The Hepatitis C virus nonstructural 5A (NS5A) protein is a hydrophilic phosphoprotein with diverse functions. The domain assignment of NS5A had been refined using a systematic in silico bioinformatics approach using DOMAC, the protein is divided into three domains and domain III is subdivided into two subdomains using ProDom and SSEP servers. The fold structure for domains II and III were predicted using the meta-server 3D-Jury. Scanning motif databases (SMART, BLOCKS, and PROSITE) gave new motifs. Two important motifs, the interleukins 1 and 8 interaction motifs, relating to NS5A function in inducing the interleukin 8 promoter, were discovered from the BLOCKS scan. Protein-protein interaction motifs were predicted as hot loops and disordered regions, corresponding to binding regions with the ds-protein kinase R, viral polymerase, and Src homology 3 signaling proteins binding motif. Other hot loops were predicted in the V3 region and in the single-stranded DNA-binding protein motif. The different mechanisms by which the NS5A protein leads to immune system signaling dysfunction points to the natural genetic engineering of this protein.