Functional analysis of viral and host components of alphavirus replicase complexes
Date
2013-10-21
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Abstract
Semliki Forest viirus (SFV) kuulub alfaviiruste perekonda, sugukond Togaviridae. Alfaviirused on olulised patogeenid ning võivad põhjustada erinevaid vaevusi nii loomadele kui ka inimestele. Looduses levivad nad valdavalt sääskede vahendusel. Lisaks oma tähtsusele patogeensuse tõttu on alfaviirused kasutusel ka bio- ning geenitehnoloogias. Alfaviirustel on positiivse polaarsusega RNA genoom, millelt transleeritakse nakatunud rakkudes polüproteiinina viiruse mittestruktuursed valgud (nsP1-nsP4), mis protsessitakse iseseisvateks valkudeks ja mis on vajalikud viiruse replikatsioonikomplekside moodustamiseks.
Antud töö tulemusena leiti, et üks viiruse mittestruktuurne valk nsP3 omab oma karboksüterminaalses otsas degradatsioonisignaali, mis kattub piirkonnaga, mis on vajalik äratundmisjärjestus viiruse mitte-struktuurse polüproteiini protsessingus, et vabastada nsP3 küljest nsP4. Lisaks leiti, et eespool degradatsioonisignaali on alfaviirustel konserveerunud kahes koopias motiiv. Edasine analüüs näitas, et vähemalt üks motiividest on vajalik, et tagada edukas nsP3-poolne G3BP1 ja G3BP2 sidumine. Antud valgud on rakus stressi graanulite moodustamiseks vajalikud komponendid, stressi graanulid on rakulisi RNAsid talletavad struktuurid ja tekivad siis, kui tingimused on rakkudele ebasoodsad. Alphaviirustega nakatunud rakud kaotavad võime moodustada stressi graanuleid ning üks mehhanismidest, kuidas see johtub, ongi nsP3 poolne G3BP1 ja G3BP värbamine viiruse replikatsioonikompleksidele.
Kuigi teame, et G3BP1 ja G3BP2 seonduvad SFV replikatsioonikompleksile, on vähe teadmisi teistest raku valkudest, mis seda samuti teevad. SFV replikatsioonikompleksid on seondunud lüsosoomidele. Söötes rakkudesse dekstraaniga kaetud magneetilised nanopartiklid jõuavad partiklid lüsosoomidesse, misjärel on võimalik korjata magneetilise sortimise läbi kokku nood rakulised vesiikulid. Kasutades kvantitatiivse proteoomika võimalusi, õnnestus tuvasta 80 valku, mis olid üleesindatud fraktsioonis, mis pärit nakatunud rakkudest. Neli valku (PCBP1, hnRNP M, hnRNP C, hnRNP K) valiti, et kinnitada metoodikat töötamist. Kõik neli kolokaliseerusid SFV replikatsioonikompleksiga. Kasutades siRNA vahendatud ekspressiooni allasurumist, ilmnes, et hnRNP M ja hnRNP C allasurumine võimendas viiruse paljunemist, kuid PCBP1 allasurumine vähendas viirusvalkude translatsiooni ning hnRNP K allasurumine suurendas viiruse RNA sünteesi.Interaktsioonipartnerite kindlaks tegemine võimaldab asuda järgmises etapis asuda leidma mooduseid, kuidas ravida alphaviiruste põhjustatud haigusi.
Semliki Forest virus (SFV) belongs to genus Alphavirus, family Togaviridae. There are many important pathogens among alphaviruses and their infection can cause various illnesses to animals and humans. In nature mosquitoes are responsible for the spread of alphaviruses. In addition, alphaviruses are used as tools in biotechnology. Alphaviruses have a genome with positive polarity that is used in infected cells to translate nonstructural polyprotein that will be processed into four subunits (nsP1-nsP4). Nonstructural proteins are needed to form replication complexes. In the present work it was discovered that one nonstructural protein nsP3 has degradation signal at its carboxyterminal end. However, it overlaps with the sequence element that acts as a signal element in the polyprotein processing resulting in the release of nsP3 from nsP4. In addition, it was found that upstream of the degradation signal nsP3 has a sequence motif in two copies. Further analysis showed that at least one of the motifs is required to bind G3BP1 and G3BP2. Those proteins participate in the formation of stress granules; stress granules store cellular RNAs if the conditions are not favourable. However, cells infected with alphaviruses loose that ability, one of the mechanisms behind the phenomenon is recruitment of G3BP1 and G3BP2 to virus replications complexes. Although we know that G3BP1 and G3BP2 bind SFV replication complexes there is little knowledge about the other cellular proteins that could do the same. SFV replication complex are bound to lysosomes. If the cells are fed with dextran-covered magnetic nanoparticles those particles end up in lysosomes allowing magnetic sorting of those cellular vesicles. By using quantitative proteomics, it was found that 80 proteins were enriched in the lysosomal fraction from infected cells. Four of them (PCBP1, hnRNP M, hnRNP C, hnRNP K) were chosen for further analysis. All four colocalized with SFV replication complexes. By using siRNA-based knockdown, it was found that knockdown of hnRNP M and hnRNP C enhanced virus replication, knockdown of PCBP1 reduced viral RNA translation and trageting of hnRNP K increased viral synhtesis.Understanding of alphavirus interactions with host is required to develop new ways to treat the disease.
Semliki Forest virus (SFV) belongs to genus Alphavirus, family Togaviridae. There are many important pathogens among alphaviruses and their infection can cause various illnesses to animals and humans. In nature mosquitoes are responsible for the spread of alphaviruses. In addition, alphaviruses are used as tools in biotechnology. Alphaviruses have a genome with positive polarity that is used in infected cells to translate nonstructural polyprotein that will be processed into four subunits (nsP1-nsP4). Nonstructural proteins are needed to form replication complexes. In the present work it was discovered that one nonstructural protein nsP3 has degradation signal at its carboxyterminal end. However, it overlaps with the sequence element that acts as a signal element in the polyprotein processing resulting in the release of nsP3 from nsP4. In addition, it was found that upstream of the degradation signal nsP3 has a sequence motif in two copies. Further analysis showed that at least one of the motifs is required to bind G3BP1 and G3BP2. Those proteins participate in the formation of stress granules; stress granules store cellular RNAs if the conditions are not favourable. However, cells infected with alphaviruses loose that ability, one of the mechanisms behind the phenomenon is recruitment of G3BP1 and G3BP2 to virus replications complexes. Although we know that G3BP1 and G3BP2 bind SFV replication complexes there is little knowledge about the other cellular proteins that could do the same. SFV replication complex are bound to lysosomes. If the cells are fed with dextran-covered magnetic nanoparticles those particles end up in lysosomes allowing magnetic sorting of those cellular vesicles. By using quantitative proteomics, it was found that 80 proteins were enriched in the lysosomal fraction from infected cells. Four of them (PCBP1, hnRNP M, hnRNP C, hnRNP K) were chosen for further analysis. All four colocalized with SFV replication complexes. By using siRNA-based knockdown, it was found that knockdown of hnRNP M and hnRNP C enhanced virus replication, knockdown of PCBP1 reduced viral RNA translation and trageting of hnRNP K increased viral synhtesis.Understanding of alphavirus interactions with host is required to develop new ways to treat the disease.
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Keywords
semliki metsaviirus, replikatsioon, mittestruktuursed valgud, geenitehnoloogia, semliki forest virus, replication, nonstructural proteins, gene technology