In planta vs viral expression of HCPro affects its binding of nonplant 21-22 nucleotide small RNAs, but not its preference for 5'-terminal adenines, or its effects on small RNA methylation.

Previous studies have found a correlation between the abilities of PVX vector-expressed HCPro variants to bind small RNAs (sRNAs), and to suppress silencing. Moreover, HCPro preferred to bind viral sRNAs of 21-22 nucleotides (nt) containing 5'-terminal adenines. This would require such viral sRNAs to have either different access to the suppressor than those of plant sequences, or different molecular properties. To investigate this preference further, we have used suppressor-competent or suppressor-deficient HCPro variants, expressed from either T-DNAs or potyvirus constructs. Then, the sRNAs generated in plants and associated with the purified HCPro variants were characterized. Marked differences were observed in the ratios of sRNAs of plant vs nonplant origin that bound to suppressor-competent HCPro, depending on the mode of its expression. Regardless of the means of expression, HCPro retained the same preference among the nonplant sRNAs of 21-22 nt for those with 5'-terminal adenines. Relative methylation levels of individual sRNAs were assessed, and the nonplant sRNAs were found to be significantly less methylated in the presence of the suppressor. Targeted binding of sRNAs based on size, 5'-terminal sequence and origin, together with affecting their methylation, could explain how HCPro counteracts silencing.