Protein interaction domains of the measles virus nucleocapsid protein (NP).

The currently accepted model for measles virus (MV) transcription and replication assumes the nucleocapsid (NP) protein to possess the ability to bind to RNA, to other NP molecules, and to the phosphoprotein (P) during ribonucleocapsid (RNP) assembly, as well as to the matrix protein (M) during virion assembly. We have cloned the MV NP open reading frame and have expressed the protein in bacteria as a fusion with glutathione-S-transferase (GST). Affinity purified GST-NP fusion protein has been used as a probe to examine the interaction of NP with [35S] methionine labeled proteins from MV-infected cells. We have demonstrated definite and specific interactions between NP and itself and between NP and P, but have been unable to demonstrate any interaction between NP and M. We have been able to provide independent confirmation of this pattern of interaction using the yeast two-hybrid assay. We have, in addition, been able to map the domains of NP involved in these interactions by assays using sets of amino- and carboxy-terminal deletion mutants of GST-NP. The NP-NP interaction domain was found to reside in the highly conserved middle and amino-terminal domains of the protein. The hyper-variable carboxy-terminus and the conserved middle domain appear to constitute separate and independent sites for the binding of P to NP. The significance of these findings in regard to MV transcription and replication is discussed.

Protein interactions entered into by the measles virus P, V, and C proteins.

Measles virus (MV) expresses at least 3 proteins from the phosphoprotein (P) cistron. Alternative translation initiation directs synthesis of the C protein from the +1 reading frame, while so-called RNA editing generates a second population of mRNAs which express the V protein from the -1 reading frame which lies within and overlaps the larger P reading frame. While the P protein has been demonstrated to be an essential cofactor for the L protein in the formation of active transcriptase complexes, the functions of the V and C proteins remain unknown. In order to investigate these functions, we have expressed the MV P, V and C proteins as GST fusions in E. coli for affinity purification and use in an in vitro binding assay with other viral and cellular proteins. The P protein was found to interact with L, NP, and with itself. These interactions were mapped to the carboxy-terminal half of the protein which is absent in the V protein. In contrast, both the V and C proteins failed to interact with any other viral proteins, but were each found to interact specifically with one or more cellular proteins. Appropriate aspects of these results were confirmed in vivo using the yeast two-hybrid system. These observations suggest that the V and C proteins may be involved in modulation of the host cellular environment within MV-infected cells. Such activity would be distinct from their previously proposed role in the possible down-regulation of virus-specific RNA transcription and replication.

Molecular analysis of HEM6 (HEM12) in Saccharomyces cerevisiae, the gene for uroporphyrinogen decarboxylase.

HEM6 (HEM12) in Saccharomyces cerevisiae encodes uroporphyrinogen decarboxylase, the fifth enzyme in the heme biosynthetic pathway. The HEM6 (HEM12) gene was cloned by complementation of heme auxotrophy of a hem6 mutant. Sequence analysis revealed an open reading frame of 1086 nucleotides. The predicted amino acid sequence of HEM6 (HEM12) shows extensive homology to those reported for uroporphyrinogen decarboxylase from mammalian sources. Expression of HEM6 (HEM12) was investigated and was found to increase two-fold in a non-fermentable carbon source. However, HEM6 (HEM12) transcription was unaffected by heme or by intermediates in the heme biosynthetic pathway. In addition, HEM6 (HEM12) expression is not regulated by the transcriptional activator complex HAP2-3-4, as has been shown for some genes encoding heme biosynthetic enzymes.