Properties and use of novel replication-competent vectors based on Semliki Forest virus

<p>Abstract</p> <p>Background</p> <p>Semliki Forest virus (SFV) has a positive strand RNA genome and infects different cells of vertebrates and invertebrates. The 5' two-thirds of the genome encodes non-structural proteins that are required for virus replication and synthesis of subgenomic (SG) mRNA for structural proteins. SG-mRNA is generated by internal initiation at the SG-promoter that is located at the complementary minus-strand template. Different types of expression systems including replication-competent vectors, which represent alphavirus genomes with inserted expression units, have been developed. The replication-competent vectors represent useful tools for studying alphaviruses and have potential therapeutic applications. In both cases, the properties of the vector, such as its genetic stability and expression level of the protein of interest, are important.</p> <p>Results</p> <p>We analysed 14 candidates of replication-competent vectors based on the genome of an SFV4 isolate that contained a duplicated SG promoter or an internal ribosomal entry site (IRES)-element controlled marker gene. It was found that the IRES elements and the minimal -21 to +5 SG promoter were non-functional in the context of these vectors. The efficient SG promoters contained at least 26 residues upstream of the start site of SG mRNA. The insertion site of the SG promoter and its length affected the genetic stability of the vectors, which was always higher when the SG promoter was inserted downstream of the coding region for structural proteins. The stability also depended on the conditions used for vector propagation. A procedure based on the <it>in vitro </it>transcription of ligation products was used for generation of replication-competent vector-based expression libraries that contained hundreds of thousands of different genomes, and maintained genetic diversity and the ability to express inserted genes over five passages in cell culture.</p> <p>Conclusion</p> <p>The properties of replication-competent vectors of alphaviruses depend on the details of their construction. In the case of SFV4, such vectors should contain the SG promoter with structural characteristics for this isolate. The main factor for instability of SFV4-based replication-competent vectors was the deletion of genes of interest, since the resulting shorter genomes had a growth advantage over the original vector.</p

Design and use of Chikungunya virus replication templates utilizing mammalian and mosquito RNA polymerase I mediated transcription

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus. It has a positive sense RNA genome that also serves as the mRNA for four non-structural proteins (nsPs) representing subunits of the viral replicase. Coupling of nsP and RNA synthesis complicates analysis of viral RNA replication. We developed trans-replication systems, where production of replication competent RNA and expression of viral replicase are uncoupled. Mammalian and mosquito RNA polymerase I promoters were used to produce non-capped RNA templates, which are poorly translated relative to CHIKV replicase generated capped RNAs. It was found that, in human cells, constructs driven by RNA polymerase I promoters of human and Chinese hamster origin performed equally well. In contrast, RNA polymerase I promoters from Aedes mosquitoes exhibited strong species specificity. In both mammalian and mosquito cells, novel trans-replicase assays had exceptional sensitivity, with up to 105-fold higher reporter expression in the presence of replicase relative to background. Using this highly sensitive assay to analyse CHIKV nsP1 functionality, several mutations that severely reduced, but did not completely block, CHIKV replicase activity were identified: (i) tagging the N-terminus of nsP1 with eGFP; (ii) mutations D63A and Y248A blocking the RNA capping; (iii) mutation R252E affecting nsP1 membrane anchoring. In contrast, a mutation in the nsP1 palmitoylation site completely inactivated CHIKV replicase in both human and mosquito cells and was lethal for the virus. Our data confirms that this novel system provides a valuable tool to study CHIKV replicase, RNA replication and virus-host interactions

Antiviral efficacy of nanomaterial-treated textiles in real-life like exposure conditions

We thank the following people and institutions for their contribution to our investigation. Toomas Varjund from TAD Logistics OÜ for providing the textiles. Ülis Sõukand from Estonian Environmental Research Center for his help with methods of chemical analysis. Estonian Research Council projects COVSG2, PRG629, PRG1496, PRG1154 and European Commission project STOP (Grant agreement ID: 101057961) for their financial support. The Center of nanomaterials technologies and research (NAMUR+) for core facility funded by project TT13 which was used conducting the research.Due to the growing interest towards reducing the number of potentially infectious agents on critical high-touch surfaces, the popularity of antimicrobially and antivirally active surfaces, including textiles, has increased. The goal of this study was to create antiviral textiles by spray-depositing three different nanomaterials, two types of CeO2 nanoparticles and quaternary ammonium surfactant CTAB loaded SiO2 nanocontainers, onto the surface of a knitted polyester textile and assess their antiviral activity against two coronaviruses, porcine transmissible gastroenteritis virus (TGEV) and severe acute respiratory syndrome virus (SARS CoV-2). Antiviral testing was carried out in small droplets in semi-dry conditions and in the presence of organic soiling, to mimic aerosol deposition of viruses onto the textiles. In such conditions, SARS CoV-2 stayed infectious at least for 24 h and TGEV infected cells even after 72h of semi-dry deposition suggesting that textiles exhibiting sufficient antiviral activity before or at 24 h, can be considered promising. The antiviral efficacy of nanomaterial-deposited textiles was compared with the activity of the same nanomaterials in colloidal form and with positive control textiles loaded with copper nitrate and CTAB. Our results indicated that after deposition onto the textile, CeO2 nanoparticles lost most of their antiviral activity, but antiviral efficacy of CTAB-loaded SiO2 nanocontainers was retained also after deposition. Copper nitrate deposited textile that was used as a positive control, showed relatively high antiviral activity as expected. However, as copper was effectively washed away from the textile already during 1 h, the use of copper for creating antiviral textiles would be impractical. In summary, our results indicated that antiviral activity of textiles cannot be predicted from antiviral efficacy of the deposited compounds in colloid and attention should be paid on prolonged efficacy of antivirally coated textiles.--//-- Alexandra Nefedova, Kai Rausalu, Eva Zusinaite, Vambola Kisand, Mati Kook, Krisjanis Smits, Alexander Vanetsev, Angela Ivask, Antiviral efficacy of nanomaterial-treated textiles in real-life like exposure conditions, Heliyon, Volume 9, Issue 9, 2023, e20067, ISSN 2405-8440, https://doi.org/10.1016/j.heliyon.2023.e20067. Published under the CC BY-NC-ND licence.Estonian Research Council projects COVSG2, PRG629, PRG1496, PRG1154; European Commission project STOP (Grant agreement ID: 101057961); the Institute of Solid State Physics, University of Latvia has received funding from EU CAMART2 project (European Union's Horizon 2020 Framework Programme H2020-WIDESPREAD-01-2016-2017 TeamingPhase2 under grant agreement No. 739508

Obatoclax Inhibits Alphavirus Membrane Fusion by Neutralizing the Acidic Environment of Endocytic Compartments

As new pathogenic viruses continue to emerge, it is paramount to have intervention strategies that target a common denominator in these pathogens. The fusion of viral and cellular membranes during viral entry is one such process that is used by many pathogenic viruses, including chikungunya virus, West Nile virus, and influenza virus. Obatoclax, a small-molecule antagonist of the Bcl-2 family of proteins, was previously determined to have activity against influenza A virus and also Sindbis virus. Here, we report it to be active against alphaviruses, like chikungunya virus (50% effective concentration [EC50] = 0.03 mu M) and Semliki Forest virus (SFV; EC50 = 0.11 mu M). Obatoclax inhibited viral entry processes in an SFV temperaturesensitive mutant entry assay. A neutral red retention assay revealed that obatoclax induces the rapid neutralization of the acidic environment of endolysosomal vesicles and thereby most likely inhibits viral fusion. Characterization of escape mutants revealed that the L369I mutation in the SFV E1 fusion protein was sufficient to confer partial resistance against obatoclax. Other inhibitors that target the Bcl-2 family of antiapoptotic proteins inhibited neither viral entry nor endolysosomal acidification, suggesting that the antiviral mechanism of obatoclax does not depend on its anticancer targets. Obatoclax inhibited the growth of flaviviruses, like Zika virus, West Nile virus, and yellow fever virus, which require low pH for fusion, but not that of pH-independent picornaviruses, like coxsackievirus A9, echovirus 6, and echovirus 7. In conclusion, obatoclax is a novel inhibitor of endosomal acidification that prevents viral fusion and that could be pursued as a potential broad-spectrum antiviral candidate.Peer reviewe

Antiviral efficacy of cerium oxide nanoparticles

The authors gratefully acknowledge the financial support by the Estonian Research Council Grants (COVSG2, PRG629, PRG1496), Estonian Centre of Excellence in Research project “Advanced materials and high-technology devices for sustainable energetics, sensorics and nanoelectronics” TK141 (2014-2020.4.01.15-0011) and University of Tartu Development Fund (PLTFYARENG53). The research was partly conducted using the NAMUR+ core facility funded by projects “Center of nanomaterials technologies and research” (2014-2020.4.01.16-0123) and TT13.Nanomaterials are prospective candidates for the elimination of viruses due to their multimodal mechanisms of action. Here, we tested the antiviral potential of a largely unexplored nanoparticle of cerium dioxide (CeO2). Two nano-CeO2 with opposing surface charge, (+) and (−), were assessed for their capability to decrease the plaque forming units (PFU) of four enveloped and two non-enveloped viruses during 1-h exposure. Statistically significant antiviral activity towards enveloped coronavirus SARS-CoV-2 and influenza virus was registered already at 20 mg Ce/l. For other two enveloped viruses, transmissible gastroenteritis virus and bacteriophage φ6, antiviral activity was evidenced at 200 mg Ce/l. As expected, the sensitivity of non-enveloped viruses towards nano-CeO2 was significantly lower. EMCV picornavirus showed no decrease in PFU until the highest tested concentration, 2000 mg Ce/l and MS2 bacteriophage showed slight non-monotonic response to high concentrations of nano-CeO2(−). Parallel testing of antiviral activity of Ce3+ ions and SiO2 nanoparticles allows to conclude that nano-CeO2 activity was neither due to released Ce-ions nor nonspecific effects of nanoparticulates. Moreover, we evidenced higher antiviral efficacy of nano-CeO2 compared with Ag nanoparticles. This result along with low antibacterial activity and non-existent cytotoxicity of nano-CeO2 allow us to propose CeO2 nanoparticles for specific antiviral applications. © 2022, The Author(s). --//-- This is an open access article Nefedova A, Rausalu K, Zusinaite E, Vanetsev A, Rosenberg M, Koppel K, Lilla S, Visnapuu M, Smits K, Kisand V, Tätte T, Ivask A., "Antiviral efficacy of cerium oxide nanoparticles", Scientific Reports (2022); 12(1):18746, doi: 10.1038/s41598-022-23465-6 published under the CC BY 4.0 licence.Estonian Research Council Grants (COVSG2, PRG629, PRG1496); Estonian Centre of Excellence in Research TK141 (2014-2020.4.01.15-0011); University of Tartu Development Fund (PLTFYARENG53); Institute of Solid-State Physics, University of Latvia has received funding from the European Union's Horizon 2020 Framework Programme H2020-WIDESPREAD-01-2016-2017-Teaming Phase 2 under grant agreement No. 739508, project CAMART2

Cross-utilisation of template RNAs by alphavirus replicases

Most alphaviruses (family Togaviridae) including Sindbis virus (SINV) and other human pathogens, are transmitted by arthropods. The first open reading frame in their positive strand RNA genome encodes for the non-structural polyprotein, a precursor to four separate subunits of the replicase. The replicase interacts with cis-acting elements located near the intergenic region and at the ends of the viral RNA genome. A trans-replication assay was developed and used to analyse the template requirements for nine alphavirus replicases. Replicases of alphaviruses of the Semliki Forest virus complex were able to cross-utilize each other's templates as well as those of outgroup alphaviruses. Templates of outgroup alphaviruses, including SINV and the mosquito-specific Eilat virus, were promiscuous; in contrast, their replicases displayed a limited capacity to use heterologous templates, especially in mosquito cells. The determinants important for efficient replication of template RNA were mapped to the 5' region of the genome. For SINV these include the extreme 5'- end of the genome and sequences corresponding to the first stem-loop structure in the 5' untranslated region. Mutations introduced in these elements drastically reduced infectivity of recombinant SINV genomes. The trans-replicase tools and approaches developed here can be instrumental in studying alphavirus recombination and evolution, but can also be applied to study other viruses such as picornaviruses, flaviviruses and coronaviruses.</p

Obatoclax inhibits alphavirus membrane fusion by neutralizing the acidic environment of endocytic compartments

As new pathogenic viruses continue to emerge, it is paramount to have intervention strategies that target a common denominator in these pathogens. The fusion of viral and cellular membranes during viral entry is one such process that is used by many pathogenic viruses including chikungunya virus, West Nile virus, influenza virus etc. Obatoclax, a small-molecule antagonist of the Bcl-2 family of proteins was previously determined to be antiviral against influenza A virus and also Sindbis virus. Here, we report it to be active against alphaviruses like chikungunya virus (EC50 = 0.03 μM) and Semliki Forest virus (SFV) (EC50 = 0.11 μM). Obatoclax inhibited viral entry processes in an SFV temperature-sensitive mutant entry assay. Neutral red retention assay revealed that obatoclax induces rapid neutralization of the acidic environment of endolysosomal vesicles and thereby, most likely inhibits viral fusion. Characterization of escape mutants revealed that mutation L369I in the SFV E1 fusion protein was sufficient to confer partial resistance against obatoclax. Other inhibitors that target the Bcl-2 family of antiapoptotic proteins neither inhibited viral entry nor endolysosomal acidification, suggesting that the antiviral mechanism of obatoclax does not depend on its anticancer targets. Obatoclax inhibited the growth of flaviviruses like Zika virus, West Nile virus and yellow fever virus, which require low pH for fusion, but not of pH-independent picornaviruses like coxasackievirus A9, echovirus 6 and echovirus 7. In conclusion, obatoclax is a novel inhibitor of endosomal acidification preventing viral fusion that could be pursued as a potential broad-spectrum antiviral candidate.</p

Chikungunya virus infectivity, RNA replication and non-structural polyprotein processing depend on the nsP2 protease's active site cysteine residue

Chikungunya virus (CHIKV), genus Alphavirus, family Togaviridae, has a positive-stand RNA genome approximately 12 kb in length. In infected cells, the genome is translated into non-structural polyprotein P1234, an inactive precursor of the viral replicase, which is activated by cleavages carried out by the non-structural protease, nsP2. We have characterized CHIKV nsP2 using both cell-free and cell-based assays. First, we show that Cys478 residue in the active site of CHIKV nsP2 is indispensable for P1234 processing. Second, the substrate requirements of CHIKV nsP2 are quite similar to those of nsP2 of related Semliki Forest virus (SFV). Third, substitution of Ser482 residue, recently reported to contribute to the protease activity of nsP2, with Ala has almost no negative effect on the protease activity of CHIKV nsP2. Fourth, Cys478 to Ala as well as Trp479 to Ala mutations in nsP2 completely abolished RNA replication in CHIKV and SFV trans-replication systems. In contrast, trans-replicases with Ser482 to Ala mutation were similar to wild type counterparts. Fifth, Cys478 to Ala as well as Trp479 to Ala mutations in nsP2 abolished the rescue of infectious virus from CHIKV RNA transcripts while Ser482 to Ala mutation had no effect. Thus, CHIKV nsP2 is a cysteine protease.Peer reviewe

Mimicking superinfection exclusion disrupts alphavirus infection and transmission in the yellow fever mosquito Aedes aegypti

Multiple viruses, including pathogenic viruses, bacteriophages, and even plant viruses, cause a phenomenon termed superinfection exclusion whereby a currently infected cell is resistant to secondary infection by the same or a closely related virus. In alphaviruses, this process is thought to be mediated, at least in part, by the viral protease (nsP2) which is responsible for processing the nonstructural polyproteins (P123 and P1234) into individual proteins (nsP1–nsP4), forming the viral replication complex. Taking a synthetic biology approach, we mimicked this naturally occurring phenomenon by generating a superinfection exclusion-like state in Aedes aegypti mosquitoes, rendering them refractory to alphavirus infection. By artificially expressing Sindbis virus (SINV) and chikungunya virus (CHIKV) nsP2 in mosquito cells and transgenic mosquitoes, we demonstrated a reduction in both SINV and CHIKV viral replication rates in cells following viral infection as well as reduced infection prevalence, viral titers, and transmission potential in mosquitoes.</p

Alfaviiruste nsP2 proteaas: nõuetest funktsionaalsuseks inhibitsioonini

Väitekirja elektrooniline versioon ei sisalda publikatsiooneAlfaviirused on umbes 70 nm diameetriga RNA genoomiga viirused (perekond Alphavirus, sugukond Togaviridae). Üks alfaviirustest on Chikungunya viirus (CHIKV), mis põhjustab haigust, millega kaasnevad piinavad liigesevalud kestavad sageli kuid peale haigestumist. Leevendamaks CHIKV haigusest põhjustatud kannatusi on oluline leida viirusvastaseid ühendeid, mis takistaksid CHIKV paljunemist rakkudes. Selleks on vaja mõista, kuidas kulgeb CHIKV elütsükkel rakkudes. nsP2 on üks CHIKV valkudest, millel on CHIKV infektsiooni ajal mitu funktsiooni. nsP2 on viiruse proteaas ja RNA helikaas, nsP2 osaleb ka raku viirusvastase immunvastuse maha surumises. Proteaasina on nsP2 see viirusvalk, mis lõikab viiruse mittestruktuurse liitvalgu individuaalseteks valkudeks. See protsess on kesksel kohal viiruse elutsükli regulatsioonis. Selle doktoritöö raames uuriti, mis on CHIKV nsP2 proteaasi nõuded funktsionaalsuseks. Leiti, et CHIKV nsP2 on tsüsteiin-proteaas, mis sarnaneb varemuuritud teiste alfaviiruste nsP2 proteaasidega. CHIKV nsP2 aminohappelises järjestuses on tsüsteiin 478 vältimatult vajalik proteaasi aktiivsuse olemasoluks ning kui see tsüsteiin või selle kõrval asuv trüptofaan 479 asendada alaniiniga, siis kaotab CHIKV nsP2 oma proteaasi aktiivsuse. Alfaviiruste mittestruktuurse liitvalgu lõikamine on ajaliselt reguleeritud protsess. Uurisime kuidas mõjutab viiruse mittestruktuursete valkude vaheliste lõikamiste toimumise kiiruse muutmine Semliki Forest viirust (SFV) ning Sindbis viirust (SINV). Leidsime, et SFV ning SINV jaoks on oluline, et viirusvalkude nsP1 ja nsP2 vaheline lõikamine ei toimuks liiga kiiresti. Kiirendatud lõikamine kahandas nende viiruste nakatamisvõimet ning vähendas viiruse RNA-de sünteesi. Viimasena osa doktoritööst moodustab ühe varem-tuntud CHIKV-inhibeeriva ühendi põhjal sünteesitud uudsete potentsiaalsete CHIKV inhibiitorite analüüs, mille tulemusena leiti ühend, mille CHIKV-vastane toime on umbes kümme korda parem kui lähteühendil.Alphaviruses (genus Alphavirus, family Togaviridae) have an RNA genome and the virions are approximately 70 nm in size. One of alphaviruses is Chikungunya virus (CHIKV), which causes a disease with debilitating joint pains that can last for months. It is important to find antiviral compounds to alleviate suffering caused by CHIKV. For that, we need to understand how the viral life cycle occurs in the cells. nsP2 is a multifunctional CHIKV protein, it is the viral protease and the RNA helicase, nsP2 also participates in the suppression of cellular immune response. As a protease, nsP2 is the viral protein, that cleaves the viral nonstructural polyprotein into individual proteins. This process plays a central role in the regulation of the viral life cycle. Within this thesis, we studied the requirements of CHIKV nsP2 for its protease activity. We found, that CHIKV nsP2 is a cysteine-protease, which is similar to previously studied alphaviral nsP2 proteases. In amino acid sequence CHIKV nsP2 must have intact cysteine 478 for its protease activity. If this cysteine or nearby tryptophan 479 is substituted with alanine, then CHIKV nsP2 loses its protease activity. Cleavage of alphaviral nonstructural polyprotein is a temporally regulated process. We studied how Semliki Forest virus (SFV) and Sindbis virus (SINV) are affected by changes in the speed of cleavages of the nonstructural polyprotein. We found, that it is important for SFV and SINV to prevent premature cleavage between viral nsP1 and nsP2. Accelerated processing of SFV or SINV nonstructural polyprotein caused decrease in infectivities and reduced viral RNA synthesis. The final part of the thesis is the analysis of novel CHIKV inhibitors. The structure of a previously known CHIKV inhibitor was modified in several ways and the anti-CHIKV properties of the novel compounds were evaluated. We identified a compound, that had approximately 10-fold increased anti-CHIKV activity in cell culture.https://www.ester.ee/record=b555640