Potato virus X (PVX) replication is precisely regulated by regulatory viral sequences and by viral and/or host proteins. In a previous study, we identified a 54-kDa cellular tobacco protein that bound to a region within the first 46 nucleotides (nt) of the 5` non-translated region (NTR) of the viral genome. Optimal binding was dependent upon the presence of an ACCA sequence at nt 10-13. To identify host factors that bind to 5` NTR elements including AC-rich sequences as well as stem-loop 1 (SL1), we used northwestern blotting and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for peptide mass fingerprinting. We screened several host factors that might affect PVX replication and selected a candidate protein, Nicotiana tabacum WRKY transcription factor 1 (NtWRKY1). We used a Tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) system to investigate the role of NtWRKY1 in PVX replication. Silencing of WRKY1 in Nicotiana benthamiana caused lethal apical necrosis and allowed an increase in PVX RNA accumulation. This result could reflect the balancing of PVX accumulation in a systemic N. benthamiana host to maintain PVX survival and still produce a suitable appearance of mosaic and mottle symptoms. Our results suggest that PVX may recruit the WRKY transcription factor, which binds to the 5` NTR of viral genomic RNA and acts as a key regulator of viral infection.