Immunology Letters 78 (2001) 195–200

Deletion of N-terminal myristoylation site of HIV Nef abrogatesboth MHC-1 and CD4 down-regulation

Bo Peng, Marjorie Robert-Guroff *Basic Research Laboratory, National Cancer Institute, National Institutes of Health, 41 Libary Dri�e, Building 41 Room d804, Bethesda,

MD 20892-5055, USA

Received 15 May 2001; received in revised form 5 July 2001; accepted 9 July 2001

Abstract

HIV-1 Nef is a desirable vaccine component because it is expressed early and abundantly during HIV infection, and containsmany CTL, T-helper cell, and B-cell epitopes. Nef, however, down-regulates MHC-1 and CD4 cell surface expression,contributing to viral escape from host immunity. To prevent Nef from down-regulating both MHC-1 and CD4 while preservingmost CTL epitopes, a panel of Nef mutants was constructed and assessed. Some mutants, as expected, modulated either MHC-1or CD4 expression. Others prevented down-regulation of both proteins but sacrificed numerous immunogenic epitopes. Deletionof 19 N-terminal amino acids including the myristoylation signal from Nef completely abrogated both MHC-1 and CD4down-regulation while preserving most CTL, T-helper and B-cell epitopes. Our results demonstrate that the myristoylation signalin the Nef protein is critical for Nef-mediated endocytosis of both MHC-1 and CD4. Non-myristoylated Nef containing a fullcomplement of CTL epitopes has greater potential as a vaccine component than wild-type Nef. © 2001 Elsevier Science B.V. Allrights reserved.

Keywords: HIV Nef; Down regulation; Myristoylation

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1. Introduction

Nef, a regulatory protein of human and simian im-munodeficiency viruses (HIV and SIV), is required forhigh-titer virus replication in vivo, and critical for thedevelopment of AIDS. Nef- deficient SIV have failed toproduce AIDS in infected rhesus macaques [1]. More-over, human long term non-progressors infected byHIV-1 isolates with a deleted or truncated nef genehave remained disease free with normal CD4 countsmore than 15 years after infection [2–4].

In addition to its biologic importance, Nef is anattractive vaccine component. It is one of the earliestand most abundantly expressed HIV proteins [5]. Thenef genes of HIV and SIV are highly conserved, sug-gesting a strong immunity to Nef would be broadlyeffective against numerous viral isolates. During naturalHIV-1 infection, Nef-specific immunity helps control

HIV infection. A cellular immune response to Nef wasassociated with protection of highly exposed heterosex-ual contacts against HIV infection [6], and CTL againstHIV antigens, including Nef, were detected in unin-fected children of HIV-infected mothers [7]. In the SIVrhesus macaque model, Nef-specific CTLs are inducedwithin days of SIV infection and contribute to thedecline of acute viremia [8]. SIV Nef immunization canalso contribute to protective efficacy. An inverse corre-lation was found between Nef-specific CTL precursorfrequency and virus load post-challenge in rhesusmacaques immunized with recombinant vaccinia ex-pressing SIV nef and subsequently challenged with SIV[9]. Overall, vaccine-induced Nef-specific CTL re-sponses should improve HIV vaccine efficacy.

Some properties of Nef, however, may significantlyreduce the efficacy of a Nef-based vaccine. Nef down-regulates CD4 and MHC-1 on the surface of host cellsby accelerating their endocytosis [10]. Both these func-tions could result in poor T-helper cell and CTL re-sponses to antigenic stimuli, leading to diminution of

* Corresponding author. Tel.: +1-301-496-2114; fax: +1-301-496-8394.

E-mail address: guroffm@exchange.nih.gov (M. Robert-Guroff).

0165-2478/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved.PII: S 0 1 6 5 -2478 (01 )00250 -4

B. Peng, M. Robert-Guroff / Immunology Letters 78 (2001) 195–200196

both cellular and humoral responses. Resistance ofHIV-infected primary T-cells expressing Nef to CTLkilling has been correlated with Nef-mediated down-regulation of MHC-1 [11].

To select an optimal Nef construct for vaccine use,we developed a panel of HIV-1 Nef mutants and inves-tigated both CD4 and MHC-I down regulation. Ourstudy demonstrates that deletion of the N-terminalmyristoylation signal of Nef completely prevents bothMHC-1 and CD4 down-regulation, while preservingalmost all CTL, T- helper and antibody epitopes. Thisnon-myristoylated Nef should be able to generate amuch greater CTL response in vivo.

2. Materials and methods

2.1. Generation of nef expression �ectors

pT7consnefhis6 contains a consensus HIV-1 nef genebased on 54 HIV-1 patient isolates [12]. The expressionvectors of NefCONSENSUS and the modified nef genes,Nef�1–19 and Nef�150–206, were constructed by cloningnef genes into a bicistronic expression vector, pIRES2-EGFP (Clontech, CA). PCR primer 1: 5�-CCGCTA-GCAACACCATGGGTGGCAAGTGGTCAAAACG-T-3� and primer 2: 5�-GAATTCTAGACTGCAG-TAATCAGCAGTCTTTGTAGTACTC-3� were usedfor NefCONSENSUS cloning. Nef�1–19 was amplifiedusing primer 2 and primer 3: 5�- CCGCTAGCAACAC-CATGAGGCGAGCTGAGCCAGCAGCAGA-3�, andNef�150–206 using primer 4: 5�-GAATTCTAGACTG-CAGTAATCACTCTGGCTCAACTGGTACTAGT-TTGAA-3� and primer 1. The amplified nef gene se-quences were inserted into the unique NheI and EcoRIrestriction enzyme sites of pIRES2-EGFP. The expres-sion vectors of Nef(AxxA)4, NefEEEE65AAAA/(AxxA)4 andNefDD175AA/E179A were generated by mutagenesis ofpNefconsIRES2-EGFP using the QuikChange site-di-rected mutagenesis kit (Stratagene, CA). All nef muta-tions were verified by DNA sequencing.

2.2. Assay of MHC-I down-regulation

MHC-I expression was analyzed by flow cytometry.293T cells were analyzed 48–72 h after transfectionwith Nef-expressing vectors using Effectane transfec-tion reagent (Qiagen, CA) as specified by the manufac-turer. The cells were stained with PE-conjugatedanti-human HLA class I antigen, clone W6/32 (Accu-rate Chemical & Scientific Co., NY), specific for theassembled class I MHC heavy chain �2–microglobulincomplex, or PE-conjugated anti-human HLA-A, B andC, clone G46-2.6 (Pharmingen, CA), specific for class IMHC heavy chain. Similar results were obtained withboth antibodies.

Fig. 1. Flow cytometric analysis of MHC-1 expression on 293T cellstransfected with NefCONSENSUS, Nef mutants, or empty control vec-tor.

2.3. Assay of CD4 down-regulation

293T cells were co-transfected with pCD4neo (a hu-man CD4 expression plasmid [13]) and the nef-expres-sion plasmids. Forty-eight hours later the cells wereharvested, washed, stained with PE-conjugated anti-CD4 monoclonal antibody (Becton Dickson), and sub-jected to FACS analysis.

3. Results

3.1. Design of mutated HIV-1 Nef proteins

A consensus Nef [12] was selected as representativeof the spectrum of Nef variants against which vaccine-

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Fig. 2. The effect of Nef mutants on down-regulation of CD4 on the surface of 293T cells. Error bars represent one standard deviation calculatedfrom three experiments.

elicited immunity would be necessary. The myristoylationsite, required for plasma membrane attachment of Nef[14] and perhaps sufficient for directing the protein to theplasma membrane [15], was targeted by deleting thecoding sequence for amino acids 1–19. Although thereis no direct evidence indicating that myristoylation is akey factor in down-regulation of MHC-1, it has beenlinked to CD4 down-regulation [13,16,17]. As endo-cytosis involves interactions among membrane-bound proteins, we postulated a simple N-terminaldeletion should prevent down regulation by both CD4and MHC-1. Moreover, mutations involving a portionof the �-helical region of Nef at amino acids 6-22 [18] havebeen implicated in MHC-1 down-regulation [19].

Highly conserved amino acids in the core ofNef, including a region with acidic charge and aproline-repeat sequence [(PxxP)4 motif] were alsotargeted. Two mutations were made: Nef(AxxA)4 andNefEEEE65AAAA/(AxxA)4. The acidic region has beenimplicated in down-regulation of both CD4 and MHC-1[13,20] and the proline repeat in down-regulation ofMHC-1 [20].

Nef interacts with a Nef-binding protein (NBP1), avacular ATPase, which facilitates the internalization ofCD4 [21]. An acidic cluster at the C-terminus of Nef(consensus DDEE) is required for the Nef–NBP1interaction. A mutation in this region, NefDD175AA/E179A,was created to test if this acidic cluster is also involvedin endocytosis of MHC-1.

Finally, a C-terminal truncation, Nef�151–206, wasprepared to define the minimal protein backbone requiredfor MHC-1 and CD4 down-regulation.

Expression levels of Nef mutants assessed by Westernblotting were high, and except for Nef�151–206 which ex-hibited a lower level, equivalent to that of NefCONSENSUS

(data not shown).

3.2. Myristoylation signal is required for Nef-induceddown-regulation of MHC-1

To assess the ability of Nef mutants to down-regulateMHC-1, a transient and transfection assay was used[22,23], followed by flow cytometry. Transfection of 293cells with NefCONSENSUS significantly reduced surfaceexpression of MHC-1 (Fig. 1). In contrast, Nef�1–19

exhibited no Nef-induced MHC-1 down-regulation, aswas also seen with the C-terminal truncated mutant,Nef�151–206. Consistent with other reports [19,20], themutation corresponding to the conserved central coreof the Nef protein, NefEEEE65AAAA/(AxxxA)4, totally im-paired down-regulation of MHC-1, while a mutationsolely in the proline repeat region, Nef(AxxA)4, onlypartially abrogated MHC-1 down-regulation.NefDD175AA/E179A, had no effect on MHC-1 down-regulation.

3.3. Mutational analyses of Nef-induced down-regulationof CD4

The modulation of CD4 cell surface expression byNefCONSENSUS or Nef mutants was evaluated using aquantitative assay [13]. The nef-expressing constructswere co-transfected with pCD4neo into 293 cells, andthe CD4 level on the surface of the cells expressing bothCD4 and EGFP was analyzed by flow cytometry. Asshown in Fig. 2, the HIV-1 NefCONSENSUS reduced CD4cell surface expression by an average of 44%. Nef�1–19

and Nef�151–206, totally abrogated Nef-induced CD4down-regulation, while NefDD175AA/E179A partially abro-gated down-regulation by 33%. The mutation targetingthe conserved central core of Nef, Nef(AxxA)4, had noimpact on CD4 down-regulation as previously reported

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Fig. 3. HIV-1 Nef, Nef mutants constructed for this study, and Nef immunogenic epitopes [28]. Mapped epitopes are located on the HIV-1HXB2

Nef sequence and are expected to be similarly mapped in NefCONSENSUS. Nef mutants are indicated in black below the sequence. CTL epitopesare marked with blue arrows; B-cell epitopes with pink arrows; T-helper epitopes with green arrows.

B. Peng, M. Robert-Guroff / Immunology Letters 78 (2001) 195–200 199

[13], however, down-regulation was partially preventedby NefEEEE65AAAA/(AxxxA)4.

4. Discussion

CTL responses play a central role in controlling viralreplication and modulating disease progression in HIVinfection [24]. Yet HIV has evolved numerous mecha-nisms for evading host immune defenses and establish-ing persistent infection. Because natural killer (NK)cells preferentially lyse cells lacking MHC-I expression,the selective down-regulation by Nef of HLA-A andHLA-B, but not HLA-C and HLA-E, is a clever meanswhereby HIV avoids both CTL- and NK cell-mediatedlysis of infected cells [25]. Nef-induced CD4 down-regu-lation is another strategy by which HIV avoids hostdefenses. CD4 is required for both activation and matu-ration of T-helper cells. IL-2 secretion by activatedCD4+ T-helper cells is necessary for generation of fullCTL effector function. Nef-induced down-regulation ofCD4 could therefore result in poor T-helper cell andCTL responses to antigenic stimuli.

Here, we attempted to design a Nef protein whichwould maintain its immunogenicity but lose its abilityto down-regulate MHC-1 and CD4. Down-regulationof MHC-1 and CD4 have been reported to involvedifferent regions of the Nef protein [14–20,23,26].These earlier studies did not examine down regulationof both CD4 and MHC-1 by the same mutants, and weexpected some mutations might affect both proteins.However, since down-regulation of both molecules re-quires anchoring of Nef at the cell membrane, andmyristoylation of Nef is a determinant for this an-choring, we tested the hypothesis that deletion of theN-terminal myristoylation signal would prevent bothCD4 and MHC-I down-regulation. Our results provethis hypothesis and extend earlier observations byshowing that removal of the Nef myristoylation site notonly prevents CD4 but also MHC-1 down-regulation.

In our study, some Nef mutants were selectivelyaffected in their abilities to modulate either MHC-1(Nef(AxxA)4; Fig. 1) or CD4 (NefDD175/AA/E179A; Fig. 2) asexpected from previous reports. Other mutations, how-ever, inclucing NefEEEE65AAAA/(AxxxA)4 and Nef�151–206

prevented down-regulation of both molecules (Figs. 1and 2). Examination of known immunogenic epitopesof Nef indicates that a significant number of CTL,T-helper, and/or B-cell epitopes would be altered as aresult of these mutations (Fig. 3), possibly reducing oreliminating CTL recognition of wild-type Nef in vivo.In contrast, the non-myristoylated Nef�1–19 should bean optimal vaccine component since it retains almost allidentified CTL, T-helper and B-cell epitopes but totallyabrogates both MHC-1 and CD4 down-regulation.Further, targeting antigens to the cytoplasm of cells can

enhance their degradation and stimulate both CTLrecognition and induction of new CTL responses whenthe molecules are administered as vaccines [27], suggest-ing a further potential benefit of a non-myristoylatedNef as a vaccine component.

Acknowledgements

We thank Dr. Warner Greene for the pCD4neoplasmid, and Dr. Brian Rogers for editorial assistance.The following reagents were obtained from the AIDSResearch and Reference Reagent Program, NationalInstitute of Allergy and Infectious Diseases:pT7consnefhis6 from Dr. Ron Swanstrom; rabbit anti-HIV-1BH10 Nef from Division of AIDS, NIAID.

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