Sofosbuvir is an inhibitor of the hepatitis C virus (HCV) NS5B ribonucleic acid (RNA)-dependent RNA polymerase, which is essential for viral replication. Sofosbuvir is a nucleotide prodrug that undergoes intracellular metabolism to form the pharmacologically active uridine analog triphosphate (GS-461203), which can be incorporated into HCV RNA by the NS5B polymerase and acts as a chain terminator. In a biochemical assay, GS-461203 inhibited the polymerase activity of the recombinant NS5B from HCV genotype 1b, 2a, 3a and 4a with a 50% inhibitory concentration (IC50) value ranging from 0.7-2.6 micrometer. GS-461203 (the active metabolite of sofosbuvir) is not an inhibitor of human deoxyribonucleic acid (DNA) and RNA polymerases, nor an inhibitor of mitochondrial RNA polymerase.

Daclatasvir stops HCV viral RNA replication and protein translation by directly inhibiting HCV protein NS5A. NS5A is critical for HCV viral transcription and translation.

Sofosbuvir directly targets the Hepatitis C virus to stop it from making copies of itself in the liver. Sofosbuvir attaches itself to the genetic information, called RNA, to block the virus from multiplying. And Daclatasvir is a direct-acting  antiviral agent (DAA) against the hepatitis C virus.