Syn-CpG-Spacer: A Panel web app for synonymous recoding of viral genomes with CpG dinucleotides.

Vertebrate genomes contain lower than expected frequencies of the CpG dinucleotide. Consequently, many vertebrate viruses have evolved to mimic this composition, possibly in order to evade host antiviral defences (Greenbaum et al., 2008). For example, the antiviral protein ZAP binds CpGs in viral single stranded RNA with specific spacing requirements (Gonçalves-Carneiro et al., 2022), though CpGs are also likely depleted in viral genomes due to other selective pressures (Forni et al., 2023). Increasing CpG abundance by synonymous recoding could facilitate attenuation of viruses without compromising their epitope antigenicity by changing non-CpG codons to alternatives containing CpG without changing the overall amino acid sequence (Gonçalves-Carneiro et al., 2022; Le Nouën et al., 2019; Sharp et al., 2023). There are three ways CpGs can be synonymously introduced in codons: at positions 1-2 for arginine (e.g. AGA → CGA), 2-3 for several amino acids (e.g. ACA → ACG), or in a 3-1 split configuration, if a subsequent codon begins with a G (e.g. ATA-GCA → ATC-GCA). Syn-CpG-Spacer is a Python progressive web app (PWA) (MDN Web Docs, 2023) made with the Panel library (Panel Development Team, 2024) that allows for consistent recoding of viral sequences and applying biologically relevant constraints. These include setting a minimum gap between CpG's, optimising for an average CpG gap, protecting cis-acting regulatory signals from modification, and modulating the A-content in the overall sequence. The app features a sequence viewer made with the Bokeh library (Bokeh Development Team, 2024) that highlights CpG dinucleotides, allowing for efficient analysis of the resulting distribution of CpGs. This is complemented by a statistical data table. Utilising Biopython (Cock et al., 2009) modules, the user can load their sequence as a FASTA file and download the outputs as an alignment in the same format. As a PWA running on Pyodide (The Pyodide development team, 2023), the code is only executed in the user's browser and they can install the app onto their machine for offline use.

RetroSnake: A modular pipeline to detect human endogenous retroviruses in genome sequencing data.

Human endogenous retroviruses (HERVs) integrated into the human genome as a result of ancient exogenous infections and currently comprise ∼8% of our genome. The members of the most recently acquired HERV family, HERV-Ks, still retain the potential to produce viral molecules and have been linked to a wide range of diseases including cancer and neurodegeneration. Although a range of tools for HERV detection in NGS data exist, most of them lack wet lab validation and they do not cover all steps of the analysis. Here, we describe RetroSnake, an end-to-end, modular, computationally efficient, and customizable pipeline for the discovery of HERVs in short-read NGS data. RetroSnake is based on an extensively wet-lab validated protocol, it covers all steps of the analysis from raw data to the generation of annotated results presented as an interactive html file, and it is easy to use by life scientists without substantial computational training. Availability and implementation: The Pipeline and an extensive documentation are available on GitHub.

Homebrew: Protocol for glassmilk-based nucleic-acid extraction for SARS-CoV-2 diagnostics.

The gold standard protocol for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection detection remains reverse transcription quantitative polymerase chain reaction (qRT-PCR), which detects viral RNA more sensitively than any other approach. Here, we present Homebrew, a low-cost protocol to extract RNA using widely available reagents. Homebrew is as sensitive as commercially available RNA extraction kits. Homebrew allows for sample pooling and can be adapted for automation in high-throughput settings. For complete details on the use and execution of this protocol, please refer to Page et al. (2022).

Homebrew: An economical and sensitive glassmilk-based nucleic-acid extraction method for SARS-CoV-2 diagnostics.

Management of COVID-19 and other epidemics requires large-scale diagnostic testing. The gold standard for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection remains reverse transcription quantitative PCR (qRT-PCR) analysis, which detects viral RNA more sensitively than any other method. However, the resource use and supply-chain requirements of RT-PCR have continued to challenge diagnostic laboratories worldwide. Here, we establish and characterize a low-cost method to detect SARS-CoV-2 in clinical combined nose and throat swabs, allowing for automation in high-throughput settings. This method inactivates virus material with sodium dodecylsulfate (SDS) and uses silicon dioxide as the RNA-binding matrix in combination with sodium chloride (NaCl) and isopropanol. With similar sensitivity for SARS-CoV-2 viral targets but a fraction of time and reagent expenditure compared with commercial kits, our method also enables sample pooling without loss of sensitivity. We suggest that this method will facilitate more economical widespread testing, particularly in resource-limited settings.

Resilient SARS-CoV-2 diagnostics workflows including viral heat inactivation.

There is a worldwide need for reagents to perform SARS-CoV-2 detection. Some laboratories have implemented kit-free protocols, but many others do not have the capacity to develop these and/or perform manual processing. We provide multiple workflows for SARS-CoV-2 nucleic acid detection in clinical samples by comparing several commercially available RNA extraction methods: QIAamp Viral RNA Mini Kit (QIAgen), RNAdvance Blood/Viral (Beckman) and Mag-Bind Viral DNA/RNA 96 Kit (Omega Bio-tek). We also compared One-step RT-qPCR reagents: TaqMan Fast Virus 1-Step Master Mix (FastVirus, ThermoFisher Scientific), qPCRBIO Probe 1-Step Go Lo-ROX (PCR Biosystems) and Luna® Universal Probe One-Step RT-qPCR Kit (Luna, NEB). We used primer-probes that detect viral N (EUA CDC) and RdRP. RNA extraction methods provided similar results, with Beckman performing better with our primer-probe combinations. Luna proved most sensitive although overall the three reagents did not show significant differences. N detection was more reliable than that of RdRP, particularly in samples with low viral titres. Importantly, we demonstrated that heat treatment of nasopharyngeal swabs at 70°C for 10 or 30 min, or 90°C for 10 or 30 min (both original variant and B 1.1.7) inactivated SARS-CoV-2 employing plaque assays, and had minimal impact on the sensitivity of the qPCR in clinical samples. These findings make SARS-CoV-2 testing portable in settings that do not have CL-3 facilities. In summary, we provide several testing pipelines that can be easily implemented in other laboratories and have made all our protocols and SOPs freely available at https://osf.io/uebvj/.

Nuclear export of plant pararetrovirus mRNAs involves the TREX complex, two viral proteins and the highly structured 5' leader region.

In eukaryotes, the major nuclear export pathway for mature mRNAs uses the dimeric receptor TAP/p15, which is recruited to mRNAs via the multisubunit TREX complex, comprising the THO core and different export adaptors. Viruses that replicate in the nucleus adopt different strategies to hijack cellular export factors and achieve cytoplasmic translation of their mRNAs. No export receptors are known in plants, but Arabidopsis TREX resembles the mammalian complex, with a conserved hexameric THO core associated with ALY and UIEF proteins, as well as UAP56 and MOS11. The latter protein is an orthologue of mammalian CIP29. The nuclear export mechanism for viral mRNAs has not been described in plants. To understand this process, we investigated the export of mRNAs of the pararetrovirus CaMV in Arabidopsis and demonstrated that it is inhibited in plants deficient in ALY, MOS11 and/or TEX1. Deficiency for these factors renders plants partially resistant to CaMV infection. Two CaMV proteins, the coat protein P4 and reverse transcriptase P5, are important for nuclear export. P4 and P5 interact and co-localise in the nucleus with the cellular export factor MOS11. The highly structured 5' leader region of 35S RNAs was identified as an export enhancing element that interacts with ALY1, ALY3 and MOS11 in vitro.

Resilient SARS-CoV-2 diagnostics workflows including viral heat inactivation.

There is a worldwide need for reagents to perform SARS-CoV-2 detection. Some laboratories have implemented kit-free protocols, but many others do not have the capacity to develop these and/or perform manual processing. We provide multiple workflows for SARS-CoV-2 nucleic acid detection in clinical samples by comparing several commercially available RNA extraction methods: QIAamp Viral RNA Mini Kit (QIAgen), RNAdvance Blood/Viral (Beckman) and Mag-Bind Viral DNA/RNA 96 Kit (Omega Bio-tek). We also compared One-step RT-qPCR reagents: TaqMan Fast Virus 1-Step Master Mix (FastVirus, ThermoFisher Scientific), qPCRBIO Probe 1-Step Go Lo-ROX (PCR Biosystems) and Luna ® Universal Probe One-Step RT-qPCR Kit (Luna, NEB). We used primer-probes that detect viral N (EUA CDC) and RdRP (PHE guidelines). All RNA extraction methods provided similar results. FastVirus and Luna proved most sensitive. N detection was more reliable than that of RdRP, particularly in samples with low viral titres. Importantly, we demonstrate that treatment of nasopharyngeal swabs with 70 degrees for 10 or 30 min, or 90 degrees for 10 or 30 min (both original variant and B 1.1.7) inactivates SARS-CoV-2 employing plaque assays, and that it has minimal impact on the sensitivity of the qPCR in clinical samples. These findings make SARS-CoV-2 testing portable to settings that do not have CL-3 facilities. In summary, we provide several testing pipelines that can be easily implemented in other laboratories and have made all our protocols and SOPs freely available at https://osf.io/uebvj/ .

The Polybasic Cleavage Site in SARS-CoV-2 Spike Modulates Viral Sensitivity to Type I Interferon and IFITM2.

The cellular entry of severe acute respiratory syndrome-associated coronaviruses types 1 and 2 (SARS-CoV-1 and -2) requires sequential protease processing of the viral spike glycoprotein. The presence of a polybasic cleavage site in SARS-CoV-2 spike at the S1/S2 boundary has been suggested to be a factor in the increased transmissibility of SARS-CoV-2 compared to SARS-CoV-1 by facilitating maturation of the spike precursor by furin-like proteases in the producer cells rather than endosomal cathepsins in the target. We investigate the relevance of the polybasic cleavage site in the route of entry of SARS-CoV-2 and the consequences this has for sensitivity to interferons (IFNs) and, more specifically, the IFN-induced transmembrane (IFITM) protein family that inhibit entry of diverse enveloped viruses. We found that SARS-CoV-2 is restricted predominantly by IFITM2, rather than IFITM3, and the degree of this restriction is governed by route of viral entry. Importantly, removal of the cleavage site in the spike protein renders SARS-CoV-2 entry highly pH and cathepsin dependent in late endosomes, where, like SARS-CoV-1 spike, it is more sensitive to IFITM2 restriction. Furthermore, we found that potent inhibition of SARS-CoV-2 replication by type I but not type II IFNs is alleviated by targeted depletion of IFITM2 expression. We propose that the polybasic cleavage site allows SARS-CoV-2 to mediate viral entry in a pH-independent manner, in part to mitigate against IFITM-mediated restriction and promote replication and transmission. This suggests that therapeutic strategies that target furin-mediated cleavage of SARS-CoV-2 spike may reduce viral replication through the activity of type I IFNs.IMPORTANCE The furin cleavage site in the spike protein is a distinguishing feature of SARS-CoV-2 and has been proposed to be a determinant for the higher transmissibility between individuals, compared to SARS-CoV-1. One explanation for this is that it permits more efficient activation of fusion at or near the cell surface rather than requiring processing in the endosome of the target cell. Here, we show that SARS-CoV-2 is inhibited by antiviral membrane protein IFITM2 and that the sensitivity is exacerbated by deletion of the furin cleavage site, which restricts viral entry to low pH compartments. Furthermore, we find that IFITM2 is a significant effector of the antiviral activity of type I interferons against SARS-CoV-2 replication. We suggest that one role of the furin cleavage site is to reduce SARS-CoV-2 sensitivity to innate immune restriction, and thus, it may represent a potential therapeutic target for COVID-19 treatment development.

Effect of complexing lipids on cellular uptake and expression of messenger RNA in human skin explants.

Messenger RNA (mRNA) represents a promising next-generation approach for both treatment and vaccination. Lipid based particles are one of the most investigated delivery systems for mRNA formulations. Here we explore how the complexing lipid affects uptake and translation of lipoplex-delivered RNA in resident cells in human skin explants and, we explore a more modular delivery system that utilizes mRNA added to pre-formed nanoparticles prior to dosing. We prepared formulations of lipoplexes with ionizable, cationic or zwitterionic lipids, externally complexed these with mRNA, and observed which cells internalized and/or expressed the mRNA over 72 h after intradermal injections into primary, human, skin explants. Using a flow cytometry panel to assess cellular phenotypes, mRNA uptake and mRNA expression, we found that, unlike other cell types, adipocytes expressed mRNA efficiently at 4 and 24 h after mRNA-lipoplex injection and contributed the greatest proportion of total RNA-encoded protein expression, despite being the lowest frequency cell type. Other cell types (epithelial cells, fibroblasts, T cells, B cells, dendritic cells, monocytes, NK cells, Langerhans cells, and leukocytes) had increasing mRNA expression over the course of 72 h, irrespective of lipoplex formulation. We observed that overall charge of the particle, but not the complexing lipid classification, was predictive for the pattern of mRNA uptake and expression among resident cell types in this model. This study provides insight into maximizing protein expression, using modular mRNA lipoplexes that are more compatible with product development, in a clinically relevant, human skin explant model.

Innate Inhibiting Proteins Enhance Expression and Immunogenicity of Self-Amplifying RNA.

Self-amplifying RNA (saRNA) is a cutting-edge platform for both nucleic acid vaccines and therapeutics. saRNA is self-adjuvanting, as it activates types I and III interferon (IFN), which enhances the immunogenicity of RNA vaccines but can also lead to inhibition of translation. In this study, we screened a library of saRNA constructs with cis-encoded innate inhibiting proteins (IIPs) and determined the effect on protein expression and immunogenicity. We observed that the PIV-5 V and Middle East respiratory syndrome coronavirus (MERS-CoV) ORF4a proteins enhance protein expression 100- to 500-fold in vitro in IFN-competent HeLa and MRC5 cells. We found that the MERS-CoV ORF4a protein partially abates dose nonlinearity in vivo, and that ruxolitinib, a potent Janus kinase (JAK)/signal transducer and activator of transcription (STAT) inhibitor, but not the IIPs, enhances protein expression of saRNA in vivo. Both the PIV-5 V and MERS-CoV ORF4a proteins were found to enhance the percentage of resident cells in human skin explants expressing saRNA and completely rescued dose nonlinearity of saRNA. Finally, we observed that the MERS-CoV ORF4a increased the rabies virus (RABV)-specific immunoglobulin G (IgG) titer and neutralization half-maximal inhibitory concentration (IC50) by ∼10-fold in rabbits, but not in mice or rats. These experiments provide a proof of concept that IIPs can be directly encoded into saRNA vectors and effectively abate the nonlinear dose dependency and enhance immunogenicity.

High resolution biosensor to test the capping level and integrity of mRNAs.

5' Cap structures are ubiquitous on eukaryotic mRNAs, essential for post-transcriptional processing, translation initiation and stability. Here we describe a biosensor designed to detect the presence of cap structures on mRNAs that is also sensitive to mRNA degradation, so uncapped or degraded mRNAs can be detected in a single step. The biosensor is based on a chimeric protein that combines the recognition and transduction roles in a single molecule. The main feature of this sensor is its simplicity, enabling semi-quantitative analyses of capping levels with minimal instrumentation. The biosensor was demonstrated to detect the capping level on several in vitro transcribed mRNAs. Its sensitivity and dynamic range remained constant with RNAs ranging in size from 250 nt to approximately 2700 nt and the biosensor was able to detect variations in the capping level in increments of at least 20%, with a limit of detection of 2.4 pmol. Remarkably, it also can be applied to more complex analytes, such mRNA vaccines and mRNAs transcribed in vivo. This biosensor is an innovative example of a technology able to detect analytically challenging structures such as mRNA caps. It could find application in a variety of scenarios, from quality analysis of mRNA-based products such as vaccines to optimization of in vitro capping reactions.

Temporal Proteomic Analysis of Herpes Simplex Virus 1 Infection Reveals Cell-Surface Remodeling via pUL56-Mediated GOPC Degradation.

Herpesviruses are ubiquitous in the human population and they extensively remodel the cellular environment during infection. Multiplexed quantitative proteomic analysis over the time course of herpes simplex virus 1 (HSV-1) infection was used to characterize changes in the host-cell proteome and the kinetics of viral protein production. Several host-cell proteins are targeted for rapid degradation by HSV-1, including the cellular trafficking factor Golgi-associated PDZ and coiled-coil motif-containing protein (GOPC). We show that the poorly characterized HSV-1 pUL56 directly binds GOPC, stimulating its ubiquitination and proteasomal degradation. Plasma membrane profiling reveals that pUL56 mediates specific changes to the cell-surface proteome of infected cells, including loss of interleukin-18 (IL18) receptor and Toll-like receptor 2 (TLR2), and that cell-surface expression of TLR2 is GOPC dependent. Our study provides significant resources for future investigation of HSV-host interactions and highlights an efficient mechanism whereby a single virus protein targets a cellular trafficking factor to modify the surface of infected cells.

[Nuclear export of viral RNAs in metazoan]. / Export nucléaire des ARN viraux chez les métazoaires.

The nuclear export of mRNAs is a complex process, involving the participaton of numerous proteins, the recruitement of which starts during the early steps of mRNAs biosynthesis and maturation. This strategy allows the cell to export only mature and non-defective transcripts to the cytoplasm where they are directed to the translational machinery. The vast majority of mRNAs is exported by the dimeric transport receptor TAP-p15, which is mainly recruited by the large multiprotein complex TREX-1. Other mRNAs that do not display all typical features of a mature transcript use variants of the TAP-p15 export pathway or recruit the alternative export receptor CRM1. Most DNA viruses, retroviruses, and influenza viruses, the mRNAs of which are synthesized in the nucleus, also use TAP-p15 and/or CRM1 to export their mRNAs. The highjacking of the cellular export machinery by viral mRNAs usually involves the presence of constitutive structural elements that directly load cellular export factors and/or viral adaptor proteins. Associated with the host export machinery, viral mRNAs escape host surveillance, are efficiently exported in the cytoplasm in order to be translated, and thus make possible the progress toward the later events of the virus life cycles.

Self-amplifying RNA SARS-CoV-2 lipid nanoparticle vaccine candidate induces high neutralizing antibody titers in mice.

The spread of the SARS-CoV-2 into a global pandemic within a few months of onset motivates the development of a rapidly scalable vaccine. Here, we present a self-amplifying RNA encoding the SARS-CoV-2 spike protein encapsulated within a lipid nanoparticle (LNP) as a vaccine. We observe remarkably high and dose-dependent SARS-CoV-2 specific antibody titers in mouse sera, as well as robust neutralization of both a pseudo-virus and wild-type virus. Upon further characterization we find that the neutralization is proportional to the quantity of specific IgG and of higher magnitude than recovered COVID-19 patients. saRNA LNP immunizations induce a Th1-biased response in mice, and there is no antibody-dependent enhancement (ADE) observed. Finally, we observe high cellular responses, as characterized by IFN-γ production, upon re-stimulation with SARS-CoV-2 peptides. These data provide insight into the vaccine design and evaluation of immunogenicity to enable rapid translation to the clinic.

The In Vitro, Ex Vivo, and In Vivo Effect of Polymer Hydrophobicity on Charge-Reversible Vectors for Self-Amplifying RNA.

RNA technology has the potential to revolutionize vaccination. However, the lack of clear structure-property relationships in relevant biological models mean there is no clear consensus on the chemical motifs necessary to improve RNA delivery. In this work, we describe the synthesis of a series of copolymers based on the self-hydrolyzing charge-reversible polycation poly(dimethylaminoethyl acrylate) (pDMAEA), varying the lipophilicity of the additional co-monomers. All copolymers formed stable polyplexes, showing efficient complexation with model nucleic acids from nitrogen/phosphate (N/P) ratios of N/P = 5, with more hydrophobic complexes exhibiting slower charge reversal and disassembly compared to hydrophilic analogues. The more hydrophobic copolymers outperformed hydrophilic versions, homopolymer controls and the reference standard polymer (polyethylenimine), in transfection assays on 2D cell monolayers, albeit with significantly higher toxicities. Similarly, hydrophobic derivatives displayed up to a 4-fold higher efficacy in terms of the numbers of cells expressing green fluorescent protein (GFP+) cells in ex vivo human skin (10%) compared to free RNA (2%), attributed to transfection enrichment in epithelial cells. In contrast, in a mouse model, we observed the reverse trend in terms of RNA transfection, with no observable protein production in more hydrophobic analogues, whereas hydrophilic copolymers induced the highest transfection in vivo. Overall, our results suggest an important relationship between the vector lipophilicity and RNA transfection in vaccine settings, with polymer biocompatibility potentially a key parameter in effective in vivo protein production.

Ornithine-derived oligomers and dendrimers for in vitro delivery of DNA and ex vivo transfection of skin cells via saRNA.

Gene therapies are undergoing a renaissance, primarily due to their potential for applications in vaccination for infectious diseases and cancers. Although the biology of these technologies is rapidly evolving, delivery strategies need to be improved to overcome the poor pharmacokinetics and cellular transport of nucleic acids whilst maintaining patient safety. In this work, we describe the divergent synthesis of biodegradable cationic dendrimers based on the amino acid ornithine as non-viral gene delivery vectors and evaluate their potential as delivery vectors for DNA and RNA. The dendrimers effectively complexed model nucleic acids at lower N/P ratios than polyethyleneimine and outperformed it in DNA transfection experiments with ratios above 5. Remarkably, all dendrimer polyplexes at N/P = 2 achieved up to 7-fold higher protein content over an optimized PEI formulation when used for transfections with self-amplifying RNA (saRNA). Finally, transfection studies utilizing human skin explants revealed an increase of cells producing protein from 2% with RNA alone to 12% with dendrimer polyplexes, attributed to expression enrichment predominantly in epithelial cells, fibroblasts and leukocytes, with minor enrichment in NK cells, T cells, monocytes, and B cells. Overall, this study indicates the clear potential of ornithine dendrimers as safe and effective delivery vectors for both DNA and RNA therapeutics.

Big Is Beautiful: Enhanced saRNA Delivery and Immunogenicity by a Higher Molecular Weight, Bioreducible, Cationic Polymer.

Self-amplifying RNA (saRNA) vaccines are highly advantageous, as they result in enhanced protein expression compared to mRNA (mRNA), thus minimizing the required dose. However, previous delivery strategies were optimized for siRNA or mRNA and do not necessarily deliver saRNA efficiently due to structural differences of these RNAs, thus motivating the development of saRNA delivery platforms. Here, we engineer a bioreducible, linear, cationic polymer called "pABOL" for saRNA delivery and show that increasing its molecular weight enhances delivery both in vitro and in vivo. We demonstrate that pABOL enhances protein expression and cellular uptake via both intramuscular and intradermal injection compared to commercially available polymers in vivo and that intramuscular injection confers complete protection against influenza challenge. Due to the scalability of polymer synthesis and ease of formulation preparation, we anticipate that this polymer is highly clinically translatable as a delivery vehicle for saRNA for both vaccines and therapeutics.

Mannosylated Poly(ethylene imine) Copolymers Enhance saRNA Uptake and Expression in Human Skin Explants.

Messenger RNA (mRNA) is a promising platform for both vaccines and therapeutics, and self-amplifying RNA (saRNA) is particularly advantageous, as it enables higher protein expression and dose minimization. Here, we present a delivery platform for targeted delivery of saRNA using mannosylated poly(ethylene imine) (PEI) enabled by the host-guest interaction between cyclodextrin and adamantane. We show that the host-guest complexation does not interfere with the electrostatic interaction with saRNA and observed that increasing the degree of mannosylation inhibited transfection efficiency in vitro, but enhanced the number of cells expressing GFP by 8-fold in human skin explants. Besides, increasing the ratio of glycopolymer to saRNA also enhanced the percentage of transfected cells ex vivo. We identified that these mannosylated PEIs specifically increased protein expression in the epithelial cells resident in human skin in a mannose-dependent manner. This platform is promising for further study of glycosylation of PEI and targeted saRNA delivery.

Foxtail mosaic virus: A Viral Vector for Protein Expression in Cereals.

Rapid and cost-effective virus-derived transient expression systems for plants are invaluable in elucidating gene function and are particularly useful in plant species for which transformation-based methods are unavailable or are too time and labor demanding, such as wheat (Triticum aestivum) and maize (Zea mays). The virus-mediated overexpression (VOX) vectors based on Barley stripe mosaic virus and Wheat streak mosaic virus described previously for these species are incapable of expressing free recombinant proteins of more than 150 to 250 amino acids, are not suited for high-throughput screens, and have other limitations. In this study, we report the development of a VOX vector based on a monopartite single-stranded positive sense RNA virus, Foxtail mosaic virus (genus Potexvirus). In this vector, PV101, the gene of interest was inserted downstream of the duplicated subgenomic promoter of the viral coat protein gene, and the corresponding protein was expressed in its free form. The vector allowed the expression of a 239-amino acid-long GFP in both virus-inoculated and upper uninoculated (systemic) leaves of wheat and maize and directed the systemic expression of a larger approximately 600-amino acid protein, GUSPlus, in maize. Moreover, we demonstrated that PV101 can be used for in planta expression and functional analysis of apoplastic pathogen effector proteins such as the host-specific toxin ToxA of Parastagonospora nodorum Therefore, this VOX vector opens possibilities for functional genomics studies in two important cereal crops.

Formation of large viroplasms and virulence of Cauliflower mosaic virus in turnip plants depend on the N-terminal EKI sequence of viral protein TAV.

Cauliflower mosaic virus (CaMV) TAV protein (TransActivator/Viroplasmin) plays a pivotal role during the infection cycle since it activates translation reinitiation of viral polycistronic RNAs and suppresses RNA silencing. It is also the major component of cytoplasmic electron-dense inclusion bodies (EDIBs) called viroplasms that are particularly evident in cells infected by the virulent CaMV Cabb B-JI isolate. These EDIBs are considered as virion factories, vehicles for CaMV intracellular movement and reservoirs for CaMV transmission by aphids. In this study, focused on different TAV mutants in vivo, we demonstrate that three physically separated domains collectively participate to the formation of large EDIBs: the N-terminal EKI motif, a sequence of the MAV domain involved in translation reinitiation and a C-terminal region encompassing the zinc finger. Surprisingly, EKI mutant TAVm3, corresponding to a substitution of the EKI motif at amino acids 11-13 by three alanines (AAA), which completely abolished the formation of large viroplasms, was not lethal for CaMV but highly reduced its virulence without affecting the rate of systemic infection. Expression of TAVm3 in a viral context led to formation of small irregularly shaped inclusion bodies, mild symptoms and low levels of viral DNA and particles accumulation, despite the production of significant amounts of mature capsid proteins. Unexpectedly, for CaMV-TAVm3 the formation of viral P2-containing electron-light inclusion body (ELIB), which is essential for CaMV aphid transmission, was also altered, thus suggesting an indirect role of the EKI tripeptide in CaMV plant-to-plant propagation. This important functional contribution of the EKI motif in CaMV biology can explain the strict conservation of this motif in the TAV sequences of all CaMV isolates.