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The RGD sequence in the cytomegalovirus DNA polymerase accessory protein can mediate cell adhesion
L C Loh1, D Locke, R Melnychuk
1Department of Microbiology, Department of Biochemistry, University of Saskatchewan, 107 Wiggins Road, Saskatoon, Saskatchewan, S7N 5E5 Canada, loh@sask.usask.ca
Abstract:
The murine cytomegalovirus (MCMV) polymerase processivity factor ppM44 (also referred to as pp50) is an abundant phosphoprotein found in MCMV-infected cells. Sequence analysis of the MCMV M44 open reading frame revealed an "RGD" motif that is also present in the human cytomegalovirus (HCMV) UL44 open reading frame. In this report, histidine-tagged M44 protein produced in Escherichia coli or the vaccinia/T7 expression system was purified to near homogeneity by metal chelation affinity chromatography using His*Bind resins. We demonstrated that recombinant M44 protein could mediate cell adhesion via its conserved "RGD" motif, because a single amino acid change (RGD to RGE) abolished cell attachment. In addition, cell adhesion was abolished in the presence of EDTA. We next showed that recombinant HCMV UL44, but not human herpesvirus type 6 p41, which lacks the RGD motif, could mediate cell adhesion in a similar manner. We also provided evidence that ppM44 was present in the culture medium during virus infection. Thus these results suggested that in addition to its primary role as the polymerase processivity factor, MCMV ppM44 may serve as a substrate for integrin-binding via its conserved RGD motif, with the potential for a novel role in the MCMV replication cycle.
Insights
Murine cytomegalovirus ppM44 protein binds cells via its RGD motif, suggesting a novel role in viral replication beyond its function as a polymerase processivity factor.
Area of Science:
- Virology
- Molecular Biology
- Cell Biology
Background:
- Murine cytomegalovirus (MCMV) polymerase processivity factor ppM44 is an abundant phosphoprotein.
- Sequence analysis revealed an RGD motif in MCMV ppM44, also found in human cytomegalovirus (HCMV) UL44.
Purpose of the Study:
- To investigate the function of the MCMV ppM44 protein.
- To determine if the RGD motif in ppM44 mediates cell adhesion.
- To explore potential roles of ppM44 in the MCMV replication cycle.
Main Methods:
- Purification of histidine-tagged M44 protein using metal chelation affinity chromatography.
- Assessing cell adhesion mediated by recombinant M44 protein.
- Mutagenesis of the RGD motif (RGD to RGE) to evaluate its role in cell attachment.
- Testing the effect of EDTA on cell adhesion.
- Evaluating cell adhesion mediated by recombinant HCMV UL44 and human herpesvirus type 6 p41.
Main Results:
- Recombinant M44 protein mediated cell adhesion through its RGD motif.
- Mutation of the RGD motif abolished cell attachment.
- EDTA treatment also abolished cell adhesion, indicating an integrin-binding mechanism.
- Recombinant HCMV UL44, but not HHV-6 p41, showed similar cell adhesion properties.
- ppM44 was detected in the culture medium during MCMV infection.
Conclusions:
- MCMV ppM44 functions as a cell adhesion molecule via its RGD motif.
- This RGD motif is conserved in HCMV UL44.
- ppM44 may act as an integrin-binding substrate, suggesting a novel role in MCMV replication.