RNA replication-independent, DNA linearization-dependent expression of reporter genes from a SARS-CoV-2 replicon-encoding DNA in human cells.

Replicons, derived from RNA viruses, are genetic constructs retaining essential viral enzyme genes while lacking key structural protein genes. Upon introduction into cells, the genes carried by the replicon RNA are expressed, and the RNA self-replicates, yet viral particle production does not take place. Typically, RNA replicons are transcribed in vitro and are then electroporated in cells. However, it would be advantageous for the replicon to be generated in cells following DNA transfection instead of RNA. In this study, a bacterial artificial chromosome (BAC) DNA encoding a SARS-CoV-2 replicon under control of a T7 promoter was transfected into HEK293T cells engineered to functionally express the T7 RNA polymerase (T7 RNAP). Upon transfection of the BAC DNA, we observed low, but reproducible expression of reporter proteins GFP and luciferase carried by this replicon. Expression of the reporter proteins required linearization of the BAC DNA prior to transfection. Moreover, expression occurred independently of T7 RNAP. Gene expression was also insensitive to remdesivir treatment, suggesting that it did not involve self-replication of replicon RNA. Similar results were obtained in highly SARS-CoV-2 infection-permissive Calu-3 cells. Strikingly, prior expression of the SARS-CoV-2 N protein boosted expression from transfected SARS-CoV-2 RNA replicon but not from the replicon BAC DNA. In conclusion, transfection of a large DNA encoding a coronaviral replicon led to reproducible replicon gene expression through an unidentified mechanism. These findings highlight a novel pathway toward replicon gene expression from transfected replicon cDNA, offering valuable insights for the development of methods for DNA-based RNA replicon applications.

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.

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.

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.

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.

Reconstitution of a telomeric replicon organized by CST.

Telomeres, the natural ends of linear chromosomes, comprise repeat-sequence DNA and associated proteins1. Replication of telomeres allows continued proliferation of human stem cells and immortality of cancer cells2. This replication requires telomerase3 extension of the single-stranded DNA (ssDNA) of the telomeric G-strand ((TTAGGG)n); the synthesis of the complementary C-strand ((CCCTAA)n) is much less well characterized. The CST (CTC1-STN1-TEN1) protein complex, a DNA polymerase α-primase accessory factor4,5, is known to be required for telomere replication in vivo6-9, and the molecular analysis presented here reveals key features of its mechanism. We find that human CST uses its ssDNA-binding activity to specify the origins for telomeric C-strand synthesis by bound Polα-primase. CST-organized DNA polymerization can copy a telomeric DNA template that folds into G-quadruplex structures, but the challenges presented by this template probably contribute to telomere replication problems observed in vivo. Combining telomerase, a short telomeric ssDNA primer and CST-Polα-primase gives complete telomeric DNA replication, resulting in the same sort of ssDNA 3' overhang found naturally on human telomeres. We conclude that the CST complex not only terminates telomerase extension10,11 and recruits Polα-primase to telomeric ssDNA4,12,13 but also orchestrates C-strand synthesis. Because replication of the telomere has features distinct from replication of the rest of the genome, targeting telomere-replication components including CST holds promise for cancer therapeutics.

Transcriptional Profile of HCV Replicon Cells after Treatment with Acetylsalicylic Acid.

OBJECTIVE: It has been demonstrated in vitro that acetylsalicylic acid (ASA) treatment halves hepatitis C virus (HCV) expression in hepatocarcinoma cells. However, the signaling pathway that promotes this ASA-induced antiviral effect has not yet been identified. AIM: The aim of this work was to identify alterations in the transcriptional profile of Huh-7-HCV-subgenomic replicon cells with vs. without ASA treatment. This comparison sheds light onto the signaling pathways and molecular mechanisms involved in the antiviral effects of ASA. METHODS: Human hepatocellular carcinoma (Huh-7) cells that express non-structural HCV proteins (Huh-7-HCV-replicon cells) were exposed to 4 mM ASA for 0, 24, 48, and 72 hours. Total RNA was isolated, and cDNA was synthesized. Transcripts were then tagged with biotin and purified. Thereafter, they were fragmented and hybridized on HG-U133 Plus 2 Gene Expression chips. Hybridization signals were captured using a GeneChip 3000 7G Scanner and analyzed via Expression Console and dChip Software. RESULTS: When exposed to ASA, hepatocarcinoma cells with non-structural HCV proteins were found to differentially regulate genes with oxidative roles in the cell. The most upregulated genes were interleukin 8 (IL-8), cytochrome P450 (CYP450), and metallothioneins (MTs), while the most downregulated genes were ribonucleotide reductases (RRs). CONCLUSION: These results show that ASA modulates the expression of genes with antioxidant functions. This suggests that ASA induces a remodeling of the antioxidant microenvironment, which may in turn interfere with the replication of HCV.

Identification of a Proline-Kinked Amphipathic α-Helix Downstream from the Methyltransferase Domain of a Potexvirus Replicase and Its Role in Virus Replication and Perinuclear Complex Formation.

Characterized positive-strand RNA viruses replicate in association with intracellular membranes. Regarding viruses in the genus Potexvirus, the mechanism by which their RNA-dependent RNA polymerase (replicase) associates with membranes is understudied. Here, by membrane flotation analyses of the replicase of Plantago asiatica mosaic potexvirus (PlAMV), we identified a region in the methyltransferase (MET) domain as a membrane association determinant. An amphipathic α-helix was predicted downstream from the core region of the MET domain, and hydrophobic amino acid residues were conserved in the helical sequences in replicases of other potexviruses. Nuclear magnetic resonance (NMR) analysis confirmed the amphipathic α-helical configuration and unveiled a kink caused by a highly conserved proline residue in the α-helix. Substitution of this proline residue and other hydrophobic and charged residues in the amphipathic α-helix abolished PlAMV replication. Ectopic expression of a green fluorescent protein (GFP) fusion with the entire MET domain resulted in the formation of a large perinuclear complex, where virus replicase and RNA colocated during virus infection. Except for the proline substitution, the amino acid substitutions in the α-helix that abolished virus replication also prevented the formation of the large perinuclear complex by the respective GFP-MET fusion. Small intracellular punctate structures were observed for all GFP-MET fusions, and in vitro high-molecular-weight complexes were formed by both replication-competent and -incompetent viral replicons and thus were not sufficient for replication competence. We discuss the roles of the potexvirus-specific, proline-kinked amphipathic helical structure in virus replication and intracellular large complex and punctate structure formation. IMPORTANCE RNA viruses characteristically associate with intracellular membranes during replication. Although virus replicases are assumed to possess membrane-targeting properties, their membrane association domains generally remain unidentified or poorly characterized. Here, we identified a proline-kinked amphipathic α-helix structure downstream from the methyltransferase core domain of PlAMV replicase as a membrane association determinant. This helical sequence, which includes the proline residue, was conserved among potexviruses and related viruses in the order Tymovirales. Substitution of the proline residue, but not the other residues necessary for replication, allowed formation of a large perinuclear complex within cells resembling those formed by PlAMV replicase and RNA during virus replication. Our results demonstrate the role of the amphipathic α-helix in PlAMV replicase in a perinuclear complex formation and virus replication and that perinuclear complex formation by the replicase alone will not necessarily indicate successful virus replication.

The A150V Polymorphism of Genotype 3 Hepatitis C Virus Polymerase Inhibits Interferon Alfa by Suppressing Protein Kinase R Activation.

BACKGROUND & AIMS: Despite recent advances in antiviral therapy for hepatitis C virus (HCV), a proportion of patients with genotype 3 (G3) HCV infection do not respond to current all oral treatment regimens. Genomic analyses have identified key polymorphisms correlating with increased resistance to direct-acting antivirals. We previously reported that amino the acid polymorphism, A150V, in the polymerase (NS5B) of G3 HCV reduces response to sofosbuvir. We now demonstrate that this polymorphism alters the response to interferon alpha. METHODS: Quantitative polymerase chain reaction, immunofluorescence, luciferase activity assay, immunoblotting, and flow cytometry were used to study the antiviral effect of interferon (IFN) on DBN G3 HCV-infected cells and G3 HCV replicons. RESULTS: We show the presence of the A150V polymorphism markedly reduces the response to IFN alpha (IC50 of S52_WT = 1.162 IU/mL and IC50 of S52_A150V = 14.45 IU/mL, 12.4-fold difference). The induction of IFN-stimulated genes in A150V replicon cells is unaffected, but nuclear localization of active protein kinase R (PKR) is reduced. Blockade of PKR activity reduced the antiviral effect of IFN on wild-type replicons, whereas augmented PKR activation promoted the antiviral effect of IFN on A150V replicons. Furthermore, we show that impaired activation of PKR in A150V replicon cells diminishes cellular apoptosis. CONCLUSIONS: These results demonstrate that polymorphisms reducing response rates to direct-acting antivirals may function beyond conferring drug resistance by modulating the intrinsic cellular antiviral response.

Segregation of four Agrobacterium tumefaciens replicons during polar growth: PopZ and PodJ control segregation of essential replicons.

Agrobacterium tumefaciens C58 contains four replicons, circular chromosome (CC), linear chromosome (LC), cryptic plasmid (pAt), and tumor-inducing plasmid (pTi), and grows by polar growth from a single growth pole (GP), while the old cell compartment and its old pole (OP) do not elongate. We monitored the replication and segregation of these four genetic elements during polar growth. The three largest replicons (CC, LC, pAt) reside in the OP compartment prior to replication; post replication one copy migrates to the GP prior to division. CC resides at a fixed location at the OP and replicates first. LC does not stay fixed at the OP once the cell cycle begins and replicates from varied locations 20 min later than CC. pAt localizes similarly to LC prior to replication, but replicates before the LC and after the CC. pTi does not have a fixed location, and post replication it segregates randomly throughout old and new cell compartments, while undergoing one to three rounds of replication during a single cell cycle. Segregation of the CC and LC is dependent on the GP and OP identity factors PopZ and PodJ, respectively. Without PopZ, replicated CC and LC do not efficiently partition, resulting in sibling cells without CC or LC. Without PodJ, the CC and LC exhibit abnormal localization to the GP at the beginning of the cell cycle and replicate from this position. These data reveal PodJ plays an essential role in CC and LC tethering to the OP during early stages of polar growth.

C19orf66 is an interferon-induced inhibitor of HCV replication that restricts formation of the viral replication organelle.

BACKGROUND & AIMS: HCV is a positive-strand RNA virus that primarily infects human hepatocytes. Recent studies have reported that C19orf66 is expressed as an interferon (IFN)-stimulated gene; however, the intrinsic regulation of this gene within the liver as well as its antiviral effects against HCV remain elusive. METHODS: Expression of C19orf66 was quantified in both liver biopsies and primary human hepatocytes, with or without HCV infection. Mechanistic studies of the potent anti-HCV phenotype mediated by C19orf66 were conducted using state-of-the-art virological, biochemical and genetic approaches, as well as correlative light and electron microscopy and transcriptome and proteome analysis. RESULTS: Upregulation of C19orf66 mRNA was observed in both primary human hepatocytes upon HCV infection and in the livers of patients with chronic hepatitis C (CHC). In addition, pegIFNα/ribavirin therapy induced C19orf66 expression in patients with CHC. Transcriptomic profiling and whole cell proteomics of hepatoma cells ectopically expressing C19orf66 revealed no induction of other antiviral genes. Expression of C19orf66 restricted HCV infection, whereas CRIPSPR/Cas9 mediated knockout of C19orf66 attenuated IFN-mediated suppression of HCV replication. Co-immunoprecipitation followed by mass spectrometry identified a stress granule protein-dominated interactome of C19orf66. Studies with subgenomic HCV replicons and an expression system revealed that C19orf66 expression impairs HCV-induced elevation of phosphatidylinositol-4-phosphate, alters the morphology of the viral replication organelle (termed the membranous web) and thereby targets viral RNA replication. CONCLUSION: C19orf66 is an IFN-stimulated gene, which is upregulated in hepatocytes within the first hours post IFN treatment or HCV infection in vivo. The encoded protein possesses specific antiviral activity against HCV and targets the formation of the membranous web. Our study identifies C19orf66 as an IFN-inducible restriction factor with a novel antiviral mechanism that specifically targets HCV replication. LAY SUMMARY: Interferon-stimulated genes are thought to be important to for antiviral immune responses to HCV. Herein, we analysed C19orf66, an interferon-stimulated gene, which appears to inhibit HCV replication. It prevents the HCV-induced elevation of phosphatidylinositol-4-phosphate and alters the morphology of HCV's replication organelle.

Expression in algae of a chimeric protein carrying several epitopes from tumor associated antigens.

Immunotherapies for cancer treatment constitute promising avenues to fight this global health issue. Algae can be used as both biofactories and delivery vehicles of vaccines; having low cost, fast growth, enhanced safety, and adjuvant effects as advantages. In the present study a multiepitope protein, called BCB, was designed as an attractive approach to develop new cancer immunotherapies. The BCB protein targets epitopes from the following tumor-associated antigens: human epidermal growth factor receptor-2 (HER2), mucin-like glycoprotein 1 (MUC1), Wilms' tumor antigen (WT1), and mammaglobin. Moreover, the BCB protein is based on the B subunit of the heat labile E. coli enterotoxin as immunogenic carrier to brake tolerance against self-antigens. A synthetic BCB-coding gene was obtained and expressed in Schizochytrium sp. using the Algevir system. The BCB protein was successfully expressed in transformed algae at levels up to 637 µg/g fresh weight, retaining the GM1-binding activity. The algae-made BCB showed reactivity towards an anti-serum against the tumor cell line 4T1; evidencing its antigenicity. Moreover the immunogenicity was evidenced in mice immunized with BCB, which developed serum IgG antibodies reacting against the 4T1 lysate. This study constitutes the first step in the development of innovative algae-based vaccines against cancer.

Study of multiple genetic variations caused by persistent hepatitis C virus replication in long-term cell culture.

The most characteristic feature of the hepatitis C virus (HCV) genome in patients with chronic hepatitis C is its remarkable variability and diversity. To better understand this feature, we performed genetic analysis of HCV replicons recovered from two human hepatoma HuH-7-derived cell lines after 1, 3, 5, 7, and 9 years in culture: The cell lines 50-1 and sO harbored HCV 1B-1 and O strain-derived HCV replicons established in 2002 and 2003, respectively. The results revealed that genetic variations in both replicons accumulated in a time-dependent manner at a constant rate despite the maintenance of moderate diversity (less than 1.8% difference) between the clones and that the mutation rate in the 50-1 and sO replicons was 2.5 and 2.9 × 10-3 base substitutions/site/year, respectively. We found that the genetic distance of both replicons increased from 7.9% to 10.5% after 9 years in culture. In addition, we observed that the guanine + cytosine (GC) content of both replicon RNAs increased in a time-dependent manner, as observed in our previous studies. Finally, we demonstrated that the high sensitivity of both replicons to direct-acting antivirals was maintained even after 9 years in culture. Our results suggest that long-term cultured HCV replicon-harboring cells are a useful model for understanding the variability and diversity of the HCV genome and the drug sensitivity of HCV in patients with chronic hepatitis C.

The Rescue and Characterization of Recombinant, Microcephaly-Associated Zika Viruses as Single-Round Infectious Particles.

Zika virus (ZIKV) is transmitted by Aedes mosquitoes and exhibits genetic variation with African and Asian lineages. ZIKV Natal RGN strain, an Asian-lineage virus, has been identified in brain tissues from fetal autopsy cases with microcephaly and is suggested to be a neurotropic variant. However, ZIKV Natal RGN strain has not been isolated; its biological features are not yet illustrated. This study rescued and characterized recombinant, single-round infectious particles (SRIPs) of the ZIKV Natal RGN strain using reverse genetic and synthetic biology techniques. The DNA-launched replicon of ZIKV Natal RGN was constructed and contains the EGFP reporter, lacks prM-E genes, and replicates under CMV promoter control. The peak in the ZIKV Natal RGN SRIP titer reached 6.25 × 106 TCID50/mL in the supernatant of prM-E-expressing packaging cells 72 h post-transfection with a ZIKV Natal RGN replicon. The infectivity of ZIKV Natal RGN SRIPs has been demonstrated to correlate with the green florescence intensity of the EGFP reporter, the SRIP-induced cytopathic effect, and ZIKV's non-structural protein expression. Moreover, ZIKV Natal RGN SRIPs effectively self-replicated in rhabdomyosarcoma/muscle, glioblastoma/astrocytoma, and retinal pigmented epithelial cells, displaying unique cell susceptibility with differential attachment activity. Therefore, the recombinant ZIKV Natal RGN strain was rescued as SRIPs that could be used to elucidate the biological features of a neurotropic strain regarding cell tropism and pathogenic components, apply for antiviral agent screening, and develop vaccine candidates.

Hepatitis C Virus RNA-Dependent RNA Polymerase Is Regulated by Cysteine S-Glutathionylation.

Hepatitis C virus (HCV) triggers massive production of reactive oxygen species (ROS) and affects expression of genes encoding ROS-scavenging enzymes. Multiple lines of evidence show that levels of ROS production contribute to the development of various virus-associated pathologies. However, investigation of HCV redox biology so far remained in the paradigm of oxidative stress, whereas no attention was given to the identification of redox switches among viral proteins. Here, we report that one of such redox switches is the NS5B protein that exhibits RNA-dependent RNA polymerase (RdRp) activity. Treatment of the recombinant protein with reducing agents significantly increases its enzymatic activity. Moreover, we show that the NS5B protein is subjected to S-glutathionylation that affects cysteine residues 89, 140, 170, 223, 274, 521, and either 279 or 295. Substitution of these cysteines except C89 and C223 with serine residues led to the reduction of the RdRp activity of the recombinant protein in a primer-dependent assay. The recombinant protein with a C279S mutation was almost inactive in vitro and could not be activated with reducing agents. In contrast, cysteine substitutions in the NS5B region in the context of a subgenomic replicon displayed opposite effects: most of the mutations enhanced HCV replication. This difference may be explained by the deleterious effect of oxidation of NS5B cysteine residues in liver cells and by the protective role of S-glutathionylation. Based on these data, redox-sensitive posttranslational modifications of HCV NS5B and other proteins merit a more detailed investigation and analysis of their role(s) in the virus life cycle and associated pathogenesis.

Four-dimensional analyses show that replication compartments are clonal factories in which Epstein-Barr viral DNA amplification is coordinated.

Herpesviruses must amplify their DNA to load viral particles and they do so in replication compartments. The development and functions of replication compartments during DNA amplification are poorly understood, though. Here we examine 2 functionally distinct replicons in the same cells to dissect DNA amplification within replication compartments. Using a combination of single-cell assays, computational modeling, and population approaches, we show that compartments initially were seeded by single genomes of Epstein-Barr virus (EBV). Their amplification subsequently took 13 to 14 h in individual cells during which their compartments occupied up to 30% of the nucleus and the nuclear volume grew by 50%. The compartmental volumes increased in proportion to the amount of DNA and viral replication proteins they contained. Each compartment synthesized similar levels of DNA, indicating that the total number of compartments determined the total levels of DNA amplification. Further, the amplification, which depended on the number of origins, was regulated differently early and late during the lytic phase; early during the lytic phase, the templates limited DNA synthesis, while later the templates were in excess, coinciding with a decline in levels of the viral replication protein, BMRF1, in the replication compartments. These findings show that replication compartments are factories in which EBV DNA amplification is both clonal and coordinated.

The role of ZAP and OAS3/RNAseL pathways in the attenuation of an RNA virus with elevated frequencies of CpG and UpA dinucleotides.

Zinc finger antiviral protein (ZAP) is a powerful restriction factor for viruses with elevated CpG dinucleotide frequencies. We report that ZAP similarly mediates antiviral restriction against echovirus 7 (E7) mutants with elevated frequencies of UpA dinucleotides. Attenuation of both CpG- and UpA-high viruses and replicon mutants was reversed in ZAP k/o cell lines, and restored by plasmid-derived reconstitution of expression in k/o cells. In pull-down assays, ZAP bound to viral RNA transcripts with either CpG- and UpA-high sequences inserted in the R2 region. We found no evidence that attenuation of CpG- or UpA-high mutants was mediated through either translation inhibition or accelerated RNA degradation. Reversal of the attenuation of CpG-high, and UpA-high E7 viruses and replicons was also achieved through knockout of RNAseL and oligodenylate synthetase 3 (OAS3), but not OAS1. WT levels of replication of CpG- and UpA-high mutants were observed in OAS3 k/o cells despite abundant expression of ZAP, indicative of synergy or complementation of these hitherto unconnected pathways. The dependence on expression of ZAP, OAS3 and RNAseL for CpG/UpA-mediated attenuation and the variable and often low level expression of these pathway proteins in certain cell types, such as those of the central nervous system, has implications for the use of CpG-elevated mutants as attenuated live vaccines against neurotropic viruses.

IL-21-based therapies induce clearance of hepatitis B virus persistence in mouse models.

Chronic hepatitis B virus (HBV) infection causes hepatitis, liver cirrhosis and hepatocellular carcinoma. Covalently closed circular DNA (cccDNA) is the sole viral transcription template and not affected by current treatment options, constituting a key determinant of HBV persistence. Novel therapeutics with demonstrable effectiveness against cccDNA are required. Methods: Previously, we established an HBV persistence mouse model using replicon plasmid derived from a clinical isolate (termed BPS) and identified IL-21 as a potent clearance-inducer. We also described another persistence mouse model based on cccDNA mimics produced in vivo termed recombinant cccDNA (rcccDNA). In this work, effectiveness of IL-21-based gene and cellular therapies was tested using these models. Results: In both models of HBV persistence, single injections with adeno-associated virus (AAV) expressing murine IL-21 highly efficiently induced clearance of both HBV markers from serum, and more importantly, BPS DNA and rcccDNA from mouse liver. Mechanistically, IL-21-induced clearance was associated with activation and liver infiltration of CD8+ T cells, and CD8 antibody injections negatively affected AAV-IL-21 effectiveness. More notably, adoptive transfer of CD8+ T cells from AAV-IL-21-cured mice engendered clearance in acceptor HBV persistence mice. Furthermore, cured mice were protected against re-challenge with long-lived memory. Most significantly, infusion of splenocytes from treatment-naïve mice stimulated ex vivo with IL-21 protein and HBV antigen could also induce clearance in treatment-naïve mice. Conclusion: These data demonstrate IL-21-based gene and cellular therapies as valid candidates for treating chronic HBV infections, with potential in removing cccDNA-harboring hepatocytes via activated CD8+ T cells accompanied by long-term protective memory.

Fine-tuning expression of begomoviral movement and nuclear shuttle proteins confers cell-to-cell movement to mastreviral replicons in Nicotiana benthamiana leaves.

Geminiviruses are a group of small plant viruses responsible for devastating crop damage worldwide. The emergence of agricultural diseases caused by geminiviruses is attributed in part to their high rates of recombination, leading to complementary function between viral components across species and genera. We have developed a mastreviral reporter system based on bean yellow dwarf virus (BeYDV) that replicates to high levels in the plant nucleus, expressing very high levels of GFP. To investigate the potential for complementation of movement function by other geminivirus genera, the movement protein (MP) and nuclear shuttle protein (NSP) from the bipartite begomovirus Bean dwarf mosaic virus (BDMV) were produced and characterized in Nicotiana benthamiana leaves. While overexpression of MP and NSP strongly inhibited GFP expression from the mastreviral reporter and caused adverse plant symptoms, optimizing the expression levels of MP and NSP allowed functional cell-to-cell movement. Hybrid virus vectors were created that express BDMV MP and NSP from mastreviral replicons, allowing efficient cell-to-cell movement comparable to native BDMV replicons. We find that the expression levels of MP and NSP must be fine-tuned to provide sufficient MP/NSP for movement without eliciting the plant hypersensitive response or adversely impacting gene expression from viral replicons. The ability to confer cell-to-cell movement to mastrevirus replicons depended strongly on replicon size: 2.1-2.7 kb replicons were efficiently moved, while 3 kb replicons were inhibited, and 3.9 kb replicons were very strongly inhibited. Optimized expression of MP/NSP from the normally phloem-limited Abutilon mosaic virus (AbMV) allows efficient movement in non-phloem cells.