Helper component proteinases (HC-Pro) are multifunctional potyvirus proteins involved in transmission of polyproteins by aphids. HC-Pro functions include suppression of antiviral RNA silencing and interfering with host symptom expression. Affinity purification-mass spectrometry was used in the present study of in vitro interactions between a recombinant Papaya ringspot virus HC-Pro protein and 23 papaya proteins extracted from the leaves of healthy and virus-infected papaya and identified by liquid chromatography–tandem mass spectrometry. Ten of the identified proteins were found to be involved in plant defense mechanisms (lipoxygenase and TIR-NBS disease resistance-like protein), photosynthesis (bisphosphate carboxylase large chain and red chlorophyll catabolite reductase), transcription (reverse transcriptase), translation (chromosome associate protein subunit h), signal transduction (membrane protein and calcium-dependent lipid-binding-like protein) and carbohydrate metabolism (sucrose-UDP glucosyltransferase and β-1,4-mannosyltransferase). In addition, the interactions between Papaya ringspot virus HC-Pro protein and host proteins may not only provide a better understanding of how viruses replicate and cause disease, but also help elucidate the molecular mechanisms underlying its various functions.