MAPPING THE TACARIBE VIRUS Z PROTEIN BINDING SITES ON THE L POLYMERASE PROTEIN

M.T. FRANZE-FERNÁNDEZ, M. WILDA, N. LÓPEZ, J.C. CASABONA.

Salamanca, España

Conferencia; XIII International Conference on Negative Strand Viruses. Salamanca, Spain, June 17th-22nd, 2006.; 2006

Negative Strand Viruses (B. Mahy, D. Kolakofsky, E. Villar)

The arenavirus Tacaribe (TacV) comprises a single phylogenetic lineage together with the four South American pathogenic producers of severe hemorrhagic disease. TacV genome encodes four proteins: the precursor of the viral glycoproteins, the nucleocapsid protein, the large L protein (2210 aminoacids) containing sequence motifs of RNA-dependent RNA polymerases of negative-strand RNA viruses and a 95-aminoacid RING-finger protein called Z. Using a reverse-genetic system we have previously demonstrated that TacV-Z protein inhibits viral RNA synthesis by direct interaction with the L polymerase (J.Virol. 77:10383-10393: 2003). To delineate the regions involved in the interaction with Z, C-terminal, N-terminal and site-directed mutations in L were analyzed for their ability to form a complex with Z as detected by coimmunoprecipitation. It was found that Z-L interactions were not disrupted by deletions of up to 550 aminoacids within the C-terminal sequence of the L molecule whereas a further 220 aminoacids truncation comprising approximately 50 residues of the C-terminal portion of L-predicted region III, reduced the interaction to about 7 % of WT L. Site-directed mutagenesis within region III identified nine aminoacids important for binding Z. Complex formation studies using L mutants with N-terminal deletions suggested a second region of interaction within aminoacids 1-292.