The implication of amino acid mutations at flavivirus NS1-2A cleavage site on NS1' protein production

Aims: The presence of a C-terminally extended form of NS1 (NS1' protein) has been previously reported in encephalitic flaviviruses, due to the presence of -1 programmed ribosomal frameshift at the N-terminal of NS2A protein. This present study is aimed to further confirm that the NS1' protein production is independent of the authentic cleavage at NS1-2A junction. Methodology and results: Six different constructs (P1-Leu, P2-Asp, P3-Phe, P3-Leu, P3-Gly and P5-8 Ala) containing various mutations at conserved and variable amino acids at C-terminal of NS1 protein were generated by site-directed mutagenesis and analysed with transient polyprotein expression assay. While analysis on the NS1-2A cleavage of the mutants exhibited extremely poor to efficient cleavage ranging from 6-89%, significant amount of NS1' being expressed in all mutants irrespective of their NS1-2A cleavage outcome. Conclusion, significance and impact study: In this analysis, we showed for the first time that the abolishment of the authentic NS1-2A cleavage in Murray Valley encephalitis virus (MVEV) did not impact on NS1' production. This observation extend on previous studies to show that NS1 and NS2A proteins are the product of NS1-2A cleavage which is catalysed by an unknown host protease while NS1' protein is a product of ribosomal frameshift, independent of the authentic cleavage at NS1-2A junction.