Though EF-hands have been found abundantly in eukaryotes and bacteria, literature reporting EF-hand or EF-hand like Ca 2+ -binding motifs in virus proteins is scarce, possibly due to lack of accurate prediction methods and robust validating methodologies. A thorough search in PubMed with the key words "EF-hand and virus" only results in 4 examples (1, 3, 5, 7, 9, 12) and the hydrophobic residues (n, blue). The predicted EF-hand from Shr (Streptococcal hemoprotein receptor, S. pyrogenes) and PlcR (phospholipase accessory protein, P. aeruginosa) are aligned with some EF-hands known to form oligomers: CaM EF3, the third EF-hand from calmodulin; TnC EF3, the third EF-hand from troponin C; PV EF3, the third EF-hand from parvalbumin; D9K EF2, the canonical EF-hand from calbindin D9K, The search patterns used for the identification of the EF-hand loop and flanking helices (Helix E and Helix F) are also shown in the bottom. ). In addition, the functions of almost 20% of these matched proteins remain uncharacterized. We hope that our prediction will serve as a prelude to more extensive searching for additional viral Ca 2+ -binding proteins that are closely associated with virus-host interacting events ( Figure  1(b) ). Rubella virus (RUB), the only member of the genus Rubivirus, in the Togaviridae family, is the causative agent of a disease called rubella or German measles. Nonstructural protein (NS) open reading frame (ORF) of RUB encodes a polypeptide precursor which is able to cleave itself into two replicase components involved in viral RNA replication. A putative EF-hand Ca 2+ binding motif of the nonstructural protease that cleaves the precursor was successfully predicted across different genotypes of RUB and determined by established grafting approach [51] . The grafted EF-loop bound to Ca 2+ and its trivalent analogs Tb 3+ and La 3+ with dissociation constants of 214, 47, and 14 μM, respectively. The NS protease containing mutations of cal-cium binding sites elimination (D1210A and D1217A) was less efficient at precursor cleavage than the wt NS protease at 35°C, and the mutant NS protease was temperature sensitive at 39°C, confirming that the Ca 2+ binding loop played a structural role in the NS protease and was specifically required for optimal stability under physiological conditions. Interestingly, the same bioinformatics algorithm that successfully predicted the Ca 2+ -binding loop in the RUB NS protease also predicted an EF-hand Ca 2+ -binding motif in nsP1 of alphaviruses (Figure 4(b) ). NsP1 is one of the four nonstructural proteins produced by alphaviruses and is involved in membrane binding and has methyl/guanylyl transferase activity.