Identification of calreticulin as a rubella virus RNA binding protein.

Abstract

Previously, we observed that sequences at the 3' end of rubella virus (RV) genomic RNA that form a stable stem-oop structure are necessary for initiation of RNA replication. A cytosolic protein found in Vero 76 cells (simian origin) specifically bound to the 3' (+)-stem4oop sequence. In the present study, we have purified the RNA binding protein and identified it as a simian homologue of human calreticulin. The purified calreticulin binds to the RV RNA with specificity similar to the protein present in cytosolic extracts. Human calreticulin antibodies recognize several forms of simian calreticulin, one of which is phosphorylated in vivo. A 2-fold increase in phosphorylation of this form of calreticulin is observed in RV-infected cells. Recombinant human calreticulin can bind RV 3' (+)-stem4oop RNA only after undergoing in vitro phosphorylation. This binding activity is abrogated by pretreatment of phosphorylated recombinant human calreticulin with alkaline phosphatase. The RV RNA was also immunoprecipitated from RV-infected UV-crosslinked Vero 76 cells by using calreticulin antibodies. Our results show that phosphorylated calreticulin is an RNA binding protein and phosphorylation is necessary for this activity. Specific binding of calreticulin to the cis-acting element of RV RNA in vivo suggests a possible role for this interaction in viral replication.Understanding the mechanism of RNA virus replication is of prime significance due to its central role in viral pathogenesis. Despite this importance, few details are known about viral replication processes in eukaryotic systems. Often hostencoded proteins are implicated in viral RNA replication (1-4), potentially contributing enzymatic, structural, or regulatory activities. However, these host factors are poorly characterized, and their functions are not well defined.

Arginylated Calreticulin at Plasma Membrane Increases Susceptibility of Cells to Apoptosis

“…CRT is a multifunctional multicompartmental protein (36), is mainly located in the ER, but also has been implicated in a variety of processes that occur outside this organelle such as in the cytoplasm (3)(4)(5)(6), in the nucleus (7,8), on the cell plasma membrane, and in other extracellular compartments (9 -17). CRT coordinates a number of cellular events from the cell surface where it has been suggested to participate in: wound healing (9, 10), thrombospondin signaling (13)(14)(15)(16), antigen presentation and complement activation (11,12), clearance of apoptotic cells (17), and immunogenicity of cancer cell death (37,38).…”

“…CRT resides mainly in the ER and is a Ca 2ϩ -binding protein that function as a Ca 2ϩ buffer and molecular chaperone, although it has also been linked to a number of processes occurring outside the ER lumen (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17), which implies other subcellular localizations for CRT other than the ER. However, the subcellular origin, biochemical features, and the precise molecular mechanism responsible for targeting CRT to the plasma membrane is not fully understood.…”

Characterization of DNA vaccines encoding the domains of calreticulin for their ability to elicit tumor-specific immunity and antiangiogenesis

“…The N domain (residues 1-180), also known as vasostatin, is extremely conserved among calreticulins from different species [13]. The N domain interacts with the DNA-binding domain of the glucocorticoid receptor in vitro [14], with rubella virus RNA [15], with α-integrin [16], and with protein disulphide-isomerase (PDI) and ER protein 57 (ERp57) [17]. The N domain of calreticulin also inhibits proliferation of endothelial cells and suppresses angiogenesis [10].…”

Ligand-specific, transient interaction between integrins and calreticulin during cell adhesion to extracellular matrix proteins is dependent upon phosphorylation/dephosphorylation events

“…It was first described as a calcium-binding protein of the sarcoplasmic reticulum of smooth-muscle cells [40,41]. Calreticulin has subsequently been shown (i) to possess a calcium-binding function [42] and chaperone activity [43] in the endoplasmic reticulum ; (ii) to have RNA binding activity [44] ; and (iii) to modify gene expression by binding to nuclear-hormone receptors [45,46]. It has also been shown that calreticulin can bind to the KXGFFKR motif within the α subunits of integrins [27] (where X indicates any residue), and that antisense oligonucleotide-mediated downregulation of calreticulin expression inhibits integrin-mediated adhesion of PC-3 and Jurkat cells [29].…”

“…The cytoplasmic domains of some integrins themselves have been shown to be phosphorylated [24] ; however, the importance of these phosphorylation events in integrin-mediated adhesion or signalling has not been fully demonstrated. While calreticulin can be phosphorylated [44], the biochemical and physiological significance of this phosphorylation is not clear, and it remains to be determined whether phosphorylation of calreticulin directly is important for its interaction with integrins. Alternatively, it is plausible that other adhesion-regulating proteins, e.g.…”