Human Immunodeficiency Virus Vectors Research Articles

Use ofcis- andtrans-Acting Viral Regulatory Sequences to Improve Expression of Human Immunodeficiency Virus Vectors in Human Lymphocytes

We compared the efficiency of human immunodeficiency virus (HIV-1) vectors that express a marker gene (chloramphenicol acetyltransferase, CAT) using different promoter elements. In one vector, CAT was expressed under the control of an internal murine leukemia virus (MuLV) long terminal repeat (LTR). In other vectors, CAT production was regulated by the HIV-1 LTR; these vectors also contained the HIV-1tatgene andpolsequences reported to exertcis-acting positive effects on reverse transcription or gene expression. Vectors employing the Tat-driven HIV-1 LTR exhibited up to 500-fold greater CAT expression in Jurkat lymphocytes or human peripheral blood mononuclear cells compared with vectors using the internal MuLV LTR element as a promoter. This difference was not due to improved packaging of the vector RNA into virions, but to an improved level of gene expression in the target cells. Target cell CAT expression was two- to threefold higher for the vector containing thepolsequences and was only slightly less than that seen for atrans-complementedenv-deleted provirus. These results indicate that defective HIV-1 vectors with efficiencies of gene transfer and expression comparable with that of HIV-1 itself are feasible.

In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector.

A retroviral vector system based on the human immunodeficiency virus (HIV) was developed that, in contrast to a murine leukemia virus-based counterpart, transduced heterologous sequences into HeLa cells and rat fibroblasts blocked in the cell cycle, as well as into human primary macrophages. Additionally, the HIV vector could mediate stable in vivo gene transfer into terminally differentiated neurons. The ability of HIV-based viral vectors to deliver genes in vivo into nondividing cells could increase the applicability of retroviral vectors in human gene therapy.

Purified GPI-Anchored CD4DAF as a Receptor for HIV-Mediated Gene Transfer

CD4 is the major cellular receptor for the human immunodeficiency virus (HIV). A hybrid gene encoding the extracellular domains of CD4, linked to the sequence encoding the membrane attachment region of the glycosylphosphatidylinositol (GPI)-anchored protein decay accelerating factor (DAF) was stably transfected into HeLa cells. The resultant cell line (T4HD) expressed GPI-anchored CD4DAF at high levels and was susceptible to gene transfer with a recombinant HIV vector. In an effort to expand the spectrum of cells susceptible to HIV gene transfer, CD4DAF was released from the surface of the T4HD cell line by detergent lysis, purified by immunoaffinity chromatography, and reincorporated into native HeLa cells. Incorporation occurred via the GPI anchor as evidenced by cleavage with phosphatidylinositol-specific phospholipase C. More than 95% of the CD4DAF-treated HeLa cells were CD4-positive by flow cytometry, and kinetic analysis demonstrated that over 75% of the fusion protein remained anchored to the cell membrane after 90 min at 37 degrees C. The purified protein retained its ability to bind the envelope protein of HIV. When incorporated, it bound fluorescein isothiocyanate (FITC)-conjugated gp120, and in its soluble form blocked transduction of CD4-positive cells incubated with an HIV-derived vector containing the Neo gene. In contrast to the T4HD cells, exposure of CD4DAF-treated cells to the Neo HIV vector yielded only transient neomycin-resistant colonies. These results suggest that endogenous synthesis of the CD4 molecule may be necessary for successful HIV genomic integration.

Human immunodeficiency virus vectors for inducible expression of foreign genes.

Tat-dependent expression of an endogenous lethal or deleterious foreign gene might be useful for abrogating the production of human immunodeficiency virus (HIV) from cells. This type of HIV-induced cellular killing, as well as other approaches to gene therapy for HIV infection, would be facilitated by simple HIV vectors that express introduced genes in a Tat-inducible manner. As part of studies to examine the feasibility of this concept, we constructed HIV-1 vectors that express the hygromycin B phosphotransferase gene (Hygr) in a Tat-dependent manner. Comparison of the efficiency of propagation of each vector indicates that sequences extending into the gag open reading frame are necessary in cis for efficient vector propagation. Southern blot analysis of genomic DNA isolated from vector-infected cells demonstrated that the vectors were capable of being propagated as expected without gross rearrangements or deletions. A fragment of the influenza A virus hemagglutinin (H5 HA) gene, capable of eliciting antibody and cytotoxic T-cell responses, was used as a marker for further characterization of the vector system. A Tat-dependent vector conferring the H5 HA+ phenotype was assayed by indirect immunofluorescence, and cells which contained but did not express the H5 HA gene were isolated. The activation of H5 HA expression following HIV infection of Tat- cells that stably contained but did not express the H5 HA construct was determined to be an efficient process.

Construction and use of a human immunodeficiency virus vector for analysis of virus infectivity

We constructed a recombinant human immunodeficiency virus (HIV) vector to facilitate studies of virus infectivity. A drug resistance gene was inserted into a gp160- HIV proviral genome such that it could be packaged into HIV virions. The HIV genome was rendered replication defective by deletion of sequences encoding gp160 and insertion of a gpt gene with a simian virus 40 promoter at the deletion site. Cotransfection of the envelope-deficient genome with a gp160 expression vector resulted in packaging of the defective HIV-gpt genome into infectious virions. The drug resistance gene was transmitted and expressed upon infection of susceptible cells, enabling their selection in mycophenolic acid. This system provides a quantitative measure of HIV infection, since each successful infection event leads to the growth of a drug-resistant colony. The HIV-gpt virus produced was tropic for CD4+ human cells and was blocked by soluble CD4. In the absence of gp160, noninfectious HIV particles were efficiently produced by cells transfected with the HIV-gpt genome. These particles packaged HIV genomic RNA and migrated to the same density as gp160-containing virions in a sucrose gradient. This demonstrates that HIV virion formation is not dependent on the presence of a viral envelope glycoprotein. Expression of a murine leukemia virus amphotropic envelope gene in cells transfected with HIV-gpt resulted in the production of virus capable of infecting both human and murine cells. These results indicate that HIV can incorporate envelope glycoproteins other than gp160 onto particles and that this can lead to altered host range. Like HIV type 1 and vesicular stomatitis virus(HIV) pseudotypes, gp-160+ HIV-gpt did not infect murine NIH 3T3 cells that bear human CD4, confirming that these cells are blocked at an early stage of HIV infection.

A synergism of plagues: “Planned shrinkage,” contagious housing destruction, and AIDS in the bronx

Techniques adapted from population and community ecology, quantitative geography, and epidemiology are applied to ecosystem and environmental index data on the Bronx in an attempt to understand the origins and potential impacts of rampant spread of human immunodeficiency virus (HIV) and its sequelae of acquired immunodeficiency syndrome (AIDS) in that borough: Recent work by Drucker and Vermund (1987), ("Estimating Prevalence of Human Immunodeficiency Virus Infection in Urban Areas with High Rates of Intravenous Drug Abuse: A Model of the Bronx in 1987," Poster presented at the Third International Conference on AIDS, June 2, 1987) estimates HIV seroprevalence levels of from 8 to 21% among men of age 25-44 in the south Bronx, at this writing, comparable to the cities of Central Africa. It is found that the "South Bronx" process of fulminating, contagious urban decay which devastated the region in the 1970s, and its associated forced population migrations, spread intravenous drug abuse, the principal HIV vector in the Bronx, from a geographically contained center in the South-Central Bronx to a virtually borough-wide phenomenon. This has significantly complicated attempts to contain HIV infection, both by shredding the social networks which are the natural vehicles for education, and by vastly enlarging the area requiring intensive targeting. Since the "planned shrinkage" municipal service cuts which triggered the "South Bronx" burnout persist, and since levels of housing overcrowding now approach those of the early 1970s in the Bronx, it is expected that a new outbreak of contagious urban decay will occur, likely again dispersing population and seriously compromising any in-place HIV control strategies. If overt AIDS itself becomes a contributor to urban deterioration in overcrowded neighborhoods susceptible to "South Bronx" process, we could then see a nonlinear ecosystem coupling between AIDS, contagious urban decay, and population shift. Elementary mathematical models are provided. Thus, in striking contrast to the middle-class male homosexual community, successful control of HIV infection in the Bronx, and by inference in other devastated ghetto communities, seems predicated on quick reestablishment of demographic stability: The tools to make the tools for control must first be reconstructed. Necessary elements of any program toward this end are briefly outlined. AIDS in the Bronx and similar areas, like tuberculosis, seems increasingly a marker disease of extreme poverty, and again like tuberculosis, seems increasingly a marker disease of extreme poverty, and again like tuberculosis, may well form an important reservoir for further spread or resurg