Small proteins called viral protein genome-linked (VPg), attached to the 5′-end of the viral RNA genome are found as common structure in the large family of picornaviruses. The replication of these viruses is primed by this VPg protein linked to a single uridylyl residue. We report a general procedure to obtain such nucleoproteins employing a pre-uridylylated tyrosine building block in an on-line solid phase-based approach. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd. The replication of picornaviruses is primed by a small so-called VPg protein linked to a uridylyl residue. We report a general procedure to obtain such nucleoproteins employing a pre-uridylylated tyrosine building block in an on-line solid phase based approach.