Biomedical subjects

E K O'Reilly

Publications and source records attributed to E K O'Reilly.

4 recordsLinked to original sources

PubMed1998

Analysis of RNA-dependent RNA polymerase structure and function as guided by known polymerase structures and computer predictions of secondary structure.

RNA-dependent RNA polymerases (RdRps) function as the catalytic subunit of the viral replicase required for the replication of all positive strand RNA viruses. The vast majority of RdRps have been identified solely on the basis of sequence similarity. Structural studies of RdRps have lagged behind those of the DNA-dependent DNA polymerases, DNA-dependent RNA polymerases, and reverse transcriptases until the recent report of the partial crystal structure of the poliovirus RdRp, 3Dpol [Hansen, J. L., et al. (1997). Structure 5, 1109-1122]. We seek to address whether all RdRps will have structures similar to those found in the poliovirus polymerase structure. Therefore, the PHD method of Rost and Sander [Rost, B., and Sander, C. (1993a). J. Mol. Biol. 232, 584-599; Rost, B., and Sander, C. (1994). Protein 19, 55-77] was used to predict the secondary structure of the RdRps from six different viral families: bromoviruses, tobamoviruses, tombusvirus, leviviruses, hepatitis C-like viruses, and picornaviruses. These predictions were compared with the known crystal structure of the poliovirus polymerase. The PHD method was also used to predict picornavirus structures in places in which the poliovirus crystal structure was disordered. All five families and the picornaviruses share a similar order of secondary structure elements present in their polymerase proteins. All except the leviviruses have the unique region observed in the poliovirus 3Dpol that is suggested to be involved in polymerase oligomerization. These structural predictions are used to explain the phenotypes of a collection of mutations that exist in several RNA polymerases. This analysis will help to guide further characterization of RdRps.

Amino Acid Sequence↗

PubMed1998

Interactions between the structural domains of the RNA replication proteins of plant-infecting RNA viruses.

Brome mosaic virus (BMV), a positive-strand RNA virus, encodes two replication proteins: the 2a protein, which contains polymerase-like sequences, and the 1a protein, with N-terminal putative capping and C-terminal helicase-like sequences. These two proteins are part of a multisubunit complex which is necessary for viral RNA replication. We have previously shown that the yeast two-hybrid assay consistently duplicated results obtained from in vivo RNA replication assays and biochemical assays of protein-protein interaction, thus permitting the identification of additional interacting domains. We now map an interaction found to take place between two 1a proteins. Using previously characterized 1a mutants, a perfect correlation was found between the in vivo phenotypes of these mutants and their abilities to interact with wild-type 1a (wt1a) and each other. Western blot analysis revealed that the stabilities of many of the noninteracting mutant proteins were similar to that of wt1a. Deletion analysis of 1a revealed that the N-terminal 515 residues of the 1a protein are required and sufficient for 1a-1a interaction. This intermolecular interaction between the putative capping domain and itself was detected in another tripartite RNA virus, cucumber mosaic virus (CMV), suggesting that the 1a-1a interaction is a feature necessary for the replication of tripartite RNA viruses. The boundaries for various activities are placed in the context of the predicted secondary structures of several 1a-like proteins of members of the alphavirus-like superfamily. Additionally, we found a novel interaction between the putative capping and helicase-like portions of the BMV and CMV 1a proteins. Our cumulative data suggest a working model for the assembly of the BMV RNA replicase.

Animals↗

PubMed1997

Analysis of the interaction of viral RNA replication proteins by using the yeast two-hybrid assay.

The yeast two-hybrid system has been a useful tool in the genetic evaluation of protein-protein interactions. However, the biological relevance of these two-hybrid interactions to viral positive-strand RNA replication has not been demonstrated. The brome mosaic virus (BMV) system has been characterized extensively both genetically and biochemically, providing numerous mutations in the BMV 1a helicase-like and 2a polymerase-like proteins. We have tested wild-type 1a and 18 insertion mutations of 1a and found a perfect correlation between the in planta phenotypes and their ability to interact with 2a in the two-hybrid system. This finding allowed further characterization of the interaction between and among the BMV viral proteins. Using the two-hybrid assay, we have found that the interaction between the helicase-like region of 1a and the N terminus of 2a is stabilized by the presence of the centrally conserved polymerase-like domain of 2a. We have also identified a novel interaction between the 1a helicase-like protein and itself. Additionally, we have found this interaction in two related tripartite RNA viruses, cowpea chlorotic mottle virus and cucumber mosaic virus. We have demonstrated that this protein-protein interaction is specific to homologous pairings of the protein.

Bromovirus↗

PubMed1995

Biochemical and genetic analyses of the interaction between the helicase-like and polymerase-like proteins of the brome mosaic virus.

Replication of the three positive-strand genomic RNAs of brome mosaic virus requires the activities of the helicase-like 1a and the polymerase-like 2a proteins. One hundred fifteen amino acids of the 2a N-terminus and the 1a helicase-like region of over 50 kDa are both necessary and sufficient for 1a-2a interaction. Requirement of the large size of the 1a helicase-like domain suggests that higher order structures might be necessary for the protein's interaction with 2a. To explore the structural properties of 1a, we used limited proteolysis of in vitro-translated 1a protein. Treatment of 1a and its deletion derivatives with papain or trypsin revealed that the C-terminal helicase-like segment of approximately 50-60 kDa is highly resistant under our assay conditions to proteolysis, while the N-terminus is rapidly degraded. All tested mutations in the helicase-like region that renders this region protease-sensitive have previously been found to be defective for RNA replication in vivo. To complement the in vitro studies, we examined the interaction of the 1a helicase-like domain and the 2a N-terminus in yeast using the two-hybrid system. Mutations previously known to disrupt 1a-2a interaction also prevented interaction in yeast. Furthermore, results from two-hybrid analysis suggest that the structural domain mapped in vitro is important for 1a-2a interaction. Finally, we found that the helicase-like proteins of three other tripartite RNA viruses also contain equivalently located protease-resistant domains.

Amino Acid Sequence↗