The 66-kDa cytoplasmic inclusion protein of tamarillo mosaic potyvirus was purified to near homogeneity using organic solvent clarification, differential centrifugation and sucrose density gradient centrifugation. ATPase and GTPase activities were shown to co-purify with the 66-kDa protein. ATPase activity was stimulated up to fivefold in the presence of 20 microM poly(A). The Km value for ATP hydrolysis (18 microM), was minimally affected upon addition of poly(A). In contrast, the Vmax value for ATP hydrolysis was increased fivefold by the addition of poly(A). Binding of RNA by the cytoplasmic inclusion protein was demonstrated by gel electrophoresis of ultraviolet cross-linked enzyme-RNA complexes. In the absence of added NTP, complexes between the cytoplasmic inclusion protein and single-stranded RNA species formed rapidly in the pH range 3-7, but not at pH 8 or 9. Binding to single-stranded RNA was markedly decreased by the addition of NaCl (10 mM), suggesting a weak association between RNA and enzyme. The cytoplasmic inclusion protein bound single-stranded RNA or partially double-stranded RNA duplexes with single-stranded overhangs of 35 bases and 81 bases, respectively, but did not bind 16-bp blunt-ended double-stranded RNA. RNA binding occurred in the absence of NTP (ATP, GTP, CTP or UTP), whereas dissociation of bound RNA occurred only in the presence of NTP. RNA duplex unwinding (helicase) activity of the enzyme was demonstrated in the presence of any of the above four NTPs using partially double-stranded RNA duplexes with 3' single-stranded overhangs. We propose that the cytoplasmic inclusion protein of tamarillo mosaic virus is an RNA helicase, which translocates in the 3' to 5' direction in an energy-dependent manner, unwinding double-stranded regions.