as viral determinants that recapitulate the enhancing effect. In conclusion, patient-derived insertions can increase HEV replication and synergistically acting viral determinants in and outside the HVR are described. These results will help to understand the underlying principles of viral adaptation by viral- and host-sequence snatching during the clinical course of infection.

New Generation Self-Replicating RNA Vaccines Derived from Pestivirus Genome.

While mRNA vaccines have shown their worth, they have the same failing as inactivated vaccines, namely they have limited half-life, are non-replicating, and therefore limited to the size of the vaccine payload for the amount of material translated. New advances averting these problems are combining replicon RNA (RepRNA) technology with nanotechnology. RepRNA are large self-replicating RNA molecules (typically 12-15 kb) derived from viral genomes defective in at least one essential structural protein gene. They provide sustained antigen production, effectively increasing vaccine antigen payloads over time, without the risk of producing infectious progeny. The major limitations with RepRNA are RNase-sensitivity and inefficient uptake by dendritic cells (DCs), which need to be overcome for efficacious RNA-based vaccine design. We employed biodegradable delivery vehicles to protect the RepRNA and promote DC delivery. Condensing RepRNA with polyethylenimine (PEI) and encapsulating RepRNA into novel Coatsome-replicon vehicles are two approaches that have proven effective for delivery to DCs and induction of immune responses in vivo.

Plant Expression of Trans-Encapsidated Chimeric Viral Vaccines with Animal RNA Replicons: An Update.

In this protocol, we outline how to produce a chimeric viral vaccine in a biosafety level 1 (BSL1) environment. An animal viral vector RNA encapsidated with tobacco mosaic virus (TMV) coat protein can be fully assembled in planta. Agrobacterium cultures containing each component are inoculated together into tobacco leaves and the self-assembled hybrid chimeric viral vaccine is harvested 4 days later and purified with a simple PEG precipitation. The viral RNA delivery vector is derived from the BSL1 insect virus, Flock House virus (FHV), and replicates in human and animal cells but does not spread systemically. A polyethylene glycol purification protocol is also provided to collect and purify these vaccines for immunological tests. In this update, we also provide a protocol for in trans co-inoculation of a modified FHV protein A, which significantly increased the yield of in planta chimeric viral vaccine.

Development of a Cell Culture Model for Inducible SARS-CoV-2 Replication.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induces direct cytopathic effects, complicating the establishment of low-cytotoxicity cell culture models for studying its replication. We initially developed a DNA vector-based replicon system utilizing the CMV promoter to generate a recombinant viral genome bearing reporter genes. However, this system frequently resulted in drug resistance and cytotoxicity, impeding model establishment. Herein, we present a novel cell culture model with SARS-CoV-2 replication induced by Cre/LoxP-mediated DNA recombination. An engineered SARS-CoV-2 transcription unit was subcloned into a bacterial artificial chromosome (BAC) vector. To enhance biosafety, the viral spike protein gene was deleted, and the nucleocapsid gene was replaced with a reporter gene. An exogenous sequence was inserted within NSP1 as a modulatory cassette that is removable after Cre/LoxP-mediated DNA recombination and subsequent RNA splicing. Using the PiggyBac transposon strategy, the transcription unit was integrated into host cell chromatin, yielding a stable cell line capable of inducing recombinant SARS-CoV-2 RNA replication. The model exhibited sensitivity to the potential antivirals forsythoside A and verteporfin. An innovative inducible SARS-CoV-2 replicon cell model was introduced to further explore the replication and pathogenesis of the virus and facilitate screening and assessment of anti-SARS-CoV-2 therapeutics.

Design of Novel Synthetic RNA Replicons Based on Emesvirus zinderi.

Self-replicating RNAs (srRNAs) are synthetic molecules designed to mimic the self-replicating ability of viral RNAs. srRNAs hold significant promise for a range of applications, including enhancing protein expression, reprogramming cells into pluripotent stem cells, and creating cell-free systems for experimental evolution. However, the development of srRNAs for use in bacterial systems remains limited. Here, we demonstrate how a srRNA scaffold from Emesvirus zinderi can be engineered into a self-encoding srRNA by incorporating the coding region of the catalytically active replicase subunit. With the help of in vitro replication assays, including an in vitro translation-coupled replication approach, we show that the resulting system enables complete replication cycles of RNA both in cis and trans, including long cargo RNAs such as tethered 5S, 16S, and 23S rRNAs. In summary, our findings suggest that these srRNAs have significant potential for fundamental research, synthetic biology, and general in vitro evolution.

A flexible 'plug and play' bicistronic construct and its application in the screening of protein expression system in Escherichia coli.

The bicistronic expression system that utilizes fluorescent reporters has been demonstrated to be a straightforward method for detecting recombinant protein expression levels, particularly when compared to polyacrylamide gel electrophoresis and immunoblot analysis, which are tedious and labor-intensive. However, existing bicistronic reporter systems are less capable of quantitative measurement due to the lag in reporter expression and its negative impact on target protein. In this work, a plug and play bicistronic construct using mCherry as reporter was applied in the screening of optimal replicon and promoter for Sortase expression in Escherichia coli (E. coli). The bicistronic construct allowed the reporter gene and target open reading frame (ORF) to be co-transcribed under the same promoter, resulting in a highly positive quantitative correlation between the expression titer of Sortase and the fluorescent intensity (R2 > 0.97). With the correlation model, the titer of target protein can be quantified by noninvasively measuring the fluorescent intensity. On top of this, the expression of reporter has no significant effect on the yield of target protein, thus favoring a plug and play design for removing reporter gene to generate a plain plasmid for industrial use.

Dissemination of the Flavivirus Subgenomic Replicon Genome and Viral Proteins by Extracellular Vesicles.

Extracellular vesicles (EVs) such as exosomes have been shown to play physiological roles in cell-to-cell communication by delivering various proteins and nucleic acids. In addition, several studies revealed that the EVs derived from the cells that are infected with certain viruses could transfer the full-length viral genomes, resulting in EVs-mediated virus propagation. However, the possibility cannot be excluded that the prepared EVs were contaminated with infectious viral particles. In this study, the cells that harbor subgenomic replicon derived from the Japanese encephalitis virus and dengue virus without producing any replication-competent viruses were employed as the EV donor. It was demonstrated that the EVs in the culture supernatants of those cells were able to transfer the replicon genome to other cells of various types. It was also shown that the EVs were incorporated by the recipient cells primarily through macropinocytosis after interaction with CD33 and Tim-1/Tim-4 on HeLa and K562 cells, respectively. Since the methods used in this study are free from contamination with infectious viral particles, it is unequivocally indicated that the flavivirus genome can be transferred by EVs from cell to cell, suggesting that this pathway, in addition to the classical receptor-mediated infection, may play some roles in the viral propagation and pathogenesis.

An optimized high-throughput SARS-CoV-2 dual reporter trans-complementation system for antiviral screening in vitro and in vivo.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is still epidemic around the world. The manipulation of SARS-CoV-2 is restricted to biosafety level 3 laboratories (BSL-3). In this study, we developed a SARS-CoV-2 ΔN-GFP-HiBiT replicon delivery particles (RDPs) encoding a dual reporter gene, GFP-HiBiT, capable of producing both GFP signal and luciferase activities. Through optimal selection of the reporter gene, GFP-HiBiT demonstrated superior stability and convenience for antiviral evaluation. Additionally, we established a RDP infection mouse model by delivering the N gene into K18-hACE2 KI mouse through lentivirus. This mouse model supports RDP replication and can be utilized for in vivo antiviral evaluations. In summary, the RDP system serves as a valuable tool for efficient antiviral screening and studying the gene function of SARS-CoV-2. Importantly, this system can be manipulated in BSL-2 laboratories, decreasing the threshold of experimental requirements.

Construction and characterization of a temperature-sensitive pRC4 replicon for Rhodococcus and Gordonia.

Temperature-sensitive plasmids are useful for genome engineering and several synthetic biology applications. There are only limited reports on temperature-sensitive plasmids for Rhodococcus and none for Gordonia. Here, we report the construction of a temperature-sensitive pRC4 replicon that is functional in Rhodococcus and Gordonia. The amino acid residues were predicted for the temperature-sensitive phenotype in the pRC4 replicon using in silico methods and molecular simulation of the DNA-binding replication protein with the origin of replication. The amino acid residues were mutated, and the temperature-sensitive phenotype was validated in Gordonia sp. IITR100. Similar results were also observed in Rhodococcus erythropolis, suggesting that the temperature-sensitive phenotype was exhibited across genera.

Alphavirus-based replicons demonstrate different interactions with host cells and can be optimized to increase protein expression.

IMPORTANCE: Alphavirus replicons are being developed as self-amplifying RNAs aimed at improving the efficacy of mRNA vaccines. These replicons are convenient for genetic manipulations and can express heterologous genetic information more efficiently and for a longer time than standard mRNAs. However, replicons mimic many aspects of viral replication in terms of induction of innate immune response, modification of cellular transcription and translation, and expression of nonstructural viral genes. Moreover, all replicons used in this study demonstrated expression of heterologous genes in cell- and replicon's origin-specific modes. Thus, many aspects of the interactions between replicons and the host remain insufficiently investigated, and further studies are needed to understand the biology of the replicons and their applicability for designing a new generation of mRNA vaccines. On the other hand, our data show that replicons are very flexible expression systems, and additional modifications may have strong positive impacts on protein expression.

[Construction of a replicative expression vector based on the porcine circovirus 2 replicon].

The antigen gene expression level of a DNA vaccine is the key factor influencing the efficacy of the DNA vaccine. Accordingly, one of the ways to improve the antigen gene expression level of a DNA vaccine is to utilize a plasmid vector that is replicable in eukaryotic cells. A replicative DNA vaccine vector pCMVori was constructed based on the non-replicative pcDNA3.1 and the replicon of porcine circovirus 2 (PCV2) in this study. An EGFP gene was cloned into pCMVori and the control plasmid pcDNA3.1. The two recombinant vectors were transfected into PK-15 cell, and the plasmid DNA and RNA were extracted from the transfected cells. Real-time PCR was used to determine the plasmid replication efficiency of the two plasmids using plasmid before and after Bcl Ⅰ digestion as templates, and the transcription level of the Rep gene in PCV2 replicon was detected by RT-PCR. The average fluorescence intensity of cells transfected with the two plasmids was analyzed with software Image J, and the transcription level of EGFP was determined by means of real-time RT-PCR. The results showed that the replication efficiency of pCMVori in PK-15 cells incubated for 48 h was 136%, and the transcriptions of Rep and Rep' were verified by RT-PCR. The average fluorescence intensity of the cells transfected with pCMVori-EGFP was 39.14% higher than that of pcDNA3.1-EGFP, and the transcription level of EGFP in the former was also 40% higher than that in the latter. In conclusion, the DNA vaccine vector pCMVori constructed in this study can independently replicate in eukaryotic cells. As a result, the expression level of cloned target gene was elevated, providing a basis for developing the pCMVori-based DNA vaccine.

Organization and replicon interactions within the highly segmented genome of Borrelia burgdorferi.

Borrelia burgdorferi, a causative agent of Lyme disease, contains the most segmented bacterial genome known to date, with one linear chromosome and over twenty plasmids. How this unusually complex genome is organized, and whether and how the different replicons interact are unclear. We recently demonstrated that B. burgdorferi is polyploid and that the copies of the chromosome and plasmids are regularly spaced in each cell, which is critical for faithful segregation of the genome to daughter cells. Regular spacing of the chromosome is controlled by two separate partitioning systems that involve the protein pairs ParA/ParZ and ParB/Smc. Here, using chromosome conformation capture (Hi-C), we characterized the organization of the B. burgdorferi genome and the interactions between the replicons. We uncovered that although the linear chromosome lacks contacts between the two replication arms, the two telomeres are in frequent contact. Moreover, several plasmids specifically interact with the chromosome oriC region, and a subset of plasmids interact with each other more than with others. We found that Smc and the Smc-like MksB protein mediate long-range interactions on the chromosome, but they minimally affect plasmid-chromosome or plasmid-plasmid interactions. Finally, we found that disruption of the two partition systems leads to chromosome restructuring, correlating with the mis-positioning of chromosome oriC. Altogether, this study revealed the conformation of a complex genome and analyzed the contribution of the partition systems and SMC family proteins to this organization. This work expands the understanding of the organization and maintenance of multipartite bacterial genomes.

Pervasive RNA folding is crucial for narnavirus genome maintenance.

A synthetic biology approach toward constructing an RNA-based genome expands our understanding of living things and opens avenues for technological advancement. For the precise design of an artificial RNA replicon either from scratch or based on a natural RNA replicon, understanding structure-function relationships of RNA sequences is critical. However, our knowledge remains limited to a few particular structural elements intensively studied so far. Here, we conducted a series of site-directed mutagenesis studies of yeast narnaviruses ScNV20S and ScNV23S, perhaps the simplest natural autonomous RNA replicons, to identify RNA elements required for maintenance and replication. RNA structure disruption corresponding to various portions of the entire narnavirus genome suggests that pervasive RNA folding, in addition to the precise secondary structure of genome termini, is essential for maintenance of the RNA replicon in vivo. Computational RNA structure analyses suggest that this scenario likely applies to other "narna-like" viruses. This finding implies selective pressure on these simplest autonomous natural RNA replicons to fold into a unique structure that acquires both thermodynamic and biological stability. We propose the importance of pervasive RNA folding for the design of RNA replicons that could serve as a platform for in vivo continuous evolution as well as an interesting model to study the origin of life.

Mutations in the non-structural protein coding region regulate gene expression from replicon RNAs derived from Venezuelan equine encephalitis virus.

Self-replicating RNA (repRNA) derived from Venezuelan equine encephalitis (VEE) virus is a promising platform for gene therapy and confers prolonged gene expression due to its self-replicating capability, but repRNA suffers from a suboptimal transgene expression level due to its induction of intracellular innate response which may result in inhibition of translation. To improve transgene expression of repRNA, we introduced point mutations in the non-structural protein 1-4 (nsP1-4) coding region of VEE replicon vectors. As a proof of concept, inflammatory cytokines served as genes of interest and were cloned in their wild type and several mutant replicon vectors, followed by transfection in mammalian cells. Our data show that VEE replicons bearing nsP1GGAC-nsP2T or nsP1GGAC-nsP2AT mutations in the nsP1-4 coding region could significantly reduce the recognition by innate immunity as evidenced by the decreased production of type I interferon, and enhance transgene expression in host cells. Thus, the newly discovered mutant VEE replicon vectors could serve as promising gene expression platforms to advance VEE-derived repRNA-based gene therapies.

Characterization of an accessory plasmid of Sinorhizobium meliloti and its two replication-modules.

Rhizobia are Gram-negative bacteria known for their ability to fix atmospheric N2 in symbiosis with leguminous plants. Current evidence shows that rhizobia carry in most cases a variable number of plasmids, containing genes necessary for symbiosis or free-living, a common feature being the presence of several plasmid replicons within the same strain. For many years, we have been studying the mobilization properties of pSmeLPU88b from the strain Sinorhizobium meliloti LPU88, an isolate from Argentina. To advance in the characterization of pSmeLPU88b plasmid, the full sequence was obtained. pSmeLPU88b is 35.9 kb in size, had an average GC % of 58.6 and 31 CDS. Two replication modules were identified in silico: one belonging to the repABC type, and the other to the repC. The replication modules presented high DNA identity to the replication modules from plasmid pMBA9a present in an S. meliloti isolate from Canada. In addition, three CDS presenting identity with recombinases and with toxin-antitoxin systems were found downstream of the repABC system. It is noteworthy that these CDS present the same genetic structure in pSmeLPU88b and in other rhizobial plasmids. Moreover, in all cases they are found downstream of the repABC operon. By cloning each replication system in suicide plasmids, we demonstrated that each of them can support plasmid replication in the S. meliloti genetic background, but with different stability behavior. Interestingly, while incompatibility analysis of the cloned rep systems results in the loss of the parental module, both obtained plasmids can coexist together.

Independent origins and evolution of the secondary replicons of the class Gammaproteobacteria.

Multipartite genomes, consisting of more than one replicon, have been found in approximately 10 % of bacteria, many of which belong to the phylum Proteobacteria. Many aspects of their origin and evolution, and the possible advantages related to this type of genome structure, remain to be elucidated. Here, we performed a systematic analysis of the presence and distribution of multipartite genomes in the class Gammaproteobacteria, which includes several genera with diverse lifestyles. Within this class, multipartite genomes are mainly found in the order Alteromonadales (mostly in the genus Pseudoalteromonas) and in the family Vibrionaceae. Our data suggest that the emergence of secondary replicons in Gammaproteobacteria is rare and that they derive from plasmids. Despite their multiple origins, we highlighted the presence of evolutionary trends such as the inverse proportionality of the genome to chromosome size ratio, which appears to be a general feature of bacteria with multipartite genomes irrespective of taxonomic group. We also highlighted some functional trends. The core gene set of the secondary replicons is extremely small, probably limited to essential genes or genes that favour their maintenance in the genome, while the other genes are less conserved. This hypothesis agrees with the idea that the primary advantage of secondary replicons could be to facilitate gene acquisition through horizontal gene transfer, resulting in replicons enriched in genes associated with adaptation to different ecological niches. Indeed, secondary replicons are enriched both in genes that could promote adaptation to harsh environments, such as those involved in antibiotic, biocide and metal resistance, and in functional categories related to the exploitation of environmental resources (e.g. carbohydrates), which can complement chromosomal functions.

Coatsome-replicon vehicles: Self-replicating RNA vaccines against infectious diseases.

Herein, we provide the first description of a synthetic delivery method for self-replicating replicon RNAs (RepRNA) derived from classical swine fever virus (CSFV) using a Coatsome-replicon vehicle based on Coatsome® SS technologies. This results in an unprecedented efficacy when compared to well-established polyplexes, with up to ∼65 fold-increase of the synthesis of RepRNA-encoded gene of interest (GOI). We demonstrated the efficacy of such Coatsome-replicon vehicles for RepRNA-mediated induction of CD8 T-cell responses in mice. Moreover, we provide new insights on physical properties of the RepRNA, showing that the removal of all CSFV structural protein genes has a positive effect on the translation of the GOI. Finally, we successfully engineered RepRNA constructs encoding a porcine reproductive and respiratory syndrome virus (PRRSV) antigen, providing an example of antigen expression with potential application to combat viral diseases. The versatility and simplicity of modifying and manufacturing these Coatsome-replicon vehicle formulations represents a major asset to tackle foreseeable emerging pandemics.

Identification of the corticotropin-releasing factor receptor 1 antagonists as inhibitors of Chikungunya virus replication using a Gaussia luciferase-expressing subgenomic replicon.

The Chikungunya virus (CHIKV), an enveloped RNA virus that has been identified in over 40 countries and is considered a growing threat to public health worldwide. However, there is no preventive vaccine or specific therapeutic drug for CHIKV infection. To identify a new inhibitor against CHIKV infection, this study constructed a subgenomic RNA replicon expressing the secretory Gaussia luciferase (Gluc) based on the CHIKV SL11131 strain. Transfection of in vitro-transcribed replicon RNA to BHK-21 cells revealed that Gluc activity in culture supernatants was correlated with the intracellular replication of the replicon genome. Through a chemical compound library screen using the Gluc reporter CHIKV replicon, we identified several compounds that suppressed CHIKV infection in Vero cells. Among the hits identified, CP-154,526, a non-peptide antagonist of the corticotropin-releasing factor receptor type-1 (CRF-R1), showed the strongest anti-CHIKV activity and inhibited CHIKV infection in Huh-7 cells. Interestingly, other CRF-R1 antagonists, R121919 and NGD 98-2, also exhibited inhibitory effects on CHIKV infection. Time-of-drug addition and virus entry assays indicated that CP-154,526 suppressed a post-entry step of infection, suggesting that CRF-R1 antagonists acted on a target in the intracellular replication process of CHIKV. Therefore, the Gluc reporter replicon system established in this study would greatly facilitate the development of antiviral drugs against CHIKV infection.