THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1993 by The American Society for Biochemistry and Molecular Biology. Inc

Vol. 268. No. 22, Issue of August 5, pp. 16519-16527.1993 Printed in U. S. A.

HIV Nucleocapsid Protein EXPRESSION IN ESCHERZCHZA COLI, PURIFICATION, AND CHARACTERIZATION*

(Received for publication, January 19, 1993, and in revised form, April 6, 1993)

Ji Chang You and Charles S. McHenry From the Department of Biochemistry, Biophysics and Genetics, University of Colorado Health Sciences Center, Denver, Colorado 80262

The single-stranded nucleocapsid protein that coats the RNA genome of human immunodeficiency virus within the virion core has been produced in Esche- richia coli and purified to homogeneity. The mature 65-amino acid protein, normally generated from the gag polyprotein precursor by HIV protease-catalyzed processing of both its amino and carboxyl termini, was produced in E. coli with authentic termini directly, without the need for processing. The protein was pu- rified 30-fold to apparent homogeneity, as determined by both amino acid analysis and SDS-polyacrylamide gel electrophoresis. Sequencing of each terminus of the purified protein indicated that no proteolytic degra- dation occurred. A molar extinction coefficient (tz80 = 8360 cm” M-’) was determined. The purified nucleo- capsid protein binds tightly to single-stranded RNA as judged by a nitrocellulose filter binding assay. A bind- ing constant (&,) of 1 % 10’ ”’ was calculated. Using fluorescence quenching of nucleocapsid protein upon RNA binding as an assay, a binding site size of seven nucleotides was determined. These results contrast to a larger 15-nucleotide site measured by others for a larger form of nucleocapsid protein-containing se- quences from its immature precursor. The possible relevance of these findings are discussed.

Human immunodeficiency virus 1 (HIV-1) is a distinct group of human retroviruses associated with the acquired immunodeficiency syndrome (AIDS) (Gallo et al., 1984; Barre- Sinoussi et d., 1983; Levy et al., 1984). In the cytoplasm of infected cells, all retroviruses undergo reverse transcription from the viral RNA to make double-stranded DNA that becomes integrated into the cellular chromosome (reviewed in Varmus and Swanstrom, 1984). Reverse transcription is initiated from a ribonucleoprotein complex found in the virion core. The HIV complex is composed of a diploid, single- stranded RNA genome, cellular tRNA primer, reverse tran- scriptases, and nucleocapsid proteins (Bolognesi et al., 1978; Fleissner and Tress, 1973; Meric et al., 1984; Vaishnav and Wong-Staal, 1991).

Full-length in vitro replication of retroviral RNA occurs efficiently only in detergent-disrupted virions or partially purified ribonucleoprotein complexes (Davis and Rueckert, 1972; Gilboa et al., 1979; Benz and Dina, 1979; Chen et al., 1980; Boone and Skalka, 1980, 1981), raising the possibility

* This work was supported by Grant R01AI26600 from the Na- tional Institutes of Health and in part by the funds from the Lucille P. Markey Charitable Tmst. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

that other viral proteins in addition to reverse transcriptase are involved in the natural replicative reaction. The major protein associated with viral genomic RNA is the nucleocapsid protein, a low molecular weight, basic protein conserved in all replication-competent retroviruses (Hiemstra et al., 1986; Henderson et al., 1981; Covey, 1986). Sequences encoding this protein are found near the 3’-end of the gag gene. The gag precursor is processed by a viral protease to yield the nucleo- capsid protein found in the virion core, the capsid protein that is more peripherally associated in the core, and the NH2- terminal myristylated matrix protein that bridges the core to the envelope. In HIV, nucleocapsid maturation is particularly complex since an additional protein, p6, not found in other retroviruses, occurs at the far carboxyl terminus of the 55- kDa gag precursor (Veronese et al., 1987). An early event is cleavage before Met377 in the precursor generating a nucleo- capsid (p15) intermediate (Henderson et al., 1988; Tritch et al., 1991). A second, relatively slow cleavage occurs to generate mature nucleocapsid (p7) and p6 (Tritch et al., 1991; Hender- son et al., 1988 Veronese et al., 1987). The p6 segment is required for release of budded particles from infected cells (Gottlinger et al., 1991). The mature nucleocapsid p7 is the only major form of nucleocapsid found in mature virions. Nucleocapsid protein binds Zn2+ (see below). Probing with “Zn2+ on membranes containing electrophoretically resolved HIV proteins shows no Zn2+ binding activity higher than that of nucleocapsid p7 (Bess et al., 1992). This protein is a 55- residue (6380 Da) protein, determined by sequencing of the isolated protein (South et al., 1990; Henderson et al., 198% the cleavage after initially reported to be only partial, is actually a quantitative cleavage).’

The multiple forms of nucleocapsid and its precursors raise the possibility that different forms are involved in specific reaction stages. The gag precursor is the central protein responsible for formation of virus-like particles, which can assemble even in the absence of genomic RNA or other retroviral proteins (Gheysen et al., 1989; Shioda and Shibuta, 1990; Levin et al., 1974; Shields et al., 1978; Crawford and Goff, 1985). The nucleocapsid domain of the gag precursor is not required for formation of virus-like particles (Meric and Spahr, 1986; Voynow and Coffin, 1985). Protease mutants form and bud noninfectious viral particles (Kohl et al., 1988; Peng et al., 1989; Gottlinger et al., 1989; Ross et al., 1991; Loeb et al., 1989; McQuade et al., 1990; Meek et al., 1990; Crawford and Goff, 1985; Oertle and Spahr, 1990; Voynow and Coffin, 1985). Proteolysis of the gag precursor and mat- uration to an infectious virus occurs after budding (Lu et al., 1979). Thus, the 55-kDa gag precursor must be the nucleocap- sid form that participates in genomic RNA selection. The RNA dimer is stabilized after budding (Stoltzfus and Snyder,

L. Henderson, personal communication.

16519

16520 HIV Nucleocapsid Protein

1975), and protease mutants unable to process gag are defec- tive in dimerization (Oertle and Spahr, 1990); thus, either mature nucleocapsid (p7) or an intermediate form (p15 in HIV) may be involved at this stage. Since mature HIV parti- cles contain only the short p7 form, this fully processed form of nucleocapsid must be the relevant participant in all stages of the replicative reaction subsequent to primer annealing.

A number of in vitro studies have been conducted on nucleocapsid purified from a variety of retroviruses. Both nucleocapsid protein (Karpel et ab, 1987; Khan and Giedroc, 1992; Davis et al., 1976; Smith and Bailey, 1979; Schulein et al., 1978; Long et al., 1980; Sykora and Moelling, 1981; Leis and Jentoft, 1983) and its full-length gag precursor (Oroszlan et al., 1976; Karpel et al., 1987) bind tightly to both RNA and single-stranded DNA nonspecifically. The dissociation con- stant observed for the noncooperative interaction of mature nucleocapsid protein with single-stranded nucleic acids is in the micromolar range after correction for overlapping binding sites along a polynucleotide chain (Karpel et al., 1987; Smith and Bailey, 1979). Binding of the gag precursor to RNA appears to be stronger (Karpel et al., 1987). The nucleic acid- binding site size of mature nucleocapsid is generally four to six nucleotides (Karpel et al., 1987; Smith and Bailey, 1979), but the binding site size is larger in vitro in the precursor form (Karpel et al., 1987). The quantity of nucleocapsid found in mature viral particles suggests approximately one nucleo- capsid protein for every four nucleotides (Karpel et al., 1987). The binding of a recombinant 71 amino acid form of HIV nucleocapsid has been reported to be 0.03 PM and moderately cooperative (Khan and Giedroc, 1992).

Analyses to determine the biochemical requirements for biologically relevant HIV nucleocapsid-RNA interactions and to understand the role of nucleocapsid in the replicative reaction require a large scale source of protein identical to that found in the virion. A 55-amino acid mature nucleocapsid protein has been purified from live virus, its sequence deter- mined, and its structure preliminarily characterized by NMR (South et al., 1990). However, virus as a source of nucleocapsid protein limits the quantity available and raises cost and safety issues for most laboratories. Recombinant forms of HIV nu- cleocapsid either as a fusion protein or as a 71-amino acid form (16 residues longer than mature nucleocapsid) have been produced and excellent characterizations for Zn2+ binding and RNA interaction performed (Fitzgerald and Coleman, 1991; Khan and Giedroc, 1992). A 72-amino acid synthetic HIV nucleocapsid has also been made (De Rocquigny et al., 1991). In our ongoing efforts to fully reconstitute the HIV replicative reaction, we have obtained a recombinant vector that ex- presses the 55-amino acid mature p7 form of nucleocapsid and report here the purification and the characterization of authentic nucleocapsid (p7) in RNA binding.

EXPERIMENTAL PROCEDURES

Materials-All cloning and restriction enzymes were purchased from New England Biolabs and used according to the manufacturer's instructions. Polymin P,2 CHES, ethanolamine, @-mercaptoethanol, and carboxypeptidase A-DFP (EC 3.4.17.1) were obtained from Sigma. Taq polymerase was purchased from Perkin-Elmer-Cetus. IPTG was purchased from Calbiochem. Bovine serum albumin was from Intergen. Spermidine-trihydrochloride was obtained from Fluka. P0ly-[8-~H]adenylic acid (532 mCi/mmol of nucleoside residue) was purchased from Amersham Corp. Nitrocellulose filters (0.45-gm pore size, 24 mm size) and the Centrex Centrifugal Microfilter were purchased from Schleicher & Schuell. The "Bead-Beater'' used for

The abbreviations used are: polymin P, polyethyleneimine; CHES, 2-[N-cyclohexylamino]ethanesulfonic acid IPTG, isopropyl- P-D-thiogalactopyranoside; PCR, polymerase chain reaction.

lysis was from Biospec Products. Electrophoresis reagents, standard protein molecular weight markers, and the Bradford protein deter- mination kit were purchased from Bio-Rad.

Chromatographic Supports and Buffers-Q-Sepharose, S-Sepha- rose, and G-50 (superfine) were obtained from Pharmacia LKB Biotechnology Inc. The following buffers were used for the purifica- tion of HIV nucleocapsid protein: buffer A (25 mM ethanolamine, pH 9.5, 10% glycerol, and 5 mM 0-mercaptoethanol), buffer B (50 mM CHES, pH 9.8, 10% glycerol, 5 mM P-mercaptoethanol, 0.1 M NaCl), buffer C (50 mM Tris-HC1, pH 7.5, 10% glycerol, 5 mM P-mercapto- ethanol). All buffers were made with distilled, deionized water first passed through a Milli-Q system.

Plasmids-pUClSARV/8A, containing the HIV gag precursor pro- tein coding sequence, was derived from a clone 8A of ARV (AIDS- associated retrovirus) as described (Sanchez-Pescador et al., 1985) and was a gift of Dr. Philip Barr. pBBMD11, an IPTG-inducible overproducer of the E. coli dnaX gene, contains the tac promoter and was constructed by M. Bradley in this laboratory. All plasmid DNAs used for cloning were isolated by the method of Holmes and Quigley (1981) and purified by cesium chloride density gradient centrifugation in the presence of ethidium bromide (Maniatis et a[., 1982). DNA concentrations were determined using the following values: 50 pg of double-stranded DNA/ml/ODZGO, 40 pg of single-stranded DNA/ml/ ODZW (Maniatis et al., 1982).

Preparation of Primers and Polymerase Chain Reaction-Two primers used for polymerase chain reaction (PCR) were chemically synthesized on a Biosearch DNA synthesizer model 8600 in the University of Colorado Oligonucleotide Synthesis Core and purified using 8 M urea-polyacrylamide gel electrophoresis as described (Hag- erman, 1985). PCR was performed according to the procedure of Saiki et al. (1988) with the purified primers and uncut pUC19ARV/BA as template, using 2 units of Taq polymerase/100 p1 of reaction. The primers used were:

B g l I I RBS Aha111 Met 5"GGG AGATCT AGGAGG TTTAAAA TAATG CAG

AGA GGCAATTTT AGG- 3' (Primer 1)

Ps tI Trm Asn 5'-GGG CTG CAG TGA ATT AGC CTG

B c l I TCT CTC AGT GCA-3' (Primer 2)

BglII, AhaIII, PstI, and Bcll are restriction enzyme cleavage sites; RBS, ribosome binding sequence; Trm, termination codon. Starting with 1 ng of template DNA (pUClSARV/BA), we obtained 153 pg of amplified DNA after 25 cycles. 100 pmol of each primer was used per reaction.

Nitrocellulose Filter Binding Assay-Binding of nucleocapsid pro- tein to polyadenylic acid was measured by filtration through nitro- cellulose filters. An aliquot of protein serially diluted with H20 was added to the binding assay buffer (10 mM Tris-HC1, pH 7.5, 10 mM NaCI, 50 Ng/ml bovine serum albumin, 0.2 mM DTT, 10 mM magne- sium acetate). The reaction was initiated by adding 5 pl of 3.76 p~ (total nucleoside) p0ly-[8-~H]A (659 cpmlpmol of nucleoside residue) to make 50 gl of final reaction volume. After 15 min of incubation at 25 "C, the total reaction sample was filtered through a 24-mm nitro- cellulose filter presoaked for 30 min in filtration buffer (10 mM Tris- HCl, pH 7.5, 10 mM NaCI) under vacuum. Filters were then washed with 50 p1 of this buffer, dried under a heat lamp, placed in 5 ml of scintillation mixture fluid (Ecoscint 0 (National Diagnostics)) and counted in a Beckman model 3801 Scintillation Counter. Total input of radiolabeled RNA was determined by spotting the same amount of RNA used for the assay on dry filters without washing. Background values obtained in the absence of protein were subtracted in all cases. One unit of activity is defined as the amount of protein required to bind 1 pmol of total nucleotides.

Partial Protein Sequencing and Amino Acid Analysis-The amino- terminal sequence of nucleocapsid protein was determined in an Applied Biosystems 477A protein sequencer at the University of Colorado Cancer Center Protein Sequencing Facility. A polyvinyli- dene fluoride membrane was submerged in 200 pl of 10 mM sodium phosphate, pH 6.8, containing 1 pg of nucleocapsid protein and incubated overnight at 4 "C. The membrane was then washed with water to remove salt, dried, and inserted into the appropriate cartridge of the sequencer. The carboxyl-terminal amino acids of nucleocapsid protein were released by digestion with carboxypeptidase A prepared as described (Ambler, 1972). Carboxypeptidase A (0.6 nmol) was

HIV Nucleocapsid Protein 16521

added to 6.6 nmol of nucleocapsid protein in a total reaction volume of 75 pl of 20 mM Tris-HC1, pH 7.8. The reaction mixture was incubated for 15 h at 25 "C and filtered through Centrex Microfilter to separate amino acids released from the undigested nucleocapsid protein and the enzyme. The resulting solution was dried, resus- pended with 20 pl of Beckman Na-S sample buffer, and subjected to amino acid analysis. All amino acid analyses were performed on a Beckman System 6300 high performance amino acid analyzer. Nor- leucine was used as an internal standard. To determine the molar extinction coefficient of nucleocapsid protein, the absorbance of a solution of nucleocapsid protein was determined on a Hewlett Pack- ard 8451A diode array spectrophotometer using protein dialyzed overnight against 10 mM potassium phosphate, pH 7.5, 1% glycerol. Aliquots of the protein in measured volumes were then hydrolyzed in acid for 22 h and subjected to quantitative amino acid analysis.

Other Methods-Protein concentrations during purification were determined by the Bradford method (1976) using y-globulin as a standard. SDS-polyacrylamide gel analysis was performed according to Laemmli (1970) with sample buffer (60 mM Tris-HC1, pH 6.8,50% glycerol, 10 mM DTT, 10 mM o-phenanthroline, 1% SDS, 0.02% bromphenol blue) and Coomassie Brilliant Blue staining. E. coli strain HBlOl was used for transformation performed as described (Maniatis et al., 1982). DNA sequences cloned into M13mp18 were determined by the dideoxy sequencing method (Sanger et al., 1977; Norrander et al., 1983).

Fluorescence Measurement-The fluorescence measurement was performed on a SLM-Aminco 48000 spectrofluorometer controlled by an IBM AT computer as previously described (Griep and McHenry, 1992). The excitation and emission band widths were set at 2 and 4 nm, respectively. To measure tryptophan fluorescence quenching of HIV nucleocapsid protein upon binding to nucleic acid, the protein (1-3 p ~ ) in 200 p1 was titrated with poly(U) in a 3 X 3-mm quartz cuvette, by adding small aliquots (2-4 pl) of a concentrated poly(U) stock solution, and was monitored for its fluorescence changes using an excitation wavelength of 290 nm and an emission wavelength of 350 nm. No correction for inner filter effects was required since the absorbance of poly(U) did not exceed 0.01 when the nucleocapsid protein was completely titrated. The measurements were performed in 10 mM Tris-HCl, pH 7.5,O.l mM 8-mercaptoethanol, 0.2% glycerol at 25 "C. The fluorescence changes were corrected for dilution of protein and the background intensity of buffer fluorescence. Poly(U) concentration was determined spectrophotometrically using an ex- tinction coefficient, CM(,,) (260 nm) = 9.2 X lo3 M" cm" (Kowalczy- kowski et al, 1981).

RESULTS

Cloning and Overproduction of HIV Nucleocapsid Protein- To provide an abundant, reproducible, safe source for nucleo- capsid protein, we developed an E. coli expression vector that directs the synthesis of nucleocapsid protein identical to that isolated from viral particles. Since HIV nucleocapsid protein is proteolytically processed from the gag precursor, synthesis of the recombinant HIV nucleocapsid protein must be initi- ated with the amino-terminal residue found in the processed protein and terminate immediately after the carboxyl termi- nus of the proteolytically processed natural protein. The presence of a methionine residue at the amino terminus of the processed mature nucleocapsid protein permitted use of the translation product without the need for any additional processing, and placement of a stop codon immediately after the processed carboxyl-terminal residue generated a protein with the correct carboxyl terminus. To accomplish this, primers for PCR were designed to amplify only the nucleo- capsid protein-coding sequence from an HIV proviral DNA clone and to place a ribosome binding sequence in front of the amino-terminal methionine and a termination codon after the desired carboxyl terminus. The recognition sequences of several restriction enzymes were also included in the noncod- ing ends of the PCR primers to generate convenient sites for cloning purposes. Both the PCR-amplified DNA and the recipient pBBMDll expression vector were digested with BglII and PstI restriction endonucleases and joined to form the HIV nucleocapsid protein expression vector pJC1. In

pJC1, the nucleocapsid protein-encoding DNA sequence is under the control of the tac promoter and subject to IPTG induction. An HIV nucleocapsid protein-producing strain was obtained by transformation of E. coli HBlOl with pJC1. Colonies that grew on ampicillin were selected, DNA isolated from them, and subjected to restriction analysis to verify the presence of the plasmid and correct orientation of the inserted DNA. DNA sequencing was performed with the cloned nu- cleocapsid gene to ensure that no mutations occurred during PCR. The nucleocapsid DNA sequence of pJCl was identical to that determined in the proviral clone (Sanchez-Pescador et al., 1985).

The production of HIV nucleocapsid protein after IPTG induction was monitored by SDS-polyacrylamide gel analysis of cell lysates obtained from cells grown in different IPTG concentrations for various times (data not shown). Maximal expression was obtained when mid-log phase cells were in- duced with 1 m M IPTG for 5 h.

Nitrocellulose Filter Binding Assay-Activity of the purified nucleocapsid protein during purification and quantitation was monitored in a nitrocellulose filter binding assay, which ex- ploited the RNA binding property of the HIV nucleocapsid protein. Under certain conditions, nucleic acids bind to nitro- cellulose filters only when they are bound to protein. If the nucleic acid is radioactive, retention of radioactivity on filters can be used to quantitate the binding protein. To determine whether our nucleocapsid protein-expressing strains overpro- duced such binding activity, we performed a titration experi- ment on radiolabeled poly(A) using cell lysates from the HIV nucleocapsid protein-producing strain, E. coli HBlOl/pJCl, and from the parental HBlOl control strain. Increasing the amount of HBlOl cell lysate did not result in an increase in binding. Titration with a cell lysate containing nucleocapsid protein resulted in a linear increase in bound poly(A). Ap- proximately 3 pmol of poly(A) (nucleotide) was bound by 1 pg of lysate protein. The efficient binding observed only with lysates from the nucleocapsid protein-producing strain sug- gested that the activity measured was due primarily to the nucleocapsid protein and not E. coli proteins. This result also indicated that the protein was sufficiently overproduced to enable detection of binding activity even in crude lysates. Thus, this assay provided a convenient means to quantitate the total activity of nucleocapsid protein in protein fractions during purification and to monitor the elution positions of the protein in each chromatographic step.

Purification of Nucfeocapsid Protein HIV nucleocapsid protein was purified 30-fold to apparent

homogeneity from E. coli HBlOl/pJCl (Table I). All opera- tions, unless otherwise noted, were conducted at 0-4 "C. Frac- tions were pooled when poly(A) binding activity was at least 50% of the peak activity.

Cell Growth-Cells were grown in 160 liters of media con- taining 2.1 kg of yeast extract, 1.4 kg of peptone, 0.5% glucose (w/v), 1.9 kg of KZHP04, 210 g of KH2P04, and 50 pg/ml

TABLE I Purification table

Fraction Total activ- Total specific activity Purification ity protein factor

units x 1 P mg units/% X 10" fold

I. Cell lysate 23.5 6,450 0.36 1 11. Polymin P 3,894

111. Ammonium sulfate 11.9 1,485 0.80 IV. Q-Sepharose 9.3 346 2.68

2.2 7.4

V. S-Sepharose 6.0 57 10.5 29.2 2.7 24 11.0 30.5 VI. G-50

16522 HIV Nucleocapsid Protein

ampicillin to mid-log phase (ODm = 0.6). Cells were then induced with a final concentration of 1 mM IPTG and grown to late log phase (OD, = 6) at 37 "C for 5-6 h with high aeration (200 liter/min, gradually increasing pressure from 2 to 14 pounds/square inch at the end). The pH was maintained between 7.0 and 7.4 by addition of concentrated NH,OH. Cells were harvested within 1 h in a continuous flow centrifuge after passing through a 70-foot stainless steel coil submerged in -2 "C liquid to cool the cells to 14 "C. Cells were weighed, resuspended in an equal weight of 50 mM Tris-HC1, pH 7.5, 10% sucrose and immediately frozen by pouring into liquid Nz.

Preparation of Cell Lysate-Frozen cells (60 g cell weight, 120 g total weight) were resuspended in 240 ml of cell lysis buffer (50 mM Tris-HC1, pH 7.8, 10% sucrose, 0.3 M NaC1, 2 mM EDTA, 10 mM spermidine-HC1) and placed in a precooled cell disrupter containing 200 ml of 0.1-mm glass beads. Cells were lysed by grinding for 1 min followed by a 10-min cooling period. This step was repeated for a total grinding time of 5 min. In preliminary experiments using lysozyme to disrupt cells, purification of nucleocapsid protein away from the in- troduced lysozyme proved very difficult because both nucleo- capsid protein and lysozyme PI values (10.8-11.0) are highly basic proteins with nearly equivalent PI values and they chromatograph similarly. Thus, we used a mechanical cell lysis method to eliminate any possible contamination by lysozyme in the purified nucleocapsid protein. The effective- ness of the mechanical cell lysis method is similar to that of the lysozyme method (yielding 3.9 x 10' and 3.5 X lo5 units/ g of cells, respectively). Lysed cells were transferred to 250- ml GSA bottles and centrifuged (15,000 X g, 4 "C, 1 h), resulting in 235 ml of supernatant (fraction I).

Polymin P Precipitation-To obtain HIV nucleocapsid pro- tein free of nucleic acids, polymin P was used to precipitate the nucleic acids leaving nucleocapsid protein in the super- natant. Polymin P (10% (w/v), pH 7.9) was slowly added to fraction I with constant stirring for 15 min to a final concen- tration of 0.5% polymin P. The solution was centrifuged (10,000 X g, 4 "C, 20 min), yielding 230 ml of supernatant (fraction 11).

Ammonium Sulfate Precipitation-(NH&S04 (0.243 g) was added to each ml of fraction I1 (40% saturation, 0 "C). After ammonium sulfate was completely dissolved, the suspension was centrifuged (10,000 x g, 4 "C, 15 rnin), and 0.285 g of (NH&304 was added to each ml (80% saturation, 0 "C) of supernatant. After further centrifugation (10,000 x g, 4 "C, 20 min), the final pellet was resuspended with 1/5 fraction I volume of buffer (50 mM Tris-HC1, pH 7.8, 10% glycerol, 0.1 mM EDTA, 5 mM &mercaptoethanol). The solution was dialyzed in Spectrapor dialysis tubing ( A I r cutoff, 3,500) against 4 liters of buffer A until the conductivity of the dialysate was equal to that of buffer A, providing fraction I11 (83 ml).

Q-Sepharose Column Chromatography-The PI of the nu- cleocapsid protein was estimated to be between l l and 11.5 using the procedure of Lampson and Tytell (1965). Thus, buffers with a high pH were used, since most cellular proteins would be expected to be negatively charged under those con- ditions and to stick to the positively charged column, while the basic nucleocapsid protein would be expected to flow through. Binding activity and stability of nucleocapsid protein were not affected at this pH.

Dialyzed fraction I11 was applied to a Q-Sepharose column (1.5 X 25 cm) equilibrated with buffer A. After washing the column with 1 column volume of buffer A, the column was developed with a linear gradient of buffer A + buffer A + 1

M NaCl (total 10 column volumes). Flow rate was maintained at 1 column volume/h with a peristaltic pump. The nucleo- capsid protein was eluted in the flow-through fraction (Fig. lA); most of the cellular protein bound and was eluted in the gradient. The activity peaks were combined and dialyzed overnight against 4 liters of buffer B, resulting in fraction IV (65 ml).

S-Sepharose Column Chromatography-Fraction IV was loaded onto a S-Sepharose column (0.6 X 10 cm) equilibrated with buffer B at a constant flow rate (2 column volume/h). The column was washed with 2 column volumes of buffer B, and the activity was eluted with a linear gradient of buffer B + buffer B + 0.6 M NaCl (total 10 column volumes). The nucleocapsid protein began to elute at 0.3 M NaCl (Fig. 1B). The peak was pooled to yield fraction V (8 ml). At this stage, nucleocapsid protein was nearly homogeneous based on SDS- polyacrylamide gel analysis.

G-50 Gel Filtration-To ensure homogeneity of the protein and to place the protein in a buffer of physiological pH, fraction V was directly applied to a G-50 gel filtration column (1.5 X 73 cm) equilibrated with buffer C at a flow rate of 15 ml/h. Fractions containing activity were pooled to result in fraction VI (16 ml) (Fig. lC), which was then divided into small aliquots, rapidly frozen in liquid N2, and stored at -70 "C. The purified nucleocapsid protein was stable when stored at -70 "C, -20 "C, or even when subjected to frequent freezing and thawing.

Characterization of Nucleocapsid Protein SDS-Polyacrylamide Gel Electrophoresis-Purified HIV nu-

cleocapsid protein (fraction VI) was denatured and analyzed by SDS-polyacrylamide gel electrophoresis. Only one band of the nucleocapsid protein was observed even when 35 pg of purified protein was loaded (Fig. 2).

Determination of the Amino- and Carboxyl-terminal Se- quences and the Amino Acid Composition-To verify that the purified HIV nucleocapsid protein does not undergo any amino- or carboxyl-terminal processing by E. coli cellular proteases, protein sequencing and amino acid analysis were performed on the purified recombinant nucleocapsid protein. Five amino acids sequenced from the amino terminus were the same as predicted from the DNA sequence. Amino acids released from the carboxyl terminus by carboxypeptidase A were also in complete agreement with the predicted sequence. Four times more asparagine (4.1 nmol) was released than alanine (1.1 nmol), indicating that asparagine is the first residue released (carboxyl-terminal amino acid). The amino- terminal (methionine) and carboxyl-terminal (asparagine) amino acids in our purified nucleocapsid protein match those of the nucleocapsid protein isolated from HIV virus (Hender- son et al., 1988l; South et al., 1990). In addition, the amino acid composition data showed an excellent correlation be- tween the amino acids expected from DNA sequence and the amino acids found in the purified nucleocapsid protein (Table 11). These results confirm that the purified nucleocapsid pro- tein is identical to that found in viral particles and that no proteolytic processing of the protein occurred in E. coli. The sequence of the HIV nucleocapsid protein inferred from the DNA sequence lacks serine, leucine, and tyrosine; these amino acids were not detected in the amino acid analysis.

Extinction Coefficient Determination-The molar concen- trations of nucleocapsid protein were determined by quanti- tation of selected stable amino acids. Values obtained from quantitative amino acid analysis were divided by the number of residues of each amino acid present in the protein to calculate the molar concentration (Table 11). The molar ex-

FIG. 1. Chromatographic steps in HIV nucleocapsid protein purifica- tion. A , Q-Sepharose column chroma- togram. E , S-Sepharose column chro- matogram. C, G-50 column chromato- gram.

HIV Nucleocapsid Protein

A.

~

/ , , I , ,

1 .o ,

0.8

0.6

0.4

0.2

Fraction

E. , 0.5 /

10 20 30 40 50 60 70 Fraction

C.

16523

NaCl (M) 0 180

140 9 e 1.

3 -

100 s a 3

6o :I: - 20

NaCl (M)

50 0

s 9 1.

40 2 $

30 g. 2 0 -

2o 1 10

10 20 30 40 50 60 70 80 Fraction

tinction coefficient of nucleocapsid protein was then deter- ferent facilities. The results agreed within 2 3 % (one standard mined by dividing the absorbance value of the same protein deviation). samples by the molar concentration of the protein. The mean Measurement of HIV Nucleocapsid Protein Binding to Hom- extinction coefficient of the HIV nucleocapsid protein was opoly(A) Using Nitrocellulose Filters-Titration of homo- determined to be 8350 cm” M” at 280 nm (Table 11), based poly(A) with nucleocapsid protein to determine the binding on amino acid analysis independently performed in two dif- constant of HIV nucleocapsid protein for its nonspecific pro-

16524 HIV Nucleocapsid Protein

kDa A B C D E

43 -

2 9 - FIG. 2. SDS-polyacrylamide gel

of the purified HIV nucleocapsid protein. The protein sample was dena- tured and subjected to electrophoresis in a 17.5% acrylamide gel and stained with Coomassie Blue as described under "Ex- perimental Procedures." Lane A, stand- ard protein molecular weight marker; lanes B, C, and E 6,12,35 pg of purified HIV nucleocapsid protein (fraction VI); lane D, no protein sample.

18.4-

14.3-

6 .2 -

3 . 0 -

TABLE I1 Amino acid composition and extinction coefficient of the HIV

nucleocapsid protein Purified HIV nucleocapsid protein in 93 pl of a buffer solution (10

mM potassium phosphate, pH 7.5, 1% glycerol) was used in determi- nation of amino acid composition and extinction coefficient. Absorb- ance was measured at 280 nm.

Amino acid Composition NC conc. c

Aspartate Threonine Glutamate Proline Alanine Valine Isoleucine Phenylalanine Lysine Arginine

6 2 7 1 3 1 1 2 7 8

nmol 13.3 4.4

16.0 2.2 6.6 2.3 2.2 4.4

16.0 17.4

WM 23.9 23.7 24.6 23.7 23.7 24.7 23.7 23.7 24.6 23.4

Average

cm" M' 8,368 8,439 8,130 8,439 8,439 8,097 8,439 8,439 8,130 8,547

8.350

tein-nucleic acid interaction resulted in a typical hyperbolic binding curve (Fig. 3A). When evaluating the binding iso- therms for proteins that interact nonspecifically with nucleic acids one must account for the binding site size resulting from protein binding to multiple equivalent sites along the nucleic acid and any cooperative interactions that occur due to pro- tein-protein interactions for proteins bound to adjacent sites. Using the binding site size ( n = 7) determined for HIV nucleocapsid protein (see below) and von Hippel's extended Scatchard analysis for nonspecific protein-nucleic acid inter- actions (Equation 10 of McGhee and von Hippel, 1974), we

" Nucleocapsid

plotted the data reported in Fig. 3A on a scatchard plot (Fig. 3B). The experimental points resulted in a straight line. An intrinsic binding constant ( K ) of 1.6 X lo6 M-' was obtained, assuming noncooperative binding.3 This binding affinity of HIV nucleocapsid protein to nonspecific nucleic acid is com- parable to the binding constants of other retroviral nucleo- capsid proteins under the ionic strength conditions used (Kar- pel et al., 1987 and references therein).

Determination of the Binding Site Size of HIV Nucleocapsid Protein on RNA Using the Intrinsic Tryptophan Fluorescence Quenching-RNA binding quenches the fluorescence of the single tryptophan residue in the HIV nucleocapsid protein, a property shared with a number of nonspecific nucleic acid- binding proteins (Kelly et al. 1976; Williams et al. 1983; Lohman et al. 1986; Karpel et al. 1987). We exploited this property to determine the binding site size of HIV nucleocap- sid protein on RNA. For such determinations to be valid, the protein concentration used should exceed the value of 1/Kw by at least 10-fold (Kowalczykowski et al., 1986). Thus, we decreased the salt concentration below that used in the nitro- cellulose titration to increase the affinity of the nucleocapsid protein for RNA. This permitted us to perform all titrations at RNA concentrations sufficiently low to avoid inner filter effects. The titration of the purified nucleocapsid protein with poly(U) resulted in quenching of tryptophan fluorescence in proportion to the amount of RNA added (Fig. 4). About 80% of the initial fluorescence value was quenched upon satura- tion. A binding site size ( n ) of 7 nucleotides/HIV nucleocapsid

The straight line observed in the Scatchard analysis with data obtained with poly(A) and nucleocapsid protein concentrations below the apparent dissociation constant suggest noncooperative binding under the solution conditions used.

FIG. 3. Binding of HIV nucleocap- sid protein to poly(A). The nitrocel- lulose filter binding assay was used to quantitate binding of HIV nucleocapsid protein to poly(A). A , titration ofpoly(A) 0.376 p~ with purified HIV nucleocapsid protein. The point at 0.1 pg represents 0.28 pM nucleocapsid protein. B, Scat- chard plot of HIV nucleocapsid protein- poly(A) using von Hippel’s extended Scatchard analysis for nonspecific pro- tein-nucleic acid interactions (McGhee and von Hippel, 1974).

HIV Nucleocapsid Protein 16525

I 1 I - ~ ~ I 0.1 02 0.3 0.4

Plotrill (m)

FIG. 4. Determination of the binding site size of HIV nucleocap- sid protein. Fluorescence measure- ments were used to determine the bind- ing site size of HIV nucleocapsid protein. The nucleocapsid protein concentration was 1.3 p ~ .

I I I I I I I I J 2 4 f 3 t E i o 12 14 16

[Nucleotidel/[Nucleocapsid protein]

16526 HIV Nucleocapsid Protein

protein was determined as the intersection of initial and final quenching slopes (indicated arrow in Fig. 4). Taking the value of 80% quenching as the saturation value and assuming that quenching is directly proportional to the amount of nucleo- capsid protein bound to RNA, a Scatchard plot was generated (not shown) and an association constant (KO) of 1 X lo8 M" was calculated for the HIV nucleocapsid protein-poly(U) in- teraction using Equation 10 of McGhee and von Hippel (1974). This very tight binding observed in these experiments in the presence of low salt (10 mM Tris-HC1) is comparable to that measured with other retroviral nucleocapsid proteins (Karpel et al., 1987).

DISCUSSION

We have constructed a vector that directs the efficient expression of the mature HIV nucleocapsid protein in E. coli. The DNA sequence encoding the nucleocapsid protein was amplified from HIV proviral DNA by PCR to obtain protein with the correct amino and carboxyl termini. After cloning into an IPTG-inducible expression vector, the nucleocapsid protein was overproduced to a level of at least 3% of the total cellular protein. In spite of its general RNA binding proper- ties, no severe inhibition of cell growth was detected. The nucleocapsid protein produced has the same sequence as the protein isolated from viral particles, as confirmed by direct sequencing of both the amino and carboxyl termini of the homogeneous purified protein. An extinction coefficient of 8350 cm" M-' a t 280 nm was determined, a value that will be useful in determining the precise concentration of nucleocap- sid protein in future experiments.

A functional RNA binding assay to monitor the nucleocap- sid protein purification indicated no significant loss of activity that might have been caused by the purification procedures and confirmed that the protein was functionally active.

The purified protein binds RNA with high affinity. The intrinsic association constants measured (2 X lo6 M-l, 10 mM Tris, 10 mM NaCl, 10 mM M$+; 10' M-', 10 mM Tris) are within the range measured for other nucleocapsid proteins. A 7-base binding site was measured by fluorescence quenching consistent with observations made with a variety of retroviral nucleocapsid proteins (Karpel et al., 1987 and references therein). However, these findings differ from measurements made with a form of HIV nucleocapsid that has an extension of 16 amino acids at the carboxyl terminus. This extension is composed of natural HIV sequences contained in precursors to the mature HIV nucleocapsid protein (Khan and Giedroc, 1992) This protein was subjected to a rigorous binding analy- sis and found to have a site size of 15. The precursor for the murine leukemia virus nucleocapsid protein binds to a 3-fold larger site than the mature protein (20 uersus 6 nucleotides) with higher affinity (Karpel et al., 1987). The ability of the gag precursor to assemble nucleic acid-free particles in the absence of nucleocapsid protein sequences (Meric and Spahr, 1986; Voynow and Coffin, 1985) suggests strong protein- protein interactions between other gag domains that can lead to cooperativity. Perhaps the larger form of HIV nucleocapsid protein that includes sequences from the p7-p6 precursor that exists in immature virions has different binding properties, important in earlier stages of viral assembly, but are processed out before the replicative reaction begins.

The replication step of HIV has provided one of the prin- cipal targets for development of antiviral agents. Our current biochemical knowledge of HIV replication comes primarily from artificial template-primers. These simplified systems have been very valuable in screening for inhibitors of the central polymerase activity of reverse transcriptase. However,

the complete replicative reaction may involve a number of specific interactions or mechanisms not required in simple homopolymeric assays so that simplified assays for screens may not reveal all useful inhibitors. It is our goal to fully reconstitute an in vitro HIV replicative reaction starting with natural long terminal repeat-containing DNA and generating a nucleoprotein particle capable of integrating into exogenous DNA. As part of these efforts, we require large quantities of nucleocapsid protein, the protein that coats the viral RNA template during the natural reverse transcription reaction. The results reported herein indicate that biochemically useful quantities of this essential viral protein can now be obtained.

Acknowledgments-We gratefully acknowledge the assistance of Julie Lippincott of the University of Colorado Cancer Center Protein Sequencing Facility for amino-terminal sequencing, Bob O'Connor of the University of Colorado Cancer Center Fermentation Facility for growing the bacteria used in this study, and Robert Binard of the University of Colorado and Jim Kenny of the Beckman Center at Stanford for the amino acid analyses.

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