Prototype Foamy Virus Integrase Displays Unique Biochemical Activities among Retroviral Integrases

Abstract

Integrases of different retroviruses assemble as functional complexes with varying multimers of the protein. Retroviral integrases require a divalent metal cation to perform one-step transesterification catalysis. Tetrameric prototype foamy virus (PFV) intasomes assembled from purified integrase and viral DNA oligonucleotides were characterized for their activity in the presence of different cations. While most retroviral integrases are inactive in calcium, PFV intasomes appear to be uniquely capable of catalysis in calcium. The PFV intasomes also contrast with other retroviral integrases by displaying an inverse correlation of activity with increasing manganese beginning at relatively low concentrations. The intasomes were found to be significantly more active in the presence of chloride co-ions compared to acetate. While HIV-1 integrase appears to commit to a target DNA within 20 s, PFV intasomes do not commit to target DNA during their reaction lifetime. Together, these data highlight the unique biochemical activities of PFV integrase compared to other retroviral integrases.

Keywords: divalent cation; integrase; prototype foamy virus; retrovirus.

Figure 1

Integration assay reaction products. DNA oligomers mimicking the viral cDNA ends (vDNA, heavy black lines) are assembled with PFV IN to form intasomes. The intasomes are added to a supercoiled plasmid (SC plasmid, light black lines) and incubated at 37 °C. The major products of integration are concerted integration (CI) where the two vDNAs of the intasome are covalently joined to the target SC plasmid. This results in a linear product with the vDNAs at each end. Additional CI to the linear product will result in shorter fragments. A minor product of intasome integration is half site integration (HSI), where only one vDNA is joined to the target DNA resulting in a tagged circle.

Figure 2

PFV intasome integration in the presence of Mg. Integration activity was assayed with a titration of (A) MgSO₄ or (B) MgCl₂. Reaction products were resolved by agarose gel electrophoresis. The gel was imaged for ethidium bromide (top) and Cy5 (center) fluorescence. Half site integration (HSI), the first concerted integration product (CI₁), and subsequent concerted integration products (CI₂) are shown. Unreacted supercoiled plasmid target DNA (SC) is visible on in the ethidium bromide image. Unreacted vDNA is only visible in the Cy5 image. The total Cy5 fluorescence in each lane was quantified and each integration product calculated as the fraction of the total fluorescence (bottom). The average of three independent experiments with at least two independent intasomes preparations is shown. Error bars indicate the standard deviation.

Figure 3

PFV intasome integration in the presence of MnCl₂ or CaCl₂. (A) Integration activity was assayed with a titration of MnCl₂ and 10 nM PFV intasomes. Reaction products were resolved by agarose gel electrophoresis. The gel was imaged for ethidium bromide (top) and Cy5 (center) fluorescence. Half site integration (HSI), the first concerted integration product (CI₁), and subsequent concerted integration products (CI₂) are shown. Unreacted supercoiled plasmid target DNA (SC) is visible on in the ethidium bromide image. Unreacted vDNA is only visible in the Cy5 image. The total Cy5 fluorescence in each lane was quantified and each integration product calculated as the fraction of the total fluorescence (bottom). The average of three independent experiments with at least two independent intasomes preparations is shown. Error bars indicate the standard deviation. (B) 10 nM PFV intasomes were assayed for integration with a titration of CaCl₂. Reaction products were resolved by agarose gel electrophoresis. The gel was imaged for ethidium bromide (top) and Cy5 (center) fluorescence. HSI and CI₁ are shown. Unreacted SC target DNA is visible on in the ethidium bromide image. Unreacted vDNA is only visible in the Cy5 image. The reaction products were <1% of the fluorescent signal in each lane and could not be accurately quantified. (C) A higher concentration of PFV intasomes, 45 nM, was assayed to confirm the presence of CI products in the presence of Ca.

Figure 4

Increasing the molar ratio of target to PFV intasomes. (A) Typical reaction conditions with 25 nM PFV intasomes are 20-fold molar excess of target sites to intasomes; (B) reducing the intasome concentration to 2.5 nM increases the molar excess of target sites to 200-fold; (C) increasing the target DNA concentration allowed for 1000-fold molar excess of target sites to 2.5 nM intasomes. Cy5 images of agarose gels show the accumulation of CI₁ and CI₂ products over time. (D) The total Cy5 fluorescence in each lane was quantified and each integration product calculated as the fraction of the total fluorescence. The average of three independent experiments with at least two independent intasomes preparations is shown. Error bars indicate the standard deviation.

Figure 5

PCA does not alter the kinetics of PFV intasome integration. PFV integration over time was assayed in the absence or presence of 5 mM PCA. As PCA has been shown to reduce aggregation of PFV intasomes, two concentrations of intasomes were assayed: 25 and 2.5 nM. Cy5 image of agarose gel with 25 nM PFV intasomes shows the accumulation of CI₁ and CI₂ products over time. The total Cy5 fluorescence in each lane was quantified and each integration product calculated as the fraction of the total fluorescence. PCA did not affect the kinetics of integration at either concentration. The average of three independent experiments with at least two independent intasomes preparations is shown. Error bars indicate the standard deviation.

Figure 6

PFV intasomes are more active in chloride buffers. (A) PFV integration over time was assayed in the presence of standard chloride buffer or an acetate buffer; (B) PFV intasomes were diluted in chloride or acetate buffer and kept on ice for variable time before the addition of target DNA and immediate transfer to 37 °C. Reaction products were resolved by agarose gel electrophoresis. The gel was imaged for ethidium bromide (top) and Cy5 (center) fluorescence. Half site integration (HSI), the first concerted integration product (CI₁), and subsequent concerted integration products (CI₂) are shown. Unreacted supercoiled plasmid target DNA (SC) is visible on in the ethidium bromide image. Unreacted vDNA is only visible in the Cy5 image. The total Cy5 fluorescence in each lane was quantified and each integration product calculated as the fraction of the total fluorescence (bottom). The average of three independent experiments with at least two independent intasomes preparations is shown. Error bars indicate the standard deviation.

Figure 7

PFV intasomes do not commit early to target DNA. Integration reactions were performed with a 3 kb and a 6 kb plasmid. Reactions were started with one plasmid and a second plasmid added at variable time later. The zero time point represents plasmids added simultaneously to the reaction. (A) The 3 kb plasmid was added first and the 6 kb plasmid added second; (B) the 6 kb plasmid was added first and the 3 kb plasmid was added second. The total Cy5 fluorescence in each lane was quantified and each integration product calculated as the fraction of the total fluorescence (bottom). The average of three independent experiments with at least two independent intasomes preparations is shown. Error bars indicate the standard deviation.