Studies on the Semliki Forest virus replicase protein nsP1
Date
2013-09-13
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Abstract
Semliki Forest viirus kuulub alfaviiruste perekonda sugukonnas Togaviridae. Alfaviirused on olulised patogeenid ning põhjustavad erinevaid vaevusi nii loomadele kui ka inimestele. Levik looduses toimub sääskede vahendusel. Lisaks tähtsusele patogeensuse tõttu on alfaviirused kasutusel ka bio- ning geenitehnoloogias. Alfaviirustel on positiivse polaarsusega RNA genoom, millelt transleeritakse viiruse replikaasi subühikud (nsP1-nsP4). Antud töö põhiline uurimisobjekt nsP1 vastutab lisaks osalemisele replikatsioonil ja transkriptsioonil replikaasi sidumise eest peremeesraku membraanidele.
Antud töö tulemusena tuvastati, et nsP1 palmitüleerimine membraaniga seondumise tugevdamiseks ei ole viirusele hädavajalik. Palmitüleerimine hõlbustab kontaktide loomist nsP1 ja viiruse polümeraasi (nsP4) vahel imetaja rakkudes. Palmitüleerimise puudumisel omandab viirus sekundaarsed mutatsioonid, mis taastavad vastavate kontaktide moodustamise võimet imetajarakkudes. Samad mutatsioonid viiruse elulemust putuka rakkudes olulisel määral ei mõjuta.
Leiti, et mittestruktuurse polüproteiini lõikamine nsP1 ja nsP2 vahelt on üks funktsionaalse replikaaskompleksi moodustumise kontrollpunktidest. 1/2 saidi lõikamise kiirendamine vähendab olulisel määral viiruse infektsioonilisust; lõikamise aeglustamisel on väiksem mõju. Mutatsioonanalüüs näitas, et 1/2 saidi lõikust mõjutab eelkõige üks nsP1 valgus lõpus asuv aminohappejääk (P5). Täiendav mutatsioon nsP2 kodeerivas piirkonnas (Q706R) taastas mutantse lõikesaidiga viiruse elujõulisuse ja liitvalgu korrektse lõikamise imetaja rakkudes, ent selline viirus ei suutnud edukalt paljuneda putuka rakkudes ning terviklikku interferoonvastust omavates imetaja rakkudes. Nendest tulemustes lähtuvalt pakuti välja hüpotees, mille kohaselt mutatsioonid 1/2 lõikesaidis ja nsP2 valgus põhjustavad viiruse P123 liitvalgu eluea pikenemise, mis takistab viiruse võimet blokeerida natiivse immuunvastuse teket. Uuritud mutatsioonide bioloogiliste efektide põhjalikumaks uurimiseks on tarvis läbi viia loomkatseid.
Semliki Forest virus is a member of the family Togaviridae genus Alpavirus. Alphaviruses are pathogens that cause illness in animals and humans. In nature mosquitos transmit the pathogen. In addition to the importance as pathogens alphaviruses are widely used as biotechnological tools. Alphaviruses have a RNA genome with positive polarity and the replicase subunits (nsP1-nsP4) are translated directly from the genome. nsP1, the protein of investigation in the current thesis, is involved in regulating the formation of a replicase complex and attaching the complex to host cell membranes. In the current thesis it was detected that the palmitoylation of nsP1 as a mean for strengthening the interaction with membranes is not indispensable for the virus. In mammalian cells palmitoylation enables direct contacts to be formed between nsP1 and the viral polymerase. If nsP1 is not palmitoylated, the virus acquires second-site mutations that rescue the ability to form contacts between these proteins. The viability of the virus in insect cells is not much affected by same compensatory mutations. The analysis of the processing requirements between nsP1 and nsP2 in the SFV ns-polyprotein highlighted its importance as one of the major regulatory points in the assembly of a functional replication complex. The accelerated processing of this site severely diminished the infectivity of the corresponding mutant genome, while a reduction in the cleavage efficiency had only a minor effect. The processing of the 1/2 site depends mostly on the amino acid residue in the terminal region of nsP1 (P5). An additional mutation (Q706R) in the nsP2 coding region rescued the viability of the virus with the mutant cleavage site and the correct polyprotein processing. The resulting viruses failed to replicate as efficiently as in mammalian cells in both insect cells and mammalian cells with an intact IFN system. It was suggested that the prolonged stabilty of polyprotein P123 that results from the mutation in the P5 position in the 1/2 cleavage site and the Q706R mutation in the nsP2 coding region contributes to the enhanced native immune response in these cells. Detailed studies using animal models are required to provide further information about the biological implications of these findings.
Semliki Forest virus is a member of the family Togaviridae genus Alpavirus. Alphaviruses are pathogens that cause illness in animals and humans. In nature mosquitos transmit the pathogen. In addition to the importance as pathogens alphaviruses are widely used as biotechnological tools. Alphaviruses have a RNA genome with positive polarity and the replicase subunits (nsP1-nsP4) are translated directly from the genome. nsP1, the protein of investigation in the current thesis, is involved in regulating the formation of a replicase complex and attaching the complex to host cell membranes. In the current thesis it was detected that the palmitoylation of nsP1 as a mean for strengthening the interaction with membranes is not indispensable for the virus. In mammalian cells palmitoylation enables direct contacts to be formed between nsP1 and the viral polymerase. If nsP1 is not palmitoylated, the virus acquires second-site mutations that rescue the ability to form contacts between these proteins. The viability of the virus in insect cells is not much affected by same compensatory mutations. The analysis of the processing requirements between nsP1 and nsP2 in the SFV ns-polyprotein highlighted its importance as one of the major regulatory points in the assembly of a functional replication complex. The accelerated processing of this site severely diminished the infectivity of the corresponding mutant genome, while a reduction in the cleavage efficiency had only a minor effect. The processing of the 1/2 site depends mostly on the amino acid residue in the terminal region of nsP1 (P5). An additional mutation (Q706R) in the nsP2 coding region rescued the viability of the virus with the mutant cleavage site and the correct polyprotein processing. The resulting viruses failed to replicate as efficiently as in mammalian cells in both insect cells and mammalian cells with an intact IFN system. It was suggested that the prolonged stabilty of polyprotein P123 that results from the mutation in the P5 position in the 1/2 cleavage site and the Q706R mutation in the nsP2 coding region contributes to the enhanced native immune response in these cells. Detailed studies using animal models are required to provide further information about the biological implications of these findings.
Description
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Keywords
semliki metsaviirus, replikatsioon, transkriptsioon (biol.), mittestruktuursed valgud, mutatsioonid, semliki forest virus, replication, transcription (biol.), nontstructural proteins, mutations