Journal of General Virology, Vol 80, 327-332, Copyright © 1999 by Society for General Microbiology

ARTICLES

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

RY Forng and CD Atreya
Division of Viral Products, Center for Biologics Evaluation and Research, US Food and Drug Administration, Bethesda, MD 20892, USA.

The rubella virus (RV)-encoded protein NSP90, which contains the retinoblastoma 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 entire 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 binding by the C --> G mutant was reduced significantly compared to that of wild-type protein. Correlating with the binding results, mutant viruses containing the LXRXE and LXGXE motifs had a reduction in replication to < 0.5% and 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-carrying virus had increased from < 0.5% to 2% of the wild- type. Sequencing of the genome of this virus revealed a nucleotide change that altered the motif from LXRXE to LXSXE, which is a known Rb- binding motif in two protein phosphatase subunits. Thus, our results clearly demonstrate that the LXCXE motif is required for efficient RV replication.

This article has been cited by other articles:

				D. N. Shepherd, D. P. Martin, D. R. McGivern, M. I. Boulton, J. A. Thomson, and E. P. Rybicki A three-nucleotide mutation altering the Maize streak virus Rep pRBR-interaction motif reduces symptom severity in maize and partially reverts at high frequency without restoring pRBR-Rep binding J. Gen. Virol., March 1, 2005; 86(3): 803 - 813. [Abstract] [Full Text] [PDF]

				S. C. Braunagel, P. A. Guidry, G. Rosas-Acosta, L. Engelking, and M. D. Summers Identification of BV/ODV-C42, an Autographa californica Nucleopolyhedrovirus orf101-Encoded Structural Protein Detected in Infected-Cell Complexes with ODV-EC27 and p78/83 J. Virol., December 15, 2001; 75(24): 12331 - 12338. [Abstract] [Full Text] [PDF]