Comprehensive Investigation of the Molecular Defect in vif-Deficient Human Immunodeficiency Virus Type 1 Virions

Replication of human immunodeficiency virus type 1 (HIV-1) inprimary blood lymphocytes, certain T-cell lines (nonpermissivecells), and most likely in vivo is highly dependent on the virallyencoded Vif protein. Evidence suggests that Vif acts late inthe viral life cycle during assembly, budding, and/or maturationto counteract the antiviral activity of the CEM15 protein andpossibly other antiviral factors. Because HIV-1 virions producedin the absence of Vif are severely restricted at a postentry,preintegration step of infection, it is presumed that such virionsdiffer from wild-type virions in some way. In the present study,we established a protocol for producing large quantities ofvif-deficient HIV-1 (HIV-1/ ${Delta}$ vif) from an acute infection of nonpermissiveT cells and performed a thorough examination of the defect inthese virions. Aside from the expected lack of Vif, we observedno apparent abnormalities in the packaging, modification, processing,or function of proteins in ${Delta}$ vif virions. In addition, we foundno consistent defect in the ability of ${Delta}$ vif virions to performintravirion reverse transcription under a variety of assay conditions,suggesting that the reverse transcription complexes in theseparticles can behave normally under cell-free conditions. Consistentwith this finding, neither the placement of the primer tRNA3Lysnor its ability to promote reverse transcription in an in vitroassay was affected by a lack of Vif. Based on the inabilityof this comprehensive analysis to uncover molecular defectsin ${Delta}$ vif virions, we speculate that such defects are likely tobe subtle and/or rare.

INTRODUCTION

Top

Introduction

Human immunodeficiency virus type 1 (HIV-1), like other lentiviruses,has a number of regulatory genes in addition to the gag, pol,and env genes that are common to all replication-competent retroviruses.The vif (for viral infectivity factor) regulatory gene of HIV-1,which is conserved in all known lentiviruses except equine infectiousanemia virus, encodes a protein that acts as a positive modulatorof viral infectivity in certain cellular environments. In particular,Vif is required for efficient replication of HIV-1 in primaryT cells and in some T-cell lines (nonpermissive cells), suchas CEM, H9, and HUT78, where it stimulates single-round infectivityon the order of 10- to 100-fold (6, 9, 19, 20, 22, 24, 53, 55,60, 63, 65, 67). However, Vif is dispensable for replicationin other T-cell lines (permissive cells), such as CEM-SS (6,19, 20, 24, 53, 55, 60, 63, 65, 67). In vivo, Vif is essentialfor pathogenic infections of simian immunodeficiency virus (SIV)in rhesus macaques, caprine arthritis encephalitis virus ingoats, and feline immunodeficiency virus in cats (16, 28, 29,34, 35, 43). Taken together, these observations suggest thatvif plays a vital role in HIV-1 replication and pathogenesisin vivo.

The exact nature of Vif function is not well established. Vifis expressed late in the viral life cycle, and in situ immunofluorescencestudies have indicated that it largely colocalizes with Gagin HIV-1-infected cells (58). In addition, subcellular fractionationexperiments have demonstrated that Vif and Gag are targetedto similar membrane-free, cytoplasmic complexes, hypothesizedto represent HIV-1 assembly intermediates (57). Whether Vifinteracts directly with Gag in these complexes is controversial(8, 31, 33, 54, 57, 68, 69). In contrast, there is compellingevidence that interactions with the host cell are essentialfor Vif function. First, Vif activity is influenced by the speciesderivation of the virus-producing cell, as evidenced by thegreater potency of HIV Vif proteins in human cells relativeto many SIV Vif proteins (62; N. C. Gaddis et al., unpublishedresults) and the noted replicative enhancement of murine leukemiavirus (a simple retrovirus that does not encode a Vif protein)in the presence of Vif (54, 62). Second, heterokaryons betweenpermissive and nonpermissive cells display the nonpermissivephenotype, suggesting that nonpermissive cells naturally expressan antiviral activity that is overcome by Vif (45, 59). Recently,we have used gene transfer experiments to show that the CEM15protein (also termed APOBEC3G [37]) is at least partially responsiblefor this antiviral activity (55); whether Vif interacts directlywith this protein is unknown. Based on this evidence, an appealingmodel is that Vif acts at the site of virus assembly, budding,and/or maturation to counteract a host antiviral mechanism andthereby impart infectivity upon progeny virions.

Virions produced in nonpermissive cells in the absence of thevif gene are impaired at a postentry step of infection and thereforefail to establish proviruses. Studies have suggested that eitherreverse transcription fails to proceed to completion or thatreverse transcripts are unstable, possibly depending on theidentity of the target cell (14, 15, 18, 46, 60, 67). Correspondingly,several groups have found ${Delta}$ vif virions to be partially defectivein endogenous reverse transcriptase (RT) assays (17, 18, 27,46, 48). Whether these findings are indicative of a specificproblem with the reverse transcription complex, structural abnormalitiesin the viral core (6, 9, 32, 48), or some other molecular defectin virions is unknown. To date, no reproducible quantitativeor qualitative difference in the protein content of wild-typeand ${Delta}$ vif virions has been reported, aside from a difference inVif incorporation (6, 11, 22, 38, 39, 42, 61) and a controversialdifference in Gag processing (6, 56). Recent work has suggested,however, that primer -genomic RNA complexes from ${Delta}$ vif virions may be unable to prime efficient reverse transcription(17).

Because HIV-1 does not replicate in nonpermissive cells in theabsence of Vif, a major obstacle for the detailed analysis of ${Delta}$ vif virions has been obtaining sufficient quantities of virus.Here, we developed a method for producing significant quantitiesof HIV-1/ ${Delta}$ vif from acute infection of nonpermissive T cells.With the exception of the expected lack of Vif, analysis ofthese ${Delta}$ vif virions revealed no noticeable difference in the quantity,processing, or posttranslational modification of any viral proteins.Additionally, the performance of ${Delta}$ vif virions in the endogenousRT reaction was not consistently diminished compared to wild-typevirions. Finally, the primer appeared to be correctly placed on purified genomes of ${Delta}$ vif virions and wasable to promote efficient initiation of reverse transcriptionin an in vitro assay. These results suggest either that thelack of Vif in ${Delta}$ vif virions is responsible for their reducedinfectivity or, in our view more likely, that the relevant defectis not easily detectable in the absence of additional insightsinto the function(s) of Vif in virus-producing cells.

MATERIALS AND METHODS

Top

Materials and Methods

Molecular clones. The HIV-1 proviral expression vectors pIIIB, pIIIB/ ${Delta}$ vif, pIIIB/ ${Delta}$ vif ${Delta}$ env,pYU-2, and pYU-2/ ${Delta}$ vif, as well as the vesicular stomatitis virus(VSV) G glycoprotein expression vector pHIT/G have been describedpreviously (21, 26, 55, 59, 63). The CCR5-expressing retroviralvector pMIGR1-P/CCR5 is a derivative of the MSCV-based pMIGR1vector (51) in which the gfp gene of pMIGR1 was replaced bythe gene for puromycin resistance from LP-M and the ccr5 genewas inserted upstream of the internal ribosome entry site.

Cells and cell lines. The human cell lines HUT78, CEM-SS-CCR5, C8166-CCR5/HIV-CAT,and 293T were maintained as previously described (21, 63). Standardretrovirus-mediated transduction of HUT78 cells with pMIGR1-P/CCR5was used to establish the HUT78-CCR5 cell line, which was thenmaintained in complete medium supplemented with 40 ng of puromycinper ml. Peripheral blood mononuclear cells (PBMCs) from healthyadult donors were purified, activated, and maintained by standardprocedures (60).

Production, purification, and quantification of viruses. Viral stocks for the analysis of HIV-1_YU-2 and HIV-1_YU-2/ ${Delta}$ vifreplication were generated by transient transfection of 293Tcells, as described previously (21). The virus was quantifiedby an enzyme-linked immunosorbent assay (ELISA) for solublep24^Gag concentration (NEN).

In order to generate high-titer virus from acutely infectedT cells for investigation of the defect in ${Delta}$ vif virions, 293Tcells were first transiently cotransfected with pHIT/G and pYU-2,pYU-2/ ${Delta}$ vif, pIIIB, pIIIB/ ${Delta}$ vif, or pIIIB/ ${Delta}$ vif ${Delta}$ env to generate pseudotypedviral stocks, as described previously (57). In experiments involvingkinetic PCR, the pseudotyped stocks were then treated with 1mg of DNase (Roche Molecular Biochemicals) per ml in the presenceof 10 mM MgCl₂ for 30 min at 37°C to minimize plasmid contaminationin the PCRs. HUT78 cells were challenged with the pseudotypedviruses by spin infection at 1,200 x g and 25°C for 2.5h. After infection, the input virus was removed by extensivewashing with phosphate-buffered saline (PBS), and the T cellswere incubated in fresh media for 24 to 36 h to allow viralproduction to reach a high level. The cells were then washedagain with PBS and placed in the appropriate medium for viralproduction. After production, the virus-containing supernatantswere centrifuged for 5 min at 500 x g and filtered through 0.45-µm-pore-sizefilters. The infectivity of the virions was determined by infectionof C8166-CCR5/HIV-CAT reporter cells as described previously(21, 60).

For some experiments, virions produced by the HUT78 cells werepurified by layering the viral supernatants onto a 20% (wt/vol)sucrose (in PBS) cushion and centrifuging them at 100,000 xg for 1.5 h at 4°C in an SW28 rotor (Beckman Instruments,Inc.). In some cases, virions were further purified by using20 to 60% (wt/vol) continuous sucrose gradients (in PBS) (22).

Analysis of replication. PBMC, HUT78-CCR5 cells, and CEM-SS-CCR5 cells were infectedwith wild-type or ${Delta}$ vif HIV-1_YU-2 stocks containing ca. 40, 5,and 10 ng of p24^Gag per ml, respectively. Cells were maintainedat ca. 2 x 10⁶ cells per ml for the PBMC infections or 1 x 10⁶cells per ml for HUT78-CCR5 and CEM-SS-CCR5 infections. At 6-dayintervals, freshly activated cells were added to the PBMC infections.Viral replication was monitored by measuring the soluble p24^Gagconcentration in the supernatants of the challenged cells.

Antibodies and Western analysis. Viral proteins from sucrose gradient-purified virions were resolvedby standard reducing sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) and transferred electrophoreticallyto nitrocellulose. Individual proteins were detected by initialincubation with the murine monoclonal antibodies 319 (Vif),24-2 (capsid [CA]), 17-3 (matrix [MA]), 4D1 (nucleocapsid [NC]),IN-2 (integrase [IN]), and MAb21 (reverse transcriptase [RT];obtained from the National Institutes of Health [NIH] AIDS Researchand Reference Reagent Program); the rabbit polyclonal antibodiesR1169 (gp120 [a generous gift of Robert Doms]) and ${alpha}$ -cyclophilinA ( ${alpha}$ -CypA); and the rat polyclonal antibody UP39 (Vpr). The membraneswere then hybridized with appropriate horseradish peroxidase-conjugatedmouse, rabbit, and rat secondary antibodies. Proteins were visualizedby enhanced chemiluminescence and autoradiography.

Two-dimensional gel analysis. For experiments involving radiolabeling of virions, HUT78 cellsinfected with wild type or ${Delta}$ vif HIV-1_YU-2 were washed severaltimes with PBS 30 to 36 h after infection and resuspended atca. 10⁶ cells per ml in labeling medium (RPMI 1640 medium lackingcysteine and methionine but containing 10% dialyzed fetal bovineserum, 1% L-glutamine, 1% penicillin-streptomycin, and 1 mCiof ³⁵S-Express [NEN] per sample). Silver stain samples weretreated similarly except that they were resuspended in regularcomplete medium instead of labeling medium. After 12 h of virusproduction, virions were purified by sucrose gradient centrifugationand lysed in isofocusing sample buffer (8 M urea, 1.7% [vol/vol]NP-40, 1.7% [vol/vol] 3/10 ampholytes [Bio-Rad], 1 mM sodiumorthovanadate [Sigma], 1 µM microcystin LR [Sigma], andComplete protease inhibitor cocktail [Roche Molecular Biochemicals]).Prior to electrophoresis, 10% (vol/vol) ß-mercaptoethanolwas added to each sample. Viral proteins were separated in twodimensions, first by nonequilibrium pH gradient electrophoresis(NEPHGE; pH 3.0 to 10) and then by standard SDS-PAGE under reducingconditions. The resolved proteins were transferred to a nitrocellulosemembrane by electroelution and visualized either by autoradiographyusing a Biomax Transcreen LE intensifying screen (Kodak) forradiolabeled samples or by silver staining for unlabeled samples.

Reversed-phase (RP)-HPLC. Virions generated in HUT78 cells were pelleted through sucrosecushions, disrupted in 8 M guanidine-HCl (Pierce) with or without50 mM dithiothreitol (DTT; Calbiochem) and fractionated by high-performanceliquid chromatography (HPLC) to isolate viral proteins. HPLCwas performed at a flow rate of 300 µl per min on a 2.1-by-100-mmPoros R2/H narrow-bore column (Boehringer Mannheim GmbH) withaqueous acetonitrile-trifluoroacetic acid solvents and a ShimadzuHPLC system equipped with LC-10AD pumps, SCL-10A system controller,CTO-10AC oven, FRC-10A fraction collector, and SPD-M10AV diodearray detector. The gradient of buffer B (0.1% trifluoraceticacid in acetonitrile) was as follows: 10 to 36.5% for 12 min,36.5 to 37% for 4 min, 37 to 41% for 7 min, 41 to 70% for 12min, and 70% for 5 min. A temperature of 55°C was maintainedduring HPLC separation. Peaks were detected by UV absorptionat 206 and 280 nm and then analyzed by sequencing with an automatedApplied Biosystems, Inc., 477 Protein Sequencer, by SDS-PAGEwith Coomassie or silver staining, and by immunoblot analysiswith enhanced chemiluminescence.

In vitro kinase assay. Virions produced in HUT78 cells were purified by sucrose gradientcentrifugation and resuspended in kinase buffer (50 mM HEPES[pH 7.5], 0.5% NP-40, 5 mM MnCl₂, 0.2 mM sodium orthovanadate[Sigma], 1 µM microcystin LR [Sigma], and Complete proteaseinhibitor cocktail without EDTA [Roche Molecular Biochemicals]).Kinase reactions were performed at 20°C for 30 min in 50µl of kinase buffer containing virus corresponding to50 ng of p24^Gag and 50 µCi of [ ${gamma}$ -³²P]ATP. The reactionswere halted by adding gel loading buffer, followed by heatingfor 5 min at 95°C. Phosphorylated proteins were resolvedby reducing SDS-PAGE, transferred to nitrocellulose, and visualizedby autoradiography with a Biomax Transcreen HE intensifyingscreen (Kodak).

Pulse-chase analysis. Approximately 30 h after infection of HUT78 cells with HIV-1_YU-2or HIV-1_YU-2/ ${Delta}$ vif, the cells were washed several times with PBSand incubated in medium without cysteine and methionine for30 min at 37°C. The cells were then pelleted, resuspendedin labeling medium (depletion medium supplemented with 0.4 mCiof ³⁵S-Express [NEN])/ml, and placed at 37°C for 15 min.After labeling, the samples were washed once in chase medium(complete RPMI 1640 medium containing 20 mM methionine and 20mM cysteine), pelleted, resuspended in complete medium, andplaced at 37°C. At 0, 1.5, 3, 6, 12, and 24 h after theaddition of chase medium, one-sixth of each sample was removed,the cells were pelleted, and the viral supernatants were filteredthrough 0.45-µm-pore-size filters. Both the cells andthe virions were then lysed in radioimmunoprecipitation assay(RIPA) buffer (final concentration: 0.1% SDS, 1% Triton X-100,1% sodium deoxycholate, 150 mM NaCl, 10 mM Tris-HCl [pH 7.5],1 mM EDTA), and p24^Gag (CA) and its precursors were immunoprecipitatedwith murine monoclonal antibody 24-4. The immunoprecipitatedproteins were separated under reducing conditions by SDS-PAGE,transferred to nitrocellulose, and visualized by autoradiographyusing a Biomax Transcreen LE intensifying screen (Kodak).

Kinetic PCR. Strong-stop and second-strand-transfer reverse transcriptionproducts were detected by kinetic (fluorescence-monitored) PCRon an ABI 7700 kinetic PCR instrument (Applied Biosystems) byusing molecular beacon technology (66). The forward primer forboth the strong-stop and second-strand-transfer reactions was5'-GCTAGCTAGGAAACCCACTGCTTA-3'. The reverse primers were 5'-GCTAGAGATTTTCCACACTGACT-3'for the strong-stop reaction and 5'-CTGCGTCGAGAGAGCTCCTCTGGTT-3'for the second-strand-transfer reaction. The molecular beaconfor both the strong-stop and second-strand-transfer reactionswas 5'-GCGAGTCACACAACAGACGGGCACACACTACTCGC-3', labeled at the5' end with the reporter fluorochrome 6-carboxyfluorescein andat the 3' end with the quencher DABCYL (4-[4'-dimethylamino-phenylazo]-benzene)(Midland Certified Reagent Company). The kinetic PCRs were performedin a 50-µl volume containing 5 µl of 10x BufferII (Applied Biosytems); 5 mM MgCl₂; 250 µM (each) dATP,dCTP, dGTP, and dTTP (Promega); 500 nM concentrations of eachprimer; a 200 nM concentration of molecular beacon; and 1.25U AmpliTaq Gold (Applied Biosystems). The cycling conditionswere 2 min at 50°C, 10 min at 95°C, and 40 cycles of15 s at 95°C and 1 min at 60°C.

Exogenous and endogenous RT assays. Exogenous RT assays were performed as previously described (62).For the endogenous RT reaction, virions produced in HUT78 cellswere pelleted in triplicate at 100,000 x g for 2 h at 4°Cin a Beckman SW28 rotor. The viral pellets were resuspendedon ice at 200 ng of p24^Gag per ml in endogenous RT buffer lackingsalt (50 mM Tris-HCl [pH 8]; 5 µg of melittin [Sigma]/ml;2 mM DTT; 2 mM magnesium acetate; 0.1 mM [each] dATP, dCTP,dGTP, and dTTP [Promega]); these pH and melittin concentrationoptima were each determined empirically (data not shown). Aliquotsof the resuspended pellets were then mixed with equal volumesof endogenous RT buffer containing various concentrations ofNaCl in duplicate. The RT reactions were incubated for 6 to8 h at 37°C and stopped by the addition of an equal volumeof 2x PCR lysis buffer (20 mM Tris-HCl [pH 8], 2 mM EDTA, 0.002%SDS, 0.002% Triton X-100). The RT products were quantified bymeans of kinetic PCR against strong-stop and second-strand-transfercDNAs. The majority of virus particles assayed by this methodwere inferred as being intact since reactions carried out inthe absence of melittin resulted in levels of cDNA synthesisthat were $~$ 10-fold lower than those seen with 5 µg of melittin/ml(at 150 mM NaCl [data not shown]).

Isolation and quantification of viral genomic RNA. Sucrose gradient-purified virions from HUT78 cells were lysedin buffer containing 10 mM Tris-HCl (pH 8), 100 mM NaCl, 1 mMEDTA, and 0.5% SDS. Genomic RNA was then isolated by performingthree phenol-chloroform extractions, followed by ethanol precipitationin the presence of 20 µg of glycogen (Roche MolecularBiochemicals) per ml as a carrier. The integrity of the genomicRNA was confirmed by Northern blotting, as described previously(55), and the level of RNA was normalized between samples bymeans of a primer extension assay. The primer 5'-GCTAGAGATTTTCCACACTGACT-3',which binds HIV-1 genomic RNA within the U5 region of the longterminal repeat, was added to the RNA samples at 1 ng per µland extended for 30 min at 37°C in RT reaction buffer (50mM Tris-HCl [pH 8]; 60 mM KCl; 3 mM MgCl₂; 10 mM DTT; 100 µM[each] dATP, dCTP, dGTP, and dTTP [Promega]; 1 U of RNasin [Promega]/µl;2.5 ng of purified HIV-1 RT [obtained from Stephen Hughes]/µl).The reactions were then heated for 20 min at 95°C to haltextension, and the primer extension products were quantifiedby means of kinetic PCR by using the strong-stop primer set.

tRNA placement assay. Analysis of the placement of the primer on the HIV-1 genomic RNA was done in a similar fashion to thatdescribed previously (3). Briefly, the primer 5'-CCCCGCACTTAATACTGACGC-3',which binds "upstream" of the primer-binding site on the HIV-1 genome, was 5' end labeled with ³²P. The labeledprimer was then mixed with the RNA samples at 1 ng per µlin RT buffer and extended for 30 min at 37°C. The cDNA productsof the primer extension were ethanol precipitated, resolvedon a denaturing 6% polyacrylamide-urea gel, and visualized byautoradiography.

tRNA extension assay. To examine the ability of the native primer to prime reverse transcription, purified HIV-1 genomic RNA-primercomplexes were incubated for 30 min at 37°C in RT buffer containing various concentrations of deoxynucleosidetriphosphates (dNTPs). Reactions were stopped by heating themfor 20 min at 95°C, and the quantity of cDNA product formedwas measured by kinetic PCR by using the strong-stop primers.

RESULTS

Top

Results

Strategy for production of ${Delta}$ vif virions. It has previously been shown that the infectivity of ${Delta}$ vif HIV-1produced in nonpermissive cells is severely reduced comparedto wild type, whereas for virions produced in permissive cells,the infectivity of wild type and ${Delta}$ vif HIV-1 is similar (6, 9,22, 24, 45, 53, 60). Therefore, when trying to determine themolecular basis for the infectivity phenotype observed in nonpermissivecells, differences between wild-type and ${Delta}$ vif virions that areseen in both nonpermissive and permissive cells are unlikelyto dictate infectivity. In this investigation, we focused onuncovering defects particular to ${Delta}$ vif virions produced in nonpermissivecells.

Because HIV-1/ ${Delta}$ vif does not replicate in nonpermissive cells,we optimized conditions to achieve a maximal output of virionsfrom an acute infection. The strategy we developed is illustratedin Fig. 1A. Briefly, wild type or ${Delta}$ vif HIV-1_YU-2 (an R5-tropicisolate) pseudotyped with the G protein of VSV was generatedby transient cotransfection of 293T (permissive) cells. Thesepseudotyped viruses then were used to spin infect HUT78 (nonpermissive)cells. After challenge, the input virus was removed by vigorouswashing of the infected T cells to prevent residual viral particlesfrom contaminating later analyses. A second round of washingwas also done closer to the time of analysis to ensure thatthe majority of virions being studied were recently assembled.Viral supernatants from the T cells were harvested for characterizationca. 36 to 48 h after infection, at which time production of ${Delta}$ vif viruses by nonpermissive cells was optimal.

View larger version (24K):
[in this window]
[in a new window]
FIG. 1. Optimization of viral production from acute infection of T cells. (A) Strategy for high-level production of HIV-1/ ${Delta}$ vif from acute infection of nonpermissive cells. See text for details. (B) Analysis of the benefits of VSV-G pseudotyping, spin infection, and use of HIV-1_YU-2 for optimal viral output after acute infection. Pseudotyped or nonpseudotyped preparations of the indicated viruses were generated by transient transfection of 293T cells and used to infect nonpermissive HUT78 cells, either by spin infection or passive diffusion. The infected cells were then washed extensively to remove residual input particles and incubated for $~$ 30 h in growth medium to allow viral production to reach a high level. The cells were then washed again and placed in fresh growth medium for $~$ 10 h. The quantity of soluble p24^Gag produced during this period was measured by ELISA.

As depicted in Fig. 1B, use of HIV-1_YU-2 instead of the X4-tropicisolate HIV-1_IIIB in this procedure resulted in a 15- to 35-foldenhancement in viral production. We suspect that much of thissubstantial improvement in yield is due to the lack of functionalCCR5 expression in HUT78 cells (41), which averts virus-celland cell-cell fusion, virus spread, and virus-induced (Env-associated)cytopathicity for the R5-tropic HIV-1_YU-2. This hypothesis issupported by the finding that deletion of the env gene of HIV-1_IIIBalso results in a significant (although not quite as great)increase in viral output (Fig. 1B). Consistent with the dependenceof HIV-1_YU-2 replication on Vif expression in nonpermissiveCCR5-expressing cells (Fig. 2), challenges of the T-cell-indicatorcell line C8166-CCR5/HIV-CAT showed that wild-type HIV-1_YU-2generated by using our protocol and HUT78 cells was generally $~$ 40-fold more infectious than ${Delta}$ vif HIV-1_YU-2 (data not shown),a finding in line with what has been observed for other HIV-1isolates (6, 9, 22, 24, 45, 53, 60). Because the initial pseudotyped ${Delta}$ vif viruses from 293T cells are as infectious as Vif-expressingviruses, the dramatic differential in infectivity of the finalvirus stocks illustrates that little, if any, of the initialinocula is carried forward into the final virus preparations.Other features of our procedure that were important for achievingoptimal yield were pseudotyping with the G protein of VSV andinoculation by spin infection rather than by passive diffusion(Fig. 1B). The ability to produce large quantities of infection-deficient ${Delta}$ vif virions enabled us to perform a number of studies that hadbeen previously problematic.

View larger version (17K):
[in this window]
[in a new window]
FIG. 2. Vif is required for replication of HIV-1_YU-2 in PBMC and HUT78-CCR5 T cells. PBMC (A), HUT78-CCR5 cells (B), or CEM-SS-CCR5 cells (C) were infected with 293T-derived stocks of HIV-1_YU-2 or HIV-1_YU-2/ ${Delta}$ vif at 40, 5, or 10 ng of p24^Gag per ml, respectively. The PBMC infections were maintained at $~$ 2 x 10⁶ cells/ml, and freshly stimulated PBMC were added every 6 days. The HUT78-CCR5 and CEM-SS-CCR5 infections were maintained at $~$ 10⁶ cells/ml. Replication was monitored by measuring the concentration of soluble p24^Gag in the supernatants of the infected cells by ELISA.

Protein composition of virions. Having optimized conditions for production of ${Delta}$ vif virions, wefirst wished to analyze whether the absence of vif results ina quantitative or qualitative change in the virion protein profile.Sucrose gradient-purified virions produced in nonpermissiveHUT78 cells were lysed, separated by standard reducing SDS-PAGE,transferred to nitrocellulose, and probed with antibodies tovarious viral and nonviral proteins (Fig. 3). As seen before,there were no obvious differences between wild-type and ${Delta}$ vifvirions in p55^Gag, CA, MA, RT, IN, or gp120 (1, 9, 17, 22, 39,44, 47, 67). In addition, the HIV-1-encoded NC and Vpr proteinsand the cellular protein CypA were found to be present in appropriatequantities in ${Delta}$ vif virions. Furthermore, hybridization of themembranes with antibodies against phosphorylated serine, threonine,and tyrosine revealed no vif-related abnormality in phosphorylationof viral proteins (data not shown). As observed previously,the only protein difference detectable in our Western analysiswas the Vif protein, which was present in wild-type but not ${Delta}$ vif virions (Fig. 3). Importantly, treatment of virions withsubtilisin, a protease that nonspecifically degrades externalviral proteins and protein contaminants (50), did not eliminatethe Vif signal in wild-type virus preparations, suggesting thatVif is in fact associated with virion cores (data not shown)(11, 39, 42). Of note, contrary to some reports (6, 56) butconsistent with others (9, 17, 22, 39, 44, 47), there was noapparent vif-related defect in the processing of the p55^Gagpolyprotein. In particular, the sizes and quantities of thep55^Gag-derived proteins MA, CA, and NC were normal in ${Delta}$ vif virions.

View larger version (33K):
[in this window]
[in a new window]
FIG. 3. Western blot analysis of wild-type and ${Delta}$ vif HIV-1 virions. Virions produced by HUT78 cells infected with HIV-1_YU-2 or HIV-1_YU-2/ ${Delta}$ vif were purified through continuous sucrose gradients, lysed, separated by SDS-PAGE, and transferred to nitrocellulose. The filters were then incubated with antibodies against the indicated proteins, followed by appropriate horseradish peroxidase-conjugated secondary antibodies. The proteins were visualized by enhanced chemiluminescence and autoradiography. The quantities of virus loaded in each lane corresponded to the indicated proteins as follows: 10 ng of p24^Gag for CA; 50 ng of p24^Gag for MA, NC, and IN; 100 ng of p24^Gag for RT, gp120, and Vpr; and 200 ng of p24^Gag for Vif and CypA.

To facilitate visualization of a broader spectrum of proteins,we next labeled virions metabolically. In addition, the sampleswere resolved according to both isoelectric point (by NEPHGE)and molecular mass (by SDS-PAGE) in hopes of achieving betterresolution and revealing differences in protein modifications.Comparison of the protein profiles of sucrose gradient-purifiedwild-type and ${Delta}$ vif virions prepared in this manner revealed themto be strikingly similar (Fig. 4A). No reproducible differencesin quantities or modifications of proteins were apparent, evenwith longer exposure times, with different pH ranges duringNEPHGE or with different concentrations of acrylamide duringSDS-PAGE (data not shown). Because metabolic labeling of proteinsdoes not allow efficient detection of proteins with long half-lives,unlabeled virions were also subjected to two-dimensional electrophoresisand silver staining (Fig. 4B). Again, no differences betweenthe proteins present in wild-type and ${Delta}$ vif virions were exposed.

View larger version (61K):
[in this window]
[in a new window]
FIG. 4. Two-dimensional gel analysis of metabolically labeled (A) or silver-stained (B) wild-type and ${Delta}$ vif HIV-1 virions. Approximately 30 to 36 h following infection of HUT78 cells with HIV-1_YU-2 or HIV-1_YU-2/ ${Delta}$ vif, the cells were metabolically labeled with [³⁵S]methionine and [³⁵S]cysteine (A) or incubated in fresh medium (B) for 12 h. Virions produced during this period were sucrose-gradient purified, lysed, and separated in two dimensions, first by NEPHGE and then by SDS-PAGE. The viral proteins were then transferred to nitrocellulose and visualized by autoradiography (A) or silver staining (B). The identity of the some spots was determined by Western blotting as described for Fig. 3.

As a further examination of the viral protein profile of ${Delta}$ vifvirions, wild-type and ${Delta}$ vif virions produced in HUT78 cells werepelleted through a sucrose cushion, disrupted, and subjectedto RP-HPLC analysis. As with the two-dimensional gel electrophoresisanalysis, the HPLC chromatograms of wild-type and ${Delta}$ vif virionswere nearly identical (Fig. 5). Minor differences did exist,for example in the CA-containing peaks. However, treatment ofvirions with subtilisin prior to RP-HPLC effectively eliminatedthese disparities (data not shown), suggesting that they weremainly attributable to contaminating serum proteins (from thegrowth medium) or cellular proteins on the exterior of virions.Further analysis of the RP-HPLC fractions by SDS-PAGE, followedby Coomassie blue staining, also revealed no major defects inthe ${Delta}$ vif virions (Fig. 5). Importantly, this RP-HPLC analysisprovided further evidence that the processing of p55^Gag is normalin ${Delta}$ vif virions, since the ratios of the p55^Gag derivatives MA,CA, NC, p6, and p1 were similar in wild-type and ${Delta}$ vif virions.In addition, side-by-side comparison of the fractions containingp6, MA, and CA on SDS-PAGE gels demonstrated that these proteinsare appropriately sized in ${Delta}$ vif virions.

View larger version (31K):
[in this window]
[in a new window]
FIG. 5. RP-HPLC examination of wild-type and ${Delta}$ vif HIV-1 virions. HIV-1_YU-2 and HIV-1_YU-2/ ${Delta}$ vif produced in HUT8 cells were pelleted through sucrose cushions, disrupted, and separated by RP-HPLC. The identity of the peaks was determined by peptide sequencing and immunoblot analysis. The protein content of each fraction was visualized by SDS-PAGE and Coomassie blue staining. Adjacent lanes on the gels show matched fractions for wild-type (green) and ${Delta}$ vif (red) viruses.

Processing of viral proteins. To address the issue of p55^Gag processing more precisely, weused pulse-chase labeling of infected cells, followed by immunoprecipitationwith a CA-specific monoclonal antibody to analyze the abilityof PR to cleave the Gag polyprotein (Fig. 6). Comparison ofthe fate of the labeled p55^Gag molecules clearly showed thatthe rate of processing of p55^Gag to CA in both the producercells (upper panels) and virions (lower panels) is unaffectedby the presence or absence of Vif. In addition, the kineticsof p55^Gag release from infected cells was very similar for wild-typeand ${Delta}$ vif viruses, suggesting that budding is normal for the ${Delta}$ vifmutant.

View larger version (43K):
[in this window]
[in a new window]
FIG. 6. Pulse-chase analysis of Gag processing. Approximately 30 h after infection of HUT78 with HIV-1_YU-2 or HIV-1_YU-2/ ${Delta}$ vif, the infected cells were washed several times with PBS and incubated in growth medium lacking cysteine and methionine for 30 min at 37°C. The cells were then labeled in growth medium containing [³⁵S]cysteine and [³⁵S]methionine for 15 min at 37°C, washed once with chase medium containing excess cold cysteine and methionine, and placed in regular growth medium. At 0, 1.5, 3, 6, 12, and 24 h after the chase, one-sixth of each culture was removed, the cells were pelleted, and both the cells and the viral supernatants were lysed in RIPA buffer. The lysates were incubated overnight in the presence of a CA-specific monoclonal antibody, and the antibody-protein complexes were captured with agarose beads conjugated to protein G. The beads were washed with RIPA buffer, and the bound proteins were solubilized in gel loading buffer, resolved by SDS-PAGE, transferred to nitrocellulose, and visualized by autoradiography. The $~$ 41-kDa band represents a Gag processing intermediate.

Virion-associated kinases. A number of kinases, including ERK2 mitogen-activated proteinkinase, a 53-kDa serine/threonine kinase, and Nm23-H1, are presentin HIV-1 virions and can phosphorylate various viral proteins(10, 12, 25, 36, 49). As is the case with mutations in vif,some of these phosphorylation events may have an effect on thefate of infection (10, 13, 25, 36). To characterize the activityof kinases in ${Delta}$ vif virions, we performed in vitro kinase assayson lysates of sucrose gradient-purified virions. The resulting³²P-labeled viral proteins were then resolved by standard SDS-PAGEand visualized by autoradiography. For both wild-type and ${Delta}$ vifvirions, three major targets of phosphorylation by the virion-associatedkinases were observed (Fig. 7). They were identified by meansof immunoprecipitation to be the HIV-1-encoded CA, MA, and NCproteins (data not shown). The pattern and extent of phosphorylationseen for the ${Delta}$ vif virions suggests that the kinases are bothpresent in normal quantities and active.

View larger version (32K):
[in this window]
[in a new window]
FIG. 7. Examination of virion-associated kinase activity. Wild-type and ${Delta}$ vif HIV-1_YU-2 virions produced in HUT78 cells were sucrose gradient purified and lysed in kinase buffer. Viral lysates corresponding to 50 ng of p24^Gag were then incubated for 30 min at 20°C in the presence of MnCl₂ and [ ${gamma}$ -³²P]ATP. Proteins that had been phosphorylated with radioactive phosphate groups during the reaction were resolved by SDS-PAGE, transferred to nitrocellulose, and visualized by autoradiography.

Activity of the reverse transcription complex. A number of labs have observed that HIV-1/ ${Delta}$ vif performs inefficientlyin the endogenous RT assay, in which virions are "stimulated"via addition of a permeabilization agent, dNTPs, and a divalentcation to reverse transcribe their genomes with the packagedprimers. To confirm this finding, we developed an assay in which strong-stop and second-strand-transfer productsof the endogenous RT reaction were quantified by kinetic (fluorescence-monitored)PCR. A notable aspect of this technology is that it facilitatesthe parallel analysis of multiple repeats of the same and similarsamples and therefore conveys an appreciation of the inherentvariability of a given assay or method of sample preparation.Initially, a series of experiments was performed to identifythe optimal permeabilization (5 µg/ml of melittin) andpH (pH 8) conditions for the reaction (data not shown). In addition,the time course of the reaction was examined, and an incubationtime of 6 to 8 h, which is within the linear range of the assay,was chosen for subsequent experiments (data not shown).

Using these conditions, the endogenous RT activity of wild-typeand ${Delta}$ vif virions produced in nonpermissive cells was comparedat various salt concentrations (Fig. 8). The results representthe average of three experiments performed with different viruspreparations. As a control, we first showed that the vif mutantvirus behaves similarly to wild type in the exogenous RT reaction(Fig. 8A), confirming the previous finding that the RT enzymeitself is fully functional in the absence of Vif (17, 27, 46).For the endogenous RT reaction, the ${Delta}$ vif virions appeared todisplay a slight (<2-fold) diminution in the accumulationof both strong-stop (Fig. 8B) and second-strand transfer (Fig.8C) cDNAs. This was also true under less-optimal experimentswhere other melittin concentrations or pHs were used (data notshown). However, we suspect that this tendency is unlikely tobe significant based on a number of issues. First, in one ofthe three experiments represented, the vif mutant was not measurablydefective in the endogenous RT assay, despite displaying a 25-foldreduction in virus infectivity compared to the wild type. Second,twofold differences between duplicates within the same experimentwere not uncommon, highlighting the intrinsic variability ofassays of this type. Third, similar variations between the wildtype and the ${Delta}$ vif mutant were sometimes observed in virions producedin permissive cells where no differences in virus infectivityare noted (e.g., CEM-SS cells) (data not shown).

View larger version (24K):
[in this window]
[in a new window]
FIG. 8. Examination of reverse transcription complexes in ${Delta}$ vif virions. (A) Exogenous RT activity of wild type and ${Delta}$ vif HIV-1_YU-2. HIV-1_YU-2 and HIV-1_YU-2/ ${Delta}$ vif produced in HUT78 cells were assayed for exogenous RT activity as described previously (62). The results represent the average of four experiments by using different virus preparations. (B and C) Endogenous RT activity of wild type and ${Delta}$ vif HIV-1_YU-2. HIV-1_YU-2 and HIV-1_YU-2/ ${Delta}$ vif produced in HUT78 cells were normalized by p24^Gag content, pelleted in triplicate, and resuspended on ice in endogenous RT buffer lacking salt at 200 ng of p24^Gag per ml. Endogenous RT buffer containing various concentrations of NaCl was then added to aliquots of the resuspended virions in duplicate, and the samples were incubated for 6 to 8 h at 37°C. The strong-stop (B) and second-strand-transfer (C) intravirion reverse transcription products were then quantified by means of kinetic PCR. The values obtained from the triplicate samples were then averaged. The results presented represent the average of three experiments with different virus preparations.

Analysis of the genomic RNA-primer complex. Recently, one group proposed that aberrant placement of and/orinitiation from the native viral primer might be responsible for the defect they observed in endogenous reversetranscription (17). However, because the placement assay thatwas employed relied on initiation from the primer to measure its position, it was not possible to distinguishbetween the two possibilities. In order to segregate placementfrom initiation, we made use of an alternative primer extension-basedplacement assay in which the position of the annealed primer is mapped by virtue of its ability to impedeDNA synthesis initiated from an "upstream" DNA primer (3). Accordingly,genomic RNA-primer complexes were extracted from wild-type and ${Delta}$ vif virions (Fig. 9A), annealed to a ³²P-labeledgag-specific primer, and incubated with recombinant HIV-1 RTin the presence of dNTPs. In the absence of the primer, the labeled primer was extended until it reached the 5' endof the genomic RNA, resulting in a 369-nucleotide fragment.However, if the primer was correctly placed, one of a number of shorter products was generated (3). A similarratio of full-length product to shorter products was observedfor wild-type and ${Delta}$ vif viruses (Fig. 9B), indicating that a similarproportion of genomic RNA-primer complexes exists in the two viruses. In addition, the shorter productswere the same lengths for both viruses, suggesting that theprimer is correctly positioned on the genome of the vif mutant.

View larger version (21K):
[in this window]
[in a new window]
FIG. 9. Analysis of genomic RNA-primer complexes. Sucrose gradient-purified HIV-1_YU-2 and HIV-1_YU-2/ ${Delta}$ vif produced in HUT78 cells were lysed, and the genomic RNA-primer complexes were purified by phenol-chloroform extraction. (A) Northern analysis of viral RNA. The purified RNA was separated by denaturing electrophoresis and hybridized with a ³²P-labeled probe against HIV-1 genomic RNA. (B) Analysis of the placement of the primer. A ³²P-labeled primer that anneals upstream of the primer-binding site on the HIV-1 genome was added to the purified genomic RNA-primer complexes and extended with recombinant HIV-1 RT in RT buffer for 30 min at 37°C. Reaction products were separated on a 6% polyacrylamide-urea gel and visualized by autoradiography. (C) Analysis of the ability of the primer to promote reverse transcription. Purified genomic RNA-primer complexes were incubated for 30 min at 37°C with recombinant HIV-1 RT in RT buffer. The resulting cDNAs were quantified by kinetic PCR against strong-stop reverse transcription products.

To examine whether efficiently primes reverse transcription of the ${Delta}$ vif virus genome, wild-type and ${Delta}$ vif genomicRNA-primer complexes were incubated with recombinant HIV-1 RT and dNTPs in the absence of added primer.The quantity of dNTPs in the reactions was varied to accountfor the possibility that a defect might only manifest itselfat lower dNTP concentrations. By using kinetic PCR to measurethe number of strong-stop cDNA copies produced in the RT reactions,the relative efficiency of priming from endogenous was determined. The data shown in Fig. 9C are one example of resultsobtained by using this assay. Here, the vif mutant was slightlymore proficient than the wild type at all dNTP concentrations.However, depending on the RNA preparation, the activity of the ${Delta}$ vif genomic RNA-complex in this assay varied from threefold below to threefold above that of the wild type.Overall, however, it appeared that the genomic RNA-primer complexes from vif mutant viruses are effective templatesfor reverse transcription.

DISCUSSION

Top

Discussion

In the present study, we developed a method for producing substantialquantities of HIV-1/ ${Delta}$ vif (typically, >100 ng of p24^Gag perml) from an acute infection of nonpermissive T cells. Theseparticles, together with matched wild-type virions, were subjectedto a detailed compositional analysis and an evaluation of theirin vitro reverse transcription phenotypes. Examination of theprotein profile of ${Delta}$ vif virions revealed no apparent abnormalityin protein incorporation, with the exception of the absenceof low levels of Vif itself (Fig. 3 to 5). In addition, therewere no obvious defects in the posttranslational modificationof viral proteins, as determined by detection with antibodiesto phosphorylated residues (data not shown), analysis of themigration patterns of the proteins on two-dimensional gels (Fig.4), and examination of the HPLC profiles of virions (Fig. 5).Also, the viral PR enzyme (Fig. 6) and the virion-associatedkinases (Fig. 7) seemed to be fully active. Analysis of theactivity of the reverse transcription complexes in ${Delta}$ vif virionsby using the endogenous RT assay likewise failed to reveal areproducible defect (Fig. 8). Individually, the RT enzyme andthe genomic RNA-primer

complex also functioned normally in in vitro assays (Fig. 8 and 9). Overall, aside fromthe presence or absence of the Vif protein itself, wild-typeand vif-defective HIV-1 virions were strikingly similar.

Our finding that cleavage of the p55^Gag polyprotein in HIV-1/ ${Delta}$ vifvirions occurs with normal kinetics (Fig. 6) and generates normalmature Gag proteins (Fig. 3 to 6) is in agreement with a numberof steady-state analyses (9, 17, 22, 39, 44, 47) and a previouskinetic analysis (39) but stands opposed to several other reportsof aberrant p55^Gag processing (6, 56). The reason for this discrepancyis not immediately obvious, but the weight of evidence now disfavorsthe idea that the absence of Vif results in reduced p55^Gag processing.Our results also contradict recent in vitro studies that havesuggested the converse notion, namely, that Vif inhibits PRaction (2, 5, 23, 40, 52). Although these investigations haveshown that Vif-derived peptides may inhibit PR in the contextof HIV-1 infection and interfere with HIV-1 replication, demonstrationof premature or excessive proteolysis of Gag during ${Delta}$ vif HIV-1infection is awaited.

Our observation that ${Delta}$ vif virions are not consistently deficientin endogenous RT assays is somewhat surprising (Fig. 8). Althoughwe did detect an approximate twofold decrease in the numberof strong-stop and second-strand-transfer reverse transcriptsgenerated by the vif mutant in some experiments, in other experimentsthe defect was negligible. Importantly, however, the infectivityof the ${Delta}$ vif virus preparations was measured in all cases andfound to be similarly diminished irrespective of performancein the endogenous RT reaction. Analogous results were obtainedwith HIV-1_IIIB (data not shown), indicating that our findingsare not specific to HIV-1_YU-2. The lack of consistent correlationbetween defects in infectivity and endogenous RT activity suggeststhat the modulation of viral infectivity by Vif does not relatedirectly to the biosynthetic capabilities of the reverse transcriptioncomplex. Of note, similar conclusions have previously been drawnon the basis of viral challenges of some T-cell lines (18, 60).Accordingly, we suspect that virions produced in the absenceof Vif have a defect in some other area, such as virion corestability (6, 9, 32, 48), that can manifest itself as a modestreverse transcription defect under some experimental conditions.

Based on the results of the endogenous RT assays (Fig. 8), itis not surprising that we found the primer to be placed properly on the genomic RNA of ${Delta}$ vif virions and tobe able to prime cDNA synthesis in vitro (Fig. 9). Notably,this result contradicts a previous study that found the placementof and/or initiation from the primer to be aberrant in the absence of Vif (17). In that study, the investigatorswere unable to distinguish between placement and the initiation of DNA synthesis. In contrast, the use ofa primer extension assay allowed us to separate the two andto show that placement of the primer is normal in ${Delta}$ vif virions (Fig. 9B). Furthermore, we demonstrated thatgenomic RNA-complexes from ${Delta}$ vif virions areable to support the synthesis of normal quantities of strong-stopcDNA in the presence of recombinant RT (Fig. 9C). Although wedo not understand the reason for these discrepancies, it isworth noting that virion RNA was purified by using guanidiniumisothiocyanate in the former report (17), whereas we used amore gentle method based on phenol-chloroform extraction.

In the present study, we again confirmed that Vif is presentin wild-type preparations of HIV-1 virions (Fig. 3) (6, 11,22, 38, 39, 42, 61, 64), even after removal of contaminatingvesicles and extravirion proteins by subtilisin treatment. Althougha consensus that Vif is associated with viral cores is generallyemerging (11, 39, 42), the functional relevance of Vif packagingremains unresolved. Arguing in favor of nonspecific incorporation,and possibly functional insignificance, are the observationsthat (i) Vif packaging levels correlate with cellular expressionlevels (61); (ii) Vif is preferentially present in immature,possibly aberrant, virions (64); and (iii) Vif is incorporatedinto murine leukemia virus particles, a virus that does notencode a Vif protein (11). Conversely, the recent finding thatgenome-deficient virions are unable to incorporate Vif (39)suggests that the reported association of Vif with RNA (17,68) may facilitate Vif packaging. However, if this is true,the aforementioned murine leukemia virus result indicates thatHIV-1 RNA is not unique in its ability to mediate incorporation.Clearly, a better understanding of Vif function will help usto address the significance of Vif packaging.

Regardless of whether Vif acts in virions, in the producer cells,or in both, its role seems to be the inhibition of one or morehost-derived antiviral factors (30, 45, 55, 59). Accordingly,one would anticipate that ${Delta}$ vif virions likely have an assayablealteration (or defect) other than the lack of Vif itself. Webelieve that the inability of the methodologies described hereto detect such a difference is most likely due either to insufficientsensitivity of the assays used or to deficiencies in our appreciationof which assays to perform. For instance, it is possible thatmetabolic labeling and silver staining (Fig. 4) are not adequateto discern the presence or absence of small quantities of anantiviral factor in virions or alterations to only a small proportionof any given virion protein.

We have recently reported that an epitope-tagged version ofCEM15, the T-cell-expressed antiviral factor that is counteractedby Vif, is incorporated in HIV-1 particles when overexpressedin virus-producing cells (55). Once antibodies are developedto endogenous CEM15, it will be important to determine whetherthis protein is differentially encapsidated into wild-type and ${Delta}$ vif virions under physiological conditions. Since CEM15 sharessignificant sequence similarity with cytidine deaminases (55),it is plausible that viral (or even cellular) RNAs (messenger,genomic, or ) or DNAs are subjected to sequence modification in nonpermissive cells in the absence of Vif. Onecan envisage a number of potential consequences for such changes.For example, the course of reverse transcription in target cellsor the fate of synthesized cDNAs could be altered. Alternatively,the coding "quality" of mRNAs in virus-producing cells couldchange, perhaps giving rise to mutant forms of cellular or viralproteins that exert dominant interfering effects on infectiousvirion production.

Interestingly, a recent analysis of HIV-1 mRNAs in infectedT cells did detect a number of guanosine-to-adenosine and cytidine-to-uridinealterations (7). Whether these changes are a result of RNA editing,and perhaps involve CEM15, or simply reflect reverse transcriptionerrors is still a matter of debate (4). Nevertheless, in viewof the potential deaminase activity of CEM15, an intriguingpossibility is that Vif protects against CEM15-mediated RNAmodifications via its ability to associate with RNA (17, 39,68). In any case, it can be expected that a molecular descriptionof CEM15 function will suggest additional aspects of ${Delta}$ vif virionstructure, composition, and activity that should be assayed.

ACKNOWLEDGMENTS

We thank Robert Doms for the gp120 polyclonal antibody and StephenHughes for the recombinant HIV-1 RT. The HIV-1 RT monoclonalantibody (MAb21) from Stephen Hughes was obtained through theAIDS Research and Reference Reagent Program, Division of AIDS,National Institute of Allergy and Infectious Diseases, NIH.

This work was supported by NIH research grants AI46246 (M.H.M.)and AI10460 (A.M.S.) and by funds from the NCI under contractNO1-CO-12400 (E.C. and L.E.H.). N.C.G. is a National ScienceFoundation predoctoral fellow. M.H.M. is an Elizabeth GlaserScientist supported by the Elizabeth Glaser Pediatric AIDS Foundation.

FOOTNOTES

* Corresponding author. Mailing address: Department of Infectious Diseases, Guy's, King's, and St. Thomas' School of Medicine, King's College London, 3rd Floor, New Guy's House, GKT Guy's Hospital, London SE1 9RT, United Kingdom. Phone: (44) 20-7955-4472. Fax: (44) 20-7955-2846. E-mail: michael.malim{at}kcl.ac.uk.

REFERENCES

Top