Publication: İnfluenza Virüs Patogenezinde Viral RNA Polimeraz Enzimi ve Konak Hücre Proteinlerinin Rolünün Araştırılması
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Date
2016-15-03
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Abstract
Bu proje çalışmasında influenza A virüsü RNA polimeraz enzimi ile ilişkili, viralreplikasyon üzerinde etkili hücesel protein faktörler belirlendi ve bu proteinlerin insan(A/WSN-H1N1) ve kuş (avian) tipi influenza A virüsü (A/Duck/Pen-H5N2) replikasyonunaetkileri moleküler düzeyde araştırıldı. Viral RNA polimeraz ile ilişkili konak proteinleri, mayaikili-hibrit metodu ile belirlendi. Bait (yem) olarak viral RNA polimeraz alt ünitelerinden PAproteini kullanıldı. HeLa ve HEK293 cDNA kitaplıklarının kullanıldığı maya ikili-hibrittestlerinde PA proteini ile ilişkili, 18 konak proteini saptandı. Bu proteinlerden EIF3L, GNB,MRPL27 ve MCM7’nin viral RNA polimeraz aktivitesini pozitif yönde etkilediği, buna karşın,MTC, FAS ve HAX1’in negatif yönde etki gösterdiği saptandı. Diğer konak proteinlerininistatistiksel olarak anlamlı bir etkileri görülmedi. Viral replikasyon açısından daha önemliolabileceği düşünülen EIF3L, GNB, MTC, ARF4 ve SNX proteinleri, siRNA teknolojilerindende yararlanılarak daha ayrıntılı çalışıldı. Bu proteinleri kodlayan genler için knock downedilen hücrelerde, GNB proteini düzeyindeki azalmanın, influenza A/Duck/Pen virüsününhem replikasyonunu hem de transkripsiyonunu aynı ölçüde inhibe ettiği görüldü. Bu proteininfluenza A/WSN virüsü replikasyonu üzerinde tam tersi etki gösterdi. ARF4, EIF3L, MTC veSNX proteinleri influenza A/Duck/Pen üzerinde virüs replikasyonunu negatif yönde etkiledi.Buna karşın hücrelerde ARF4, EIF3L ve MTC proteinlerindeki azalma, influenza A/WSNvRNA sentezini istatistiksel olarak anlamlı düzeyde arttırdı. Birkilte immün-çöktürme ve GSTile çöktürme deneyleri influenza virüsü PA proteini ile EIF3L, GNB ve SNX proteinleriarasında direkt bir ilişki olduğunu gösterdi.Elde edilen veriler influenza virüs patogenezinde viral RNA polimeraz enziminin PAalt ünitesinin ve ilk kez bu çalışmada virüsü ile ilişkili olduğu saptanan konak proteinlerininbüyük bir önem taşıdığını ortaya koydu. Farklı influenza A virüsü tiplerinin insanları dakapsayacak şekilde yeni konak organizmalara adaptasyonunda, virüsün hücreleretutunmasında etkili olan HA proteini ve hücre reseptörleri kadar, hücre içerisinde PA proteiniile konak proteinleri arasındaki ilişkinin de çok önemli olduğu sonucuna varıldı.
In this Project, the host proteins associated with influenza A virus RNA polymerase wereidentified and the effect of these proteins on replication of human (A/WSN-H1N1) and aviantype (A/Duck/Pan-H5N2) influenza viruses was investigated at the molecular level. The hostproteins having relationship with viral RNA polymerase were defined with yeast two-hybridassay. PA protein subunit of RNA polymerase, was used as a bait. 18 host proteins wereidentified with yeast two-hybrid screening in cDNA libraries of HeLa and HEK293. EIF3L,GNB, MRPL27 and MCM7 upregulated the influenza RNA polymerase activity, whereas MTCFAS and HAX1 downregulated. Other candidate proteins did not show statistically significanteffects on RNA polymerase. EIF3L, GNB, MTC, ARF4 and SNX proteins, considered to bemore important in terms of virus replication, were studied in more detail. In knock down cellsfor genes coding candidate proteins, the decrease of GNB protein inhibited both transcriptionand replication of influenza A/Duck/Pen. In contrast, this protein showed the stimulatoryeffect on replicaition of influenza A/WSN. ARF4, EIF3L MTC and SNX showed inhibitoryeffects on influenza A/Duck/Pen replication. In contrast, reduction of ARF4, EIF3L and MTCprotein levels in the cells, significantly accelerated vRNA synthesis of influenza A/WSN. Theco-immunoprecipitation and GST pull down assays demonstrated direct interactions betweeninfluenza PA protein and the host proteins EIF3L, GNB and SNX.The results obtained in this project revealed that influenza RNA polymerase subunitPA and host proteins, which showed to be associated with PA protein at the first time, havegreat significance for influenza pathogenicity. It was concluted that relationship between thehost proteins and PA have a greate importance as well as HA protein, major viral surfaceantigen and the structure of cell receptors for adaptation of influenza viruses to the new hostorganisms including human.
In this Project, the host proteins associated with influenza A virus RNA polymerase wereidentified and the effect of these proteins on replication of human (A/WSN-H1N1) and aviantype (A/Duck/Pan-H5N2) influenza viruses was investigated at the molecular level. The hostproteins having relationship with viral RNA polymerase were defined with yeast two-hybridassay. PA protein subunit of RNA polymerase, was used as a bait. 18 host proteins wereidentified with yeast two-hybrid screening in cDNA libraries of HeLa and HEK293. EIF3L,GNB, MRPL27 and MCM7 upregulated the influenza RNA polymerase activity, whereas MTCFAS and HAX1 downregulated. Other candidate proteins did not show statistically significanteffects on RNA polymerase. EIF3L, GNB, MTC, ARF4 and SNX proteins, considered to bemore important in terms of virus replication, were studied in more detail. In knock down cellsfor genes coding candidate proteins, the decrease of GNB protein inhibited both transcriptionand replication of influenza A/Duck/Pen. In contrast, this protein showed the stimulatoryeffect on replicaition of influenza A/WSN. ARF4, EIF3L MTC and SNX showed inhibitoryeffects on influenza A/Duck/Pen replication. In contrast, reduction of ARF4, EIF3L and MTCprotein levels in the cells, significantly accelerated vRNA synthesis of influenza A/WSN. Theco-immunoprecipitation and GST pull down assays demonstrated direct interactions betweeninfluenza PA protein and the host proteins EIF3L, GNB and SNX.The results obtained in this project revealed that influenza RNA polymerase subunitPA and host proteins, which showed to be associated with PA protein at the first time, havegreat significance for influenza pathogenicity. It was concluted that relationship between thehost proteins and PA have a greate importance as well as HA protein, major viral surfaceantigen and the structure of cell receptors for adaptation of influenza viruses to the new hostorganisms including human.
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Keywords
Biyoloji, Farmakoloji ve Eczacılık, Hücre Biyolojisi, Mikrobiyoloji, Viroloji