Alphaviral nsP2 protease: from requirements for functionality to inhibition
Date
2023-05-09
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
Abstract
Alfaviirused 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.
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.
Description
Väitekirja elektrooniline versioon ei sisalda publikatsioone
Keywords
Alphavirus, Chikungunya virus, RNA viruses, viral proteins, replication