An in vitro assay for hepatitis C virus NS3 serine proteinase.

Hepatitis C virus (HCV) encodes a polyprotein of which the majority of nonstructural proteins are matured by the viral serine proteinase located in the N terminus of NS3. Intracellular studies using recombinant vaccinia virus have shown that both NS3 and its cofactor NS4A are required to enhance processing at the NS3-dependent cleavage sites. We developed an in vitro (cell-free) assay in which the HCV serine proteinase was shown to be enzymatically active, by mixing lysates of cells expressing either the serine proteinase or a nonstructural protein substrate.

Substrate determinants for cleavage in cis and in trans by the hepatitis C virus NS3 proteinase.

Processing of the hepatitis C virus polyprotein is accomplished by a series of cotranslational and posttranslational cleavages mediated by host cell signalases and two virally encoded proteinases. Of these the NS3 proteinase is essential for processing at the NS3/4A, NS4A/4B, NS4B/5A, and NS5A/5B junctions. Processing between NS3 and NS4A occurs in cis, implying an intramolecular reaction mechanism, whereas cleavage at the other sites can also be mediated in trans.

Kinetic and structural analyses of hepatitis C virus polyprotein processing.

Recombinant vaccinia viruses were used to study the processing of hepatitis C virus (HCV) nonstructural polyprotein precursor. HCV-specific proteins and cleavage products were identified by size and by immunoprecipitation with region-specific antisera. A polyprotein beginning with 20 amino acids derived from the carboxy terminus of NS2 and ending with the NS5B stop codon (amino acids 1007 to 3011) was cleaved at the NS3/4A, NS4A/4B, NS4B/5A, and NS5A/5B sites, whereas a polyprotein in which the putative active site serine residue was replaced by an alanine remained unprocessed, demonstrating that the NS3-encoded serine-type proteinase is essential for cleavage at these sites.

Nonstructural protein 3 of the hepatitis C virus encodes a serine-type proteinase required for cleavage at the NS3/4 and NS4/5 junctions.

We have studied processing of the nonstructural (NS) polyprotein of the hepatitis C virus. A series of cDNAs corresponding to predicted NS2/3/4 or NS3/4 regions were constructed, and processing of the polyproteins was studied in an in vitro transcription-translation system. We report that a catalytically active serine-type proteinase is encoded by the NS3 region.

Progression of early steps of human immunodeficiency virus type 1 replication in the presence of an inhibitor of viral protease.

We have evaluated a possible role for human immunodeficiency virus type 1 protease during early steps of replication. For these studies, a specific inhibitor of human immunodeficiency virus protease, Ro31-8959, was used. Synthesis of viral cDNA, its integration into cellular DNA, and its transcription were determined during a one-step, acute infection of MT-4 cells.