A method for producing high-titer, helper-free infectious retroviruses is disclosed which employs a novel strategy that uses transient transfection of new retroviral producer cell lines, ecotropic line BOSC 23 and amphotropic line CAK 8, both of which cell lines and their precursor cell lines are disclosed. Because of the advantages over stable packaging cell lines, the BOSC 23 and CAK 8 transient transfection systems greatly facilitate and extend the use of helper-free retroviral vectors. The cell lines and corresponding methods possess wide application in both the medical and biotechnical fields, including gene therapy. These potential applications are disclosed and illustrated.

https://www.pnas.org/content/pnas/90/18/8392.full.pdf

Production of high titer helper-free retroviruses by transient transfection

During progression of the Acquired Immune Deficiency Syndrome (AIDS), the human immunodeficiency virus type 1 (HIV-1) is harbored in CD4+ T cells, which act as the primary reservoir for the virus. In vitro, HIV-1 requires activated T cells for a productive infection; however, in vivo, the number of circulating T cells in the activated state that are potential targets for HIV-1 infection is low. We have investigated the ability of HIV-1 to infect resting T cells, and the consequences of such an infection. T cell activation was not required for HIV-1 infection; however, viral DNA was unable to integrate in resting T cells and was maintained extrachromosomally. Subsequent T cell activation allowed integration of extrachromosomal forms and led to a productive viral life cycle. Extrachromosomal forms of viral DNA were found to persist for several weeks after infection of resting T cells and, following T cell activation, these forms maintained their ability to integrate and act as a template for infectious virus. Several lines of evidence, including temporal analysis of HIV-1 replication and analysis of an HIV-1 integrase deletion mutant, indicated that extra-chromosomal HIV-1 DNA genomes were transcriptionally active. These results are compatible with a model whereby HIV-1 can persist in a non-productive extra-chromosomal state in resting T cells until subsequent antigen-induced or mitogen-induced T cell activation, virus integration and release. Thus agents that induce T cell activation may control the rate of HIV-1 replication and spread during AIDS progression.

HIV-1 replication is controlled at the level of T cell activation and proviral integration.

Immunoglobulin and T-cell-receptor genes are assembled from component gene segments in developing lymphocytes by a site-specific recombination reaction, V(D)J recombination The proteins encoded by the recombination-activating genes, RAG1 and RAG2, are essential in this reaction, mediating sequence-specific DNA recognition of well-defined recombination signals and DNA cleavage next to these signals Here we show that RAG1 and RAG2 together form a transposase capable of excising a piece of DNA containing recombination signals from a donor site and inserting it into a target DNA molecule The products formed contain a short duplication of target DNA immediately flanking the transposed fragment, a structure like that created by retroviral integration and all known transposition reactions The results support the theory that RAG1 and RAG2 were once components of a transposable element, and that the split nature of immunoglobulin and T-cell-receptor genes derives from germline insertion of this element into an ancestral receptor gene soon after the evolutionary divergence of jawed and jawless vertebrates

Transposition mediated by RAG1 and RAG2 and its implications for the evolution of the immune system

The P210bcr/abl protein is associated with virtually every case of human chronic myelogenous leukemia. Unlike the related P160gag/v-abl oncogene product of Abelson murine leukemia virus, P210bcr/abl does not transform NIH 3T3 fibroblasts. To assess whether P210bcr/abl might transform hematopoietic cell types, retroviral constructs encoding P210bcr/abl were used to infect the bone marrow-derived interleukin 3-dependent Ba/F3 cell line. As for P160gag/v-abl, cell lines expressing P210bcr/abl were growth factor independent and tumorigenic in nude mice. No evidence for autocrine production of interleukin 3 by factor-independent cell lines was found. These experiments establish that P210bcr/abl can transform hematopoietic cell types to tumorigenicity.

https://www.pnas.org/content/pnas/85/23/9312.full.pdf

Transformation of an interleukin 3-dependent hematopoietic cell line by the chronic myelogenous leukemia-specific P210bcr/abl protein

We have developed a versatile program for the analysis of nucleic acid and protein sequences on the IBM Personal Computer. The program is interactive and self-instructing. It contains all the features generally found in sequence analysis programs on large computers, including extensive homology routines, as well as new procedures for the entry of sequence data. The program contains facilities to store and utilize the entire Nucleic Acid Sequence Data Bank. We have devised a new algorithm to find restriction enzyme sites, which allows our microcomputer program to find all sites on a small plasmid for 100 different enzymes in 1 to 2 minutes.

A comprehensive sequence analysis program for the IBM personal computer.

By screening a library of unintegrated, circular Moloney murine leukemia virus (M-MuLV) DNA cloned in lambda phage, we found that approximately 20% of the M-MuLV DNA inserts contained internal sequence deletions or inversions. Restriction enzyme mapping demonstrated tht the deleted segments frequently abutted a long terminal repeat (LTR) sequence, whereas the inverted segments were usually flanked by LTR sequences, suggesting that many of the variants arose as a consequence of M-MuLV DNA molecules integrating within their own DNA. Nucleotide sequencing also suggested that most of the variant inserts were generated by autointegration. One of the recombinant M-MuLV DNA inserts contained a large inverted repeat of a unique M-MuLV sequence abutting an LTR. This molecule was shown by nucleotide sequencing to have arisen by an M-MuLV DNA Molecule integrating within a second M-MuLV DNA molecule before cloning. The autointegrated M-MuLV DNA had generally lost two base pairs from the LTR sequence at each junction with target site DNA, whereas a four-base-pair direct repeat of target site DNA flanked the integrated viral DNA. Nucleotide sequencing of preintegration target site DNA showed that this four-base-pair direct repeat was present only once before integration and was thus reiterated by the integration event. The results obtained from the autointegrated clones were supported by nucleotide sequencing of the host-virus junction of two cloned M-MuLV integrated proviruses obtained from infected rat cells. Detailed analysis of the different unique target site sequences revealed no obvious common features.

J Hoffman

Papers

Intramolecular integration within Moloney murine leukemia virus DNA.