The preference of MuLV for the selection of tRNAPro as a replication primer was investigated by altering the primer-binding site (PBS) to be complementary to tRNALys,3. MuLV-based vectors with a PBS complementary to tRNALys,3 were found to be approximately 2-fold less infectious than vectors with the wild-type PBS complementary to tRNA Pro. MuLV with a PBS complementary to tRNALys,3 was replication competent and maintained the PBS during early stages of in vitro culture. Upon extended culture, PBS were isolated which were complementary to tRNAArg. A second MuLV was generated in which the region upstream of the PBS which is predicted to form an RNA stem loop structure was altered so that the nucleotide sequence within the loop would be complementary to the anticodon of tRNALys,3. The virus with both the U5 and PBS complementary to tRNALys,3 was also replication competent. Upon extended in vitro culture though, this virus reverted to utilize tRNA Lys1,2. Analysis of the infectivity and replication of the wild-type and mutant viruses revealed that tRNAPro was the preferred tRNA for high-level replication. Viruses with a PBS complementary to tRNAArg or tRNALy1,2 replicated at levels approximately 30% and 10% as effective as the wild-type virus, while virus with a PBS complementary to tRNALys,3 had the slowest replication kinetics and least infectivity. Comparison of the virion tRNA content of the wild-type and mutant viruses revealed similar ratios with respect to levels of tRNAPro, tRNA Arg and tRNALys. Modeling of the U5-PBS region revealed that the predicted RNA structure for the virus that selected tRNAArg was more similar to the wild type virus that uses tRNAPro than the virus which use tRNALys1,2 or tRNALys,3; the virus that uses tRNALys,3 had the most profound disruption in the predicted RNA structure. The results of these studies demonstrate that MuLV has evolved to preferentially select tRNAPro for high-level replication and are discussed with respect to common features of the primer selection process between MuLV and other retroviruses. © 2005 Elsevier Inc. All rights reserved.