Genetic editing of HBV DNA by monodomain human APOBEC3 cytidine deaminases and the recombinant nature of APOBEC3G - PubMed (original) (raw)

Genetic editing of HBV DNA by monodomain human APOBEC3 cytidine deaminases and the recombinant nature of APOBEC3G

Michel Henry et al. PLoS One. 2009.

Abstract

Hepatitis B virus (HBV) DNA is vulnerable to editing by human cytidine deaminases of the APOBEC3 (A3A-H) family albeit to much lower levels than HIV cDNA. We have analyzed and compared HBV editing by all seven enzymes in a quail cell line that does not produce any endogenous DNA cytidine deaminase activity. Using 3DPCR it was possible to show that all but A3DE were able to deaminate HBV DNA at levels from 10(-2) to 10(-5)in vitro, with A3A proving to be the most efficient editor. The amino terminal domain of A3G alone was completely devoid of deaminase activity to within the sensitivity of 3DPCR ( approximately 10(-4) to 10(-5)). Detailed analysis of the dinucleotide editing context showed that only A3G and A3H have strong preferences, notably CpC and TpC. A phylogenic analysis of A3 exons revealed that A3G is in fact a chimera with the first two exons being derived from the A3F gene. This might allow co-expression of the two genes that are able to restrict HIV-1Deltavif efficiently.

PubMed Disclaimer

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Monodomain A3A and A3H can hyperedit HBV DNA.

A) 3DPCR amplification of HBV DNA from on A3A and A3H/pCayw co-transfections of QT6 using a 12°C gradient across the heating block. M denotes molecular weight markers, while pv refers to the empty expression vector. Primer-dimers represent the low molecular weight band. B) Mutation matrices compared to the plus strand as reference where n indicates the number of bases sequenced. C) Mutation matrix for the pCayw plus plasmid vector control.

Figure 2

Figure 2. Monodomain variants of A3G are stably expressed.

A) Boxes denote each of the eight exons; the shaded 6th exon harbours the zinc finger motif, HAEXnPCX2C. Shaded exon 3 encodes the singular HPEXnPCX2C variant. The V5 tag is shown as a black box. B) Confocal immunofluorescence of the three constructs expressed in QT6 cells. DAPI stained cells are on the right, V5 tagged constructs were revealed with x-labelled monoclonal to the V5 tag (centre), while the merged photos are given to the right.

Figure 3

Figure 3. Uniquely the C-terminal domain of A3G is catalytically active.

A) 3DPCR amplification using an 11°C gradient in denaturation temperature across the heating block. M denotes molecular weight markers, pv the empty expression vector, the white asterisks denote hyperedited HBV DNA, while the white triangles indicate PCR products containing internal deletions (confirmed by sequencing of cloned products). B) Mutation matrices for A3G and A3Gc on QT6 cells where n indicates the number of bases sequenced. C) The inter-conversion of G→A is related to the concentration of A3G co-transfected.

Figure 4

Figure 4. Proline 66 is not responsible for the inactive N-terminal Zn finger domain of A3G.

A) PCR amplification of HBV DNA from a variety of co-transfection experiments at 95°C and the restrictive temperature of 88°C. M denotes molecular weight markers; C-, amplification control without DNA; C+, pCayw alone; pv the empty expression vector as negative control; A2, APOBEC2 as another negative control. Primer-dimers represent the low molecular weight band. B) Mutational analysis of the cloned 95°C PCR products and those of the positive A3G control at 88°C.

Figure 5

Figure 5. Human A3G and A3Gc hot and cold spots are highly correlated.

A) Individual editing frequencies across the minus strand target cytidine residues for A3G and A3Gc co-transfections of Huh7 cells. These represent the fraction of A3 edited sequences bearing an edited C at a given position. B–D) Highly correlated site-specific editing frequencies for A3G, A3Gc/HBV co-transfections of QT6 and Huh7 cells. The site-specific editing frequencies calculated for a given data set are correlated with the site-specific editing frequencies from another data set at the level of each C residue. The identities of the data sets are given on the x and y-axes. Least-mean squared fits to the data are also given.

Figure 6

Figure 6. Cytidine deamination dinucleotide context varies as a function of the target sequence.

A) Cytidine editing of the minus DNA strand of HBV by six A3s, the horizontal line indicating the expected values based on the sequence composition of the target. B) Cytidine editing of the minus DNA strand of HIV by six A3s, the horizontal line indicating the expected values based on the sequence composition of the target. Asterisks denote statistically significant deviations from the expected value (p<0.001).

Figure 7

Figure 7. HIV-1Δ_vif_ editing by 6 A3 deaminases.

A) 3DPCR of cDNA harvested 18 h post-infection of CEMx174 cells. Virus stocks were made using QT6 cells, white asterisks represent the PCR product cloned and sequenced. B) Mutation statistics for the HIV-1 hypermutants.

Figure 8

Figure 8. Phylogenic analysis of A3 exons indicates past intergenic recombination.

All Neighbor-Joining trees are drawn to the same scale. Numbers at branching nodes are percentages of 1000 bootstrap replications - only bootstrap values >40 are given. A3H was excluded was excluded from the exon 1 and 8 trees for lack of significant homology. A3B was excluded from the exon 1 tree because it is only 71 bases long and led to an even less robust tree. Nonetheless, as it differs from the A3DE sequence by only 2 bases, it is shown in parentheses. The anomalous A3G sequences are highlighted in red. Another case of possible recombination is highlighted in sky blue as the differing (3A-3, 3B-6, 3G-6) and (3A-4, 3G-7) 3B-7) topologies are supported by strong bootstrap values. After gap stripping, the lengths of each alignment were as follows: exon 1, 89 bp; exons 2+5, 135 bp; exons 3+6, 256 bp; exons 4+7, 113 bp and exon 8, 240 bp.

Figure 9

Figure 9. Colour coded representation of A3 coding exons.

Colour denotes generally similar overall clustering and hides finer detail. Coding regions are drawn to scale. As the iMet of A3H is within exon 2, exon1 is not shown. As it is not possible in an a posteriori analysis to distinguish between recombination and rate heterogeneity, while some clusters are not supported by strong bootstrap values, the schematic should not be overly interpreted.

Similar articles

Cited by

References

    1. Jarmuz A, Chester A, Bayliss J, Gisbourne J, Dunham I, et al. An anthropoid-specific locus of orphan C to U RNA-editing enzymes on chromosome 22. Genomics. 2002;79:285–296. - PubMed
    1. Harris RS, Bishop KN, Sheehy AM, Craig HM, Petersen-Mahrt SK, et al. DNA deamination mediates innate immunity to retroviral infection. Cell. 2003;113:803–809. - PubMed
    1. Lecossier D, Bouchonnet F, Clavel F, Hance AJ. Hypermutation of HIV-1 DNA in the absence of the Vif protein. Science. 2003;300:1112. - PubMed
    1. Liddament MT, Brown WL, Schumacher AJ, Harris RS. APOBEC3F properties and hypermutation preferences indicate activity against HIV-1 in vivo. Curr Biol. 2004;14:1385–1391. - PubMed
    1. Mangeat B, Turelli P, Caron G, Friedli M, Perrin L, et al. Broad antiretroviral defence by human APOBEC3G through lethal editing of nascent reverse transcripts. Nature. 2003;424:99–103. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources