Modulation of activity of Moloney murine leukemia virus preintegration complexes by host factors in vitro

Abstract

We have explored the requirements for host proteins in the integration of Moloney murine leukemia virus (MoMuLV) cDNA in vitro. Following infection, it is possible to lyse cells and obtain preintegration complexes (PICs) capable of integrating the MoMuLV cDNA into an added target DNA in vitro (intermolecular integration). PICs can be stripped of required proteins by gel filtration in high-salt buffers (600 mM KCI), allowing the nature of the removed factors to be investigated by in vitro reconstitution. In a previous study of human immunodeficiency virus type 1 (HIV-1) PICs, the host protein HMG I(Y) was found to be able to restore activity to salt-stripped PICs. In contrast, salt stripping and reconstitution of MoMuLV PICs led to the proposal that a host factor is important for a different activity, blocking integration into the cDNA itself (autointegration). In this report, we investigated reconstitution of salt-stripped MoMuLV PICs and found that addition of cellular extract from uninfected NIH 3T3 cells could block autointegration and also restore intermolecular integration. Isolation of the intermolecular integration-complementing activity yielded HMG I(Y), as in the HIV-1 case. However, HMG I(Y) could not block autointegration, implicating a different host factor in this process. Additionally, when MoMuLV PICs were partially purified but not salt stripped, the intermolecular integration activity was reduced but could be stimulated by the addition of any of several purified DNA binding proteins. In summary, three activities were detected: (i) the intermolecular integration cofactor HMG I(Y), (ii) an autointegration barrier protein, and (iii) stimulatory DNA binding proteins.

FIG. 1

Inferred pathway for intermolecular integration and autointegration. MoMuLV cDNA (thin lines) and target DNA (thick lines) are indicated. The 5′ ends (small solid circles) and 3′ ends (arrows) are indicated. II, integration intermediate.

FIG. 2

Analysis of intermolecular integration and autointegration products by restriction enzyme digestion. (A) Map of the MoMuLV genome and sizes of DNA fragments from substrates (viral cDNA and φX174 RFI), integration product (II), and autointegration products (circular) expected to be detected by the LTR probe. (B to D) Southern blot analyses of integration products generated by PICs partially purified by low-ionic-strength precipitation (lanes 1 and 2) or salt-stripped PICs (lanes 3 and 4) in the absence (−) or presence (+) of linearized target DNA (B) or circular target DNA (C and D). DNAs were digested with HindIII (C) or BamHI (D) prior to electrophoresis.

FIG. 3

Integration-complementing activity and autointegration barrier activity of extracts of NIH 3T3 cells and DNA binding by the integration-complementing activity. (A) Reconstitution of salt-stripped MoMuLV PICs with cytoplasmic or nuclear extract. Lane 1 shows the activity of PICs purified by low-ionic-strength precipitation. The partially purified PICs were salt stripped and reconstituted with 10 μl of buffer alone (lane 2), cytoplasmic extract (10, 2, and 0.4 μl [lanes 3 to 5, respectively]) or nuclear extract (10, 2, and 0.4 μl [lanes 6 to 8, respectively]). Each reaction mixture was adjusted to standard solution conditions prior to addition of target DNA and incubated at 37°C for 30 min. The percentages of intermolecular integration activity (II%) and autointegration activity (circular%) were measured with a Molecular Dynamics PhosphorImager. (B) DNA binding by the integration-complementing activity. Salt-stripped PICs were reconstituted with 10 μl of buffer only (lane 1), 10 μl of nuclear extract (lane 2), 10 μl of extract depleted by cellulose (lane 3), single-stranded DNA-cellulose (lane 4), or double-stranded DNA-cellulose (lane 5).

FIG. 4

Analysis of the site of action of nuclear extract by order of addition. Two orders of addition were compared: (i) addition of nuclear extract to salt-stripped PICs and then addition of target DNA (lanes 2 to 4) or (ii) addition of nuclear extract to target DNA and then addition of salt-stripped PICs (lanes 5 to 7). The amounts of nuclear extract added per reaction mixture are indicated above the autoradiogram. Lane 1 (Buffer) contained no added protein. The amounts of reactants were as follows: φX174 DNA, 2.6 × 10⁻⁹ M; HMG I(Y) in 10 μl of nuclear extract, 1.1 × 10⁻⁸ M; and cDNA in PICs, 8.3 × 10⁻¹³ M. The number of binding sites for HMG I(Y) in φX174 DNA is not known but is likely to be large.

FIG. 5

Fractionation of HMG proteins of NIH 3T3 cells identified HMG I(Y) as the intermolecular integration-complementing activity. (A) Analysis of the activity of HMG fractions eluted from a Mono-S column. The HMG proteins were extracted from uninfected NIH 3T3 cells (3T3 extract) with 5% perchloric acid and loaded onto a Mono-S column. Proteins were then eluted with a NaCl concentration gradient. Fractions 8 to 28 were collected and assayed for complementing activity. Salt-stripped PICs were incubated with buffer only (lane 1), 2 μl of nuclear extract (lane 2), or fractions 8 to 28 (lanes 3 to 13). (B) Analysis of the Mono-S fractions by SDS–15% polyacrylamide gel electrophoresis. The gel was stained with Coomassie brilliant blue. Molecular mass (M.W.) standards (in kilodaltons) are shown in lane 1. (C) Western blot analysis of Mono-S fractions with an anti-HMG I(Y) antibody.

FIG. 6

Reconstitution of salt-stripped PICs with purified proteins. Salt-stripped PICs were reconstituted with the indicated purified protein at the concentrations indicated over the gels. All the reactions were performed under the standard reaction conditions in a total volume of 110 μl. BSA, bovine serum albumin.

FIG. 7

Stimulation of the intermolecular integration activity of partially purified but not salt-stripped PICs by purified DNA binding proteins. The PICs were purified by hypotonic lysis of cells followed by gel filtration through Sepharose CL-4B in 150 mM KCl. The PICs were then mixed with the purified protein at the concentrations indicated. All the reactions were performed under standard reaction conditions in a total volume of 110 μl. BSA, bovine serum albumin.