Dual inhibitory effects of APOBEC family proteins on retrotransposition of mammalian endogenous retroviruses

Abstract

We demonstrated previously that the cytosine deaminase APOBEC3G inhibits retrotransposition of two active murine endogenous retroviruses, namely intracisternal A-particles (IAP) and MusD, in an ex vivo assay where retrotransposition was monitored by selection of neo-marked elements. Sequencing of the transposed copies further disclosed extensive editing, resulting in a high load of G-to-A mutations. Here, we asked whether this G-to-A editing was associated with an impact of APOBEC3G on viral cDNA yields. To this end, we used a specially designed quantitative PCR method to selectively measure the copy number of transposed retroelements, in the absence of G418 selection. We show that human APOBEC3G severely reduces the number of MusD and IAP transposed cDNA copies, with no effect on the level of the intermediate RNA transcripts. The magnitude of the decrease closely parallels that observed when transposed copies are assayed by selection of G418-resistant cells. Moreover, sequencing of transposed elements recovered by PCR without prior selection of the cells reveals high-level editing. Using this direct method with a series of cytosine deaminases, we further demonstrate a similar dual effect of African green monkey APOBE3G, human APOBEC3F and murine APOBEC3 on MusD retrotransposition, with a distinct extent and site specificity for each editing activity. Altogether the data demonstrate that cytosine deaminases have a protective effect against endogenous retroviruses both by reducing viral cDNA levels and by introducing mutations in the transposed copies, thus inactivating them for subsequent rounds of retrotransposition. This dual, two-step effect likely participates in the efficient defense of the cell genome against invading endogenous retroelements.

Figure 1

Rationale of the retrotransposition assay. (A) Schematic representation of the retrotransposition assay and experimental procedure for detection of retrotransposition. A retroelement marked with the neo reporter gene driven by its own promoter (Pr) and placed in backward orientation (gray boxes) is introduced by transfection into HeLa cells, together with or without an APOBEC expression vector. The neo gene is rendered inactive by the presence of a forward intron, which is spliced out of the RNA intermediate, resulting in a functional gene after reverse transcription and integration, allowing detection of retrotransposition. Cell RNA and DNA are extracted before G418 selection, as indicated. (B) Structure of the neo gene in the marked elements before and after retrotransposition, with the specific primers used for quantitative PCR analysis and the size of the expected PCR fragments indicated. (C) Quantitative PCR analysis of retrotransposed marked elements: (upper) ethidium bromide-stained agarose gel of the PCR products obtained using the primers shown in (B) and cellular DNA from Hela cells transfected with a marked IAP, with the 643 bp fragment specific for the retrotranscribed spliced copies and the 725 bp fragment for the non-transposed parental copies indicated; (lower) plot of the intensity of the 643 bp fragment against PCR cycle number; inset, linear fit using a semi-log scale, yielding an increment value per cycle of 1.75. The 725 bp band, as well as other spurious bands (e.g. ∼0.4 and ∼0.9 kb), were of variable intensity depending on the experiments, sometimes being hardly detectable (see text and Figure 2). The presence of such parasite bands clearly illustrates why standard real-time qPCR could not be used.

Figure 2

Effects of hA3G on the amount of MusD, IAP and L1 transposed copies and intermediate RNAs. (A) Quantitative analysis of the effects of human hA3G on the number of retrotransposed MusD, IAP and L1 neo-marked elements. HeLa cells were transfected with 1.5 µg of the indicated marked retrotransposon, in the presence or absence of 1.5 µg of hA3G expression vector. DNA was extracted 2 days post-transfection, and PCR was carried out with 0.5 µg of cellular DNA. The results are represented as in Figure 1C, with the band of the expected size (643 bp) for the retrotransposed copies indicated with black arrowheads. Data points in the intensity/cycle plots are the means of duplicate determinations, and each curve is representative of at least three independent experiments with a mean fold reduction of 13.6 ± 3.4 for MusD (n = 5), 3.4 ± 1.5 for IAP (n = 3) and no significant difference for L1 (n = 3). (B) hA3G has no effect on the retroelement transcript levels. RNAs were extracted 2 days post-transfection from the same cell populations as in (A) and reverse transcribed in vitro. Quantification of the cDNAs was performed as in (A). Data points in the plots are the means of duplicate determinations and the curves are representative of two independent experiments.

Figure 3

Differential effects of cytosine deaminases on the retrotransposition of MusD. Analysis of the effect of human hA3G, hAID, hA1, hA2, hA3B, hA3C, hA3F, African green monkey agmA3G and murine mA3 on MusD retrotransposition. (A) Measurement of the number of G418^R clones after selection in G418-containing medium of the cells co-transfected with the marked MusD and the expression vector for the indicated cytosine deaminase; values are given as the percentages of the corresponding values obtained in the absence of cytosine deaminase expression vector (no APO). Data are the means ± SD of at least three independent experiments. (B) Quantitative analysis as in Figure 2 of the number of retrotransposed MusD copies, in the absence of G418 selection. DNA was extracted from cells transfected as in (A). Values of the intensity of the specific 643 bp PCR fragment, measured after 30 cycles of amplification, are expressed as the percentages of the corresponding values in the absence of cytosine deaminase. Data points are the means ± SD of at least three independent experiments.

Figure 4

Differential effect of cytosine deaminases on G-to-A hypermutations. (A) Upper panels: Two-entry tables showing nucleotide substitution preferences for MusD mutations detected in the absence of cytosine deaminase (no APO) or in the presence of hA3G, hAID, hA1, hA2, hA3B, hA3C, hA3F, agmA3G and mA3. At day 2 post-transfection, retrotransposed MusD copies were amplified by PCR, cloned and sequenced (643 bp neo fragment). Clones (20–25) were sequenced under each condition (only 16 for the ‘no APO’ control). N, total number of bases sequenced. Lower panels: Histograms showing the number of G-to-A mutations per individual 643 bp sequence in the retrotransposed MusD elements. (B) Number of mutations induced by each cytosine deaminase in the retrotransposed MusD copies, per 10 000 nt. Open bars represent the total number of mutations for 10 000 sequenced nucleotides and black bars the number of G-to-A mutations. (C) Target sequence preference of hA3G, hA3F, agmA3G and mA3. The influence of the 3′ neighboring nucleotides for the G-to-A mutations identified in the retrotransposed MusD copies is illustrated.

Figure 5

Model for the dual effect of APOBEC activity on retrotransposition. Active endogenous proviruses—such as MusD—produce endogenous retroviral particles, whose RNA is reverse transcribed into single-strand DNA that is sensitive to APOBEC deoxycytidine deaminase activity. Uracil-containing cDNA (either single-strand or double-strand) is then degraded before integration, possibly by cellular UNGs, a process which reduces the number of proviral copies prone to cause insertional mutagenesis (first ‘line of defense’). The proviral copies that have escaped degradation can integrate but disclose G-to-A mutations that render them defective for subsequent retrotransposition cycles (second ‘line of defense’).