Coronavirus
RNA synthesis is performed by a multienzymatic replicase complex together with cellular factors. This process requires the specific recognition of
RNA cis-acting signals located at the ends of the viral genome. To identify cellular
proteins involved in coronavirus
RNA synthesis, transmissible
gastroenteritis coronavirus (TGEV) genome ends, harboring essential cis-acting signals for replication, were used as baits for
RNA affinity
protein purification. Ten
proteins were preferentially pulled down with either the 5' or 3' ends of the genome and identified by proteomic analysis. Nine of them, including members of the heterogeneous
ribonucleoprotein family of
proteins (hnRNPs), the
poly(A)-binding protein (PABP), the p100 transcriptional co-activator
protein and two aminoacyl-
tRNA synthetases, showed a preferential binding to the 3' end of the genome, whereas only the
polypyrimidine tract-binding protein (PTB) was preferentially pulled down with the 5' end of the genome. The potential function of the 3' end-interacting
proteins in virus replication was studied by analyzing the effect of their silencing using a TGEV-derived replicon and the infectious virus. Gene silencing of PABP,
hnRNP Q, and
glutamyl-prolyl-tRNA synthetase (EPRS) caused a significant 2 to 3-fold reduction of
viral RNA synthesis. Interestingly, the silencing of
glyceraldehyde 3-phosphate dehydrogenase (GAPDH), initially used as a control gene, caused a 2 to 3-fold increase in
viral RNA synthesis in both systems. These data suggest that PABP,
hnRNP Q, and EPRS play a positive role in
virus infection that could be mediated through their interaction with the viral 3' end, and that GAPDH has a negative effect on
viral infection.