Bernard Avalosse

NS-1 and NS-2 proteins may act synergistically in the cytopathogenicity of parvovirus MVMp

The interaction of parvovirus minute virus of mice (prototype strain, MVMp) with simian virus 40 (SV40)-transformed human cells (NB-E) was investigated by means of transfection with MVMp molecular clones derived from the infectious recombinant plasmid (pMM984). pMM984 inhibits stable transformation of NB-E cells to geneticin resistance (G418R) upon cotransfection with the selectable pSV2neo plasmid. We show here that this inhibition is not merely caused by a repression of marker gene expression from the SV40 early region promoter in pSV2neo and rather is likely to reflect the cytotoxic action of the parvovirus. Starting from plasmid pMM984, defined mutations were introduced into the genome of MVMp and more particularly into sequences coding for the NS-1 and/or NS-2 nonstructural proteins. In this way we could show that the NS-1 protein is necessary for the inhibition of transformation to G418R and that the NS-2 protein acts synergistically to enhance this effect. Moreover, results obtained with different viral mutants indicate that the inhibitory action of NS-1 on stable transformation can be dissociated from the ability of this protein both to transactivate the parvoviral p39 promoter of the capsid protein-encoding region and to drive parvoviral DNA amplification. Altogether these data point to a probable direct toxicity of MVMp nonstructural proteins for permissive host cells.

XSIP1, a Xenopus zinc finger/homeodomain encoding gene highly expressed during early neural development.

We have isolated a Xenopus homologue of the zinc finger/homeodomain-containing transcriptional repressor Smad-interacting protein-1 (SIP1) from mouse. XSIP1 is activated at the early gastrula stage and transcription occurs throughout embryogenesis. At the beginning of gastrulation, XSIP1 is strongly expressed in prospective neurectoderm. At the neurula stage, XSIP1 is highly expressed within the neural plate but weakly in the dorsal midline. At later stages of development transcripts are detected primarily within the neural tube and neural crest. In the adult, XSIP1 expression is detected at variable levels in several organs.

Gene therapy for cancer

This review looks at various gene therapy strategies that are currently being investigated either in experimental studies or in clinical trials. These approaches attempt to either enhance the antitumor immune response of the host, express conditional toxins specifically in tumor cells, reverse the transformed phenotype of tumor cells, or protect normal tissues against the toxicities of conventional treatments.

Cellular contaminants of adeno-associated virus vector stocks can enhance transduction.

Transduction efficiency of different types of recombinant (r)AAV-2 based vectors preparations markedly differed, with apparently no correlation with the replicative titers. Using HeLa cells as target for transduction, 105 and 30 infectious units were necessary to observe one transductant using respectively cesium-chloride-purified rAAV and crude lysates of producer cells obtained by sonication. The purified vectors were however able to transduce HEK-193 cells efficiently, but transgene expression was detected with some delay compared with crude lysates. The unexpected high transduction efficiency of sonicated crude lysates was due to virally mediated gene transfer, since similar sonicated crude lysates, but with no AAV rep and cap genes, did not lead to detection of transgene products after incubation with HeLa cells. Furthermore, sonicated cellular extracts of 293 or 293/T cells given in trans stimulate transduction of HeLa cells by purified rAAV. In contrast, neither extracts from the adenovirus E1-transformed 911 cell line, nor from other cell lines not harboring any adenovirus gene, had enhancing effect on rAAV-mediated transduction. These data suggest that 293 sonicated extracts contain factors which stimulate rAAV-mediated transduction of cells that are normally poorly transduced and offer a system to identify such factors and to characterize further the steps limiting the transfer of gene by AAV vectors.

The genome structure of a new chicken virus identifies it as a parvovirus

The nucleic acid of chicken parvovirus-like particles showed sensitivity to DNase and S1 nuclease treatment and resistance to digestion with RNase. Viral DNA readily served as a template for self-primed conversion in vitro into a double-stranded form of about 5200 base pairs. There was no evidence for encapsidation of strands of opposite polarities. These findings confirm the taxonomic classification of chicken parvovirus-like particles as fowl parvovirus type 1 within the Parvovirus genus of the Parvoviridae.

Method for concentrating and purifying recombinant autonomous parvovirus vectors designed for tumour-cell-targeted gene therapy

Recent work has highlighted the use of parvoviruses as potential vectors for tumour-cell-targeted gene therapy. The oncotropic properties of the prototype strain of minute virus of mice (MVMp) suggest that this virus might be a useful vehicle for introducing selectively therapeutic genes, e.g. lymphokine or suicide genes, into tumour cells and preferentially expressing them. But the low titre of recombinant virus stocks (10(5)-10(6) infectious units per ml) and their high level of contamination by cell proteins make it practically impossible to evaluate their efficacy in in vivo systems. A technique is described for producing cellular contaminant-free stocks of recombinant virus particles, with titres up to 5 x 10(8) IU/ml.

Xath2, a bHLH gene expressed during a late transition stage of neurogenesis in the forebrain of Xenopus embryos.

We have identified a Xenopus bHLH gene, Xath2, which is the homologue of the murine MATH-2/NEX-1 gene, using a functional expression screening approach. Overexpression of this gene in neurula embryos induces the expression of the N-tubulin neuronal marker but does not stimulate the expression of the X-ngnr-1 and NeuroD proneural genes. Expression of Xath2 begins in stage 32 embryos and is restricted to the dorsal telencephalon. Within the neuroepithelium of the dorsal telencephalon, Xath2 expression is detected in postmitotic cells located more laterally than those expressing several other related bHLH neuronal regulators.

Cloning and Sequencing of Defective Particles Derived from the Autonomous Parvovirus Minute Virus of Mice for the Construction of Vectors with Minimal cis-Acting Sequences

ABSTRACT The production of wild-type-free stocks of recombinant parvovirus minute virus of mice [MVM(p)] is difficult due to the presence of homologous sequences in vector and helper genomes that cannot easily be eliminated from the overlapping coding sequences. We have therefore cloned and sequenced spontaneously occurring defective particles of MVM(p) with very small genomes to identify the minimalcis-acting sequences required for DNA amplification and virus production. One of them has lost all capsid-coding sequences but is still able to replicate in permissive cells when nonstructural proteins are provided in trans by a helper plasmid. Vectors derived from this particle produce stocks with no detectable wild-type MVM after cotransfection with new, matched, helper plasmids that present no homology downstream from the transgene.

A novel MVMp-based vector system specifically designed to reduce the risk of replication-competent virus generation by homologous recombination.

Recent work highlights the potential usefulness of MVM-based vectors as selective vehicles for cancer gene therapy (Dupont et al, Gene Therapy, 2000; 7: 790–796). To implement this strategy, however, it is necessary to develop optimized methods for producing high-titer, helper-free parvovirus stocks. Recombinants of MVMp (rMVMp) are currently generated by transiently co-transfecting permissive cell lines with a plasmid carrying the vector genome and a helper plasmid expressing the capsid genes (replaced with a foreign gene in the vector genome). The resulting stocks, however, are always heavily contaminated with replication-competent viruses (RCV), which precludes their use in vivo and particularly in gene therapy. In the present work we have developed a second-generation MVMp-based vector system specifically designed to reduce the probability of RCV generation by homologous recombination. We have constructed a new MVMp-based vector and a new helper genome with minimal sequence overlap and have used the degeneracy of the genetic code to further decrease vector–helper homology. In this system, the left homologous region was almost completely eliminated and the right sequence overlap was reduced to 74 nt with only 61% homology. We were thus able to substantially reduce (∼200 ×), but not completely eliminate, generation of contaminating viruses in medium-scale rMVMp preparations. Since the remaining sequence homology between the new vector and helper genomes is weak, our results suggest that contaminating viruses in this system are generated by nonhomologous recombination. It is important to note, unlike the autonomously replicating helper viruses produced from the first-generation vector/helper genomes, the contaminating viruses arising from the new packaging system cannot initiate secondary infection rounds (so they are not ‘replication-competent viruses’). Our findings have important implications for the design of new MVMp-based vectors and for the construction of trans-complementing packaging cell lines.

Mutagenesis at putative apurinic sites in alkylated single-stranded DNA of parvovirus H-1 propagated in human cells

The treatment of parvovirus H-1, a single-stranded DNA virus, with ethylnitrosourea immediately prior to infection of human cells, resulted in both virus mutagenesis and lethality (immediate hits). The incubation of treated virus, prior to inoculation, under conditions promoting the release of alkylated bases, slightly reduced the mutagenicity of ethylnitrosourea but significantly increased its killing effect (delayed hits). In untreated cells, the appearance of one apurinic/apyrimidinic site in viral DNA correlated with the formation of approximately one delayed lethal hit per virus. Cells which had been sublethally UV irradiated prior to infection, were able to overcome about 20% of the delayed lethal hits inflicted to ethylnitrosourea-treated H-1. This UV-enhanced reactivation was accompanied by viral mutagenesis and was not observed for immediate lethal hits. Therefore, UV irradiation of human cells appears to trigger a conditioned recovery response which might alleviate a block to the replication of single-stranded DNA containing apurinic sites, allowing these noncoding lesions to direct mutagenesis. UV-irradiated cells also displayed a mutator phenotype towards untreated parvovirus H-1. In contrast, ethylnitrosourea failed to induce human cells to cause mutagenesis of undamaged viral DNA, although it enhanced their ability to reactivate damaged virus.