Proteolytic cleavage analysis at the Murray Valley encephalitis virus NS1-2A junction

S Addis, Eva LEE, J Bettadapura, Mario Lobigs

Research output: Contribution to journalArticle

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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. Methods and 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. Conclusion: 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.
Original languageEnglish
Article number144
Pages (from-to)1-11
Number of pages11
JournalVirology Journal
Volume12
DOIs
Publication statusPublished - 2015

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Murray Valley encephalitis virus
Dengue Virus
Flavivirus
Polyproteins
Amino Acid Substitution
Site-Directed Mutagenesis
Endoplasmic Reticulum
Amino Acid Sequence
Peptide Hydrolases
Clone Cells
Viruses
Amino Acids
Mutation

Cite this

Addis, S ; LEE, Eva ; Bettadapura, J ; Lobigs, Mario. / Proteolytic cleavage analysis at the Murray Valley encephalitis virus NS1-2A junction. In: Virology Journal. 2015 ; Vol. 12. pp. 1-11.
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Proteolytic cleavage analysis at the Murray Valley encephalitis virus NS1-2A junction. / Addis, S; LEE, Eva; Bettadapura, J; Lobigs, Mario.

In: Virology Journal, Vol. 12, 144, 2015, p. 1-11.

Research output: Contribution to journalArticle

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AU - LEE, Eva

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AU - Lobigs, Mario

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AB - 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. Methods and 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. Conclusion: 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.

KW - DNA Mutational Analysis

KW - Dengue Virus/physiology

KW - Encephalitis Virus, Murray Valley/genetics

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KW - Protein Processing, Post-Translational

KW - Viral Proteins/genetics

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