In vitro murine leukemia retroviral integration and structure fluctuation of target DNA

Abstract

Integration of the retroviral genome into host DNA is a critical step in the life cycle of a retrovirus. Although assays for in vitro integration have been developed, the actual DNA sequences targeted by murine leukemia retrovirus (MLV) during in vitro reproduction are unknown. While previous studies used artificial target sequences, we developed an assay using target DNA sequences from common MLV integration sites in Stat5a and c-myc in the genome of murine lymphomas and successfully integrated MLV into the target DNA in vitro. We calculated the free energy change during folding of the target sequence DNA and found a close correlation between the calculated free energy change and the number of integrations. Indeed, the integrations closely correlated with fluctuation of the structure of the target DNA segment. These data suggest that the fluctuation may generate a DNA structure favorable for in vitro integration into the target DNA. The approach described here can provide data on the biochemical properties of the integration reaction to which the target DNA structure may contribute.

Figure 1. In vitro integration using retroviral LTRs.

(A) The 5′ and 3′ LTR of MLV proviral DNA (red line) was used after removal of other elements encoding gag, pol, pro and env. The sequence shown displays the MLV LTR in the form integrated into the host DNA. The target DNA (grey line) was ligated into the pCR2.1 TOPO plasmid vector (black line). Arrowheads next to the proviral DNA sequence represent the processed ends. After incubation of retroviral and target DNA with integrase, proviral DNA was integrated into the target sequence or plasmid. The integration site was then sequenced. (B) The MLV integration site hot spot in the lymphoma genome of SL/Kh mice is represented by the red square. The utilized target sequences M0-M4 are shown below. M0 is identical to the native Stat5a sequence. Red letters in the sequence indicate the most frequent sites of integration in hematopoietic tumors as previously reported by us .

Figure 2. In vitro MLV-LTR integration into Stat5a.

The vertical axis to the left represents the number of integrations into each nucleotide in M0 (native Stat5a), modified sequences M1-M4, and control same length random sequences R1-R5. These sequences are 400-bp in length, portions of which are shown in Figure 1. The horizontal axis represents the bases 1105–1153 in the Stat5a gene. The sequences shown are the junction of the target sequence and 5′- MLV LTR when the MLV is inserted at nucleotide 1130. (A) Integration sites identified with the in vitro assay using sequences M0-M4. Black circles (L) represent the number of mice suffering from lymphomas resulting from MLV integration into the individual nucleotides shown. The number of integrations into nucleotide 1130 per 2000 integrations was significantly greater with sequences M0 and M1 than with sequences M2-M5 (*P<0.05). (B) Integration sites identified with the in vitro assay using the 5 random sequences (R1-R5) inserted into the plasmid DNA.

Figure 3. Target sequence length and integration.

(A) Presumed secondary structure of the top strand generating a cruciform in the presence of 60 mM of MgCl₂, as predicted using the M-fold program . (B) Absolute value of the Gibbs' free energy change during DNA folding and generation of a cruciform by the top strand. Arrows represent the marginal points in which the lengths are threshold values of the free energy change. (C) Number of integrations into nucleotide 1130 in the top strand cruciform. Arrows represent the marginal points in which the lengths are the threshold value. Arrows correspond well to the positions of those in (B) (n = 6; mean ± s.d.). The square of the correlation coefficient for the absolute energy value shown in (B) and the number of integrations shown in (C) was 0.838.

Figure 4. In vitro integration in buffers of varying MgCl₂ concentration.

(A) Electrophoresis of plasmid DNA with (upper) or without (lower) the target sequence DNA. Supercoiled DNA was electrophoresed at 0 min, at 30 min, and at 60 min after incubation. Arrows and arrowheads indicate fragments corresponding to peaks shown in (B). (B) Electropherogram of the plasmid with the target sequence and the plasmid alone at incubation for 0 min and 30 min. (C) Graph showing the relative area of the electrophoretic signal of supercoiled plasmid DNA with (red) or without (blue) target Stat5a DNA at 60 min after incubation in buffer containing various concentrations of MgCl₂ (unit mM, n = 6; mean ± s.d.). (D) Graph showing the total number of integration sites within the target Stat5a DNA (red) and the number of integrations at nucleotide 1130 (blue) at 60 min after incubation in buffer containing various concentrations of MgCl₂ (unit mM, n = 6; mean ± s.d.). (E) Sample photo of a secondary structure when using a buffer containing 60 mM and 30 mM of manganese dichloride (mM). Supercoiled DNA (in the upper photo) and globular DNA (in the lower photo) are displayed.

Figure 5. In vitro integration using the c-myc promoter sequence and buffers containing variable concentrations of MgCl₂.

(A) Thermodynamic analysis of the presumed cruciform structure from the c-myc promoter sequence DNA (GENEBANK M12345, No. 711-980) in the presence of 60 mM MgCl₂. Black arrows indicate the previously reported integration sites –. Red arrows indicate representative in vitro integration sites. A box indicates the hot spot segment. (B) Graph showing the relative area of supercoiled plasmid DNA with (target +) or without target c-myc DNA (target −) at 60 min after incubation in buffer containing various concentrations of MgCl₂ (unit mM, n = 6; mean ± s.d.). (C) Graph showing the total number of integration sites within the target c-myc DNA and the number of integration into the hot spot at 60 min after incubation in buffer containing various concentrations of MgCl₂ (unit mM, n = 6; mean ± s.d.).