Journal: Journal of Virology
Article Title: Infectious Molecular Clones with the Nonhomologous Dimer Initiation Sequences Found in Different Subtypes of Human Immunodeficiency Virus Type 1 Can Recombine and Initiate a Spreading Infection In Vitro
Figure Lengend Snippet: (A) Recombination between heterologous RNA during replication results in four proviral forms, the two parental forms, NL4-3Δ pol and NL4-3Δ env , and two recombinant forms, wild-type NL4-3 (NL4-3WT) and NL4-3Δ pol/Δenv . Arrows denote oligonucleotides used for PCR amplification (Rec1, Rec2, DIS1, and DIS2) and hybridization (Std*, Pol *, and ENV *) and the direction of sequence complementarity. Oligonucleotides marked with asterisks are probes for detecting PCR products. Hatch marks denote unrepresented sequences in HIV-1. Long terminal repeat (LTR) and structural genes are shown. (B) Provirus resulting from a single-cycle infection of SupT1 cells. Wild-type NL4-3 DIS (CG) and virus generated by cotransfection of NL4-3Δ pol DIS (CG) and NL4-3Δ env DIS (CG) were used to infect SupT1 cells. Lysates of infected cells were amplified with primers Rec1 and Rec2, and proviral sequences were identified by hybridization with Std*, Pol *, and ENV * probes. Molecular weights of the PCR products confirm expected recombination products. (C) DNA recombination during virus production. Hirt supernatants from 293 cells transfected with no DNA, pNL4-3Δ env DIS (CG), pNL4-3Δ env DIS (CG) and NL4-3Δ pol DIS (CG), NL4-3Δ pol DIS (CG), or NL4-3 DIS (CG) were amplified with primers Rec1 and Rec2, and proviral sequences were identified by hybridization with the Std* probe. Lysate of SupT1 cells infected (Inf) with virus generated by cotransfection of NL4-3Δ pol DIS (CG) and NL4-3Δ env DIS (CG) was amplified as a control. WT, wild type.
Article Snippet: The PCR mixture contained 250 nM primers Rec1 and Rec2, 350 μM deoxynucleoside triphosphate, 1.75 mM MgCl2 , 50 mM Tris-HCl (pH 9.2), 16 mM (NH4 )2 SO4 , and 3.5 U of polymerase (Boehringer Mannheim).
Techniques: Recombinant, Polymerase Chain Reaction, Amplification, Hybridization, Sequencing, Infection, Generated, Cotransfection, Transfection