Oligomerization of the influenza virus nucleoprotein: Identification of positive and negative sequence elements

被引:63
作者
Elton, D [1 ]
Medcalf, E [1 ]
Bishop, K [1 ]
Digard, P [1 ]
机构
[1] Univ Cambridge, Dept Pathol, Div Virol, Cambridge CB2 1QP, England
基金
英国惠康基金;
关键词
D O I
10.1006/viro.1999.9818
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
The RNA genome of influenza virus is encapsidated by the virus nucleoprotein (NP) to form ribonucleoprotein (RNP) structures of defined morphology. These structures result from the ability of NP to oligomerise and to bind single-strand RNA. To characterise NP oligomerization, we developed a binding assay using immobilised NP fusion proteins and in vitro translated NP. This system was used to estimate a dissociation constant for NP-NP contacts of 2 x 10 M-7. Analysis of NP deletion mutants identified three sequence elements important for oligomerization. Two regions corresponding to the middle and C-terminal thirds of the polypeptide were identified as the minimal sequences capable of promoting NP-NP contacts. However, the C-terminal 23 amino-acids of NP inhibited oligomerization, as their removal increased self-association 10-fold. Single codon changes identified amino acids important for the function of these regions. Alanine substitution of R199 decreased binding affinity threefold, whereas alteration of R416 had a more drastic effect, reducing binding 10-fold. In contrast, mutation of F479 increased self-association fivefold. Mutations altering NP oligomerization affected the ability of the polypeptides to support influenza virus gene expression in an in vivo assay. Decreased oligomerization activity correlated with decreased transcriptional function. However, mutations that increased self-association also decreased transcription competence. This indicates that NP contains both positive and negative sequence elements involved in oligomerization and is consistent with the importance of NP-NP contacts for the formation of a transcriptionally active RNP. (C) 1999 Academic Press.
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页码:190 / 200
页数:11
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