title: High-Frequency Illegitimate Strand Transfers of Nascent DNA Fragments During Reverse Transcription Result in Defective Retrovirus Genomes.
abstract: Two strand transfers of nascent DNA fragments during reverse transcription are required for retrovirus replication. However, whether strand transfers occur at illegitimate sites and how this may affect retrovirus replication are not well understood.
  
  
  
  The reverse transcription was carried out with reverse transcriptases (RTs) from HIV-1, HIV-2, and murine leukemia virus. The nascent complementary DNA fragments were directly cloned without polymerase chain reaction amplification. The sequences were compared with the template sequence to determine if new sequences contained mismatched sequences caused by illegitimate strand transfers.
  
  
  
  Among 1067 nascent reverse transcript sequences, most of them (72%) matched to the template sequences, although they randomly stopped across the RNA templates. The other 28% of them contained mismatched 3'-end sequences because of illegitimate strand transfers. Most of the illegitimate strand transfers (81%) were disassociated from RNA templates and realigned onto opposite complementary DNA strands. Up to 3 strand transfers were detected in a single sequence, whereas most of them (93%) contained 1 strand transfer. Because most of the illegitimate strand-transfer fragments were generated from templates at 2 opposite orientations, they resulted in defective viral genomes and could not be detected by previous methods. Further analysis showed that mutations at pause/disassociation sites resulted in significantly higher strand-transfer rates. Moreover, illegitimate strand-transfer rates were significantly higher for HIV-2 RT (38.2%) and murine leukemia virus RT (44.6%) than for HIV-1 RT (5.1%).
  
  
  
  Illegitimate strand transfers frequently occur during reverse transcription and can result in a large portion of defective retrovirus genomes.
authors.affiliation: *School of Life Sciences, Jilin University, Changchun, China; †National Engineering Laboratory for AIDS Vaccine, Jilin University, Changchun, China; ‡Key Laboratory for Molecular Enzymology and Engineering, Jilin University, Changchun, China; and §Duke Human Vaccine Institute, Duke University Medical Center, Durham, NC.
authors.forename: Xiaojun
authors.initials: X
authors.lastname: Li
authors.forename: Peihu
authors.initials: P
authors.lastname: Fan
authors.forename: Chunlai
authors.initials: C
authors.lastname: Jiang
authors.forename: Tonghui
authors.initials: T
authors.lastname: Ma
authors.forename: Xianghui
authors.initials: X
authors.lastname: Yu
authors.forename: Wei
authors.initials: W
authors.lastname: Kong
authors.forename: Feng
authors.initials: F
authors.lastname: Gao
mesh_terms: D004261:DNA Replication; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D004277:DNA, Single-Stranded / Q000096:biosynthesis* / Q000235:genetics*; D016679:Genome, Viral / Q000235:genetics*; D015497:HIV-1 / Q000201:enzymology / Q000235:genetics*; D015498:HIV-2 / Q000201:enzymology / Q000235:genetics*; D009052:Leukemia Virus, Murine / Q000201:enzymology / Q000235:genetics*; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D048348:Reverse Transcription; D014779:Virus Replication
chemical_list: D018076:DNA, Complementary; D004277:DNA, Single-Stranded; D012194:RNA-Directed DNA Polymerase