Gene Therapy (1999) 6, 660–666  1999 Stockton Press All rights reserved 0969-7128/99 $12.00 http://www.stockton-press.co.uk/gt Inhibition of HIV-1 by an anti- single-chain variable fragment (SFv): delivery by SV40 provides durable protection against HIV-1 and does not require selection

M BouHamdan1, L-X Duan1, RJ Pomerantz1 and DS Strayer1,2 1The Dorrance H Hamilton Laboratories, Center for Human Virology, Division of Infectious Diseases, Department of Medicine; and 2Department of Pathology, Anatomy and Cell Biology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA, USA

Human immunodeficiency virus type I (HIV-1) encodes the SFv-IN was confirmed by Western blotting and several that are packaged into virus particles. Inte- immunofluorescence staining, which showed that Ͼ90% of grase (IN) is an essential retroviral enzyme, which has SupT1 T-lymphocytic cells treated with SV(Aw) expressed been a target for developing agents to inhibit virus repli- the SFv-IN without selection. When challenged, cation. In previous studies, we showed that intracellular HIV-1 replication, as measured by HIV-1 p24 expression of single-chain variable fragments expression and syncytium formation, was potently inhibited (SFvs) that bind IN, delivered via retroviral expression vec- in cells expressing SV40-delivered SFv-IN. Levels of inhi- tors, provided resistance to productive HIV-1 infection in bition of HIV-1 infection achieved using this approach were T-lymphocytic cells. In the current studies, we evaluated comparable to those achieved using simian-virus 40 (SV40) as a delivery vehicle for anti-IN (MLV) as a transduction vector, the major difference being therapy of HIV-1 infection. Prior work suggested that deliv- that transduction using SV40 did not require selection in ery using SV40 might provide a high enough level of trans- culture whereas transduction with MLV did require selec- duction that selection of transduced cells might be tion. Therefore, the SV40 vector as gene delivery system unnecessary. In these studies, an SV40 expression vector represents a novel therapeutic strategy for gene therapy to was developed to deliver SFv-IN (SV(Aw)). Expression of target HIV-1 proteins and interfere with HIV-1 replication.

Keywords: HIV-1; SV-40; gene therapy; intracellular immunization

Introduction of cell types from humans and other mammals and expresses its genes in them. Recombinant, replication- Various techniques have been developed to express deficient SV40-derived vectors may express transgenes recombinant constructs within cells, in culture, in animal stably in cell lines, in primary cultures, and in vivo.4–6 The 1 models and in humans. The application of molecular virus is stable to manipulation and can be made to high genetics to human biology and disease has improved our titer (у1010 infectious units (IU)/ml), and further concen- understanding of and ability to treat a variety of disease trated if needed. states. Retroviral gene delivery vectors based on onco- HIV-1, as a member of the lentivirus family, has a com- , such as Moloney murine leukemia virus plex viral life cycle and utilizes multiple cellular and (MLV), have been the most commonly used vectors for virally encoded regulatory proteins to tightly control its 2,3 gene transfer into the host cell . MLV has been replication.7 The essential retroviral enzymes, reverse used to deliver therapies for diverse diseases, including transcriptase (RT), ribonuclease H (RNaseH), protease cancer, inherited genetic disorders and infection with (PR) and integrase (IN), lack cellular counterparts and HIV-1. However, onco-retroviral vectors have limitations have been used as targets for developing agents that that often include a need to select cultured cells to enrich inhibit virus replication.8–10 Despite considerable for those cells expressing the transgene. Such selection advances in anti-RT and PR chemotherapy, genetic generally necessitates transduction ex vivo, followed by changes in the virus can confer drug resistance.11 This cumbersome selection in culture. problem has led to proposals for alternative therapeutic We have devised a gene transfer system based on sim- strategies in which host cells may be genetically altered 4 ian virus-40 (SV40) as a vector. SV40 infects a wide range or engineered to confer long-lasting protection against virus infection or replication.12,13 Several such strategies are currently being reported and applied to the inhibition Correspondence: DS Strayer, Department of Pathology, Anatomy and Cell Biology, Jefferson Medical College, Thomas Jefferson University, Philadel- of HIV-1 replication. They include exploitation of trans- phia, Pennsylvania 19107, USA dominant-negative mutant HIV-1 protein expression, Received 14 July 1998; accepted 28 October 1998 viral antisense oligonucleotide sequences, specific Inhibition of HIV-1 by SV40-delivered anti-integrase SFv M BouHamdan et al 661 ribozymes, HIV-1 transactivated suicide genes and intra- cellular against several different HIV-1- specific proteins.14–26 Recently, we reported that intracellular expression of SFv moieties targeted to RT, IN and Rev strongly inhibited HIV-1 replication in human cells.15–20,23,24,27 An early event in the life cycle of all retroviruses, including HIV-1, is integration of a double-stranded proviral DNA into the host cell genome. This step is necessary for pro- ductive viral replication.28 In natural infection, linear viral DNA contained within pre-integration complexes is the direct precursor of the integrated proviral genome.29 In previous studies, we demonstrated that anti-IN SFv #33, driven by the cytomegalovirus immediate–early pro- moter (CMV-IEP) and delivered by MLV to selected human T-lymphocytes, effectively inhibited HIV-1 repli- cation.20 To assess intracellular immunization as a tool for gene therapy of HIV-1 infection further, IN was tar- geted for specific blockade by the same anti-IN intracellu- lar SFv expression construct, but delivered using an SV40 vector. The effectiveness of this construct in inhibiting HIV-1 was tested in a CD4+ human T lymphoma cell line. Figure 1 Schematic map of the SV40-derivative construct used to express We show here that an SV40-derived vector can trans- SFvIN#33. The genome of SV(Aw), the SV40-derivative used to transduce duce human T-lymphocytic cells to express the protein the SFvIN#33, is illustrated here. This virus was constructed as described SFv-IN effectively without selection. Furthermore, HIV-1 in Materials and methods, and in general according to the approach out- replication, as measured by HIV-1 p24 antigen expression lined in previous publications.4 Briefly, in this virus, the SFvIN#33 cDNA and syncytia formation, was inhibited in SupT1 is driven by the CMV-IEP, which is in turn immediately downstream T-lymphocytic cells expressing SFv-IN. from the SV40-EP. The latter overlaps the SV40 , and thus cannot be entirely deleted from the viral genome. The late virus genes (VP1, VP2, VP3) are intact in this construct, as are the SV40 late pro- moter, enhancer and early and late polyadenylation signals. The construc- Results tion of SV(HBS), the control SV40 virus used in these studies, was reported previously.30 Construction of viruses expressing SFvIN#33 and HBsAg proteins A map of SV(HBS) has been reported.30 This recombinant SV40 virus was used as a negative control. Construction of pSLXCMV-SFvIN#33, which expresses SFvIN#33, has been described previously.20 Briefly, sequences encoding the variable light (V1) and variable heavy (Vh) chains of the anti-IN monoclonal antibody (Mab) were cloned from a murine hybridoma cell-line’s RNA. After ligation of V1 and Vh chains into a single fragment, by utilizing the flexible linker (GGGGS)3, the SFv fragment was cloned into an SV40 expression vector downstream of cytomega- lovirus immediate–early promoter (CMV-IEP). The orien- tations and structures of open reading frames were veri- fied by DNA sequencing. A map of the SV40 vector containing the SFvIN#33 (SV(Aw)) driven by CMV-IEP, and used for all these studies, is shown in Figure 1.

Detection of SFvIN#33 by Western blotting Lysates were prepared from 106 SupT1 cells transduced as described with SV(Aw) or with MLV-SFvIN#33, or mock-transduced. These proteins were electrophoresed using SDS-PAGE, blotted to PVDF membranes, probed with anti-mouse IgG antibody and visualized as described in Materials and methods. As a positive con- Figure 2 Expression of anti-IN SFv in SV(Aw)- and MLV-SFvIN#33- trol, recombinant SFvIN#33 produced in E. coli was used. transduced cells. SupT1 cells were treated with SV(Aw), once at MOI Results demonstrated that both selected MLV-SFvIN#33- of 10, without selection. Protein from 106 cells, harvested 4 days after transduced SupT1 cells and unselected SV(Aw)-trans- transduction, was electrophoresed on SDS-PAGE, blotted to PVDF mem- # branes and probed with anti-mouse IgG to visualize expression of the duced cells expressed the SFvIN 33 transgene (Figure 2). # Levels of transgene expression were comparable in SFvIN 33. In parallel, protein from an equal number of selected, MLV- SFvIN#33-transduced cells, was electrophoresed, blotted and visualized unselected SV(Aw)-transduced cells and in selected similarly. For both preparations, SFvIN#33 expressed in E. coli was the MLV-SFvIN#33-transduced cells (Figure 2). The larger positive control. Equal numbers of non-transduced SupT1 cells were the molecular size bands seen here in the extracts from negative control. Inhibition of HIV-1 by SV40-delivered anti-integrase SFv M BouHamdan et al 662 SV(Aw)-transduced cells may represent either splice vari- cells, compared to the same SFv delivered by MLV- ants of the original transcript or ribosomal reading SFVIN#33. HIV-1 challenge studies were performed

through translational stop signals. using virus strain NL4–3. Control SupT1 cells were untreated, or treated with SV(HBS). It is of note that cell SV40 delivery of SFvIN#33 and expression in growth curves and viability of SupT1 cells were mammalian cells unaltered by transduction with either of these SV40- To obtain transfectants expressing SFvIN#33 and HBsAg, derived vectors, as described above. The infectivity recombinant SV40-based viruses were prepared from assays were performed with transduced, mixed SupT1 COS-7 packaging cells and SupT1 cells were treated with cell populations. For comparison, MLV-SFvIN#33-trans- these viruses as described in Materials and methods. duced, selected SupT1 cells were challenged in parallel. After infection with SV(Aw) or SV(HBS), the cells were For HIV-1 challenge experiments, unselected and cultured for 2 weeks without selection and then analyzed uncloned SFvIN#33-transduced SupT1 cells, HBSAg by immunostaining. Expression of SFvIN#33 was SupT1 cells and non-transduced SupT1 cells were treated

detected using an anti-mouse IgG. The SV(Aw)-treated with two different doses of HIV-1NL4–3 (0.05 pg/ml and SupT1 cells express the transgene, SFvIN#33 (Figure 3). 0.5 pg/ml of p24 antigen equivalents). Replication of SupT1 cells treated with SV(HBS) were used as controls HIV-1 in these cultures was evaluated by quantifying the and did not express detectable SFvIN#33 (Figure 3). The levels of HIV-1 p24 antigen released into the culture anti-IN-SFv protein was principally detected in the cyto- medium (Figure 4). At the lower MOI, only very low lev- plasm of transduced cells. els of HIV-1 p24 antigen were observed in the super- These results are important for several reasons. First, natants of SV(Aw)-transduced, unselected SupT1 cells these data demonstrate that SV40 can transduce SupT1 challenged with HIV-1NL4–3. Similar protection was seen cells to express the anti-IN SFv efficiently. Secondly, the in selected SupT1 cells transduced with MLV-SFvIN#33, efficiency of SV40 transduction is very high: more than that had been selected following transduction to assure 90% of cells stain positively for SFvIN#33 expression. sustained transgene expression. These data indicate vir- Unlike our experience with MLV transduction of the tually complete protection from HIV-1 by SV40 transduc- same SFv (approximately 30% of cells were positive for tion of SFvIN#33, at levels comparable to those achieved SFvIN#33 expression), selection of the SV40-treated using the same anti-IN-SFv delivered with MLV- SupT1 cells for transgene expression in culture was not SFVIN#33. This protection was evident throughout the required to achieve very high rates of transduction. time-course of our studies, up to and including 24 days Finally, SV40 transduction did not slow the rate of cell after challenge. proliferation significantly. Cell recovery, tested by coun- Control non-transduced and SV(HBS)-transduced cells ting of trypan blue-negative cells, was comparable in showed no inhibition of HIV-1 replication, as demon- SV40-treated and MLV-treated cultures (data not shown). strated by dramatic initial increases in HIV-1 p24 antigen Thus, SV40 transduction was not demonstrably toxic to (Figure 4). On day 24 after infection, SupT1 SFvIN#33 these cells. mixed cell populations showed complete inhibition of HIV-1 p24 antigen production, as compared with the Inhibition of HIV-1 replication in human T-lymphoid cells non-transduced SupT1 cells and cells expressing HBsAg. that express the anti-IN SFv: comparative effects of At the higher MOI, little or no protection from HIV-1 MLV and SV40 transduction challenge was seen in cultures expressing SFvIN#33 We then tested whether intracellular expression of transduced by either MLV-SFvIN#33 or SV40 (data not SFvIN#33 delivered by SV40 to SupT1 cells could prevent shown). HIV-1 replication in this line of susceptible T-lymphoid Microscopically, SV(Aw)-transduced SupT1 cells

Figure 3 Analysis of SFv protein expression in transduced SupT1 cells. Transduced SupT1 cells were fixed and immunostained with goat anti-mouse polyclonal IgG, using a FITC-conjugated rabbit anti-goat IgG. SupT1 cells transduced with SV(HBS) (a) were used as negative controls. SupT1 cells transduced with SV(Aw) (b, c) demonstrated mainly cytoplasmic staining. Magnification X100 (a, b) and ×400 (c). Inhibition of HIV-1 by SV40-delivered anti-integrase SFv M BouHamdan et al 663 Discussion

We have previously reported that anti-IN SFv#33 signifi- cantly inhibited HIV-1 replication, when delivered to SupT1 cells using a retroviral vector, followed by selec- tion for SFv-expressing cells. We now report that anti-IN SFv#33 delivery, using SV40 as a transduction vehicle, is equally effective as MLV SFvIN#33 in protecting SupT1 cells from HIV-1 infection and that this delivery system has the distinct advantage that its effectiveness does not require selection for transgene-expressing cells before challenge with HIV-1. SV40 was chosen as a delivery vehicle to compare with MLV in these studies for several reasons. SV40 infects virtually all mammalian nucleated cell types, and high titer SV40-derived gene transfer vectors are stable and are easily produced, with expression of SV40-delivered transgenes persisting for long periods in vivo.4,5 Also, a single transduction treatment of normal human or simian bone marrow progenitor cells results in long-term car- Figure 4 Inhibition of HIV-1 replication in SFvIN-transduced T-lymph- oid cells. SupT1 cells were transduced with SV(Aw), or SV(HBS) (mixed riage of the transgene, most likely via integration (DS Strayer et al, preliminary observations). SV40 also trans- cellular populations). These SupT1 cells were challenged with HIV-1NL4–3 virions with identical quantities of HIV-1 p24 antigen, overnight. HIV-1 duces resting and dividing cells with about equal replication was quantified by assaying HIV-1 p24 antigen levels in the efficiency: unstimulated peripheral blood mononuclear culture supernatants by ELISA. These graphs are representative of two cells are easily transduced ex vivo, as are hepatocytes, independent experiments. neurons and other non-cycling cells in vivo,5,6 (M Zern and D Strayer, in preparation; R Chowdhury and D Strayer, in preparation). This is of importance, as non- activated T-lymphocytes and non-proliferating showed weak cytopathic effects when infected with HIV- monocyte/macrophages are critical cellular reservoirs for 1NL4–3 as assessed by observing syncytium formation and HIV-1 in vivo. cell death (Figure 5). Even when cultures were chal- The SV40-derived constructs applied to these studies 4 lenged at high HIV-1NL4–3 MOI, cells transduced with SV are replication-incompetent in cells that lack SV40 Tag. (Aw) showed delayed syncytium formation at assay In addition, the viral large T antigen is the major target points up to 18 days after challenge (Figure 5). These of the immune system in eliminating cells infected with results suggest that intracellular anti-IN SFv expression wild-type (wt) SV40,31–35 suggesting that Tag-deleted, protected cells against the cytopathic effects of HIV-1. On replication-defective SV40-derivative viruses should be day 21 after infection, non-transduced SupT1 cells or minimally immunogenic. We have confirmed this

SV(HBS)-transduced SupT1 cells, infected with HIV-1NL4–3 hypothesis: such vectors can be administered to normal, started to die. By comparison, SupT1 cells transduced immunocompetent animals many times but do not elicit with SFvIN#33 showed normal cell growth. Eventually, detectable neutralizing antibody versus SV40.30 however, challenge at 0.5 pg/ml doses of HIV-1NL4–3 The combination of lack of immunogenicity and high overcame the protective activity of SFvIN#33, whether levels of durable transduction efficiency of SV40 for bone delivered by SV40 or by MLV (not shown). marrow-derived cells, suggested that this virus might

Figure 5 Anti-IN SFv inhibition of cytopathic effects of HIV-1 infection. The microscopic morphology (syncytium formation and cell death) of SupT1 cells infected with syncytium-inducing strain HIV-1NL4–3 (0.5 pg/ml) after 15 days after infection is shown. (a) SupT1 non-transduced cells; (b) mixed cell populations expressing HBsAg; (c) mixed cell populations transduced with SV(Aw) and expressing anti-IN SFv. All photomicrographs are ×400. Inhibition of HIV-1 by SV40-delivered anti-integrase SFv M BouHamdan et al 664 deliver anti-HIV-1 gene therapeutics effectively. To test anti-retroviral gene therapy represents a potentially use- this possibility, we developed a recombinant SV40 vector ful new class of anti-retroviral agents. This concept could to deliver the SFvIN#33, which had already been shown also further assist in analyzing the molecular events to inhibit HIV-1 infection when it was delivered by within the lentiviral life cycle. MLV.20 Cytoplasmic SFvIN#33 blocks HIV-1 infection some- what more efficiently than a nucleus-targeted Materials and methods SFvIN#33.20 Our current immunostaining results suggest that the SFvIN#33 localizes predominantly in the cyto- Plasmids and viral expression constructs plasm. Using SV40 to express the anti-IN SFv#33 did not The HIV-1 molecular clone used in this study was pNL4–3. affect the localization of the protein, which is still cyto- This strain was obtained from the AIDS Reagent Reposi- plasmic. In this report, we demonstrate that intracellular tory (NIH). HIV-1NL4–3 is T-tropic viral strain with highly expression of an anti-HIV-1 IN-SFv decreases HIV-1 rep- cytopathic effects. # lication in T-lymphocytic cells. This finding is significant The construct, MLV vector containing anti-IN 33 # 20 in that it suggests a new and previously unexplored (pSLXCMV-SFvIN 33), has been described previously. # avenue in attempts to inhibit HIV-1 replication before The IN 33 SFv binds to the non-specific DNA binding integration of viral DNA into the host genome. site in HIV-1 IN, and strongly inhibits HIV-1 replication Transduction using onco-retroviral vectors has certain in transduced human T-lymphocytic cells. limitations. Firstly, one must often select the cells Construction of recombinant SV40 derivative viruses expressing the gene of interest by using a neomycin- for gene transfer has been described previously in prin- 4 resistance gene (neor). Secondly, although they may vary ciple. The recombinant SV40-derived virus, SV(Aw), was in host cell range depending on how they are packaged, made similarly and will be described briefly. The wild- oncoretroviruses usually infect a limited range of cells type (wt) SV40 genome cloned into pBR322 was provided and these cells must be dividing at the time of infection. as plasmid pBSV-1 from Janet Butel (Baylor College of Finally, the efficiency of transduction with MLV vectors Medicine). The large T antigen gene was excised from in our hands is relatively low. In contrast, SV40 infects this plasmid and replaced by a polylinker. Into this poly- a wide range of cells and has a very high transduction linker, an expression construct containing cytomegalo- efficiency. Our results show that Ͼ90% of cells treated virus intermediate–early promoter (CMV-IEP) plus the # with SV(Aw) express anti-IN SFv#33 after transduction cDNA for SFvIN 33 was cloned, to yield and continued culture. This is an advantage that could pBSV(CMV)Aw. be very useful for transduction of primary cells. It has The control virus used for these studies was SV(HBS). been reported that SV40 can infect such cells as hemato- The production and characterization of this virus have 30 poietic and peripheral blood mononuclear cells been reported. Briefly, the gene for B surface (PBMC).5,36 The authors in the former report used the antigen (HBsAg) was cloned into the Tag-deleted SV40 SV40-based pseudoviral system for transfer of the human genome, immediately downstream from two tandem MDR1 gene. Highly efficient gene transfer into several SV40 early promoters. The resulting plasmid was important cell types, including fresh unmanipulated pri- pSV5(HBS). mary human bone marrow cells and PBMC, was shown.5,36 We have also found high efficiency gene trans- Production of SV40 derivative viruses fer to bone marrow-derived cells using SV40, as The techniques used in generating viruses from such described above (DS Strayer et al, in preparation). plasmids as pSV5(HBS) and pBSV(CMV)Aw have been Of importance, most if not all anti-HIV-1 gene thera- described.4 Briefly, the virus genome was excised from peutics can be overwhelmed by utilizing relatively high the carrier plasmid and purified by agarose gel electro- challenge MOIs dramatically in vitro. This observation phoresis. It was then recircularized and transfected into was repeated in our studies: SFvIN#33 delivered by both COS-7 cells, the packaging cell-line used for these studies. SV40 and MLV was highly effective in inhibiting HIV-1 No helper virus was used: the COS cells supply the at challenge 0.05 pg/ml of p24 antigen, but did not dra- necessary Tag in trans. These virus stocks were prepared matically alter the course of HIV-1 infection at challenge as cell lysates. After initial virus preparations were made 0.5 pg/ml of p24 antigen. This finding may be problem- in this fashion, expanded stocks were prepared by atic for inhibiting HIV-1 in the interstices of lymphatic infecting COS-7 cells with virus from the original prep- tissues of infected individuals, where HIV-1 concen- aration. Further transfections are not done. Resulting trations may be higher than those inhibited by the thera- viruses are replication incompetent.4 peutic approaches described here.37 In such cases, mul- Virus stocks are titered by in situ PCR as described pre- tiple simultaneous therapeutic modalities or multiple viously.38 This provides for direct enumeration of infec- administrations may be necessary to provide effective tious units in a preparation. protection. The ability to deliver functional anti-HIV-1 SFv-IN, Cell cultures using SV40 as vector represents a novel and potentially The COS-7 packaging cell line was maintained in Dulbec- useful technology that could contribute to the efficient co’s modified Eagle’s medium (DMEM) + 10% fetal calf inhibition of HIV-1 replication. The high efficiency of serum (FCS, Hyclone, Logan, UT, USA). The same cul- SV40 transduction, the lack of need for selection, the ture conditions were used for 293T. SupT1, a CD4+ ability of SV40 to transduce a wide range of target cells human T-lymphocyte cell line susceptible to HIV-1 infec- and the potential for multiple administrations in vivo, all tion, was grown in RPMI-1640 medium + 10% FCS.4 All combine with the effectiveness of SFvIN#33 delivery by the cells were grown at 37°C in humidified incubator

SV40 demonstrated here, to suggest that SV40-delivered with 5% CO2. Inhibition of HIV-1 by SV40-delivered anti-integrase SFv M BouHamdan et al 665 HIV-1 viral stocks NaCl, 0.5% NP-40, 1 mm PMSF, 10 mg/ml leupeptin, 293T cells were transfected by a standard calcium-phos- 2 mg/ml aprotinin, 10 mg/ml pepstatin). Protein lysate phate method (Promega, Madison, WI, USA). 1 × 106 was loaded on a 12% SDS-polyacrylamide gel, electroph- 293T cells were plated in 10 cm dishes, transfected with oresed and blotted to a PVDF membrane (Schleicher and 10 mg of pNL4–3 DNA and then incubated in DMEM con- Schuell, Keene, NH, USA). The membrane was blocked taining 10% FCS (growth medium) for 7 h. The medium with 5% skim milk for 2 h, treated with goat polyclonal was then removed and replaced with fresh DMEM anti-mouse IgG overnight at 4°C, and then with horserad- growth medium + 10% FCS. Virus-containing super- ish peroxidase (HRP)-conjugated rabbit anti-goat IgG natants were collected at 48 h. The quantity of virus antibody for 2 h at room temperature. Signal was present in transfected cell supernatants was determined detected using chemiluminescence reagent (DuPont by measuring HIV-1 p24 antigen levels, using an NEN, Wilmington, DE, USA). enzyme-linked immunosorbant assay (ELISA) (Cellular Products). Viral stocks were assayed for their infectious titers on CEM T-cells, and these data were used to Acknowledgements calculate MOI in each experiment. The authors wish to thank Ms Rita M Victor and Ms Transduction of T-lymphocytic cells Brenda O Gordon for excellent secretarial assistance. The Procedures for infecting SupT1 cells with MuLV- technical assistance of Mr Joe Milano was invaluable for SFvIN#33, and for selecting transgene-expressing trans- these experiments. Dr Janet S Butel kindly supplied duced cells, have been reported elsewhere in detail.20 For pBSV-1, from which all SV40-derived constructs were transduction using SV40-derived vectors, SupT1 cells made. The collaboration and input of Dr Harris were treated with SV(Aw), carrying the cDNA for Goldstein, Albert Einstein College of Medicine, and of Dr SFvIN#33, or SV(HBS), carrying the gene for R Paul Johnson, Harvard Medical School, are gratefully surface antigen (HBsAg) for 24 h at an MOI of 10. This acknowledged. These studies were supported in part by step was repeated twice, but at MOI of 3, on sequential USPHS grants AI41399 and RR13156 to DSS and AI38666 days. No selection was used. The SV40-treated SupT1 and AI36557 to RJP. cells were then cultured for 2 weeks.

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