Frequent dual initiation of reverse transcription in murine leukemia virus-based vectors containing two primer-binding sites.
Retroviruses package two copies of viral RNA into each virion. Although each RNA contains a primer-binding site for initiation of DNA synthesis, it is unknown whether reverse transcription is initiated on both RNAs. To determine whether a single virion is capable of initiating reverse transcription more than once, we constructed a murine leukemia virus-based vector containing a second primer-binding site (PBS) derived from spleen necrosis virus and inserted the green fluorescent protein gene (GFP) between the two PBSs. Initiation of reverse transcription at either PBS results in a provirus that expresses GFP. However, initiation at both PBSs can result in the deletion of GFP, which can be detected by flow cytometry and Southern blotting analysis. Approximately 22-29% of the proviruses formed deleted the GFP in a single replication cycle, indicating the minimum proportion of virions that initiated reverse transcription on both PBSs. These results show that a significant proportion of MLV-based vectors containing two PBSs have the capacity to initiate reverse transcription more than once.
Voronin
Yegor A
YA
HIV Drug Resistance Program, National Cancer Institute-Frederick, Frederick, MD 21702, USA.
Pathak
Vinay K
VK
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D004261:DNA Replication; D005786:Gene Expression Regulation; D005822:Genetic Vectors / Q000235:genetics*; D009052:Leukemia Virus, Murine / Q000235:genetics*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012313:RNA / Q000235:genetics / Q000378:metabolism*; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic
C034477:RNA primers; D012367:RNA, Viral; D012313:RNA