David W. McCourt

Production of yellow fever virus proteins in infected cells: identification of discrete polyprotein species and analysis of cleavage kinetics using region-specific polyclonal antisera.

Flavivirus proteins are produced by translation of a single long open reading frame and a complex series of cotranslational and post-translational proteolytic cleavages. To study these processing events in yellow fever virus (YF)-infected cells, polyclonal antisera recognizing C, prM, E, NS1, NS2B, NS3, NS4B, and NS5 were generated using peptide and fusion protein immunogens. Evidence suggests that production of the structural protein precursors involves rapid cotranslational processing consistent with signalase cleavages. The synthesis of the NS1 glycoprotein involves cleavage of polyprotein precursors (t1/2 approximately 10 minutes) which probably contain portions of the NS2A gene product. Endoglycosidase F treatment or labeling in the presence of tunicamycin suggests that YF prM and NS1 each have two N-linked oligosaccharides. NS2B is produced without any identifiable precursors or associated polyprotein species. Processing of the NS3-4-5 region is complex and occurs rapidly. A series of polyproteins can be detected whose molecular weights correlate with the cleavage sites defined by available N-terminal amino acid sequence data. However, convincing precursor-product relationships between these polyproteins and the mature NS3 and NS5 proteins could not be demonstrated. In contrast, NS4B appears to be produced by cleavage of a discrete precursor believed to be NS4AB. N-terminal sequence data for the putative NS4AB product has tentatively defined the NS3-4A cleavage site. A scheme for in vivo processing of the YF polyprotein is presented and discussed.

Yellow fever virus proteins NS2A, NS2B, and NS4B: identification and partial N-terminal amino acid sequence analysis

A series of fusion proteins corresponding to the hydrophobic ns2 and ns4 regions of yellow fever virus (YF) were generated in Escherichia coli using trpE fusion vectors. Antisera to ns2 and ns4 region fusion proteins recognize virus-specific proteins of 15 and 27 kDa, respectively. N-terminal amino acid sequence analysis of the 27-kDa protein indicates that the N-terminus of YF NS4B immediately follows a signalase-like cleavage site. Additional sequence data generated by microsequence analysis of labeled proteins immunoprecipitated with mouse hyperimmune antisera have identified the 15-kDa protein as NS2B and an additional 20-kDa viral protein as NS2A. Comparison of the sequences adjacent to the N-termini of these viral proteins suggests that three distinct types of cleavage events are involved in processing the hydrophobic YF ns2 and ns4 regions. These include cleavage after a short side chain amino acid to generate the N-terminus of NS2A, cleavage after two arginine residues to produce the N-terminus of NS2B, and a cleavage site consistent with the specificity of signalase to generate the N-terminus of NS4B. Analysis of virus-specific protein patterns in several different mammalian cell lines and in Aedes albopictus cells suggests that the same cleavage sites are used in different hosts. These findings are discussed in relation to the processing of flavivirus polyproteins.

A simple in situ cyanogen bromide cleavage method to obtain internal amino acid sequence of proteins electroblotted to polyvinyldifluoride membranes

We report a simple method to obtain internal amino acid sequences from larger proteins electroblotted to polyvinyldifluoride membranes. To demonstrate this method, immunoglobulin heavy and light chains are separated by gel electrophoresis and electroblotted to the membrane. The separated chains, immobilized to the membrane, are cleaved in situ by cyanogen bromide and the resulting fragments are subsequently eluted from the membrane. The fragments are separated by gel electrophoresis, electroblotted and subjected to gas-phase microsequence analysis.

Purification and sequence analysis of two rat tissue inhibitors of metalloproteinases

Two protein inhibitors of metalloproteinases (TIMP) were isolated from medium conditioned by the clonal rat osteosarcoma line UMR 106-01. Initial purification of both a 30-kDa inhibitor and a 20-kDa inhibitor was accomplished using heparin-Sepharose chromatography with dextran sulfate elution followed by DEAE-Sepharose and CM-Sepharose chromatography. Purification of the 20-kDa inhibitor to homogeneity was completed with reverse-phase high-performance liquid chromatography. The 20-kDa inhibitor was identified as rat TIMP-2. The 30-kDa inhibitor, although not purified to homogeneity, was identified as rat TIMP-1. Amino terminal amino acid sequence analysis of the 30-kDa inhibitor demonstrated 86% identity to human TIMP-1 for the first 22 amino acids while the sequence of the 20-kDa inhibitor was identical to that of human TIMP-2 for the first 22 residues. Treatment with peptide:N-glycosidase F indicated that the 30-kDa rat inhibitor is glycosylated while the 20-kDa inhibitor is apparently unglycosylated. Inhibition of both rat and human interstitial collagenase by rat TIMP-2 was stoichiometric, with a 1:1 molar ratio required for complete inhibition. Exposure of UMR 106-01 cells to 10(-7) M parathyroid hormone resulted in approximately a 40% increase in total inhibitor production over basal levels.

Purification and characterization of bovine interstitial collagenase and tissue inhibitor of metalloproteinases

In this report we describe the purification of bovine interstitial collagenase and provide information on its substrate specificity, kinetic parameters of catalytic activity, and amino terminal protein sequence. In addition, we present a simplified protocol for the purification of bovine tissue inhibitor of metalloproteinases (TIMP). Collagenase was purified by sequential chromatography through heparin-Sepharose, DEAE-Sepharose, and green-agarose, resulting in a product that was greater than 95% pure as judged by polyacrylamide electrophoresis. Typical of other interstitial collagenases, the isolated bovine protein was activated by protease and organomercurial treatment. It also demonstrated a kinetics and substrate specificity similar to those of human collagenase. TIMP was purified by sequential chromatography through heparin-Sepharose and DEAE-Sepharose followed by reverse-phase HPLC. The purified protein had a size, N-terminal sequence, and inhibitor activity similar to those of other mammalian TIMPs. Partial peptide sequences suggested that bovine collagenase and TIMP have strong sequence homology to their human homologues.

Hepatitis C Virus Polyprotein Processing

Although HCV has been classified in the flavivirus family, little is known about HCV polyprotein processing. Expression studies utilizing a nearly full-length strain cDNA clone and various truncated derivatives have mapped at least nine cleavage products. These include the putative virion capsid protein (C), two envelope glycoproteins (El and E2), and six nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A and NS5B). Two HCV-encoded proteinases important for non-structural region processing were identified and studied by deletion analyses and site-directed mutagenesis. A serine proteinase domain located in the N-terminal one third of the NS3 protein was found to be necessary for four downstream cleavages. Cleavage at the 2/3 site appeared to be autocatalytic and mediated by a novel overlapping proteinase consisting of NS2 and the NS3 serine proteinase domain. Cleavage sites for both proteinases have been localized by N-terminal sequence analysis.

A new polymorphic determinant on HLA-DQ molecules

In man, the immune response genes are located within the HLA-D/DR region, and the gene products, the Ia antigens, are expressed on B lymphocytes, monocytes, and a percentage of null cells and activated T lymphocytes. We recently identified a human Ia antigen, K19, which appeared to be limited in its expression to B lymphocytes, and to be preferentially expressed on the more mature cells within this population. This work was facilitated by a monoclonal antibody. HK-19, which recognized a monomorphic determinant of this Ia molecule. We now report the characterization of a second monoclonal antibody, HK-13, which recognized the same molecule as HK-19, but only on cells from some individuals. The greater affinity of HK-13 allowed more complete characterization of the K19/K13 molecule. This characterization included cytofluorography, two-dimensional gel electrophoresis, tryptic peptide mapping, and partial N-terminal amino acid sequencing, and indicated that K19 and K13 were epitopes on HLA-DQ (DC) molecules. The pattern of reactivity of HK-13 on a panel of typing cells did not correlate with any of the known HLA-DQ polymorphic determinants. Thus, HK-13 is a new polymorphic determinant of the HLA-DQ series.