Endogenous origins of HIV-1 G-to-A hypermutation and restriction in the nonpermissive T cell line CEM2n

Abstract

The DNA deaminase APOBEC3G converts cytosines to uracils in retroviral cDNA, which are immortalized as genomic strand G-to-A hypermutations by reverse transcription. A single round of APOBEC3G-dependent mutagenesis can be catastrophic, but evidence suggests that sublethal levels contribute to viral genetic diversity and the associated problems of drug resistance and immune escape. APOBEC3G exhibits an intrinsic preference for the second cytosine in a 5'CC dinucleotide motif leading to 5'GG-to-AG mutations. However, an additional hypermutation signature is commonly observed in proviral sequences from HIV-1 infected patients, 5'GA-to-AA, and it has been attributed controversially to one or more of the six other APOBEC3 deaminases. An unambiguous resolution of this problem has been difficult to achieve, in part due to dominant effects of protein over-expression. Here, we employ gene targeting to dissect the endogenous APOBEC3 contribution to Vif-deficient HIV-1 restriction and hypermutation in a nonpermissive T cell line CEM2n. We report that APOBEC3G-null cells, as predicted from previous studies, lose the capacity to inflict 5'GG-to-AG mutations. In contrast, APOBEC3F-null cells produced viruses with near-normal mutational patterns. Systematic knockdown of other APOBEC3 genes in an APOBEC3F-null background revealed a significant contribution from APOBEC3D in promoting 5'GA-to-AA hypermutations. Furthermore, Vif-deficient HIV-1 restriction was strong in parental CEM2n and APOBEC3D-knockdown cells, partially alleviated in APOBEC3G- or APOBEC3F-null cells, further alleviated in APOBEC3F-null/APOBEC3D-knockdown cells, and alleviated to the greatest extent in APOBEC3F-null/APOBEC3G-knockdown cells revealing clear redundancy in the HIV-1 restriction mechanism. We conclude that endogenous levels of APOBEC3D, APOBEC3F, and APOBEC3G combine to restrict Vif-deficient HIV-1 and cause the hallmark dinucleotide hypermutation patterns in CEM2n. Primary T lymphocytes express a similar set of APOBEC3 genes suggesting that the same repertoire may be important in vivo.

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).

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.