title: Proteolytic cleavage analysis at the Murray Valley encephalitis virus NS1-2A junction.
abstract: BACKGROUND
  Our understanding of the proteolytic processing events at the NS1-2A junction in the flavivirus polyprotein has not markedly progressed since the early work conducted on dengue virus (DENV). This work identified an octapeptide sequence located immediately upstream of the cleavage site thought to be important in substrate recognition by an as yet unknown, endoplasmic reticulum-resident host protease. Of the eight amino acid recognition sequence, the highly conserved residues at positions P1, P3, P5, P7 and P8 (with respect to N-terminus of NS2A) are particularly sensitive to amino acid substitutions in terms of DENV NS1-NS2A cleavage efficiency; however, the role of the octapeptide in efficient NS1 and NS2A production of other flaviviruses has not been experimentally addressed.
  
  
  RESULTS
  Using site-directed mutagenesis at the NS1-2A cleavage site of Murray Valley encephalitis virus (MVEV), we confirmed the dominant role of conserved octapeptide residues for efficient NS1-2A cleavage, while changes at variable and the P1' residues were mostly tolerated. However, digressions from the consensus cleavage motif derived from studies on DENV were also found. Thus, comparison of the impact on cleavage of mutations at the NS1-2A junction of MVEV and DENV showed virus-specific differences at both conserved and variable residues.
  
  
  CONCLUSIONS
  We show, with subgenomic expression and infectious clone-derived mutants of MVEV that conserved residues in the flavivirus octapeptide motif can be replaced with a different amino acid without markedly reducing cleavage efficiency of NS1 and NS2A.
authors.affiliation: John Curtin School of Medical Research, The Australian National University, Canberra, ACT, Australia. khadijah@umt.edu.my.|School of Fundamental Science, Universiti Malaysia Terengganu, 21030, Kuala Terengganu, Terengganu, Malaysia. khadijah@umt.edu.my.
authors.forename: Siti Nor Khadijah
authors.initials: SN
authors.lastname: Addis
authors.affiliation: Faculty of Education, Science, Technology and Mathematics, University of Canberra, Canberra, ACT, Australia. eva.lee@canberra.edu.au.
authors.forename: Eva
authors.initials: E
authors.lastname: Lee
authors.affiliation: Emerging Viruses & Inflammation Research Group, Institute for Glycomics, Griffith University, Nathan, QLD, Australia. j.bettadapura@griffith.edu.au.
authors.forename: Jayaram
authors.initials: J
authors.lastname: Bettadapura
authors.affiliation: Australian Infectious Diseases Research Centre, School of Chemistry and Molecular Biosciences, The University of Queensland, St Lucia, QLD, Australia. m.lobigs@uq.edu.au.
authors.forename: Mario
authors.initials: M
authors.lastname: Lobigs
mesh_terms: D004252:DNA Mutational Analysis; D003716:Dengue Virus / Q000502:physiology; D018068:Encephalitis Virus, Murray Valley / Q000235:genetics / Q000502:physiology*; D016297:Mutagenesis, Site-Directed; D020815:Polyproteins / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D020815:Polyproteins; D014764:Viral Proteins