THE NTP-BINDING MOTIF IN COWPEA MOSAIC-VIRUS-B POLYPROTEIN IS ESSENTIAL FOR VIRAL REPLICATION

We have assessed the functional importance of the NTP-binding motif (N TBM) in the cowpea mosaic virus (CPMV) B-RNA-encoded 58K domain by cha nging two conserved amino acids within the consensus A and B sites (GK SRTGK500S and MDD545, respectively). Both Lys-500 to Thr and Asp-545 t o Pro substitutions are lethal as mutant B-RNAs were no longer replica ted in cowpea protoplasts. Transiently produced mutant proteins were n ot able to support trans-replication of CPMV M-RNA in cowpea protoplas ts in contrast to transiently produced wild-type B proteins. Therefore loss of viral RNA synthesis was a result of a protein defect rather t han an RNA template defect. Mutant B polyproteins were correctly proce ssed in vitro and in vivo and the regulatory function of the 32K prote in on processing of B proteins was not affected by these mutations. Si nce regulation of processing by the 32K protein depends on interaction with the 58K domain, the mutations in the NTBM apparently do not inte rfere with this interaction. The Asp-545 to Pro substitution left inta ct the binding properties of the 84K precursor of the 58K protein, wit h respect to ATP-agarose, whereas the Lys-500 to Thr substitution decr eased the binding capacity of the 84K protein, suggesting that the Lys -500 residue is directly involved in ATP binding. The Lys-500 to Thr s ubstitution in the 58K domain resulted in an altered distribution of v iral proteins, which failed to aggregate into large cytopathic structu res as observed in protoplasts infected with wild-type B-RNA. However viral proteins containing the Asp-545 to Pro substitution showed a nor mal distribution in protoplasts.