Unraveling the intricate nature of the alphavirus RNA replicase
Date
2023-06-30
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Abstract
Alfaviirused on RNA genoomiga viiruste perekond, mille hulka kuulub olulisi inimeste haigustekitajaid. Imetajaid, linde ja roomajaid nakatavad alfaviirused võib jagada kaheks, millest esimesed põhjustavad artriidi-tüüpi sümptomeid ning teised neuroloogilisi haiguseid. Meditsiiniliselt on üks olulisemaid alfaviiruseid chikungunya viirus, mis põhjustab inimestes palavikku, löövet ning liigesevaegusi. Liigesevaegused võivad areneda kuude või isegi aastate pikkuseks artriidiks. Iga RNA genoomiga viiruse elutsükli keskne osa on RNA genoomi replikatsioon. Replikatsioon ehk viiruse genoomi paljundamine on seetõttu ka üks põhilisi elemente, mille vastu on suunatud vaktsiinide ning viirusvastaste vahendite loomine. Hoolimata alfaviiruste laiast levikust inimeste seas ei ole tänini saadaval heakskiidetud vaktsiine või viirusvastaseid vahendeid. Seetõttu on oluline uurida erinevate alfaviiruste RNA replikatsiooni toimumise põhimõtteid ning nõudeid. Alfaviiruste genoomi replikatsiooni viivad läbi neli spetsiaalset valku, mida kutsutakse replikaasi valkudeks. Nendest replikaasi valkudest moodustatakse rakkudes kompleksid, millesse koondatakse viiruse uute genoomide tootmine. Selles doktoritöös uuriti kümne erineva alfaviiruse näitel kõnealuste replikatsioonikomplekside moodustumist. Aktiivsete replikatsioonikomplekside moodustumise eelduseks on, et alfaviiruste replikaasid tunnevad rakus ära enda genoomi, seonduvad sellega ning alles siis on võimelised seda paljundama. Me näitasime, et mõnede alfaviiruste replikaasid suudavad inimese rakkudes ära tunda võõraste viiruste genoomidele omaseid järjestusi ning replitseerivad neid efektiivselt, samas kui osade viiruste replikaasid näitasid üles tugevat spetsiifilisust enda genoomi suhtes. Lisaks näitasime, et funktsionaalseid alfaviiruste replikatsioonikomplekse on võimalik moodustada replikaasi valkudest, mis pärinevad erinevatelt alfaviirustelt (st, moodustatakse hübriidsed replikaasid). Kasutades selliseid hübriidseid replikaase tuvastasime, millised replikaasi valgud oma genoomile spetsiifilisi elemente enne replikatsiooni alustamist ära tunnevad ning määravad, kas seda genoomi replitseerida või mitte. Samuti näitasime, et matriits-järjestusi, mis sisaldavad alfaviiruste genoomidele spetsiifilisi elemente ning ekspresseerivad reportereid, on võimalik aktiveerida reaalse viirusnakkuse korral. Selliseid biosensoreid hakatakse viirusnakkuse korral rakkudes tootma, mille tagajärjel on nakatunud rakke võimalik visuaalselt tuvastada rohelise värvuse järgi. Siin töös kogutud informatsiooni abil õnnestus viimaks määrata alfaviiruste replikaasi keskse osa, nsP4 valgu aktiivse subühiku 3D struktuur ning uurida selle struktuursete elementide olulisust mutageneesi abil.
Selle doktoritöö raames läbi viidud uurimused annavad uut informatsiooni alfaviiruste replikatsiooni kohta. Hübriidsete replikatsioonikomplekside moodustumine annab aimu, mis võib toimuda alfaviiruste replikatsioonil juhul, kui ühte rakku on nakatanud mitu erinevat alfaviirust. Alfaviiruste replikaaside võime ära tunda võõrastele viirusgenoomidele spetsiifilisi elemente aitab pilgu heita ka alfaviiruste genoomide võimalikule rekombinatsioonile ning evolutsioonile.
Alphaviruses are a genus of viruses with a positive-strand RNA genome and include multiple important human pathogens. Alphaviruses that infect mammals, birds and reptiles can be largely divided into two groups. First, viruses that cause illness characterized by arthralgia and second, viruses that cause neurological diseases. One of the most important alphaviruses medically is chikungunya virus, which can be characterized in humans by symptoms like fever, rash and joint pain with the possibility of persisting into months- or even years-long arthritis. The central part in the life cycle of every virus with an RNA genome is the RNA replication. Thus, the RNA replication, i.e., the propagation of the RNA genome, is one of the main targets for the generation of vaccines and antivirals. Despite the wide distribution of alphaviruses among humans, no licenced vaccines or antiviral drugs are available. This emphasizes the importance of acquiring detailed information on the fundamental principles of the RNA replication. The replication of alphavirus genomes is performed by four specific proteins, called the replicase proteins. These replicase proteins assemble into functional replication complexes that carry out the propagation of the RNA genome. In this thesis, the formation of the replication complexes was studied on the example of 10 different alphaviruses. The prerequisite for the formation of active replication complexes is the replicase proteins recognizing their genome, binding it and only then they can initiate the replication. Here, we discovered that some alphavirus replicases can recognize and replicate RNA sequences originating from different alphaviruses, whereas some replicases demonstrated high specificity toward their own genome. Furthermore, we found that functional replication complexes can be formed from replicase proteins which originate from different viruses (i.e., hybrid replicases). Using these hybrid replicases we demonstrated which replicase proteins account for recognizing the virus genome specific elements and determine whether to replicate the genome or not. In addition, we showed that template constructs which include alphavirus genome specific elements and encode for reporter proteins are activated in cells that are infected with relevant alphaviruses. Thus, such biosensors are expressed in cells upon alphavirus infection and the infection of cells can be confirmed by visually observing the infected cells turning green. Finally, using the information obtained in this thesis, we participated in a study which resulted in acquiring the 3D structure of the central part of the alphavirus replicase, the catalytic subunit of the nsP4, and studied the importance of some structurally key elements using structure guided mutagenesis. Studies included in this thesis offer new information on the RNA replication of alphaviruses. The possibility of forming active hybrid replication complexes sheds light on alphavirus replication in cases where multiple viruses have infected a single cell. The ability of the alphavirus replicases to recognize RNA elements of different viruses offers insights into the possible recombination of the alphavirus genomes and, more generally, the evolution of alphaviruses.
Alphaviruses are a genus of viruses with a positive-strand RNA genome and include multiple important human pathogens. Alphaviruses that infect mammals, birds and reptiles can be largely divided into two groups. First, viruses that cause illness characterized by arthralgia and second, viruses that cause neurological diseases. One of the most important alphaviruses medically is chikungunya virus, which can be characterized in humans by symptoms like fever, rash and joint pain with the possibility of persisting into months- or even years-long arthritis. The central part in the life cycle of every virus with an RNA genome is the RNA replication. Thus, the RNA replication, i.e., the propagation of the RNA genome, is one of the main targets for the generation of vaccines and antivirals. Despite the wide distribution of alphaviruses among humans, no licenced vaccines or antiviral drugs are available. This emphasizes the importance of acquiring detailed information on the fundamental principles of the RNA replication. The replication of alphavirus genomes is performed by four specific proteins, called the replicase proteins. These replicase proteins assemble into functional replication complexes that carry out the propagation of the RNA genome. In this thesis, the formation of the replication complexes was studied on the example of 10 different alphaviruses. The prerequisite for the formation of active replication complexes is the replicase proteins recognizing their genome, binding it and only then they can initiate the replication. Here, we discovered that some alphavirus replicases can recognize and replicate RNA sequences originating from different alphaviruses, whereas some replicases demonstrated high specificity toward their own genome. Furthermore, we found that functional replication complexes can be formed from replicase proteins which originate from different viruses (i.e., hybrid replicases). Using these hybrid replicases we demonstrated which replicase proteins account for recognizing the virus genome specific elements and determine whether to replicate the genome or not. In addition, we showed that template constructs which include alphavirus genome specific elements and encode for reporter proteins are activated in cells that are infected with relevant alphaviruses. Thus, such biosensors are expressed in cells upon alphavirus infection and the infection of cells can be confirmed by visually observing the infected cells turning green. Finally, using the information obtained in this thesis, we participated in a study which resulted in acquiring the 3D structure of the central part of the alphavirus replicase, the catalytic subunit of the nsP4, and studied the importance of some structurally key elements using structure guided mutagenesis. Studies included in this thesis offer new information on the RNA replication of alphaviruses. The possibility of forming active hybrid replication complexes sheds light on alphavirus replication in cases where multiple viruses have infected a single cell. The ability of the alphavirus replicases to recognize RNA elements of different viruses offers insights into the possible recombination of the alphavirus genomes and, more generally, the evolution of alphaviruses.
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Väitekirja elektrooniline versioon ei sisalda publikatsioone
Keywords
Alphavirus, RNA, replication