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Comparative Study
. 2008 Jul;82(13):6536-45.
doi: 10.1128/JVI.00554-08. Epub 2008 Apr 30.

Comparison of G-to-A mutation frequencies induced by APOBEC3 proteins in H9 cells and peripheral blood mononuclear cells in the context of impaired processivities of drug-resistant human immunodeficiency virus type 1 reverse transcriptase variants

Stefanie Andrea Knoepfel  1 , Nadine Christina Salisch, Peter Michael Huelsmann, Pia Rauch, Hauke Walter, Karin Jutta Metzner
Affiliations
Comparative Study

Comparison of G-to-A mutation frequencies induced by APOBEC3 proteins in H9 cells and peripheral blood mononuclear cells in the context of impaired processivities of drug-resistant human immunodeficiency virus type 1 reverse transcriptase variants

Stefanie Andrea Knoepfel et al. J Virol. 2008 Jul.

Abstract

APOBEC3 proteins can inhibit human immunodeficiency virus type 1 (HIV-1) replication by inducing G-to-A mutations in newly synthesized viral DNA. However, HIV-1 is able to overcome the antiretroviral activity of some of those enzymes by the viral protein Vif. We investigated the impact of different processivities of HIV-1 reverse transcriptases (RT) on the frequencies of G-to-A mutations introduced by APOBEC3 proteins. Wild-type RT or the M184V, M184I, and K65R+M184V RT variants, which are increasingly impaired in their processivities, were used in the context of a vif-deficient molecular HIV-1 clone to infect H9 cells and peripheral blood mononuclear cells (PBMCs). After two rounds of infection, a part of the HIV-1 env gene was amplified, cloned, and sequenced. The M184V mutation led to G-to-A mutation frequencies that were similar to those of the wild-type RT in H9 cells and PBMCs. The frequencies of G-to-A mutations were increased after infection with the M184I virus variant. This effect was augmented when using the K65R+M184V virus variant (P < 0.001). Overall, the G-to-A mutation frequencies were lower in PBMCs than in H9 cells. Remarkably, 38% +/- 18% (mean +/- standard deviation) of the env clones derived from PBMCs did not harbor any G-to-A mutation. This was rarely observed in H9 cells (3% +/- 3%). Our data imply that the frequency of G-to-A mutations induced by APOBEC3 proteins can be influenced by the processivities of HIV-1 RT variants. The high number of nonmutated clones derived from PBMCs leads to several hypotheses, including that additional antiretroviral mechanisms of APOBEC3 proteins other than their deamination activity might be involved in the inhibition of vif-deficient viruses.

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Figures

FIG. 1.
FIG. 1.
Replication kinetics of HIV-1 NL4-3 and HIV-1 NL4-3Δvif harboring different RT variants in H9 and MT4 cells. Shown are the results of a representative example of three independent experiments. Supernatants from uninfected cells served as negative controls and are indicated by crosses. (A) H9 cells were infected with HIV-1 NL4-3 (black squares), HIV-1 NL4-3M184V (black circles), HIV-1 NL4-3M184I (black diamonds), HIV-1 NL4-3Δvif (open squares), HIV-1 NL4-3ΔvifM184V (open circles), and HIV-1 NL4-3ΔvifM184I (open diamonds). (B) MT4 cells were infected with all six HIV-1 variants listed above, HIV-1 NL4-3K65R+M184V (black triangles), and HIV-1 NL4-3ΔvifK65R+M184V (open triangles). Supernatants were collected daily or every second day and assayed for viral p24 antigen by enzyme-linked immunosorbent assay.
FIG. 2.
FIG. 2.
Total mutation frequencies of viral DNA do not differ in H9 cells and PBMCs infected with HIV-1 NL4-3 harboring different RT variants. (A) H9 cells and PBMCs were infected with HIV-1 NL4-3 containing vif and the different RT variants, wild-type RT, M184V, M184I, and K65R+M184V. After the second round of infection, viral DNA was isolated and 466 nucleotides of the env gene were amplified, cloned, and sequenced. The percentages of HIV-1 env clones carrying the indicated numbers of mutations are shown. The numbers of clones analyzed are given. Two independent experiments using H9 cells were performed, and PBMCs from two different donors, A and B, were used. The infection with HIV-1 NL4-3K65R+M184V was only performed in PBMCs from donor B. (B) Preferences of nucleotide substitutions in HIV-1 env clones in H9 cells and PBMCs infected with HIV-1 NL4-3 harboring the different RT variants. The total numbers of mutations are indicated.
FIG. 3.
FIG. 3.
Impact of impaired processivities of HIV-1 RT variants and the cell type on the G-to-A mutation frequencies induced by APOBEC3 proteins after infection with HIV-1 NL4-3Δvif variants. Shown are summarized results of two independent experiments using H9 cells and of independent experiments using PBMCs from four different donors, A to D. Cells were infected with HIV-1 NL4-3Δvif, HIV-1 NL4-3ΔvifM184V, and HIV-1 NL4-3ΔvifM184I. PBMCs from donors B to D were also infected with HIV-1 NL4-3ΔvifK65R+M184V. Viral DNA was isolated after the second round of infection, and 466 nucleotides of the env gene were cloned, sequenced, and analyzed. (A) The percentages of HIV-1 env clones carrying the indicated numbers of G-to-A mutations are shown. The numbers of clones analyzed are given. (B) Preferences of nucleotide substitutions in HIV-1 env clones in H9 cells and PBMCs infected with HIV-1 NL4-3Δvif harboring different RT variants. The total numbers of mutations are indicated. (C) Comparison of G-to-A mutation frequencies in HIV-1 env sequences harboring G-to-A mutations and generated from H9 cells and PBMCs after infection with HIV-1 NL4-3Δvif, HIV-1 NL4-3ΔvifM184V, HIV-1 NL4-3ΔvifM184I, and HIV-1 NL4-3ΔvifK65R+M184V. Error bars show standard deviations. WT, wild type. Asterisks represent statistical significance within both groups, i.e., H9 cell- and PBMC-derived viral env sequences. *, P < 0.05; ***, P < 0.001.
FIG. 4.
FIG. 4.
G-to-A mutation spectrum of viral DNA in H9 cells and PBMCs. Shown are nucleotides 7057 to 7522 of the 466-bp env fragment of HIV-1 NL4-3, which was cloned and sequenced. The V3 and V4 loops are underlined, and all first guanine residues of the GG motifs, which are preferentially targeted by APOBEC3G, are depicted in bold. Given in red are the percentages of guanine residues mutated to adenine in a total of 290 HIV-1 env sequences obtained by the infection of H9 cells (95 clones) and PBMCs from four different donors (195 clones) harboring at least one G-to-A mutation. Percentages of G-to-A mutations within GA motifs, which are the preferred motifs of APOBEC3F, are depicted in black when more than 5% of those motifs have been mutated.
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