Synthesis of full-length viral DNA in CD4-positive membrane vesicles exposed to HIV-1. A model for studies of early stages of the hiv-1 life cycle.
CD4-positive membrane vesicles (MV) were isolated under isotonic conditions from human T lymphoblastoid cells MT-2 and CEM and tested for their ability to support reverse transcription of viral RNA upon exposure to human immunodeficiency virus, type 1 (HIV-1). MV contained cytoplasms as confirmed by the presence of mitochondrial DNA but were devoid of chromosomal DNA. Virus binding and vesicle lysis assays revealed that 4-19% (depending upon virus dose) of MV-bound HIV-1 entered the vesicles. HIV-1 internalized in MV was able to initiate and complete viral DNA synthesis as determined by the detection of products of reverse transcription using polymerase chain reaction amplification of viral DNA using regions present in early (strong stop) transcripts and full-length double-stranded molecules. Viral DNA was undetectable in MV exposed to HIV-1 at 0 degrees C, in MV exposed to UV-inactivated virus at 37 degrees C, or after exposure to intact virus at 37 degrees C in the presence of reverse transcriptase inhibitors 2',3'-dideoxycytidine and a tetrahydroimidazo[4,5,1-jk](1,4)-benzodiazepin-2-(1H)-thione derivative, indicating that viral DNA detected in HIV-1-exposed MV was synthesized de novo. Kinetic studies revealed that HIV-1 DNA synthesis in MV was very rapid; full-length viral DNA was detected within 15 min of exposure at 37 degrees C, and the DNA levels increased 90-fold after 1 h and declined thereafter. Strong stop viral DNA was 10-fold more abundant than full-length DNA after 1 h at 37 degrees C, indicating that 10% of input viral genomes are fully transcribed in MV within this time frame. This system preserves the critical features of intact CD4-bearing cells to permit studies of HIV-1 entry, uncoating, and reverse transcription of viral RNA.
Simm
M
M
Molecular Virology Laboratory, St. Luke's-Roosevelt Hospital Center and College of Physicians & Surgeons, Columbia University, New York, New York 10019, USA. djv@columbia.edu
Pekarskaya
O
O
Volsky
D J
DJ
D015496:CD4-Positive T-Lymphocytes / Q000821:virology*; D002462:Cell Membrane / Q000821:virology; D004261:DNA Replication; D004279:DNA, Viral / Q000096:biosynthesis*; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D015497:HIV-1 / Q000502:physiology*; D006801:Humans; D007700:Kinetics; D016133:Polymerase Chain Reaction; D014779:Virus Replication
D004279:DNA, Viral