Functional analysis of the orthobunyavirus nucleocapsid (N) protein

Bunyaviruses;Viral proteins;Nucleoproteins

Eifan, Saleh A. ; Elliott, Richard Michael ; Elliott, Richard Michael

University:University of St Andrews

Department:Biology (School of)

关键词: Bunyaviruses;    Viral proteins;    Nucleoproteins;   

Others  :  https://research-repository.st-andrews.ac.uk/bitstream/handle/10023/542/Saleh%20A%20Eifan%20PhD%20thesis.pdf?sequence=6&isAllowed=y

来源: DR-NTU

【 摘 要 】

Bunyamwera virus (BUNV) is the prototype of the family Bunyaviridae. It has atripartite genome consisting of negative sense RNA segments called large (L),medium (M) and small (S). The S segment encodes the nucleocapsid protein (N) of233 amino acids. The N protein encapsidates all three segments to formtranscriptionally active ribonucleoproteins (RNPs). The aim of this project was todetermine the domain map of BUNV N protein.To investigate residues in BUNV N crucial for its functionality, random and site-specific mutagenesis were performed on a cDNA clone encoding the BUNV Nprotein. In total, 102 single amino acid substitutions were generated in the BUNV Nprotein sequence.All mutant N proteins were used in a BUNV minigenome system to compare theiractivity to wt BUNV N. The mutant proteins displayed a wide-range of activity, fromparental-like to essentially inactive. The most disruptive mutations were R94A,I118N, W134A, Y141C, L177A, K179I and W193A.Sixty-four clones carrying single substitutions in the BUNV N protein were used in theBUNV rescue system in an attempt to recover viable mutant viruses. Fiftyrecombinant mutant viruses were rescued and 14 N genes were nonrescuable.The 50 mutant viruses were characterized by: titration, protein labelling, westernblotting, temperature sensitivity and host-restriction. Mutant viruses displayed a wide range of titers between 10³-10⁸ pfu/ml, and three different plaque sizes large,medium and small. Protein labelling and western blotting showed that mutations inthe N gene did not affect expression of the other viral genes as much as affecting Nprotein expression. It was demonstrated that single amino acid substitutions couldalter N protein electrophoretic mobility in SDS- PAGE (e.g. P19Q and L53F).Temperature sensitivity tests showed that recombinant viruses N74S, S96S, K228Tand G230R were ts, growing at 33˚C but not at 37˚C or 38˚C, while the parental virus grew at all temperatures. Using the northern blotting technique, mutant viruses N74Sand S96G were shown to have a ts defect in genome-synthesis (late replicationstep), while mutant viruses K228T and G230R had a ts defect in antigenome-synthesis (early replication step).Host-restriction experiments were performed using 5 different cell lines (Vero-E6,BHK-21, 2FTGH-V, A549-V and 293-V). Overall, the parental virus grew similarly inall cell lines. Likewise, the majority of mutant viruses follow this pattern except mutantvirus Y23A. It showed a 100-fold reduction in titer in 2FTGH-V cells. Comparing theratios of intracellular and extracellular particles revealed that only 15% of the totalvirus particles of mutant Y23A was released as extracellular particles compared to30% of the parental virus.Fourteen N genes were nonrescuable. They were characterized by (i) their activity inthe BUNV minigenome system, (ii) their activity in BUNV packaging assay, (iii) theirability to form multimers, (iv) their ability to interact with L protein, and (v) their impacton RNA synthesis.In summary, BUNV N protein was shown to be multi-functional and involved in theregulation of virus transcription and replication, RNA synthesis and assembly, viainteractions with the viral L polymerase, RNA backbone, itself or the viralglycoproteins.