In Vivo Association of Calreticulin with RVRNA. To explore
whether the in vitro association of calreticulin with the RV 3'
(+)-SL RNA has a physiological relevance to RV infection,
we examined the in vivo association of calreticulin with RV
RNA (Fig. 5). Ribonucleoprotein (RNP)-RV RNA complexes
were immunoprecipitated with calreticulin specific
antibody from lysates of UV-crosslinked RV-infected cells
and from samples to which synthetic RV RNA was added,
whereas UV-crosslinked RNP complexes from RV-infected
cells transiently expressing FLAG-Cal were immunoprecipitated
with FLAG peptide antibodies (IBI/Eastman Chemical).
The RV RNA present in the RNP complexes was
detected by RT-PCR, followed by digestion of the DNA with
Nhe I. Nhe I digests ofthe 161-bp RT-PCR DNA revealed 98-
and 63-bp DNA fragments only in samples containing the RV
RNA (Fig. 5, lanes 1-6).