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

MAXIMILIANO WILDA, NORA LOPEZ, CRISTINA MARINO, JUAN C. CASABONA, JOSE M. DELFINO , AND MARIA T.FRANZE-FERNANDEZ

JOURNAL OF VIROLOGY

Año: 2006

0022-538X

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 (aa) ] and a 95-aa RING-finger protein called Z. Using a minigenome rescue assay we have previously demonstrated that TacV-Z protein inhibits viral RNA synthesis by direct interaction with the L polymerase protein ( J.Virol. 77:10383-10393 : 2003 ). Sequence alignment among L proteins of negative-strand RNA viruses shows conserved domains of which domain III contains the conserved motifs A, B, C, and D , thought to  form the module containing the active site in RNA synthesis. For mapping the Z binding sites on L we performed C-terminal and N-terminal deletions and site-directed mutagenesis and analyzed the L-mutants ability to form a complex with Z as detected by coimmunoprecipitation. Results showed that Z-L interaction was not disrupted by deletions of up to 550 aa at the C-terminus of L whereas a further 220 aminoacids truncation , which included approximately the last 50 residues of predicted region III,  reduced the interaction to about 7 % that of WT L. Site-directed mutagenesis within region III led to identify two aminoacids in motif A (residues # 1188 and 1189) and one in motif C (residue # 1329) important for binding Z. Complex formation studies using L mutants with N-terminal deletions defined a second region within aa 156 and 292 required for Z-L interaction.