New insights into alphaviral nsP2 functions
Date
2023-01-12
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Abstract
Alfaviirused (sugukond Togaviridae) on sfäärilise ümbrisega virionide ja positiivse polaarsusega RNA genoomiga viirused, mis enamasti levivad lülijalgsete vektorite vahendusel. Paljud alfaviirused on olulised inimeste patogeenid, sealhulgas ulatuslikke puhanguid põhjustav Chikungunya viirus (CHIKV), Austraalias leviv Ross River viirus (RRV), Ameerikas leviv ida hobuste entsefaliidi viirus (EEEV) ja ka Eestis leiduv Sindbis viirus (SINV). Alfaviiruste põhjustatud haigused sõltuvad viiruse liigist ja selle päritolust,kus üldistatult põhjustavad Vana Maailma alfaviirused palaviku, löövet ja artriiti, samal ajal kui Uue Maailma alfaviirused on sageli kõrge patogeensusega põhjustades neuroloogilisi haiguseid, sealhulgas entsefaliiti.
Alfaviiruste RNA genoom kodeerib nelja viiruse RNA replikaasi allühikut, mis toodetakse liitvalgust eellase (polüproteiini) kujul. Liitvalk lõigatakse neljaks valmis replikaasi valguks viiruse nsP2 proteaasi abil. Viimastel aastakümnetel läbiviidud uurimistööd on võimaldanud välja selgitada viiruse replikaasi kompleksi kui ka selle komponentide peamised funktsioonid ning meie arusaamine viiruse valkude struktuuridest on oluliselt täienenud. Ka need uurimised on näidanud, et alfaviiruse infektsiooni üks esimesi ja kõige olulisemaid sündmusi on replikaasi valkude eellase lõikamine valmis valkudeks. Eelvalgu lõikamised toimuvad kindlas järjekorras ja onkindlalt ajastatud, mis määrab ära selle, kas vabanevad valgud moodustavad toimiva RNA replikaasi või mitte. Seega kujutab eelvalgu lõikamise protsessi läbi viiv nsP2 endast RNA replikatsiooni käivitamise ja efektiivsuse regulaatorit. Samas on tegemist multifunktsionaalse valguga, mis on lisaks RNA sünteesi reguleerimisele oluline nii raku viirusvastaste kaitsereaktsioonide aktiveerimisel kui ka nende mahasurumiseks. Sellest tulenevat mõjutavad muudatused nsP2 valgus, sealhulgas punktmutatsioonid valgu funktsionaalselt olulistes regioonides, oluliselt viiruse bioloogilisi omadusi. Seejuures ei ole mitmete nsP2 aktiivsuste taga seisvad molekulaarsed mehhanismid lõpuni selged. Küll aga on selgunud, et nsP2 funktsioone mõjutavad interaktsioonid alfaviiruse ülejäänute replikaasi valkude nsP1, nsP3 ja nsP4-ga.
Käesoleva uurimistöö raames selgitati välja ja analüüsiti mitmeid nsP2 valgu ja RNA replikaasi seni tundmatuid funktsioone. Töö peamised tulemused saab kokku võtta järgnevalt:
1. alfaviiruste genoomide alusel konstrueeritud trans-replikatsiooni süsteemid võimaldavad uurida nsP2 valgu funktsioone, mis on seotud viiruse RNA replikaasi kompleksi moodustamisega, aga ka selle moodustamise blokeerimise ja/või selle aktiivsuse mahasurumisega. Viimati nimetatud aktiivsused on olulised viiruste superinfektsiooni blokeerimisel ja tõenäoliselt ka viiruse replikatsiooni ajalisel reguleerimisel. CHIKV ja SINV trans-replikatsiooni süsteeme kasutades tehti kindlaks, et superinfektsiooni blokeerimise puhul on võtmesündmuseks ühe viiruse replikaasi eellase lõikamine teise viiruse nsP2 poolt ja et seda mõjutavad mutatsioonid nii sihtmärk-liitvalgus kui ka nsP2 valgus;
2. leiti, et individuaalse nsP2 süntees sääse (vektorputuka) rakkudes surub maha alfaviiruse replikatsiooni aktiivsust. Seda nähtust ei põhjusta ainult nsP2 proteaasne aktiivsus – olulised on ka nsP2 proteaassest aktiivsusest sõltumatud mehhanismid. See, milline on konkreetsete mehhanismide osakaal, sõltub nii viiruse RNA replikaasi kui ka selle moodustamist inhibeeriva nsP2 valgu päritolust. Homoloogse viiruse replikatsiooni mahasurumisel domineerib nsP2 proteaassest aktiivsustest sõltuv mehhanism, kus nsP2 poolt teostatud replikaasi eelvalgu lõikamine 2/3 saidist välistab funktsionaalse RNA replikaasi moodustumise. Mõne alfaviiruse (SINV) puhul on nsP2 proteaassest aktiivsusest sõltuv mehhanism ka peamiseks mehhanismiks millega surutakse alla heteroloogsete alfaviiruste replikaaside moodustamist;
3. nii SINV kui ka CHIKV nsP2 valk takistab heteroloogsete alfaviiruste RNA replikatsiooni ka proteaassest aktiivsusest sõltumatute mehhanismide abil, kus CHIKV nsP2 puhul on need peamisteks meetoditeks heteroloogsete alfaviiruste replikatsiooni mahasurumisel. Selle inhibeerimise täpne põhjus on hetkel teadmata, küll aga näitasime, et erinevad mutatsioonid nsP2 valgus mõjutavad taolise inhibeerimise efektiivsust. Need andmed viitavad sellele, et proteaassest aktiivsusest sõltumatu replikatsiooni inhibeerimine saavutatakse tõenäoliselt mitmete paralleelsete mehhanismide abil;
4. väga patogeensele ja seetõttu vähe uuritud EEEV-le konstrueeritud trans-replikastiooni süsteem osutus kõrgelt efektiivseks nii inimese kui ka sääse rakkudes. Samuti osutus võimalikuks EEEV replikaasi eelvalku (P1234) kodeeriva järjestuse jagamine kaheks (P123 + nsP4) ja kolmeks (nsP1+P23+nsP4) komponendiks. See võimaldas uurida kas ja kuidas replikaasi komponentide vahekord mõjutab EEEV RNA replikaasi aktiivsust. Läbiviidud katsed näitasid, et erinevalt seni uuritud alfaviirustest sõltub EEEV RNA replikaasi aktiivsus teda moodustavate komponentide optimaalsest vahekorrast ja et RNA replikaasi katalüütilise allühiku (nsP4) ülehulk vähendab EEEV RNA replikaasi aktiivsust. Selline aktiivsuse langus tuleneb sellest, et väheneb nii rakkude hulk milles RNA replikatsioon aktiveeritakse, kuid ka RNA replikatsiooni aktiivsus nendes rakkudes. Hetkel pole veel selge kas tegemist on EEEV RNA replikaasi unikaalse omadusega või leidub ka teisi sarnaste omadustega alfaviiruste replikaase;
5. uuriti RRV loodusliku isolaadi RRV 2528 omadusi. Leiti, et see viiruse isolaat põhjustab väga tugevat tüüp-I interferoonide vastust. Viiruse genoomi analüüs tõi välja, et RRV 2528 erineb teistest vähemal määral interferoonide tootmist indutseerivatest RRV isolaatidest mitmete mutatsioonide poolest, millest paljud paiknevad just nsP2 valgus Näitasime, et need mutatsioonid mõjutavad RRV nakkuse käigus toimuvat interferoonide tootmist, kuid ei mõjuta viiruse replikaasi eelvalgu protsessimist valmis valkudeks, viiruse võimet suruda maha raku valgusünteesi ega viiruse struktuurvalkude sünteesi. Seega ei tulene suurenenud tüüp-I interferoonide vastus defektist RRV 2528 RNA replikatsioonikomplekside moodustamisel või võimetusest maha suruda raku üldist geeniekspressiooni.
Over the last decades, the intensive studies of factors/activities responsible for multiple aspects of alphavirus infection have been performed. In particular, our understanding about structures and functions of viral RNA replicase and its components has significantly increased. Novel findings emphasize that one of the first and most essential event in alphavirus infection is processing of the ns polyprotein carried out by its nsP2 region and an individual nsP2; this process not only ensures the release of functional replicase subunits but also determines would these proteins form the active RCs or not. Thus, it is increasingly evident that nsP2 is one of the “main driving forces” of successful RNA replication. Furthermore, due to its versatile functions and various activities nsP2 is involved in other aspects of infection. It is one of the key determinants associated with activation as well as counteracting of antiviral response in infected cells. Therefore, different modifications of the protein, including point mutations, often have drastic impact on alphavirus infection. However, much of the precise mechanisms of P2 action remain enigmatic. What is clear is that nsP2 does not act alone, its activities are modulated by other components of viral replicase. The current study allowed us to identify and confirm new functions and properties of nsP2 and alphavirus RNA replicase. The general conclusions of this study can be presented as follows: 1. Alphavirus trans-replicase systems can be applied as a tool for studies of functions of nsP2 associated with inhibition of RC formation/activity. These functions are essential for SIE and are likely related to these used to regulate RNA replication in the alphavirus infected cells. Using trans-replicase systems of CHIKV and SINV, it was found that the key event in SIE is targeting of replicase precursor (P1234) by an individual nsP2 protein and that this ability of nsP2 can be altered by mutations present in its functionally important regions. 2. It was found that synthesis of an individual (free) nsP2 in mosquito cells has an inhibitory effect on the alphavirus RC formation/functionality. This is not, however, a result of a single mechanism but results from combination of nsP2 protease-activity dependent mechanism and protease-activity independent mechanisms. The level and dominant mode of inhibition of alphavirus RNA replication depends form the virus, source of free nsP2 and substitutions present in this protein. The protease-activity mediated mechanism is important for suppression of replication of matching virus and relies mostly on ability of nsP2 to cleave 2/3 site in ns polyprotein. For some viruses such as SINV it is also dominant mechanism used to suppress formation of RNA replicases of heterologous alphaviruses. 3. nsP2 of SINV and CHIKV can inhibit formation/activity of RNA replicase of heterologous alphavirus using protease-activity independent mechanisms; for nsP2 of CHIKV this is the dominant mechanism to suppress activity of RNA replicases of heterologous alphaviruses. The precise details of protease-activity independent mode of action of nsP2 remain unknown; however, it is clear that this property can be enhanced by introduction of certain mutations into nsP2. It is likely that the protease-activity independent inhibitory effect originates not from a single mechanism but from several mechanisms. 4. Trans-replicase of highly pathogenic EEEV was found to be highly active in human and mosquito cells. Splitting of construct of EEEV P1234 expression into two (P123 and nsP4) or three (nsP1, P23 and nsP4) expression construct allowed analysis of requirements of active RC formation. It was found that activity of EEEV RNA replicase depends from correct ratio of P123 (or nsP1+P23) component to nsP4 component and that in contrast to previously studied alphaviruses an excess of nsP4 reduced activity of EEEV RNA replicase. The reduction was due to the decrease of a number of cells were RNA replication was initiated as well as to the reduced RNA replicase activity in such cells. It remains unclear, is this property unique for EEEV RNA replicase. 5. Natural isolate RRV 2528 was found to be a prominent inducer of type-I IFN expression. This property was associated with specific amino acid substitutions in the nsP2 encoded by this isolate. None of these substitutions or their combination affected ability of RRV to induce shutdown of cellular protein synthesis or level of viral structural proteins expression. Similarly, no effect on the processing of P1234 was detected. Combined, these findings indicate that the excessive type-I IFN induction was not due to the lack of the ability to induce general shutdown of cellular gene expression or due to the defects in RC formation.
Over the last decades, the intensive studies of factors/activities responsible for multiple aspects of alphavirus infection have been performed. In particular, our understanding about structures and functions of viral RNA replicase and its components has significantly increased. Novel findings emphasize that one of the first and most essential event in alphavirus infection is processing of the ns polyprotein carried out by its nsP2 region and an individual nsP2; this process not only ensures the release of functional replicase subunits but also determines would these proteins form the active RCs or not. Thus, it is increasingly evident that nsP2 is one of the “main driving forces” of successful RNA replication. Furthermore, due to its versatile functions and various activities nsP2 is involved in other aspects of infection. It is one of the key determinants associated with activation as well as counteracting of antiviral response in infected cells. Therefore, different modifications of the protein, including point mutations, often have drastic impact on alphavirus infection. However, much of the precise mechanisms of P2 action remain enigmatic. What is clear is that nsP2 does not act alone, its activities are modulated by other components of viral replicase. The current study allowed us to identify and confirm new functions and properties of nsP2 and alphavirus RNA replicase. The general conclusions of this study can be presented as follows: 1. Alphavirus trans-replicase systems can be applied as a tool for studies of functions of nsP2 associated with inhibition of RC formation/activity. These functions are essential for SIE and are likely related to these used to regulate RNA replication in the alphavirus infected cells. Using trans-replicase systems of CHIKV and SINV, it was found that the key event in SIE is targeting of replicase precursor (P1234) by an individual nsP2 protein and that this ability of nsP2 can be altered by mutations present in its functionally important regions. 2. It was found that synthesis of an individual (free) nsP2 in mosquito cells has an inhibitory effect on the alphavirus RC formation/functionality. This is not, however, a result of a single mechanism but results from combination of nsP2 protease-activity dependent mechanism and protease-activity independent mechanisms. The level and dominant mode of inhibition of alphavirus RNA replication depends form the virus, source of free nsP2 and substitutions present in this protein. The protease-activity mediated mechanism is important for suppression of replication of matching virus and relies mostly on ability of nsP2 to cleave 2/3 site in ns polyprotein. For some viruses such as SINV it is also dominant mechanism used to suppress formation of RNA replicases of heterologous alphaviruses. 3. nsP2 of SINV and CHIKV can inhibit formation/activity of RNA replicase of heterologous alphavirus using protease-activity independent mechanisms; for nsP2 of CHIKV this is the dominant mechanism to suppress activity of RNA replicases of heterologous alphaviruses. The precise details of protease-activity independent mode of action of nsP2 remain unknown; however, it is clear that this property can be enhanced by introduction of certain mutations into nsP2. It is likely that the protease-activity independent inhibitory effect originates not from a single mechanism but from several mechanisms. 4. Trans-replicase of highly pathogenic EEEV was found to be highly active in human and mosquito cells. Splitting of construct of EEEV P1234 expression into two (P123 and nsP4) or three (nsP1, P23 and nsP4) expression construct allowed analysis of requirements of active RC formation. It was found that activity of EEEV RNA replicase depends from correct ratio of P123 (or nsP1+P23) component to nsP4 component and that in contrast to previously studied alphaviruses an excess of nsP4 reduced activity of EEEV RNA replicase. The reduction was due to the decrease of a number of cells were RNA replication was initiated as well as to the reduced RNA replicase activity in such cells. It remains unclear, is this property unique for EEEV RNA replicase. 5. Natural isolate RRV 2528 was found to be a prominent inducer of type-I IFN expression. This property was associated with specific amino acid substitutions in the nsP2 encoded by this isolate. None of these substitutions or their combination affected ability of RRV to induce shutdown of cellular protein synthesis or level of viral structural proteins expression. Similarly, no effect on the processing of P1234 was detected. Combined, these findings indicate that the excessive type-I IFN induction was not due to the lack of the ability to induce general shutdown of cellular gene expression or due to the defects in RC formation.
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Keywords
Alphavirus, RNA viruses, viral proteins, replication