Effects of homology length in the repeat region on minus-strand DNA transfer and retroviral replication.
Homology between the two repeat (R) regions in the retroviral genome mediates minus-strand DNA transfer during reverse transcription. We sought to define the effects of R homology lengths on minus-strand DNA transfer. We generated five murine leukemia virus (MLV)-based vectors that contained identical sequences but different lengths of the 3' R (3, 6, 12, 24 and 69 nucleotides [nt]); 69 nt is the full-length MLV R. After one round of replication, viral titers from the vector with a full-length downstream R were compared with viral titers generated from the other four vectors with reduced R lengths. Viral titers generated from vectors with R lengths reduced to one-third (24 nt) or one-sixth (12 nt) that of the wild type were not significantly affected; however, viral titers generated from vectors with only 3- or 6-nt homology in the R region were significantly lower. Because expression and packaging of the RNA were similar among all the vectors, the differences in the viral titers most likely reflected the impact of the homology lengths on the efficiency of minus-strand DNA transfer. The molecular nature of minus-strand DNA transfer was characterized in 63 proviruses. Precise R-to-R transfer was observed in most proviruses generated from vectors with 12-, 24-, or 69-nt homology in R, whereas aberrant transfers were predominantly used to generate proviruses from vectors with 3- or 6-nt homology. Reverse transcription using RNA transcribed from an upstream promoter, termed read-in RNA transcripts, resulted in most of the aberrant transfers. These data demonstrate that minus-strand DNA transfer is homology driven and a minimum homology length is required for accurate and efficient minus-strand DNA transfer.
Dang
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Department of Microbiology and Immunology, School of Medicine, West Virginia University, Morgantown, West Virginia 26506, USA.
Hu
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D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004279:DNA, Viral / Q000096:biosynthesis* / Q000235:genetics*; D005822:Genetic Vectors; D051379:Mice; D008979:Moloney murine leukemia virus / Q000235:genetics* / Q000502:physiology; D008980:Moloney murine sarcoma virus / Q000235:genetics* / Q000502:physiology; D016133:Polymerase Chain Reaction; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D020079:Terminal Repeat Sequences / Q000235:genetics*; D014158:Transcription, Genetic; D014779:Virus Replication
D004279:DNA, Viral; D012367:RNA, Viral