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. 1999 Sep;37(9):2943–2951. doi: 10.1128/jcm.37.9.2943-2951.1999

FIG. 1.

FIG. 1

HIV-1 sequences in cloning vectors for antiretroviral resistance testing. (A) pJM11ΔGPR is used to clone HIV-1 sequences of Gag p7/p1 and Gag p1/p6 cleavage sites and PR (GPR). pJM11ΔGPR and the HIV-1 PCR product amplified with insert PCR primers Apa1988 and Sse2835 (in gray) are digested with restriction enzymes ApaI and Sse8387I and subsequently are ligated with T4 DNA ligase. Recombinant clones are cotransfected with p83-10 (6) into MT-2 cells to produce infectious virus. A polylinker (not shown) including the only XbaI and BamHI restriction sites in the plasmid substitutes for the deleted HIV-1 sequence in each of the vectors depicted in Fig. 1. Plasmid sequences are not shown for this or any other vector. (B) pJM14ΔRT is used to clone HIV-1 RT. pJM14ΔRT and the HIV-1 DNA PCR product amplified with insert PCR primers 2574U29 and 4120L35 (in gray) are digested with restriction enzymes XmaI and PacI and subsequently are ligated with T4 DNA ligase. Recombinant clones are cotransfected with p83-10 (6) into MT-2 cells to produce infectious virus. (C) pJM13ΔGPRT is used to clone HIV-1 sequences encoding Gag p7/p1 and Gag p1/p6 cleavage sites, PR, and RT (GPRT). pJM13ΔGPR and pJM20ΔGPRT are used to clone an HIV-1 PCR product amplified with the same insert PCR primers, 5CAI1964B and 3CAI4155LIG (in gray). pJM13ΔGPRT and the PCR product are digested with ApaI and PacI and subsequently are ligated with T4 DNA ligase. Recombinant clones are cotransfected with p83-10 (6) into MT-2 cells to produce infectious virus. (D) pJM20ΔGPRT is used to clone HIV-1 sequences encoding Gag p7/p1 and Gag p1/p6 cleavage sites, PR, and RT (GPRT). pJM20ΔGPRT is used for HIV-1 sequence-specific UDG cloning. It is digested with BamHI and then amplified with vector PCR primers 3CAV1982 and 5V4142 (see Fig. 2). Vector and insert (in gray) PCR products are digested with UDG and directly used to transform E. coli. Recombinant clones are cotransfected with pJM31ΔGPRT into MT-2 cells to produce infectious virus. (E) pJM31ΔGPRT is used to generate either pooled or molecularly cloned infectious HIV-1 with patient specimen-derived sequences of Gag p7/p1 and Gag p1/p6 cleavage sites, PR, and RT (GPRT). pJM31ΔGPRT digested with BamHI and the HIV-1 insert PCR product amplified with primers 1811U24 and 4522L24 (in gray) is directly cotransfected into MT-2 cells to produce infectious virus as a pool of different variants. A reconstructed pJM20GPRT plasmid is cotransfected with pJM31ΔGPRT into MT-2 cells to produce a cloned infectious virus.