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The Journal of Immunology, 1998, 160: 1489-1496.
Copyright © 1998 by The American Association of Immunologists

Cells Transfected with a Non-Neutralizing Antibody Gene Are Resistant to HIV Infection: Targeting the Endoplasmic Reticulum and Trans-Golgi Network

Paul Zhou1,*, Simoy Goldstein{dagger}, Krishnakumar Devadas*, Deepanker Tewari* and Abner Louis Notkins*

* Experimental Medicine Section, Oral Infection and Immunity Branch, National Institute of Dental Research and Immunodeficiency Virus Section, and {dagger} Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Plasmids containing single chain Fv (scFv) non-neutralizing human anti-HIV-1 gp41 Ab cDNA, with or without endoplasmic reticulum (ER) or trans-Golgi network (TGN) retention signals, were constructed. Stable transfectants expressing these scFvs then were generated from COS-7 cells and HIV-1-susceptible CD4+ human T cells (Jurkat). scFv without a retention signal was secreted from cells, whereas scFv with an ER or TGN retention signal remained primarily within targeted intracellular compartments. The expression of scFv, scFv-ER, and scFv-TGN did not adversely affect the appearance of uninfected cells, as measured by growth rate or CD4 expression. Pulse-chase experiments revealed that the t1/2 of scFv-ER and scFv-TGN within cells was greater than 24 h and less than 9 h, respectively. The scFv-ER and scFv-TGN bound HIV gp160, and the scFv-ER-gp160 and the scFv-TGN-gp160 complexes were stable within HIV-infected transfectants. Further studies revealed that the maturation processing of gp160 into gp120 and gp41 was blocked in the scFv-ER transfectants, but not in the scFv-TGN transfectants. Moreover, HIV replication, as measured by p24, was inhibited by up to 99% in cells transfected with scFv-ER or scFv-TGN, but was not inhibited in cells transfected with the secretory form of scFv. It is concluded that the targeting of non-neutralizing anti-HIV-1 Abs to specific intracellular compartments blocks HIV replication and represents a potential therapeutic strategy for protecting uninfected lymphopoietic stem cells from HIV-1-infected patients.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
In nature, Abs are made by B lymphocytes, secreted, and exert their action extracellularly. By rDNA techniques, cDNA constructs can now be made that encode functionally active single chain Fv (scFv)2 Ab fragments. These constructs can be used to transfect a variety of different cells. The incorporation of retention signals into these cDNA constructs gives the encoded protein the added ability of being targeted to specific intracellular compartments such as the endoplasmic reticulum (ER) or the nucleus (1). Recently, several groups have succeeded in expressing scFv molecules within cells (2, 3, 4, 5, 6, 7, 8, 9, 10). These scFvs markedly inhibited the function of the intracellular proteins to which they were directed.

HIV infects T cells and macrophages. Once inside cells, HIV is no longer accessible to neutralizing Ab, making it an ideal candidate for intracellular scFv gene therapy. The HIV envelope glycoprotein, which is an oligomer composed of extracellular gp120 and transmembrane gp41, is required for infectivity. gp120 is responsible for the adsorption of virions to the CD4 receptor (11, 12, 13) and coreceptors (CKR-5 and fusin) (14, 15, 16, 17) on the host cell. gp41 mediates the fusion between viral and host cell membranes (12, 13). In addition, HIV-1 gp120 and gp41, on the surface of HIV-1-infected cells, can interact with the CD4 and fusin receptors on the surface of uninfected cells to form syncytia, which results in cell death (12, 13). gp160 is synthesized in the ER, where it is also glycosylated and oligomerized. The oligomer then moves to the Golgi network. It is in the Golgi network that gp160 is cleaved into gp120 and gp41 by subtilisin-like cellular endoproteases (18, 19, 20). Noncovalently linked, gp120 and gp41 are then expressed on the cell surface (21). Besides its involvement in membrane fusion, gp41 was found to contain the hydrophobic heptad sequence repeat for oligomerization (22), have calcium-binding capability (23), interact with HIV-1 p17 (matrix protein) through its cytoplasmic tail during the virus-budding process (24), and be involved in the pathogenesis of AIDS dementia by elevating immunologic nitric oxide synthase (25). gp41, thus, has multiple roles in the life cycle of HIV infection and might be a good target for intracellular Ab intervention.

Marasco et al. have successfully targeted neutralizing anti-HIV-1 gp120 scFv into ER and found that it could inhibit HIV-1 replication (2, 26). To extend this envelope-based gene therapeutic approach, we generated several scFvs derived from a human mAb against HIV-1 gp41. The hybridoma secreting this Ab was prepared from peripheral B lymphocytes of an HIV-1-positive patient. The Ab is of high affinity with a Kd value of 4 x 10-10 M, but fails to neutralize HIV-1 infection in vitro (27). The present experiments were initiated to determine 1) whether non-neutralizing anti-HIV-1 gp41 scFvs could be targeted into the ER or trans-Golgi network (TGN) compartment; and 2) if so, whether they could bind to the corresponding Ag(s) and exert anti-HIV activity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Oligonucleotide primers

The oligonucleotide primers for generating scFv, scFv-ER, and scFv-TGN were as follows: I, 5'-agcaagcttcaggtgcagctgcagcaatg-3'; II, 5'-cgctcccaccacctccagagccaccgccacctgcagagacagtgaccattgt-3'; III, 5'-ggaggtggtgggagcggtggcggcggatct gccgagctcatgacccagtct-3'; IV, 5'-gcgaagctttcgtttgatctccagtttgg-3'; V, 5'-cagaagcttaccatgaaacatctgtggttcttc-3'; VI, 5'-cagaagctttcatcgtttgatctccagtttggt-3'; VII, 5'-cagaagctttcgaagctcgtccttctcgcttttgatctccagtttgg-3'; VIII, 5'-atcaaacgaaccagtgcagaaagcagc-3'; IX, 5'-cagaagcttactaagttttaggttcaaacgttg-3'; X, 5'-gcactggttcgtttgatctccagtttggt-3'.

For bacterial expression of scFv, primers I and II were used to amplify the VH sequence, and primers III and IV to amplify the VL sequence of anti-HIV-1 gp41 Ig genes. At the 5' ends of primers II and III, a short 30- or 31-nucleotide sequence (italicized) with 16 overlapping nucleotides between them was introduced (underlined). After sewing them together by overlapping PCR, the sequence encoding a (Gly-Gly-Gly-Gly-Ser)3 linker was introduced between VH and VL.

Primers II, III, V, and VI were used to generate scFv in mammalian cells. The same overlapping PCR strategy was used as described above. scFv-ER was generated with primers V and VII by using the above mammalian cells expressing scFv as a template. scFv-TGN was generated with primers V, VIII, IX, and X. Primers VIII and IX were used to amplify a 192-bp sequence encoding the transmembrane domain and cytoplasmic tail of TGN38 protein (28). Primers V and X were used to amplify scFv. At the 5' end of primers VIII and X, 17 overlapping nucleotides were introduced (underlined) for sewing scFv and TGN together by overlapping PCR.

Construction and expression of scFv in bacteria

To generate scFv of anti-HIV-1 gp41 Ab genes, total RNA was isolated from human hybridoma T15G1, and the first strand of cDNA was synthesized (29). Based upon the published sequence (27), the VH and VL coding sequence of the heavy and the light chain of Ig genes were amplified with two pairs of oligonucleotide primers I, II, III, and IV, with the HindIII site introduced at the 5' ends of primers I and IV. At the 5' ends of primers II and III, a short 30- or 31-nucleotide sequence with 16 overlapping nucleotides between them was introduced (see primers). The amplified VH and VL fragments then were sewn together with a sequence encoding a linker (Gly-Gly-Gly-Gly-Ser)3 by overlapping PCR (30). The overlapping PCR-amplified scFv product was ligated into a TA vector system for sequence analysis (Invitrogen Corp., San Diego, CA). Conditions for regular PCR and overlapping PCR were as described (29, 30).

The insert containing the scFv sequence then was recloned into the HindIII site of the bacterial expression vector pET25b (Novagen Madison, WI) in both sense and antisense orientations. The resulting plasmids were transformed into the bacterium BL21(DE)3. IPTG induction, protein expression, and purification of scFv from the soluble fraction of the bacterial lysate were conducted according to the manufacturer’s instructions.

Construction and expression of scFv, scFv-ER, and scFv-TGN in mammalian cells

To construct scFv for mammalian cell expression, we first searched the literature for the consensus sequence encoding the leader signal of human VH-IV, since anti-HIV-1 gp41 Ig is a member of this family. Based upon the consensus sequence (31), primer V was synthesized, which contained a HindIII site, Kozak motif, and a nucleotide sequence corresponding to the first 21 nucleotides of the leader signal. Using the same first strand cDNA as a template, the VH fragment was amplified with primers V and II. The VL fragment was amplified with primers III and VI. VH and VL then were linked together by overlapping PCR, and the resulting scFv fragment was ligated into a TA vector for sequence analysis.

To construct scFv-ER, primer VII, which had an additional SEKDEL-coding sequence, was synthesized. Using the above scFv as a template, scFV-ER was PCR amplified with primers V and VII and ligated into a TA vector for sequence analysis.

Since the original cDNA sequence of TGN38 was reported from rat hepatoma cells (28), to construct scFv-TGN, total RNA was isolated and the first strand of cDNA was synthesized from rat liver, as described. A 192-bp sequence encoding the transmembrane domain and cytoplasmic tail, including the YQRL retention signal of TGN38 protein, was PCR amplified with primers VIII and IX. At the 5' end of primer VIII, an additional nine nucleotides, corresponding to the 3' end of the VL sequence, were added to perform overlapping PCR with scFv. At 5' end of primer IX, a HindIII site was introduced and the original anticodon ctt was changed to ttt (see the primers) to eliminate a HindIII site in the coding sequence. scFv, with an additional eight nucleotides corresponding to the 5' end of the transmembrane domain of TGN38, was PCR amplified with the above primer V and a new primer X using the scFv as a template. scFv-TGN then was generated with overlapping PCR by sewing the TGN and scFv fragments together. The resulting scFv-TGN was ligated into a TA vector for sequence analysis. The inserts containing scFv, scFv-ER, and scFv-TGN then were recloned into the HindIII site of the mammalian expression vector pRC/CMV/TCRenh (32).

To generate stable transfectants, the resulting plasmids, designated pRC/CMV/TCRenh/scFv, pRC/CMV/TCRenh/scFv-ER, pRC/CMV/TCRenh/scFv-TGN, and pRC/CMV/TCRenh vector alone, were linearized at an XmnI site. Twenty micrograms of linearized DNA per construct were mixed with 106 COS-7 or Jurkat cells (American Type Culture Collection, Rockville, MD) in 0.8 ml of RPMI 1640 (Life Technologies). Electroporation was performed at the capacitance of 960 µF and 300 V/0.4 cm for Jurkat cells, and at the capacitance of 960 µF and 240 V/0.4 cm for COS-7 cells. Stable transfectants were generated by G418 selection (Life Technologies) (1.5 mg/ml for Jurkat and 0.6 mg/ml for COS-7) for 2 to 3 wk and then by limiting dilution.

Enzyme-linked immunosorbent assay

HIV-1 gp160 (Intracell Cambridge, MA), thyroglobulin (Sigma Chemical Co., St. Louis, MO), and lysozyme (Sigma Chemical Co.) were coated onto 96-well Microtiter plates (Dynatech Labs., Chantilly, VA) at a concentration of 10 µg/ml (100 µl/well) at 4°C overnight. Serially diluted scFv was added and incubated at 4°C for 2 h. Plates then were washed twice with PBS containing 0.5% Tween, incubated with anti-herpes simplex virus Tag Ab (Novagen) for 2 h, and then with horseradish peroxidase-conjugated anti-mouse IgG serum (Kpl) for another hour at room temperature before being stained with 3,3', 5,5' tetramethylbenzidine substrate (Kpl).

Immunoprecipitation

To study scFv expression in transfectants, Jurkat lines containing vector alone (designated J-V), Jurkat lines containing pRC/CMV/TRCenh/scFv (designated J-scFv), Jurkat lines containing pRC/CMV/TRCenh/scFv-ER (designated J-scFv-ER), and Jurkat lines containing pRC/CMV/TRCenh/scFv-TGN (designated J-scFv-TGN) were lysed and immunoprecipitated. Briefly, 2 x 106 cells were incubated with 1.2 ml of methionine-free RPMI 1640 medium supplemented with 10% FBS, 2 mM L-glutamine, and 1 mM sodium pyruvate for 30 min, and then metabolically labeled with [35S]methionine (125 µCi/ml; DuPont NEN, Boston, MA) for 4 h. After labeling, supernatants were collected, and the cells were washed three times with HBSS and solubilized with lysis buffer (10 mM Tris-HCl, pH 7.4, 1% Nonidet P-40 (w/v), 150 mM NaCl, 1 mM EDTA, 1 mg/ml BSA, and 1 mM PMSF). The radioactivity of labeled proteins in cell lysates was determined by TCA precipitation. scFv, scFv-ER, and scFv-TGN were immunoprecipitated with Kappalock-Sepharose (Upstate Biotechnology, Lake Placid, NY). The latter is a 32-kDa recombinant protein that recognizes epitopes on the framework region of the V{kappa} light chain. The precipitated samples then were electrophoresed on 14% SDS/PAGE gels. The gels were treated with EN3HANCE (DuPont NEN) and dried before autoradiography.

For the pulse-chase experiments, 10 x 106 cells of HIV-1 noninfected or infected J-V, J-scFv, J-scFv-ER, and J-scFv-TGN lines were incubated with 3 ml of methionine-free medium for 30 min and then metabolically labeled with [35S]methionine (150 µCi/ml; DuPont NEN) for 1 or 2 h, as indicated. After labeling, the cells were washed three times with HBSS and cultured in 3 ml of DMEM medium supplemented with 10% FBS, 2 mM L-glutamine, and 1 mM sodium pyruvate for various times. At each time point, 2 x 106 cells were collected and solubilized with the lysis buffer. TCA precipitation, immunoprecipitation, SDS/PAGE, and autoradiography were performed as described above.

Immunofluorescence staining and FACS analysis

To study intracellular scFv expression, COS-7 lines containing vector alone (designated C-V), COS-7 lines containing pRC/CMV/TRCenh/scFv-ER (designated C-scFv-ER), and COS-7 lines containing pRC/CMV/TRCenh/scFv-TGN (designated C-scFv-TGN) were seeded onto culture chamber slides (Nunc) and incubated at 37°C for 1 or 2 days. Cells were fixed with 95% ethanol and 5% acetic acid at -20°C for 10 min, washed twice with PBS, blocked with 5% goat serum for 1 h, and then incubated with rabbit anti-human {kappa}-chain Ab (Boehringer Mannheim Corp., Indianapolis, IN) for 1 h at room temperature. FITC-conjugated goat anti-rabbit IgG (Sigma Chemical Co.) was used as the second Ab to stain the cells. The coverslips were mounted, and slides were examined with a fluorescence microscope and photographed. FACS analysis of expression of CD4 was the same as described (32).

HIV-1 infection, p24 assay, and syncytial formation

J-V, J-scFv, J-scFv-ER, and J-scFv-TGN lines (1 x 106 cells) were mixed with HIV-1, strain IIIB (0.001 multiplicity of infection), or zidovudine (AZT)-resistant primary isolate 5AO12 (0.005 multiplicity of infection) (33), in a final volume of 0.5 ml, and incubated at 37°C for 2 h. Cells then were washed three times with HBSS and resuspended in 5 ml of DMEM medium supplemented with 10% FBS, 2 mM L-glutamine, and 1 mM sodium pyruvate, and incubated at 37°C for 21 days. At intervals of 3 to 4 days, 2.5 ml of the culture supernatant was collected and replaced with the same amount of fresh medium. Viral particles in culture supernatants were measured by ELISA for HIV-1 p24 gag protein (DuPont NEN), according to the manufacturer’s instructions. Viability of cells and number of syncytia were counted by trypan-blue staining using light microscopy.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of scFvs in bacteria and their Ag-binding activity

Human anti-HIV-1 gp41 scFv cDNA was constructed as described in Materials and Methods and expressed in bacteria. The bacterial lysate was electrophoresed on a 12% SDS/PAGE gel and stained with Coomassie blue. As seen in Figure 1GoA, a major 36-kDa band, corresponding to the expected size of scFv, was detected only in bacteria induced with IPTG containing the scFv sense construct, and not in bacteria containing the scFv antisense construct. The specificity of reactivity of scFv for HIV-1 gp160, as measured by ELISA, is illustrated in Figure 1GoB. scFv reacted with HIV-1 gp160, but not with thyroglobulin or lysozyme.



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  		FIGURE 1. Expression and Ag binding of anti-HIV-1 gp41 scFv in bacteria. A, Bacterial lysate subjected to 12% SDS/PAGE and stained with Coomassie blue. Sense (S) and antisense (AS) constructs induced with IPTG. B, Ag-binding specificity of scFv isolated from the soluble fraction of the bacterial lysate, as measured by ELISA.

 
Expression of scFv in Jurkat transfectants

Jurkat cells were transfected with vectors carrying scFv, scFv-ER, or scFV-TGN (Fig. 2GoA), and stable cell lines were generated. The degree of expression of scFvs in the Jurkat transfectants was determined by immunoprecipitation. As seen in Figure 2GoB, more than 90% of the scFv without retention signals was secreted in the culture supernatants (i.e., cell line J-scFv). In contrast, scFv-ER and scFV-TGN were detected only in the cell lysate, indicating that the ER and TGN retention signals were operative.



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  		FIGURE 2. Expression of human anti-HIV-1 scFvs in Jurkat transfectants. A, Schematic representation of the scFv, scFv-ER, and scFv-TGN proteins. B, Expression of scFv, scFv-ER, and scFv-TGN proteins in culture supernatants and cell lysates in stably transfected Jurkat cells. Jurkat cells transfected with vector (J-V), scFv (J-scFv), scFv-ER (J-scFv-ER), or scFv-TGN (J-scFv-TGN). C, Pulse-chase analysis of scFv, scFv-ER, scFv-TGN proteins in cell lysates of stably transfected Jurkat cell lines. Chase time in hours.

 
To determine the stability of the scFvs inside cells, pulse-chase experiments were performed. Figure 2GoC shows that scFv-ER was very stable, with a t1/2 greater than 24 h. scFv-TGN was moderately stable, with a t1/2 less than 9 h. In contrast, scFv had a very short t1/2 of less than 1 h, suggesting that without retention signals, scFv is very quickly secreted into the extracellular fluid. To further evaluate the stability of the scFv, scFv-ER, and scFv-TGN, cells were treated with brefeldin A (BFA), which is known to cause a rapid redistribution of proteins from Golgi to ER (34). Treatment with BFA extended the t1/2 of scFv-TGN from less than 9 h to well over 22 h (data not shown), arguing that the difference in stability between scFv-ER and scFv-TGN was not due to the different retention signals, but to the compartment to which the scFv was targeted.

Intracellular expression of scFv-ER and scFv-TGN

To further localize scFv-ER and scFv-TGN within cells, immunofluorescence staining of COS-7 transfectants expressing scFv-ER and scFv-TGN was performed (Fig. 3Go). COS-7 cells transfected with vector alone and stained with anti-scFv Ab served as the negative control (Fig. 3GoA). Cells transfected with scFv-ER (Fig. 3GoB) and scFv-TGN (Fig. 3GoC) showed strong staining in the perinuclear region of the cytosol, with no appreciable staining in the nucleus or on the cell surface. The staining pattern of the scFv-ER-transfected cells was ring shaped, whereas that of the scFv-TGN-transfected cells was crescent shaped, patterns characteristic of molecules distributed in the ER and TGN, respectively (28). These findings, together with the results of the pulse-chase (above) and HIV-1 gp160/gp120 experiments (below), argue that the retention signals KDEL and YQRL are targeting the scFvs into the ER and TGN, respectively.



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  		FIGURE 3. Immunofluorescence localization of scFv-ER and scFv-TGN proteins in stably transfected COS7 cells. COS7 cells transfected with vector alone (A), scFv-ER (B), or scFv-TGN (C) were fixed, permeabilized, incubated with rabbit anti-human {kappa} Ab, and stained with FITC-conjugated goat anti-rabbit IgG Ab. The stained cells were photographed and digitally scanned (photomicrographs x200).

 
Inhibition of HIV-1 replication in CD4+ T cell lines expressing scFv-ER and scFv-TGN

To see whether the expression of scFvs can alter CD4 expression and cell growth in the transfectants, we tested cell surface expression of CD4 by FACS analysis. Figure 4Go shows that expression of CD4 on cell surface was similar in cells transfected with scFv, scFv-ER, and scFv-TGN, as compared with cells transfected with vector alone. The growth rate of the cells also was similar (data not shown).



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  		FIGURE 4. Surface expression of CD4 in representative Jurkat cell lines transfected with scFv (J-scFv), scFv-ER (J-scFv-ER), scFv-TGN (J-scFv-TGN), or vector alone (J-V), and stained with anti-CD4 Ab or isotype-matched irrelevant mouse IgG1 Ab as the control.

 
To see whether the expression of scFvs renders Jurkat cell transfectants resistant to HIV-1 replication, J-V, J-scFv, J-scFv-ER, and J-scFv-TGN cell lines were infected with HIV-1 IIIB or AZT-resistant primary isolate 5AO12. As seen in Table IGo, HIV-1 IIIB infection was strongly inhibited in all J-scFv-ER and J-scFv-TGN cell lines, as measured by the level of HIV-1 p24 in culture supernatants. Inhibition was apparent as early as day 7 postinfection and continued throughout the experiment. Supernatants collected on days 14 and 17 showed 90 to 99% reduction in HIV-1 replication in J-scFv-ER and J-scFv-TGN lines as compared with the J-V control line. On day 21, one J-scFv-ER (J-scFv-ER-6) and one J-scFv-TGN (J-scFv-TGN-3) cell line still showed strong (>90%) to moderate (>40%) inhibition of HIV-1 replication, respectively. In contrast to the resistance of transfectants expressing scFv-ER and scFv-TGN, transfectants expressing the secretory form of scFv (cell line J-scFv) were susceptible to HIV-1 infection. The experiment was repeated three additional times and with two other JV control cell lines with similar results. Similar results also were obtained with the AZT-resistant primary isolate, 5AO12 (33) (Table IGo).


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  		Table I. Level of p24 in scFv transfectants infected with HIV-1

 
Inhibition of HIV-1-induced syncytial formation by scFv-ER and scFv-TGN

HIV-1-induced syncytia was detected as early as 7 days postinfection, and peaked at 10 to 14 days in Jurkat cells transfected with scFv, scFv-ER, scFv-TGN, or vector alone. However, as seen in Figure 5Go, the size and number of syncytia were substantially larger in the J-V- and J-scFv-transfected cell lines (individual syncytia contained several hundred cells) (Fig. 5Go, A and B) as compared with J-scFv-ER- and J-scFv-TGN-transfected cells (individual syncytia contained less than 16 cells in the J-scFv-ER line and less than 32 cells in the J-scFv-TGN line) (Fig. 5Go, C and D). There were 5 to 6 times more HIV-1-induced syncytia in the J-V and J-scFv cell lines as compared with the J-scFv-ER and J-scFv-TGN cell lines (Fig. 5GoE).



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  		FIGURE 5. Syncytia induced by HIV-1 at 10 days postinfection in Jurkat cells stably transfected with vector alone (J-V) (A), scFv (J-scFv) (B), scFv-ER (J-scFv-ER) (C), or scFv-TGN (J-scFv-TGN) (D). Photographs were taken at x320 magnification. Number of syncytia at 10 days postinfection determined by light microscopy (E).

 
Coimmunoprecipitation of scFvs with HIV-1 gp160

To test the Ag-binding capacity of scFv-ER and scFv-TGN in HIV-1-infected cells, pulse-chase experiments were performed, followed by immunoprecipitation with Kappalock-Sepharose at 3 wk postinfection. As seen in Figure 6Go, immunoprecipitation of scFv-ER resulted, at each of the time points tested, in the coprecipitation of HIV-1 gp160. The scFv-ER/gp160 complex was stable within cells for at least 24 h. Figure 6Go also shows that immunoprecipitation of scFv-TGN resulted in the coprecipitation of HIV-1 gp160. However, the amount of gp160 that coprecipitated with scFv-TGN was considerably less than that with scFv-ER, perhaps due to the lower concentration of scFv-TGN that was available (Figs. 2Go, B and C). Alternatively, less gp160 may have been available to scFv-TGN, since it is known that most of the gp160 is processed in the cis-Golgi compartment before reaching the TGN compartment (18). Surprisingly, we were not able to coprecipitate HIV-1 gp41 with either scFv-ER or scFv-TGN, indicating that the amount of HIV-1 gp41 generated in these cells was too low for detection. We also performed reverse coimmunoprecipitation with an anti-HIV-1 gp120 mAb (see below). Most of the time we could coimmunoprecipitate both scFv-ER and scFv-TGN with gp160, but the intensity of scFv-ER and scFv-TGN bands was much weaker (data not shown).



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  		FIGURE 6. Pulse-chase analysis of coimmunoprecipitation of scFv and HIV-1 gp160 in stably transfected Jurkat cells infected with HIV-1. scFv proteins in cell lysates were immunoprecipitated by Kappalock-Sepharose. Jurkat cells transfected with vector (J-V), scFv-ER (J-scFv-ER), or scFv-TGN (J-scFv-TGN). Pulse chase was performed at 21 days postinfection. The pulse time was 2 h. Chase time in hours.

 
Effect of scFv-ER and scFv-TGN on the maturation of HIV-1 envelope proteins

To study the effect of scFv-ER and scFv-TGN on the maturation of HIV-1 envelope proteins, the stably transfected cell lines J-V, J-scFv, J-scFv-ER, and J-scFv-TGN were infected with HIV-1. Pulse-chase experiments were performed with anti-HIV-1 gp120 Ab (Fig. 7Go). In all cases, comparable amounts of gp160 were present at zero time (i.e., the end of the 1-h pulse). At 2 h into the chase, gp120 resulting from the cleavage of gp160 was present in the J-V, J-scFv, and J-scFv-TGN cell lines, but barely detectable in the J-scFv-ER cell lines. These experiments argue that the binding of scFv-ER to HIV-1 gp160 results in the retention of the scFv-ER/HLV-1 gp160 complex within the ER, and significantly delays the conversion of gp160 to gp120 and gp41.



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  		FIGURE 7. Effect of scFvs on maturation of HIV-1 envelope proteins. Pulse-chase analysis of HIV-1 gp160 and gp120 in HIV-1-infected Jurkat cells stably transfected with vector alone (J-V), scFv (J-scFv), scFv-ER (J-scFv-ER), or scFv-TGN (J-scFv-TGN). The analysis was performed at 21 days postinfection, and anti-HIV-1 gp120 mAb was used to immunoprecipitate HIV-1 gp160 and gp120 from cell lysates. The pulse time was 1 h. Chase time in hours.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Recently, scFvs expressed intracellularly have been used successfully to inhibit HIV replication (2, 3, 5, 6, 7). In this study, we describe the construction and properties of an scFv derived from a non-neutralizing human mAb directed against HIV-1 gp41 (27). By molecularly linking the anti-HIV-1 gp41 scFv with appropriate retention signals, we were able to direct the scFv to two different intracellular compartments, the ER and the TGN. scFv-TGN was targeted to the TGN by incorporating the nucleotide sequence YQRL into the construct. YQRL is known to be responsible for TGN retention (35, 36), but had not been used previously to target scFv Ab. TGN plays an important role in protein sorting, including the separation of: secretory proteins from proteins destined for the lysozyme compartment; regulatory proteins from constitutive proteins; and polarized cell proteins destined for different domains of the plasma membrane (37, 38, 39, 40). Our experiments showed that the anti-HIV-1 scFv-TGN construct expressed protein in the TGN that reacted with HIV-1 gp160. In contrast, anti-HIV-1 scFv-ER was targeted to the ER by incorporating KDEL, a known ER retention signal (2, 8, 9, 10, 41), into the construct. The anti-HIV scFv-ER construct expressed protein in the ER that reacted with HIV-gp160. Both constructs inhibited HIV replication, although more scFv protein was detected in cells transfected with the ER construct than the TGN construct. In part, this may be due to the fact that the t1/2 of scFv Ab was longer in cells transfected with constructs containing the ER than the TGN retention signal. scFv Ab targeted to the ER could be distinguished from scFv Ab targeted to the TGN based on the immunofluorescence staining patterns (Fig. 3Go, B and C), the conversion of gp160 to gp120 (Fig. 7Go), the t1/2 of the scFvs (Fig. 2GoC), and the response to treatment with BFA (data not shown). In contrast to scFv constructs with retention signals, scFv constructs without retention signals expressed scFv Ab that was largely secreted from the cell and failed to inhibit HIV replication within the cell.

scFv-ER and scFv-TGN appear to use different mechanisms to inhibit HIV-1 replication and syncytium formation. It is known that during the maturation of the envelope protein, the precursor protein gp160 is first synthesized, glycosylated, and oligomerized in the ER, and then moves to the Golgi network. It is in the Golgi network that gp160 is cleaved into gp120 and gp41 by subtilisin-like cellular endoproteases (19, 20). In this study, we showed that the conversion of HIV-1 gp160 to gp120 was blocked in the ER-targeted transfectants, suggesting that anti-HIV-1 gp41 scFv-ER binds to gp160 in the ER. This would prevent or slow the movement of gp160 from the ER to the Golgi network. In the TGN-targeted transfectants, the rate of HIV-1 gp120 synthesis appeared to be nearly the same as that found in the control cells. This suggests that scFv-TGN does not prevent the maturation conversion of HIV-1 gp160 into gp120 and gp41, but precisely how scFv-TGN inhibits HIV-1 replication and syncytial formation remains unclear.

Neutralizing as well as non-neutralizing Abs are generated in patients during the course of HIV infection (42). The major immunodominant epitopes for neutralizing Abs in many, but not all, primary isolates show a high rate of mutation, particularly in the V3 loop of HIV-1 gp120 (43, 44). Thus, it may be difficult to develop useful intracellular scFvs from neutralizing Abs that will have broad application against a variety of HIV strains. Non-neutralizing Abs, on the other hand, may not exert selective force for viral mutation in vivo. It is therefore possible that epitopes recognized by non-neutralizing Abs may be more conserved among viral isolates than epitopes recognized by neutralizing Abs. In this context, gp41 contains the most conserved regions of HIV-1 envelope proteins (44, 45). The demonstration in this study that an anti-HIV-1 gp41 Ab that had no neutralizing activity when secreted outside cells had HIV-inhibitory activity when expressed inside cells, provides a potential therapeutic strategy for HIV treatment based on constructing scFvs that act by blocking the assembly of intracellular HIV rather than neutralizing extracellular HIV.

In conclusion, it is becoming possible to molecularly engineer and express intracellular Ab genes directed against a variety of preselected proteins. The capacity of the expressed Abs to bind to and block the action of specific intracellular proteins provides a powerful tool for elucidating the function of these proteins. The demonstration here that scFvs directed against gp41 inhibit HIV replication raises the possibility that intracellular Abs also may be therapeutically useful by introducing them into human hemopoietic stem cells from HIV-infected individuals, so that when these stem cells mature into HIV-susceptible CD4+ T cells, the intracellular anti-HIV-1 scFvs will protect them from HIV infection. The targeting of different anti-HIV scFv genes to different compartments within a single cell to block different HIV gene products may achieve even more complete and longer-term inhibition of HIV replication. This possibility is now under investigation.


   Acknowledgments
 
The authors thank Drs. Michael S. Lan and Gang Peng for reviewing this manuscript, Dr. George Chen for FACS analysis, and Ms. Janice Solomon for skillful preparation of this manuscript. The fresh isolate HIV-1 5AO12 was obtained through AIDS Research and Reference Reagent Program, Division of AIDS, National Institute on Allergy and Infectious Diseases, National Institutes of Health, from Dr. Douglas Richman.


   Footnotes
 
1 Address correspondence and reprint requests to Dr. Paul Zhou, Experimental Medicine Section, Oral Infection and Immunity Branch, National Institute of Dental Research, National Institutes of Health, Building 30, Room 114, Bethesda, MD 20892. Back

2 Abbreviations used in this paper: scFv, single chain Fv; AZT, zidovudine; BFA, brefeldin A; ER, endoplasmic reticulum; IPTG, isopropylthio-ß-galactoside; TGN, trans-Golgi network; TRCenh, T cell receptor enhancer. Back

Received for publication July 16, 1997. Accepted for publication October 10, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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