A previously unidentified host protein protects retroviral DNA from autointegration - PubMed (original) (raw)

A previously unidentified host protein protects retroviral DNA from autointegration

M S Lee et al. Proc Natl Acad Sci U S A. 1998.

Abstract

Integration of a DNA copy of the viral genome into a host chromosome is an essential step in the retrovirus life cycle. The machinery that carries out the integration reaction is a nucleoprotein complex derived from the core of the infecting virion. To successfully integrate into host DNA, the viral DNA within this complex must avoid self-destructive integration into itself, a reaction termed autointegration. We have previously shown [Lee, M. S. and Craigie, R. (1994) Proc. Natl. Acad. Sci. USA 91, 9823-9827] that viral nucleoprotein complexes isolated from Moloney murine leukemia virus-infected cells exhibit a barrier to autointegration. This autointegration barrier could be destroyed by stripping factors from the complexes and subsequently restored by incubation with a host cell extract, but not by incubation with an extract of disrupted virions. We have now used this autointegration barrier reconstitution assay to purify the host factor from uninfected NIH 3T3 fibroblasts. It is a single polypeptide of 89 aa that does not match any previously identified protein. The identity of the protein was confirmed by expressing it in Escherichia coli and demonstrating the activity of the heterologously expressed protein in the reconstitution assay.

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Figures

Figure 1

Figure 1

Outline of the autointegration barrier reconstitution assay. (I) Auto-INC, the substrate for the reconstitution reaction, was prepared by disruption of the autointegration barrier from the INC with high salt treatment, followed by removal of free components through gel filtration. (II) The autointegration barrier was reconstituted onto the purified auto-INC by addition of various BAF-containing fractions. (III) The extent of reconstitution reactions was measured by the integration assay that simultaneously analyzes intermolecular integration and autointegration.

Figure 2

Figure 2

Purification of the barrier-to-autointegration factor (BAF). (_A_I, _B_I, and _C_I) The silver-stained SDS/polyacrylamide gels (16% run in Tricine buffer). (_A_II, _B_II, and _C_II) The autoradiograms of the Southern blots showing the BAF activity as described in Fig. 1. _A_I and _A_II for the Mono Q column; _B_I and _B_II for the Superdex-200 column; _C_I and _C_II for the Phenyl superose column. Fx, column fractions; FT, flow-through from the column; L, sample load to the column. The load to the Mono Q column (_A_II, lanes 2 and 3) as well as the load to the Superdex-200 (_B_II, lanes 2–4) was titrated by 2-fold increments in the activity assay. The BAF activity is represented by conversion of the 5.6-kb band and smear (autointegration products) to the 11.0-kb band (intermolecular integration products) (_A_II, _B_II, and _C_II).

Figure 3

Figure 3

Nucleotide sequence and deduced amino acid sequence of the ORF of the BAF-encoding cDNA. The four internal peptide sequences that were obtained by partial amino acid sequencing are underlined.

Figure 4

Figure 4

Expression of functional BAF in Escherichia coli. (A) Overexpression, solubilization, and purification of the murine BAF. E. coli cells with the pET-15b recombinant plasmid containing BAF-ORF were harvested before (lane 1) or after (lanes 2–5) IPTG induction. Lanes: 1 and 2, whole cell lysates in 4% SDS; 3, soluble lysates; 4, guanidine hydrochloride-soluble portion of the insoluble fraction; 5, the purified BAF after removal of the His-tag and reverse-phase chromatography; 6, molecular mass standards. Samples were analyzed by SDS/PAGE, followed by Coomassie blue staining. (B) The recombinant BAF is active in the autointegration barrier reconstitution assay. Lanes: 1, no addition; 2–4, the purified BAF in 2, 10, and 50 ng in each reconstitution reaction.

Figure 5

Figure 5

DNA bridging by BAF and its potential role in blocking autointegration. Linearized double-stranded øX174 DNA (_Pst_I digest of øX174 replicative form I DNA, 30 μM in nucleotides) was preincubated with various concentrations of BAF (0–0.8 μM of monomers) for 10 min on ice. (A) Aggregation of DNA by BAF. One aliquot of each preincubation mixture was centrifuged at 15,000 × g for 10 min. After addition of SDS, the DNA remaining in the supernatants was analyzed by agarose gel electrophoresis and stained with ethidium bromide. (B) Binding of BAF can make a target DNA unavailable for integration. INCs were added to a second set of preincubation mixture aliquots (lanes 2–6) and assayed for their integration activity into the preincubated target DNA. Intermolecular integration reaction products (P1, ≈14 kbp) were separated from unreacted substrate (S, ≈9 kbp) by agarose gel electrophoresis and visualized by Southern blotting and hybridization. (C) Naked DNA is a preferred target for integration. Linear 2.7-kbp DNA (_Bst_NI digest of øX174 replicative form I DNA, 30 μM in nucleotides) was added to a third set of aliquots of linear 5.4-kbp øX174 DNA that had been preincubated with the various concentrations of BAF. Integration reactions were initiated by addition of INCs (lanes 2–6) and analyzed as described in B except that P2 (≈12 kbp) represents integration into the naked target DNA. Lanes 1 for B and C are INCs without addition of target DNA. (D) Intermolecular aggregation of DNA by BAF. Bridging of the DNA by BAF results in the formation of a network. (E) Intramolecular bridging by BAF may compact the viral DNA.

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