Oncoselective transduction of CD80 and CD86 in tumor cell lines using an autonomous recombinant parvovirus.

BACKGROUND: The aim of this study was to enhance selectively the immunostimulatory properties of tumor cells. Based on their oncotropic properties, we used autonomous recombinant parvoviruses to transduce the genes coding for the constimulatory molecules CD80 (B7-1) or CD86 (B7-2) specifically into tumor cells without transducing normal cells. MATERIALS AND METHODS: After infection of tumor cells by these viruses, surface expression of CD80 and CD86 molecules was assessed by FACS and enhancement of immunostimulatory properties was assessed in alloreactions with G-10 purified T cells. RESULTS: Infection of normal and transformed cells with recombinant MVM- B7-1 or B7-2 viruses leads to expression of costimulatory molecules only by tumor cells and confers on them the capacity to sensitize naive T cells in vitro. CONCLUSION: This approach should ultimately lead to selective expression of costimulatory molecules in tumor tissues in vivo without affecting normal cells.

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.

Cruciform structure of a DNA motif of parvovirus minute virus of mice (prototype strain) involved in the attenuation of gene expression.

It has previously been reported that the region between nucleotides 259 and 383 immediately downstream from the P4 early promoter of parvovirus minute virus of mice, prototype strain (MVMp) is responsible for transcriptional attenuation. Attenuation results from the premature pausing of RNA polymerase II within this sequence (designated to as att) and seems to depend on potential RNA secondary structure. To assess the attenuation capacity of att under near physiological conditions, the early transcription unit of MVMp was replaced by the chloramphenicol acetyltransferase reporter gene under control of the early P4 promoter, in the presence or absence of att. The resulting recombinant vectors were encapsidated in parvovirus particles and replicated in cells after co-infection with the wild-type virus. The att fragment reduced the rate of expression of the reporter gene by approximately threefold, confirming previously reported data from transfection experiments performed in the same cellular system. This attenuation factor is unexpectedly high, considering that the 'readthrough' fold of the nascent viral transcript is thermodynamically more stable than the 'attenuation' configuration. In an attempt to elucidate this point, we sought for the presence of secondary structures in the template DNA molecule. In vitro nuclease probing of viral dsDNA revealed that the att fragment had a cruciform configuration with both complementary strands folding into the computer-predicted stem-loop 'attenuation' structure. These observations lead us to propose that the secondary structure of the DNA template may prompt the formation of the 'attenuation' stem-loop in nascent mRNAs by bringing corresponding self-complementary sequences into close proximity.

Use of an autonomous parvovirus vector for selective transfer of a foreign gene into transformed human cells of different tissue origins and its expression therein.

In this work, we report the transduction of a chloramphenicol acetyltransferase (CAT) reporter gene into a variety of normal and transformed human cells of various tissue origins. The vector used was MVM/P38cat, a recombinant of the prototype strain of the autonomous parvovirus minute virus of mice (MVMp). The CAT gene was inserted into the capsid-encoding region of the infectious molecular clone of MVMp genome, under the control of the MVM P38 promoter. When used to transfect permissive cells, the MVM/P38cat DNA was efficiently replicated and expressed the foreign CAT gene at high levels. By cotransfecting with a helper plasmid expressing the capsid proteins, it was possible to produce mixed virus stocks containing MVM/P38cat infectious particles and variable amounts of recombinant MVM. MVM/P38cat viral particles were successfully used to transfer the CAT gene and to express it in a variety of human cells. Both viral DNA replication and P38-driven CAT expression were achieved in fibroblasts, epithelial cells, T lymphocytes, and macrophages in a transformation-dependent way, but with an efficiency depending on the cell type. In transformed B lymphocytes, however, the vector was not replicated, nor did it express the CAT gene.

Lack of a detectable effect of capsid proteins on the cell-dependent activity of parvovirus MVMp promoters.

Deletion of upstream elements from the early P4 promoter of parvovirus MVM (minute virus of mice, prototype strain MVMp) has a differential effect on its activity, depending on the host cell considered. This indicates that upstream motifs participate in the control of promoter P4 functioning and are responsive to factors which are, at least in part, cell-type specific. In contrast with other viral systems, the capsid proteins of MVMp had no detectable effect on gene expression driven by either the early P4 or late P38 promoter of MVMp in permissive and non-permissive cells.

Inhibition of heterologous DNA replication by the MVMp nonstructural NS-1 protein: identification of a target sequence.

The nonstructural protein NS-1 of minute virus of mice (MVMp), an autonomous parvovirus, trans-inhibits the replication of a chimeric plasmid containing the SV40 origin of replication (ori) embedded in the MVMp genome. It appears that a 157-bp 5' proximal sequence of MVMp DNA is sufficient, in the presence of NS-1, to cause the inhibition of DNA replication driven by the SV40 ori placed on the same molecule. This effect is not dependent on the orientation of the MVMp target sequence and results from both a reduced level of utilization of SV40 ori and the blockage of progressing replication forks at the level of the target. Furthermore, replication driven by Epstein-Barr virus origin (oriP) is trans-inhibited by MVMp but this inhibition does not require the presence of parvoviral sequences in cis. On the basis of sequence homologies between EBV oriP and MVMp 5' terminal sequence, it is proposed that the direct or indirect interaction of NS-1 with parvovirus-like sequences present in heterologous viral and possibly also cellular genomes may result in an inhibition of DNA replication.

Initiation of transcription from the minute virus of mice P4 promoter is stimulated in rat cells expressing a c-Ha-ras oncogene.

Transformation of FR3T3 rat fibroblasts by a c-Ha-ras oncogene but not by bovine papillomavirus type 1 is associated with an increase in the abundance of mRNAs from prototype strain MVMp of infecting minute virus of mice, an oncosuppressive parvovirus. This differential parvovirus gene expression correlates with the reported sensitization of ras- but not bovine papillomavirus type 1-transformed cells to the killing effect of MVMp (N. Salomé, B. van Hille, N. Duponchel, G. Meneguzzi, F. Cuzin, J. Rommelaere, and J. Cornelis, Oncogene 5:123-130, 1990). Experiments were performed to determine at which level parvovirus expression is up-regulated in ras transformants. An MVMp "attenuation" sequence responsible for the premature arrest of RNA elongation was either placed or not placed in front of the chloramphenicol acetyltransferase gene and brought under the control of MVMp early promoter P4. Although the MVMp attenuator reduced P4-driven chloramphenicol acetyltransferase expression, the extent of attenuation was similar in normal and ras-transformed cells. Moreover, the analysis of P4-directed viral RNAs in MVMp-infected cultures by RNase protection and nuclear run-on assays also revealed a transcription elongation block of a similar amplitude in both types of cells. In addition, the stabilities of the three major parvoviral mRNAs did not vary significantly between normal and ras-transformed cells. Hence, it is concluded that the ras-induced increase in the accumulation of parvoviral mRNAs is mainly controlled at the level of transcription. Consistently, the TATA motif of the P4 promoter proved to have a differential photoreactivity when tested by in vivo UV footprinting assays in ras-transformed versus normal cells.

Programmed killing of human cells by means of an inducible clone of parvoviral genes encoding non-structural proteins.

Although its dependence on the target cell type is well established, the cytopathogenicity of parvoviruses has remained elusive to date as far as its mechanism is concerned. However, indirect evidence suggested that parvoviral non-structural (NS) proteins may be the cytotoxic effectors. In order to test this hypothesis, a molecular clone of parvovirus MVMp was modified, by replacing the P4 promoter of the NS transcription unit by the glucocorticoid-inducible promoter of the mouse mammary tumour virus. Clones of neoplastic human cells that had incorporated this construct and that were induced to produce NS proteins by dexamethasone, showed a cytopathic effect and eventually died. Our data strongly suggest that the intracellular accumulation of parvoviral NS products jeopardizes the survival of the cells, which cannot be detected unless a threshold protein concentration is reached. Interestingly, a cell variant could be isolated which resisted dexamethasone-induced killing, although it was fully inducible for the production of NS proteins. This variant was also unusually resistant to infection with MVMp virions, thus confirming the essential role played by the NS proteins in the parvoviral cytotoxicity and indicating that the cytocidal activity of the parvoviral NS products is modulated by cellular factors that may vary from one cell to another.

Sensitization of transformed rat fibroblasts to killing by parvovirus minute virus of mice correlates with an increase in viral gene expression.

Cultures of established rat fibroblasts transformed by the avian erythroblastosis virus were more susceptible to the cytopathic effect of the autonomous parvovirus minute virus of mice, prototype strain (MVMp), than were their untransformed homologs. This effect could be ascribed to the presence of a greater fraction of cells that were sensitive to the killing action of MVMp in transformed cultures than in their normal parents. Yet, transformed and normal lines were similarly efficient in virus uptake, DNA amplification, and capsid protein synthesis. In contrast, transformants accumulated 2.5- to 3-fold greater amounts of all three major MVM mRNA species and nonstructural protein than did their normal progenitors. Thus, in this system transformation-associated sensitization of cells to MVMp appears to correlate primarily with an increase in their capacity for the expression of the viral transcription unit which encodes nonstructural proteins and is controlled by the P4 promoter. Consistently, a reporter gene was expressed at a higher level by transformed versus normal cultures, when placed under the control of the MVM P4 promoter. As infectious MVMp was produced in larger amounts by transformed cultures, a late step of the parvoviral cycle, such as synthesis, encapsidation of progeny DNA, or both, was also stimulated in the transformed cells.