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.

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Sequences at the 5' and 3' ends of the rubella virus (RV) genomic RNA can potentially form stable stem-loop (SL) structures that are postulated to be involved in virus replication. We have analyzed the function of these putative SL structures in RNA translation by constructing chimeric chloramphenicol acetyltransferase (CAT) RNAs, flanked either by both 5'and 3'-terminal sequence domains from the RV genome or several deletion derivatives of the same sequences. After in vitro transcription of chimeric RNAs, the translational efficiencies of these RNAs were compared by the rabbit reticulocyte lysate translation system. For in vivo translation studies, the level of CAT activity was measured for chimeric RV/CAT RNAs expressed in transfected cells by the adenovirus major late promoter. Both in vivo and in vitro translation activities of the chimeric RNAs revealed that the presence of 5' and 3' SL sequences of RV RNA, in correct (+) orientation and context [5'(+)SL and 3'(+)SL, respectively] was necessary for efficient translation of chimeric RV/CAT RNAs. The presence of the RV 5'(+)SL sequence had the primary enhancing effect on translation. To identify host proteins which interact with the 5'(+)SL which may be involved in RV RNA translation, RNA gel-shift and UV cross-linking assays were employed. Two host proteins 59 and 52 kDa in size, present in cytosolic extracts from both uninfected and RV-infected cells, specifically interacted with the RV 5'(+)SL RNA. Direct binding comparisons between wild-type and mutant 5'(+)SL RNAs demonstrated that sequences in and around the * Corresponding author. t Present address: Laboratory of Pulmonary and Molecular Immunology, National Heart, Lung, and Blood Institute, Bethesda, MD 20892. sequence termed the internal ribosomal entry site or ribosomal landing pad (8,12,28,33,34). Within this domain of highly complex secondary structure, several structural features, a polypyrimidine tract flanked by two extended SL structures, profoundly influence translation of poliovirus proteins (11). The SL structure present immediately 5' of the polypyrimidine tract must be preserved for both poliovirus translation and 7106