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. 2015 Apr 30:12:39.
doi: 10.1186/s12977-015-0167-3.

Key determinants of target DNA recognition by retroviral intasomes

Erik Serrao  1 , Allison Ballandras-Colas  2 , Peter Cherepanov  3   4 , Goedele N Maertens  5 , Alan N Engelman  6
Affiliations

Key determinants of target DNA recognition by retroviral intasomes

Erik Serrao et al. Retrovirology. .

Abstract

Background: Retroviral integration favors weakly conserved palindrome sequences at the sites of viral DNA joining and generates a short (4-6 bp) duplication of host DNA flanking the provirus. We previously determined two key parameters that underlie the target DNA preference for prototype foamy virus (PFV) and human immunodeficiency virus type 1 (HIV-1) integration: flexible pyrimidine (Y)/purine (R) dinucleotide steps at the centers of the integration sites, and base contacts with specific integrase residues, such as Ala188 in PFV integrase and Ser119 in HIV-1 integrase. Here we examined the dinucleotide preference profiles of a range of retroviruses and correlated these findings with respect to length of target site duplication (TSD).

Results: Integration datasets covering six viral genera and the three lengths of TSD were accessed from the literature or generated in this work. All viruses exhibited significant enrichments of flexible YR and/or selection against rigid RY dinucleotide steps at the centers of integration sites, and the magnitude of this enrichment inversely correlated with TSD length. The DNA sequence environments of in vivo-generated HIV-1 and PFV sites were consistent with integration into nucleosomes, however, the local sequence preferences were largely independent of target DNA chromatinization. Integration sites derived from cells infected with the gammaretrovirus reticuloendotheliosis virus strain A (Rev-A), which yields a 5 bp TSD, revealed the targeting of global chromatin features most similar to those of Moloney murine leukemia virus, which yields a 4 bp duplication. In vitro assays revealed that Rev-A integrase interacts with and is catalytically stimulated by cellular bromodomain containing 4 protein.

Conclusions: Retroviral integrases have likely evolved to bend target DNA to fit scissile phosphodiester bonds into two active sites for integration, and viruses that cut target DNA with a 6 bp stagger may not need to bend DNA as sharply as viruses that cleave with 4 bp or 5 bp staggers. For PFV and HIV-1, the selection of signature bases and central flexibility at sites of integration is largely independent of chromatin structure. Furthermore, global Rev-A integration is likely directed to chromatin features by bromodomain and extraterminal domain proteins.

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Figures

Figure 1
Figure 1
Sequence logos and dinucleotide step analysis of integration sites with 4 bp TSDs. (A-D) The height of each individual base at a given position is proportional to the frequency of the corresponding nucleotide within the sequences represented by the logos, and the height of each stack of base logos reflects the level of conservation at that position. (E) Percent YR utilization across the integration sites from panels A-D is shown relative to the calculated random value of 22% (dotted gray horizontal line). (F) Same as in panel E, except that the graph depicts RY utilization across the integration sites. Statistical analysis of panel E and F results are shown in Additional file 2: Figure S2 panels A and B, respectively. (G and H) The percent of YR (panel G) and RY (panel H) enrichment for each virus compared to random.
Figure 2
Figure 2
Sequence logos and dinucleotide step analysis of integration sites from retroviruses that yield 5 bp TSDs. Sequence logos for HIV-1 (A), EIAV (B), SIV (C), and Rev-A (D). (E) YR step analysis for the integration sites depicted in panels A-D. (F) Same as in panel E, except RY dinucleotide frequencies were calculated. Statistical analyses of panel E and F results are depicted in Additional file 2: Figure S2 panels C and D, respectively. Percent YR and RY enrichment for each virus compared to random is shown in (G) and (H), respectively. Other labeling is as in Figure 1.
Figure 3
Figure 3
Sequence logos and YR/RY dinucleotide selectivities of viral integration sites with 6 bp TSDs. Sequence logos are shown for conglomerate integration sites of ASLV (A), HERV-K (B), HTLV-1 (C), and MMTV (D). (E) YR frequency utilization across the integration sites of viruses depicted in panels A-D. (F) Same as in panel E, except the plot is for RY dinucleotide utilization. Statistical analyses of panel E and F results are shown in Additional file 2: Figure S2 panels E and F, respectively. The percent YR and RY enrichment for each virus compared to random is in (G) and (H), respectively. Other labeling is as in Figure 1.
Figure 4
Figure 4
Sequence logos for PFV and HIV-1 integration sites in nucleosome-free versus chromatinized tDNA. (A) The logo illustrates the average nucleotide sequence of the first 50 nucleotides of center-aligned nucleosomal DNA sequences isolated from chicken erythrocytes [68]. (B) PFV integration sites derived from virus-infected cells [92,93]. (C) Integration sites from recombinant PFV intasomes and deproteinized cellular DNA. (D) HIV-1 integration sites from virus-infected cells [87]. (E) Concerted HIV-1 integration sites from recombinant HIV-1 IN and naked pGEM9Zf(−) plasmid DNA [33].
Figure 5
Figure 5
Flexibility profiles for PFV and HIV-1 integration sites in nucleosome-free versus chromatinized tDNA. (A and B) YR and RY frequency charts, respectively, for PFV integration sites into deproteinized genomic DNA (PFV in vitro) and from virus infection. Vertical dotted black line represents central dinucleotide step(s), and horizontal dotted grey line represents the MRC frequency of YR/RY utilization. (C and D)) Bar graphs illustrating the percent YR and RY enrichment, respectively, at the central dinucleotide step relative to MRC values. (E and F) YR and RY frequency charts, respectively, for HIV-1 integration sites into naked plasmid DNA (HIV-1 in vitro) and from virus infection. (G and H) Bar graphs illustrating the percent YR and RY enrichment, respectively, at the central dinucleotide steps compared to MRC.
Figure 6
Figure 6
BRD4 protein and interaction with Rev-A IN. (A) Schematic of human BRD4 isoform C (NCBI reference sequence NP_055114.1) highlighting various protein domains and the 426–720 fragment used in this study. (B) Ni-NTA pull-down of purified BRD4462–720 by His6-tagged Rev-A IN. Migration positions of standards (in kDa) are labeled to the left. Lanes 1–4: 20% reaction input of the indicated proteins. Lanes 5 and 6: the indicated BRD4462–720 protein was incubated with Ni-NTA beads in the absence of Rev-A IN. Lanes 7 and 8: the indicated BRD4462–720 protein was incubated with Rev-A IN-bound beads. The gel is representative of results obtained from three independent experiments.
Figure 7
Figure 7
Concerted integration activity of Rev-A IN. (A) Ethidium bromide stained image of Rev-A IN integration reactions in the presence of increasing concentrations of indicated BRD4462–720 protein. Migration positions of standards (in kb) are shown to the left, and the positions of single vDNA end, or half-site, and concerted vDNA integration products, as well as supercoiled (s.c.) and open circular (o.c) forms of the plasmid tDNA, are to the right. Lanes 1 and 2: Rev-A IN and tDNA incubated without vDNA or with vDNA, respectively. Lanes 3–6: increasing concentrations (0.05, 0.15, 0.35, 0.5 μM) of BRD4462–720 incubated with Rev-A IN plus vDNA and tDNA. Lanes 7–10: same as lanes 3–6 but with BRD4462–720/L630E. (B) Strand transfer activities for three independent experiments ± standard error of the mean, measured by quantification of DNA band intensity. The results were normalized to the level of Rev-A IN concerted integration activity in the absence of BRD4462–720 proteins, which was set to 100%. P values of <0.05 and <0.01 are indicated by * and **, respectively, as determined by one-tailed t-test.
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