Mutations in the retinoblastoma protein-binding LXCXE motif of rubella virus putative replicase affect virus replication

The rubella virus (RV)-encoded protein NSP90, which contains the retinoblas toma protein (Rb)binding motif LXCXE, interacts with Rb and RV replication is reduced in cells lacking Rb. Whether the LXCXE motif of RV NSP90 itself is essential for Rb binding and virus replication is not known. Therefore, in the present study, the functional role of this motif was investigated by site-directed mutagenesis in a plasmid from which infectious RV RNA can be produced, Three critical mutations in the motif, two substitutions at the conserved cysteine residue (C --> G and C --> R) and a deletion of the enti re motif, were created. A cell-free translated NSP90 C terminus polypeptide containing the deletion did not bind to Rb and a polypeptide carrying the C --> R substitution had barely detectable binding affinity for Rb, Rb bind ing by the C --> G mutant was reduced significantly compared to that of wil d-type protein, Correlating with the binding results, mutant viruses contai ning the LXRXE and LXGXE motifs had a reduction in replication to < 0.5% an d 47% of the wild-type, respectively, while deletion of the motif was found to be lethal. By the first serial passage, replication of the LXRXE-carryi ng virus had increased from < 0.5% to 2% of the wildtype, Sequencing of the genome of this virus revealed a nucleotide change that altered the motif f rom LXRXE to LXSXE, which is a known Rb-binding motif in two protein phosph atase subunits, Thus, our results clearly demonstrate that the LXCXE motif is required for efficient RV replication.