The potyvirus cylindrical inclusion (CI) protein, an RNA helicase required for genome replication, was analyzed genetically using alanine-scanning mutagenesis. Thirty-one mutations were introduced into the CI protein coding region of modified tobacco etch virus (TEV) genomes expressing either beta-glucuronidase or green fluorescent protein reporters. Twelve of the mutants were replication-defective in protoplast inoculation assays. Among the 19 replication-competent mutants, several possessed cell-to-cell or long-distance movement defects in tobacco plants. Two mutants, AS1 and AS8, were restricted to single cells in inoculated leaves despite genome amplification levels that were equivalent to that of parental virus. Other mutants, such as AS9 and AS14, were able to move cell to cell slowly but were debilitated in long-distance movement. These data provide genetic evidence for a direct role of CI protein in potyvirus intercellular movement, and for distinct roles of the CI protein in genome replication and movement. In combination with high-resolution ultrastructural analyzes and previous genetic data, these results support a model in which CI protein interacts directly with plasmodesmata and capsid protein-containing ribonucleoprotein complexes to facilitate potyvirus cell-to-cell movement.