Proc. Natl. Acad. Sci. USAVol. 93, pp. 12519-12524, October 1996Medical Sciences

Amphipathic domains in the C terminus of the transmembraneprotein (gp4l) permeabilize HIV-1 virions: A molecularmechanism underlying natural endogenous reverse transcription

(lentivirus lytic peptide/reverse transcription inhibitors/polyamines/deoxyribonucleoside triphosphates)

Hui ZHANG, GEETHANJALI DORNADULA, PRASAD ALUR, MARK A. LAUGHLIN, AND ROGER J. POMERANTZ*The Dorrance H. Hamilton Laboratories, Center for Human Virology, Division of Infectious Diseases, Department of Medicine, Jefferson Medical College,Thomas Jefferson University, 1020 Locust Street, Suite 329, Philadelphia, PA 19107

Communicated by Hilary Koprowski, Thomas Jefferson University, Philadelphia, PA, August 15, 1996 (received for review May 21, 1996)

ABSTRACT Reverse transcription of HIV-1, without de-tergent or amphipathic peptide-induced permeability of theviral envelope, has been demonstrated to occur in the intactHIV-1 virion. In this report, we demonstrate that the amphi-pathic domains in the C terminus of the transmembraneglycoprotein (gp4l) account for the natural permeability ofthe HIV-1 envelope to deoxyribonucleoside triphosphates, thesubstrates for DNA polymerization. In addition, nonphysi-ological deoxyribonucleoside triphosphates, such as 3'-azido-3'-deoxythymidine 5'-triphosphate and 3'-deoxythymidine 5'-triphosphate, can also penetrate the viral envelope, incorpo-rate into, and irreversibly terminate reverse transcripts. As aresult, viral infectivity is potently inhibited. Since the lenti-viral envelope with these newly demonstrated characteristicscan serve as a delivery pathway for anti-reverse transcriptionagents, we propose a unique strategy to prevent HIV-1 inter-and, possibly, intrahost transmission.

HIV-1 virions serve as important agents for inter- and intra-host transmission of retroviral nucleic acids. Our previousworks have demonstrated that virions of lentiviruses are alsobiochemically active particles. Intravirion DNA synthesis,termed "natural endogenous reverse transcription" (NERT),can take place even in a physiological microenvironment. Theretroviral reverse transcriptase is capable of using low quan-tities of dNTPs, either packaged from the viral-producing cellsor existing in the physiological microenvironment. As a result,the virions that have initiated NERT have higher infectivityupon quiescent target cells, and subsequently could play animportant role in HIV-1 transmission and pathogenesis (1).However, an important question remains to be clarified: As theretroviral envelope is derived from cellular membranes, howcould dNTPs, which have been shown to not penetrate cellularmembranes in physiological conditions, pass through the ret-roviral envelope?

It has been demonstrated that the composition of HIV-1envelope membranes are different from cellular membranes(2, 3). Membrane-associated HIV-1-specific proteins, such asthe transmembrane envelope glycoprotein gp4l, or pl7, couldmake viral envelopes different from the cellular membranes inseveral aspects, including permeability. The membrane bind-ing domains in the C terminus of HIV-1 gp4l have uniquecharacteristics. There are two amphipathic domains in thisregion, termed lentivirus lytic peptides-1 and -2 (LLP-1 andLLP-2), that could strongly bind to membranous structures(4-8). Palmitoylation of the cysteine residues upstream ofthese domains can enhance this binding (9). Synthesized LLP-1peptides can lyse either prokaryotic or eukaryotic cells whenadded to culture (6, 10). Further, when synthesized LLP-1

peptides are mixed with planar phospholipid bilayer mem-branes, pore formation is observed (11). Electrical conductionexperiments indicated that when the density of these peptideson the membrane reached the same density as gp4l in the viralenvelope, the pore size can be so large that even a short peptidemay pass through (11). If gp4l, containing this fragment, wasexpressed on the cellular membrane of prokaryotic cells, itwould permeabilize the membrane to several compounds (12).Because gp4l forms tetramers and reaches a relatively higherdensity in the viral envelope than in cellular membranes(13-15), it should have an even stronger influence upon theintegrity of viral envelopes, as compared with cellular mem-branes. Thus, we assumed that these amphipathic domains inthe C terminus of gp4l may bind to the viral envelope,decrease the stability of the membrane bilayer, and make theseretroviral envelopes permeable to dNTPs. To test this hypoth-esis, we created deletion mutants in the C terminus of gp4l inan infectious clone of HIV-1 and tested the affect of thedeletion of LLP domain(s) upon NERT.

MATERIALS AND METHODSPlasmids and Mutagenesis. HIV-gpt is derived from the

HIV-lHXB2 clone. Its env gene was deleted by replacing theNdeI/BglII fragment (nucleotides 6404-7620) with the gptgene driven by a simian virus 40 promoter. The HIV-1 env genewas expressed from the plasmid HXB2-env. The murineleukemia virus (MLV) env gene was expressed from theplasmid simian virus-A-MLV-env (16). The pNL4-3 constructexpresses a wild-type HIV-1 virion with all open readingframes intact (17). All plasmids were obtained from theNational Institutes of Health AIDS Research and ReferenceReagent Program.To generate a deletion of the LLP-1 region (nucleotides

8696-8782) in the pNL4-3 clone, a PCR-based mutagenesismethod was adapted (18). Four primers were synthesized: gpl,5'-TAGTGAACGGATCCTTAG-3' (sense, 8457-8474, con-taining a BamHI site); gp2, 5'-TAGGTCTCGAG ATACT-GC-3' (antisense, 8880-8897, containing a XhoI site); gp3,5'-GCAGTAGCTGAGGGGACAGATTAAGATGG-GTGGCAAGTGGTC-3' (sense); and gp4, 5'-GACCACT-TGCCACCCATCTTAATCTGTCCCCTCAGCTACTGC-3'(antisense). The primers gp3 and gp4 are completely comple-mentary to each other and contain both 5' and 3' flankingsequences of LLP-1 but no LLP-1 sequence itself (deletion ofLLP-1). A fragment was isolated, and utilized to replace the

Abbreviations: RT, reverse transcription; ERT, endogenous RT;NERT, natural ERT; dNTP, deoxyribonucleoside triphosphate; LLP,lentivirus lytic peptides; MLV, murine leukemia virus; AZT, 3'-azido-3'-deoxythymidine; ddT, 3'-deoxythymidine; CEM, CD4+ T-lymphocyte cells; RD, rhabdomyosarcoma; AZTTP, AZT 5'-triphosphate; ddTTP, ddT 5'-triphosphate.*To whom reprint requests should be addressed.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

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BamHI/XhoI segment in pNL4-3. The final product, pNL4-3with deletion of the LLP-1, was entitled pNL4-3/LLP1. Tointroduce a stop codon (TAA) into the env open readingframe, to truncate both LLP-1 and LLP-2 sections, a primergpla: 5 '-TAGTGAACGGATCCTaAG-3' (containingBamHI) was synthesized. PCR was performed with gpla/gp2as primers and pNL4-3 as a template. The PCR fragment wasused to replace the BamHI/XhoI section in pNL4-3. The finalproduct, pNL4-3 with a stop codon (8471-8473) upstream ofboth LLP-1 and LLP-2 regions, but without influence on theoverlapping rev gene expression, was referred to as pNL4-3ALLP1 +2.

Preparation of Peptides. Melittin and maganin II werepurchased from Sigma. LLP-1 (Arg-Val-Ile-Glu-Val-Leu-Gln-Ala-Ala-Tyr-Arg-Ala-Ile-Arg-His-Pro-Arg-Arg-Ile-Arg-Gln-Gly-Leu-Glu-Arg-Ile-Leu) and analog 1 peptide (Arg-Val-Ile-Glu-Val-Leu-Gln-Ala-Ala-Tyr-Glu-Ala-Ile-Arg-Glu-Pro-Glu-Arg-Ile-Arg-Gln-Gly-Leu-Glu-Arg-Ile-Leu) weresynthesized and purified as described (6, 10).

Transfections. To produce HIV-1 virions, the infectiousclones of wild-type or mutated HIV-1 were transfected intohuman rhabdomyosarcoma (RD) cells using the calcium-phosphate coprecipitation method as described previously(19).Endogenous RT (ERT). The analysis ofERT was performed

by two methods: a direct-labeling methodology and quantita-tive DNA-PCR, as described previously (1).

Viral Infectivity Assays. HIV-1NL43 virions in the freshsupernatant of virus-producing cells (CEM; CD4+ T-lymphocytic line) were mixed with various reagents, as men-tioned in the figure legends. After incubation at 37°C for 4 hr,the mixtures were placed onto a 20% sucrose-TN solution andvirions were pelleted in a Ti6O rotor at 90,000 x g for 1 hr. Forvarious experiments, the mixtures were subsequently dividedinto (i) quantitative DNA-PCR to detect intracellular RT asdescribed (1, 21) and (ii) determination of infectivity titer asdescribed (1).

RESULTSHIV-1 Envelope Proteins Play an Important Role in the

Permeability of Virions to dNTPs. To investigate whetherenvelope proteins of HIV-1 account for the permeability ofvirions to dNTPs, an HIV-1 construct without the env gene,HIV-1-gpt, was used to transfect RD cells to produce viralparticles, with or without the HIV-1 env gene, or containingthe MLV-env gene, carried by separate plasmids. The virionswere harvested and normalized by HIV-1 p24 antigen andgenomic RNA semiquantitative dot blot analysis (not shown).ERT was driven by adding 100 ,uM each of dGTP, dATP, anddCTP and 1 ,uM [32P]dTTP/dTTP (1). As shown in Fig. LA,viral particles with HIV-1 envelope proteins were significantlymore permeable to [32P]dTTP than viral particles lackingenvelope proteins, and consequently, significantly more denovo viral DNA was synthesized. PCR analysis also indicatedsignificantly more viral gag DNA synthesis (#30-fold), if thevirion particles contained HIV-1 envelope proteins (Fig. 1B).The significant increment of permeability to dNTPs seems tobe a unique feature for HIV-1 envelope proteins, because useof MLV envelope proteins did not make viral particles signif-icantly more permeable to dNTPs (Fig. 1 A and B).Amphipathic Domains in the C Terminus of HIV-1 gp4l

Permeabilize the Viral Envelope to dNTPs. We next tested thepermeability of the HIV-1 envelope to dNTPs, in the absenceof the LLP domains in the C terminus of gp4l. pNL4-3,pNL4-3ALLP1, and pNL4-3ALLP1+2 were transfected intoRD cells to produce viral particles (Fig. 2A). The virions wereharvested and also normalized. ERT was first examined in theabsence of any synthesized peptides. The deletion of the LLP-1and LLP-1 plus LLP-2 decreased the permeability to dNTPs,

A c

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HlV-gptHIV-gpt +HXB2 envHIV-gpt + SV-A-MLV-env5000 --l50050 w- HIV-1 DNA Copies50

FIG. 1. The envelope protein of HIV-1 permeabilizes virions todNTPs and allows dNTP incorporation into intravirion reverse tran-scripts. HIV-gpt, with or without the HIV-1 env gene or MLV-env geneexpression constructs, were cotransfected into RD cells. The virionswere pelleted and normalized. (A). The normalized virions (100 ng ofHIV-1 p24 antigen equivalents), by both HIV-1 p24 antigen and RNAdot blot analyses, from each aliquot were mixed with ERT reactionbuffer: 100 ,uM each of dATP, dCIP, and dGTP, plus 1 ,uM[32P]dTTP/dTTP, without polyamines, DDT, detergent, and amphi-pathic peptide (1). The reaction was allowed to proceed at 37°C for 15hr. The de novo synthesized viral DNA was extracted and electro-phoresis was performed on alkaline-denatured gels, followed byautoradiography. The density of 32P-labeled nascent viral DNA wasquantitated with a PhosphorImager (Molecular Dynamics). The rel-ative density was calculated by comparing the HIV-gpt (1.0) vs. theother virions. (B) The normalized virions (1 ng HIV-1 p24 antigenequivalents) from each aliquot were mixed with ERT reaction buffer:100 ,uM dNTPs, without polyamines, DDT, detergent, amphipathicpeptide, and isotope. The reaction was allowed to proceed at 37°C for15 hr. The virions were then immunocaptured and the de novosynthesized viral DNA was extracted. PCR was performed to amplifygag DNA using the SK38/SK39/SK19 primer and probe system asdescribed previously (20, 21). A standard curve of HIV-1 proviralDNA is illustrated on the right. These autoradiographs are represen-tative of at least two independent experiments.

thus decreasing de novo intravirion DNA synthesis, eitherdetected by the direct-labeling method or quantitative PCR(Fig. 2 B and C).To investigate whether a synthesized LLP-1 peptide could

interact with the viral envelope and perturb its stability, weadded a synthesized LLP-1 peptide into the ERT reactionsystem. As controls, other peptides, such as analog 1 of LLP-1,melittin, and maganin-2 were also added into the reactionbuffer, respectively. Of note, melittin, a 26-amino acid amphi-pathic peptide, has been routinely used in ERT reactionsystems to make retroviral particles permeable to dNTPs(22-25). As indicated in Fig. 3 A and B, the LLP-1 peptide, aswell as melittin, can make the envelope of virions with adeletion of the LLP-1 domain in the C terminus of gp4lsignificantly more permeable to dNTPs (compare lane 2 tolanes 3 and 5 in Fig. 3A and B). The active concentrations ofLLP-1 to alter dNTP permeability are in the range of 0.5-4,uM. At 2 ,uM, the affect reached a maximum, while higherconcentrations (>5 ,uM) of the LLP-1 peptide significantlydecreased the viral DNA synthesis (data not shown). This maybe due to complete lysis of the viral envelope, and thus,disruption of the virion. The analog 1 peptide, in which threeof the positively charged amino acid residues have beenreplaced with negatively charged residues, was much lesseffective in permeabilizing the viral envelope (Fig. 3A and B,lane 6). Interestingly, maganin 2, an amphipathic helical

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TM domain LLP-2 LLP- I ApNL4-3

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FIG. 2. Deletion of the LLP domains in the C terminus of HIV-1gp4l decreased the [32P]dTTP incorporation into intravirion reverse

transcripts. (A) Schematic maps for plasmids pNL4-3, pNL4-3ALLP1,and pNL4-3ALLP1+2 are illustrated (TM, transmembrane). PlasmidspNL4-3, pNL4-3ALLP1, and pNL4-3ALLP1+2 were transfected intoRD cells. The virions were pelleted and normalized by HIV-1 p24antigen. The genomic RNA from each aliquot of virions was furtherextracted and quantitated by dot blot. The normalized virions wereanalyzed by the direct labeling methodology (B), and by quantitativeDNA-PCR (C) (1, 20, 21). These autoradiographs are representativeof at least two independent experiments.

peptide, which usually lyses prokaryotic cells but not eukary-otic cells (26), also made the viral envelope permeable todNTPs (Fig. 3 A and B, lane 4).Exogenous Triphosphates of Anti-RT Nucleoside Analogs

Inhibit NERT. As previously demonstrated (1), polyaminescould significantly enhance HIV-1 NERT, even at very lowconcentration of dNTPs (Fig. 4A, lane 2). Fig. 4 indicates thatHIV-1 NERT stimulated by polyamines, in the presence ofdNTPs at physiological concentrations, can be potently inhib-ited by exogenous triphosphate chain-terminators, AZTTP or

ddTTP, respectively (Fig. 4A, lanes 3 and 4). As controls, thenucleoside precursors, 3'-azido-3'-deoxythymidine (AZT) and3'-deoxythymidine (ddT), did not inhibit NERT (Fig. 4A, lanes5 and 6). Conversely, deletion of the LLP-1 domain in the Cterminus of gp4l blocked not only exogenous dNTPs, but alsothe triphosphate terminators' passage through the viral enve-

lope. As a result, intravirion RT driven by polyamines andendogenous dNTPs was not affected by triphosphate termi-nators in LLP-1-mutant HIV-1 virions (Fig. 4B, lanes 3 and 4).

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FIG. 3. Synthesized amphipathic peptides can complement thedeletion of the LLP to permeabilize HIV-1 virions. (A) Direct-labelinganalysis for ERT. The normalized virions (100 ng of HIV-1 p24antigen equivalents) produced by plasmids, pNL4-3 and pNL4-3ALLP1, were mixed with the ERT reaction buffer, with a variety ofsynthesized peptides: melittin (5 ,uM), maganin II (5 ,uM), LLP-1 (2,uM), and analog 1 peptide (2 ,uM). (B) Quantitative DNA-PCR forERT. These autoradiographs are representative of at least two inde-pendent experiments.

Termination of Intravirion HIV-1 RT Potently InhibitsIntracellular RT and Viral Infectivity. When chain termina-tors are incorporated into viral reverse transcripts, theoreti-cally, RT cannot be further extended, even if unincorporatedinhibitors have been removed. To test whether inhibition ofNERT by exogenous triphosphate chain terminators, in thepresence of polyamines, at concentrations found in humanseminal fluid (1), and dNTPs at physiological concentrations,would also alter intracellular RT, HIV-1 virions that weretreated with exogenous triphosphates of anti-reverse trans-criptase nucleoside analogs were allowed to infect replicatingCEM cells. Fig. SA indicates that intracellular HIV-1 RT was

potently inhibited, when NERT was efficiently initiated bypolyamines and dNTPs at physiological concentrations andinhibited by AZTTP or ddTTP (lanes 4 and 5). If NERT wasnot efficiently initiated by polyamines, the inhibition effect byAZTTP or ddTTP was not significant (Fig. 5A, lane 8). Tomake certain that the reagents were completely washed off andthe inhibition effect of AZTTP and ddTTP was not due tocontamination, as a control, AZT or ddT were also mixed withvirions and washed off by similar procedures. As shown in Fig.5A, treating the virions with AZT or ddT did not affect NERTand, subsequently, did not alter intracellular RT (lanes 6 and7).We further examined the infectivity titer of HIV-1 virions,

if NERT was initiated and inhibited by chain terminatortriphosphates. Fig. SB demonstrates that the viral infectivitytiter upon replicating cells was inhibited by 16- to 32-fold, as

compared with controls. Alternatively, these data demon-strated that -94-97% (15/16 to 31/32) of infectious HIV-1virions had initiated NERT, if stimulated with polyamines atphysiological concentration of dNTPs, and would be inacti-vated by exogenous chain terminator triphosphates.

DISCUSSIONHIV-1, as well as other lentiviruses, has a long intracytoplas-mic envelope domain. Deletions in this region can affect viralinfectivity, assembly, or cell fusion (27-31). In the case ofsimian immunodeficiency virus, a mutation to introduce a stop

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FIG. 4. Intravirion HIV-1 reverse transcripts can be terminated bytriphosphates of anti-reverse transcriptase nucleoside analogs. (A)HIV-1NL4-3 virions (1 ng HIV-1 p24 antigen equivalents) were mixedwith the ERT reaction buffer (1) containing: 50 nM dNTPs, poly-amines (spermine 3 mM, spermidine 0.1 mM), with or withoutAZTTP (5 ,uM; Moravek Biochemicals, La Brea, CA), ddTTP (5 ,uM;Sigma), AZT (5 ,uM; Sigma), and ddT (5 ,uM; Sigma). Of note, lane1 contains virions treated only with dNTPs (50 nM), without poly-amines (spermine and spermidine). (B) HIV-1NL4-3 virions and HIV-1NL4-3ALLP virions were mixed with the ERT reaction buffer contain-ing: 50 nM dNTPs, polyamines, with or without AZTTP (5 ,LM). Thereaction was allowed to proceed at 37°C for 4 hr. The virions were thenimmunocaptured and the viral DNA was extracted. PCR was per-formed to amplify U3 and the gag DNA with U31/U32/U33 andSK38/SK39/SK19 primer and probe system, respectively, as describedpreviously (20, 21). This autoradiograph is representative of at leasttwo independent experiments. S.S., spermine and spermidine.

codon that truncates this domain near the C terminus, isaccompanied by an enhancement of viral replication in humancell cultures (32). However, if simian immunodeficiency vi-ruses with this mutation are injected into monkeys, the mu-tation reverts. The simian immunodeficiency virus strains witha long envelope C terminus in gp4l would dominate in vivo,suggesting that the C terminus of gp4l is required for efficientviral replication in vivo (33). In this report, we demonstratethat the C terminus of gp4l in HIV-1 plays an important rolein making the viral envelope permeable to dNTPs, which canassure intravirion RT and continuous utilization of dNTPs, inits physiological microenvironments. This progression may beslow, and full-length viral DNA may not always be completedwithin the virion, however, intravirion RT is important forHIV-1 to establish an infection of quiescent cells. It has beendemonstrated that virions harboring high levels of intravirionDNA are better able to infect quiescent cells, probably due toa more efficient usage of the synthesis machinery for RT thatexists in the virions themselves and less dependence upon the

c1 _ -aA

FIG. 5. Intracellular RT and infectivity titers of HIV-1 wereinhibited by treating the virions with triphosphates of chain termina-tors and polyamines. HIV-1NL4-3 virions were treated with: Lanes: 1,heat-inactivation (HI) at 56°C for 30 min; 2, 50 nM dNTPs; 3, 50 nMdNTPs and polyamines (3 mM spermine and 0.1 mM spermidine); 4,50 nM dNTPs and polyamines plus AZTTP (5 AM); 5, 50 nM dNTPsand polyamines plus ddTTP (5 ,M); 6,50 nM dNTPs and polyaminesplus AZT (5 ,uM); 7,50 nM dNTPs and polyamines plus ddT (5 ,uM);8, 50 nM dNTPs plus AZTTP (5 ,uM). After incubating at 37°C for 4hr, the reagents were washed off by passing the virions through a 20%sucrose solution with TN buffer. The virions were normalized byHIV-1 p24 antigen. The virion aliquots were then divided: (A)Quantitative PCR for intracellular HIV-1 RT: Virions treated withvarious reagents were allowed to infect CEM cells. At 12 hr postin-fection, DNA was extracted from CEM cells with a "quick lysis"methodology and quantitative PCR was performed with the M667/M661/SK31 primer and probe [amplifies RU5, primer binding site, 5'noncoding region (RU5-PBS-5NC)] (1). (B) Viral infectivity: TheCEM cells were infected by serially diluting the HIV-1 virions at 37°Cfor 4 hr and the unbound virions were washed off. The cells were thencultured and HIV-1 p24 antigen expression was quantitated by ELISA.The autoradiograph and graph are representative of at least twoindependent experiments. S.S., spermine and spermidine.

degrading synthesis machinery in quiescent target cells (1).The studies in the present report demonstrate the molecularmechanisms underlying this newly described stage in thelentiviral life-cycle, NERT or intravirion RT (1).

Theoretical studies indicated that the a-helix of LLP-1 andLLP-2 displays an extraordinary high degree of amphiphicityand could bind to viral envelopes in two ways: (i) spanning themembranous bilayer to form channel-like structures or (ii)binding to the inner face of the membrane (34). The structure

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formed by LLP-1 and LLP-2 to make viral envelopes perme-able to dNTPs remains to be clarified. However, as deletionsof LLP-1 alone can significantly decrease entry of dNTPs intothe viral particles, and the addition of LLP-1 peptides caneffectively increase this permeability, these findings demon-strate that LLP-1 should play an important role in permeabi-lizing the viral envelope. It is notable that, in MLV virions,intravirion RT takes place inefficiently unless the exogenousdNTP concentration approaches very high levels (5 mM) (19).This may be due to the lack of typical amphipathic domains inthe C terminus of the MLV transmembrane protein (6). TheLLP-1 peptide can permeabilize an HIV-1 envelope to dNTPsat 2 ,uM, which is lower than the concentrations (10-100 ,uM)required to lyse both prokaryotic and eukaryotic cells (6, 10).Compared with the cellular membrane, the HIV-1 envelopehas some unique features. It has been shown that the protein-to-lipid ratio (weight), and cholesterol-to-phospholipid molarratio in the HIV-1 envelope are higher than that in cellularmembranes, and that its lipid composition is more ordered (2,3). Further, unlike cellular membranes, there is almost no newsupplements for the lipid envelope of cell-free virions. Thus,the fluidity of a viral envelope is limited. These features oflentiviral envelopes could make their membranous structuresmore accessible to perturbation by LLP.

Permeability to triphosphates yields a nascent and importanttarget to inhibit intravirion RT, and, subsequently, irreversiblyinactivate HIV-1 virions. The nucleoside analog RT-inhibitorshave played an important role in combating HIV-1 infection.However, only nucleoside precursors are now used becausetriphosphates of nucleoside analogs cannot pass through cel-lular membranes, in normal situations. The efficiency ofphosphorylation is a problem for these precursors. In somequiescent cells, phosphorylation is not efficient (35, 36).Conversely, phosphorylation for some agents is not sufficient,even in replicating cells. For example, ddTTP has been shownto inhibit HIV-1 RT, as effectively as AZTTP in a cell-freereaction system (37). However, because of a much lowerefficiency of phosphorylation, the antiviral effect of its pre-cursor, ddT, is much less than AZT and other dideoxyribo-nucleosides (38). Our data demonstrate that ddTTP is stilleffective in preventing HIV-1 replication, if it is utilized toterminate intravirion reverse transcripts. Further, as phos-phorylation is not required, inhibition ofNERT by the triphos-phates of nucleoside analogs might protect quiescent cells fromHIV-1 infection.

Save for barrier methods, there are no other approvedapproaches available to effectively prevent sexual transmissionof HIV-1. As demonstrated in this report, incorporation ofchain terminators into intravirion HIV-1 reverse transcriptscan potently decrease HIV-1 NERT, and subsequently, de-crease the viral infectious titer even in actively replicatingtarget cells. As demonstrated previously, in the presence ofdNTPs at physiological concentrations, polyamines canstrongly stimulate the NERT process within infectious HIV-1virions (1). As the physiological concentrations of polyaminesare very high in human seminal fluids (39), and intravirionHIV-1 RT occurs efficiently in seminal fluids (1), administra-tion of triphosphates of nucleoside analogs in the genital tractor intestines might potently inactivate at least 94-97% ofinfectious HIV-1 virions. Given that the major target cells forsexual transmission of lentiviruses in the submucosal tissue ofthe genital tract are quiescent dendritic or mononuclear cells(40, 41), and intravirion reverse transcripts are important forHIV-1 to establish infection in quiescent cells (1), terminatingintravirion reverse transcripts could further decrease viralinfectivity upon these quiescent cells in vivo. Based upon theseconsiderations, we propose that a novel strategy to directlyinactivate HIV-1 virions would be by terminating intravirionreverse transcripts. This approach may be quite useful inpreventing either heterosexual and/or homosexual transmis-

sion of HIV-1. In this approach, triphosphates of nucleosideanalogs might directly act as a virucidal agents, which could bereadily coadministered with spermicidal agents.

We thank Ronald C. Montelaro and Timothy Beary for providingpeptides, Didier Trono for helpful;discussions, and Rita M. Victor andBrenda 0. Gordon for excellent secretarial assistance. This work wassupported in part by U.S. Public Health Service Grants AI33810,A138666, and AI36552 to R.J.P.

1. Zhang, H., Dornadula, G. & Pomerantz, R. J. (1996) J. Virol. 70,2809-2824.

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