Figure 1.
CEM2n is a near-diploid, non-permissive T cell line.
(A) Flow cytometric analysis of fixed CEM2n (red) and CEM4n (gray) cells stained with propidium iodide. (B) Contour plot of CD4 and CXCR4 levels in CEM2n. (C) HIV spreading infection profiles in CEM2n as monitored by periodic infection of an LTR-GFP reporter cell line, CEM-GFP. (D) Giemsa-banding karyotype of a representative CEM2n metaphase spread. Red arrows indicate typical lesions in lymphoblastic leukemia.
Figure 2.
Construction and characterization of A3G-Null CEM2n cells.
(A) A3G exon 3 targeting strategy. LA, left homology arm; SA, splice acceptor; IRES, internal ribosomal entry site; Neo, G418 resistance gene; pA, poly adenylation signal; RA, right homology arm; yellow triangles, loxP sites. (B) A3 mRNA expression profiles of the indicated cells relative to parental CEM2n (mean and s.d. shown for triplicate experiments). (C) Immunoblots of A3G, A3F, and tubulin (TUB) in the indicated cells. (D) Infectivity of Vif-deficient HIV produced using the indicated cell lines following a single replicative cycle (mean and s.d. shown for p24-normalized triplicate experiments). (E) 3D-PCR profiles of HIV gag-pol and cellular MDM2 targets within genomic DNA of infected CEM-GFP reporter cells. (F) HIV G-to-A mutation profiles of proviruses originating in the indicated cell types. The mutation frequency at each dinucleotide is illustrated as a pie chart wedge (n≥15 kb per condition).
Table 1.
Gene targeting statistics in CEM2n.
Table 2.
Mutation summary.
Figure 3.
Construction and characterization of A3F-Null CEM2n cells.
(A) A3F exon 3–4 targeting strategy. LA, left homology arm; SA, splice acceptor; IRES, internal ribosomal entry site; Neo, G418 resistance gene; pA, poly adenylation signal; RA, right homology arm; yellow triangles, loxP sites. (B) A3 mRNA expression profiles of the indicated cells relative to parental CEM2n (mean and s.d. shown for triplicate experiments). (C) Immunoblots of A3F, A3G, and tubulin (TUB) in the indicated cells. (D) Infectivity of Vif-deficient HIV produced using the indicated cell lines following a single replicative cycle (mean and s.d. shown for p24-normalized triplicate experiments). (E) 3D-PCR profiles of HIV gag-pol and cellular MDM2 targets within genomic DNA of infected CEM-GFP reporter cells. (F) HIV G-to-A mutation profiles of proviruses originating in the indicated cell types. The mutation frequency at each dinucleotide is illustrated as a pie chart wedge (n≥15 kb per condition).
Figure 4.
Construction and characterization of A3F-Null/A3-Knockdown CEM2n cells.
(A) Levels of each indicated A3 mRNA in CEM2n or A3F-null cells transduced with shNS, shA3B, shA3C, shA3D, shA3G, or shA3H constructs (mean and s.d. shown for triplicate experiments). (B) Immunoblots of A3G and tubulin (TUB) in CEM2n or A3F-null cells stably transduced with the indicated shRNA-expressing lentivirus. (C) Infectivity of Vif-deficient HIV produced using the indicated transduced cell pool and reported using the CEM-GFP system (mean and s.d. shown for p24-normalized triplicate experiments; in some instances, the error is nearly indistinguishable from the histogram bar outline). (D) 3D-PCR profiles of HIV gag-pol and cellular MDM2 targets within genomic DNA of infected CEM-GFP reporter cells. (E) HIV G-to-A mutation profiles of proviruses originating in the indicated cell types. The mutation frequency at each dinucleotide is illustrated as a pie chart wedge (n≥15 kb per condition). Pie charts were generated for those conditions with ≥1 mutation per kb analyzed. Mutation numbers for all conditions can be found in Table 2 and Table S1.
Figure 5.
Characterization of independent knockout and knockdown clones.
(A) Levels of each indicated A3 mRNA in CEM2n, A3G- and A3F-null derivatives, and CEM-SS (relative to TBP; mean and s.d. shown for triplicate experiments). (B) Levels of each indicated A3 mRNA in CEM2n or A3F-null cells transduced with shNS, shA3D, or shA3G constructs (relative to TBP; mean and s.d. shown for triplicate experiments). (C) Single-cycle infectivity of Vif-deficient HIV produced in parallel in the indicated cell lines (mean and s.d. shown for p24-normalized triplicate experiments). (D) The kinetics of Vif-proficient (blue diamonds) and Vif-deficient (red squares) HIV spreading infection in the indicated cell lines. Numbers distinguish independent clones.