Virology 405 (2010) 556–567

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Fundamental differences between the nucleic acid chaperone activities of HIV-1nucleocapsid protein and Gag or Gag-derived proteins: Biological implications

Tiyun Wu a, Siddhartha A.K. Datta b, Mithun Mitra a, Robert J. Gorelick c, Alan Rein b, Judith G. Levin a,⁎a Laboratory of Molecular Genetics, Program in Genomics of Differentiation, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes ofHealth, Building 6B, Room 216, 6 Center Drive, Bethesda, MD 20892-2780, USAb HIV Drug Resistance Program, National Cancer Institute-Frederick, Frederick, MD 21702-1201, USAc AIDS and Cancer Virus Program, SAIC-Frederick, Inc., National Cancer Institute-Frederick, Frederick, MD 21702-1201, USA

⁎ Corresponding author. Fax: +1 301 496 0243.E-mail address: levinju@mail.nih.gov (J.G. Levin).

0042-6822 Published by Elsevier Inc.doi:10.1016/j.virol.2010.06.042

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Article history:Received 27 April 2010Returned to author for revision 16 May 2010Accepted 23 June 2010Available online 23 July 2010

Keywords:Nucleic acid chaperoneHIV-1GagNucleocapsid proteinCapsid proteinReverse transcriptionNucleic acid-driven multimerizationRetrovirusMinus-strand transfer

The HIV-1 Gag polyprotein precursor has multiple domains including nucleocapsid (NC). Although matureNC and NC embedded in Gag are nucleic acid chaperones (proteins that remodel nucleic acid structure), fewstudies include detailed analysis of the chaperone activity of partially processed Gag proteins andcomparison with NC and Gag. Here we address this issue by using a reconstituted minus-strand transfersystem. NC and NC-containing Gag proteins exhibited annealing and duplex destabilizing activities requiredfor strand transfer. Surprisingly, unlike NC, with increasing concentrations, Gag proteins drastically inhibitedthe DNA elongation step. This result is consistent with “nucleic acid-driven multimerization” of Gag and thereported slow dissociation of Gag from bound nucleic acid, which prevent reverse transcriptase fromtraversing the template (“roadblock” mechanism). Our findings illustrate one reason why NC (and not Gag)has evolved as a critical cofactor in reverse transcription, a paradigm that might also extend to otherretrovirus systems.

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ublished by Elsevier Inc. Open access under CC BY-NC-ND license.

Introduction

The human immunodeficiency type 1 (HIV-1) Gag polyproteinprecursor is the only viral protein required for assembly of virus-likeparticles (VLPs) (reviewed in Freed, 1998; Swanstrom andWills, 1997;Vogt, 1997). Monomeric Gag is a 55-kDa multi-domain protein, whichcontains (from the N- to C-terminus) matrix (MA), capsid (CA), spacerpeptide 1 (SP1), nucleocapsid (NC), spacer peptide 2 (SP2), and p6(Henderson et al., 1992) (reviewed in Adamson and Freed, 2007; Freed,1998; Turner and Summers, 1999; Vogt, 1997) (Fig. 1A). Each of thedomains contributes to the overall activity of Gag and to its unique role inHIV-1 replication. For example, the MA domain is responsible for Gagtargeting to the plasma membrane during virus assembly and also bindsRNA (Adamson and Freed, 2007; Chukkapalli et al., 2010; Shkriabai et al.,2006). CA is a protein interaction domain that facilitates Gag multi-merization (Datta et al., 2007a,b; Gamble et al., 1997); the NC domainbinds RNA, is required for genomic RNA packaging and primer placement(Darlix et al., 1995; Kleiman and Cen, 2004; Levin et al., 2005; Rein et al.,1998), has a role in viral RNA dimerization (Darlix et al., 1990; Feng et al.,1999; Liang et al., 1998), and interacts with the Bro 1 domain of the host

protein Alix during virus assembly (Dussupt et al., 2009; Popov et al.,2009); and the p6 domain is needed for virus release and interactionwithhost factors in the ESCRT pathway (Adamson and Freed, 2007). During orshortly after budding of virions from the cell, the viral protease (PR) isactivated and Gag is cleaved into the virus structural proteins. Cleavageoccurs sequentially and in a highly ordered manner (Adamson andFreed, 2007; Freed, 1998; Swanstrom and Wills, 1997; Vogt, 1997).

ThematureNC protein, which is released in a late cleavage reaction(Swanstrom and Wills, 1997), plays a major role in assuring thespecificity and efficiency of reverse transcription (reviewed in Bampiet al., 2004; Darlix et al., 1995; Levin et al., 2005; Rein et al., 1998;Thomas and Gorelick, 2008) and is also important for other events inthe virus life-cycle including maturation of the genomic RNA dimer(Feng et al., 1996; Muriaux et al., 1996) and integration of proviral DNAinto the host genome (Carteau et al., 1999; Thomas et al., 2006). NC'sfunction in virus replication is correlated with its ability to act as anucleic acid chaperone. This activity allows NC to catalyze nucleic acidconformational changes that result in the most thermodynamicallystable structures (Tsuchihashi andBrown, 1994; reviewed in Levin et al.,2005; Rajkowitsch et al., 2007; Rein et al., 1998). The NC domain in Gagalso has nucleic acid chaperone activity and asmentioned above, its rolein viral DNA synthesis is to mediate primer placement, i.e., annealing ofthe tRNA3

Lys primer to the viral RNA genome, a reaction that occurs

Fig. 1. HIV-1 Gag proteins and reconstituted model system used for assay of minus-strand transfer. (A) Schematic representation of WT and mutant HIV-1 Gag proteins as well as Gag-derived cleavageproducts. GagΔp6 (Campbell and Rein, 1999)was used as the source ofWTGag. The asterisk inGagWMindicates that residues Trp316 andMet317 in theCTD of CAweremutated to Ala (Datta et al., 2007b). Partial deletion of the MA domain in GagΔ16-99 (Datta and Rein, 2009; Gross et al., 2000) is shown as a dashed line. Each of the domains in Gag islabeled and represented by open, closed, or gray rectangles. (B) Gel analysis of purified Gag and Gag-derived proteins used in this work. The proteins were analyzed on a 4–12% gradientSDS–polyacrylamide gel. The amounts of protein loaded on the gel were as follows: MA, 40 μg; CA, 20 μg; MACA, 20 μg; CANC, 20 μg; GagΔ16-99, 25 μg; Gag WM, 25 μg; GagΔp6, 20 μg.Molecularmarkerswere runon the samegel toverify themolecularmassof theHIV-1proteins and thepositionsof themarkers are shownto the left of thegel image. For eachof the testproteins,the residue numbers in Gag and the respective molecular mass values were as follows: MA (1–131, 14.8 kDa); CA (132–362, 25.6 kDa); MACA (1–362, 40.3 kDa); CANC (132–431, 33.5 kDa);GagΔ16-99 (1–15 and 100–447, 40.3 kDa); GagWM (1–447, 49.9 kDa); and Gag (1–447, 50.0 kDa). Note that p6 is not present in any of the Gag proteins (seeMaterials andmethods section,“Purificationof proteinsused in this study”). (C) Schematic diagram illustrating the reconstituted systemused for assayofminus-strand transfer (Heilman-Miller et al., 2004). Thediagramshowsannealingof the94 complementarybases in theR regions of theRNA148acceptor and 33P-labeledDNA128. The54-nt sequence fromU3 in acceptorRNAserves as the template forRT-catalyzedDNA extension. The final DNA transfer product is 182 nt. The diagrams in A and C are not drawn to scale.

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during virus assembly (Cen et al., 1999, 2000; Feng et al., 1999; Huanget al., 1997; Kleiman and Cen, 2004).

Previous studies of the chaperone activities of Gag and NC have notgenerally included detailed comparison with the activities of partiallyprocessed Gag proteins. To address this issue, we examined the activity ofwild-type (WT) and mutant Gag and Gag-derived proteins to determinehow chaperone activity is affected by removing one or more domainsfrom Gag. Experimentally, our approach was to use our reconstitutedminus-strand transfer assay system, which models the NC-dependenttransfer of (−) strong-stop DNA [(−) SSDNA] to the 3′ end of the viralRNA genome, in a reaction facilitated by annealing of the complementaryrepeat regions in the RNA and DNA substrates (Basu et al., 2008; Levin etal., 2005; Thomas and Gorelick, 2008). This system represents a rigoroustest for chaperone function (Guo et al., 1997; Heilman-Miller et al., 2004)and can also be used to examine the nucleic acid chaperone activity ofproteins other than HIV-1 NC (Zúñiga et al., 2010).

Here, our results demonstrate that only Gag and partially processedGag proteins containing the NC domain have nucleic acid chaperoneactivity. Although the activities of these proteins share some propertiesin common with mature NC, there are also fundamental differences.Thus, like NC, Gag and Gag-derived proteins exhibit annealing andduplex destabilizing activities that are required for minus-strandtransfer. Surprisingly, as the protein concentration is increased, NCstimulates strand transfer, whereas Gag proteins drastically inhibitstrand transfer (specifically, the DNA elongation step) by apparentlyblocking reverse transcriptase (RT) movement along the template. Werefer to this inhibition as the “roadblock mechanism”. Moreover,endogenous RT (ERT) assays of minus-strand transfer with detergent-treated HIV-1 virions havingmutations at one ormore PR cleavage sites(Wyma et al., 2004) give results consistent with data obtained with thereconstituted system. Collectively, our studies provide additionalinsights into the question of why mature NC (and not a precursor) has

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evolved as a critical cofactor that facilitates efficient and specific HIV-1reverse transcription.

Results

Nucleic acid chaperone activity of Gag and Gag WM proteins facilitateminus-strand transfer

In the present study, we set out to determine functional differencesbetween the nucleic acid chaperone activities of NC embedded withinlarger precursors and mature NC. Our approach was to investigate theactivities of unprocessed Gag and partially processed Gag proteins and tocompare these activitieswith that ofNC (Figs. 1A andB). For thiswork,weused our reconstituted minus-strand transfer assay system (Fig. 1C),which represents an especially sensitive readout for chaperone function(Guo et al., 1997; Heilman-Miller et al., 2004). This system measures (i)annealing of the RNA 148 acceptor to (−) SSDNA (i.e., DNA 128), whichrequires nucleic acid aggregation activity aswell as helix destabilization ofthe highly structured complementary trans-activation response element(TAR) stem-loops at the 3′ ends of the repeat regions in (−) SSDNA and

Fig. 2. Effect of HIV-1 NC and Gag proteins onminus-strand transfer. (A) Gel analysis. Reactionconcentrations of HIV-1 NC (lanes 2 to 5). In parallel sets of reactions, DNA 128 was incubateconcentrations of WT Gag (lanes 7 to 12) or Gag WM (lanes 14 to 19). The protein concentratrepresents a control reaction inwhich acceptor and NC or Gagwere omitted. Only SP products aBar graphs showing the percentage (%) of minus-strand transfer product (B) or SP products (protein, open bars; HIV-1 NC, closed bars; Gag, gray bars; Gag WM, hatched bars; C, cross-hat

acceptor RNA; (ii) RT-catalyzed elongation of annealed (−) SSDNA, usingthe 54-nt U3 region present in the acceptor as the template, to give a 182-nt transfer product; and (iii) a competing reaction, known as self-priming(SP), inwhich fold-back structures at the3′endof (−) SSDNA(inducedbythe presence of the DNA TAR stem-loop) are extended by RT, forming aheterogeneous mixture of dead-end SP products (Beltz et al., 2005;Driscoll and Hughes, 2000; Guo et al., 1997, 2000; Heilman-Miller et al.,2004; Lapadat-Tapolsky et al., 1997; Levin et al., 2005 and referencestherein). In these experiments, “Gag” refers to the protein known asGagΔp6 (Campbell and Rein, 1999).

To evaluate the chaperone activities of HIV-1 Gag and Gag WM, amutant with poor protein dimerization activity (Datta et al., 2007a,b),we tested the effect of increasing protein concentration on minus-strand transfer (Fig. 2). A similar set of reactions with HIV-1 NC wasincluded as a positive control. The strand transfer and SP productswere resolved by polyacrylamide gel electrophoresis (PAGE) (Fig. 2A)and were quantified by PhosphorImager analysis of the gel data (Figs.2B and C, respectively). SP products were identified by comparisonwith the bands formed in a control reaction (C), which lacks acceptorand NC (Guo et al., 1997; Heilman-Miller et al., 2004).

mixtures were incubated for 60 min in the absence (No, lane 1) or presence of increasingd with RNA 148 for 60 min in the absence (No, lanes 6 and 13) or presence of increasingions for each set of reactions are indicated at the bottom of the figure. The lane labeled Cre formed under these conditions (Guo et al., 1997; Heilman-Miller et al., 2004). (B and C)C) synthesized as a function of NC, Gag, or Gag WM protein concentrations. Symbols: noched bar.

Fig. 3. Stimulation of the minus-strand annealing reaction by HIV-1 NC, Gag, and Gag WM proteins. Reaction mixtures were incubated for 30 min at 37 °C under the conditionsdescribed inMaterials andmethods. Bar graphs show the % of DNA 128 that was annealed in the absence (No, open bars) or presence of increasing concentrations of NC (closed bars),Gag (gray bars), or Gag WM (hatched bars).

Table 1Apparent dissociation constants for binding of NC and Gag to 20-nt ssDNA and ssRNAoligonucleotides.

Protein Kd (nM)

DNA RNA

NC 143±12a 108±23Gag 9±1 8±1Gag WM 20±0.1 23±1

a The error determinations represent the SD.

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In accordwith previous results (Guo et al., 1997; Heilman-Milleret al., 2004), increasing concentrations of NC dramatically stimulat-ed synthesis of the strand transfer product and severely inhibitedSP (Figs. 2B and C, compare lanes 2 to 5 with lane 1). Gag and GagWM also exhibited strand transfer activity in a dose-dependentmanner, but significant activity occurred over a very narrow range(0.08–0.12 μM) (lanes 9 and 10 and lanes 16 and 17, respectively).At Gag and Gag WM concentrations that support strand transfer(i.e., up to 0.12 μM), inhibition of SP was more modest than thatobserved with NC (Fig. 2C), implying that the duplex destabilizingactivity of Gag is not as effective as that of NC. At higherconcentrations of Gag proteins, SP was drastically reduced, but asexplained below, this is due to a general reduction in RT-catalyzedDNA extension. Gag WM facilitated strand transfer only slightlymore efficiently than Gag (Fig. 2B, compare lanes 16 and 17 withlanes 9 and 10). This would suggest at first glance that the oligomericstatus of these proteins might be the same under the conditions of thisassay (for further discussion of this point, see below.)

Comparison of the NC, Gag, and GagWMresults showed that a higherconcentration of NCwas needed to achieve maximal strand transfer thanwas required for the Gag proteins (3.2 μM NC vs. 0.12 μM Gag or GagWM). However, the percent strand transferwith NCwas ~1.5-fold higherthan the values obtained for reactions with the two Gag proteins.Curiously, in contrast to NC's behavior (Fig. 2B, lanes 4 and 5), when theconcentration of Gag orGagWMwas raised, e.g., to 0.23 μM(Fig. 2B, lanes11 and 18, respectively), there was a substantial reduction in strandtransfer activity. At 0.46 μM, strand transfer activity was completelyinhibited (Fig. 2B, lanes 12 and 19, respectively). In additional reactionswith 0.46 μM Gag and increasing concentrations of RT from 20 to 50 nM(in our assay we use 10 nM), strand transfer was slightly increased, butnot to an appreciable extent (e.g., 20 nM RT, b10%; 50 nM RT, 15%)(data not shown).

Since the minus-strand transfer assay requires both annealing andDNA elongation for a positive readout (Fig. 1C), it was important todetermine which of these steps might be affected by increasedconcentrations of Gag and Gag WM.We therefore measured annealingalone as a function of protein concentration (Fig. 3). The data showedthat for NC, Gag, and Gag WM, increasing the protein concentrationresulted in a greater extent of annealing. In contrast to the strandtransfer results, annealing with both Gag proteins did not lead to areduction in the percent annealed product, even at the concentration(0.46 μM) at which strand transfer was completely abolished (Fig. 2).Moreover, heat annealing of RNA 148 to DNA 128 followed by additionof increasing concentrations of Gag plus a constant amount of RT led toresults similar to those presented in Fig. 2B, i.e., inhibition of strandtransfer (data not shown).

Taken together, these findings clearly demonstrate that theobserved inhibition of strand transfer is caused by an inhibition ofRT-catalyzed DNA extension and not annealing. Thus, when strandtransfer was inhibited (concentrations of Gag and Gag WM above0.12 μM), the reduction in SP (Fig. 2C, lanes 12 and 19) was due to ageneral reduction of polymerase activity and not to the helixdestabilizing activity of the Gag proteins. In addition, as will bediscussed in detail below, the data suggest that binding of Gagproteins to single-stranded (ss) nucleic acids creates a “roadblock”,which significantly reduces the ability of RT to traverse the template.

The results of Fig. 3 also show that with NC and the two Gagproteins, the annealing reaction reached an end point of ~60% in eachcase. However, the amount of Gag or Gag WM required for maximalactivity (0.16 μM) was considerably lower than the amount of NCneeded (between 1.6 and 3.2 μM). To account for this difference, weused FA to measure the nucleic acid binding affinity of Gag and NC toss 20-nt DNA and RNA oligonucleotides (Table 1). In accord with thedata of Fig. 3, we found that under our conditions, Gag was bound tonucleic acid ~15-fold more efficiently than NC (Table 1), e.g., theapparent dissociation constant (Kd) values for binding to the DNAoligonucleotide were 9±1 nM (Gag) vs. 143±12 nM (NC). The Kd

values for GagWM (with DNA, 20±0.1) were similar to those of Gag,as expected from the data of Fig. 3. Note that in each case, the Kd

values for binding to the RNA and DNA substrates were very similar.

Relative contributions of MA and NC to Gag nucleic acid chaperoneactivity

Like NC, the MA domain in Gag also binds nucleic acids (Adamsonand Freed, 2007; Vogt, 1997). It was therefore of interest to ascertainwhether the MA domain contributes to Gag nucleic acid chaperoneactivity. To address this question, we initially assayed the strandtransfer activity of three proteins lacking the NC domain (Fig. 4A), i.e.,

Fig. 4.Minus-strand transfer activity of HIV-1 MA, CA, and MACA proteins in the absence or presence of HIV-1 NC added in trans. 33P-labeled DNA 128 was incubated with RNA 148for 60 min in the absence or presence of increasing concentrations of MA (2 to 5), CA (7 to 10), and MACA proteins (12 to 15). (A) and (B) Bar graphs show the % of minus-strandtransfer product synthesized as a function of the protein concentrations of MA, CA, andMACA in the absence (A) or presence of (B) 0.92 μMNC added in trans. Symbols: open bars, noprotein; gray bars, NC only; and closed bars, increasing concentrations of MA, CA, and MACA (concentrations indicated at the bottom of the figure).

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MA (lanes 1 to 5), CA (lanes 6 to 10), and MACA (lanes 11 to 15). MAand CA had no detectable nucleic acid chaperone activity atconcentrations similar to those used for Gag and Gag WM. MACAhad a very small stimulatory effect at the highest concentrations of theprotein (lanes 14 and 15), but the level of activity was too low to beconsidered significant.

We also performed an assay to see if these proteins added in transmight affect NC-mediated strand transfer activity. NC (0.92 μM) wasadded to reactions containing increasing amounts of the threeproteins (Fig. 4B). The first bar in each panel (lanes 1, 6, and 11)represents activity with NC alone. NC activity was unchanged in thepresence of the three proteins, although in the case of MA and to alesser extent MACA, there was a very small reduction in NC activitywhen the concentrations of these two proteins were raised to 0.46 μM(lanes 5 and 15).

Next we assayed the activity of two proteins that contain the NCdomain: (i) GagΔ16-99, which lacks a region in MA enriched withbasic residues; and (ii) CANC, which is missing all of MA (Fig. 1A). Theresults in Fig. 5A demonstrate that both of these proteins retained theability to promote minus-strand transfer in a dose-dependentmanner, although the most significant activity was in a narrowrange of protein concentration. Thus, for GagΔ16-99, maximal activitywas between 0.23 and 0.46 μM (lanes 6 and 7); this activity wassomewhat lower than that achieved with the optimal concentrationfor Gag (0.12 μM) (Fig. 2B, lane 10). At a higher concentration ofGagΔ16-99 (0.92 μM) (lane 8), the reduction in strand transferactivity wasmodest, unlike the complete abolition of this activity with0.46 μM Gag (Fig. 2B, lane 12). The CANC protein behaved more likeGag and stimulated maximal strand transfer between 0.12 and0.23 μM (lanes 13 and 14). Moreover, adding higher concentrationsof CANC (0.46 and 0.92 μM, lanes 15 and 16 respectively) completely

eliminated strand transfer activity. In contrast to Gag or Gag WM,neither the CANC nor the Gag Δ16-99 mutant was able to inhibit SP(Fig. 5B). Since minus-strand transfer is inhibited at 0.46 and 0.92 μMCANC, the reduction in SP products at these concentrations (lanes 15and 16) probably reflects a general reduction in polymerase activity.

Collectively, the data in Figs. 4A and 5 demonstrate that the majordeterminant for nucleic acid chaperone activity is the NC domain inWT Gag and truncated Gag-derived proteins. While the MA domainappears to contribute to chaperone activity in the context of Gag, theabsence of MA in an NC-containing partially processed Gag proteinsuch as CANC has a relatively small effect on strand transfer activity inour system.

Kinetics of strand transfer and annealing

To further quantify differences in the nucleic acid chaperoneactivities of Gag, CANC, and GagΔ16-99, we investigated the kineticsof minus-strand transfer and annealing in a series of reactionscontaining three different concentrations of each protein (0.06, 0.12,and 0.23 μM) (Fig. 6). The optimal concentrations were 0.12 μM for Gag(Fig. 6A–1) and CANC (Fig. 6B–1), whereas for GagΔ16-99, it was0.23 μM(Fig. 6C–1). Note thatmaximal strand transfer (about 40%)wasachievedby30 minwith these concentrations and remainedunchangedwith further incubation for as long as 240 min (data not shown).

The rates of minus-strand transfer were calculated from theexperimental data for reactions with 0.12 μM(Table 2, middle column).Of the three proteins, Gag had the highest kobs value (line 1). The rate forCANC was only slightly reduced (1.3-fold) relative to the rate for Gag(compare lines 1 and 2). In contrast, the rate for GagΔ16-99 (line 3)was5-fold lower than the rate for Gag (compare lines 1 and 3). However, atthe optimal concentration for GagΔ16-99 (0.23 μM), the rate of strand

Fig. 5. Effect of GagΔ16-99 and CANC proteins on minus-strand transfer. Reaction mixtures were incubated for 60 min in the absence (No, open bars, 1 and 9) or presence ofincreasing concentrations of GagΔ16-99 (cross-hatched bars, 2 to 8) and CANC (dark gray bars, 10 to 16). The concentrations (μM) of GagΔ16-99 and CANC, respectively, were thesame as those given for Gag in the legend to Fig. 2, except for the last points, i.e., at 0.92 μM. (A) and (B) Bar graphs. The % of minus-strand transfer product (A) and SP products (B)synthesized was plotted as a function of GagΔ16-99 and CANC protein concentrations.

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transfer was calculated as 0.125±020 min−1, i.e., 1.7-fold lower thanthe rate for Gag and 1.4-fold lower than the rate for CANC. These resultsindicate that when reactions contained optimal concentrations for eachprotein, the rates were similar.

We also assayed the kinetics of annealing alone for each of the threeproteins. In each case, a concentration of 0.23 μM gave the greatestamount of annealed product: almost 70% for Gag and CANC (Figs. 6A-2and B-2, respectively) and over 60% for GagΔ16-99 (Fig. 6C-2). Incontrast to the strand transfer results, the maximal annealing levelswere reachedwithin 1 to 5 min. At a lower concentration, 0.12 μM, theGag reaction was significantly more efficient than the CANC andGagΔ16-99 reactions.

The rates of minus-strand annealing were determined at aconcentration of 0.12 μM (Table 2, right column), since at a higherconcentration, the kinetics for Gag annealing were too fast to measure(Fig. 6A-2). Interestingly, for all three proteins, the rates of annealingwere significantly higher than the rates of strand transfer (Table 2,compare middle column with right column). Gag had the highest rateof annealing among the three proteins (Table 2, right column, line 1).The rates for CANC and GagΔ16-99 were 2-fold and 5.4-fold lower,respectively, than the rate for Gag (Table 2, right column, compareline 1 with lines 2 and 3).

Collectively, these results demonstrate that raising the proteinconcentration of Gag leads to increased stimulation of both the rateand final level of annealing, consistent with the end point data in Fig. 3and in contrast with the strand transfer data (Figs. 2A and B).Although the rates for strand transfer were lower than the rates forannealing, it is interesting that in both assays, the ratio of the rate forGag to the rates for either CANC and GagΔ16-99 were very similar,with activity ranked as GagNCANCNGagΔ16-99 (Table 2).

Activity of Gag and partially processed Gag proteins in ERT assays

In addition to assays of Gag and Gag-derived proteins in ourreconstituted system, it was of interest to measure the activity of suchproteins in a viral setting. A series of HIV-1 mutants having mutationsat specific PR cleavage sites in Gagwas constructed by the Aiken group(Wyma et al., 2004). These mutants include (i) MA/CA, (ii) MA/SP1,(iii) MA/NC, and (iv) MA/p6 (uncleaved Gag) (Fig. 7). The absence ofan arrow at a site present inWT Gagmeans that cleavage is blocked atthat site in themutant (Fig. 7). Note that these mutations do not affectcleavage of the Pol moiety in Gag-Pol and RT is expressed as themature protein.

Initial efforts to analyze synthesis of viral DNA products in cellsinfected with these virions were unsuccessful, since the amounts ofDNA detected were very low and therefore highly variable. However,it was possible to perform ERT assays with detergent-treated virionsand to measure minus-strand transfer efficiency, i.e., the amount ofextended minus-strand DNA (U3-U5) divided by the amount of (−)SSDNA (R-U5), by qPCR analysis of the DNA products (Table 3).Interestingly, for thosemutants able to generatemature NC, i.e., MA/CAand MA/SP1, minus-strand transfer efficiency essentially matched thatobserved for WT. The MA/NC virus, which contains uncleaved NC, had55% of WT activity. MA/p6 (uncleaved Gag) exhibited ~30% of WTactivity, presumably reflecting a low level of strand transfer facilitatedby Gag. This is consistent with our finding in the reconstituted systemthat under some conditions, small amounts of the strand transferproduct could be detected in reactions containing Gag and an increasedconcentration of RT (data not shown). In addition, some of the Gagactivity could be due to the fact that in addition to uncleaved Gag,mutant virions were shown to contain some MA/NC (Wyma et al.,

Fig. 6. Kinetics of minus-strand transfer and annealing in the presence of Gag, CANC, and GagΔ16-99. Reaction mixtures were incubated from 1 to 60 min (minus-strand transfer) or1 min to 30 min (annealing) at 37 °C with different concentrations of Gag (A), CANC (B), or GagΔ16-99 (C). The % of minus-strand transfer product (A-1, B-1, C-1) and annealedproduct (A-2, B-2, C-2) were plotted against time of incubation. Symbols: Minus-strand transfer, closed symbols; annealing, open symbols. Protein concentrations were as follows:no protein, diamonds, dashed lines; 0.06 μM, squares, 0.12 μM, inverted triangles; and 0.23 μM, circles. Only one set of minus protein values for either theminus-strand transfer (A-1,B-1, C-1) or annealing reactions (A-2, B-2, C-2) is shown. Note that in this series of experiments, the values for Gag-mediated minus-strand transfer at a concentration of 0.23 μMwere close to baseline, similar to what was seen at 0.46 μM in Fig. 2. This is due to the fact that a different Gag preparation was used here. We have found that there is some variationbetween preparations, but the extent of variation is relatively small and does not affect the overall conclusions.

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2004). (All of the other cleavage mutants contain only the predictedproteins (Wyma et al., 2004).) The negative control for theseexperiments was the viral mutant PRD25G (Ott et al., 2000) with amutation in the PR active site.

Taken together, these results demonstrate that Gag and partiallyprocessed Gag proteins with the NC domain are able to promoteminus-strand transfer in detergent-treated virus particles as well as ina reconstituted system with purified components. In virions wheremature NC could be formed, activity was not affected by the presenceof a partially processed Gag fragment, consistent with the results ofFig. 4B.

Discussion

The goal of the present study was to determine the effect on thenucleic acid chaperone activity of HIV-1 NC when it is embedded inGag or partially processed Gag proteins. A positive result in the assaywe use (Fig. 1C) is dependent upon the characteristic activities of anucleic acid chaperone: (i) ability to facilitate annealing and weakdestabilization of nucleic acid secondary structure and (ii) rapidnucleic acid binding/dissociation kinetics (Cruceanu et al., 2006a,b;Darlix et al., 2007; Levin et al., 2005; Rein et al., 1998). Here, we showfor the first time, that Gag has both annealing and duplex

Table 3Minus-strand transfer efficiency in ERT assays.

Virus Minus-strand transfer efficiency (%)a,b

WT 102±7MA/CA 99±27MA/SP1 96±10MA/NC 55±6MA/p6 29±3PRD25G

c 8±0.4

a Minus-strand transfer efficiency expressed as a percentage of U3-U5/R-U5 copies.b Numbers to the left of the “±” are the percent efficiencies and those to the right are

the standard deviations of values from two independent experiments.c pNL4-3 with an active site mutation that inactivates PR.

Table 2Rates of strand transfer or annealing (kobs values)a.

Proteinb Rates of strand transfer (min−1) Rates of annealing (min−1)

Gag 0.22±0.027 0.75±0.059CANC 0.17±0.027 0.38±0.094GagΔ16-99 0.044±0.003 0.14±0.032

a Rates were determined by fitting the data to a single exponential equation.b The concentration of each protein was 0.12 μM.

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destabilization activities since it is able to stimulate strand transfer andreduce SP. NC reaches a higher end point value for strand transfer andsuppresses SP more efficiently than Gag, suggesting that NC's duplexdestabilization activity is more effective than that of the precursorprotein. However, relatively high concentrations of NC are required formaximal activity (Fig. 2).

The results alsodemonstrate that theNCdomain inGag is crucial for itschaperone function in strand transfer, in accord with studies on primerplacement with Gag (Cen et al., 2000; Feng et al., 1999; Guo et al., 2009)and Gag-derived proteins (Chan et al., 1999; Roldan et al., 2005). In vitro,mature NC also promotes primer placement (Cen et al., 2000; Feng et al.,1999; Hargittai et al., 2001; Iwatani et al., 2007; Prats et al., 1988; Rong etal., 1998; Tisné et al., 2001); for more detailed discussion and references,see Levin et al., 2005). Thus, CANC or GagΔ16-99, which have an NCdomain, but are lacking all or a large portion ofMA, respectively, facilitatestrand transfer (Figs. 5 and 6), whereas proteins without an NC domain,e.g., MA, CA, and MACA do not (Fig. 4). The more effective chaperoneactivity of Gag in comparison to GagΔ16-99 in our assay system suggeststhat in the context ofWT Gag, the NC domain binds and concentrates thenucleic acid, which allows additional binding by MA, thereby enhancingchaperone activity (Datta et al., 2007a). Studies of GagWMsuggest that itadopts a variety of compact conformations, allowing the MA and NCdomains to be near each other (Datta et al., 2007a). More recent workprovides evidence that nucleic acid interaction with highly basic residuesin theMA domain of Gag, in addition to interactions with the NC domain,regulates Gag binding to lipid membranes (Chukkapalli et al., 2010). Thedifference in the activities of Gag andCANCmight bedue to the lack ofMAin CANC and/or different folds of the CA N-terminal domain (NTD) in thetwo proteins (von Schwedler et al., 2003).

In reactions that only measure the annealing step in minus-strandtransfer, Gag, Gag WM, and NC all reach the same end point, but amuch higher concentration of NC is needed compared with theconcentrations of the other two proteins (Fig. 3). This observation isconsistent with FA analysis showing that Gag and Gag WM havesignificantly greater affinity for binding to ssDNA or RNA than NC(Table 1) and also with earlier data (Cruceanu et al., 2006b; Roldan etal., 2004; Roldan et al., 2005). Interestingly, both the extent and rate ofGag annealing are also higher than the corresponding values for CANC

Fig. 7. Minus-strand transfer efficiency in ERT reactions with virions containing HIV-1WT Gag or partially processed Gag proteins. HIV-1 WT Gag and Gag mutants blocked atPR cleavage sites. The schematic diagram shows Gag and partially processed Gagproteins resulting from mutation of cleavage sites (Wyma et al., 2004). The arrowsindicate the PR cleavage sites inWTGag. The absence of an arrow at a site present inWTGag indicates that cleavage at that site is blocked in the mutant.

and GagΔ16-99 (Fig. 6; Table 2). This difference in annealing activitymay account, at least in part, for the fact that these two proteins areunable to inhibit SP (Fig. 5), which occurs only when (−) SSDNAexists in an unannealed form (Guo et al., 1997). Note that in both theannealing and strand transfer assays the order of activity isGagNCANCNGagΔ16-99.

A major finding of this study is the observation that when theconcentrations of Gag or Gag WM are ≥0.23 μM, DNA elongation issignificantly reduced or abolished completely (Fig. 2). This phenom-enon was also observed with CANC and GagΔ16-99, although theeffect was less dramatic with the deletion mutant (Fig. 5). Indeed, acommon feature of proteins exhibiting concentration-dependentinhibition of polymerization is that they all contain the CA, SP1, andNC domains. Interestingly, the blocking effect is correlated with thebehavior of Gag in single-molecule DNA stretching experiments(Cruceanu et al., 2006b), a technique that gives information on nucleicacid binding kinetics (Williams et al., 2009). In studies of Gag and NC,Cruceanu et al. (Cruceanu et al., 2006a,b) demonstrated that NC has ahigh on-off rate, whereas Gag, once bound to the ssDNA or RNAtemplate, dissociates very slowly. Indeed, even with relatively lowconcentrations of Gag, it can act as a roadblock to DNA extension,whereas fully processed NC allows RT to continue elongating the DNAor RNA primer (Figs. 2A and B). Thus, Gag lacks a critical property ofhighly functional nucleic acid chaperones such as HIV-1 NC (Cruceanuet al., 2006a). Recently, it was reported that the poor chaperoneactivity of HTLV-1 NC is also correlated with slow dissociation kinetics(Qualley et al., 2010). In addition, Iwatani et al. (Iwatani et al., 2007)found that a roadblock mechanism could explain the deaminase-independent inhibitory effect of APOBEC3G on DNA polymerizationreactions catalyzed by HIV-1 RT (Bishop et al., 2008; Iwatani et al., 2007).

These considerations led us to ask: (i) What causes Gag's slowdissociation from bound nucleic acid and the resulting roadblock toRT-catalyzed DNA extension? and (ii) Why are there negligibledifferences between GagΔp6 and Gag WM activity in the strandtransfer assay? In addressing these questions, it is important to notethat in the absence of nucleic acid, Gag multimerizes at proteinconcentrations in the μM range, whereas 95% of Gag WM ismonomeric (Datta et al., 2007a). However, in the presence of nucleicacid (the condition that prevails in our assays), both Gag and GagWMbind to nucleic acid with similar affinity (Table 1) and can engage inspecific protein–protein interactions, mediated by the CA domain(Datta et al., 2007a; Stephen et al., 2007). We refer to this as “nucleicacid-driven multimerization”. Indeed, the fact that Gag WM canassemble into VLPs in vivo, albeit with lower efficiency and higherheterogeneity when bound to nucleic acid (Datta et al., 2007a),suggests that this protein can recapitulate basic protein–proteininteractions necessary for VLP formation. Thus, both Gag and GagWMcan effectively inhibit strand transfer by the roadblock mechanism. Inprinciple, such a mechanism could inhibit premature initiation ofreverse transcription. However, at present, it is not knownwhether RTis released from Gag-Pol before PR cleavage of Gag or whether HIV-1Gag-Pol can catalyze reverse transcription in the infected cell.

564 T. Wu et al. / Virology 405 (2010) 556–567

In earlier work, Roldan et al. (2005) showed thatwhen a tRNA3Lys-viral

RNA duplex was annealed by GagΔp6 or a mini-Gag protein, which ineach case remained in the reaction mixtures throughout the incubation,elongationof the tRNAprimerwaseither completelyoralmost completelyinhibited, respectively. In their system, DNA extension following anneal-ing was more efficient with a mini-Gag dimerization mutant than withWTmini-Gag (Roldan et al., 2005), a result that contrastswith ourfindingthat Gag and GagWMhave very similar activities (Figs. 2 and 3). Gag andmini-Gag caneach interactwithnucleic acids, resulting inprotein–proteininteractions.However, it is likely that themini-Gag–mini-Gag interactionsare not as stable as the Gag–Gag interactions observed in our study, sincethe mini-Gag construct is missing the NTD of CA, which contributes tostabilization of the protein multimers. Thus, the monomeric mini-Gagmutant has greater activity than theWT protein, whereas the strength ofthe Gag–Gag interactions even with Gag WM is sufficient to block DNAelongation.

Another issue that we address is the behavior of Gag and partiallyprocessed Gag proteins in our reconstituted strand transfer systemand the effects of such proteins in a viral setting. For this work, wetook advantage of a series of HIV-1 viral mutants that are blocked atone or more Gag cleavage sites and do not have conical cores (Wymaet al., 2004). Since attempts to assay viral DNA products synthesizedin infected cells were unsuccessful, we chose to measure ERT activityin detergent-treated virions (Fig. 7; Table 3). In this assay, the lack ofconical cores per se does not block reverse transcription (Kaplan et al.,1994; Tang et al., 2001) (Thomas, J.A. and Gorelick, R.J., unpub. obs.).The results of the ERT assays are consistent with the data from thereconstituted system. In particular, we note that transdominanteffects of partially processed Gag proteins, which could potentiallyinhibit NC activity in our experiments, are not observed in either assay(Fig. 4B; Table 3), in contrast to the situation with virus-infected cells(Checkley et al., 2010; Lee et al., 2009; Müller et al., 2009; Rulli et al.,2006). Thus, transdominant inhibition of viral DNA synthesis by Gagcleavage proteins occurs only in a cell-based system where a block inGag cleavage is closely linked to (i) the assembly of aberrant viralcores and/or (ii) improper uncoating of cores following virus entryinto the cell.

In summary, we have demonstrated that there is a majordifference between the nucleic acid chaperone activities of NCembedded in HIV-1 Gag and Gag-derived proteins and those ofmature NC. Although all of these proteins have annealing and helixdestabilizing activities, the activity of Gag proteins is less effectivethan that of mature NC. Thus, unlike NC, Gag dissociates slowly frombound nucleic acid, a consequence of nucleic acid-driven Gagmultimerization that occurs even at relatively low protein concentra-tions. This in turn leads to drastic inhibition of DNA elongation. Werefer to this inhibition as the roadblock mechanism. It will beinteresting to determine whether this mechanism is used moregenerally by other retroviruses. In conclusion, our data help to explainwhy NC and not Gag is a critical cofactor for viral DNA synthesis andillustrate how the presence of NC in a multi-domain protein like Gagmodulates the nature of its nucleic acid chaperone activity.

Materials and methods

Materials

DNA oligonucleotides were obtained from Lofstrand (Gaithers-burg, MD) and Integrated DNA Technologies (Coralville, IA). T4polynucleotide kinase, proteinase K, SUPERaseIN, and Gel LoadingBuffer II were obtained fromApplied Biosystems (Foster City, CA). [γ-33P]ATP (3,000 Ci/mmol) was purchased from PerkinElmer (Shelton, CT). Allof the columns used for protein purification were purchased from GEHealthcare Life Sciences (Piscataway, NJ). HIV-1 RT was obtained fromWorthington (Lakewood, NJ). The sequence of NC and the nucleic acidsused in this study were derived from the HIV-1 pNL4-3 clone (GenBank

accession no. AF324493) (Adachi et al., 1986). Sequences of the purifiedGag and Gag cleavage proteins were derived fromHIV-1 BH10 (GenBankaccession no. M15654.1) (Campbell and Rein, 1999; Ratner et al., 1985),except for GagΔ16-99, which contains sequences from HIV-1 NL4-3 andBH10 (SpeI site in CA to 3′ end is BH10) (Gross et al., 2000).

Purification of proteins used in this study

Gag and Gag cleavage products (Fig. 1A) were expressed in BL21(DE3)pLysS cells (Stratagene). Cells expressing HIV-1 matrix (MA)were induced at 37 °C for 4 h with 1 mM isopropyl-beta-D-thioga-lactoside and were lysed in buffer A (20 mM Tris–HCl, pH 7.4, 10 mM2-mercaptoethanol, and 1 mMphenylmethylsulfonyl fluoride [Sigma-Aldrich]) containing 150 mM NaCl. After centrifugation at 12,000×gfor 15 min to remove cellular debris, theMA proteinwas purified fromthe total lysate by taking a 40–70% ammonium sulfate cut. The proteinwas dialyzed against buffer A containing 150 mM NaCl. After dialysis,ammonium sulfate was added to 40% saturation and the protein waschromatographed on a Butyl Sepharose column. Fractions containingthe protein were dialyzed against buffer A with 50 mMNaCl and wererun on an SP® Sepharose column. The purified protein was stored at−80 °C in buffer A with 150 mMNaCl and 10% Glycerol. HIV-1 CA andMACA were purified as described in references (von Schwedler et al.,1998; Yoo et al., 1997), respectively.

Since HIV-1 Gag expressed in E. coli is extensively degraded bybacterial proteases (Campbell and Rein, 1999), the stably expressedprotein known as GagΔp6 was used instead of authentic Gag in assaysfor minus-strand transfer. Note that in addition to missing the p6domain, GagΔp6 is not myristoylated at its N-terminus (Campbell andRein, 1999). GagΔp6 proteins with additional changes were asfollows: (i) GagΔ16-99, missing residues 16–99 in the MA domain(Datta and Rein, 2009; Fäcke et al., 1993; Gross et al., 2000) and (ii)Gag WM, GagΔp6 with Trp316 and Met317 (i.e., Trp184 and Met185in CA, respectively) changed to Ala, which causes a defect in Gagdimerization (Datta et al., 2007a,b). GagΔp6, the two GagΔp6-derivedproteins, and CANC (Campbell and Vogt, 1995) were purified byphosphocellulose affinity chromatography as described previously(Datta et al., 2007a; Datta and Rein, 2009). In the final purificationstep, the proteins were subjected to further chromatography onSuperose 12 (GagΔp6, GagΔ16-99, Gag WM, CANC) or Superdex 75(MA, MACA, CA) gel filtration columns to eliminate RNase and DNasecontamination. Analysis of the Gag and Gag-derived protein prepara-tions by SDS–PAGE indicated that the proteins were at least 90% pure(Fig. 1B). The residue numbers in Gag and the respectivemolecularmassvalues are given in the legend to Fig. 1B. Recombinant HIV-1 NC wasprepared as described previously (Carteau et al., 1999; Wu et al., 1996).

In vitro minus-strand transfer assay

The minus-strand transfer assay was performed as describedpreviously (Heilman-Miller et al., 2004) with several changes. Sincemaximal activity was obtained at 1mMMg2+ in reactions with eitherNC (Wu et al., 2007) or Gag (data not shown), we used 1 mMMg2+ inall of the polymerase reactions. In addition, 0.5 U of SUPERaseIN wasadded per 20-μl reaction. HIV-1 NC or Gag and Gag-derived proteinswere added at the concentrations specified in the legends to thefigures. Reaction mixtures (final volume, 20 μl) also contained DNA128 ((−) SSDNA) labeled at its 5′ end with 33P, using the 32P labelingprotocol described in Guo et al. (1995), RNA 148 (acceptor RNA), andHIV-1 RT, each at a final concentration of 10 nM. Incubation was at37 °C for the indicated times. Termination of the reactions, electro-phoresis in denaturing gels, and PhosphorImager analysis wereperformed as described (Wu et al., 2007). In addition to the transferproduct, dead-end products caused by SP of fold-back structures atthe 3′ end of (−) SSDNA (Beltz et al., 2005; Driscoll and Hughes, 2000;Guo et al., 1997, 2000; Heilman-Miller et al., 2004; Lapadat-Tapolsky

565T. Wu et al. / Virology 405 (2010) 556–567

et al., 1997; Levin et al., 2005) and references therein) were alsoformed. The percentage of minus-strand transfer product synthesizedwas calculated by dividing the amount of transfer product by totalDNA (transfer product plus SP products and remaining DNA 128),multiplied by 100. The percentage of SP products synthesized wascalculated in a similar manner. The data for strand transfer and forannealing (see below) represent the average of results obtained in atleast three independent experiments. Error bars shown in the figuresrepresent the standard deviation.

Annealing assay

33P-labeled DNA 128 (0.2 pmol) was incubated at 37 °C with 0.2pmol of RNA 148 in buffer containing 50 mM Tris–HCl (pH 8.0) and75 mM KCl for the indicated times in the absence or presence of Gagproteins (final volume, 20 μl), as described (Wu et al., 2007). The finalconcentration of the nucleic acid substrates was 10 nM.

Fluorescence anisotropy (FA) experiments

Equilibrium binding of HIV-1 NC and Gag to a 20-nt AlexaFluor-488-labeled ss DNA oligonucleotide (JL936, 5′-Alex488-AGCTGCTTTTTGCCTGTACT-3′) and a 20-nt fluorescein-labeled ssRNAoligonucleotide (JL943, 5′-Fl-AGCUGCUUUUUGCCUGUACU-3′) wasmeasured using FA. The oligonucleotides contained the 20-nt ssRNAor DNA sequence from the extreme 3′ terminus of U3. JL936 (purifiedby high performance liquid chromatography) and JL943 (purified byPAGE) were obtained from Integrated DNA Technologies (Coralville,IA) and Dharmacon, Inc. (Lafayette, CO), respectively. FA measure-ments were performed in Corning® 384-well low volume blackpolystyrene NBSTM microplates (Corning, NY) using a SpectraMax®

M5multimodemicroplate reader (Molecular Devices, Sunnyvale, CA).JL936 or JL943 (each 20 nM) were incubated with increasingconcentrations of NC or Gag in 50 mM Tris–HCl (pH 8.0), 75 mM KCl,1 mM MgCl2, and 1 mM DTT. Samples were excited at 485 nm andemission intensities at both parallel and perpendicular planes werecollected at 525 nm. The resulting plot of anisotropy vs. proteinconcentration was fit using a one-site binding model (Iwatani et al.,2007) to obtain the Kd value.

Virus production

Viruses were derived by transfection of 293 T cells in 100-mmculture dishes, using the CaPO4/DNA coprecipitation method (Gra-raham and van der Eb, 1973), as described previously (Thomas et al.,2008). Plasmids for the production of the MA/CA, MA/SP1, MA/NC,and MA/p6 mutant viruses (Wyma et al., 2004) were a generous giftfrom Christopher Aiken, Vanderbilt University Medical Center, Nash-ville, TN. The PR mutant, PRD25G, was a generous gift from David Ott,SAIC-Frederick, Inc., NCI-Frederick, Frederick, MD (Ott et al., 2000).TheWTHIV-1 plasmid pNL4-3 (Adachi et al., 1986)was obtained fromMalcolm Martin (NIAID, NIH) through the AIDS Research andReference Reagent Program, Division of AIDS, NIAID, NIH.

ERT assays

Viral supernatant fluids (30 ml) were treated with DNase I andwere then pelleted through a 5-ml 20% (w/v) sucrose cushion (in PBSwithout Ca2+ or Mg2+) in a Beckman SW32 rotor at 144,000×g for 1 hat 4 °C. The pellets were resuspended and subjected to ERT asdescribed previously (Thomas et al., 2008). Reverse transcriptionproducts, R-U5 (representative of (−) SSDNA products), and U3-U5(representative ofminus-strand transfer products)were quantified byqPCR, as described previously (Buckman et al., 2003).

Acknowledgments

We thank Christopher Aiken for the Gag cleavage mutants, DavidOtt for the HIV-1 PR mutant PRD25G, Malcolm Martin for the pNL4-3clone, which was obtained from the NIH AIDS Research and ReferenceReagent Program, and Hans Luecke, who generously allowed us to usehis Spectromax M5 instrument for the Kd determinations. We are alsoindebted to Christopher Jones, Ioulia Rouzina, and Karin Musier-Forsyth for valuable discussion and for sharing unpublished data. Thiswork was supported by the Intramural Research Program of the NIH(Eunice Kennedy Shriver National Institute of Child Health andHuman Development [J.G.L.] and the National Cancer Institute, Centerfor Cancer Research [A.R.]) and was funded in part with federal fundsfrom the National Cancer Institute under contract numbers N01-CO-12400 and HHSN261200800001E (R.J.G.). The content of thispublication does not necessarily reflect the views or policies of theDepartment of Health and Human Services, nor does mention of tradenames, commercial products, or organizations imply endorsement bythe U.S. government.

Authors Judith G. Levin and Alan Rein wish to dedicate this paperto the memory of Brenda I. Gerwin, a dear friend and colleague.

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