Structural and functional interactions between measles virus nucleocapsid protein and cellular heat shock protein
Cells respond to stress by producing heat shock proteins (HSPs), particularly the major inducible 72kDa protein (Hsp72). Although HSPs are generally viewed as protecting cells against injury, data has also shown that elevated cellular Hsp72 promotes damage induced by measles virus (MV) in vitro. Specifically, Hsp72 enhances MV transcription and replication activity, resulting in increased viral antigen expression, cytopathic effect, and infectious viral progeny release. Hsp72-dependent stimulation of MV transcription reflects functional interactions between Hsp72 and an 8 amino acid (aa) binding motif within the extreme C-terminus of the nucleocapsid protein (N), a major structural component of the nucleocapsid. Nucleocapsid consists of viral genomic RNA packaged by N, polymerase cofactor (P), and the viral encoded RNA-dependent RNA polymerase (L). The nucleocapsid activity can be measured using minireplicons in which the viral genomic coding sequence is replaced by chloramphenical acetyltransferase (CAT) RNA. Using this approach, we identified the C-terminal 24 amino acids of N protein as a negative regulatory domain of MV transcription. Addition of Hsp72 by transfection significantly stimulates viral transcription/replication activity mediated by N. Mutations in the Hsp72 binding motif based upon naturally occurring sequence polymorphisms can diminish Hsp72 stimulation of MV minireplicon reporter gene expression. Loss of functional interaction correlates to the loss of low affinity binding interaction. Binding interactions were monitored in real time using surface plasmon resonance technology (BIAcore). These same mutations, when incorporated into the N protein of recombinant infectious virus, also abrogate Hsp72-dependent infection phenotypes. Incorporation of a non-functional Hsp72 binding motif reduced the stimulatory effect of pre-conditioning upon viral transcription, cytopathic effect (CPE), and cell-free infectious viral progeny release. However, loss of a functional binding motif doesn’t abrogate the stimulatory effect of pre-conditioning on viral genome levels. N protein lacking the Hsp72 binding motif maintains the ability to form a stable complex with Hsp72 by both nucleocapsid-Hsp72 particle isolation as well as N-Hsp72 co-immunoprecipitation. BIAcore analysis of the last 125 aa of N protein (Ntail) and Hsp72 interaction confirmed the existence of additional Hsp72 binding domains. The relevance of high affinity interactions to Hsp72-dependent stimulation of MV genome replication remains to be established.
School:The Ohio State University
School Location:USA - Ohio
Source Type:Master's Thesis
Keywords:hsp72 morbillivirus nucleocapsid protein binding motif reverse genetics biacore
Date of Publication:01/01/2004