Structure-guided insights into the functions of CHIKV nsP2
Date
2024-01-10
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Abstract
Alfaviirused on positiivse polaarsusega RNA genoomi ja ümbrisega viirused mis on vastutavad paljude inimeste ja loomade haiguste eest. Üks meditsiiniliselt olulisemaid alfaviiruseid on chikungunya viirus (CHIKV). Selle viiruse põhjustav chikungunya palavik on äge haigus millega tavaliselt kaasneb lööve ja liigesevalud, ja millele sageli järgneb kuid või isegi aastaid kestev krooniline faas. Viimase kahekümne aasta jooksul on CHIKV põhjustanud miljoneid nakkusjuhtumeid enam kui 50-s riigis ja temast on saanud ülemaailmse tähtsusega patogeen. Litsentseeritud ravimid CHIKV põhjustatud haiguse raviks puuduvad.
Alfaviiruse genoomse RNA replikatsioon sõltub RNA sünteesi ja modifikatsiooni protsessist mida viivad läbi viiruse poolt kodeeritud mitte-struktuurvalgud (nsP-d). nsP2 valku peetakse RNA replikatsiooni võtmevalguks ja peamiseks regulaatoriks. Siiski olid nsP2 valgu ensümaatiliste aktiivsuste ja muude funktsioonide omavahelised seosed ebapiisavalt uuritud. Käesolev uuring selgitas välja nsP2 valgus struktuuri mis võimaldab paremini mõista selle valgu funktsioone alfaviiruse replikatsioonis. Samuti näitasime, et nsP2-d saab kohandada täiendavate viiruse replikatsiooni jaoks oluliste funktsioonide täitmiseks. Selle uurimistöö peamised järelduse on järgnevad:
1: Tuvastasime kriitilised aminohapped jäägid, mis vahendavad „stacking“ interaktsioone nsP2 valgu ja CHIKV genoomi 3' otsa vahel. Nende interaktsioonide takistamine muudab viiruse paljunemisvõimetuks.
2: Paindlik domeenide vaheline regioon, mis ühendab nsP2 valgu N-otsa poolset helikaasi ja C-otsa poolset proteaasi piirkondi, on CHIKV replikatsiooni jaoks samuti hädavajalik. Ainult väikesed muudatused selles piirkonnas, nagu ühe aminohappejäägi eemaldamine ja kuni 10 aminohappejäägi lisamine, ei mõjuta oluliselt viiruse replikatsiooni. Suuremad muudatused, nagu kolme või viie aminohappejäägi eemaldamine, inaktiveerivad viiruse.
3: G3BP/Rin seondumise motiivide sisestamine nsP2 paindlikesse (domeenide vahelistesse) piirkondadesse oli mitte ainult viiruse poolt talutav vaid ka funktsionaalselt efektiivne - sisestatud järjestus suutis toetada mutantse CHIKV replikatsiooni nii imetajate kui ka putukarakkudes. Samad motiivid taastasid viiruse, mille nsP3 valgus puudusid G3BP seondumise motiivid, võime paljuneda imetaja rakkudes.
Alphaviruses are enveloped viruses with positive-strand RNA genomes that are responsible for many human and animal diseases. One of the most medically important alphaviruses is chikungunya virus (CHIKV), which can cause chikungunya fever, an acute febrile disease typically accompanied by rash and debilitating and chronic arthralgia that can last for months or even years. Since its first massive outbreak at 2004, CHIKV has caused millions of cases in over 50 countries and has become a re-emerging pathogen of global importance. However, there are no licensed drugs to treat the disease caused by CHIKV infection. Alphavirus genomic RNA replication depends on the process of RNA synthesis and modification carried out by non-structural proteins (nsPs) encoded by viral RNA. nsP2 is considered as the main driving force and master-regulator of RNA replication. However, the coordination of multiple enzymatic activities and other functions of nsP2 was poorly understood. The current study provided structural basis for better understanding of the roles of nsP2 in the alphavirus replication process; it also indicated that nsP2 can be engineered to perform additional functions required for virus replication. The main conclusions of this study could be illustrated as follows: 1: We identified the crucial residues that formed the stacking interactions between nsP2 and the RNA molecule corresponding to conserved 3’ end of CHIKV genome. Disruption of these interactions was found to have detrimental effect on viral RNA replication. 2: The flexible interdomain linker, connecting the N-terminal helicase and C-terminal protease regions of nsP2, was found to be essential for CHIKV replication as well; only small changes such as the deletion of one amino acid residue and the insertion of up to 10 amino acids residues were tolerated without significant impact on viral replication. However, the deletion of three or five residues of linker was lethal for CHIKV. 3: The insertion of G3BP/Rin binding motifs into the flexible regions of nsP2 (in the linker region or between different domains of nsP2 protease) was not only tolerated but the inserted sequence was able to support replication of mutant CHIKV in both mammalian and insect cells. The same insertion could also restore the infectivity of viruses lacking G3BP binding motifs in nsP3 in mammalian cells.
Alphaviruses are enveloped viruses with positive-strand RNA genomes that are responsible for many human and animal diseases. One of the most medically important alphaviruses is chikungunya virus (CHIKV), which can cause chikungunya fever, an acute febrile disease typically accompanied by rash and debilitating and chronic arthralgia that can last for months or even years. Since its first massive outbreak at 2004, CHIKV has caused millions of cases in over 50 countries and has become a re-emerging pathogen of global importance. However, there are no licensed drugs to treat the disease caused by CHIKV infection. Alphavirus genomic RNA replication depends on the process of RNA synthesis and modification carried out by non-structural proteins (nsPs) encoded by viral RNA. nsP2 is considered as the main driving force and master-regulator of RNA replication. However, the coordination of multiple enzymatic activities and other functions of nsP2 was poorly understood. The current study provided structural basis for better understanding of the roles of nsP2 in the alphavirus replication process; it also indicated that nsP2 can be engineered to perform additional functions required for virus replication. The main conclusions of this study could be illustrated as follows: 1: We identified the crucial residues that formed the stacking interactions between nsP2 and the RNA molecule corresponding to conserved 3’ end of CHIKV genome. Disruption of these interactions was found to have detrimental effect on viral RNA replication. 2: The flexible interdomain linker, connecting the N-terminal helicase and C-terminal protease regions of nsP2, was found to be essential for CHIKV replication as well; only small changes such as the deletion of one amino acid residue and the insertion of up to 10 amino acids residues were tolerated without significant impact on viral replication. However, the deletion of three or five residues of linker was lethal for CHIKV. 3: The insertion of G3BP/Rin binding motifs into the flexible regions of nsP2 (in the linker region or between different domains of nsP2 protease) was not only tolerated but the inserted sequence was able to support replication of mutant CHIKV in both mammalian and insect cells. The same insertion could also restore the infectivity of viruses lacking G3BP binding motifs in nsP3 in mammalian cells.
Description
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Keywords
Chikungunya viirus, alfaviirused, mittestruktuursed valgud, replikatsioon, Chikungunya virus, Alphavirus, nonstructural proteins, replication