HIV-1 Proprotein Processing As a Target for Gene Therapy

P Cordelier1, MA Zern2 and DS Strayer1 1Department of Pathology, Jefferson Medical College, Philadelphia, PA, USA; and 2Department of Medicine, University of California, Davis, CA, USA

The central role of endoconvertases and HIV-1 protease 40 000 TCID50 (MOI ¼ 0.04). By Western blot analyses, the

(HIV-1 PR) in the processing of HIV proproteins makes the delivered a1AT inhibited cellular processing of gp160 to design of speciﬁc inhibitors important in anti-HIV gene gp120 and decreased HIV-1 virion gp120. SV(AT) inhibited Gag therapy. Accordingly, we tested native a1 antitrypsin (a1AT) processing of p55 as well. Furthermore, high levels of delivered by a recombinant simian virus-40-based vector, uncleaved p55Gag protein were detected in HIV virus particles SV(AT), as an inhibitor of HIV-1 proprotein maturation. Cell recovered from SV(AT)-transduced cells lines and primary lines and primary human lymphocytes were transduced with lymphocytes. Thus, delivering a1AT using SV(AT) to human SV(AT) without selection and detectable toxicity. Expression lymphocytes strongly inhibits replication of HIV-1, most likely of a1AT was conﬁrmed by Northern blotting, immunoprecipi- by inhibiting the activities both of the cellular serine proteases tation and immunostaining. SV(AT)-transduced cells showed involved in processing gp160 and of the aspartyl protease, Gag no evidence of HIV-1-related cytopathic effects when HIV-1 PR, which cleaves p55 . a1AT delivered by SV(AT) challenged with high doses of HIV-1NL4-3. As measured by may represent a novel and effective strategy for gene HIV-1 p24 assay, SV(AT)-transduced cells were protected therapy to interfere with HIV replication, by blocking a stage from HIV-1NL4-3 at challenge dose of 40 000 TCID50 (MOI ¼ in the virus replicative cycle that has until now been 0.04). In addition, peripheral blood lymphocytes treated with inaccessible to gene therapeutic intervention. SV(AT) were protected from HIV doses challenge up to Gene Therapy (2003) 10, 467–477. doi:10.1038/sj.gt.3301891

Gag Keywords: a1 antitrypsin; SV40; gp160; p55

Introduction terminus of the basic consensus sequence Arg–X–Lys/ Arg–Arg.16,17 This sequence is recognized by the kexin/ The physical structure of virions and the replicative cycle subtilisin-like serine proteases.18 The cellular serine of HIV-1 provide many potential targets for gene therapy. protease, furin, was the ﬁrst of this group that was To date, several RNA and protein agents have been shown to catalyze proteolytic maturation of both HIV-1 introduced into susceptible cells and tested as inhibitors and inﬂuenza virus envelope glycoproteins.19 Recent of HIV-1 replication. These studies have included data suggest that other proteases may also be involved in dominant negative mutants of HIV structural (Gag,1 such processing (for review see Ref 20). Env2) or regulatory (Rev,3 Tat,4 PR5) proteins, TAR6 and HIV-1 protease (PR) is an aspartyl protease that is RRE7 RNA decoys molecules for the viral proteins (Tat, needed to process the gag precursor polyprotein into REV), antisense RNAs,8 antibodies directed against Rev,9 several smaller proteolysis products.21 Just as cellular gp12010 or Tat,11 and ribozyme designed to cleave at serine protease activity is fundamentally important to different conserved sites of the HIV-1 genome.12,13 These envelope, viral aspartyl protease activity is required for approaches all target phases of HIV-1 replication before gag proprotein processing: both processes are necessary virus assembly. Alternatively, gp160 proteolytic proces- to generate infectious viral progeny.19,22 Thus, gene sing can be inhibited after expression of an ER-retained delivery to inhibit proteolytic cleavage of envelope and CD4 chimera in primary human T cells.14 gag precursors could represent a new approach for gene We describe here a different approach to anti-HIV therapy to interfere with HIV-1 infectivity. genetic therapy, one that inhibits proteolytic processing In the present work, we evaluated the ability of a of HIV-1 capsid and envelope proteins necessary in the native human protein, a1 antitrypsin (a1AT), to block

HIV late replicative cycle. HIV envelope glycoproteins maturation of HIV-1 proproteins. a1AT, a 53 kDa serum control viral tropism and promote membrane fusion. protein is the main physiologic inhibitor of neutrophil 15 23 They are synthesized as inactive 160 kDa precursors, elastase and other enzymes. a1AT thus controls and activated by endoproteolytic cleavage at the carboxyl proteolysis associated with tissue injury and represents an important modulatory defense against proteolytic tissue damage. Correspondence: Dr DS Strayer, Department of Pathology, Jefferson Medical College, 1020 Locust Street, Room 251, Philadelphia, PA 19107, We have developed a gene transfer system based on 24 USA simian virus-40 (SV40) as a vector. SV40 infects a wide Received 1 September 2002; accepted 4 March 2002 range of cell types from human and other mammals, Inhibiting HIV-1 proprotein processing P Cordelier et al 468 ensuring the expression of its genome. Recombinant, Treatment: T-antigen deleted, replication-deﬁcient, SV40-derived vectors deliver stable transgene expression with high a efﬁciency and without selection to cell lines and in 24–26 SV(HBS) SV(AT) SV(AT) Mock primary cell cultures, and in vivo. rSV40 vectors can Mock Mock be concentrated to high titer (41012 infectious units α-1AT (IU)/ml), stably integrate into the host genome, do not (1.3 kb) require helper viruses, and do not elicit cellular or 27 humoral responses in animals. SupT1 PBL HepG2 We delivered native human a1AT, a protease inhibitor, to human lymphocyte cell lines and primary human b Treatment: lymphocyte cultures using an rSV40-based vector SupT1 + (SV(AT)). We demonstrate that SV(AT) strongly inhibits SV(AT) replication and spread of HIV-1 by blocking two mock necessary steps in HIV-1 proprotein processing: it blocks i.p. : AT 83 kDa cellular serine proteases that are involved in processing α1AT (54kDa) of gp160 to gp120 and gp41, and, surprisingly, it also 42 kDa decreases the activity of HIV-1 PR, an aspartyl protease Blot: AT that cleaves p55Gag within the budding viral particles. Figure 1 Delivery of a1AT in SV(AT)-transduced human lymphocytic cells. (a) SupT1 cells and normal primary human PBL were transduced as described in Materials and Methods with SV(AT). Control cells were Results transduced with SV(HBS), or mock transduced. 15 mg of total RNA was harvested from these cells 5 days later, electrophoresed, and then hybridized Detection of a1AT in lymphocytic cells transduced with with a1AT cDNA probe. RNA (1 mg) from HepG2 cells was used as a SV(AT) positive control for hybridization (right). (b) SupT1 cells were transduced

Prior to examining the ability of a1AT to alter HIV-1 as described in Materials and Methods with SV(AT), or mock transduced. replication in T cell lines and peripheral blood lympho- Four weeks later, 500 mg of total proteins was subjected to immunoprecipitation for a1AT. After protein separation by SDS/PAGE, gels were cytes (PBL), we assessed the ability of SV(AT) to blotted to a PVDF membrane, then analyzed using an antibody directed transduce these cells. Thus, SupT1 cells and normal against a1AT. Results are representative of three immunoblots performed primary human PBL were transduced as described in on three separate sets of transduction and immunoprecipitation. Materials and methods with SV(AT). In our experience, the rSV40 treatment protocol used routinely transduces 495% of these cells, without selection.28 Transduction of infectivity assay. For HIV-1 challenge experiments, these cells with SV(HBS) was used as a negative control; unselected a AT-transduced SupT1 cells, SV(HBS)-trans- mock-transduced HepG2 cells, expressing high levels of 1 duced SupT1 cells and mock-transduced SupT1 cells endogenous a AT, were used as a positive control. 1 were treated with different amounts of HIV-1 . Samples of 15 mg of total cell RNA were electrophoresed, NL4-3

blotted and hybridized with a human a1AT-specific Inhibition of HIV-1-induced cytopathic effects by a1AT cDNA probe. Expression of human a1AT was seen in SV(AT)-transduced cells but not in mock-transduced Microscopically, SV(AT)-transduced SupT1 cells showed cells, or in SV(HBS)-transduced cells (Figure 1a). A 1.3 kb no evidence of HIV-1-related cytopathic effects when transcript, observed in the SV(AT)-transduced cells, challenged with HIV-1NL4-3 (Figure 3). That is, HIV- corresponds in size to the native a1-AT detected in the induced syncytia and cell death were not seen. On day 18 positive control cell line, HepG2. In addition, we found after challenge, mock-transduced SupT1 cells or that a1-AT protein was easily detected after immuno- SV(HBS)-transduced SupT1 cells started to die. By precipitation of total cell proteins from SV(AT)-trans- comparison, SupT1 cells transduced with a1AT continue duced SupT1 cells (Figure 1b). To illustrate the to proliferate normally. Thus, a1AT expression protected transduction efficiency of SV(AT) vector in HIV-1- cells against the cytopathic effects of HIV-1. susceptible cells, SupT1 cells were transduced with SV(AT), or SV(HBS) as a negative control. Unselected Inhibition of HIV-1 replication by a1AT cells were tested for AT expression by immunofluores- Replication of HIV-1 in these cultures was evaluated by cence microscopy 2 weeks later. Over 98% of SV(AT)- measuring levels of HIV-1 p24 antigen released into the

treated cells expressed a1AT (Figure 2a). Control cells culture medium. HIV-1 p24 analyses demonstrated that

transduced with SV(HBS) did not express a1AT. The a1AT conferred protection from infection at challenge growth and viability of transduced cells were unaltered doses of 4000 and 40 000 TCID50 of HIV-1NL4-3 (0.004 and by transduction, as assayed by counting of trypan-blue- 0.04 MOI of HIV-1NL4-3, respectively) whereas cells negative cells (Figure 2b). expressing HBsAg and mock-transduced SupT1 cells permitted high levels of HIV-1 p24 replication (Figures

Inhibition of HIV-1 by a1AT in human T-lymphocytic cell 4a and b). lines

We then tested whether expression of a1AT delivered by a1AT inhibition of HIV-1 in primary human cell cultures

SV40 to SupT1 cells could prevent HIV-1NL4-3 cytotoxicity Successful transduction and protection from HIV-1 in and replication in this line of susceptible human T- susceptible primary human cells is essential for gene lymphoid cells. Mock-transduced SupT1 cells alone and therapy to be practicable. The higher the levels of

unselected cells expressing a1AT or HBsAg were used for transduction and protection, the more likely that this

Gene Therapy Inhibiting HIV-1 proprotein processing P Cordelier et al 469 a Treatment goal can be achieved. We conﬁrmed that transduction of SupT1 + unstimulated PBL transferred expression of a1AT by Northern analysis of RNA from SV(AT)-transduced fresh SV(HBS) SV(AT) PBL as described above (Figure 1). It should be emphasized that these primary PBL preparations were not stimulated at the time of transduction. Thus, these data reﬂect transduction of unstimulated PBL, although IL-2 was subsequently added to maintain these cells. The

extent of a1AT-mediated inhibition of HIV-1 replication was tested by challenging SV(AT)-transduced PBL, SV(BUGT)-transduced PBL and mock-transduced PBL

with 4000 and 40 000 TCID50 of HIV-1NL4-3 (0.004 and 0.04

MOI of HIV-1NL4-3 , respectively). The SV40 transduction control for PBL was the SV(BUGT) vector because we have found that stimulated PBL from HBsAg-immune donors eliminate SV(HBS)-transduced cells.25 PBL treated with SV(AT) showed strong resistance to HIV-1 challenge, compared to parental mock-transduced cells or PBL expressing BUGT protein (Figures 4c and d).

a AT inhibits gp160 maturation b 125 1 Endoproteolytic cleavage of the HIV-1 envelope glycoprotein is essential for viral infectivity. Inhibiting gp160 cleavage to gp120 and gp41 should in theory prevent virus propagation. Maturation of gp160 depends in part on the activity of cellular serine proteases, also called 100 endoconvertases. We hypothesized that, as a protease

inhibitor—or serpin – a1AT may interfere with HIV-1 replication by blocking cleavage of gp160. To assess this possibility, unselected and uncloned SV(AT)-transduced and SV(HBS)-transduced SupT1 cells were treated with 75 4000 TCID50 (0.004 MOI) of HIV-1NL4-3. Cells were lysed 7 Control rSV40-transduced PBL days later. Western blot analyses for gp160-related Control rSV40-transduced SupT1 products showed high levels of gp120 in HIV-1- SV(AT)-transduced PBL challenged SV(HBS)-transduced SupT1 cells, compared

% of mock-transduced cell proliferation % of mock-transduced SV(AT)-transduced SupT1 to SV(AT)-transduced SupT1 cells (Figure 5, left panel). 50 That is, gp160 was efficiently cleaved intracellularly to 012 3 4 5 6 7 8 yield gp120 in SupT1 cells expressing HBsAg. On the Days post transduction other hand, gp160 was the predominant envelope glycoprotein molecular species detected in SV(AT)- Figure 2 Expression and lack of toxicity of a1AT delivered by SV(AT) in T cells. (a) SupT1 cells were transduced with SV(AT) (right) or SV(HBS)- transduced SupT1 cells: its processing to gp120 was transduced with SV(HBS) (left) then stained 2 weeks later with goat anti- efficiently inhibited by transduction with SV(AT). a 1AT, followed by Alexa Fluir 546-Donkey anti-goat IgG. Results are To confirm the inhibitory effect of a1AT on HIV-1 representative of two immunostaining performed on two separate sets of envelope glycoprotein processing in primary cell cul- transduction. (b) SupT1 cells and normal primary human PBL were transduced as described in Materials and methods with SV(AT). Control tures, SV(AT)-transduced and SV(BUGT)-transduced cells were transduced with SV(HBS) or SV(BUGT). Cell proliferation was PBL were treated with 10 000 TCID50 (0.01 MOI) of assayed by counting of trypan-blue-negative cells. Results are mean 7 HIV-1NL4-3. Cells were lysed 7 days later. Inhibition of s.e.m. of n¼4 experiments. gp160 conversion to gp120 by SV(AT) was also seen in

Mock SV(HBs) SV(AT)

Figure 3 SV(AT) inhibition of cytopathic effects of HIV-1. SupT1 cells were transduced with SV(AT) or SV(HBS), or mock-transduced. They were then challenged with 400 TCID50 of syncytia-inducing strain HIV-1NL4-3. After 8 days of infection, syncytia formation was abundant in mock-transduced or SV(HBS)-transduced cells, but cell fusion was absent in cultured transduced with SV(AT). Representative syncytia are highlighted with arrows.

Gene Therapy Inhibiting HIV-1 proprotein processing P Cordelier et al 470 a b 3500 SupT1 cells 3000 SupT1 cells Mock Mock 3000 2500 SV(HBS) SV(HBS) 2500 SV(AT) SV(AT) 2000 2000 1500 1500 1000 HIV-1 p24 antigen (ng/ml) HIV-1 1000 p24 antigen (ng/ml) HIV-1

500 500

0 0 0 2 4 6 8 10 12 14 16 18 20 22 0 2 4 6 8 10 12 14 16 18 20 22 Days post challenge with 4000 TCID 50 Days post challenge with 40 000 TCID50 (0.004 MOI) HIV-1 NL4-3 (0.04 MOI) HIV-1NL4-3

c 20 d PBL Mock 25 PBL Mock SV(BUGT) SV(BUGT) 15 20 SV(AT) SV(AT)

15 10

10 HIV-1 p24 antigen (ng/ml) HIV-1 HIV-1 p24 antigen (ng/ml) HIV-1 5 5

0 0 0 2 4 6 8 10 12 14 16 18 20 0 2 4 6 8 10 12 14 16 18 20 22

Days post challenge with 4000 TCID50 Days post challenge with 40 000 TCID50 (0.004 MOI) HIV-1NL4-3 (0.04 MOI) HIV-1NL4-3 Figure 4 Inhibition of HIV-1 replication in SV(AT)-transduced T cells. Cultures of 106 mock-transduced SupT1 cells and SupT1 transduced with SV(AT) 6 or SV(HBS) were challenged with 4000 (a) or 40 000 (b)TCID50 of HIV-1NL4-3.10 mock-transduced PBL and PBL transduced with SV(AT) or SV(BUGT)

were challenged with 4000 (c) or 40 000 (d) TCID50 of HIV-1NL4-3. HIV-1 replication was quantitated by assaying HIV-1 p24 antigen levels in the culture supernatants by ELISA at the time points indicated after challenge. These graphs are representative of three independent experiments. Performing the Student t-test on individual data points indicated statistically signiﬁcant differences (Po0.01) in p24 levels between SV(AT) transduced and all control groups for assay points 12 days post-challenge.

these primary cell cultures, conﬁrming the results progeny from SV(AT)-transduced SupT1 cells (Figure 6, obtained with SV(AT)-transduced SupT1 cells (Figure 5, left panel). The latter virus particles only carried the right panel). Taken together, these data demonstrate that uncleaved envelope glycoprotein gp160 detectably.

SV(AT) delivery of a1AT efficiently inhibited HIV-1 gp160 These findings were confirmed after infection of processing. SV(AT)-transduced and SV(BUGT)-transduced PBL with

These findings were confirmed by studying gp120 40 000 TCID50 (0.04 MOI) of HIV-1NL4-3. Cell culture content of virus particle progeny from SV(AT)-trans- supernatants were harvested at day 7 post-infection and duced cells. For this purpose, unselected and uncloned subjected to ultracentrifugation for virion purification as SV(AT)-transduced and SV(HBS)-transduced SupT1 cells described in Material and methods. Analyses by Western

were treated with 10 000 TCID50 (0.01 MOI) of HIV-1NL4- blot for envelope glycoprotein (Figure 6, right panel)

3. Cell-free culture supernatants were harvested 7 days confirmed the findings with SupT1 cells: gp160 was the post-challenge, and subjected to ultracentrifugation for predominant envelope glycoprotein molecular species virion purification as described in Materials and meth- detected in SV(AT)-transduced PBL virus particle pro- ods. geny. gp120 was the sole envelope species detectable present Inhibition of gp160 cleavage should also result in in virus particle progeny recovered from SV(HBS)- decreased gp41, in both virions and cells. Therefore, 106 transduced cells, but no gp120 was detected in virus SupT1 cells were transduced with SV(AT) or SV(HBS),

Gene Therapy Inhibiting HIV-1 proprotein processing P Cordelier et al 471 Treatment Western blot analysis of these protein preparations revealed decreased gp41 (Figure 7, lower panel), as well as gp120 (Figure 7, upper panel) as a result of SV(AT) treatment of the HIV-1-challenged cells.

a1AT inhibits HIV-1 PR activity NO HIV-1 SV(AT) + control SV(HBS) SV(AT) SV(BUGT) a 202 kDa We tested whether the protease inhibitor 1AT could also interfere with HIV-1 protein processing by blocking HIV- gp160 1 gag processing elicited by the retroviral aspartyl 121 kDa gp120 protease HIV-1-PR. To assess this possibility, unselected and uncloned SV(AT)-transduced and SV(HBS)-trans-

duced SupT1 cells were treated with 4000 TCID50 (0.004

MOI) of HIV-1NL4-3. Cells were lysed 7 days later. Equal 70 kDa Lamin A amounts of total cell proteins were separated by SDS– PAGE and analyzed by Western blot for p55Gag. Using a polyclonal antibody specific for HIV-1 p24 that recognized p24 and p24-containing Gag proteins, we observed Supt1 cells PBL a 55 kDa protein in HIV-1-challenged SV(AT)-transduced Figure 5 SV(AT) inhibition of cellular gp160 processing. SupT1 cells (left SupT1 cells but not in HIV-1-challenged SV(HBS)- panel) and PBL (right panel) were mock transduced or transduced with the transduced SupT1 cells (Figure 8, left panel). To confirm rSV40 vectors indicated, and then challenged with 4 000 TCID50 of HIV-1. the inhibitory effect of a1AT on HIV-1 proprotein Cells were lysed 7 days later. After separation by SDS/PAGE, gels were processing, SV(AT)-transduced and SV(BUGT)-trans- blotted to a PVDF membrane, then analyzed using an antibody directed duced PBL were treated with 10 000 TCID (0.01 MOI) against HIV-1 gp120. gp41 visualization was not permitted by protein size 50 exclusion. Filters were reprobed using an antibody directed against Lamin of HIV-1NL4-3. Cells were lysed 7 days later, and equal A. The positive control is a mixture of purified gp120+gp160 from the NIH amounts of total cell proteins were separated by SDS– AIDS Research Reference Reagent Program. Results are representative of PAGE. These Western blot analyses confirmed the results five immunoblots performed on five separate sets of transduction. obtained with SV(AT)-transduced SupT1 cells, ie that transduction with SV(AT) inhibited the proteolytic processing of p55Gag (Figure 8, right panel). We did not then challenged with 10 000 TCID50 HIV-1. After 7 days detect p24Gag in proteins made from HIV-1-challenged of culture, HIV virions were purified from culture cells, even though the method of protein preparation supernatants, and proteins were prepared from these employed would have allowed such visualization. This virions and the transduced cells that produced them. result is consistent with the conclusion that PR cleavage of p55Gag occurs principally within the budding HIV virions. Treatment These findings were extended by the study of the p55Gag content of SV(AT)-transduced cells virus particle progeny. For this purpose, unselected SV(AT)-transduced and SV(HBS)-transduced SupT1 cells were treated NO HIV-1 SV(AT) + control SV(HBS) SV(AT) SV(BUGT) 202 kDa cellular virion proteins proteins gp160 121 kDa gp120 Treatment: HIV + HIV +

70 kDa Lamin A SV(AT) no HIV SV(AT) SV(HBS) SV(HBS) mw (kDa) 160 120

Supt1 PBL virions virions 41 Figure 6 Detection of gp120 in HIV-1 virions. SupT1 cells (left panel) and PBL (right panel) were mock transduced or transduced with the rSV40 vectors indicated, and then challenged with 10 000 and 40 000 TCID50 of Figure 7 Effect of SV(AT) on gp41 in HIV-1 virions and infected cells. HIV-1, respectively. HIV-1 virions recovered from cellular supernatants SupT1 cells (106) were transduced with SV(AT) or SV(HBS), and then were purified and lysed 7 days later. Proteins were separated by SDS/ challenged with 10 000 TCID50 of HIV-1. HIV-1 virions recovered from PAGE, blotted to a PVDF membrane, and then analyzed using an antibody cellular supernatants were purified and lysed 7 days later. Simultaneously, directed against HIV-1 gp120. gp41 visualization was not permitted by cellular proteins were prepared by lysing the cells in these cultures. protein size exclusion. Filters were reprobed using an antibody directed Proteins, 15 mg/lane, were separated by SDS/PAGE, blotted to a PVDF against Lamin A. The positive control is a mixture of purified gp120 + membrane, and then analyzed using an antibody directed against HIV-1 gp160 from the NIH AIDS Research Reference Reagent Program. Results gp120 (upper panel) and gp41 (lower panel). Proteins from SupT1 cells are representative of five immunoblots performed on five separate sets of that were not challenged with HIV were electrophoresed in the center lane. transduction. The sizes of the key HIV glycoprotein bands are indicated (arrows).

Gene Therapy Inhibiting HIV-1 proprotein processing P Cordelier et al 472 Treatment Treatment SV(AT) SV(AT) SV(AT) SV(BUGT) SV(HBS) SV(AT) SV(BUGT) SV(HBS)

81 kDa

p55Gag 81 kDa 52 kDa 52 kDa p55Gag

70 kDa Lamin A 20 kDa p24Gag

SupT1 cells PBL Figure 8 SV(AT) inhibition of cellular p55Gag cleavage, SupT1 cells (left panel) and PBL (right panel) were mock-transduced or transduced with the 70 kDa Lamin A rSV40 vectors indicated, and then challenged with 10 000 and 40 000

TCID50 of HIV-1, respectively. Cells were lysed 7 days later. Equal amounts of proteins were separated by SDS/PAGE, blotted to a PVDF membrane, and then analyzed by Western blot using a polyclonal antibody SupT1 virions PBL virions directed against HIV-1 p24. Filters were reprobed using an antibody directed against Lamin A to illustrate equal loading of protein. Results are Figure 9 Detection of p55Gag protein in HIV-1 virions recovered from representative of four immunoblots performed on four separate sets of SV(AT)-transduced cells. SupT1 cells (left panel) and PBL (right panel) transduction. were mock transduced or transduced with the rSV40 vectors indicated, and then challenged with 10 000 and 40 000 TCID50 of HIV-1, respectively. HIV-1 virions recovered from cellular supernatants were purified and lysed with 10 000 TCID (0.01 MOI) of HIV-1 . Cell-free 7 days later. Equal amounts of protein were separated by SDS/PAGE, 50 NL4-3 blotted to a PVDF membrane, and then analyzed by Western blot using culture supernatants were harvested at day 7 post- polyclonal antibody directed against HIV-1 p24. Filters were reprobed challenge, and subjected to ultracentrifugation for virion using an antibody directed against Lamin A to illustrate equal loading of purification as described in Materials and methods. protein. Results are representative of three immunoblots performed on Equal amounts of total viral protein were separated by three separate sets of transduction. SDS–PAGE and analyzed by Western blotting. High levels of uncleaved p55Gag protein were detected in virus particle progeny recovered from cells expressing a AT, of a highly specific stable adduct between the active site 1 30 compared to SV(HBS)-transduced SupT1 cells (Figure 9, serine of elastase and the residue Met358 in the serpin. upper left panel). Moreover, conversion of p55Gag into To be infectious, HIV-1 virus requires viral glycopro- virion p24 was strongly inhibited in SV(AT)-transduced tein maturation by host cell proteins, probably by cells, compared to SV(HBS)-transduced cells. Western endoproteases in the Golgi apparatus. Env proprotein blot analysis using a specific HIV-1 p24 antibody showed is organized as a trimer, and envelope structures are that high levels of HIV-1 p24 were only detectable in derived from a gp160 kDa precursor, gp160, which is cleaved into a gp120 external surface envelope species virions recovered from SV(HBS)-transduced SupT1 cells, 16 but not in those recovered from cultures of SV(AT)- and gp41, a transmembrane glycoprotein. The virion transduced cells (Figure 9, lower left panel). These results gp120, located at the virus surface, interacts with were confirmed by Western blot analyses of virus particle lymphocyte CD4 receptor to initiate the entry of HIV-1 into CD4-postive cells.31. Expression of gp160 in different progeny from PBL expressing a1AT (Figure 9, right panel). These data strongly suggest that, whether directly mammalian cell types and in yeast demonstrated that the or indirectly, a AT inhibits the activity of the HIV-1 PR, intracellular gp160-processing activities are ubiquitously 1 32 an aspartyl protease. expressed. Mammalian convertases such as furin cleave the envelope precursors.19 Based on these ob-

servations, the catalytic site of a1AT was exchanged with the consensus sequence recognized by convertases. The

engineered serpin (a1AT Portland, a1PDX) was shown to Discussion 33,34 be a potent inhibitor of furin. a1PDX was shown to

a1AT is a member of the superfamily of inhibitors block the processing of HIV-1 gp160 in transfected cells,

referred to as serpin (serine protease inhibitor). a1AT is and the activity of furin, as well as other proteases. produced and exported by the liver, is found in its active Recently, a1PDX has been reported to interfere with HIV- form primarily in the plasma, and accounts for more 1 replication, after stable transfection in the CD4-positive than 1% of plasma mass (for review see Ref 23). The Jurkat cells.35 Inhibition of the cytopathic effect of HIV-1 primarily function of this serpin is to regulate the and of virus replication appeared to correlate with the remodeling of the extracellular matrix: to achieve this capacity of the engineered serpin to interfere with the

goal, a1AT neutralizes neutrophil elastase activity by maturation of gp160 to gp120 and gp41. Recently, a1AT acting as a suicide substrate for the target protease.29 has been reported as a candidate circulating HIV-1 36 Protease inactivation by a1AT results from the formation inhibitor in vivo.

Gene Therapy Inhibiting HIV-1 proprotein processing P Cordelier et al 473 Here, we report that native human a1AT delivered by 1 glycoprotein processing, a process that is known to an rSV40 vector strongly inhibits HIV-1 replication in require the activity of cellular endoproteases. Thus, a vitro. We observed that a1AT expression after SV(AT) treatment based on using native a1AT to inhibit HIV-1 transduction protected cells against the cytopathic effects replication may be useful despite the virus’ ability to of HIV-1. The results of HIV-1 p24 analysis demonstrated mutate. that a1AT conferred protection to SupT1 cells from HIV- We tested whether the protease inhibitor a1AT could

1NL4-3 challenge doses of 4000 and 40 000 TCID50 (0.004 also interfere with HIV-1 protein processing by blocking and 0.04 MOI, respectively), whereas cells expressing HIV-1 p55Gag proteolytic maturation elicited by the HBsAg and mock-transduced SupT1 permitted high retroviral aspartyl protease HIV-1 PR. HIV-1 PR is levels of HIV-1 replication at these doses. Further encoded within the pol gene of the virus and is conﬁrmation was obtained by demonstrating that responsible for maturation of the nascent viral particle. SV(AT) effectively transduced and protected unstimu- The HIV-1 viral gag and gag/pol are expressed as lated human PBL. For comparison, the single-chain Fv polyprotein. Excision of individual enzymes and struc- (SFv) antibody IN #33 against HIV-1 integrase delivered tural proteins is felt to be performed by the aspartyl by both rSV40 and retroviral vectors was highly effective protease, HIV-1 PR, and largely occurs in budding in inhibiting HIV-1 replication at challenge doses of 40 virions.21 Unexpectedly, Western blot analyses showed Gag TCID50, but its protective efﬁciency was less evident at that SV(AT) inhibited processing of p55 in SupT1 cells higher challenge doses: it did not alter the course of HIV- and in primary human lymphocytes; high levels of 28 Gag 1 infection at challenge doses of 400 TCID50. We have uncleaved p55 protein accumulated within cells and yet to identify a challenge dose of HIV-1 that can were detected in virus particle progeny recovered from completely overwhelm the protective effect of a1AT. these cells after transduction with SV(AT).

Preliminary experiments showed a 60% reduction of These results strongly suggest that a1AT inhibits HIV-1 replication in SupT1 cells transduced with SV(AT) activity of HIV-1 PR, an aspartyl protease. The mechan- challenged with 0.3 MOI of HIV-1NL4-3, compared to ism(s) by which a1AT inhibits HIV-1 PR requires further control cultures (data not shown). investigation. Such promiscuity in a1AT inhibitory

The strength of the protective effect of a1AT may rest functions has already been reported: a1AT was shown in the fact that its principal target is cellular, not viral, to be a competitive inhibitor of renin,43 an enzyme protein. Increasing the HIV viral challenge dose is belonging to the aspartyl protease family, and the unlikely to compensate for a block in cellular protein – observed a1AT-related decreases in HIV-1 PR activity in this case serine protease – activity. We hypothesized might represent comparable cross-reactivity. Other pos- that the serpin a1AT would interfere with HIV-1 sibilities exist as well. For example, a cellular serine replication by blocking endoproteolytic activation of protease may be necessary for the activation of HIV-1 PR. gp160. Western blot analyses showed that gp160 was Alternatively, the virus assembly and colocalization of the predominant molecular species envelope glycopro- HIV-1 PR protein and p55Gag may also involve one or tein detected in SV(AT)-transduced Supt1 cells and more as yet undefined serine protease activities. These primary human lymphocytes: its processing to gp120 possibilities are not mutually exclusive, and are being was efficiently inhibited by transduction with SV(AT). investigated. Furthermore, HIV virus particles recovered from Crucial activities in which HIV relies on a cellular SV(AT)-transduced SupT1 cells and primary human enzyme function may represent points in the virus lymphocytes only contained the uncleaved envelope replicative cycle that are less susceptible to being glycoprotein gp160 detectably. Taken together, these circumvented by HIV mutations. They may therefore results showed that a1AT delivered by SV(AT) to point to vulnerabilities in HIV replication that could be lymphocytes inhibited cellular processing of gp160 to attractive targets for anti-HIV therapeutics. gp120, and decreased HIV-1 virion gp120. Since gp120 is The use of recombinant replication-deficient SV40- important in syncytia formation and in HIV-1 infectivity, derived vectors allows stable expression of transgene these findings may explain protection of SV(AT)-trans- with high efficiency and without selection in cell lines, duced cells from both events. Inhibition of gp160 primary cell cultures, and in vivo; rSV40s stably integrate processing is known to generate viral particles unable into the host genome and are effective vehicles whether to activate the necessary fusion between the viral particle or not target cells are dividing.24–26 Furthermore, rSV40s and the plasma membrane of the target cell.19 do not elicit cellular or humoral immune responses in 27 Interestingly, for some viruses, a direct correlation animals. The fact that a1AT occurs naturally in human between the efficiency of virus proprotein cleavage by blood makes immune responses against a1AT highly cellular endoproteases and virulence can be drawn.37,38 unlikely. The ultimate test for this inhibitor will be its

Native a1AT may provide an alternative to the peptides transfer in CD34+ hematopoietic, to allow continuous mimicking the cleavage region of gp160. Despite their repopulation of a patient’s immune system with such efficiency, these latter inhibitors have had toxic side genetically altered cells. For such an approach to be effects, limiting their use as antiviral agents.39 practicable, a conditional promoter that is HIV-1-specific, In AIDS therapy, the rate of drug-induced mutation of such as the HIV-1 LTR, should regulate expression of 40–42 HIV-1 is still a major problem. HIV-1 proteins evolve a1AT. We have used this promoter efficiently to deliver quickly in response to measures intended to inhibit their conditional expression of other transgenes,43 and are activities. However, the virus requires certain cellular currently devising such a construct to deliver a1AT. functions in order to generate infectious progeny. These In conclusion, delivering a1AT to human lymphocytic cellular functions are unlikely to be rescued by virus cell lines or primary human lymphocytes using SV(AT) mutation and thus may be important targets for anti-HIV as a vector strongly inhibits HIV-1 replication and therapeutics. Our results showed that a1AT inhibits HIV- spread. The likely mechanism for this effect is direct

Gene Therapy Inhibiting HIV-1 proprotein processing P Cordelier et al 474 inhibition of cellular serine proteases that process gp160. with 2 mML-glutamine and Earle’s BSS adjusted to In addition, the activity of HIV-1 PR, an aspartyl contain 1.5 g/l sodium bicarbonate, 0.1 mM non-essential protease, that is principally active within budding amino acids, 1.0 mM sodium pyruvate, streptomycin

virions, is also inhibited. a1AT delivered by rSV40 may (100 mg/ml), penicillin (200 units/ml), gentamicin therefore represent a novel and promising gene ther- (10 mg/ml) and 10% (vol/vol) fetal bovine serum. All apeutic approach, both to interfere with HIV-1 replica- the cells were grown at 371C in humidiﬁed incubator

tion, and to study the involvement of serine proteases in with 5% CO2. HIV-1 replication. Source and preparation of peripheral blood lymphocytes Materials and methods Venous blood from HIV-1-negative individuals without Plasmids and viral expression constructs identifiers was obtained through the Thomas Jefferson The molecular clone of HIV-1 used in these studies was University blood donor center. Peripheral blood mononuclear cells (PBMC) were prepared by Histopaque-1077 pNL4-3. This strain was obtained from the NIH AIDS Reagent Repository and was passaged and titered density separation (Sigma). The isolated PBMC were cultured overnight in complete RPMI. Cell preparations according to standard techniques. HIV-1 NL4-3 is a T- tropic viral strain with highly cytopathic effects. enriched in PBL were obtained after separation from plastic adherent mononuclear cells and macrophages. Construction of recombinant SV40 derivative viruses (rSV40) for gene transfer has been described previously.24 For simplicity, these adherent cell-depleted PBMC preparations are referred to as PBL. PBL were maintained in The construct pT7ma1AT, containing a modified a1AT cDNA, has been described previously, as has SV(AT), the complete RPMI in the presence of natural human purified interleukin II (20 units/ml, GIBCO) at 371Cin rSV40 vector derived from it.44 Briefly, the 5.24 kb SV40 genome (kind gift of Dr Janet Butel, Baylor College of a humidified CO2 incubator. Medicine) was modified by excising the genes for large T antigen (Tag) and small T antigen (tag), without disturb- Transduction ing the SV40 early promoter and the SV40 polyadenyla- For transduction with SV40-derived virus, SupT1 cells tion signal. The late viral genes, VP1, VP2 and VP3, and PBL were treated for 24 h at an MOI of 10. This which encode capsid proteins, also remain. The human treatment was repeated twice, but at an MOI of 3, on

a1AT cDNA was inserted downstream of the SV40 early sequential days. No selection was used. The rSV40- promoter, driven by the cytomegalovirus immediate treated cells were then cultured for 5 days before further early promoter (CMV-IEP). Control viruses for these manipulation. For the purposes of transduction, PBL studies have been reported: SV(HBS) and SV(BUGT), were cultured without added IL-2. Following transduc- which respectively carry the cDNA for hepatitis B tion, PBL were cultured with IL-2 as described. surface antigen (HBsAg) and human bilirubin-UDP- 44,45 glucuronosyl-transferase. Detection of a1AT by Northern blot Total RNA from parental cells alone and cells expressing s Production of rSV40 derivative virus a1AT or HBsAg was extracted using RNAeasy Mini Kit The techniques used to generate rSV40 viruses from such (Qiagen). mRNAs were detected by Northern hybridiza- 24 plasmids as pT7ma1AT have been described. Briefly, tion analysis as previously described employing a the virus genome was excised from the carrier plasmid human a1AT cDNA probe. Samples of 15 mg of total and purified by agarose gel electrophoresis. It was then RNA were electrophoresed in a 1% agarose/formalde- recircularized and transfected into COS-7 cells, the hyde gel, transferred on a nylon filter (Nytran Super packaging cell line used for these studies. No helper Charge, Schleicher and Schuell), UV cross linked with a virus was used: COS-7 cells supply the necessary Tag in Stratalinker oven (Stratagene), and baked for 2 h at 801C. trans. Virus stocks were prepared as cell lysate then The filters were prehybridized in 50% formamide, 5 Â purified as described elsewhere.24 Resulting viruses are SSPE, 20 mg/ml denaturated salmon sperm DNA, 5 Â replication incompetent. Virus stock is titered by in situ Denhardt’s, 0.1% SDS at 421C for 8 h. The filters were PCR as reported.46 subsequently hybridized under the same conditions with a a1AT cDNA probe labeled with a-32P-dCTP by a Cell culture random DNA labeling kit (Roche Molecular) at 421C The COS-7 packaging cell line was maintained in overnight. After hybridization, the filters were washed Dulbecco’s modified Eagle’s medium (DMEM) with under high stringency conditions in 0.1 Â SSC 0.1% SDS 2mML-glutamine adjusted to contain 1.5 g/l. sodium at 651C for 30 min and the signals were visualized with bicarbonate, 4.5 g/l glucose, 1.0 mM sodium pyruvate, the use of a phosphoimager (Molecular Dynamics Storm streptomycin (100 mg/ml), penicillin (200 units/ml), gen- 840). tamicin (10 mg/ml) and 10% (vol/vol) fetal calf serum. The same culture conditions were used for 293T cells. HIV-1 viral stocks SupT1 CD4-positive human T-lymphocyte cell line 293T cells were transfected using calcium phosphate susceptible to HIV-1 infection was grown in RPMI-1640 (Promega). 1 Â 106 293T cells were plated in 10 cm

medium, streptomycin (100 mg/ml), penicillin dishes, transfected with 10 mg of pNL4-3 DNA, then (200 units/ml), gentamicin (10 mg/ml) and 10% (vol/ incubated in DMEM containing streptomycin (100 mg/ vol) fetal bovine serum (complete RPMI). The human ml), penicillin (200 units/ml), gentamicin (10 mg/ml) and hepatocellular carcinoma cell line HepG2 was grown in 10% (vol/vol) fetal calf serum for 7 h. The medium was Dulbecco’s modiﬁed Eagle’s minimum essential medium then removed and replaced with fresh DMEM containing

Gene Therapy Inhibiting HIV-1 proprotein processing P Cordelier et al 475 streptomycin (100 mg/ml), penicillin (200 units/ml), gen- 5% milk ), blots were incubated overnight at 41C with a tamicin (10 mg/ml) and 10% (vol/vol) fetal calf serum. polyclonal anti-a1AT antibody (Sigma) (1:200). Secondary Virus-containing supernatants were collected at 48 h. antibodies goat anti-rabbit (Pierce) horseradish perox- Infectious virus present in transfected cell supernatants idase-conjugated antibodies (1:10 000) were added, and was quantitated by standard techniques on SupT1 cells blots were incubated for 1 h at room temperature. Each to determine the 50%-tissue culture infective dose antibody incubation was followed by four washes in PBS 47 (TCID50) of HIV-1 per milliliter of fluid. (pH 7.4)/0.1% Tween-20. Chemiluminescence (ECL Plus, Amersham) was performed as directed by the manufac- HIV-1 infectious assays turer. The HIV-1-susceptible T cells (SupT1,PBL) that expressed a the 1AT, HBsAg or BUGT proteins were maintained in Immunostaining for a AT expression complete RPMI for 5 days before HIV-1 challenge four 1 50 x 103 SV(HBS)-transduced SupT1 and cells expressing hours before infection of the cells with HIV-1 ,1 Â NL4-3 a AT proteins are transferred 2 weeks post-transduction 106 mock-transduced cells and cells expressing a AT, 1 1 to four-chambered glass slides. Cells are fixed with PBS/ HBsAg or BUGT were plated in 1 ml of medium per well 4% PFA pH 7.4 at room temperature for 20 min, in a 12-well plate. Cells were infected with various permeabilized using PBS/0.1% NP40 for 10 min at room TCID of cell-free HIV-1 overnight. The cells were 50 NL4-3 temperature, and blocked overnight in normal donkey washed with prewarmed, serum-free medium and serum at 41C. Slides are treated for 1 h at 371C with a maintained in growth medium. Every 3 days, half of 1:200 dilution of polyclonal goat anti-a AT (Sigma) for 1 the supernatant was changed and aliquoted for HIV-1 1 hat371C. After extensive washes with PBS, cells are p24 analyses. Every 3 days, cells were split in a 1:2 ratio incubated with a 1:1000 dilution of Alexa Fluir 546 to maintain a cell density of approximately 1 x 106/ml. labeled donkey anti-goat IgG (Molecular Probe) for 1 h at The HIV-1 p24 antigen levels in supernatant were 371C. After extensive washes in PBS, cells are mounted determined by ELISA. The infectious doses of HIV-1 and analyzed by epifluorescence microscopy. used in these experiments are expressed as TCID50 per 1 x106 cells per ml of culture. Western blot analysis Protein isolation from virions 5x106 mock-transduced SupT1 and PBL, and cells 6 5 Â 10 SupT1 cells and PBL, either mock-transduced or expressing a1AT, HBsAg or BUGT proteins, were treated expressing a1AT, HBsAg or BUGT proteins, were for 1 week with 4000 and 10 000 TCID50 of HIV-1NL4-3, infected with 10 000 and 40 000 TCID50 of HIV-1NL4-3, respectively. After one wash in PBS, cells were lysed on respectively. The cells were washed with prewarmed, ice in 500 ml of lysis buffer (50 mM Tris, pH 7.4/150 mM serum-free medium and maintained in complete med- NaCl/1% Nonidet P-40/1% sodium deoxycholate/1 Â ium. Cell-free culture supernatants (5 ml) were harvested protease inhibitor mix (Sigma)/0.5 mM sodium orthova- at day 7 post infection, and subjected to ultracentrifuga- nadate/0.02% sodium azide) for 5 min. The protein tion at 40 000 g for 120 min at 41C. Virus pellets were suspension was transferred to a microfuge tube and resuspended and lysed on ice in 1 ml of lysis buffer was spun for 5 min at 10 000 g to remove cell debris. The (50 mM Tris, pH 7.4/150 mM NaCl/1% Nonidet P-40/1% protein suspension was subsequently concentrated using sodium deoxycholate/1 Â protease inhibitor mix (Sig- centricon centrifugal filter devices (Millipore). Centricon ma)/0.5 mM sodium orthovanadate/0.02% sodium YM-10 (cut-off value 10 kDa) were used for p55GAG azide) for 15 min. The protein suspension was subse- Western studies, whereas YM-50 (cut-off value 50 kDa) quently concentrated using centricon centrifugal filter were used for gp120 Western studies. For gp120 Western devices (Millipore). Centricon YM-10 (cut-off value 10 blots, proteins were separated by 5% TB PAGE SDS-free kDa) were used for p55GAG Western studies, whereas (Bio-Rad). For p55Gag Western blots, proteins were YM-50 (cut-off value 50 kDa) were used for gp120 separated by 4–20% Ready SDS/PAGE (Bio-Rad) and Western studies. transferred to a PVDF-Plus membrane (Osmonics, Minnetonka, MN,USA). After overnight blocking (PBS Immunoprecipitation of a1AT (pH 7.4)/0.1% Tween-20 containing 5% milk), blots were 5 Â 106 mock-transduced SupT1 and cells expressing incubated with mouse anti-gp120 ID6 antibody (ARRRP a1AT proteins were lysed on ice in 500 ml of lysis buffer # 2343) (1:1000) or sheep anti-p24 antibody (ARRRP #287) (50 mM Tris, pH 7.4/150 mM. NaCl/1% Nonidet P-40/1% (1:500) in PBS (pH 7.4)/0.1% Tween-20 containing 1% Sodium deoxycholate/1 Â protease inhibitor mix (Sig- milk for 3 h at room temperature. Secondary antibodies ma)/0.5 mM sodium orthovanadate/ 0.02% sodium of rabbit anti-mouse or goat anti-sheep (Pierce) horse- azide) for 5 min. The protein suspension was transferred radish peroxidase-conjugated antibodies (1:10 000) were to a microfuge tube and was spun for 20 min at 15 000 Â added, and blots were incubated for 1 h at room g to remove cell debris. 500 mg of total cell proteins was temperature. Each antibody incubation was followed incubated with 1/10 dilution of polyclonal anti-a1AT by four washes in PBS (pH 7.4)/0.1% Tween-20. antibody (Sigma) overnight at 41C under agitation. Chemiluminescence (ECL Plus, Amersham) was per- Protein A sepharose was then added for 3 h at 41C formed as directed by the manufacturer. It should be under agitation. Immune complexes were extensively emphasized that to facilitate clear resolution of gp160 washed with lysis buffer, then boiled for 5 min. Proteins and gp120 bands, smaller amounts of total proteins from were separated by 4–20% Ready SDS/PAGE (Bio-Rad) SV(HBS)-transduced, or later SV(BUGT)-transduced cells and transferred to a PVDF-Plus membrane (Osmonics, and viral progeny were loaded per lane than from Minnetonka, MN, USA). After room temperature block- SV(AT)-transduced cells and viral progeny. To test for an ing for 1 hour (PBS (pH 7.4)/0.1% Tween-20 containing equal protein loading of the various lanes, p55Gag

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Gene Therapy