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The Herpes simplex virus (HSV) amplicon was designed in our laboratory, as a defective virus vector, capable of shuttle delivery of DNA sequences and genes from prokaryotic to eukaryotic cells, tissues or organs. The HSV vector was termed "amplicon" to delineate the fact that it carried the cloned sequences of interest, within amplified concatemeric defective genomes, packaged in HSV virions. Employing the replication functions of their helper virus, the amplicons are wide tropic vectors, capable of entry, replication, and gene expression in varied types of cells, both in vitro, and in vivo. In this brief review, the amplicons will be revisited, beginning with studies of the naturally occurring defective genomes, where many of the virologic aspects of the system were established. Properties of the amplicon system and its components, will be enlightened. The cis acting functions required for amplicon propagation will be briefly described. This will follow with examples of our studies of prokaryotic, viral and eukaryotic DNA sequences, which were amplified and expressed within HSV defective genomes. Studies designed to examine the involvement of helper viruses will be described, including our analyses of host shutoff mutant helper viruses. Finally, recent studies of other laboratories will be reviewed, with emphasis on what appears to be the use of amplicons as neurotropic vectors, towards potential gene therapy.
The effect of hematopoietic stem cell age on leukemogenesis in vitro was tested in nonrecharged, corticosterold-supplemented NIH Swiss [N:NIH(S)] mouse long-term bone marrow cultures infected with Friend murine leukemia virus of anemia-inducing strain (F-MuLV-A) or spleen focus-forming virus (SFFV) [Rauscher murine leukemia virus (R-MuLV)], a pseudotype virus derived by rescue of the SFFV genome from SFFV-Balb/3T3 clone A31 nonproducer cells with clonal helper R-MuLV. Cultures at 33 degrees C derived from 10-day-old or adult mouse marrow generated colony-forming unit culture granulocytic macrophage (CFUc) progenitor cells for over 20 weeks and colony-forming unit spleen cells for 14 weeks and generated permanent granulocytic leukemia cell lines after infection with F-MuLV-A at week 1, 2, or 4 but not at week 8. Leukemia lines were of granulocyte phenotype whether induced by F-MuLV-A or SFFV (R-MuLV) and synthesized myeloperoxidase and lysozyme but were restricted in ability to generate superoxide in response to phorbol myristate acetate stimulation. Cultures (31 degrees C) infected with temperature-sensitive (ts) helper virus mutant pseudotypes of SFFV as well as SFFV (R-MuLV) generated granulocytic leukemia lines, whereas only SFFV (R-MuLV) pseudotype virus-infected cultures became leukemic at 37 degrees C. R-MuLV wild type or ts mutant helper virus infection alone increased cell proliferation and numbers of CFUc but did not generate leukemia. These data indicated that gene(s) specific to F-MuLV-A or a virus rescued from SFFV-Balb/3T3 clone A31 nonproducer cells are required for transformation in vitro of a hematopoietic stem cell present in early but absent in late bone marrow cultures.
Macrophage (M phi) process dengue type 2 virus (DV) antigen and present it to B cells leading to their clonal expansion and transmit DV-specific helper cytokine (HF) signal to B cells. The present study was, therefore, undertaken to investigate the binding of DV or HF-pulsed M phi with 51Cr- labelled B lymphocytes. The binding was dependent on the number of DV antigen positive M phi or dose of HF, period (peak with DV at 24 h and with HF at 4 h) and temperature of incubation. The binding was significantly increased by treating DV-pulsed M phi with DV-specific helper T cells or HF. Glutaraldehyde fixed M phi (which do not process DV antigen (Rizvi et al. 1989)) had poor binding capacity. The binding was DV- or HF-specific as it was inhibited by treatment of M phi with specific antisera. The findings thus show that M phi and B lymphocytes are actively bridged via DV antigen and HF to effectively transmit the antigen-specific signal.
Adeno-associated virus is a defective DNA virus, requiring the presence of a helper virus in order to replicate. In this paper we consider its origin in light of several observations, most notably the following: its own replication inhibits that of the helper virus; its DNA structure resembles that of transposable (moveable) elements; and extrachromosomal circles of DNA, about the size of adeno-associated virus DNA, have been found recently in eukaryotic cells. We have arrived at a hypothesis consisting of two main ideas: (1) that cells may use transposable DNA as a mechanism of defense against viral attack, and (2) that adeno-associated virus may have evolved directly from this cellular defense mechanism.
Retroviral vectors infect primate bone marrow cells and express in vivo the transferred genes (the human ADA gene and the bacterial gene for neomycin resistance). The SAX vector appears to express human ADA at normal levels, but the infection efficiency is low (less than 1%) so that the gene product is only detectable in the peripheral blood at low levels. Vector expression disappears after 5 months (except for occasional T cells), presumably due to a failure to infect a renewal stem cell. While the level of ADA expression obtained in primates would not appear to be sufficient to correct outright the disease caused by ADA deficiency, it is possible that T-cell progenitors in the marrow will have a selective advantage. T cells expressing an ADA vector would then able to expand and potentially restore immune function. Unfortunately, this hypothesis will go untested until an animal model for ADA deficiency is found or a human clinical trial is performed. At present, consideration of gene therapy as a treatment for ADA deficiency would only be appropriate if all conventional forms of treatment were unsuccessful. If such a scenario should present itself, the critical question becomes one of safety, to both the patient and those in contact with the patient. We have begun to address the safety issues associated with gene therapy. Five animals exposed to replication-competent retrovirus during bone marrow transplantation show no evidence of helper virus, with a mean follow-up of 18.3 months. Four animals injected with replication-competent helper virus cleared the virus rapidly and, after the initial clearance, have shown no evidence of retroviremia, with a mean follow-up of 5.2 months. Our preliminary findings suggest that murine retorviruses do not cause a productive infection in vivo. These results, combined with the availability of better producer cell lines free of helper virus, are encouraging, and suggest that the risk of clinical disease from murine retrovirus introduced by a gene therapy protocol should be small. Unfortunately, high infection efficiency and long-term vector expression still must be obtained before retroviral-mediated gene transfer can be considered as first-line therapy for ADA deficiency.
Defective herpes simplex virus type 1 vectors (HSV amplicons) have been used as vehicles for efficiently delivering foreign genes into non-dividing cells such as neutrons in vitro and in vivo. This system is useful for studying neuronal physiology and may have potential for human gene therapy of neuronal disorders. The preparation of infectious amplicon particles is normally achieved by transfecting amplicon plasmid DNA, which contains the HSV replication origin and packaging signal, into mammalian cell lines followed by infection of the cells with HSV helper virus. This allows for replication and packaging of both viral and amplicon plasmid DNA. To improve the packaging efficiency of amplicons, several parameters involved in the packaging process were investigated. By introducing the SV40 DNA replication origin into an amplicon plasmid and prereplicating it before HSV infection, it was demonstrated that the existing amount of amplicon DNA prior to infection in the cells is not a rate-limiting step during HSV packaging. In addition, it was shown that the yield of the packaged amplicon particles can be improved by: (1) using a relatively small amount of HSV helper virus up to multiplicity of infection (m.o.i.) equal to 0.1 at infection; (2) infecting with HSV helper virus at 2 or 3 days post-transfection; and (3) passaging the initial packaged amplicon stocks 1-2 times on fresh host cells.
Naturally occurring defective interfering RNAs (DI-RNAs) and satellite RNAs greatly reduce the accumulation of their helper virus in vivo, but often modulate symptom expression in an unpredictable manner. Deletion mutants Nc/S, Na/M and Sa/Nc + M/S, derived from brome mosaic virus (BMV) RNA-2, failed to replicate when co-inoculated with BMV RNAs-1 and -2 to barley protoplasts. However, the inoculum RNA corresponding to these deletion mutants was extremely stable and could have been mistaken for plus-strand progeny had minus-strand progeny analysis been omitted. These results accentuate the need for such tests in evaluating the ability of mutant viral sequences to replicate. One of the mutants, Nc/S, effectively interfered with the accumulation of BMV RNAs-1 and -2 in barley protoplasts. This non-replicating interfering RNA was termed NRI RNA-2 Nc/S. When present with RNAs-1 and -2 at low inoculum amounts (1 microgram), NRI RNA-2 Nc/S reduced replication of RNA-2, the parental RNA, by 63% and preferentially interfered with minus-strand RNA accumulation. At higher levels (4 micrograms), it completely displaced replication of both RNAs-1 and -2. Mutations eliminating translation of a truncated p2a protein from NRI RNA-2 Nc/S did not alleviate the interference effect, demonstrating that a defective replicase protein was not responsible for the decreased accumulation of genomic RNA. At an NRI RNA: genomic RNA inoculum molar ratio of 1:1, NRI RNA-2 Nc/S reduced the accumulation of all helper virus RNAs by 55%. Since this reduction was seen for both wild-type RNA-3 and delta SGP RNA-3, a deletion mutant of RNA-3 that lacks the subgenomic promoter necessary for coat protein expression, it was evident that the effective interference mediated by NRI RNA-2 Nc/S was not mitigated by encapsidation. The ability of the NRI RNAs to mimic satellite DI RNAs in depressing helper virus replication suggests that their expression in transgenic plants may provide a new and widely applicable approach for inducing resistance to viral infection.
The biological activity of a molecularly cloned DNA of a rat endogenous C-type leukaemia helper virus, RHHV, was assessed by intranuclear microinjection into normal rat kidney cells (NRK153). Release of rat C-type leukaemia helper viruses by the microinjected cells was examined by superinfection of Kirsten-transformed non-producer cells (K-NRK). Immediate release of helper leukaemia viruses at a very low level was observed only in the NRK153 m3 . 5/cir cells microinjected with the supercoiled form of RHHV DNA in toto, suggesting that the circular form of the virus DNA might have expedited the replication and expression of virus particles. Genome rescue experiments were also performed by co-cultivating the microinjected NRK153 m cells carrying various linear RHHV DNAs, in toto or of subgenomic sizes, with K-NRK cells. The results indicated that both the total and the 5.8 to 6.2 kb DNA fragment proximal to the 5' terminus of the cloned RHHV 8.8 kb DNA were able to rescue successfully a transforming replication-competent pseudotype virus. Subgenomic DNA fragments derived from the centre or the 3' end of the RHHV DNA were ineffective in the genome rescue experiments.
Injection i.v. of avian erythroblastosis virus (AEV) strain ES4 causes a high incidence of leukemia and the death of most of the inoculated chicks within 2 weeks. As found earlier, the virus is defective for replication and transforms bone marrow cultures in vitro, and surprisingly, also chick embryo fibroblasts. Inoculation of transformed AEV cells negative for virus production into newborn chicks induced the formation of sarcomas only, whereas cells superinfected with helper virus induced the formation of erythroblastosis in addition to sarcomas. The helper virus alone caused neither sarcomas nor erythroblastosis during the experimental period. These findings were explained by the hypothesis that AEV-induced erythroblastosis develops more rapidly than do AEV-induced sarcomas and that animals receiving i.v. injections die of the leukemia before sarcomas become detectable. The observation that animals receiving i.m. injections of AEV developed sarcomas at the site of injection strongly supports this concept. Most of the animals that received i.m. injections also developed an erythroblastosis that was delayed, however, in comparison to the animals receiving i.v. injections. Our data also suggest that the erythroblastosis induced by AEV does not suppress the formation of sarcomas in the same animal.
Using a defective interfering Sendai virus stock (DIH4) freed of nondefective helper virus, we found that the closely related parainfluenza viruses 1 and 3 could substitute for the Sendai virus helper in replicating DIH4, creating chimeric nucleocapsids. The morbillivirus measles and the rhabdovirus VSV could not substitute. When DIH4 is incubated intracellularly for 5 days in the absence of help, the ability of PIV3 to rescue DIH4 at this time depended on fresh Sendai virus polymerase. The PIV3 polymerase apparently can only copy the chimeric template, but not that wrapped in the homologous Sendai NP protein. These results suggest that the cis-acting RNA sequences important for genome replication, e.g., the promoter and the encapsidation site, have been conserved among these viruses, but that the interactions between the polymerase and the template protein NP are unique for each virus.
We have constructed a set of packaging cell lines useful for the generation of helper-free recombinant retroviruses with amphotropic and ecotropic host ranges. To eliminate the problems of transfer of packaging functions and helper virus formation encountered with the previously available packaging systems, two mutant Moloney murine leukemia virus-derived proviral genomes carrying complementary mutations in the gag-pol or env regions were sequentially introduced into NIH 3T3 cells by cotransformation. Both genomes contained a deletion of the psi sequence necessary for the efficient encapsidation of retroviral genomes into virus particles and additional alterations at the 3' end of the provirus. We show that the resulting packaging cell lines psi CRIP and psi CRE can be used to isolate clones that stably produce high titers (10(6) colony-forming units/ml) of recombinant retroviruses with amphotropic and ecotropic host ranges, respectively. More importantly, we demonstrate that viral producers derived from the packaging cell lines do not transfer the packaging functions, or yield helper virus, even under conditions where existing packaging cell lines can be shown to yield transfer of packaging functions and/or helper virus. These properties of the psi CRIP and psi CRE packaging lines make them particularly valuable reagents for in vivo gene transfer studies aimed at cell lineage analysis and the development of human gene replacement therapies.
Expression of specific peptide epitopes on the surface of virions has significant potential for studying viral biology and designing vectors for targeted gene therapy. In this study, an HSV-1 amplicon plasmid expressing a modified glycoprotein C (gC), in which the heparan sulfate binding domain was replaced with a His-tag, was used in generating HSV-1 virions. Western blot analysis demonstrated the presence of modified gC in the purified virions. The amplicon vectors were packaged using a gC-, lacZ+ helper virus to generate a mixture of high-titer helper virus (lacZ+) and amplicon vectors (GFP+), which expressed modified gC in the virion envelope. His-tagged virions bound to 293 6H cells expressing a cell surface pseudo-His-tag receptor four-fold more efficiently than to parental 293 cells and also proved more effective than wild-type virus in binding to both cell types. Binding resulted in productive infection by the modified virions with expression of reporter genes and cytopathic effect comparable to those of wild-type virions. Thus, not only can HSV-1 tropism be manipulated to recognize a non-herpes simplex binding receptor, but it is also possible to increase the infective capacity of the vectors beyond that of the wild-type virus via specific ligand receptor combinations.
We have employed repeat units of herpes simplex virus (HSV) defective genomes to derive a cloning-amplifying vector (amplicon) that can replicate in eucaryotic cells in the presence of standard HSV helper virus. The design of the HSV amplicon system is based on the previous observation that cotransfection of cells with helper virus DNA and seed monomeric repeat units of HSV defective genomes results in the regeneration of concatemeric defective genomes composed of multiple reiterations of the seed repeats. Cotransfection of cells with helper virus DNA and chimeric repeat units containing bacterial plasmid pKC7 DNA resulted in the generation of defective genomes composed of reiterations of the seed HSV-pKC7 repeats. These chimeric defective genomes were packaged into virus particles and could be propagated in virus stocks, with the most enriched passages containing more than 90% chimeric defective genomes. Furthermore, monomeric chimeric repeat units could be transferred back and forth between bacteria and eucaryotic cells. A derivative vector constructed so as to contain several unique restriction enzyme sites could be potentially employed in the introduction of additional viral or eucaryotic DNA sequences into eucaryotic cells.
Amplicon vectors incorporate genetic elements from Herpes simplex virus (HSV) in a plasmid form which is packaged into virions in the presence of a replication-defective helper virus. We constructed a new amplicon vector, pHermes-tet-lacZ, that carries the bacterial beta-galactosidase (lacZ) gene under the control of a minimal promoter preceded by a heptameric tetracycline operator. The minimal promoter element is activated by a tetracycline-responsive hybrid protein, the gene for which is also present in the vector. This amplicon was propagated in parallel in two different permissive cell lines, E5 and 2-2, in the presence of two helper viruses, d120 and 5dl1.2, respectively. The viral stocks produced were injected into the hippocampal region of the mouse brain, where strong localized expression of the transgene developed in the granular cell layer of the dentate gyrus with limited cytotoxicity. The transgene expression could be repressed by a factor of 10 after administration of tetracyclines. The repression level depended on the helper virus present in the injected viral stock. The in vivo regulation of transgene expression conferred by the tetracycline responsive element improves the flexibility of amplicon vectors as tools for gene transfer into the brain.
The distribution of the helper component (HC) protein of potato virus Y in tissues and cells of infected plants was studied by immunoblotting and immunogold labelling techniques. This HC protein was found in leaf blade and vein tissue but not in the petiole of leaves. In infected cells, the protein was localized in rod-shaped cytoplasmic inclusions known as amorphous inclusions.
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Classical swine fever virus (CSFV) strain WB82, isolated from a wild boar in 1982, induced a distinct cytopathic effect (CPE) in primary swine testicle cell culture and in most of the porcine cell lines. This strain of CSFV was found to be composed of two biotypes. cytopathogenic (cp) CSFV, as a minor population, and noncytopathogenic (noncp) CSFV, as a major population. The noncp CSFV (designated strain WB82/E+) was obtained by biological cloning, and it showed the exaltation of Newcastle disease virus phenomenon. In Northern blot analysis and RT-PCR assay, CSFV RNA with a subgenomic (sg) length was detected in addition to full-length viral RNA only in the cells in which a CPE had been revealed. These RNAs represent the genomes of typical defective interfering (DI) particles because of the strict dependence on a complementing helper virus and interference with replication of the helper virus. The sg RNA, which exhibits the genomes of the DI particles, lacked the nucleotides of the viral genomic region from Npro to NS2 (4764 bases). When extracted sg RNA was transfected to the cells infected with the WB82/E+ strain, a distinct CPE was observed. Interestingly, the CPE was observed in cells infected with other heterologous noncp CSFV ALD and GPE- strains by sg RNA transfection. The results suggested that these noncp CSFVs act as helper viruses for the replication of sg RNA (DI particles). It was also shown that the cytopathogenicity of strain WB82 is caused by apoptosis.