Transduction of cellular sequence by a human immunodeficiency virus type 1-derived vector.

During studies examining the rate of human immunodeficiency virus type 1 (HIV-1) mutation in a single cycle of replication, the 5' long terminal repeat of one progeny provirus was found to contain an insertion of 147 bp including an entire tRNA sequence as well as an additional 66 bp insertion of nonviral origin. Database searches revealed that 65 of 66 bp aligned with the human CpG island sequence found on chromosomes 6, 14, and 17. Therefore it seems probable that it is of human cellular sequence origin and was transduced by HIV-1. This is the first demonstration that HIV-1 can capture a cellular sequence. The site of integration of the parental provirus was mapped to chromosome 1p32.1. Sequence with homology to the transduced CpG island was not found on chromosome 1, suggesting that the transduced cellular sequence was not linked to the site of viral integration.

Role of B cells as antigen-presenting cells in vivo revisited: antigen-specific B cells are essential for T cell expansion in lymph nodes and for systemic T cell responses to low antigen concentrations.

Studies in B cell-deficient mice generated by continuous injection of anti-mu antibodies (muSM) showed that T cell priming in lymph nodes was dependent on antigen presentation by B cells. This concept has recently become controversial since a wide range, from complete deficiency to near normal T cell responses, was reported in studies carried out with B cell-deficient mice generated by gene disruption (muMT). In this study we show that in the absence of B cells, T cell responses are greatly reduced in all the available muMT mouse strains although responses in muMT of the C57BL/6 background (which were used for most studies with muMT) were much more variable and could reach up to 42% of control. In contrast, T cell responses in muMT --> F(1) bone marrow chimeras which have the same phenotype as muMT were totally impaired, suggesting a principle difference between mice developing without B cells (muMT mice) and muSM which are made B cell deficient only after birth. Normal T cell priming was completely restored by reconstitution of muMT and muMT --> F(1) mice with syngeneic B cells. Interestingly, only B cell populations containing antigen-specific B cells were capable of reconstituting T cell responses. Monoclonal B cells taken from Ig transgenic mice could not reconstitute responses to an irrelevant antigen. We also found that B cells were also required for systemic T cell priming when antigen concentrations were limiting but were not required for priming (for T cell help) when mice were immunized with a high antigen dose.

An inducible packaging cell system for safe, efficient lentiviral vector production in the absence of HIV-1 accessory proteins.

Lentiviral vectors based on human immunodeficiency virus type 1 (HIV-1) possess the ability to deliver exogenous genes to both dividing and nondividing cells and to subsequently establish a stable provirus in these target cells, which can allow long-term expression of the transferred gene. Herein we describe a stable packaging cell line that is devoid of HIV-1 tat, vif, vpr, vpu, and nef. In order to avoid any risk of cytotoxicity associated with constitutive expression of HIV-1 protease or the VSV-G envelope protein, transcription of the packaging and envelope constructs was tightly controlled by employing the ecdysone-inducible system. Using this cell line, we have been able to consistently generate concentrated pseudotyped vector virus stocks with titers in the range of 10(8) IU/ml, which can efficiently transduce actively dividing and growth-arrested cells in vitro. This novel packaging cell line for lentiviral vectors facilitates the production of high-titer virus stocks in the absence of replication-competent virus and provides us with an important tool for use in future gene transfer studies.

A gene therapy approach for treating T-cell-mediated autoimmune diseases.

Experimental autoimmune encephalomyelitis (EAE) is a demyelinating disease of the central nervous system (CNS) that serves as a model for multiple sclerosis (MS) in humans. In mice, EAE is mediated by Th1 type CD4(+) T cells specific for various myelin proteins which migrate from the periphery to the CNS. Removal or blocking of CD4(+) cells before or shortly after disease induction was shown to prevent disease onset and/or disease progression but also results in general immune suppression. Most treatment regimens for autoimmune diseases currently rely on general suppression of the T-cell compartment most commonly by steroids. In this paper, an experimental, gene therapy-based model is presented in which susceptible mice are made resistant to EAE induction by specifically down-regulating an autoreactive T-cell population. By using a retroviral gene transfer protocol, normal B cells were genetically modified to constitutively express the SJL-specific proteolipid (PLP) encephalitogenic determinant and then adoptively transferred into syngeneic hosts. To ensure appropriate presentation of the exogenous encephalitogenic peptide in association with MHC class II, the encephalitogenic sequence was fused to a lysosomal targeting sequence. Adoptive transfer of syngeneic B cells expressing the PLP encephalitogenic determinant into normal, naive, genetically susceptible mice induced PLP-specific unresponsiveness and completely protected the majority (62% and 83% using an intermediate and a high titer retroviral vector, respectively) of the animals from EAE induction. The remaining animals had a delayed disease onset and/or lower disease severity. All protected mice expressed the exogenous gene in the spleen as detected by reverse transcriptase-polymerase chain reaction.

High rate of recombination throughout the human immunodeficiency virus type 1 genome.

The diploid nature of human immunodeficiency virus type 1 (HIV-1) indicates that recombination serves a central function in virus replication and evolution. Previously, while examining the nature of obligatory primer strand transfers during reverse transcription, a high rate of recombination was observed at the ends of the viral genome within the viral long terminal repeats, prompting the following question: does recombination occur at a high rate throughout the genome? To address this question, two vectors based upon different strains of HIV-1 were utilized. The vectors were comprised predominantly of autologous HIV-1 sequence and were approximately the same size as the parental genome. The proviral progeny of heterodimeric virions were analyzed after a single cycle of replication, and the sequence heterogeneity between the two strains allowed direct examination of recombination crossovers. The results obtained indicate that HIV-1 undergoes approximately two to three recombination events per genome per replication cycle. These results imply that both HIV-1 RNAs are typically utilized during reverse transcription and that recombination is an important aspect of HIV-1 replication.

Long-term contribution to the myeloid compartment by lineage-committed stem cells.

The current paradigm concerning the kinetics of hematopoiesis is that only the most primitive pluripotential bone marrow stem cells can support prolonged hematopoiesis whereas more differentiated, lineage-committed stem cells can only contribute to a particular lineage for a limited period of time. In this study, we present evidence that in mice, the spleen contains a long-lived myeloid-committed stem cell population(s) that continuously replenishes the mature myeloid lineage for at least 9 months. After myeloid-specific retroviral-mediated gene transfer, the exogenous gene could be detected in thioglycollate-induced macrophages and granulocytes by Southern blot analysis and by in situ polymerase chain reaction on an individual cell basis. The targeted stem cell population does not repopulate the bone marrow in secondary recipients and did not give rise to cells other than cells of the myeloid lineage. It therefore represents the first nonpluripotential stem cell population capable of replenishing a hemopoietic lineage for a long period of time. The ability to target a myeloid-specific stem cell could facilitate gene therapy of congenital disorders of the myeloid system such as lysosomal storage diseases. It also offers a unique opportunity to assess the immunologic consequences of expressing an exogenous gene of choice exclusively in the myeloid lineage.

The nature of human immunodeficiency virus type 1 strand transfers.

The diploid nature of human immunodeficiency virus type 1 (HIV-1) suggests that recombination serves a central function in virus replication and evolution. A system was developed to examine HIV-1 strand transfers, including the obligatory DNA primer strand transfers as well as recombinational crossovers during reverse transcription. Sequence heterogeneity between different strains of HIV-1 was exploited for examining primer transfer events. Both intra- and intermolecular primer transfers were observed at similar frequencies during minus-strand DNA synthesis, whereas primer transfers during plus-strand DNA synthesis were primarily intramolecular. Sequence analysis of long terminal repeats from progeny proviruses also revealed a high rate of homologous recombination during minus-strand synthesis, corresponding to an overall rate of approximately three crossovers per HIV-1 genome per cycle of replication. These results imply that both viral genomic RNAs serve as templates during HIV-1 reverse transcription and that primer strand transfers and recombination may contribute substantially to the rapid genetic variation of HIV-1.

Regulated lentiviral packaging cell line devoid of most viral cis-acting sequences.

Packaging cell lines derived from human immunodeficiency virus-1 (HIV-1) are promising tools for in vivo somatic cell gene therapy protocols due to the ability of lentiviruses to infect nondividing cells. We describe here the generation of a safe, stable HIV-1 packaging cell line capable of expressing all of the HIV-1 structural, enzymatic, and regulatory proteins but lacking majority of the cis-acting sequences. The use of an inducible expression system circumvented the cytotoxic and cytostatic effects associated with the expression of some of the HIV-1 viral proteins. Reverse transcriptase activity was detectable in the supernatant from the stable packaging line 1 day after induction, while vector titers peaked 5 days postinduction. Vector titers of up to 3.5 x 10(4) infectious units/ml (IU/ml) were maintained through 8 months following the establishment of the cell line. Lineage-specific gene delivery can be achieved from this established cell line as viral stocks obtained specifically infect CD4(+) target cells. Moreover, this cell line provides a safe and easy to use system for screening of drugs that inhibit HIV-1 replication.

N2- and C8-substituted oligodeoxynucleotides with enhanced thrombin inhibitory activity in vitro and in vivo.

2'-Deoxyguanosine (G) analogues carrying various hydrophobic substituents in the N2 and C8 positions were synthesized and introduced through solid-phase synthesis into 15-mer oligodeoxynucleotide, GGTTGGTGTGGTTGG, which forms a chairlike structure consisting of two G-tetrads and is a potent thrombin inhibitor. The effects of the substitutions at N2 and C8 of the G-tetrad-forming G residues on the thrombin inhibitory activity are relatively small, suggesting that these substitutions cause relatively small perturbations on the chairlike structure formed by the oligodeoxynucleotide. Introduction of a benzyl group into N2 of G6 and G11 and naphthylmethyl groups into N2 of G6 increased the thrombin inhibitory activity, whereas other substituents in these positions had almost no effect or decreased the activity. Particularly, the oligodeoxynucleotide carrying a 1-naphthylmethyl group in the N2 position of G6 showed an increase in activity by about 60% both in vitro and in vivo. Substitutions on the N2 position of other G residues had little effect or decreased the activity. Introduction of a relatively small group, such as methyl and propynyl, into the C8 positions of G1, G5, G10, and G14 increased the activity, presumably due to the stabilization of a chairlike structure, whereas introduction of a large substituent group, phenylethynyl, decreased the activity, probably due to the steric hindrance.

Discontinuous plus-strand DNA synthesis in human immunodeficiency virus type 1-infected cells and in a partially reconstituted cell-free system.

Human immunodeficiency virus type 1 (HIV-1) replication requires conversion of viral RNA to double-stranded DNA. To better understand the molecular mechanisms of this process, we examined viral DNA synthesis in a simple cell-free system that uses the activities of HIV-1 reverse transcriptase to convert regions of single-stranded HIV-1 RNA to double-stranded DNA in a single incubation. This system recapitulated several of the required intermediate steps of viral DNA synthesis: RNA-templated minus-strand polymerization, preferential plus-strand initiation at the central and 3' HIV-1 polypurine tracts, and DNA-templated plus-strand polymerization. Secondary sites of plus-strand initiation were also observed at low frequency both in the cell-free system and in cultured virus. Direct comparison of viral and cell-free products revealed differences in the precision and selectivity of plus-strand initiation, suggesting that the cell-free system lacks one or more essential replication components. These studies provide clues about mechanisms of plus-strand initiation and serve as a starting point for the development of more complex multicomponent cell-free systems.

Use of single-cycle analysis to study rates and mechanisms of retroviral mutation.

Retroviruses evolve at rapid rates. This allows them to escape immune surveillance, thwarts vaccine development, and leads to rapid emergence of drug-resistant virus. Information regarding the retroviral mutation rates and the underlying mechanisms of mutagenesis will undoubtedly expedite the development of strategies to combat retroviral-mediated diseases. In this review, we discuss how the unique retroviral life cycle can be adapted such that retroviral variation can be studied in a single cycle of replication. By limiting replication to a single cycle, retroviral mutation rates can be directly measured, and the consequences of mutations can be observed. In addition, retroviral recombination rates as well as the nature of primer strand transfer during reverse transcription can be studied using this system. Molecular analysis of the spectrum of mutations arising during a single cycle of virus replication also sheds light on the mechanisms of mutagenesis and retroviral replication.

A murine model for genetic manipulation of the T cell compartment.

The expression of exogenous genes in long-lived primary T cells is potentially beneficial for the treatment of various diseases including cancer, AIDS, genetic defects of the lymphoid compartment, and systemic enzyme deficiencies such as hemophilia. One approach for genetic modification of T cells is to introduce therapeutic genes into hematopoietic stem cells that would give rise to cells of the lymphoid lineage. Efficient gene transfer and expression in stem cells is often problematic, however. A more direct approach is to introduce the genes into mature primary T lymphocytes since the transferred genes can be maintained and expressed for long periods by long-lived peripheral T cells. In this report, we describe the adoptive transfer into SCID mice of both murine and human primary T cells that have been efficiently transduced with exogenous genes. Primary murine T cells transduced with a retroviral vector containing the human adenosine deaminase (ADA) gene persisted for at least 5 months in lymphoid organs of SCID mice, continuously expressing the exogenous gene. Primary human T cells were also used as target cells for transfer of the beta-galactosidase (lacZ) gene. Expression of the lacZ gene could be detected in over 20% of the transduced primary T cells before adoptive transfer into SCID mice. Transduced human T cells were injected into SCID mice intraperitoneally (ip), and the beta-galactosidase activity could be detected in cells collected from peritoneal exudate washes of recipient mice 6 weeks post-injection. These results demonstrate the availability of a murine model in which the long-term effects of expression of exogenous genes in both murine and human T cells can be tested.

Inducible human immunodeficiency virus type 1 packaging cell lines.

Packaging cell lines are important tools for transferring genes into eukaryotic cells. Human immunodeficiency virus type 1 (HIV-1)-based packaging cell lines are difficult to obtain, in part owing to the problem that some HIV-1 proteins are cytotoxic in a variety of cells. To overcome this, we have developed an HIV-1-based packaging cell line which has an inducible expression system. The tetracycline-inducible expression system was utilized to control the expression of the Rev regulatory protein, which in turn controls the expression of the late proteins including Gag, Pol, and Env. Western blotting (immunoblotting) demonstrated that the expression of p24gag and gp120env from the packaging cells peaked on days 6 and 7 postinduction. Reverse transcriptase activity could be detected by day 4 after induction and also peaked on days 6 and 7. Defective vector virus could be propagated, yielding titers as high as 7 x 10(3) CFU/ml, while replication-competent virus was not detectable at any time. Thus, the cell line should enable the transfer of specific genes into CD4+ cells and should be a useful tool for studying the biology of HIV-1. We have also established an inducible HIV-1 Env-expressing cell line which could be used to propagate HIV-1 vectors that require only Env in trans. The env-minus vector virus titer produced from the Env-expressing cells reached 2 x 10(4) CFU/ml. The inducible HIV-1 Env-expressing cell line should be a useful tool for the study of HIV-1 Env as well.

A nickel chelate microtiter plate assay for six histidine-containing proteins.

Protein purification has been made significantly easier by the use of affinity tags that can be genetically engineered at either the amino- or carboxyl-terminus of recombinant proteins. One of the most widely used tags is six consecutive histidine residues or 6His tag. These residues bind with high affinity to metal ions immobilized on chelating resins even in the presence of denaturing agents and can be mildly eluted with imidazole. We report the methodology for the immobilization of six histidine-containing proteins onto microtiter plates. A derivative of nitrilotriacetic acid (NTA) was prepared. This derivative, N,N-bis[carboxymethyl]lysine (BCML), was easily coupled to a maleic anhydride-activated polystyrene microtiter plate. The plate was then charged with Ni2+ for the capture of the 6His-tagged proteins. Using two different recombinant proteins with the 6His tag at either the N- or C-terminus, we demonstrated that the binding to the Ni(2+)-NTA plate was specific for six histidine-containing proteins. Proteins lacking the 6His tags did not bind to the plate. The plate was used in a modified enzyme-linked immunoabsorbent assay format to quantitate protein concentrations and determine the affinity of protein-ligand interactions. The technology can also be extended to include high-throughput screening assays for antagonists of protein-protein interactions.

Mechanisms of retroviral mutation.

Retroviruses, like other RNA viruses, mutate at very high rates (0.05-1 mutations per genome per replication cycle) and exist as complex genetically heterogeneous populations ('quasispecies') that are ever changing. De novo mutations are generated by inherently error-prone steps in the retroviral life cycle that introduce base substitutions, frame shifts, genetic rearrangements and hypermutations.

Genetic rearrangements occurring during a single cycle of murine leukemia virus vector replication: characterization and implications.

Retroviruses evolve at rapid rates, which is presumably advantageous for responding to selective pressures. Understanding the basic mutational processes involved during retroviral replication is important for comprehending the ability of retroviruses to escape immunosurveillance and antiviral drug treatment. Moreover, since retroviral vectors are important vehicles for somatic cell gene therapy, knowledge of the mechanism of retroviral variation is critical for anticipating untoward mutational events occurring during retrovirus-medicated gene transfer. The focus of this report is to examine the spectrum of genomic rearrangements arising during a single cycle of Moloney murine leukemia virus (MoMLV) vector virus replication. An MoMLV vector containing the herpes simplex virus thymidine kinase (tk) gene was constructed. MoMLV vector virus was produced in packaging lines, and target cells were infected. From a total of 224 mutant proviruses analyzed, 114 had gross rearrangements readily detectable by Southern blotting. The remaining proviruses were of parental size. PCR and DNA sequence analysis of 73 of the grossly rearranged mutant proviruses indicated they resulted from deletions, combined with insertions, duplications, and complex mutations that were a result of multiple genomic alterations in the same provirus. Complex hypermutations distinct from those previously described for spleen necrosis virus and human immunodeficiency virus were detected. There was a correlation between the mutation breakpoints and single-stranded regions in the predicted viral RNA secondary structure. The results also confirmed that the tk gene is inactivated at an average rate of about 8.8% per cycle of retroviral replication, which corresponds to a rate of mutation of 3%/kbp.

Both anti-CD11a (LFA-1) and anti-CD11b (MAC-1) therapy delay the onset and diminish the severity of experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is a demyelinating disease of the central nervous system (CNS) induced in rodents by activated CD4+ T cells specific for various myelin proteins such as myelin basic protein and proteolipid protein. The disease is characterized by breach of the blood-brain barrier, perivascular infiltration of leukocytes into the CNS, local inflammation and demyelination in the form of plaques. In this study, we evaluated the effect of administration of antibodies to two members of the beta 2 integrin sub-family of adhesion molecules, CD11a and CD11b, on the onset and progression of EAE. CD11a and CD11b are involved in cell-cell interactions leading to T cell and macrophage extravasation to inflammatory sites and T cell activation. Our results show that anti-CD11a antibodies could completely block the induction of EAE and anti-CD11b antibodies significantly delayed the onset and diminished the severity of clinical signs of EAE even when injections were initiated at the first appearance of clinical signs.

A peripheral blood-derived monolayer supports long-term cultures of human CD4+ and CD8+ T lymphocytes.

An in vitro culture system has been developed for the long-term maintenance of primary, human peripheral blood and umbilical cord blood T lymphocytes, which does not rely on the use of stimulatory cytokines, antigen, or mitogens. In these cultures, a monolayer of adherent cells, some spindle-shaped and some resembling macrophages, developed within a week. All adherent cells were positive for the extracellular matrix proteins laminin and fibronectin, the intermediate filament vimentin, and for the surface markers major histocompatibility complex class II, platelet-endothelial cell adhesion molecule l (CD31), and E-Selectin (ELAM-1; CD62E). They were negative for the leukocyte common antigen (CD45), the macrophage marker MO-2 (CD14), muscle-specific actin, and Factor VIII-related antigen. These monolayers supported the maintenance of nonadherent, resting, mature T cells for up to 3 months, and these cells retained their ability to respond to mitogens and allogeneic cells. Both CD4+ and CD8+ cells were supported. The proportion of CD4+ and CD8+ cells remained unchanged after 3 months in culture. We have also used T cells from 2-month-old cultures as target cells for retroviral vector-mediated gene transfer. Up to 30% of the long-term T cells expressed the transferred lacZ gene after infection with a retroviral vector. The infection efficiency was similar to that obtained for fresh peripheral blood T cells, indicating that the long-term-cultured cells might be suitable for certain gene therapy applications.

Mutations of conserved cysteine residues in the CWLC motif of the oncoretrovirus SU protein affect maturation and translocation.

The envelope glycoprotein complex is composed of two polypeptides, an external heavily glycosylated polypeptide (SU) and a membrane-spanning protein (TM). Together they form a heterodimer on the surface of the virion. These proteins are synthesized in the form of a polyprotein precursor which is glycosylated and proteolytically processed during its maturation in the secretory pathway. A highly conserved stretch of four amino acids, CWLC, has been identified in most known oncoretroviral SU proteins, about two-thirds of the distance from the amino terminus. To study the significance of this sequence for the structure and/or function of SU, cysteine to serine mutations were made in reticuloendotheliosis virus strain A. Initial studies showed that substitution of either one or both cysteines resulted in the production of noninfectious virus. Furthermore, immunoprecipitations and pulse-chase analysis demonstrated that the mutants yielded envelope polyprotein precursors which were stable. However, the polyprotein precursors were not proteolytically processed into SU and TM, and immunoprecipitations indicate that the immature polyproteins form aggregates, suggesting that the mutations interfere with proper folding. Although not proteolytically processed, at least one of the mutant glycoproteins appeared to be efficiently transported to the cell surface. These studies indicate that changing either cysteine residue abrogates viral infectivity by affecting folding, inhibiting normal maturation of the envelope glycoproteins.