Abstract
A two-hybrid system was used to study interaction in vivo between the nucleocapsid protein (NP) and the phosphoprotein (P) of human parainfluenza virus type 3 (HPIV-3). Two plasmids, one containing the amino terminus of P fused to the DNA-binding domain of the yeast transactivator, GAL4, and the other containing the amino terminus of NP fused to the herpesvirus transactivator, VP16, were transfected in COS-1 cells along with a chloramphenicol acetyltransferase (CAT) reporter plasmid containing GAL4 DNA-binding sites. A specific and high-affinity interaction between NP and P was observed as measured by the activation of the CAT gene. Mapping of the domains in P (603 amino acids) involved in the association with NP revealed that NH2-terminal 40 and COOH-terminal 20 amino acids are important for such association. Interestingly, a stretch of NH2-terminal amino acids as short as 63-403 interacted with NP more than the wild type, reaching greater than 2.5-fold as measured by the CAT assay. These results suggest that a domain is present in P that negatively regulates its interaction with NP. Deletion of NH2-terminal 40 and COOH-terminal 160 amino acids of NP reduced the CAT activity by more than 95%. These results underscore the important differences between negative strand RNA viruses with respect to interactions between these two viral proteins involved in gene expression.
Highlights
From the Wepartment of Biology, Case Western Reserve University, Cleveland, Ohio 44106 and the §Department of Molecular Biology, Research Institute, The Cleveland Clinic Foundation, Cleveland, Ohio 44195
One containing the amino terminus ofP fused to the DNA-binding domain of the yeast transactivator, GAIA, and the other containing the amino terminus of nucleocapsid protein (NP) fused to the herpesvirus transactivator, VP16, were transfected in COS-I cells along with a chloramphenicol acetyltransferase (CAT) reporter plasmid containing GAIA DNA-binding sites
To gain insight into the replication process of human parainfluenza virus type 3 (HPIV-3), we have studied the formation of the Np· P complex, which is the essential component involved in the switch from transcription to replication
Summary
From the Wepartment of Biology, Case Western Reserve University, Cleveland, Ohio 44106 and the §Department of Molecular Biology, Research Institute, The Cleveland Clinic Foundation, Cleveland, Ohio 44195. Deletion of NH2-terminal 40 and COOH-terminal 160 amino acids of NP reduced the CAT activity by more than 95% These results underscore the important differences between negative strand RNA viruses with respect to interactions between these two viral proteins involved in gene expression. The requirements of the formation of both the L·P complex and the Np·P complex in transcription and replication, respectively, have been shown in vitro and in vivo for vesicular stomatitis virus (VSV), a prototype negative strand RNA virus [6,7,8] This has been demonstrated in Sendai virus, a paramyxovirus, by expression of the recombinant proteins in the cell [5, 9]. The precise mechanism and the roles played by the respective complexes in the switch from transcription to replication remain unclear
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