In this study we have performed a mutational analysis of the cowpea mosaic comovirus (CPMV) genome-linked protein VPg to discern the structural requirements necessary for proper functioning of VPg. Either changing the serine residue linking VPg to RNA at a tyrosine or a threonine or changing the position of the serine from the N-terminal end to position 2 or 3 abolished virus infectivity. Some of the mutations affected the cleavage between the VPg and the 58K ATP-binding protein in vitro, which might have contributed to the lethal phenotype. RNA replication of some of the mutants designed to replace VPg with the related cowpea severe mosaic comovirus was completely abolished, whereas replication of others was not affected or only mildly affected, showing that amino acids that are not conserved between the comoviruses can be critical for the function of VPg. The replicative proteins of one of the mutants failed to accumulate in typical cytopathic structures and this might reflect the involvement of VPg in protein–protein interactions with the other replicative proteins.