title: Functional Analysis of West Nile Virus Proteins in Human Cells.
abstract: West Nile Virus (WNV) lineage 2 strains have been responsible for large outbreaks of neuroinvasive disease in the United States and Europe between 1999 and 2012. Different strains in this lineage have previously been shown to produce either severe or mild neuroinvasive disease in mice. Phylogenetic and amino acid comparisons between highly or less virulent lineage 2 strains have demonstrated that the nonstructural (NS) gene(s) were most variable. However, the roles of some of the NS proteins in virus life cycle are unknown. The aim of this chapter is to describe simple computational and experimental approaches that can be used to: (1) explore the possible roles of the NS proteins in virus life cycle and (2) test whether the subtle amino acid changes in WNV NS gene products contributed to the evolution of more virulent strains. The computational approaches include methods based on: (1) sequence similarity, (2) sequence motifs, and (3) protein membrane topology predictions. Highlighted experimental procedures include: (1) isolation of viral RNA from WNV-infected cells, (2) cDNA synthesis and PCR amplification of WNV genes, (3) cloning into GFP expression vector, (4) bacterial transformation, (5) plasmid isolation and purification, (6) transfection using activated dendrimers (Polyfect), and (7) immunofluorescence staining of transfected mammalian cells.
authors.affiliation: Department of Tropical Medicine, Medical Microbiology and Pharmacology, John A. Burns School of Medicine, University of Hawaii at Manoa, 651 Ilalo Street, BSB 320, Honolulu, HI, 96813, USA. pakieli@hawaii.edu.|Pacific Center for Emerging Infectious Diseases Research, John A. Burns School of Medicine, University of Hawaii at Manoa, Honolulu, HI, USA. pakieli@hawaii.edu.|Department of Molecular Biosciences and Bioengineering, University of Hawaii at Manoa, Honolulu, HI, USA. pakieli@hawaii.edu.
authors.forename: Pakieli H
authors.initials: PH
authors.lastname: Kaufusi
authors.affiliation: Department of Tropical Medicine, Medical Microbiology and Pharmacology, John A. Burns School of Medicine, University of Hawaii at Manoa, 651 Ilalo Street, BSB 320, Honolulu, HI, 96813, USA.|Department of Molecular Biosciences and Bioengineering, University of Hawaii at Manoa, Honolulu, HI, USA.
authors.forename: Alanna
authors.initials: A
authors.lastname: Tseng
authors.affiliation: Department of Tropical Medicine, Medical Microbiology and Pharmacology, John A. Burns School of Medicine, University of Hawaii at Manoa, 651 Ilalo Street, BSB 320, Honolulu, HI, 96813, USA.|Pacific Center for EmergingInfectious Diseases Research, John A. Burns School of Medicine, University of Hawaii at Manoa, Street 651 Ilalo Street, BSB 320G, Honolulu, HI, 96813, USA.
authors.forename: Vivek R
authors.initials: VR
authors.lastname: Nerurkar
mesh_terms: D000818:Animals; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D019295:Computational Biology / Q000379:methods*; D019143:Evolution, Molecular; D057809:HEK293 Cells; D006801:Humans; D009154:Mutation; D010802:Phylogeny; D010957:Plasmids / Q000235:genetics; D020539:Sequence Analysis, Protein; D017423:Sequence Analysis, RNA; D014709:Vero Cells; D017361:Viral Nonstructural Proteins / Q000235:genetics* / Q000378:metabolism*; D014902:West Nile virus / Q000145:classification / Q000235:genetics* / Q000472:pathogenicity*
chemical_list: D017361:Viral Nonstructural Proteins
keywords: Cloning; Computation; Immunofluorescence; Protein topology; Sequence similarity; Transfection; WNV