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Proteolytic processing and membrane association of tomato ringspot nepovirus rna-1-encoded proteins Wang, Aiming


Tomato ringspot nepovirus (TomRSV) encapsidates a bipartite, messenger sense RNA genome. RNA-1 is likely to encode all the viral proteins required for RNA replication. The expression and processing strategies of the RNA-1-encoded polyprotein (PI) were studied. It was found that PI is intramolecularly proteolytically processed by a virus-encoded protease at five cleavage sites to release six end products, i.e. XI, X2, NTB, VPg, Pro and Pol. All PI cleavage sites were identified. The genomic organization of TomRSV RNA-1 is distinct from that of the closely related comoviruses and of other nepoviruses. In TomRSV, two mature proteins (XI and X2) are released from the region upstream of NTB whereas only one mature protein is released from the corresponding region in all other characterized nepo- and comoviruses. Viral RNA replication is thought to be carried out by a replication complex which is secured on cellular membranes by a virus-encoded protein. To test if the NTB-VPg protein which contains a predicted trans-membrane domain is the anchor protein, association of this protein with microsomal membranes was studied in vitro. It was found that the NTB-VPg protein is an integral membrane protein and that the predicted transmembrane domain at the C-terminus of NTB is required for the membrane-association. The association of the NTB-VPg protein with microsomal membranes results in the generation of glycosylated and signal peptidase-processed proteins. The importance of these modifications for viral RNA replication needs to be elucidated. The molecular variability of the RNA-1-encoded replication-related viral proteins including VPg, Pro and Pol, and of the RNA-2-encoded coat protein among five TomRSV isolates was studied. It was found that the nucleotide sequences and the deduced amino acid sequences of RNA-1 were more conserved than those of RNA-2. Amino acid substitutions were not found in all conserved motifs identified previously in the protease and polymerase domains. Furthermore, the VPg amino acid sequence was identical in all isolates studied despite some variations at the nucleotide sequence level. The above results are discussed in light of the possible strategies for processing of the PI polyprotein and for the formation of the replication complex on cellular membranes.

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