Kevin W. Ryan

Separate domains of sendai virus P protein are required for binding to viral nucleocapsids

The role of Sendai virus P protein in viral RNA synthesis involves association with the nucleocapsid template. There is evidence that the carboxyl-terminal region of P protein is responsible for this association (K. W. Ryan and D. W. Kingsbury, 1988, Virology 167, 106-112). To define the P protein sequences involved more precisely, deletions were generated in a cDNA clone of the P gene. Proteins synthesized in vitro from these altered P genes were mixed with extracts from infected cells to determine if they could attach to nucleocapsids. Under conditions where full-size P protein was able to bind, a protein comprising the 95 carboxyl-terminal residues of P protein (Sendai virus X protein) did not bind. This indicated that other P protein residues were required, in addition to the 95 residues at the carboxyl-terminal end. To locate these other residues, P genes were constructed with overlapping deletions of sequences encoding the carboxyl-terminal 40% of the protein. Analysis of these deleted proteins revealed that the necessary residues were in two separate binding domains, amino acids 345 to 412 and 479 to 568 (the carboxyl-terminus). Deletion of the 66 residues between these regions did not affect attachment. Therefore, the formation of a functional binding site requires residues within two separate regions of P protein.

Two noncontiguous regions of sendai virus p protein combine to form a single nucleocapsid binding domain

Binding of Sendai virus P protein to viral nucleocapsids requires amino acids in two separate regions of P protein. Both required regions are near the carboxyl terminus, and they are separated by a region which is expendable for binding (K. W. Ryan and A. Portner, 1990, Virology 174, 515-521). To examine the topography of these regions in the folded P protein molecule we mapped the epitopes present in several undenatured P proteins with overlaping deletions near their carboxyl termini. The epitopes recognized by two monoclonal antibodies were each composed of both protein regions necessary for binding, indicating that these two regions are each required at some point during the folding of P protein. To determine if these protein regions interact directly in forming the nucleocapsid binding domain, we constructed a deleted P gene which encodes a protein comprising only these two regions with all other P protein sequences deleted. This protein was able to bind to nucleocapsids, demonstrating that these two regions alone are sufficient to form the nucleocapsid-binding domain. In addition, this protein formed the folded epitopes comprising the two nucleocapsid-binding regions, indicating that the two regions interact directly with each other to form a single folded structure. The involvement of this binding domain in viral mRNA synthesis was examined by testing the ability of each monoclonal antibody to inhibit the in vitro transcription activity of full-size P protein. Several antibodies to epitopes near the binding domain were found to be potent inhibitors of viral transcription, showing that these regions contribute to P proteins role in mRNA synthesis.

Carboxyl-terminal region of sendai virus P protein is required for binding to viral nucleocapsids

The Sendai virus P protein is a component of the viral nucleocapsid, where it participates in RNA synthesis. To identify domains of the protein involved in nucleocapsid recognition, deleted P protein molecules were generated from a cDNA clone of its gene. In vitro transcription of the complete gene and translation of the transcript generated a protein with electrophoretic mobility and immunoreactivity indistinguishable from those of authentic P protein. The in vitro product bound specifically to nucleocapsids when mixed with extracts from infected cells. However, a product lacking only 30 carboxyl-terminal amino acid residues (5% of the molecule) did not bind. Residues within a 195 amino acid region, adjacent to and overlapping by one amino acid with the carboxyl-terminal 30 residues, were also required for binding. No other protein region was required. Therefore, the 224-residue region which includes the carboxyl terminus appears to contain the nucleocapsid attachment site, and the 30 terminal residues either form part of the site or are required to maintain an active conformation.

A plasmid that improves the efficiency of foreign gene expression by intracellular T7 RNA polymerase.

To facilitate the construction of recombinant plasmids for expressing cloned genes with T7 RNA polymerase supplied by recombinant vaccinia virus, a plasmid expression vector was designed by combining parts of plasmids pTZ18R, pBluescript II KS+, and pAR2529. The 3043-bp plasmid pTF1 has a T7 RNA polymerase promoter, multiple cloning site for insertion of foreign genes, and a T7-specific transcription termination signal. Plasmid pTF1 had several advantages compared with the reference plasmid pAR2529, including more efficient replication in bacteria, greater flexibility in the insertion and subcloning of foreign genes, and increased efficiency of liposome-mediated introduction into cultured cells for expression of the foreign gene.

Expression of cDNA encoding the sendai virus hemagglutinin-neuraminidase gene: Characterization of wild-type and mutant gene products

Cloned cDNA encoding the Sendai virus (SV) hemagglutinin-neuraminidase (HN) envelope glycoprotein was expressed in cultured cells in two ways: (I) infection with HN-expressing recombinant vaccinia virus, or (II) transfection with a plasmid with T7 promoter and termination sequences flanking the HN gene, with intracellular T7 RNA polymerase supplied by coinfection with recombinant vaccinia virus that expresses the enzyme. The HN expressed was indistinguishable from the authentic SV protein in antigenicity, cell surface location, and formation of oligomeric structures. In addition, HN expressed from cDNA functioned normally in both hemadsorption and neuraminidase activities. The usefulness of cDNA expression for analyzing HN structure and function was evaluated by mutating the HN cDNA and observing the consequences for HN protein activity. Since previous work indicated that the lysine residue at position 461 is important for the neuraminidase activity of HN, we used site-directed mutation to produce HN protein with this lysine residue changed to glutamic acid. The mutated HN had neuraminidase activity with significantly increased thermal stability, indicating that residue 461 may be essential to the proteins conformation.

Selective interference with P protein binding to paramyxovirus nucleocapsids

We previously observed that some anti-nucleoprotein monoclonal antibodies were able to displace P protein from Sendai virus (SV) nucleocapsid cores. The current work extends that observation by showing that such antibody-mediated P displacement is not unique to Sendai virus nucleocapsids, but can also occur with nucleocapsids of a related human respiratory pathogen. Anti-NP antibody prevents binding of SV P protein to nucleocapsids from human parainfluenza virus type 1 (PIV1) or from SV. Antibody also prevents binding of PIV1 P protein to PIV1 nucleocapsids, but not to SV nucleocapsids. We have also examined the stoichiometry of antibody interference with P binding, to determine how large a nucleocapsid region can be protected from P binding by a single antibody molecule. We found that approximately 40 antibody molecules per nucleocapsid complex can block attachment of most P protein. This indicates that a single antibody molecule can prevent P binding to a region representing about 65 nucleoprotein monomers on the nucleocapsid core.