Expression of a Modified Form of CD4 Results in the Release of an Anti-HIV Factor-Derived from the Sequence

This information is current as Irene Zaldívar, María Ángeles Muñoz-Fernández, Balbino of October 1, 2021. Alarcón and Ester San José J Immunol published online 24 June 2009 http://www.jimmunol.org/content/early/2009/06/24/jimmuno l.0802944 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published June 24, 2009, doi:10.4049/jimmunol.0802944 The Journal of Immunology

Expression of a Modified Form of CD4 Results in the Release of an Anti-HIV Factor-Derived from the Env Sequence1

Irene Zaldívar,* María A´ ngeles Mun˜oz-Ferna´ndez,† Balbino Alarco´n,2* and Ester San Jose´*

We have studied the inhibitory effect of a CD4 chimera (CD4␧15) on HIV replication. This chimera is retained in the endoplasmic reticulum and traps the HIV envelope precursor gp160, preventing its maturation. Retroviral expression of the chimera strongly inhibited HIV replication even when it is expressed by only a minority of the T cell population. This protective effect on bystander nontransduced cells is mediated by a soluble factor that we identified as a fragment of HIV gp120 envelope protein and accord- ingly, we named this factor Env-derived antiviral factor (EDAF). Biochemical and immunoreactivity data show that EDAF is comprised of the gp120 C3-C5 regions and indeed, a recombinant protein bearing this sequence reproduces the anti-HIV prop- erties of EDAF. Surprisingly, three tryptic peptides derived from EDAF are homologous but not identical with the corresponding sequences of the HIV isolate used to generate EDAF. We propose that EDAF results from an alternative intracellular processing of the Env protein provoked by its association to CD4␧15 and the selection of the best fitted Env protein sequences contained Downloaded from within the HIV isolate. The presence of EDAF improves the therapeutic potential of the CD4␧15 gene and it opens new possibilities for antiviral treatment and vaccine development. The Journal of Immunology, 2009, 183: 0000–0000.

he therapeutic strategies currently used to combat HIV ing cells to express genes with anti-HIV activity has been proposed infection were developed in the early 1990s. In general, as a potential treatment for AIDS patients, and a steady stream of T they rely upon the inhibitory influence of at least three gene-based interventions using different strategies have been de- http://www.jimmunol.org/ different small molecules on one or two enzymes vital for HIV scribed in cell culture experiments (8, 9). replication: and protease. The combination of The life cycle and replication of HIV begins when the HIV these potent drugs, known as highly active antiretroviral therapy envelope glycoprotein (gp120) recognizes the CD4 molecule and (HAART),3 has greatly improved the prognosis for patients with the CCR5 or CXCR4 coreceptors (10). The envelope glycoproteins HIV infection (1–3). However, HAART does not eradicate the are synthesized as a 160 kDa precursor in the endoplasmic retic- virus, which remains latent in cellular reservoirs, implying that this ulum (ER), and this precursor is proteolytically processed by cel- therapy must be administered continually. If we also consider the lular proteases in the Golgi apparatus (11) to produce mature severe side effects of the drugs and the emergence of HIV drug- gp120 and present in the virion. The processing of gp160 is resistant mutants (4–6), it is clearly worthwhile searching for al- inefficient due to the formation of intracellular complexes with by guest on October 1, 2021 ternative anti-HIV strategies. CD4 and only 10–15% of the precursor molecule is processed and Molecules that specifically block the binding of the HIV enve- exported to the plasma membrane. The excess of both proteins is lope protein (gp120) to its receptors, as well as the fusion process, degraded in lysosomes (12) and in the case of CD4, by a mecha- constitute a new class of receptor-based therapeutic agents for HIV nism dependent on the HIV-1 protein Vpu (13). type 1 (HIV-1) infection and gp120-mediated pathogenesis (7). In We have already shown that the expression of a CD4 chimera addition to inhibitors of entry and fusion, is another that is retained in the ER, CD4␧10, inhibits HIV-1 replication (14). alternative approach to HAART. The idea of genetically modify- The interaction of gp160 with CD4␧10 appears to provoke its retention in the ER, thereby preventing its maturation and export to the Golgi apparatus. This chimera is composed of the complete *Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones CD4 molecule with 10 aa of the CD3␧ ER retention signal ap- † Científicas-Universidad Auto´noma de Madrid, and Laboratorio de Inmuno-Biología, pended to its C terminus. Stable expression of CD4␧10 in T cell Hospital Universitario Gregorio Maran˜o´n, Madrid, Spain lines protected them from the cytopathogenic effects of HIV. Fur- Received for publication September 12, 2008. Accepted for publication May 7, 2009. thermore, transduction of human T lymphoblasts from seropositive The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance individuals with retroviral particles derived from the Moloney vi- ϩ with 18 U.S.C. Section 1734 solely to indicate this fact. rus inhibited the depletion of the CD4 population. However, 1 This work was supported by Grants SAF2003-04137 and SAF2006-01391 from the these studies were performed in suboptimal conditions, using a “Comisio´n Interministerial de Ciencia y Tecnología,” Grants 08.2/0027.1/2003 and retroviral vector that did not allow the transduced cells to be fol- S-SAL-0159-2006 from the “Comunidad de Madrid,” and Grants QLK2 2000-01040 4 and LIFE/STREP/03/0594 from the “Fundacio´n Rodríguez Pascual” and from the lowed, and with low titer retroviral supernatants (10 PFU/ml). We European Union. The institutional support of “Fundacio´n Ramo´n Areces” to the Cen- have studied a bicistronic vector that coexpresses a longer version tro de Biología Molecular is also acknowledged. of the CD4 chimera containing the full ER retention signal of 2 Address correspondence and reprint requests to Dr. Balbino Alarco´n, Centro de CD3␧ (CD4␧15) (15) and a GFP marker. Using these vectors, we Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas- ␧ Universidad Auto´noma de Madrid, Madrid 28049, Spain. E-mail address: balarcon@ show that CD4 15 expression in a small percentage of T cells cbm.uam.es exerts a potent antiviral effect on bystander nontransduced cells. 3 Abbreviations used in this paper: HAART, highly active antiretroviral therapy; This result has permitted us to identify an antiviral factor that is EDAF, Env-derived antiviral factor; ER, endoplasmic reticulum; MOI, multiplicity of released by the CD4␧15-expressing T cells upon infection with infection; HA, hemagglutinin; MS, mass spectrometry; HIV-1, HIV type 1. HIV. We have identified and characterized this new antiviral fac- Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 tor, which is the main focus of this study.

www.jimmunol.org/cgi/doi/10.4049/jimmunol.0802944 2 ANTIVIRAL EDAF

Materials and Methods Purification and characterization of the antiviral factor by Retroviral vectors and plasmids chromatography The pCIE15 and pD15 constructs were obtained by cloning CD4␧15 into Size exclusion. A 250 ml Sephacryl S-200 HR column (Amersham) was the bicistronic retroviral vectors pCIE (pLZR-CMV-gfp) (16, 17) provided calibrated with a commercial kit (Amersham), 20 ml of the antiviral su- by Dr. A. Bernard (Centro Nacional de Biotecnología, Madrid, Spain) and pernatant was loaded onto the column and then 20-ml aliquots were MP91 provided by Dr. D. von Laer (Institute for Biomedical Research, collected. Frankfurt, Germany) (18). To express the fragments of gp120, they were Con A-Sepharose column. A total of 2 ml of antiviral supernatant was cloned into the pSR␣-HA vector that harbors a leader sequence (19). For incubated with 500 ␮l of a Con A-Sepharose slurry (Sigma-Aldrich) and some experiments that required purification, construct III was tagged at the the soluble factor was eluted with increasing concentrations of ␣-methyl- C terminus with a stretch of 6 histidine residues. mannopyranoside (Sigma-Aldrich). Cells and Abs Cation exchange column. The antiviral supernatant was passed through an Econo-Pac High S Cartridge (Bio-Rad). The column was washed with All the CD4ϩ T lymphoblastic cell lines used in the infection experiments MOPS 20 mM (pH 7.4), and the bound proteins were eluted with increas- (Jurkat, PM1, and MT2) were obtained from the American Type Culture ing sodium chloride concentrations. Collection. The cell clone C10 was obtained through the limiting dilution of MT2 cells transduced with the pCIE15 construct and selected for GFP Immunoprecipitation and Western blot analysis expression. These cells were maintained in RPMI 1640 plus 5% heat- COS-7 cells were transfected by electroporation with either the empty vec- inactivated FBS. Peripheral mononuclear cells from healthy donors were tor (pSR␣-HA) or with the pSR␣-HA vector expressing a C-terminal hexa- isolated using Ficoll-Hypaque (Amersham Biosciences) that were stimu- ␣ ␮ histidine tagged construct III (pSR -HA-III-His6). The COS cells were lated with 1 g/ml phytohematogglutinin (Sigma-Aldrich) and recombi- lysed for 30 min on ice in 10 mM Tris-HCl (pH 7.6), 150 mM NaCl, 2 mM nant IL-2 (100 U/ml; Roche) at 37°C for 48–72 h, and propagated in EDTA, and 0.5% Brij 96 (Sigma-Aldrich) 48 h after transfection in the complete medium in the presence of 100 U/ml IL-2. presence of protease inhibitors (1 ␮M leupeptin, 1 ␮M aprotinin, 1 mM Downloaded from The polyclonal anti-gp160 antiserum was provided by G. del Real (In- PMSF, and 10 mM iodoacetamide). The cleared lysates, as well as the vestigacio´n y Tecnología Agraria y Alimentaria, Madrid, Spain) and it was supernatant from transfected COS cells, were precipitated overnight at 4°C raised against the purified gp160 protein. Abs against different regions of with Ni-NTA agarose beads (Qiagen). The precipitates were washed and gp120 (C1-ARP 3076, C3-ARP 3051, C4-ARP 388, C5-ARP 3221, V3- incubated with a lysate of 8 ϫ 106 Jurkat cell lysates overnight at 4°C. The EVA 331), anti-p24 (ARP 313), and anti-p17 (ARP 3057) were obtained beads were then washed, boiled in Laemmli buffer, resolved on a 10% from the National Institutes of Health AIDS Research and Reference Re- SDS-PAGE gel under reducing conditions, and transferred to nitrocellulose agent Program. The PE-coupled anti-human CD4, CD69, CD25, and

membranes (Bio-Rad). The membranes were subsequently incubated with http://www.jimmunol.org/ CXCR4 Abs were purchased from BD Pharmingen. The H-370 anti-CD4 anti-CD4 or anti-gp160 Abs for1hatroom temperature, followed by Ab used to probe Western blots was from Santa Cruz Biotechnology, and peroxidase-labeled goat anti-rabbit IgG (1/7000; Pierce) for 30 min at room the Leu-3a anti-CD4 Ab labeled with APC was purchased from BD temperature, and Ab binding was visualized by ECL (SuperSignal West Biosciences. Dura Extended Duration Substrate; Pierce). HIV strains and infection In other experiments, the purified construct III protein bound to Ni-NTA beads was eluted with 200 mM imidazole and used in competition exper- The T tropic HIV-1 strain NL4.3 (20) was provided by Dr. J. Alcamí iments with the Leu-3a anti-CD4 Ab. Briefly, MT2 cells were incubated (Instituto de Salud Carlos III, Madrid, Spain), whereas the CBL23 HIV-2 with a 1/10 dilution of the eluted protein III at 4°C for 30 min. Afterward, strain was obtained from the National Institutes of Health AIDS Research the cells were stained with Leu-3a directly labeled with APC and analyzed and Reference Reagent Program. All strains were grown in MT2 cells and in a FACSCalibur (BD Biosciences) flow cytometer. their titers were determined using the end point dilution method (20, 21). by guest on October 1, 2021 The Ba-L M tropic strain (National Institutes of Health AIDS Research and Results Reference Reagent Program) was grown on blood human monocytes. In- ␧ fection experiments were conducted as previously described (14, 21). Viral Expression of CD4 15 does not affect the surface expression of replication was monitored by measuring the HIV-1 p24 Ag in the super- endogenous CD4 or T cell activation natant of the cultures by ELISA (Innogenetics) and by visualizing the cy- Because the CD4␧15 chimera could potentially interact with en- topathic effect produced by the virus. Viral load was measured by RT-PCR amplification of gag using Amplicor HIV-1 (Roche). dogenous CD4 in transduced T cells, impairing both CD4 ex- pression at the plasma membrane and CD4ϩ T cell function, we Cell transfections and transductions assessed whether transduction of CD4␧15 influenced CD4 ex- Vectors were produced and transduced as previously described (17), pression. MT2 cells transduced with pCIE15, a vector coex- and transient transfection of COS-7 and Jurkat cells was achieved by pressing CD4␧15 and GFP, expressed similar levels of surface electroporation. CD4 to these of nontransduced cells or cells transduced with the Activation experiments empty vector (Fig. 1A). Furthermore, CD4 expression was iden- tical in GFP-negative and GFP-positive cells. Expression of the Ag stimulation of the TCR complex was conducted by incubating the ␧ CH7C17 derivative of Jurkat T cells at 37°C in the presence of HLA-DR1- CXCR4 HIV coreceptor was also unaltered by CD4 15 trans- transfected DAP murine fibroblasts (DAP-DR1) at an APC to T cell ratio duction (Fig. 1A). of 1:1. APCs were preincubated overnight at 37°C with increasing con- To assess whether transduction of CD4␧15 exerts a negative centrations of influenza hemagglutinin (HA) peptide 307–319 in RPMI effect on CD4 function during T cell activation, we transduced the 1640 medium supplemented with 1% FBS. The cells were stimulated for ϩ 24 h to detect CD69 and for 48 h to detect CD25. After stimulation, the CD4 CH7C17 T cell line and followed its activation in response cells were transferred to ice-cold PBS, washed twice, and stained with Abs to a MHC class II-presented Ag. The CD25 and CD69 activation against CD69 and CD25 directly labeled with PE. The samples were an- markers were induced in CD4␧15-transduced cells at levels com- alyzed in a FACSCalibur (BD Biosciences) flow cytometer, and the mean parable to the control cells transduced with the empty vector (Fig. fluorescence intensity was measured at each point for the green cells ␧ 1B). Furthermore, the release of IL-2 into the culture supernatant (CD4 15- or mock-transduced cells). The supernatants from these cultures ␧ (24 h after stimulation) were analyzed to detect IL-2 by ELISA (Cultek). in response to Ag was not affected by CD4 15 expression (Fig. 1C). These results indicate that CD4␧15 expression does not in- Purification of the antiviral factor by density gradients terfere with TCR-driven T cell activation. The culture supernatant from CD4␧15-transduced and HIV-infected MT2 cells was ultracentrifuged at 100,000 ϫ g for 90 min at 4°C. The pellet was Expression of CD4␧15 exerts a protective effect on bystander resuspended in 1.5 ml of PBS and layered on a discontinuous 6–18% nontransduced T cells gradient of iodixanol (OptiPrep; Invitrogen). Ultracentrifugation was per- formed in a 70.1 Ti rotor (fixed angle) at 250,000 ϫ g for 90 min at 4°C, To study the potential anti-HIV effect of the CD4␧15 chimera, the and the fractions were taken from the top of the tube. human MT2 and Jurkat T cell lines, as well as human blood T cell The Journal of Immunology 3

CD4␧15 was also stronger than expected in primary T cells in- fected with the X4 and R5 strains, given that only 25% of the cells were transduced (Fig. 2, A and B). Furthermore, we detected a dose-dependent effect, both in terms of p24 release and of CD4ϩ T cell protection, when human T lymphoblasts expressing CD4␧15 with different efficiencies were infected with the 1936 clinical iso- late (Fig. 2C). Indeed, the percentage of CD4ϩ T cells recovered to the levels of uninfected cells. The strong reductions obtained in HIV replication and the complete protection of the cell population when only a minority of the cells was transduced suggests the existence of a protective bystander effect on nontransduced cells. An alternative explanation is that the number of transduced cells was underestimated and that the percentages of cells expressing CD4␧15 was higher than that of GFP-positive cells detected by flow cytometry. To exclude this possibility and to demonstrate the existence of a bystander effect, mixed cultures of CD4␧15-express- ing C10 cells (a cell clone from CD4␧15-transduced MT2 cells) and parental MT2 cells were infected with HIV. Complete protec- tion against HIV replication could be observed even when as few as 10% of the cell population expressed CD4␧15 (Fig. 2D). This Downloaded from result unambiguously demonstrated that the expression of CD4␧15 in a small population of T cells exerted a protective bystander effect on nontransduced cells. Cells expressing CD4␧15 and infected with HIV produce a

soluble factor that prevents the replication of HIV in http://www.jimmunol.org/ nontransduced cells Because T cells acquired resistance to HIV-1 when only a few of them carried the therapeutic CD4␧15 protein, a soluble factor that offers protection to untransduced cells may be released. To deter- mine whether this might be the case and to exclude the need for cell-to-cell contact, experiments were performed in culture plates with two chambers separated by a 0.4-␮m pore membrane that permits the diffusion of macromolecules but not of cells (Boyden FIGURE 1. The presence of CD4␧15 in T cells does not affect the ex- by guest on October 1, 2021 pression of CD4 and CXCR4, or T cell activation. A, MT2 cells transduced chambers). Transduced and untransduced cells were infected and or not with CD4␧15 or the empty vector (GFP) were stained with PE- incubated in these chambers (as shown in Fig. 3), and HIV repli- labeled Abs against CD4 and CXCR4. Flow cytometry analysis is shown cation in the MT2 cells in the lower chamber was strongly reduced in two colors. B, The CH7C17 T cell line was transduced with CD4␧15 or when CD4␧15-expressing cells were grown in the upper chamber the empty vector, and afterward, the cells were stimulated with the DAP- rather than parental MT2 cells (Fig. 3). This result was indepen- DR1 APC line loaded with increasing concentrations of the HA peptide. dent of the viral isolate (NL4.3, HIV-1; CBL23, HIV-2) or of the After 24 and 48 h, the cells were recovered and stained with Abs against T cell line (MT2, PM1) used. Therefore, CD4␧15-expressing cells CD69 and CD25, respectively. CD69 and CD25 expression are shown as appear to release an antiviral factor that diffuses into the lower the mean fluorescence intensity. C, The supernatants from the same ex- periments described in B were tested for the presence of IL-2 by ELISA. chamber, protecting nontransduced cells from HIV infection. The antiviral factor is a medium-sized, positively charged and lymphoblasts, were transduced with the CD4␧15-expressing con- particle-associated glycoprotein structs pCIE15 or pD15. Although the efficiency of transduction To characterize the antiviral factor, we first determined whether was not always 100% (Fig. 2A), we tested the susceptibility of the the antiviral factor was constitutively secreted by CD4␧15-ex- transduced cell population to HIV infection. Transduced cell pop- pressing cells or whether it was released upon infection. We found ulations were infected with the T-tropic (X4) strain NL4.3 or with that the antiviral factor was present in the supernatant of MT2 cells the M-tropic (R5) Ba-L strain of HIV-1 at low multiplicity of transduced with CD4␧15 and infected with HIV but not in that of infection (MOI, 10Ϫ3 or 10 ng, respectively). When HIV replica- nontransduced or noninfected cells (Fig. 4A). Hence, both CD4␧15 tion was measured by the levels of p24 Ag in the culture super- expression and HIV infection are required for release of this an- natant, transduction of CD4␧15 protected both cell lines against tiviral factor. Furthermore, the antiviral factor appears to be re- HIV-1 infection during the experiment (Fig. 2B). Interestingly, leased late into the supernatant as the maximal activity was not there was a 10,000-fold reduction in p24 release from MT2 cells detected until 24 h postinfection (Fig. 4B). (Fig. 2B), even if only 33% of the cell population was transduced To characterize the antiviral factor, the supernatant of CD4␧15- (Fig. 2A). A similar effect was also observed in Jurkat cells when transduced MT2 cells infected with HIV was subjected to different only 56% of the cells were transduced (Fig. 2, A and B). A pro- treatments. The antiviral factor appears to be resistant to temper- tective effect was also detected when MT2 cells were infected with ature as heating the supernatant to 56° or 70°C did not affect its the primary ME46 isolate (group M, subtype B), the HIV-2 capacity to inhibit HIV replication (Fig. 4C). Because there was a CBL-23 laboratory strain and the chimeric SIV-HIV virus SHIV 2- to 20-fold difference in the p24 concentration between the upper 86.9 strain (data not shown), suggesting that CD4␧15 expression chamber where the antiviral factor is produced and the lower has a broad ranging anti-HIV effect. The protection exerted by chamber into which the antiviral factor diffuses (Fig. 3), we thought 4 ANTIVIRAL EDAF Downloaded from http://www.jimmunol.org/

FIGURE 2. Expression of CD4␧15 exerts a protective effect on bystander nontransduced cells. A, MT2 and Jurkat T cell lines and human T lymphoblasts were transduced with the pCIE15 and pD15 retroviral vectors expressing CD4␧15. The efficiencies of the transduction are shown by flow cytometry according to GFP expression. B, The same cells as in A were infected at a MOI of 0.001 with the T-tropic (X4) strain NL4.3 or with 10 ng of the M-tropic by guest on October 1, 2021 (R5) strain Ba-L. After several days in culture, the p24 Ag in the supernatant was analyzed by ELISA. A representative experiment of 5 is shown. C, Different populations of CD4␧15-transduced T lymphoblasts were infected at a MOI of 0.001 with the 1936 clinical isolate. The release of the p24 Ag was evaluated 7 days postinfection and it is represented according to the percentage of GFP-positive cells, as well as showing the percentage of living CD4ϩ T cells at this time point. D, Nontransduced MT2 cells and the CD4␧15-expressing C10 clone were mixed at the ratios indicated (i.e., 10% means that 10% of the cells in the culture before infection were C10 and 90% were MT2) and they were infected at an MOI of 0.001 with NL4.3. The course of infection was analyzed according to the p24 released and the viral load. A representative experiment of three is shown. that the antiviral factor could be a slow-diffusing large molecule or than in the cleared supernatant, suggesting that the antiviral factor is, particle. Indeed, when the culture supernatant was ultracentrifuged at or is associated to, a particle (Fig. 4D). 100,000 ϫ g and the antiviral activity concentrated in the pellet rather We tested the antiviral activity of the factor after fractioning the supernatant through a cation exchange column, a lectin column (Con A) and a size exclusion column. Maximal antiviral activity was detected in fractions that corresponded to a positively charged protein eluted with 100 mM NaCl (Fig. 4E), to a glycosylated protein eluted with 200–500 mM ␣-methyl mannoside (Fig. 4F), and to a protein of 25–43 kDa (Fig. 4G).

Identification of the antiviral factor as a fragment of gp120 We used two different methods to identify the antiviral factor based on the properties described above. In the first approach we performed a FIGURE 3. A soluble antiviral factor is released by CD4␧15-expressing series of ultracentrifugations based on the fact that the antiviral factor cells infected with HIV. Transwell experiments with different T cell lines and concentrates in the pellet (Fig. 4D). After subjecting the culture su- viral isolates. MT2 and PM1 cells transduced with retroviral vectors express- pernatant of HIV-infected clone C10 to centrifugation at 100,000 ϫ g, ing CD4␧15 (pCIE15 and pD15) were infected at a MOI of 0.001 with the the pellet was resuspended and centrifuged on an iodixanol density NL4.3 and CLB23 isolates of HIV-1 and HIV-2, respectively, and placed in the indicated chamber of the Boyden chambers. In all cases, both cell popu- gradient, where the antiviral activity concentrated in the intermediate lations (present in the upper and the lower chambers) were infected. The density fractions (fractions 5–7, Fig. 5A). As a control, the culture course of infection was followed by the formation of syncytia and p24 release supernatant of nontransduced HIV-infected MT2 cells was fractioned (in pg/ml) 7 days after infection. Levels of syncytia are not detectable (Ϫ), detect- following the same protocol and no protective effect was detected in able (ϩ), and abundant (ϩϩ). A representative experiment of three is shown. any of these fractions (Fig. 5A). The Journal of Immunology 5

Unfortunately, silver staining after SDS-PAGE of the principal protective fractions from C10 cells did not reveal specific protein bands (data not shown) and thus, the antiviral factor was further purified by ultracentrifuging the fractions from the first density gradient with antiviral activity in a second iodixanol density gra- dient. The fractions from this second gradient were tested for an- tiviral activity and analyzed by SDS-PAGE and silver staining. Maximal protection was coincident with the presence of a 32–35 kDa protein doublet in fraction 10 (Fig. 5B). When both bands were trypsin digested and analyzed by manual interpretation of mass spectrometry (MS)/MS spectra, the only reliable sequence obtained corresponded to a heptapeptide (STWNGTR). This pep- tide had 100% sequence identity with the C3-V4 boundary of gp120 from 93 HIV isolates (Fig. 5C and data not shown). The second approach to purify the antiviral factor involved the serial application of the three chromatography techniques already tested: size exclusion, lectin affinity, and cation exchange. After this last column, the protective and a nonprotective fraction were

resolved by SDS-PAGE and stained with SYPRO-Ruby. Four ap- Downloaded from parently specific bands were detected in the protective fraction (Fig. 5D), of which three corresponded to contaminants (keratins and trypsin). The relevant band (band 2) yielded two peptides of interest that were identified by BLAST analysis as highly homol- ogous to sequences present in region C3 of gp120, or at the V3-C3

boundary in 75 HIV isolates (Fig. 3E, and data not shown). http://www.jimmunol.org/ Because the two different approaches coincided in identifying peptides corresponding to sequences present in the gp120 envelope protein of HIV, the antiviral factor produced by cells expressing the CD4␧15 chimera and infected with HIV appeared to be a frag- ment of gp120. To demonstrate this hypothesis, we first deter- mined whether the gp120 fragment identified by MALDI-TOF was only produced by CD4␧15-transduced cells. We found that a ϳ35

kDa protein recognized by an anti-gp120 polyclonal antiserum was by guest on October 1, 2021 concentrated in the intermediate fractions of iodixanol gradients obtained from culture supernatants of CD4␧15-transduced MT2 FIGURE 4. Characterization of the soluble antiviral factor. A, Clone C10 was infected at a MOI of 1 with isolate NL4.3 and the culture super- cells infected with HIV (Fig. 6A, bottom). However, this protein natant from these cells, as well as that from uninfected C10 and MT2 cells, was not detected in the culture supernatant of nontransduced MT2 was collected 24 h later. These supernatants were added at a 1/4 dilution to cells infected with HIV, even though a thick band corresponding to MT2 cells infected with isolate NL4.3 at a MOI of 0.001, and Ag p24 full-length gp120 was present in the heaviest fractions (Fig. 6A, release was measured 7 days postinfection. Control: MT2 infected in the top). The 35-kDa gp120 fragment was also detected in the culture same conditions without adding any supernatant. B, Clone C10 was in- supernatant of both PM1 and Jurkat cells transduced with CD4␧15 fected at a MOI of 1 and the supernatant was collected at the times indi- (Fig. 6B). At this point, we named this gp120 fragment from Env- cated after infection. The protective effect was analyzed as in A. C, Culture supernatant from MT2 cells transduced with CD4␧15 and infected with derived antiviral factor (EDAF). isolate NL4.3 at a MOI of 1 was collected 24 h postinfection and heated to To study the composition of EDAF in terms of the regions of 56° or 70°C for 30 min. The heated supernatants were tested in the pro- gp120 that make it up, a panel of region-specific mAbs was assayed tection assay as in A. D, The 24-h supernatant from clone C10 infected with in immunoblots after fractionation on iodixanol gradients. We found isolate NL4.3 at a MOI of 1 was ultracentrifuged at 100,000 ϫ g. The pellet that although an anti-C1 Ab reacted with gp120 but not with EDAF, and the cleared supernatant were assayed for antiviral activity as in A.To an anti-C3 Ab and an anti-C4 Ab reacted equally well with both characterize the antiviral factor, the 24-h supernatant from MT2 cells trans- proteins. An anti-V3 mAb reacted with the upper part of the gp120 duced with pCIE15 (60% GFP-positive) and infected with isolate NL4.3 at a MOI of 1 was fractionated on different columns: strongly acidic cation fragment but not with the lower part, suggesting that the doublet ob- exchanger (sequentially eluted with increasing concentrations of sodium served in some gels (e.g., Figs. 5B and 6A, bottom) corresponded to chloride) (E), Con A-Sepharose 4B (sequentially eluted with increasing fragments of gp120 in which the V3 sequence was present or absent concentrations of ␣-methyl mannopyranoside) (F), and gel filtration (G). As- (see Fig. 6C for a summary of the Ab reactivity of EDAF). Because terisk indicates the positions of m.w. standards. All fractions obtained were EDAF pelleted at 100,000 ϫ g, we thought it might form part of a analyzed in the protection assay and all the data are presented as the mean and defective HIV particle, lighter than the full virus (Fig. 4D). However, SD of experiments performed in triplicate. There are some fractions that when Abs against the p24 protein and the p17 matrix protein did not added to MT2 cells in the protection assay produced higher p24 levels than the react with the fractions that contained EDAF (Fig. 6C). Furthermore, control with no fraction added. This is most likely due to an excess of infective HIV in the culture supernatant of EDAF-producing cells (despite the heat the HIV genome was not detected in these fractions by PCR (data not inactivation). To produce EDAF, cells were infected at high multiplicity, shown). These results, suggested that EDAF is not associated to viral whereas for the protection assay, a MOI of 0.001 was used and therefore, little particles or pseudoparticles and thus, the appearance of EDAF in the residual infective virus could produce a difference in such fractions. A repre- pellet may indicate that some of these gp120 fragments could form sentative experiment of three to five experiments is shown. aggregates. 6 ANTIVIRAL EDAF Downloaded from http://www.jimmunol.org/

FIGURE 5. Purification and identification of the antiviral factor. A, The culture supernatant of MT2 cells (f) and clone C10 (Ⅺ) infected at a MOI of 1 was ultracentrifuged at 100,000 ϫ g. The pellet was resuspended and ultracentrifuged on iodixanol density gradients, and the fractions were evaluated in the protection assay. B, The protective fraction obtained from a first gradient was subjected to a second ultracentrifugation in iodixanol density gradient. The fractions were resolved in a 10% SDS-PAGE gel and the proteins were silver stained. A specific doublet of 31–35 kDa detected in fraction 10 is indicated. C, The two protein bands from the previous gel were analyzed by MALDI-TOF and a heptapeptide was assigned by manual interpretation of MS/MS spectra. The homology with gp120 from different HIV isolates is shown. Isolate 1-AAL93522, isolate 2-AAL93512, isolate 3-AAC55434, and isolate 4-AAL9351. D, The culture supernatant from MT2 cells transduced with pCIE15 (60% GFP-positive) and with isolate NL4.3 infected at a MOI of 1 was

serially passed through three different columns under the conditions optimized in Fig. 4: 1) size exclusion, fractions 6 and 7; 2) Con A, fractions 6 and 7; and 3) by guest on October 1, 2021 cation exchange, fraction 4. The protective fraction from the last column, together with a nonprotective one, were separated in a 10% SDS-PAGE gel and stained with SYPRO-Ruby. The bands marked were analyzed by MALDI-TOF. E, Protein band 2 yielded two peptide sequences that were assigned by manual interpretation of MS/MS spectra. BLAST analyses of these sequences with gp120 from different HIV isolates are shown: isolate 5-ABY27422, isolate 6-ABV00711, isolate 7-ABU42771, isolate 8-ABU42787, and isolate 9-AAB17208.

A recombinant protein corresponding to the C3-C5 sequence of differences in glycosylation or to the presence of C-terminal se- gp120 mediates the antiviral effects of EDAF quences from region C5 in construct III not present in EDAF. According to the information gathered from MS/MS and the im- By comparing the sequences of gp120 and the EDAF frag- munoreactivity displayed, EDAF must contain the C3 and C4 but ment (C3-C5) we ventured that the soluble factor might inhibit not the C1 sequences, and it may or may not contain V3. Consid- the interaction of CD4 with gp120. This idea is consistent with ering these restrictions and the size of EDAF (ϳ35 kDa), we in- the fact that the majority of the amino acids of gp120 that in- serted four gp120-derived constructs into an expression vector, teract with CD4 are present in EDAF (Fig. 7D). To test this with or without the V3 region and the gp41 fusion peptide, to hypothesis, recombinant protein III purified from both the cul- determine whether any of them possessed antiviral activity (Fig. ture supernatant and lysate of transfected COS cells was incu- 7A). The fact that the Abs used against the C5 sequence was not bated with Jurkat T cell lysates, showing clearly that CD4 (from informative (Fig. 6C), prevented a more precise definition of the the Jurkat cell lysate) associated with the recombinant protein recombinant protein. The four constructs were transiently trans- III (Fig. 7E). Furthermore, purified construct III also blocked fected into COS cells and the culture supernatants of these cells the staining of MT2 cells with the Leu-3a CD4 mAb known to were assayed for antiviral activity. Unlike the other constructs, compete with gp120 for CD4 binding (Fig. 7F) (22). Together, construct III presented significant anti-HIV activity (Fig. 7B), these data show that EDAF binds CD4 and they suggest that it which was also manifested when construct III was transiently interferes with HIV infection by competing with the gp120- transfected in Jurkat T cells before they were infected with HIV CD4 interaction. (Fig. 7C). Hence, the sequence comprising C3-C5 appears to have anti-HIV activity reinforcing the idea that the antiviral factor pro- Discussion duced by HIV-infected CD4␧15-expressing T cells is a fragment Because the endoproteolytic maturation of the Env precursor of gp120 consisting of the last third of the protein sequence. The protein is a crucial step in the production of viral particles (23), difference in size and mobility of the recombinant protein III (Fig. our initial aim was to develop a strategy in which the stable ex- 7B) when compared with EDAF (Fig. 6, A and B) could be due to pression of a CD4 chimera permanently retained in the ER blocks The Journal of Immunology 7

FIGURE 6. Immunological characterization of EDAF. A, Iodixanol

density fractions obtained as in Fig. 5A were separated in a 10% SDS- Downloaded from PAGE gel, transferred to nitrocellulose membranes and incubated with a polyclonal anti-gp120 antiserum that identified the 35-kDa gp120 frag- ment. The reactivity of the anti-gp120 antiserum with fractions bearing the gp120 present in nontransduced infected MT2 cells is also shown, although these cells do not produce the 35-kDa gp120 fragment. B, The same as A with culture supernatants of PM1 and Jurkat cells transduced with CD4␧15 and infected with HIV. C, Reactivity in Western blots of protecting frac- http://www.jimmunol.org/ tions with Abs specific for different HIV proteins and gp120 regions.

maturation of gp160. Indeed, this key step in the viral cycle is an increasingly attractive target for inhibitor design (24). Because the replication of different HIV strains was strongly inhibited in the presence of CD4␧15 (including laboratory strains, clinical isolates, X4 and R5 strains), it would appear that this construct has good by guest on October 1, 2021 potential as a therapeutic gene. However, more interestingly the FIGURE 7. Evaluation of the antiviral activity of recombinant EDAF. A, ␧ CD4 15 exerts a protective bystander effect on nontransduced Organization of the variable and constant regions of the immature gp160 gly- cells. Indeed, the protective bystander effect of CD4␧15-express- coprotein, and the positions of gp120 corresponding to the three tryptic pep- ing cells is mediated by the release of a soluble antiviral factor tides identified are indicated. The gp120 regions contained in constructs I-IV upon infection with HIV. are shown. Construct I- V3-C5 sequence; construct II- V3-C5 sequence and Several antiviral factors have been described, including chemo- fusion peptide; construct III- C3-C5 sequence; construct IV- C3-C5 sequence kines like CCL5 (25) and cytokines such as IFN-␣ (26). An anti- and fusion peptide. B, Constructs I-IV or empty vector (␾) were transfected viral factor produced by activated CD8ϩ T cells (CD8 antiviral into COS cells and the culture supernatant was collected 48 h postinfection and factor) was first described several years ago (27), although its mo- tested in a protection assay. Data presented are the mean and SD of an exper- iment performed in triplicate. Level of protein expression in immunoblots with lecular characterization is still pending (28, 29). Another anti-HIV ϩ an anti-HA Ab is illustrated (inset). C, Jurkat cells were transiently transfected soluble factor is produced by CD4 T cells from nonprogressing with either the empty vector (control) or with construct III. At 48 hours after seropositive patients (30) and it is also thought that T cells infected transfection, cells were infected with isolate NL4.3 at a MOI of 0.1 and 0.5 and with attenuated virus, such as vif-defective HIV, release a soluble the p24 Ag release was measured 5 days postinfection. D, Ribbon diagram antiviral factor (31). The antiviral factor produced by CD4␧15- representing the tertiary structure of the gp120 monomer. The C3-C5 sequence expressing T cells is released after HIV infection, suggesting that is highlighted in cyan, whereas the CD4 contact sites are shown in red. E, it is either induced or encoded by the viral genome. Using two Construct III bearing a hexahistidine tag or an empty vector (␾) were trans- different biochemical approaches to purify the antiviral factor, we fected into COS cells, and the cell lysates and culture supernatants were in- have identified this factor as a fragment of gp120. According to the cubated with Ni-NTA beads. Afterward, the beads were incubated with a Ju- sequence of three tryptic peptides identified, its immunoreactivity rkat cell lysate and the precipitate was analyzed in Western blots probed with an anti-CD4 Ab. F, Purified protein III obtained as in E and eluted with 200 using domain-specific Abs, and the size of the factor (ϳ35 kDa), mM imidazole was preincubated with MT2 cells on ice before the Leu-3a APC the fragment must correspond to a polypeptide included in the last anti-CD4 Ab was added for 30 min at 4°C. The cells were analyzed by flow third of the gp120 sequence. Furthermore, transfection of a recom- cytometry. MT2 cells treated with purified protein III (red line), MT2 cells binant protein comprising the C3-C5 sequence of gp120 confers treated with empty vector (blue line), and untreated MT2 cells (gray line) are antiviral activity. These results positively identify the antiviral fac- shown. G, Alignment of the three tryptic peptides from EDAF with the gp120 tor as a gp120 fragment, which we have named EDAF. sequence of the NL4.3 isolate. A dash indicates identity and an asterisk indi- The C3-C5 fragment comprises most of the outer domain of cates homology. gp120 and part of the bridging sheet (32–34). Significantly, 21 of the 26 aa of gp120 that make contact with CD4 are present in recognition of the CD4 cell receptor by HIV. Indeed, this factor EDAF. Therefore, most CD4-interacting regions in gp120 are binds to CD4 and it competes with Leu-3a, an anti-CD4 Ab that present in EDAF and thus, we predict that it could interfere with recognizes the gp120-binding site in CD4 (22). 8 ANTIVIRAL EDAF

The mechanism by which EDAF is produced in cells express- 2. Gulick, R. M., J. W. Mellors, D. Havlir, J. J. Eron, C. Gonzalez, D. McMahon, ing the CD4␧15 chimera is more puzzling. Because CD4␧15 D. D. Richman, F. T. Valentine, L. Jonas, A. Meibohm, E. A. Emini, and J. A. Chodakewitz. 1997. Treatment with indinavir, zidovudine, and lamivudine interacts with the gp160 precursor in the ER and this interaction in adults with human immunodeficiency virus infection and prior antiretroviral inhibits the normal processing to gp120 and gp41 (14), it is therapy. N. Engl. J. Med. 337: 734–739. ␧ 3. Hammer, S. M., K. E. Squires, M. D. Hughes, J. M. Grimes, L. M. Demeter, likely that the gp160-CD4 15 complex is processed abnor- J. S. Currier, J. J. Eron, Jr., J. E. Feinberg, H. H. Balfour, Jr., L. R. Deyton, et al. mally. This processing perhaps takes place in a compartment 1997. A controlled trial of two nucleoside analogues plus indinavir in persons other than the trans-Golgi network where gp160 proteolysis is with human immunodeficiency virus infection and CD4 cell counts of 200 per cubic millimeter or less. AIDS Clinical Trials Group 320 Study Team. N. Engl. believed to occur (35). Our preferred hypothesis is that by com- J. Med. 337: 725–733. plexing to gp160, CD4␧15 drives this protein into a proteolytic 4. Schmit, J. C., L. Ruiz, B. Clotet, A. Raventos, J. Tor, J. Leonard, J. Desmyter, compartment, perhaps the lysosome, where gp160 is degraded E. De Clercq, and A. M. Vandamme. 1996. Resistance-related mutations in the ␧ HIV-1 protease gene of patients treated for 1 year with the protease inhibitor except for the regions that most closely contact CD4 15, which ritonavir (ABT-538). AIDS 10: 995–999. would be sterically protected. Accordingly, EDAF would result 5. Condra, J. H., D. J. Holder, W. A. Schleif, O. M. Blahy, R. M. Danovich, from the fingerprinting of CD4␧15 onto gp120. Indeed, the ob- L. J. Gabryelski, D. J. Graham, D. Laird, J. C. Quintero, A. Rhodes, et al. 1996. Genetic correlates of in vivo viral resistance to indinavir, a human immunode- served variability in the size of purified EDAF and the presence ficiency virus type 1 protease inhibitor. J. Virol. 70: 8270–8276. of more than one protein band could be the consequence of the 6. d’Arminio Monforte, A., L. Testa, F. Adorni, E. Chiesa, T. Bini, G. C. Moscatelli, mechanism by which EDAF is generated, resulting from partial C. Abeli, S. Rusconi, S. Sollima, C. Balotta, et al. 1998. Clinical outcome and predictive factors of failure of highly active antiretroviral therapy in antiretrovi- proteolytic cleavage. Interestingly, although a BLAST analysis ral-experienced patients in advanced stages of HIV-1 infection. 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