Adenovirus early gene regulation and the adeno-associated virus helper effect.
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Adenoviridae↗
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In vivo replacement of the helper virus in Bryan's strain of Rous sarcoma virus by AMV and RPL12 viruses.
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Animals↗
Evidence for helper-independent murine sarcoma virus. II. Differences between the ribonucleic acids of clone-purified leukemia virus, helper-independent and helper-dependent sarcoma viruses.
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Animals↗
[Use of infectious and oncogenic viruses as helpers for defective Rous virus].
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Animals↗
Biological behavior of tumors and associated retroviremia in cats inoculated with Snyder-Theilen fibrosarcoma virus and the phenomenon of tumor recurrence after primary regression.
The fate of tumors and associated retroviremia was studied in 111 cats infected with the Snyder-Theilen strain of feline sarcoma virus (FeSV). Tumors appeared at the site of inoculation within 7 to 10 days. A retroviremia, due mainly to the associated feline leukemia virus helper virus (FeLV-helper), developed at the same time as tumors. Of the cats, 44 developed progressively growing tumors and therefore had to be killed, and 67 developed tumors that regressed. There was a strong correlation between the persistence of the accompanying retroviremia and the growth of the tumors. The 44 cats with progressively growing fibrosarcomas remained retroviremic until death. Conversely, 53 of the 67 cats with solitary, regressing tumors were only transiently retroviremic. Tumor regression in these cats paralleled the disappearance of retrovirus from the blood. The fate of tumors and retroviremia was not always the same, however. Twelve cats remained persistently retroviremic after all signs of gross tumors disappeared. Two other kittens became nonviremic within 20 days after inoculation, yet tumors continued to grow and even metastasize for another 3 to 5 weeks before regressing. Fibrosarcomas recurred 3 weeks to 8 months later in 8 of 12 persistently retroviremic cats with regressed tumors. Although the blood and bone marrow from these cats contained predominantly FeLV-helper, tumor cells yielded both FeSV and FeLV-helper. Of 53 animals, 3 developed recurrent fibrosarcomas 5 weeks to 8 months after all signs of tumors and retroviremia had disappeared. Cells cultured from these tumors appeared initially like normal fibroblasts and were virus nonproducers. After one to three passages in culture, however, cells became malignantly transformed and replicated both FeSV and FeLV-helper. Cultures of the bone marrow from these and other nonviremic cats with regressed tumors yielded only FeLV-helper.
Animals↗
Herpes simplex virus 1 (HSV-1) helper co-infection affects the distribution of an amplicon encoded protein in glia.
HSV-1 derived amplicons expressing cytoplasmic beta-galactosidase (pA-SF1), or plasma membrane targeted TIMP-Thy1 (pA-TT1), were used to transduce glial cells in vitro. By monitoring the expression of reporter genes from both amplicons and helper virus, we determined that many cells were infected by both particles. In glial cells infected only by pA-SF1 beta-galactosidase immunoreactivity was restricted to the cytoplasm; co-infection with helper HSV-1 (wild type), resulted in additional nuclear beta-galactosidase immunoreactivity. Co-infection of cells with amplicon pA-TT1 and helper virus did not affect the plasma membrane localization of TIMP/Thy1. Thus, co-infection with wild type helper virus altered the localization of an amplicon encoded cytoplasmic, but not plasma membrane protein.
Cell Membrane↗
Abnormalities in myelopoietic regulatory interactions with acidic isoferritins and lactoferrin in mice infected with Friend virus complex: association with altered expression of Ia antigens on effector and responding cells.
The regulation of myelopoiesis was evaluated in B6D2F1 mice inoculated with Friend virus complex (spleen focus-forming virus plus helper virus) or helper virus alone by analyzing acidic isoferritin (AIF) and lactoferrin (LF) interactions with target cells. Under normal conditions, AIF suppresses colony and cluster formation by an Ia-antigen-positive cycling subpopulation of mouse granulocyte-macrophage progenitor cells (CFU-GM). Under the same conditions, the release of AIF-inhibitory activity and granulocyte-macrophage colony stimulatory factors (GM-CSF) from an Ia-antigen-positive subpopulation of monocytes and macrophages is suppressed by LF. Within one to two days after inoculation in vivo with Friend virus complex or helper virus, mouse CFU-GM become insensitive in vitro to suppression by purified human AIF as well as crude mouse AIF, and by four days, bone marrow, spleen, and thymus cells of these mice release much greater quantities of AIF-inhibitory activity than the cells from mice injected with control medium. The Friend virus complex itself has no influence in vitro on CFU-GM from normal mice. In addition, the release of AIF-inhibitory activity from bone marrow, spleen, and resident peritoneal cells and the release of GM-CSF from resident peritoneal cells of mice infected with Friend virus complex are not suppressed by LF. The inability of AIF to suppress colony formation by bone marrow and spleen CFU-GM from mice infected with Friend virus complex is associated with the loss of Ia (I-A subregion) antigens from CFU-GM, even though CFU-GM are in cycle. The nonresponsiveness of bone marrow, spleen, and peritoneal cells from these mice to LF suppression of AIF release and the inability of LF to influence GM-CSF release from peritoneal cells is associated with loss of Ia antigens from these cells. The above abnormalities are similar to the defects noted using cells from patients with leukemia. These results suggest that mice infected with Friend virus complex can serve as a model for investigating abnormalities in cell regulation and their relationships to disease progression.
Animals↗
Effects of exogenous, nonleukemogenic, ecotropic murine leukemia virus infections on the immune systems of adult C57BL/6 mice.
Mouse AIDS (MAIDS) develops in mice infected with a mixture of replication-competent ecotropic and mink lung cell focus-inducing murine leukemia viruses and an etiologic replication-defective virus. Helper viruses are not required for induction of MAIDS, but the time course of disease is accelerated in their presence. To understand the possible contributions of ectropic murine leukemia viruses to MAIDS pathogenesis, we biologically cloned a series of viruses from the MAIDS-inducing LP-BM5 virus mixture. These viruses were examined for replication in tissues of infected mice and for effects on the immune system. All virus stocks replicated efficiently in mice. Infected animals showed slight lymphadenopathy and splenomegaly due primarily to B-cell proliferation associated with differentiation to immunoglobulin secretion resulting in twofold increases in serum immunoglobulin M levels; however, B-cell responses to helper T-cell-independent antigens were increased rather than decreased as in MAIDS. Analyses of CD8+ T-cell function showed that cytotoxic T-lymphocyte responses to alloantigens were comparable in control and infected mice. Finally, we showed that infection resulted in enhanced expression of transcripts for interleukin-10, interleukin-4, and gamma interferon. These cytokines can all contribute to B-cell activation and may promote the expansion of a target cell population for the MAIDS defective virus.
Animals↗
Helper virus-free transfer of human immunodeficiency virus type 1 vectors.
Recombinant vectors based on the type 1 human immunodeficiency virus (HIV-1) can be used to deliver genes into cells expressing the HIV receptor, CD4. We have used a transient RNA packaging system to compare the safety and efficacy of HIV-1 vector transduction by wild-type and replication-deficient helper viruses. Helper virus-free vector transfer was consistently achieved when the helper virus gag-pol and env genes were expressed from separate plasmids such that two recombination events were required to form an infectious genome. Other forms of attenuation, such as deletion of the 5' phi region, were inadequate to prevent helper virus transmission. Vector transduction by the wild-type and non-replicating helper viruses occurred with comparable efficiency except in instances where efficient vector RNA expression was dependent upon transactivating factors supplied by the helper virus. These data demonstrate the feasibility of safe gene transfer using HIV-1 vectors.
Base Sequence↗
Friend virus-infected long-term bone marrow cultures produce colony stimulating factor dependent and independent granulocyte-macrophage progenitor cells for over four years in vitro.
C3H/HeJ mouse long-term bone marrow cultures infected at initiation with a cloned polycythemic strain of Friend spleen focus forming virus in a cloned N-tropic murine leukemia virus helper virus coat, persistently produced: colony-forming unit spleen (CFUs) for 55 weeks that formed macroscopic spleen colonies in syngeneic or allogeneic C57B10.Br/J mice; and L-cell or WEHI-3 cell conditioned medium-dependent granulocyte-macrophage colony forming unit culture (GM-CFUc); and morphologically normal granulocytes for over 245 weeks. Colony stimulating factor (CSF)-independent colony forming progenitor cells were first detectably produced in vitro at 75 weeks, and when subcultured generated karyotypically distinct permanent factor-independent tumorigenic cell lines. Nonadherent cells removed from long-term marrow cultures at 19 but not at 77 weeks reconstituted donor origin hematopoiesis in C57B10.Br/J mice as measured by B-cell lineage surface immunoglobin allotype. Nonadherent cells removed at 77 weeks produced lethal splenomegaly and marrow infiltration with culture origin cells in C57B10.Br/J mice. Despite generation of clonal malignant cell lines, L-cell DSF (CSF-1, M-CSF) responsive GM-CFUc that were simultaneously produced over 4 years in the same long-term marrow cultures, grew to 7 day colonies in semisolid medium and terminally differentiated. Thus, adherent stromal cells in Friend virus-infected long-term bone marrow cultures simultaneously support CSF-responsive and malignant CSF-independent hematopoietic progenitor cells.
Animals↗
Spontaneous mutagenesis of a plant potyvirus genome after insertion of a foreign gene.
The RNA genome of tobacco etch potyvirus (TEV) was engineered to express bacterial beta-glucuronidase (GUS) fused to the virus helper component proteinase (HC-Pro). It was shown previously that prolonged periods (approximately 1 month) of TEV-GUS propagation in plants resulted in the appearance of spontaneous deletion variants. Nine deletion mutants were identified by nucleotide sequence analysis of 40 cDNA clones obtained after polymerase chain reaction amplification. The mutants were missing between 1,741 and 2,074 nucleotides from TEV-GUS, including the sequences coding for most of GUS and the N-terminal region of HC-Pro. This region of HC-Pro contains determinants involved in helper component activity during aphid transmission, as well as a highly conserved series of cysteine residues. The deletion variants were shown to replicate and move systemically without the aid of a helper virus. Infectious viruses harboring the two largest HC-Pro deletions (termed TEV-2del and TEV-7del) were reconstructed by subcloning the corresponding mutated regions into full-length DNA copies of the TEV genome. Characterization of these and additional variants derived by site-directed mutagenesis demonstrated that deletion of sequences coding for the HC-Pro N-terminal domain had a negative effect on accumulation of viral RNA and coat protein. The TEV-2del variant possessed an aphid-nontransmissible phenotype that could be rescued partially by prefeeding of aphids on active HC-Pro from another potyvirus. These data suggest that the N-terminal domain of HC-Pro or its coding sequence enhances virus replication or genome expression but does not provide an activity essential for these processes. The function of this domain, as well as a proposed deletion mechanism involving nonhomologous recombination, is discussed.
Amino Acid Sequence↗
Mutational analysis of the Uukuniemi virus (Bunyaviridae family) promoter reveals two elements of functional importance.
We have performed an extensive mutational analysis of the proposed promoter region of the phlebovirus Uukuniemi (UUK), a member of the Bunyaviridae family. This was achieved by using a recently developed RNA polymerase I (Pol I)-driven reverse genetics system (R. Flick and R. F. Pettersson, J. Virol. 75:1643-1655, 2001). Chimeric cDNAs containing the coding region for the reporter chloramphenicol acetyltransferase (CAT) in an antisense orientation were flanked by the 5'- and 3'-terminal nontranslated regions of the UUK virus-sense RNA (vRNA) derived from the medium-sized (M) RNA segment. The chimeric cDNAs (Pol I expression cassettes) were cloned between the murine Pol I promoter and terminator, and the plasmids were transfected into BHK-21 cells. CAT activity was determined after cotransfection with viral expression plasmids encoding the RNA-dependent RNA polymerase (L) and the nucleoprotein (N) or, alternatively, after superinfection with UUK virus helper virus. Using oligonucleotide-directed mutagenesis, single point mutations (substitutions, deletions, and insertions) were introduced into the viral promoter region. Differences in CAT activities were interpreted to reflect the efficiency of mRNA transcription from the mutated promoter and the influence on RNA replication. Analysis of 109 mutants allowed us to define two important regulatory regions within the proximal promoter region (site A, positions 3 to 5 and 2 to 4; site B, positions 8 and 8, where underlined nucleotides refer to positions in the vRNA 3' end). Complementary double nucleotide exchanges in the proximal promoter region, which maintained the possibility for base pairing between the 5' and 3' ends, demonstrated that nucleotides in the two described regions are essential for viral polymerase recognition in a base-specific manner. Thus, mere preservation of panhandle base pairing between the 5' and 3' ends is not sufficient for promoter activity. In conclusion, we have been able to demonstrate that both ends of the M RNA segment build up the promoter region and are involved in the specific recognition by the viral polymerase.
Animals↗
Cloning of the rat endogenous helper leukemia virus DNA sequence and expression of the helper activity encoded by the cloned DNA sequence in normal rat kidney cells by microinjection.
By the use of recombinant DNA technology and microinjection in cultured cells, the molecular genetic elements involved in the evolution of a retrovirus with the multipotential to infect, transform and replicate in host cell, have been critically examined in this investigation. Recently we have identified and purified the integrated and proviral DNA sequences specific for two rat endogenous helper leukemia viruses, WR- RaLV , originated from a chemically induced wild rat fibrosarcoma, and RHHV , isolated from a chemically induced rat hepatoma, HTC-H1 (1). By using a multidisciplinary approach combining restriction endonuclease analysis, reverse phase V-column chromatography, agarose gel electrophoresis, Southern blot transfer and filter nucleic acid hybridization, we were able to demonstrate that the rat helper leukemia viral DNA sequence was approximately 8.4-8.8 kb. The 8.8 kb RHHV DNA was molecularly cloned via the EK-1 certified vector pBR 322 plasmid into E. coli RRI cells. A successful recombinant clone, 8/32, that carried one entire RHHV 8.8 kb DNA sequence was mapped by restriction endonuclease analyses. Restricted DNA fragments of various sizes throughout the complete RHHV genome were isolated and purified for intranuclear microinjection into normal rat kidney cells. Release of type C infectious helper virus in these microinjected cells was investigated by superinfection on K-NRK, Kirsten sarcoma transformed non-producer cells. Recombination of the helper viral DNA sequence, en toto or of subgenomic sizes, carried in microinjected cells, with the sarcomagenic DNA sequence, carried in K-NRK cells, was also studied by genome-rescue and cell-transformation experiments. Our observations led to the conclusion that all critical genetic elements including the 5' LTR helper DNA sequence, gag, pol, and env genes, encoded for the biological activity of the type C helper virus resided within the 6.0 kb proximal to the 5' terminus of the endogenous rat type C helper virus DNA. They proved vitally essential for the recombination with the Src sequence during the evolution of an infectious, transforming and replication-competent retrovirus.
Animals↗
Multiple steps are required for the induction of tumors by Abelson murine leukemia virus.
Helper virus-free Abelson murine leukemia virus (A-MuLV) was used to induce monoclonal pre-B-cell tumors in mice. The clonality, patterns of immunoglobulin heavy-chain gene rearrangement, tumorigenicity, and v-abl oncogene expression in individual preleukemic and leukemic colonies were compared. Our results indicate that A-MuLV preleukemic cells with low or undetectable tumorigenic potential give rise to leukemic cells with high tumorigenic potential by a process of subclone selection. The levels of v-abl oncogene product in preleukemic and leukemic cell populations were not significantly different. These results suggest that an additional event(s) unrelated to the level of the v-abl protein product is required for A-MuLV-transformed cells to become fully malignant.
Abelson murine leukemia virus↗
[Comparison of the immune response to gene transfer into the rat brain with helper or helper virus-free HSV-1 vectors].
OBJECTIVE: This study was conducted to compare the immune response to gene transfer with helper virus or helper virus-free vectors. METHODS: Two vector stocks were delivered by stereotactic injection into the striatum and then the immune response markers including TCR, CD4, CD8, MHC-I and MHC-II were examined. RESULTS: The data showed that the immune response to either vector stock reached maximal levels by 4 to 7 days after gene transfer. However, the maximal levels of all markers to the helper virus-free stock were 3 to 10 folds lower than the markers to the helper virus stock. The immune response to the helper virus-free stock declined rapidly to be close to the levels of PBS control. CONCLUSION: The immune response to the helper virus-free stock is significantly less than the response to the vector stock that contains helper virus.
Animals↗
Human immunodeficiency virus type 2 lentivirus vectors for gene transfer: expression and potential for helper virus-free packaging.
In addition to the long-term expression of the transgene provided by all retroviral vectors, lentiviruses present the opportunity to transduce nondividing cells and potentially achieve regulated expression. The development of lentiviral vectors requires the design of transfer vectors to ferry the transgene with efficient encapsidation of the transgene RNA and with full expression capability, and of a packaging vector to provide packaging machinery in trans but without helper virus production. For both vectors, a knowledge of packaging signal is required-the signal to be included in the transfer vector but excluded from the packaging vector. Among the human lentiviruses, human immunodeficiency virus type 1 and type 2 (HIV-1 and HIV-2), we think HIV-2 is better suited for gene transfer than HIV-1. It is less pathogenic and thus safer during design and production; its desirable nuclear import and undesirable cell-cycle arrest functions are segregated on two separate genes. In HIV-1 infection, it is less likely to recombine with the resident HIV-1, and it may itself downregulate HIV-1 expression. Evidently, elements located both upstream and downstream of the splice donor site in the leader sequence participated in RNA encapsidation and these sequences appeared necessary and sufficient. Deletion of both sequence elements resulted in a dramatic curtailment of RNA encapsidation and helper virus production. This was accompanied by some but acceptable loss of gene expression capability. The helper virus-free phenotype and expression capability of the double mutant was maintained upon replacement of its 3' long terminal repeat with a minigene cassette containing a transcriptional termination signal and a drug resistance marker gene. Deletion of the splice donor site itself had a dramatic negative effect on gene expression, supporting the important role of this element in the life of RNA.
Cell Line↗
Helper virus-free herpes simplex virus-1 plasmid vectors for gene therapy of Parkinson's disease and other neurological disorders.
Vectors based on herpes simplex virus type 1 (HSV-1) have potential for gene therapy of neurological disorders. HSV-1 plasmid vectors (amplicons) contain only approximately 1% of the 150-kb HSV-1 genome and have been packaged into virus particles by using a helper virus. We have demonstrated that HSV-1 plasmid vectors which express tyrosine hydroxylase can cause long-term biochemical and behavioral recovery in the 6-hydroxydopamine rat model of Parkinson's disease. Furthermore, we and others have used HSV-1 plasmid vectors which express a wide range of genes that affect neuronal physiology. Because of the pathogenicity of the HSV-1 helper virus, however, the use of this vector system has been limited to studies in animal models or primary cultures of neural cells. Thus, to increase the safety of HSV-1 plasmid vectors, we recently developed a helper virus-free packaging system that may facilitate studies on neuronal physiology and potential therapeutic applications.
Animals↗
Limiting dilution cultures reveal latent influenza virus-specific helper T cells in virus-primed mice.
Although lymphokine producing helper T cells are suspected to play an important role in the development of anti-viral cytotoxicity, they have not previously been demonstrable in influenza virus-primed mice unless the mice have first been pretreated with cyclophosphamide. It has been assumed that cyclophosphamide pretreatment was required to block the activities of suppressor cells which would otherwise interfere with helper T cell priming. We have developed a limiting dilution assay (LDA) for estimating influenza virus-specific T cell precursors for IL-2 secretion ("pHTL"), and have found that equal numbers of pHTL develop in virus-primed mice regardless of cyclophosphamide pretreatment. This result suggests that the cyclophosphamide-sensitive regulatory cells do not act in vivo to prevent helper cell priming, but rather act in vitro to oppose expression of helper cell activity. Comparison of helper cell function in conventional (high density) cultures and in LDA cultures reveals an Lyt-2+ cell which prevents development of cytotoxic function in conventional assays, but which fails to affect the outcome of the LDA experiments. We found that the pHTL population consists almost entirely of Lyt-2- cells, and that mice primed with the Type A strain, PR/8, develop pHTL responsive to a wide range of influenza viruses, including a Type B strain. Both results modify conclusions suggested by conventional assay methods. Since the LDA experiments seem to be relatively resistant to regulatory interactions which can influence the outcome of standard assay cultures, they can supply information about the characteristics of the pHTL itself that would otherwise be difficult to obtain.
Animals↗