Exeen M. Morgan

Complete sequence of the sendai virus NP gene from a cloned insert

A DNA molecule representing all but the three terminal bases of the Sendai virus nucleoprotein (NP) gene, copied from viral mRNA, was inserted into pBR322. The NP insert comprised 1673 bases. The first AUG protein initiation codon, at position 65, began an open reading frame of 1551 bases, encoding a protein of 517 amino acids with an amino acid composition corresponding to previously published data. The NP gene sequence determined in the present work is similar to that described by Shioda et al. [ Nucl . Acids Res. 11, 7317 (1983)], but there are 14 amino acid differences that probably reflect differences in virus strains. The predicted secondary structure of the NP molecule and the locations within that structure of potential protease cleavage sites are in accord with structural domains previously defined by controlled protease digestion.

Antibodies against Sendai virus L protein: distribution of the protein in nucleocapsids revealed by immunoelectron microscopy

Antibodies against the L protein of Sendai virus were made by immunizing rabbits with a synthetic peptide representing a carboxyl-terminal region of the protein predicted from the base sequence of its gene. These antibodies were used to localize the L protein in viral nucleocapsids by electron microscopy. Immunogold labeling revealed that L protein molecules were distributed in clusters along nucleocapsids, suggesting that L molecules act cooperatively in viral RNA synthesis. Immunogold double-labeling showed that all L clusters were associated with clusters of P molecules. We believe that this morphological association reflects the functional cooperation of the L and P proteins in viral RNA synthesis.

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.

Sequence of the sendai virus L gene: open reading frames upstream of the main coding region suggest that the gene may be polycistronic

The sequence of the L gene of Sendai virus, encompassing 6799 nucleotides, has been determined, completing the primary sequence of the entire virus genome. An open reading frame beginning at position 569 codes for a basic protein of 2048 amino acids with an estimated Mr of 231,608. No nucleotide sequence similarities with the analogous L gene of vesicular stomatitis virus were observed. However, comparison of the deduced amino acid sequences of both proteins revealed a conserved 18 amino acid sequence that may have functional significance. Two additional overlapping reading frames which precede the L protein sequence could encode proteins with MrS of 6474 and 14,026, suggesting that the gene is polycistronic.

Reovirus enzymes that modify messenger RNA are inhibited by perturbation of the lambda proteins

Abstract When pyridoxal phosphate (PLP) binds reversibly to a select population of reovirus λ protein molecules, the transcriptase activity in virus cores is inhibited [Morgan, E. M., and Kingsbury, D. W. (1980), Biochemistry 19, 484–489]. We now report that each of the enzymes involved in post-transcriptional modifications of virus mRNA molecules (nucleotide phosphohydrolase, guanylyltransferase, and both methyltransferases) is also inhibited reversibly by PLP. This supports the view that reovirus mRNA transcription and modification are accomplished by a topographically related group of enzyme molecules and suggests that these enzymatic activities reside in one or more of the λ protein species. PLP did not interact with the RNA binding sites of the methyltransferases or guanylyltransferase, with the GTP binding site of guanylyltransferase, or with the nucleotide binding site of the phosphohydrolase, as shown by the inability of these substrates to compete with PLP in kinetic assays or to block PLP-directed reductive alkylation of the λ proteins. However, kinetic data suggested that PLP interacts with the AdoMet binding sites of the reovirus methyltransferases.