The site directed mutagenesis was carried out by PCR based method [20] using appropriate sense and anti sense primers with desired changes. The oligonucleotide primers used for site-directed mutagenesis (SDM) were custom made from Sigma. Mutagenic DNA primers (table 1) were designed to create alanine substitution at residues K189 and D159 in GxxxxGKT and DExx motifs of rNSs respectively. The presence of the desired mutations was screened initially by the gain or loss of a restriction site and was confirmed by DNA sequencing.