Minireviews

HIV Accessory Proteins: EmergingTherapeutic Targets

Roger H. Miller and Nava SarverTargeted Interventions Branch, Basic Sciences Program, Division ofAIDS, National Institute of Allergy and Infectious Diseases, NationalInstitutes of Health, Bethesda, Maryland, U.S.A.

HIV ACCESSORY PROTEINSIn addition to the capsid (gag), polymerase (pol),and envelope (env) genes common to all retrovi-ruses, human immunodeficiency virus type 1(HIV-1) possesses six additional genes (i.e., nef,rev, tat, vif, vpr, and vpu) that encode regulatoryproteins (Fig. 1). Four of these proteins, Nef, Vif,Vpr, and Vpu, were originally termed "accesso-ry" proteins due to the fact that their function invitro appeared to be nonessential for HIV repli-cation (reviewed in Ref. 1). However, it is nowevident that these proteins play an importantrole in viral replication in vivo, and some may beintimately involved in HIV pathogenesis and dis-ease progression.

The following review is based on a recentsymposium (HIV Accessory Proteins: Therapeu-tic Opportunities, February 2, 1995, Washington,DC) focusing on the therapeutic opportunitiesafforded by HIV accessory proteins.

NEFNef, a viral protein expressed early in infection,possesses two distinct functions: the ability toenhance HIV replication and the ability to down-regulate the T cell surface CD4 molecule (re-viewed in Ref. 2). Enhancement of virus replica-tion results from the presence of virion-associated Nef that increases the infectivity ofcell-free virus. This was revealed by studies com-paring the replication capabilities of infectiousprovirus plasmids and virus particles in CEM

Address correspondence and reprint requests to: NavaSarver, Targeted Interventions Branch, Basic Sciences Pro-gram, Division of AIDS, NLAID, NIH, Bethesda, MD 20892-7620.

Copyright 1995, Molecular Medicine, 1076-1551/95/$10.50/0Molecular Medicine, Volume 1, Number 5, July 1995 479-485

cells (a human T cell line) in a single-cycle assaywhere subsequent rounds of infection were in-hibited by the addition of neutralizing antibody.Transfection of CEM cells with identical amountsof either a nef deletion (A) mutant or wild-type(wt) proviral DNA yielded similar expression lev-els of the HIV capsid protein p24. However,when CEM cells were infected with equal inoc-ula of Anef and wt virus (standardized for theirp24 content), Anef virus yielded 3- to 5-fold lessp24 than did the wt HIV- 1. Thus, the presence ofparticle-associated Nef in wt virus resulted in anincrease in p24 production in infected CEM cell.

The mechanism whereby Nef confers repli-cative advantage on the virus is not yet estab-lished. Comparison of virions with or withoutNef revealed no difference in reverse tran-scriptase (RT) activity, viral binding, or viral en-try into host cells. The reduction in infectivity ofAnefvirus does not seem to reflect on an inabilityto degrade CD4 (see below). When cells engi-neered to express a truncated CD4 molecule,lacking virtually the entire cytoplasmic domainrequired for Nef-induced CD4 degradation, wereinfected with Anefor wt HIV, attenuation of Anefvirus growth was still observed.

The severe combined immunodeficientmouse implanted with human thymus and livertissue (SCID-thy/liv) is an important animalmodel for studying HIV infection and pathogen-esis. Mice are generated by surgical transplanta-tion of human fetal liver and thymus fragmentsunder the kidney capsule. After several months,the implanted tissues become fused together toform a graft, which is histologically and physio-logically indistinguishable from a normal humanthymus. Direct inoculation of the graft with HIVresults in infection of human CD4-positive cellsand precursor CD4/CD8-double positive cells.

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HIV-1 PROVIRAL GENOME

S;P.

POL

GAG F

PR

RT

ENV

REV EXII

IVPI

ITAT EX)

'PU

IN

FIG. 1. Diagrammatic representation of the HIV-1 genome.The long terminal repeats (LTRs), divided into the unique 3' (U3), repeat (R), and unique 5' (U5) domains, flankthe protein encoding regions of the virus genome (light blue). The large precursor proteins encoded by the capsid(GAG), polymerase (POL), and envelope (ENV) genes are proteolytically cleaved as depicted by the vertical lines.The GAG protein is cleaved into the smaller proteins p17, p24, and p6; the POL protein is cleaved into the pro-tease (PR), reverse transcriptase (RT), and integrase (IN) proteins; and the ENV protein, with a signal peptide(S.P.) sequence at the amino terminus, is cleaved into the gp 120 (surface) and gp4l (transmembrane) proteins.Exons (EX) 1 and 2 of the rev and tat genes as well as the genes encoding the accessory proteins are highlighted bycolor.

The SCID-thy/liv model was used to evaluate theeffect of HIV regulatory proteins on CD4-positivecells. CEM cells were transfected with infectiousplasmids with deletions in the nef, vif, vpr, or vpugenes, and the virus produced was titered onactivated human peripheral blood lymphocytes(PBLs). SCID-thy/liv mice were then inoculatedwith equal amounts of infectious virus. Suchanalysis revealed a hierarchy in the abilities ofthe four deletion mutant viruses to replicate andinduce CD4 depletion in the graft relative toinfection with wt HIV as follows: Avpr > Avpu >Avif> Anef. Deleting the nefgene was thus moredeleterious to virus replication than deleting anyof the other regulatory genes studied.

Another well-established function of Nef isits ability to down-regulate CD4, a surface mol-ecule crucial for helper T cell signaling and aprincipal cellular receptor for HIV. When nefgene constructs were expressed in CD4 positivecells, the half-life of CD4 was found to decreasefrom 24 to 4-6 hr. While expression of CD4 wasunaffected, once the protein reached the cyto-plasmic membrane Nef induced rapid endocyto-sis followed by lysosomal degradation of CD4 (3).

Recent work indicates that Nef may disrupt theinteraction between CD4 and the p56Ick tyrosinekinase (4). Site-directed mutagenesis of the cy-toplasmic tail of CD4 (the binding site of p56lckand Nef) reveal that mutated CD4 moleculeswith decreased responsiveness to Nef also havereduced capacity to associate with p561ck. Aminoacids 407 and 410 within a hydrophobic domainof CD4 are especially important. Thus, it is likelythat Nef, or a Nef-recruited factor, competes withp561ck for a binding site on the cytoplasmic tail ofCD4 to disrupt CD4/p561ck interaction, therebyinducing CD4 endocytosis and its subsequentdegradation.

VPUVpu is a regulatory protein encoded by HIV- 1 butnot by HIV-2 or most of the simian immunode-ficiency viruses. This phosphorylated transmem-brane protein has two distinct and separablefunctions in the HIV replication cycle: enhance-ment of viral export from infected cells and deg-radation of CD4 in the endoplasmic reticulum

5' LTR 3' LTR5

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(ER). The requirement for Vpu in particle releaseis cell-type specific. Pulse-chase experiments uti-lizing constructs expressing HIV p6 Gag (a pro-cessed Gag peptide derived from the carboxyl-terminus of p55 Gag) and/or Vpu indicate thatthe latter facilitates particle release in HeLa butnot in COS cells. This finding implies the pres-ence of a Vpu-interacting protein in some cellsbut not in others. Additional support for a cellu-lar Vpu-interacting protein comes from a studyshowing that Vpu increases the release of heter-ologous retrovirus particles.

Using the yeast two-hybrid system a Vpubinding protein, or UBP, was identified. The pro-tein, approximately 69 kD in size, is encoded bya 2.9-kb cellular transcript expressed in a varietyof human tissues and cells, including PBLs. Par-tial analysis indicates that UBP shares certainsequence similarities with the immunophilin su-perfamily of proteins, which includes the cyclo-philins and the FK506 binding proteins (FKBPs).It is still unclear what role UBP plays in mediat-ing Vpu function(s) and whether UBP/Vpu in-teraction is required for HIV replication.

Insight into the mechanism of Vpu-mediatedviral export comes from solution NMR experi-ments demonstrating that the transmembranedomain of oligomeric Vpu has the potential toform an ion conductive membrane pore. Exper-iments in Xenopus oocytes verified the ion chan-nel activity of synthetic Vpu. Interestingly,FKBP12, a member of the immunophilin familyrelated to UBP, also interacts with a cellular pro-tein involved in ion channel formation. Whilemutation of the transmembrane domain of Vpuresulted in the loss of Vpu-mediated ion channelformation activity and caused a reduction in vi-rus release, there was no effect on CD4 degrada-tion, indicating that these two functions of Vpuare separate.

The mechanism for Vpu-mediated CD4 deg-radation differs from that described for Nef. InHIV-infected cells, gp 160 Env and CD4 formcomplexes that become sequestered in the roughER, preventing further transport of gp 160 andCD4 to the cellular membrane. Vpu reverses thisinhibition of gp 160 sequestering by selectivelydegrading CD4 (5). Site-directed mutagenesis ofVpu suggests that both the anchor and cytoplas-mic domains of the protein are required for thisprocess.

As indicated, both the enhancement of viralsecretion and the induction of CD4 degradationby Vpu involve distinct molecular mechanisms.It is not clear at this time whether blocking either

function alone will have an impact on viralgrowth and pathogenesis or whether both func-tions will need to be targeted for maximal ther-apeutic benefit.

VIFSeveral independent studies have shown that theVif protein significantly increases the infectivityof HIV- 1 particles. The importance of this proteinis underscored by the observation that virtuallyall lentiviruses examined (the single exceptionbeing equine infectious anemia virus) possess avif gene. While Vif was previously thought to befound only in the cytoplasm of infected cells (6),recent experiments have revealed the existenceof Vif in mature virions. Studies with anti-Vifantibodies show that Vif exists within the viruscore rather than on the virus surface, and it isestimated that 20 Vif molecules are encapsidatedper virion.

Within the cytoplasm of infected cells, Vifexists both as soluble and as membrane-associ-ated forms. Site-directed mutagenesis of vif re-vealed that the basic amino acids at the carboxylterminus of the molecule (amino acids 157-160and 173-184) are important for association withcell membranes. Interestingly, pretreatment ofmembranes with trypsin abolishes the ability ofVif to bind, suggesting that Vif interacts with amembrane-associated protein which stably an-chors it to the membrane surface.

Again, these studies indicate an associationbetween an HIV- 1 regulatory protein, in this caseVif, with a cellular protein(s). Such proteins arelikely to be required for the proper functioning ofthe regulatory proteins in viral replication andmay provide additional targets for therapeuticintervention. One advantage in targeting a cel-lular "partner" over a viral gene product is that itobviates potential emergence of HIV escape mu-tants resistant to specific anti-viral agents.

In additional studies of the role of Vif in virusreplication, Vif mutant virions purified fromCEM cells (but not those purified from SupTIcells) were shown to exhibit a major defect inendogenous reverse transcriptase (RT) activity.Radiolabeled nucleotides were incorporated pri-marily into low molecular weight DNA frag-ments in the mutant virus. This suggests that ayet unidentified component of the virus coreinvolved in reverse transcription is a target forVif function. Although Vif does not effect RNAdimerization, the level of unprocessed p55 Gag

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in Avif virions was elevated compared with thatin wt virions. At the present time, it is not clearwhat this effect on Gag processing means withregards to the role of Vif in HIV infectivity.

A second function of Vif involves reorgani-zation of cytoskeletal elements, consisting largelyof microtubules and intermediate filaments.While microtubules are composed of actin ortubulin, intermediate filaments are exclusivelymade of several proteins, notably vimentin. Ex-periments examining the cellular localization ofVif in HeLa cells transfected with an expressionplasmid revealed that Vif associates specificallywith intermediate filaments comprised of vimen-tin. Treatment of HeLa cells with brefeldin A, adrug that mediates the trafficking of membraneproteins from the ER to the Golgi, produced pe-rinuclear caps of vimentin filaments. Vif ex-pressed in HeLa cells colocalized specifically tosuch structures following drug treatment. WhileVif does not affect the phosphorylation or solu-bility of vimentin, it causes a dramatic reorgani-zation of the cytoskeleton. Vif may, therefore,also function to enhance the movement of thevirion to the nucleus by reorganizing the cellularcytoskeletal elements. One hypothesis is that af-ter the virion enters the host cell and uncoatingbegins, Vif attaches to vimentin and initiates thereorganization of intermediate filaments. As-sisted by a vimentin-associated factor capable ofmoving along the filaments, the Vif-containingviral pre-integration complex is efficiently trans-ported to the nucleus via a "three-dimensional"process, rather than a "two-dimensional" pro-cess in the absence of Vif. Conceivably, therapeu-tic agents that block Vif's ability to interact withvimentin are likely to interfere with the trans-port of the pre-integration complex to the nu-cleus, and dramatically affect the ability of HIV toefficiently infect lymphocytes.

VPRVpr exists as an oligomer (possibly a tetramer) inthe nucleus of infected cells. Vpr is incorporatedinto virions via a specific mechanism involvingp6 Gag (7). Virus strains encoding Vpr replicatefaster and to higher levels than strains unable toencode a functional Vpr protein. Structural anal-ysis of Vpr has focused on two fronts: determin-ing the location of the nuclear localization signal(NLS) that facilitates transport of the nascentprotein into the cell nucleus, and identifying thesequence(s) responsible for Vpr oligomerization

(see below). Typical NLS elements consist of astretch of positively charged amino acids. TheNLS of Vpr is atypical in that it is located in anegatively charged domain at the amino termi-nus of the molecule. The Vpr NLS resides in adomain coincident with a region required for theinteraction of the protein with a 180-kD cellularfactor that may facilitate transport of Vpr into thenucleus. Recombinant Vpr expressed in Esche-richia coli is capable of forming oligomeric struc-tures and preliminary work indicates that aminoacids at the amino terminus of the molecule arerequired for oligomerization. Mutagenesis exper-iments mapped the crucial domain to amino ac-ids 36-43. Speculations as to the purpose of Vproligomerization include expanding the capabili-ties of the small (i.e., 14 kD) monomeric proteinand/or increasing the packaging efficiency of theprotein.

The role of Vpr as an integral component ofthe virus may be explained by its profound bio-logical effect on cells; Vpr was shown to regulatecell proliferation as well as cell differentiation(8,9). As Vpr is released into the cell duringuncoating of the virion, it is presumed to "pre-pare" an environment favorable to HIV replica-tion and gene expression. In one study, an infec-tious molecular HIV clone was altered to expressthe murine Thy protein, a cell surface moleculethat can serve as a convenient marker for mon-itoring virus gene expression. Using a dual stain-ing method to analyze concomitantly the cellcycle status and the infection status of individualcells, infection with the altered HIV recombinantwas shown to induce the accumulation of cellsarrested in G2 in a variety of cell lines (PBL,SupTl, HeLa, and COS cells). In contrast, mockinfected cells had twice as many cells in Gl thanin G2. When the vpr gene was mutated, therecombinant infectious HIV could no longer in-duce G2 arrest. Interestingly, the vpr mutant vi-rus was still capable of spreading and inducingsyncytia. Another study examined the effects ofbaculovirus-expressed Vpr on tumor cell prolif-eration. While purified Vpr caused a dramaticinhibition of proliferation, latently infected celllines treated with Vpr exhibited reactivation ofvirus replication. Such effects were the samewhether recombinant Vpr or Vpr purified fromHIV-infected sera were used. Overall, these stud-ies implicate Vpr in inducing a long-lasting in-crease in cellular permissiveness to HIV replica-tion by modulating the differentiation stage ofthe host cell.

These studies are consistent with recent find-

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ings demonstrating that viruses containing anintact vpr gene are unable to establish a chronicinfection of cultured lymphocytes due to thedeath of the infected cells (10). This effect isobserved late in infection and occurs even inPBLs transfected with a provirus with a func-tional vpr gene and a defective env gene. Sincecell-to-cell spread does not occur under thesecircumstances, cell death must be mediated viaVpr synthesized de novo from the integrated pro-virus and not from import of virion-associatedVpr into the cell. Combined with other results,the data are consistent with the hypothesis thatVpr either prevents cells from continued prolif-eration or induces their terminal differentiation.The latter could explain why primary macro-phages, which are differentiated cells, are capa-ble of long-term expression of HIV without sig-nificant cell death. Additional experiments arenecessary to determine the relevance of the invitro observations to HIV replication in vivo.

How does Vpr exert such profound effects oncells? A recent report suggests that Vpr is linkedto the glucocorticoid steroid pathway (11). Anewly discovered cellular protein which associ-ates with Vpr provides the link to this importantregulatory pathway. Vpr-interacting protein 1(Rip-i) is a 41 -kD cytosolic protein that is ex-pressed in a wide variety of tissues and cells,including those that support HIV replication.Rip- I is translocated to the nucleus by both Vpror glucocorticoid receptor (GR) -II-stimulatingsteroids. The finding that Vpr and Rip-I coim-munoprecipitate with the human GR provides apotential biochemical mechanism for Vpr's activ-ity and suggests the possible use of anti-glucocor-ticoid agents as inhibitors of HIV replication.

PROSPECTS FOR THERAPYAn emerging theme is that Nef, Vif, Vpr, and Vpuare involved in crucial aspects of HIV replicationand release, cell susceptibility to infection, andmodulation of signal transduction (Table 1). It isalso apparent that several of these regulatoryproteins share common functions mediated bydifferent mechanisms, such as the downregula-tion of CD4 by both Nef and Vpu. While Nefpromotes the internalization of surface CD4 andsubsequent degradation in lysosomal vesicles,Vpu targets CD4 sequestered in the ER via aCD4-gp 160 complex. Why does the virus devotetwo gene products to CD4 down-regulation? Ithas been shown that removal of the HIV receptor

from the cell surface benefits HIV replication bypreventing superinfection and by enhancing therelease of progeny virions; additional studies willdetermine whether CD4 down-regulation isimportant in other aspects of HIV infection ofhumans.

One of the most intriguing findings pre-sented at the meeting was that the HIV regula-tory proteins interact with specific cellular factorsto perform their functions. Whatever the precisemechanisms involved, Nef, Vif, Vpr, and Vpu andtheir associated cellular proteins afford highlypromising yet underexploited therapeutic oppor-tunities. Conceivably, targeting cellular factorsmay be more effective than targeting viral fac-tors. This premise is founded on the observationthat cellular genes replicate with considerablymore fidelity than HIV genes, thereby providingconserved targets for therapeutic interventions.Thus, the serious and prevalent problem associ-ated with the evolution of virus variants resistantto the inhibitory effect of the drug(s) could bealleviated. Although a downside to targeting cel-lular factors is the potential cellular toxicity, itshould be possible, nonetheless, to strike a clin-ical balance with significant antiviral effect andmanageable toxicity. Importantly, it is possiblethat a synergistic inhibitory effect may beachieved in targeting the viral and cellular pro-teins simultaneously using combination thera-pies. Overall, efforts to exploit these dual targetsshould be explored in devising therapies againstHIV infection which may be used alone or incombination with other anti-HIV therapeuticstrategies.

Understanding the mode of action of the HIVregulatory proteins and their associated cellularfactors opens a window for deciphering the in-tricate mechanism(s) associated with HIV-medi-ated cellular dysfunction and dysregulation. Thisknowledge, in turn, is imperative for developingeffective therapies against HIV infection, eitherby identification of specific inhibitory agents inbiochemical screening assays, a rational drug de-sign approach driven by structure-function rela-tionship, or by novel therapeutic strategies (e.g.,gene-based inhibition).

ACKNOWLEDGMENTSWe thank the following investigators for theirpresentations at the meeting, some of which aresummarized herein: Grace Aldronvandi, UCLASchool of Medicine and Jonsson Comprehensive

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TABLE 1. HIV accessory proteins

InteractingMolecular CellularWeight Cellular Virus Factor(s) Known/Putative(kD) Modifications Location Association (size, kD) Functions

Nef 27 Myristoylated Cytoplasm Yes Ick (56)a * Increases virioninfectivity

* Down-regulatessurface CD4

Vif 23 ? Cytoplasm Yes (?)b * Increases virioninfectivity

* Reorganizescytoskeletalelements

Vpr 14 ? Nucleus Yes Rip-I (41)C * Promotescellulardifferentiation

RIP (180)d 0 Arrests cells inG2 phase of cellcycle

* Interacts withglucocorticoidsteroid pathway

* Prevents cellproliferationduring chronicinfection

Vpu 16 Phosphorylated Cytoplasm ? UBP (69)e 0 Enhances viralexport frominfected cells viaion channelformation

* Promotes CD4degradation inER

aNef appears to compete for a CD4 binding site with the p561ck tyrosine kinase to induce endocytosis of CD4.bVif may interact with two uncharacterized cellular factors: (i) a membrane-associated protein which stably anchors Vif on themembrane surface and (ii) a vimentin-associated factor capable of moving a Vif-containing pre-integration complex along inter-mediate filaments.'Rip-1 is a cytosolic protein that is translocated to the nucleus by Vpr. Rip-I coprecipitates with Vpr and the glucocorticoid recep-tor.dRIP is also reported to be involved in Vpr nuclear transport but the exact relationship of Rip-I and RIP has not yet been estab-lished. The fact that both Rip-I and RIP appear to be involved in Vpr nuclear transport has led to the speculation that the latterrepresents a multimeric form of Rip-i.eUBP shares similarities with the immunophilin family of proteins that includes the cyclophilins and the FK506 binding proteins.It has been speculated that UBP may be involved in the ion channel formation capability of Vpu.

Cancer Center; Dana Gabuzda, Dana Farber Can-cer Institute; John Guatelli, UCSD and the SanDiego Veterans Affairs Medical Center; M. AbdulJabbar, The Cleveland Clinic Foundation; JohnKappes, UAB; Nathaniel Landau, Aaron Dia-mond AIDS Research Center; Jon Marsh, Na-tional Institute of Mental Health; Antonio Pan-

ganiban, University of Wisconsin; VincentePlanelles, UCLA School of Medicine and JonssonComprehensive Cancer Center; Ulrich Schubert,National Institute of Allergy and Infectious Dis-eases; Jacek Skowronski, Cold Spring HarborLaboratories; Klaus Strebel, National Institute ofAllergy and Infectious Diseases; Didier Trono,

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The Salk Institute for Biological Studies; RobertVigne, INSERM; David Weiner, University ofPennsylvania; and Lingjun Zhao, University ofKansas Medical Center. We also thank OpendraSharma for helpful suggestions on the programand Carl Dieffenbach for critical reading of themanuscript.

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