No abstract

We have previously provided evidence that amino acid substitutions within the N-terminal portion of the helper component-proteinase (HC-Pro) from tobacco vein mottling virus (TVMV), in particular at Lys-307, not only affect the aphid transmission activity of HC-Pro but also have a significant effect on TVMV virulence. In the present study amino acids which differ in their charge properties were substituted at position 307. A highly basic residue was required to retain helper component activity and virulence. Deletion and insertion mutagenesis in the 5' terminus of the HC-Pro gene suggested that this RNA domain may be an essential element for TVMV infectivity. Replacement of the TVMV HC-Pro gene with that from another potyvirus, zucchini yellow mosaic virus, maintained infectivity and aphid transmissibility of the chimeric virus, although symptoms were attenuated. Our results suggest that, in addition to its importance in aphid transmission, the HC-Pro gene may be of general importance in regulating virulence of potyviruses, possibly by interaction of these sequences with the host.

No abstract

Amino acids near the N terminus of the coat protein of tobacco vein mottling virus were deleted or altered by site-directed mutagenesis to determine the effect on aphid transmissibility of the virus. Deletion of a three amino acid sequence Asp-Ala-Gly, which is conserved in aphidtransmissible potyvirus isolates, abolished transmission. The mutation Ala --Thr in this triplet drastically reduced transmission, whereas the mutation Asp --Asn had no effect, and the mutation Asp -' Lys consistently reverted to the wild-type residue. The mutation Lys--Glu, in the residue adjacent to the glycine ofthe triplet, drastically reduced transmission, whereas the mutation Gin -+ Pro, seven residues downstream from the glycine had no effect. Comparison of the sequences of other potyviruses suggests that the presence ofa glycine residue at the third position of the Asp-Ala-Gly triplet is critical for aphid transmissibility and that certain changes in the residues adjacent to this position abolish or greatly reduce aphid transmissibility.

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.