Recombinant Human Immunodeficiency Virus Type 1 Nucleocapsid ...

Vol. 267, No. 10, Issue of April 5, pp. 6689-6695, 1992 Printed in U.S A.

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1992 by The American Society for Biochemistry and Molecular Biology , Inc

Recombinant Human Immunodeficiency Virus Type 1 Nucleocapsid (NCp7)Protein Unwinds tRNA* (Received for publication, August 5, 1991)

Raza Khanand David P. GiedrocS From the Department of Biochemistry and Biophysics, Texas A & M University, College Station, Texas 77843-2128

The nucleocapsid protein (NC) of all animal retrovi- The gag gene of all known retroviruses encodes a multiruses, encoded by the gag gene, is the major structural functional polyprotein precursorproduct which has been protein of the core ribonucleoprotein complex, bound shown toplayfundamental roles invirus genomic RNA to genomic RNA in maturevirions. NC is also thought encapsidationand replication (1, 2). These RNAbinding to play one or more accessory roles in reverse tran- activities are mediated by the nucleocapsid (NC)’ domain of scription. Mature NC (~7”‘)from human immunodefi- the precursor or processed NC subunit, respectively (1-3). ciency virus type 1 (HIV-1) is a 71-amino acid, basic Correctencapsidation involves formation of the genomic protein which contains twoCysaHis Zn(I1) retroviral- RNA dimer, composed of two identical 35 S viral RNA moltype zinc finger domains. Herein, we describe the sub- ecules, and requires both major and minor cis-acting RNA cloning and expression of HIV-1 NC, denoted NC71, elements found toward the 5‘ end of the genome (1, 4-6). from aninducible phageT7 RNA polymerase promoter Genomic dimerizationappearstoaidinthe switchfrom in Escherichia coli. Purified NC71 can be reversibly genomic RNA translation of gag precursor polypeptides to reconstituted with 2 g-at Zn(I1) determined by atomic genomic packaging and virus assembly in the production of absorption. Ultraviolet circulation dichroism spectros-progeny virus (7). I n vitro, viral NC from several retroviral copy has been used to characterize the complexes be- sources appears able to promote dimerization (oligomerizatween highly purified NC71 and the RNA homopoly- tion) of viral genomic fragments containing major cis-acting nucleotide poly(A) and E. coli tRNA”’”“. On poly(A), packaging signals (7-10). In the initiation of proviral cDNA Znz NC71 is characterized by an apparent site size n = synthesis by reverse transcriptase, NC from both HIV-1 and 15 f 3 nucleotides and high affinity (Kapp= 3 X 10’ MoMLV have been hypothesizedto promote the base-pairing M-’) and moderately cooperative ( O = 170 f 25)bindor annealing of the cognate replication primer tRNA onto ing. A mixture of E. coli tRNA species (tRNAmixed) was genomic viralfragmentscontainingthe complementary used to probe the conformational changes induced in primer binding site (7-12). tRNA upon binding of HIV-1 NC71. Two structural NCfromall animalretrovirusescontainsone or two forms of tRNAmixed,which differ in their degree of conserved Cys-Xaa2-Cys-Xaa4-His-Xaa4-Cys retroviral-type tertiary structure, were assayed for susceptibility their zinc-finger Cys3Hismotifs (13,14),which can be reconstituted to denaturation by NC71. Five molar monomer equiv- with Zn(II), Cd(II), and Co(I1) i n vitro (15-18) (see Fig. 1). alents of NC71 are required to denature the “inactive” These domains are thus reminiscent of “zinc-finger” domains tRNA in the absence of Mg2+. A Zn(I1)-free, oxidized in other nucleic acid binding proteins (19). The role or funcform of NC71 was also shownto unwind inactive tRNA tional significance of metalbinding by NC in the virion with the same efficiency and stoichiometry. The de- remains poorly defined. Mutagenesis of Zn(I1) coordinating tailed spectral changes which occur on NC-induced ligands and adjacent aminoacids in infectious proviral clones denaturation closely mimic temperature-induced defrom anumber of retroviruses results in a varietyof replicative naturation of inactive tRNA””“. The prototype helix- defects. The most severe completely block genomic encapsidestabilizing protein, T4 gene 32 protein, is unable to dation, while less severe mutations result inreduced levels of unwind this formof tRNA under the sameconditions. correctly dimerized genomic RNA (20-26). Interestingly, all The stoichiometry of unwinding of inactive tRNA by suchmutants show dramatically reduced infectivities (by NC71 is consistent with the sitesize determined with 210”-fold), apparently independent of the amountof genomic poly(A). An “active” form of tRNAmixed, prepared by RNA packaged (20-26). These studiessuggest that thisregion thermal denaturation and refolding of the inactive of NC is important in RNArecognition at some point in the form with Mg“’, proved less susceptible to both tem- retroviral life cycle, and/or plays a structural role in eventual perature and NC71-induced unwinding. The mecha- formation of the reverse transcriptionally active ribonucleonistic implications of these findings on the reported protein complex (cf. Ref. 27). It is generallyaccepted that biochemical activities of RNA:RNAannealing and rep-processed NC is bound nonspecifically in a histone-like conlication primertRNA positioning byNC are discussed. densation of the diploid RNA genome in mature virus prep* This work was supported by the Texas Agricultural Experiment arations (27). These functional activities are consistent with Station, Biomedical Research Support Group funds to the College of the biochemical activities of purified NC. Zn(I1) coordination Agriculture at TexasA & M, and National Institutes of Health Grant however, appears to have little affect on this type of binding GM42569. This work is in partial fulfillment of the requirements of i n vitro (18,28,29). Itwas recently demonstrated that neither the M S . degree (to R. K.). The costs of Publication of this article dimerization of genomic RNA fragments nor tRNA annealing 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. $ Recipient of American Cancer Society Junior Faculty Research Award JFRA-270. To whom correspondence should headdressed.

The abbreviations used are: NC, nucleocapsid; ss, single-stranded; Na,EDTA, trisodium ethylenediaminetetraacetic acid; MoMLV, Moloney murine leukemia virus; HIV-l, humanimmunodeficiency virus; kh, kilobase(s); hp, basepair(s); DTT,dithiothreitol.

6689

6690

tRNA Unwinding by HIV-1 Nucleocapsid Protein

by purified MoMLV NC in vitro requires a functional Zn(I1) coordination chelate (lo), in contrast to anearlier report (9). Inthis report, we presentexperiments directed toward elucidating the mechanism of RNARNA annealing by HIV1 NC. The mode by which NC interacts with model singlestranded as well as duplex and tertiary structure-containing RNA molecules of defined structure must be determined before experiments with more complex retroviral RNAs are undertaken.Herein, we describe the subcloning, bacterial overexpression, and purification of mature, correctly processed HIV-1 NC2 (30, 31), denoted NC71, and CD' spectroscopic studies aimed at identifying the types of complexes NC71 forms with the model ssRNA polynucleotide, poly(A), and two structural forms of tRNA. We conclude that NC71 is a potent RNA double-helix unwinding protein which derives from its ability to bind stoichiometrically and cooperatively to single-stranded nucleic acids.

to the lac and phage T7 promotors to make pIBInc7l.hivl (3.1 kb). The 228-bp NdeI-BamHI fragment was then excised from pIBnc7l.hivl and subcloned into the same sites of expression vector PET-3b (37) behind the phage T7 promoter to create pT7nc7l.hivl. Both strandsof the insert in pT7nc7l.hivl which encodes NC71 were sequenced using the dideoxy chain termination method and were shown to exactly match the published sequence of pBH10-R3 (36). NC71 Purification-pT7nc71.hivl was freshly transformed into E. coli BL21(DE3)/pLysS (37) to ampicillin and chloramphenicol resistance. Asingle colony was chosen and grown in 3 ml of Luria broth supplemented with 100 pg/ml ampicillin for 12 h, 37 "C. The culture was then diluted 10-fold and allowed to grow to A 6 =~0.7. Subsequent 10-fold dilutions of the exponentially growing cultures were made until thedesired preparative culturevolume was obtained, e.g. 6 liters. At no time prior to preparative induction was the culture allowed to grow beyond AsW = 1.0. In a typical large-scale experiment, 6-9-liter cultures taken to A600 = 0.5-0.7 in Fernbach shaking flasks at 37 "C were induced with 0.4 mM isopropyl-1-thio-/3-D-galactopyranoside, and allowed to grow for 2-3 h prior to harvest. The cells were pelleted by centrifugation at 5000 rpm, 5 min, washed with ice-cold water, and stored frozen at -20 "C until use. All subsequent manipulations were done at 4 "C unless otherwise indicated, using freshly degassed EXPERIMENTALPROCEDURES (Nz-purged) buffers. The cell pellet was allowed to warm to room temperature briefly and lysis buffer (10 mM Tris-HC1, pH 8.0, 50 mM Materials NaCl, 20 mM dithiothreitol (DTT), 20 mM MgCI,, 4 mMCaC12, 50 All buffers were prepared with doubly distilled and deionized Milli- p M ZnClz, 0.1 mM phenylmethylsulfonyl fluoride, 20 pg/ml DNase) Q water. ssDNA-cellulose was prepared as described (32). 5,5'-Di- was added at a ratio of 10 ml of buffer/g wet weight cells to complete thiobis(nitrobenzoic acid), isopropyl-1-thio-8-D-galactopyranoside,the resuspension of the cells. This suspension was gently stirred at and chromatographically purified DNase were obtained from Sigma. 37 "C for 30 min, solid NaCl was added to 0.8 M, incubated an Poly(A) was purchased from the Midland Certified Reagent Company additional 30 min, 37 "C, and then centrifuged at 6000 rpm for 10 (Midland, TX) and used following exhaustive dialysis into 10 mM min. This low-speed lysis pellet contained most of the NC71. The Tris-HC1, 0.1 mM Na,EDTA, 0.1 M NaCl, pH 8.1. tRNA'"Ixd from NC71 was then solubilized by dissolving the low-speed pellet in 20% Escherichia coli MRE 600, obtained from Boehringer-Mannheim (v/v) trifluoroacetic acid and 6 M guanidinium hydrochloride in 10 (Indianapolis, IN), was used following phenokchloroform extraction, mM Tris-HC1, pH 8.0 (40). This fraction was loaded directly onto a precipitation from ethanol, and exhaustive dialysis against 10 mM 2.5 X 10-cm CIScolumn (Waters preparative CIS,125-A pore, 55-105 Tris-HC1, 0.1 mM Na3EDTA, 0.05 M NaC1, pH 8.1. This is the p~ particle size) and washed with 0.05% trifluoroacetic acid, and "inactive" tRNAmixed preparation (33). An aliquot of this material (2.2 then with 0.05% trifluoroacetic acid, 10% acetonitrile. The NC71 X 10" M molecules) was heated to 75 "C for 2 min followed by a fraction was eluted by a stepto 0.05% trifluoroacetic acid, 40% quick-cooling on ice (33). The CD spectrum of this material was acetonitrile. The proteaseinhibitor phenylmethylsulfonyl fluoride indistinguishable from the starting material. The "active" tRNAmixed was added to 0.1 mM, and this solution was lyophilized to dryness. was prepared by adding MgCI, to 10 mM, followed by heating to 60 "C This acidified NC71 fraction was dissolved in 20 ml of 100 mM Trisfor 5-6 min and quick-cooled on ice (33). Molecular biologicals were HCI, pH 8,O.l M DTT, 0.2 mM ZnCl?, 50 mM NaCI, and loaded onto obtained from either New England Biolabs (Boston, MA), Boehrina ssDNA-cellulose column (1.5 X 15 cm) equilibrated with 100 mM ger-Mannheim, or Promega (Fisher Scientific). RecombinantT4 gene Tris-HC1, pH 8, 0.01 M DTT, 0.2 mM ZnCIZ,50 mM NaCl. After 32 protein lacking the C-terminal 49 amino acids (g32P-A) was washing, the column was developed with a 100-ml linear gradient purified from the overproducing plasmid, pPLg32-A.wt (32), and from 50 mM to 1 M NaCl in the same buffer. Generally, two distinct purified essentially as described for the wild-type protein (32). fractions of NC71 were eluted from the column during the NaCl gradient (Fig. 1A). N-terminal sequencing and amino acid analysis revealed that the first fraction was enriched in two proteolytic prodMethods ucts of NC71, corresponding to NC 11-71 and NC 4-71, resulting Plasmid Constructions-All molecular biological methods were car- from cleavage following Arg" and Arg, respectively (Fig. 1B). The ried out according to standardmethods (35). The plasmid pBH10-R3 second, tighter binding fraction was primarily authentic 1-71 with (kindly provided by H. Z. Streicher, National Cancer Institute, and variable amounts of NC71 4-71. In some cases, to further purify NC used with permission) was the source of the coding region for HIV-1 1-71 and to remove adventitious Zn(II), the ssDNA-cellulose NC71 NC (36). A 4.2-kb 5' EcoRI fragment was cloned into the EcoRI site pool was subjected to dialysis against 10 mM Tris-HC1, pH 7.6, 50 of pUC8 to make pEcoB. A 2.2-kb HindIII-KpnI fragment containing mM NaCl, 2 mM 0-mercaptoethanol, and was subsequently loaded the 3' one-third of the gag open reading frame and 5' two-thirds of ontoa TSK-Gel CM-650M carboxymethyl-Sephadex (1 X 9 ml) the pol open reading frame from pEcoB was then cloned between the column equilibrated with 20 mM Tris-HCI, pH 7.6, 50 mM NaC1, 0.1 same restrictionsites of pIBI24 (IBI, New Haven, CT)to make mM DTT. Following a wash with equilibration buffer, the column pIBI.HK22. This fragment was then purified and used as a template was developed with a step to 0.2 M NaCI, and a 25/25 ml linear 0.2/ with mutagenic for 25 cycles of the polymerase chainreaction 0.5 M NaCl gradient in the same buffer. The major pool fraction, primers GG300 (5'-CAGCTACCATACATATGCAGAGAGGC-3') eluting last in the NaCl gradient (at -0.4 M NaCl), was greater than and GG400 ( 5 ' - T G G A T C C G G T C T G C m T A A A A A T T C C C - 95% pure by amino acid analysis and N-terminal sequencing. NC71 TGGCCTTCCC-3'). The 5'GG300 primer introduces aNdeI restric- was exhaustively dialyzed under Nz against50 mM sodium phosphate, tion site (underlined) which overlaps the initiatorprotein methionine pH 6.8,O.l M NaCl and stored under N, atmosphere at -80 "C. The codon, and exactly corresponds to Met378in thegag polyprotein ~ 5 5 " ~ . 280/260 ratio exceeded 1.6 for all preparations. The molar extinction GG400 introduces a TAG stop codon following the codon for Phe448 coefficient of NC71 was estimated by UV absorption underdenaturing (boldface), as well as a BamHI restriction site further to the 3' side conditions to be ezS0 = 13,000 M" .cm", calculated from the net Trp of the stop codon (underlined). These manipulations produce a trans- (ez8,, = 5600 M" .cm" X 2) and Tyr (eZ80 = 1800 M".cm" X 1) lational reading frame which should encode a 71-amino acid NC contribution. Protein concentration quantitated by amino acid analyprotein. The crude polymerase chain reactionproduct was then sis gave similar results. All three forms of HIV-1 NC (1-71,4-71, and treated with Klenow fragment and dNTPs to form blunt ends, and 11-71) contained 2.0 k 0.2 g.at nondialyzable Zn(I1) by atomic cloned into theSmaI site of pIBI24 in the opposite orientation relative absorption (Perkin-Elmer 2380 operating in the flame mode) and 5.9 k 0.3 Cys upon titration with 5,5'-dithiobis(nitrobenzoic acid). A One report (40) suggests that there is an additional C-terminal metal-free form of NC was prepared by dialyzing NC against lOmM processing event in the 71 amino acid protein at intheMN Tris-HC1, 0.1 M NaC1, 5% glycerol, pH 8.0, 10 mM EDTA, followed strain HIV-1 sequence (cf. Fig. 1B) to make a 55-amino acid NC by dialysis against 10 mM Tris-HC1,O.l M NaCI, 5% glycerol, pH 8.0. protein. The significance of this cleavage is unknown. This sample was devoid of Zn(I1) (50.1 g.at Zn(I1)) and contained

tRNA Unwinding by HIV-1 Nucleocapsid Protein no reactive Cys by 5,5'-dithiobis(nitrobenzoic acid) titration. CD Spectroscopy-Near UV-CD spectra were collected on a Jasco C-600 spectrapolarimeter in a 10-mm pathlength rectangular cuvette (volume = 1.65-1.7 ml) thermostatted a t 25 f 0.1 "C in 10 mM TrisHCI, 0.1 mM DTT, pH8.3 (buffer A), 0.1 M NaCI, with or without 10 mM MgCI,. CD spectra of poly(A) were recorded from 300-240 nm at the indicated concentrations of poly(A), quantitated by = 10,300 M (nucleotide)".cm"(38). Foreach acquisition,two scans were digitally averaged at a scan rate of 20 nm/min, 2-s time constant, 1nm bandwidth, and 20-mdeg full scale. CD spectra of tRNAmixed were recorded from 310-210 nm with the same acquisition parameters as used for poly(A). In some cases, four spectra recorded from 270 to 250 nm were digitally averaged. [tRNA"''xed]was determined with = 4.8 X 10" M (molecule)".cm" or 19.9 (mg molecule.ml")" (34). Use of this molar extinction coefficient gives a mean weight residue ellipticity [O],,, a t 262 nm (assuming average values of 320 g/mol nucleotide and 75 nucleotides per tRNA) for "inactive" tRNA"'""' of 1.74 X lo4 deg.cm2.dmol" and 1.95 X lo4 deg.cm2.drnol-l for "activated" tRNA,both within 5% of theirpublished values(34). In titrations of poly(A) and tRNAwith protein, aliquotsof protein were added directly to the CD cuvette and continuously stirredfor 2 min. T h e stirring was then ceased, and the CD spectrum was recorded as indicated. During the course of multiple scans, or upon rescanning 10-20 min later, the CD spectrum of the protein-RNA complex did not significantly change (50.5 mdeg), evidence that equilibrium had been reached. [NC71] was determined by UV absorption as described above. Free NC71 is devoid of optical activity in the 300-240 nm region at the protein concentrationsused forthe binding experiments, and does not contribute to the CD spectrum of the complex. In the g32P-A/tRNA titration experiments, the small (56%) spectral contribution of the aromatic CD of g32P-A wasdigitally substracted from the UV-CD spectra of g32P-A-tRNA mixtures to obtain the correctedspectrum of thetRNA.g32P complex. All nucleicacid spectra were then corrected for dilution and the resulting corrected CD spectrum obtained. Fractional saturation (0)at the ith addition S on the positive Cotton of NC71 [NC71l1 to poly(A) W ~ calculated peak (264 nm) from 0 = (mdegi - mdegi,i,)/(mdegfi.,l - mdegini,). Similar results were obtained with the negative Cotton band a t 248 nm (data not shown). The error bars on all points reflect the cumulative effects of the signal-to-noise level of the optical activity of the complex following the ith addition of protein, the RNA alone, and the fully saturated complex. Theoretical McGhee-von Hippel (39) isotherms were calculated with given values of Ki (intrinsic monomer affinity), w (cooperativity parameter), andn (occluded site size) from Equation 1 (39) as follows,

6691

4NC71 PUUL

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FIG. 1. A , purification of HIV-1 NC71 by ssDNA-cellulose chromatography. toand t = 2 h, total cellular lysates of E. coli BL21(DE3)/ induction; LOAD ( S ) ,protein pT7nc7l.hivl before ( t o ) and after (2 h) fraction loaded onto ssDNA-cellulose column; 12-35, fractions eluted fromssDNA-cellulosecolumnwitha linear 0.05 to 1.0 M NaCl gradient. See"Experimental Procedures" for details. B, deduced primary structure of recombinant HIV-1 NC71.

authentic 1-71, with trace amounts of NC 4-71, was characterized in this study. Authentic NC71 as well as the two proteolyzed forms of NC, all contain -2 g-at Zn(I1) by atomic absorption and 6 cysteine thiols upon titration with DTNB. A metal-freeform of NC71, prepared by dialysis against EDTA, resultsin facile oxidation of most or all of the sulfhydryls, not unexpected based on previous findings with a fusion protein NCpI5(16). Analytical gel filtration chromatography reveals that both forms of NC71 cochromatograph with RNaseTI, theexpected position of the monomeric or dimeric form of the protein. This suggests that in the metal-free oxidized form of NC71, most or all of the Cys in NC71 participate in intramolecular disulfide bonds (28). One- and two-dimensional 'H NMR spectra of Znn NC71 show extensive similaritiesand chemical V/LF = K,b,(l - nv){[2w(l - n.]/[(2w - 1)(1 - nv) shift dispersion of previously published spectra of Znz HIV-1 + Y + R)]}"" X ( [ l - (n + 1)v + R]/[2(1 - n v ) ] j 2 (1) NC 1-55 from virions (40), suggesting that both the recombinant and viral molecules have very similarconformations where R = { [ l - (n + l ) v J 2 + 4wv(l - nv)}", and Y (binding density) (data not shown). The far-UV CD spectrum ofZnn NC71 = O/n and LF = Lr - Lg, where LF is free ligand [NC71] and LR is curves were superimposed shows the following transitions: a t 238 nm, -500 deg-cm2. the bound NC71, LR = Y. [poly(A)]~. These on the experimental data. The site size n was determined independ- dmol"; 221 nm, +1400 deg.cm*.dmol"; 203 nm, -9300 deg. cm2.dmol-*; and 189 nm, +lo00 deg cm2.dmol". Analysis of ently at low [NaCI] and was assumed not to change with [NaCI]. Several nonstoichiometric titrations were subjected to nonlinear these data suggest little standard (Y or p structure, and indileast-squares regression using the program NONLIN for Macintosh cates thatnonregular turn andcoil secondary structures must (Robelko Software) in order to obtain estimates of w and Kiwith n predominate. These CD spectral features are roughly similar fixed at 15. The error given in these values reflects the statistical to those previously found for NC preparations from HIV-1 65% confidence limits. and otherretroviral sources (16-18). NC71 Binds Cooperatiuely t o Poly(A)-Complex formation RESULTS between NC71 and the homopolymer poly(A) was monitored Bacterial Expression and Characterization of Mature HIVl by diminution of the intense Cotton bands in UV the circular NCp7,NC71-Fig. 1A shows that when pT7nc7l.hivl is trans- dichroism spectrum of p01y(A)~(see Fig. 3A, inset). NC71 formed into E. coli BLPl(DES)/pLysS, expression of a protein unstacks or otherwise reduces the electronic coupling of adof apparent molecular mass less than 11 kDa is observed jacent bases of the helical poly(A) single strand; similar strucfollowing induction of T7 RNA polymerase. NC71 has a tural changes characterize bindingby T4 gene 32 protein (41) predicted molecular mass of 8300 daltons. A typical chromatography run of the ssDNA-cellulose column is also shown It proved problematic to measure NC71-polynucleotide complex (Fig. 1A). Note that at least two major fractions of NC are formation by monitoring the quenchingof NC71 protein Trp fluoreseluted from ssDNA-cellulose a t different[NaCl] over the cence since free NC71 exhibited a variable time-dependent loss in course of elution with a NaCl gradient (see "Experimental fluorescence quantum yield a t low [NaCI] and protein concentrations required for these experiments. In addition, while the residual fluoProcedures"). These two NC fractions differ in their primary rescence of the Zn(II)? complex was strongly quenched by poly(A) structures in the N-terminal region (see "Experimental Pro- binding, the fluorescence of the metal-free apoprotein was quenched cedures"). The tightest bindingmaterial, which contained much less so (D. Giedroc, unpublished results). ~~

Unwinding tRNA

6692

0.7

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by HIV-1 Nucleocapsid Protein

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FIG. 2. A , stoichiometric binding by HIV-1 NC71 to poly(A) in buffer A, 0.1 M NaCl, pH 8.3, 25 "C. [Poly(A)] = 1.37 X M nucleotide. The CD intensity at 264 nm was recorded as a function of R, the molar ratio of total [NC71] to total poly(A) nucleotide, and converted to fractional saturation (see "Experimental Procedures"). Theapparent site-size n = [p0ly(A)],/[NC71]~equals 1/R at the stoichiometric point (indicated by the arrow) and is n = 13.6 from this experiment. Inset, CD spectra from 270 to 250 nm of 1.32 X lo-' M poly(A) recorded in the presence of increasing amounts of NC71. B, binding of NC71 in buffer A, 0.29 M NaCl, 25 "C, [poly(A)] = 1.32 X lo-' M. The solid lines are theoretical isotherms calculated from the McGhee-von Hippel model (39) with the product K;.w held constant at 4.42 X lo6 "' with K, and w varied reciprocally: curue a, K, = 8.8 X 10' M-', w = 500; curve b, K, = 2.6 X lo3 M-', w = 170; curue c , K, = 7.36 X lo3 M-', w = 60; n was set to15 in all simulations. Curve b was obtained from a nonlinear least squares two-parameter fit of the experimental datatoEquation 1 with 65% confidence intervals indicated K, = 2600 f 400 M-' and w = 170 f 25. C, binding isotherms collected at three NaCl concentrations in buffer A containing 0.1 M (O), 0.21 M (O), and 0.292 M (m), 25 "C. The solid lines

and other helix-destabilizing proteins (42). NC71 binding is tight (Kapp=3 X lo7 M-') and stoichiometric at low [NaCl] (100 mM) (Fig. 2A). Multiple titrations carried out at different total concentrations of poly(A) ranging from 13 to40 X M indicate that saturation is obtained atR = 0.067 f 0.011, or a ratio of one NC71 monomer per 15 k 3 nucleotides ( n = 15 f 3). This protein-RNAcomplex is completely dissociable with increasing [NaCl], indicative of an electrostatic contribution to the binding process (see below) (43). Thebinding affinity can bereduced by increasing the [NaCl] such thatnonstoichiometric binding isotherms canbe quantitatively analyzed. A representative titration performed under such conditions (0.29 M NaC1,25 "C) (Fig. 2 B ) is clearly sigmoidal, suggestive of moderate cooperativityassociated with NC71 binding. Nonlinear least squaresregression analysis of this titration allows estimation of intrinsic affinity of the NC71 monomer (K,) and cooperativity ( w ) of binding, with n determined above, modeled with the large ligand cooperative overlap binding model of McGhee and von Hippel (39). The best-fit isotherm (curue b ) specifies the following parameters: Kapp= Ki.w = 4.4 X lo5 "I, with moderate cooperativity ( w = 170 f 25) and n fixed at 15. Theoretical isotherms calculated by holding the product K,. w constant with K; and w varied reciprocally reveal that w values %fold greater ( w = 500, curue a ) or %fold less ( w = 60, curue c ) than w = 170 do not fit the experimental dataas well. Proteins which associate withnucleic acids can derive much of their bindingfree energy froman increase in entropywhich occurs upon the release of cations thermodynamically associated with the nucleic acid lattice in the formation of electrostatic interactions(43). The salt-dependenceof the binding of NC71 to poly(A) was estimated by carrying out titrations like those described at various [NaCl] with all other buffer components held constant (Fig. 2C). Herewe collect the data for three representative titrations carried out a t 0.1,0.21, and M. The smooth 0.29 M NaClwith[poly(A)] held a t 1.3 X curves are the best-fit isotherms described by the McGheevon Hippel model with w and n held at 170 and 15, respectively, and Kigiven by 1.4 X lo4, 4800, and 2600 M" at 0.1, 0.21, and 0.29 M NaCl. The inset plots the NaCl dependence in the form of a plot of log (Ki.w ) uersw log [NaCl] or a loglog plot (43). The NC7l/poly(A)complex shows a moderate Na+ dependence with d o g (K;.w)/dlog [NaCl] = -3.8 f 0.7. Since the contribution of anion release from NC71 to this salt-dependence has not yet been determined but may be significant (cf. Ref. 44), this slope corresponds to 14.5 f 1.0 Na+ ionsreleased from poly(A) upon the bindingof a NC71 monomer (43).The binding affinity estimated at 1 M NaC1, Kapp(l N ~ ) + ,remains significant, =3 X IO3 M-', suggesting that electrostatic interactions, while important, are supplemented by a significant nonelectrostatic free energy term. NC71 Can Denature tRNA-The optical activity of tRNA in the near UV-CD spectrum originates with A-like the duplex regions of the molecule (34). Two structural forms of a mixture of E. coli tRNA (lysine, phenylalanine, serine, and valinespecific) were prepared which differ in the amount of tertiary structure present (33, 34). An "inactive" form was prepared by dialyzing tRNA ina buffercontaining low monovalent salt concentration (50 mM NaCl) and no Mg2f. An "activated" tRNA formwas prepared from this materialby heat-denaturrepresent theoretical isotherms described by the following parameters: n = 15, w = 170 for all three curves with K, = 1.4 X lo5, 4800, and 2600 M-', for the 0.1, 0.21, and 0.29 M NaCl titrations, respectively. Inset, dlog (KL.w)/dlog[NaCl] (log-log plot) of the [NaCl] dependence of K,. w obtained from data in the main body of the figure.


6693

ation, followed by refolding in the presence of 10 mMMgC12 at elevated temperature (33). The complete UV-CD spectra of these two forms indicate that the activated form is characterized by a positive Cotton band centered at 263 nm of slightly greater (11%) intensity than the inactiveconformer, coupled with a more negative transition centered at 212 nm (Fig. 3A). These features mirror those reported previously (34). The activated form is much less susceptible to thermal denaturation than the inactive tRNA (Fig. 3A, inset), additional evidence that the Mg2+ complex has greater tertiary structure content (34). Fig. 3B compares the relative efficacy of Zn2NC71 and recombinant bacteriophage T 4 g32P-A, a C-terminal deletion product of the prototype helix-destabilizing protein, T4 gene 32 protein, of denaturing both formsof tRNAmixed. This fragment, unlike native g32P itself, is able to efficiently unwind duplex regions in natural DNAs, provided a single-stranded nucleationsiteispresent (38). NC71 induces a complete collapse of the Cotton effect in the nearUV-CD spectrum of the inactive tRNA conformerat NC7l/tRNAnucleotide ratio -m (R) of ~0.065. Thisrepresents physical evidence that NC71 P 0.5 can denature orunwind tRNA. The optical activity 262 at nm v) -m of the M P form, in contrast, is reduced by only 40-50%, even when twice as much NC71 is added. G32P-A is com2 0.3 pletely unable to reduce or otherwise alter the CD spectrum ?! U of protein of either form of tRNA over thesamerange 0.1 concentration. The Cotton band of tRNA in the proteintRNA complex is fully restored to itsuncomplexed intensity -0.1 by increasing the [NaCl] to greater than 0.7 M (data not 0 0.04 0.08 0.16 0.12 0.2 shown). The spectral changes which result from NC7l-inR, [protein], / [tRNAnucleotide], duced denaturation of tRNA closely mimic the temperature1.1 induced denaturation of the molecule. These changes can be summarized by an initialred shift in the positive Cotton band, 0.9 followed by gradual diminution of the positive and negative 0 CD bands (spectra not shown). Fig. 3C compares the efficiency of tRNA unwinding by E 0.7 4NC71 with g32P-A with the data expressed on a per tRNA m molecule basis (taking anaverage of 75 nucleotides per E. coli & 0.5 iij tRNA molecule). From this plot it clear is that 5-6 monomer m equivalents of NC71 are required to unwind inactive tRNA. 0 0.3 G32P-A is far less effective; similar results were obtained for 2 native g32P (data not shown). Also shown is the unwinding LL 0.1 of tRNA by the zinc-free and thiol-oxidized form of NC71. T h e relative efficacies of tRNA unwinding by Znn and apoa result consistent with oxidized NC appear indistinguishable, -0.1 0 2 4 6 8 10 12 the poly(A) titrations of the apo-oxidized protein (data not [protein], / [tRNA molecule], shown; see also Refs. 18, 29). Although the binding of NC71 a sigmoidal binding curve, totRNAappearstoresultin suggestive of cooperativity and potentially consistent with the FIG. 3. A , UV CD spectra of inactive (curue a, 7.94 X lo-' mg/ml molecules) and Mg+-activated(curue b, 7.55 X lo-* mg/ml molecules) poly(A) data (Fig. 2B), the relationship between fractional E. coli tRNA""xedin buffer A, 0.1 M NaCl, with (active) and without change in the tRNA CD spectrum at a single wavelength (262 (inactive) 10mM MgC12,25 "C. Inset, temperature-dependent unfoldnm) and fractional saturation of the tRNAmolecule by NC71 ing of active (0)and inactive (0) tRNA (1.5 X lo-' mg/ml). B , HIVis not expected to be linear, since theoverall shape of the CD 1 NC71 (0,m) is much more efficient than phage T4 g32P-A (0,O) in denaturing inactive (closed symbols) and active (open symbols) E. spectrum is changed significantly upon NC71 binding (data coli tRNAmixed.The fractional signal change in the ellipticity at 262 not shown). nm is plotted against R, the molar of ratio total [NC71] to total tRNA 0)

L

DISCUSSION

Darlix and coworkers (7-12) have recently reconstituted a replication complex capable of accurate minus strand strong stop cDNA synthesis in vitro from a variety of retroviral systems. Thereaction minimally requires a 5' primer binding site-containing genomic RNA fragment, tRNA primer, purifiedreverse transcriptase, and processed NC, which upon formation of a 16-18-bp heteroduplex, primes cDNA synthesis (1, 7-12). Although proviralsynthesis occurred in the absence of added NC, addition of a few molecules of NC per

.,

M molecules, 7.0 X M nucleotide. [tRNA] as follows: 5.3 X nucleotides (m); 4.0 X M molecules, 3.0 X M nucleotides (0); 6.8 X M molecules, 5.1 X M nucleotides (0); 5.7 X M molecules, 4.3 X M nucleotides (0). C, fractional ellipticity at Zn2 262 nm plotted as a function of [protein],/[tRNA molecules],. M molecules; 0, g32P-A, 6.8 X M NC71, [tRNA] = 5.3 X molecules; 0, apo-oxidized NC71, [tRNA] = 6.1 X lo-? M molecules.

viral RNA strand appeared to greatly increase the yield of cDNA products (11).Other bona fide single-strand binding proteins including E. coli recA and phage T4 gene 32 protein (g32P) proved inactive in thisactivity (7-12). All physiochem-

6694


ical studies published to datesuggest that matureNC purified uitro is zinc-independent The precise role of Zn(I1)from manyretroviral sources binds preferentiallyto ss nucleic binding by NC or thepolyprotein continues to remain undeacids (18, 28, 29). It is well established that stoichiometric fined, and awaits identification of a Zn(I1)-dependent viral unwinding by helix-destabilizing proteins, like T4 gene 32 RNA ligand(s) (e.g. derived from thecis-acting encapsidation protein (as opposed to catalytic unwinding by helicases), can locus) (48) and/or detailed physiochemical characterization promote intermolecular RNA duplex formation (annealing) of the gag polyprotein itself or intermediate processed forms by destabilizing intramolecular duplex regions (38, 42). An- of gag (cf. Ref. 12). other aspect of this activity is the facilitation by helix-destaRecombinant ZnzNC71 bindsto ss nucleic acidswith bilizing proteins of correct intramolecular base-pairing by moderate cooperativity, w = 170 f 25 (39). Previouslyreported melting out incorrect structures. In contrast,a direct anneal- binding properties of viral and synthetic NC preparations ing activity, like that of arecombinase, for example (45), from other retroviralsources are generally indicative of relarequires that the local concentration of complementary single- tively weak and weakly or noncooperative binding (w = 1-20) stranded sequences be greatlyincreased. With NC71, this to homopolymers a t low salt (Kapp = lo6M-') and a moderately could potentially be achieved with morethan one RNA bind- smaller site size, n = 5 & 1 (18, 28, 29). These NC molecules ing site per NC monomer, or NC-NC protein interactions. previously characterized however, differ from HIV-1 NC in Recently, mammalian heterogeneous nuclear ribonucleopro- that in HIV-1 and other complex retroviruses, thenucleocaptein Al, a ssRNA binding protein, was shown to facilitate sid domain of gag is further processed just C-terminal to the intermolecular basepairing of RNA (46), apparently through second Cys-His region, to yield a p6 C-terminal subunit of a facilitated annealing rather than an unwinding mechanism. unknown function (30). The nucleic acid binding properties Recombinant HIV-1 NC71 has the intrinsic capacity to of this fragment or thatof the plfINC(16) progenitor remain unwind or denature duplex regionsof tRNA ina stoichiometric unreported, but may well differ from mature NC71. Indeed, fashion, by virtue of its affinityfor ssRNA. Analytical exper- the first viralproteolytic cleavage event in HIV-1 maturation iments with the partially double-stranded DNA and RNA occurs at the mature N terminus of NC71, followed by liberalternating copolymers (d[poly(A-T)] and r[poly(A-U)]) fur- ation of the C-terminal p6 fragment (31).Thus the108-amino ther confirms this.4 The occluded site size of 15 nucleotides acid p7-p6peptide is transiently present prior to mature NC71 per NC monomer determined with poly(A) predicts that one in virions; this suggests the possibility that the nucleic acid tRNA molecule could bind up to five to six NC monomers. binding activities thatwe observe for NC71 may be regulated This binding would occur concomitant with a collapse of the by the processing at the p7-p6 junction (49). This type of CD spectrum which derives fromthe A-form duplexstructure. regulation is not without precedent. The C-terminal region of This is consistent with the stoichiometrywethat observe with T4 gene 32 protein provides a kinetic barrier for unregulated the inactive tRNA conformer, indicating that the binding is DNA duplex unwinding, the equilibrium binding parameters quite tight(Fig. 3C). T4 g32P is unable tounwind either form unchanged (38). of tRNA (Fig. 3C). Thus, it is not necessary to invoke NCIt is of interest to compare the conformational changes RNA specificity to explain why g32P proved inactive in the induced in tRNA by NC71 with those induced by another primer tRNA annealing assay(8). helix-destabilizing protein,mammalianUP1 (33, 42), now The Mg2+ form of tRNA is less susceptible to NC-induced known to be a proteolytic fragment of the hnRNPA1 protein unwinding, with approximately 4 0 4 0 % of the optical activity (50). UP1 was previously shown to bind to theinactive form relatively resistant to NC binding. However, even this level of yeast tRNAL""-3and maximally depress the UV-CD Cotton of unwinding may be sufficient for intermolecular tRNAband by ~ 4 0 4 0 % Addition . of 0.01 M MgC1, to the UP1primer binding site duplex formation since 'H NMR studies inactive tRNA complex quickly brought about return of the of tRNA melting under similar solution conditionsgenerally tRNA spectrum to that indistinguishablefrom the native, reveal that the tertiary structural basepairs and the anticodon fully folded active tRNAL""-3.UP1 thus facilitates the renaand acceptor helices open first, followed by complete denaturation of tRNA from an inactive to anactive conformer in turation of these helices, then the T\kChelix, and finally the the presence of M$+; these two conformers are nominally D helix (47). Thus the region that would be complementary separated by a very large free energy barrier (33). The prest o the viral primer binding site, e.g. the tertiary structural ence of M$+, by virtue of stabilizing the nativeform of tRNA, contacts at the elbow of "L" and the acceptor helix in the must bring aboutdissociation of the transientlyformed UP1. Mg2+ form, become single-stranded first. It is possible that tRNA complex since UP1 has much lower affinity for duplex these are theregions that NC is mostefficient at ~ n w i n d i n g . ~structures (33). A kinetic model was developed (42), whereby Finally, given the site size for NC71 of 15 f 3 nucleotides, UP1bindstransientlytothe inactive form, reducing the potentially as few as one or perhaps two bound NC71 moleenergy barrier to renaturation. UP1 therefore reduces the cules would be required to sufficiently denature even the lifetime of thedenaturedintermediate,inpromotingthe active M$+. tRNA conformer. correct intramolecularbasepairing. Complete collapse of CD signal of the inactive tRNA conWe find that NC71 can completely depressthe Cottoneffect former is observed, indicative of uniform denaturation of a of inactive tRNA, andalso shows someability to bind to and mixed population of tRNA molecules (Fig. 3B). Thus, the unwind the active or M$+ form, unlike UP1. Furthermore, unwinding by NC71 is unlikely to be specific for the replicaaddition of20 mM MgZf to the preformed NC7l-inactive tionprimer packagedbyHIV-1,tRNALy"3 (12, 36). The tRNA complex formed in theabsence of Mg2+only results in reverse transcriptase may play a larger role in primer tRNA ) of the opticalactivity even after selectivity (11). The unwinding reaction clearlydoes not a partial ( ~ 2 0 % return extended periods of incubation time (data not shown). It is require Zn(I1) coordination by NC71 (Fig. 3C),consistent possible that NC71 denatures tRNA and so in doing, forms a with a recent study which showed the tRNA positioning in R. Khan, unpublished results. In the annealing experiments previouslyreported (7-12), itis unclearif the tRNA molecule contained the full complement of tertiary structure.

NC molecules which lack the highly basic N-terminal region (amino acids 1-10 in NC 11-71; cf. Fig. IC) have a 10-20-fold lower affinity for both poly(A) and tRNA"'xed,if one assumes the same site sizes as for 1-71 molecule (D. Giedroc and R. Khan, unpublished results).


6695

21. Fu, X., Katz, R. A., Skalka, A. M., and Leis, J. (1988)J. Biol. Chem. 263,2140-2145 22. Gorelick, R. J., Nigida, S. M., Jr., Bess, J. W., Jr., Aurthur, L. O., Henderson, L. E., and Rein, A. (1990)J. Virol. 64, 32073211 23. Meric, C., and Goff, S. P. (1989)J. Virol. 63, 1558-1568 24. Meric, C., Gouilloud, E., and Spahr, P.-F. (1988)J. Virol. 62, 3328-3333 25. Meric, C., and Spahr, P.-F. (1986)J. Virol. 60,450-459 26. Dupraz, P., Oertle, S., Meric, C., Damay, P., and Spahr, P.-F. (1990)J. Virol. 64,4978-4987 27. Chen, M.-J., Garon, C. F., and Papas, T. S. (1980)Proc. Natl. Acad. Sci. U. S. A. 77,1296-1300 28. Jentoft, J. E., Smith, L. M., Fu, X., Johnson, M., and Leis, J. (1988)Proc. Natl. Acad. Sci. U. S. A. 85,7094-7098 29. Karpel, R. L., Henderson, L. E., and Oroszlan, S. (1987)J.Biol. Chem. 262,4961-4967 Acknowledgments-We thank JohnDenu and Huawei Qiu for their assistance with the NONLIN for Macintosh program and Elizabeth 30. Henderson, L. E., Benviste, R. C., Sowder, R. C., Schultz, A. M., and Oroszlan, S. (1988)UCLA Symp. Mol. Cell. Biol. 71, 135Burns for performing some of the titration experiments as well as 147 help with the purification of NC71. 31. Tritch, R. J., Cheng, Y.-S. E., Yin, F. H., and Erickson-Vitanein, S. (1991)J . Virol. 65,922-930 REFERENCES 32. Giedroc, D. P., Qiu, H., Khan, R., King, G.C., and Chen, K. 1. Dickson, C., Eisenmann, R., Fan, J., Hunter, E., and Teich, N. (1991)Biochemistry 31,765-774 (1985)in RNA Tumor Viruses (Weiss, R., Teich, N., Varmus, 33. Karpel, R. L., and Burchard, A. C. (1980)Biochemistry 19,4674H., and Coffin, J., eds) Ed. 2,Vol. 1, pp. 513-648,Cold Spring 4682 Harbor Laboratory, Cold Spring Harbor, NY 34. Willick, G . , Oikawa, K., and Kay, C. M. (1973)Biochemistry 12, 2. Nissen-Meyer, J., and Abraham, A. K. (1980)J. Mol. Biol. 142, 899-905 19-28 35. Maniatis, T., Fritsch, E. F., and Sambrook, J . (1982)Molecular 3. Leis, J., Baltimore, D., Bishop, J. 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NC71. tRNA complex which, in contrast to the UP1complex, is resistant to M e dissociation, with the result that intrumolecular basepairing within tRNA is slow. If a complementary RNA molecule is present, intermolecular annealing would appear favored(7-12). This resistance might lie on the observed cooperativity of binding by NC71 (Fig. 2 B ) , relative to UP1, which binds noncooperatively to nucleic acids (51). Quantitative studies of the interaction of NC71 with a single recombinant tRNA species, as well as other partially doublestranded model RNAs of defined structurearerequired to further elucidate the molecular aspects of this and other NCRNA complexes, the formation of which appear important for one or more stages in retroviral replication.

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