Mutational Analysis of the C-Terminal Gag Cleavage Sites in Human Immunodeficiency Virus Type 1

Human immunodeficiency virus type 1 (HIV-1) Gag is expressedas a polyprotein that is cleaved into six proteins by the viralprotease in a maturation process that begins during assemblyand budding. While processing of the N terminus of Gag is strictlyrequired for virion maturation and infectivity, the necessityfor the C-terminal cleavages of Gag is less well defined. Toexamine the importance of this process, we introduced a seriesof mutations into the C terminus of Gag that interrupted thecleavage sites that normally produce in the nucleocapsid (NC),spacer 2 (SP2), or p6^Gag proteins. Protein analysis showed thatall of the mutant constructs produced virions efficiently upontransfection of cells and appropriately processed Gag polyproteinat the nonmutated sites. Mutants that produced a p9^NC/SP2 proteinexhibited only minor effects on HIV-1 infectivity and replication.In contrast, mutants that produced only the p8^SP2/p6 or p15^NC/SP2/p6protein had severe defects in infectivity and replication. Toidentify the key defective step, we quantified reverse transcriptionand integration products isolated from infected cells by PCR.All mutants tested produced levels of reverse transcriptionproducts either similar to or only somewhat lower than thatof wild type. In contrast, mutants that failed to cleave theSP2-p6^Gag site produced drastically less provirus than the wildtype. Together, our results show that processing of the SP2-p6^Gagand not the NC-SP2 cleavage site is important for efficientviral DNA integration during infection in vitro. In turn, thisfinding suggests an important role for the p9^NC/SP2 speciesin some aspect of integration.

INTRODUCTION

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INTRODUCTION

The Gag polyprotein is the major structural component of retrovirusesand is the only viral protein strictly required for particleformation by orthoretroviruses (reviewed in references 8 and13). During assembly and budding, human immunodeficiency virustype 1 (HIV-1) Gag is cleaved by the viral protease, liberatingsix mature proteins from the Gag polyprotein (13, 45), p15^MA,p24^CA, SP1 (spacer peptide 1, also known as p2), p7^NC, SP2 (alsoknown as p1), and p6^Gag. The organization of HIV-1 Gag and itsmature cleavage products is presented in Fig. 1. The newly liberatedmature Gag proteins then complete the maturation process byrearranging the interior core structure of the virion, observableby electron microscopy as a transition from the doughnut-shapedimmature core morphology to a cone-shaped core of the maturevirus (13, 15, 42, 45). By this maturation process, the proteinswithin Gag and Gag-Pol shift roles from parts of a polyproteinthat is dedicated to assembly to individual proteins that functionin the infection process.

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FIG. 1. Proviral constructs. Diagrams of the Gag regions of the various pNL4-3 constructs used in this study are presented. The mature proteins within Gag and the minor cleavage sites (denoted by dotted lines in SP2 and p6^Gag) are indicated on the NL4-3 wild-type construct map. The region mutated is presented for each mutant with the expected product highlighted in black. A dotted line indicates the C-terminal partial protease cleavage site (after position 36) in p6^Gag.

Gag processing occurs in an ordered fashion (37, 41, 48). Theinitial cleavage of Gag appears to take place between SP1 andNC, liberating MA-CA-SP1 (p40^Gag) and NC-SP2-p6^Gag (p15^NC/SP2/p6)as intermediary products. Processing at this site and at theothers in the p40^Gag partial cleavage product is essential forproper maturation and virion infectivity (19, 26, 36). In contrast,the importance of processing at the C-terminal sites in Gag,NC-SP2-p6^Gag, is not altogether clear. In vitro, processingat the SP2-p6^Gag site can be dependent on the RNA-binding sequencesin the NC protein (44). Alteration of the SP2-p6^Gag site bymutating both the P₁ and P₁' positions produced virions withmostly p8^SP2/p6 with some p15^NC/SP2/p6 that exhibited a replicationdefect in T cells (54). Mutations altering the two proline residuesin the middle of SP2 eliminated viral replication (23). Nevertheless,p9^NC/SP2 has been found to be more efficient than p7^NC at promotingreverse transcriptase (RT)-associated functions in vitro (3,27). Still, it is not clear how important the processing ofNC, SP2, and p6^Gag is for the infection process.

To better understand the requirement for proteolytic processingof the C-terminal p15^NC/SP2/p6 region of Gag, we produced aseries of NL4-3 constructs with mutations designed to blockprocessing at these cleavage sites. The results showed thatwhile processing of the NC-SP2 site is dispensable for infectivity,cleavage at the SP2-p6^Gag site is required for efficient integrationby the infecting virus.

MATERIALS AND METHODS

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MATERIALS AND METHODS

DNA mutagenesis. The pNL4-3 infectious molecular clone of HIV-1 (1) (GenBankaccession no. AF324493) was used for these studies and alteredby site-directed mutagenesis using PCR-based methods, eitherby direct amplification with a mutagenic primer or by two roundsof amplification using the overlap extension procedure (24).The NL4-3 mutants constructed included the following: NC-N55D,with nucleotide 2083 changed from A to G, resulting in an asparagine-to-asparticacid substitution at NC position 55; NC-N55I, with nucleotide2084 changed from A to T, resulting in an asparagine-to-isoleucinesubstitution at NC position 55; NC-SP2, with nucleotide 2084changed from A to G, resulting in an asparagine-to-serine substitutionat NC position 55 that was combined with another mutation, nucleotide2089 changed from T to C, resulting in an isoleucine-to-threoninesubstitution at SP2 position 5; SP2-F16S, with nucleotide 2131changed from T to C, resulting in a phenylalanine-to-serinesubstitution at SP2 position 16; p15, a combination of the NC-SP2and SP2-p6^Gag mutants; p6-L1D, with nucleotides 2133 to 2136changed from TCTT to CGAT, resulting in an aspartic acid-to-leucinesubstitution at p6^Gag position 1 and a serine-to-arginine substitutionat polymerase (Pol) position 17; p6-L1K, with nucleotides 2133to 2136 changed from TCTT to CAAA, resulting in an asparticacid-to-lysine substitution at p6^Gag position 1 and an alterationof Pol codons serine 17 to glutamine and serine 18 to threonine;p6-Q2D, with nucleotides 2136 to 2139 changed from TCAG to CGAC,resulting in a glutamine-to-aspartic acid substitution at p6^Gagposition 2 and an alteration of Pol serine 17 to leucine andserine 18 to arginine. After construction, the regions of DNAthat were PCR amplified were sequenced to confirm the mutationsand to rule out the possibility of any additional changes introducedduring the PCR-mediated mutagenesis process.

Cell culture. 293T human embryonic kidney, HeLa-CD4-LTR-lacZ (HCLZ) (18),JC53BL-13 (also known as TZM-bl) (49), and HOS human osteosarcomacell line cells were cultured in Dulbecco's modified Eagle'smedium, while H9 human T-cell leukemia cells were cultured inRPMI 1640. All media were supplemented with 2 mM L-glutamine,100 U per ml penicillin, 100 µg per ml streptomycin, andeither 10% (vol/vol) fetal bovine serum (293T, JC53BL-13, HCLZ,and H9) or 5% (vol/vol) fetal bovine serum and 5% (vol/vol)calf serum (HOS). All cell culture products were obtained fromInvitrogen, Inc. (Carlsbad, CA). Transient transfections of293T cells were carried out by using calcium phosphate transfection(20) or TransIT-293 (Mirus Bio Corp., Madison, WI). The HIV-1infection assays using either HCLZ or JC53BL-13 cells were carriedout as previously described (18). Briefly, the cells were infectedwith dilutions of virus and the assay was developed for ß-galactosidaseactivity by staining with 5-bromo-4-chloro-3-indolyl-ß-galactoside48 h postinfection. Infected cells, those staining blue, wereobserved by light microscopy and counted to score infectionevents. The ability of viruses to replicate on H9 cells wascarried out as previously described (18). Briefly, H9 cellswere exposed to 10-fold dilutions of virus and the cultureswere monitored periodically for infection by the presence ofand increase in RT activity in the culture medium over a 6-weekculture period. Samples that were 10 times the background leveland greater than 1/100 of the peak level of the undiluted wild-typecontrol were considered positive for infection.

Protein analysis. Isolation of virions by density centrifugation and immunoblotanalysis were performed as previously described (33). Primarygoat antiserum against p7^NC (goat 77), p24^CA (goat 81), andp17^MA (goat 83), as well as rabbit antiserum against p6^Gag (DJ-30552),were obtained from the AIDS Vaccine Program, NCI-Frederick,Frederick, MD. Mouse antibody against RT, NEA-9304001EA, wasobtained from Perkin-Elmer (Wellesley, MA). Proteins were detectedby developing blots with either horseradish peroxidase-conjugatedanti-goat, anti-rabbit (both from Biochain Institute, Hayward,CA), or anti-mouse (Life Technologies, Inc., Gaithersburg, MD)secondary antibody and the Immun-star horseradish peroxidasesubstrate kit (Bio-Rad, Hercules, CA) on LumiFilm (Roche AppliedScience, Indianapolis, IN). Reversed-phase high-pressure liquidchromatography (HPLC) and matrix-assisted laser desorption ionizationtime-of-flight (MALDI-TOF) mass spectrometry were carried outas previously described (33).

Real-time PCR of reverse transcription products and proviral DNA. Assays for reverse transcription and integration products withinHOS cells after infection were carried out as previously described(25). Briefly, wild-type and mutant viruses pseudotyped withthe vesicular stomatitis virus G surface glycoprotein (VSV-G)were generated by calcium phosphate cotransfection of 293T cellsfollowed by a repetitive washing procedure to remove plasmidDNA. The virus stocks were then used to infect HOS cells, andtotal DNA was isolated from the cells 24 h postinfection. ForHIV-1 Env-mediated infections, virus stocks were prepared bycalcium phosphate transfection and then treated with DNase toremove carryover plasmid from transfections: supernatants wereexposed to 10 U/ml of DNase (Sigma-Aldrich, St. Louis, MO) in4 mM MgCl₂ at 37°C for 1 h. H9 cells (3 x 10⁶) were infectedin a 24-well plate using Virofect R/L Magnetofection reagent(OZ Biosciences, Inc., Marseilles, France) according to themanufacturer's instructions using the recommended magnetic plate(OZ Biosciences). Analyses of early (strong stop; R-U5) andlate reverse transcription DNA products (plus-strand transfer;R-5' untranslated region [UTR]) and 2-LTR junction productswere carried out as previously described (5). The proviral DNAassay (Alu-LTR) was performed as described elsewhere (46). Negativecontrol infections with heat-inactivated (65°C for 20 min)aliquots of the virus stocks were used to assess the levelsof carryover plasmid DNA from the transfection. The quantitiesof carryover plasmid DNA were typically less than 0.1% of thatobtained from live virus.

RESULTS

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RESULTS

To examine the importance of the processing of the p15^NC/SP2/p6precursor, we produced a series of mutant constructs in full-lengthpNL4-3 designed to eliminate Gag proteolytic processing in theC terminus of p7^NC and the N terminus of SP2 to generate virusesthat contain p9^NC/SP2, p8^SP2/p6, or p15^NC/SP2/p6 (Fig. 1). Onepotential complication to this approach is that, in additionto these primary cleavage sites, there are minor secondary sitesin this region of Gag (Fig. 1) that can be cleaved in maturevirions (21, 22, 33). One of these minor cleavages occurs afterSP2 amino acid 5, potentially cleaving within a p9^NC/SP2 orp15^NC/SP2/p6 species produced by our cleavage site mutants.To eliminate this possibility, we mutated the P₁ amino acidof this site (SP2 amino acid 5, Gag position 437) (Fig. 1) inconstructs that had mutations either at the NC-SP2 site (NC-SP2in Fig. 1) or at both the NC-SP2 and SP2-p6^Gag sites (p15 inFig. 1).

A complication in genetically blocking protease cleavage isthe difficulty in predicting what sequence substitutions atthe protease cleavage site will eliminate protease recognitionin the viral mutants. General selection rules based on proteasecrystal structure and studies with peptides suggest that insertionof hydrophilic and charged residues at the cleavage site interruptsprocessing (39, 52, 53). Nevertheless, our experience has shownthat some of the mutations suggested by these rules do not alwaysprevent cleavage when placed in the context of the virus. Usingthese rules as well as our previous observations, we introduceda series of mutations into the C terminus of Gag. Contrary toexpectations, some of the mutated cleavage sites were eithercompletely or mostly cleaved in virions despite a poor predictedsubstrate site: preliminary immunoblotting with p7^NC antiserumshowed that NC-SP2 site mutants containing isoleucine, histidine,and lysine in the P₁ position were processed by protease, whilethose containing serine and aspartic acid were not cleaved (datanot shown). Also, an initial p6^Gag immunoblot assay revealedthat a serine in the P₁' position of SP2-p6^Gag was also processed,while aspartic acid and lysine substitutions were not cleaved.

The NL4-3-based constructs selected for further study are displayedin Fig. 1. All of the NC and SP2 mutants were constructed sothat the Gag mutations did not alter the overlapping Pol readingframe or alter the frameshift slippery site (which overlapsthe wobble codon of the asparagine at P₁ of the NC-SP2 site).The p6^Gag mutants had changes in the Pol frame at the beginningof the p6^Pol protein (also referred to as the preprotease ortransframe protein). These secondary mutations did not alterprocessing of Gag at other sites or reverse transcriptase incorporation(presented below).

To study the mutant virions, preparations were produced by transfectingeach molecular clone into 293T cells and then isolating virionsby density centrifugation for protein analyses. To examine anyimpact the mutations might have on virus production and processingof the N-terminal Gag proteins (i.e., those in the p40^Gag partialcleavage product), equal amounts of virion preparations (byvolume) were analyzed by immunoblotting with p24^CA antiserum.The results showed that the mutants produced nearly equivalentlevels of virions, indicating that they exhibit no overt assemblydefects. The virion samples also did not contain appreciableamounts of p55^Gag or partially processed products; thus, themutations in the C terminus of Gag did not alter the overallprocessing of p24^CA or p17^MA. Since changes to the middle ofSP2 can alter Gag processing and Gag-Pol incorporation (23),we detected the Pol protein product in the samples by strippingthe blot and exposing it to an antibody against RT. The blotshowed that the relative intensities of the RT signals presentin the mutant samples compared to those of Gag were similarto the wild-type sample (Fig. 2). Hence, the amount of Pol incorporationdoes not appear to be significantly altered by these mutations.

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FIG. 2. Immunoblots of mutant virion preparations. p24^CA, RT, p7^NC, and p6^Gag immunoblots of equal volumes of virion preparations are presented. Samples are identified above the respective lanes. sssDNA, a virus preparation produced from transfected, sheared salmon sperm DNA as a mock control. Molecular masses, as calculated by relative mobilities, and identities of bands are indicated at the margins of the blots. Due to the poor retention of wild-type p6^Gag, a 10-fold-longer exposure is provided to the right of the second p6^Gag immunoblot to show the migration of wild-type p6^Gag.

A p7^NC immunoblot analysis of the virions revealed that mutatingthe NC-SP2 cleavage site had various effects on processing.The NC-N55I sample contained a band that was similar to thewild type, p7^NC, as well as a readily detectable minor bandthat migrated higher in the gel, likely p9^NC+SP2. Therefore,processing apparently still occurred, though inefficiently,at the NC-SP2 site in this mutant. Only one band was presentin the NC-N55D and NC-SP2 virion samples, migrating at a positionhigher than the wild-type band, consistent with the presenceof the p9^NC/SP2 species that would be produced by blocking theNC-SP2 cleavage site. As expected, the SP2-F16S, p6-L1D, p6-L1K,and p6-Q2D mutants contained a normal-sized p7^NC band (Fig.2), as the NC-SP2 site was not mutated in these constructs.The p15 mutant sample contained a band that migrated much higher,consistent with the expected p15^NC/SP2/p6 product.

To examine the status of p6^Gag in the mutants, the blot wasstripped and exposed to p6^Gag antiserum. This revealed thatthe samples of the NC-N55I, NC-N55D, and NC-SP2 mutants containeda wild-type-sized p6^Gag band. (The wild-type p6 bands are lessintense due to the nature of p6^Gag, a protein that is relativelyhydrophilic and does not efficiently transfer from the gel ontopolyvinylidene difluoride membranes [33].) The SP2-F16S, p6-L1D,p6-L1K, and p6-Q2D mutant samples contained two higher-migratingbands on the blots. Since the C terminus of p6^Gag is alternativelyprocessed after p6^Gag residue 36 (Gag position 483) (Fig. 1)(21, 22, 33), the former is likely an SP2-p6^Gag product whilethe latter is an apparent SP2-p6^Gag product missing its extremeC terminus. The p15 mutant contained a much higher migratingband that coincided with the band in the p7^NC immunoblot, confirmingthe presence of both p7^NC and p6^Gag in this species. In additionto the p15^NC/SP2/p6 product, there were weaker bands that correspondedto those observed for the SP2-p6^Gag mutants, indicating thatthere was a small amount of processing at the NC-SP2 site. Together,these results indicate that we have produced a functional seriesof cleavage site mutants.

HPLC and mass spectrometry analysis. Considering the relatively small size of the mutant proteinsand the potential for minor cleavages in this region, we confirmedthe identities of the p7^NC, SP2, and p6^Gag products by isolatingthem from virion preparations with reversed-phase HPLC and identifiedthem with a subsequent MALDI-TOF mass spectrometry analysis(Fig. 3; Table 1). These data generally confirmed the immunoblotdata (Fig. 2). Nearly all of the NC in the NC-N55D mutant waspresent as a p9^NC/SP2 product (Fig. 3; Table 1), though thesevirions did contain a small amount of p7^NC (less than approximately5%, based on relative absorbance at 206 nm) (Fig. 3; Table 1).(There is also another peak in the HPLC that elutes at a similartime as the p9^NC/SP2 peak that corresponds to ubiquitin [Ub][Fig. 3], which is incorporated into virions [34, 35, 43].)The NC-SP2 mutant contained only p9^NC/SP2 with no detectablep7^NC (Fig. 3; Table 1). Thus, NC-SP2 processing appears to becompletely blocked in this mutant. NC-N55I virions displayedan intermediate phenotype (Fig. 3), containing a mixture ofp7^NC and p9^NC/SP2 (approximately 40% based on relative absorbance)(Fig. 3; Table 1), in agreement with our prior blotting data(Fig. 2). Mass spectrometry revealed that the p7^NC in this mutantvirus, while appearing normal-sized by immunoblotting, containedadditional mass that is consistent with the addition of a phenylalaninefrom the N terminus of SP2. Likewise, the SP2 protein was missingmass that would account for this residue. Thus, the mutationappears to have altered processing at this site in two ways,either moving the cleavage site over one residue, making p7^NC+F,or preventing cleavage entirely (Fig. 3). This underscores theimportant of biochemical analyses of cleavage site mutants.

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FIG. 3. HPLC analysis of mutants. The complete HPLC chromatogram for wild-type NL4-3 virions is shown at the top of the figure with the region that is presented for the mutants indicated by arrows. The identities of the Gag proteins, determined by Coomassie brilliant blue-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, protein sequencing, and mass spectrometry, are identified above each respective peak. The mutant proteins as identified by MALDI-TOF mass spectrometry are shaded in black. The p6^Gag-containing species that are missing the C-terminal 16 amino acids due to cleavage after residue 36 are indicated by a "-cf" notation, e.g., p6^-cf, in the identifying superscript. The C-terminal fragment itself was labeled p6^cf when observed.

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TABLE 1. Mass spectrometry data for cleavage site products

As expected, the SP2-p6^Gag cleavage site mutants, SP2-F16S,p6-L1D, p6-L1K, and p6-Q2D, contained only the p8^SP2/p6 productwith the expected mass (Table 1), though we did observe a truncatedform (Fig. 3, p8^-cf) that matched the expected size for a p8^SP2/p6product with a C-terminal cleavage at p6 residue 36. This specieswas previously observed in our immunoblot data (Fig. 2). Additionally,we also could detect the C-terminal 16-amino-acid protein ofp6^Gag in this sample (Fig. 3, cf).

Analysis of the p15 mutant virions detected the p15^NC/SP2/p6species (Fig. 3; Table 1) and two proteins with masses consistentwith the expected N- and C-terminal fragments for a cleavagein the C-terminal tail (Fig. 3, p15^-cf and cf, respectively).In addition, we did find a slight amount of p8^SP2/p6 (Fig. 3).Together, these data confirm the immunoblot data and furtherdemonstrate that there was no unexpected processing at any minorsites.

Mutant infectivity and replication. The ability of the HIV-1 mutants examined above to carry outa single round of infection was tested by using JC53bl-13 cellsin an LTR-lacZ Tat complementation assay (18). Their abilityto perform multiple rounds of replication in H9 cells was determinedusing an endpoint titer method. The results (Table 2) showedthat the NC mutants NC-N55D, NC-N55I, and NC-SP2 produced single-roundtiters and H9 endpoint tissue culture infective doses that wereonly somewhat lower than those containing wild-type NC. Thus,even though both the NC-N55D and NC-SP2 mutants contain mostlyuncleaved p9^NC+SP2, they had only modest reductions in the infectivityand replication assays. Also, the NC-N55I virions, which containpartially or miscleaved p7^NC, maintained high relative infectivities.These results show that the cleavage of p7^NC from SP2 is notessential for replication.

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TABLE 2. Infectivity and replication of mutants

In contrast, the single-round infectivities of the SP2-p6^Gagcleavage site mutants, SP2-F16S, p6-L1D, p6-L1K, and p6-Q2D,were at least 2 logs lower than wild-type levels. The effecton replication was even more dramatic: the titers for thesemutants were $≤$ 10^–6 of the titer of the wild type. Similarto the SP2-p6^Gag cleavage site mutants, the infectivity andreplication competence of the p15 mutant were also reduced tolevels near those of the SP2-p6^Gag mutants. Therefore, thesedata indicate that cleavage of SP2 from p6^Gag is important inHIV-1 replication.

Analysis of infection products. To determine the replication block for the SP2-p6^Gag and p15mutants, we examined the ability of VSV-G-pseudotyped mutants(NC-N55D, NC-SP2, SP2-F16S, and p15) to carry out reverse transcriptionand integration steps in HOS cells by detecting key DNA productsinside the cell with real-time PCR 24 h postinfection. Processivityof reverse transcription, measured by conversion of early (strongstop; R-U5) to late (plus-strand transfer; R-5' untranslatedregion [UTR]) reverse transcription products (RT^N), was onlyslightly reduced for the NC-ND, SP2-F16S, and p15 mutants whencompared to the wild-type samples (a representative experimentis shown in Fig. 4, early-late RT^N; full data sets are presentedin Tables S1 to S3 of the supplemental material). The NC-SP2mutant produced reverse transcriptase products essentially ata wild-type efficiency, consistent with its infectivity phenotype(Fig. 4; see also Tables S1 to S3). Therefore, a gross failurein carrying out reverse transcription was not the major defectin the SP2-F16S and p15 mutants.

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FIG. 4. Reverse transcription and integration products assayed by real-time PCR. Representative results of a quantitative real-time PCR analysis are presented as percent conversion between two representative steps in the infection process. Results for VSV-G-mediated infection are presented as black bars, and data from HIV-1 Env-mediated infections are in white. Parameters presented are indicated next to each graph. Typical samples analyzed contained extract from about 1.1 x 10⁴ cells. For the wild type, the target quantities detected were 3.2 x 10⁵ copies of R-U5, 1.33 x 10⁵ copies of R-5'UTR, 3.3 x 10⁴ copies of provirus, and 4.8 x 10³ copies of 2-LTR circles. The amounts of provirus in H9 cells infected with HIV-1 Env-containing virus were below the level of detection and thus were omitted. Full data sets of experiments are presented in Tables S1 to S6 in the supplemental material.

Another aspect assayed was the production of 2-LTR circles (representativedata shown in Fig. 4, late RT^N-2-LTR circles). A small amountof the newly reverse transcribed DNA forms 2-LTR circles asa dead-end by-product that is produced during infection. Thisis commonly used as an indicator that plus-strand synthesiswas completed and that the resulting viral DNA was exposed tonuclear enzymes. The PCR analysis showed that 2-LTR formationfor the SP2-F16S and the p15 mutants was one-half to one-thirdof the wild-type value and that of the NC cleavage site mutants(Fig. 4; see also Tables S1 to S3 in the supplemental material).While this is somewhat reduced, the differences do not accountfor the greater than 6-log difference in replication observedbetween these viruses.

Unlike the two other steps, integration, as measured by an Alu-LTR-basedPCR assay, was drastically reduced in the cells infected withthe SP2-F16S and p15 mutants (representative data shown in Fig.4, late RT^N-provirus). The analysis showed that the conversionof late reverse transcription product (R-5'UTR) to provirus(Alu-LTR) was greatly reduced for these mutants, to less than3% of the wild-type levels (Fig. 4; see also Table S1 in thesupplemental material). Thus, the primary defect in the SP2-F16Sand p15 mutants appears to be a severe defect in integration.

Even though both VSV-G- and HIV-1 Env-mediated infection ofcells with NL4-3 produces reverse transcription products atthe same efficiency in unperturbed cells (47), VSV-G pseudotypingcan overcome various blocks to HIV reverse transcription duringinfection that are induced by genetic mutants and cellular inhibitors(2, 40). Thus, the use of VSV-G could possibly mask a reversetranscription defect in our mutants. To address this possibility,we infected H9 cells using our mutants without VSV-G pseudotyping(NC-N55D, NC-SP2, SP2-F16S, p6-Q2D, and p15), thereby relyingon CD4/gp120-mediated infection. The results essentially mirroredthose obtained from the VSV-G/HOS cell experiments (Fig. 4;see also Tables S4 to S6 in the supplemental material): themutants had only minor decreases in reverse transcription (early-lateRT^N) and a modest defect in nuclear accessibility (late RT^N-2LTR circle). Neither defect accounts for the gross defect inreplication. The amounts of provirus for these experiments werebelow the level of detection due to the reduced number of infectionevents with the HIV-1 Env-mediated infections. Overall, theseresults demonstrated that the mutants carry out reverse transcriptionrelatively efficiently. Thus, the defect in infection is ata later step, immediately prior to or during integration.

DISCUSSION

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DISCUSSION

Our results show that removal of SP2 from p7^NC is not requiredfor efficient HIV-1 replication. In contrast, processing atthe SP2-p6^Gag cleavage site is required for efficient HIV-1replication. These latter results are consistent with a replicationdefect observed by Yu et al. with a different SP2-p6^Gag cleavagemutant (54). Our real-time PCR data revealed that the primarydefect caused by blocking SP2-p6^Gag processing was at or justbefore the integration step. It is unclear how blocking theSP2-p6^Gag site could affect integration. The loss of free SP2does not appear to play a direct role, as our p9^NC/SP2 mutantswere infectious despite containing no detectable SP2. The Polpolyproteins appear to be incorporated into virions at near-wild-typelevels (Fig. 2; immunoblotting results for protease are notshown), and so it is unlikely that there is a simple IN-relatedexplanation. Therefore, the production of either p6^Gag or p9^NC-SP2,both of which are not produced by the SP2-p6^Gag or p15 mutants,could be required for integration. The p6^Gag protein has notbeen implicated in any postentry/infection events and is locatedoutside the mature core in virions; thus, it is unlikely tointeract with the RNA or integrase (50). In contrast, the NCprotein has already been implicated in promoting efficient integration(5, 16, 17, 46). Thus, it is possible that a block in the productionof p9^NC-SP2 is responsible for the defect in integration. Similarto the results presented here, our previous results from geneticstudies support some role for NC at or just before the integrationstep: HIV-1 mutants with CCHC NC zinc finger substitutions toother nonretroviral motifs, CCCC and CCHH, maintain genomicRNA incorporation and exhibit only somewhat reduced levels ofreverse transcription, yet have a severe block in integration(5, 16, 17, 46).

How NC, potentially as a p9^NC/SP2 species, could be requiredfor integration is not clear. NC could be acting as a somewhatlabile component of the integration complex or as a protectivefactor for the cDNA ends, presumably by binding to them (5,17, 46). In vitro, p7^NC and especially p9^NC/SP2 have been foundto increase the efficiency of concerted integration (7, 14,38). Interestingly, p9^NC/SP2 can condense double-stranded DNAinto dense protein/DNA complexes much more efficiently thaneither p7^NC or p15^NC/SP2/p6 (32), suggesting that p9^NC/SP2 mightspecifically associate with the viral cDNA. Alternatively, p9^NC/SP2could play a role in nuclear localization of the preintegrationcomplex, although our experiments were conducted on dividingcell cultures. Despite all of these indirect supporting data,neither NC nor p9^NC/SP2, also called NCp7 or NC[1-72] (11),is routinely observed in isolated preintegration complexes (30).Detectable amounts of p9^NC/SP2 do not appear to be present inHIV-1 cores (50): nearly all of the NC appears to be p7^NC witha very minute amount of p15^NC/SP2/p6. Also, in our years ofbiochemical analyses of wild-type HIV-1 virions, we have notfound much evidence for p9^NC/SP2 in virions (21) (data not shown).However, it is possible that only a small amount, two to fourmolecules per virion bound to the cDNA ends, might be present,levels below the sensitivity of our past and current proteinanalyses.

Alternatively, a p9^NC/SP2 species could act transiently at theassembly/maturation step by assisting the correct formationof the mature virus, perhaps placing IN in the correct positionwithin the viral core. Recent reports of in vitro experimentshave implicated p9^NC/SP2 in the maturation process (31, 32).Theoretically, p9^NC/SP2 would not have to be present in thecore to assist the integration process in this way.

From our data and other processing site mutation studies (19,26, 42, 51), it appears that the only Gag proteolyic cleavagesite that is not strictly required for replication in vitrois the NC-SP2 junction. Unlike NC, the proteins in the N-terminalregion of Gag play mostly structural roles. Therefore, havingadditional sequences attached to them due to incomplete processinglikely causes defects in their fairly constrained protein-proteininteractions. However, NC primarily functions in RNA bindingand chaperone activity (4, 10, 28), roles that apparently cantolerate additional sequences attached to p7^NC.

One question that remains unanswered is the following: why isp7^NC produced if p9^NC/SP2 is sufficient for replication? Itis important to consider that in cell culture with limited roundsof replication, we may not detect minor replication inefficienciesthat would apply a selective pressure for the maintenance ofthis site over the many cycles of replication that occur inan infected individual (9). Indeed, sequential passaging ofthe NC-N55D and NC-SP2 mutants results in a restoration of theNC-SP2 site within four passages (4 weeks) (data not shown),supporting this hypothesis. Furthermore, some protease inhibitor-resistantviruses isolated from patients have sequence changes at boththe NC-SP2 and the SP2-p6^Gag sites that appear to restore normallevels of processing by the mutant protease (6, 12, 29, 55).Thus, processing at both sites appears to be important for long-termviral fitness in vivo.

Overall, our findings are consistent with a role for NC in promotingthe integration process, possibly as a p9^NC/SP2 species. Thepotential involvement of NC in integration suggests furtherstudies to clarify its role in this process, e.g., either adirect or indirect interaction of p9^NC/SP2 with the preintegrationcomplex or its providing an organizing function during the maturationphase of assembly.

ACKNOWLEDGMENTS

We thank Laurie Queen and Teresa Shatzer for technical assistanceand Lou Henderson for the inspiration to examine this topic.

This project has been funded in whole or in part with federalfunds from the National Cancer Institute, National Institutesof Health, under contract no. NO1-CO-12400.

The content of this publication does not necessarily reflectthe views or policies of the Department of Health and HumanServices, nor does mention of trade names, commercial products,or organization imply endorsement by the U.S. Government.

FOOTNOTES

* Corresponding author. Mailing address: AIDS Vaccine Program, SAIC-Frederick, Inc., National Cancer Institute at Frederick, Frederick, MD 21702-1201. Phone: (301) 846-5723. Fax: (301) 846-5588. E-mail: ott{at}ncifcrf.gov

Published ahead of print on 18 July 2007.

Supplemental material for this article may be found at http://jvi.asm.org/.

REFERENCES

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