Structure-based mutational analysis of the yellow fever virus non-structural protein 3 helicase

Karla Ann Combs, Purdue University

Abstract

Flaviviruses, small, enveloped positive sense RNA viruses, cause significant human disease in over 100 countries worldwide. Yellow fever virus (YFV), the prototype member of the flavivirus genus, causes symptoms ranging from general malaise and fever to hemorrhaging leading to death. The RNA genome codes for ten proteins: three structural (C, prM and E) that compose the virus particle and seven non-structural (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) necessary for viral genome replication. Yellow fever virus NS3, a 623 amino acid protein, has an N-terminal protease domain followed by a helicase domain that resides in the C-terminal 440 amino acids. However, the role of the helicase within the viral life cycle is currently unknown but it is presumed to unwind a duplex of RNA critical to genome replication. Preliminary results are presented here on residues that lie within the RNA binding groove that may have an effect on helicase activity in vivo. Amino acids within the helicase have not only been implicated in replication, but more recently have been suggested to be involved in the process of producing infectious virus particles. Here the role of helix 10, a thirteen amino acid amphipathic helix (608-620) residing in the third crystallographic domain of the yellow fever virus NS3 helicase, was investigated in replication and virus production. A chimera was constructed in which the amino acids of yellow fever helix 10 were replaced with the amino acids of dengue 2 (DEN2). Using a replicon based system along with YFV cDNA it was determined that the replication of this chimera was delayed by 24 hours, but reached wild type levels. Interestingly, even though there were wild type levels of replication, the chimera was deficient in virus particle production. Two mutations within the YFV helix, K617E and E620A, had a similar delay in replication but produced small plaques, indicating that the delay in replication was not completely responsible for the lack of plaques. Unlike other mutations observed to have wild type levels of replication in the absence of plaque formation, the virus deficiency could not be trans-complemented and a reduction in the amount of subviral particles released was observed. These differences indicate that the residues within helix 10 affect a different part of the infectious virus assembly pathway than what has been observed before.

Degree

Ph.D.

Advisors

Kuhn, Purdue University.

Subject Area

Molecular biology|Virology

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