SV40 Vectors Research Articles

784. Trans-Activated IFN-α2 Delivered to T Cells by a Recombinant SV40 Vectors Inhibits Early Stages in the HIV-1 Replicative Cycle

784. Trans-Activated IFN-α2 Delivered to T Cells by a Recombinant SV40 Vectors Inhibits Early Stages in the HIV-1 Replicative Cycle

1277. Functional Proteins Can Be Secreted from Cells Transduced with Recombinant SV40 Vectors Carrying the cDNAs for Those Proteins

1277. Functional Proteins Can Be Secreted from Cells Transduced with Recombinant SV40 Vectors Carrying the cDNAs for Those Proteins

In vitro-packaged SV40 pseudovirions as highly efficient vectors for gene transfer.

A procedure for in vitro packaging of plasmid DNA in recombinant SV40 capsid proteins was developed by Sandalon et al. (1997). Here, we report the highly efficient transduction into different human, murine and monkey cell lines using a scaled-up protocol for producing SV40 pseudovirions, packaged in vitro, carrying the human multidrug-resistance gene MDR1 encoding P-glycoprotein (P-gp) or the green fluorescent protein reporter gene (GFP) under control of SV40 and cytomegalovirus (CMV) promoters. The percentage of expressing cells was proportional to the number of transducing particles, with close to 100% of cells transduced at optimal ratios of transducing particles to cells. The ability to confer multidrug resistance was evaluated by measuring dye efflux and cell-surface expression in infected cells. The relative level of expression of P-gp driven by the different promoters varied among different cell lines. In human lymphoblastoid cells, which express high levels of major histocompatibility complex (MHC) class I (a surface receptor for SV40), constructs that carry an intron yield the highest expression. Our experiments further demonstrate that MDR1 and GFP expression driven by these promoters is transient; however, transduced cells remain MDR1-positive if selected in colchicine. Thus, the SV40 vectors are well suited to situations in which only short-term expression is required or expression is selected, such as for bone marrow protection during chemotherapy.

Generation of recombinant SV40 vectors for gene transfer.

Generation of recombinant SV40 vectors for gene transfer.

A Packaging System for SV40 Vectors without Viral Coding Sequences

SV40 vectors have been used as expression vectors for mammalian cells since the early 1980s. More recently, they have been used as gene transfer vectors in mice and in human peripheral blood cells. Here we described a system for packaging SV40 vectors without viral coding sequences. Recombinant adenovirus-expressing SV40 capsids can effectively package plasmids that contain the SV40 replication origin. The final yield of infectious SV40 vector is about 3 × 105, with a SV40 to adenoviral vector ratio of about 1000:1. Helper adenoviruses can be effectively heat-inactivated with no effect on the infectivity of SV40 vectors.

Evidence from lateral mobility studies for dynamic interactions of a mutant influenza hemagglutinin with coated pits.

Replacement of cysteine at position 543 by tyrosine in the influenza virus hemagglutinin (HA) protein enables the endocytosis of the mutant protein (Tyr 543) through coated pits (Lazarovits, J., and M. G. Roth. 1988. Cell. 53:743-752). To investigate the interactions between Tyr 543 and the clathrin coats in the plasma membrane of live cells, we performed fluorescence photobleaching recovery measurements comparing the lateral mobilities of Tyr 543 (which enters coated pits) and wild-type HA (HA wt, which is excluded from coated pits), following their expression in CV-1 cells by SV-40 vectors. While both proteins exhibited the same high mobile fractions, the lateral diffusion rate of Tyr 543 was significantly slower than that of HA wt. Incubation of the cells in a sucrose-containing hypertonic medium, a treatment that disperses the membrane-associated coated pits, resulted in similar lateral mobilities for Tyr 543 and HA wt. These findings indicate that the lateral motion of Tyr 543 (but not of HA wt) is inhibited by transient interactions with coated pits (which are essentially immobile on the time scale of the lateral mobility measurements). Acidification of the cytoplasm by prepulsing the cells with NH4Cl (a treatment that arrests the pinching-off of coated vesicles from the plasma membrane and alters the clathrin lattice morphology) led to immobilization of a significant part of the Tyr 543 molecules, presumably due to their entrapment in coated pits for the entire duration of the lateral mobility measurement. Furthermore, in both untreated and cytosol-acidified cells, the restrictions on Tyr 543 mobility were less pronounced in the cold, suggesting that the mobility-restricting interactions are temperature dependent and become weaker at low temperatures. From these studies we conclude the following. (a) Lateral mobility measurements are capable of detecting interactions of transmembrane proteins with coated pits in intact cells. (b) The interactions of Tyr 543 with coated pits are dynamic, involving multiple entries of Tyr 543 molecules into and out of coated pits. (c) Alterations in the clathrin lattice structure can modulate the above interactions.

Fibroblasts transfected with Torpedo acetylcholine receptor beta-, gamma-, and delta-subunit cDNAs express functional receptors when infected with a retroviral alpha recombinant.

Torpedo californica acetylcholine receptor (AChR) alpha-, beta-, gamma-, and delta-subunit cDNAs were each stably introduced into muscle and/or fibroblast cell lines using recombinant retroviral vectors and viral infection, or using SV-40 vectors and DNA-mediated cotransfection. The expressed proteins were characterized in terms of their molecular mass, antigenicity, posttranslational processing, cell surface expression, stability in fibroblasts, stability in differentiated and undifferentiated muscle cells, and ability (of alpha) to bind alpha-bungarotoxin (BuTx). We demonstrated that the alpha, beta, gamma, and delta polypeptides acquired one, one, two, and three units of oligosaccharide, respectively. If all four subunits were expressed in the same cell, fully functional cell surface AChRs were produced which had a Kd for BuTx of 7.8 X 10(-11) M. In contrast, subunits expressed individually were not detected on the surface of fibroblasts and the Kd for BuTx binding to individual alpha polypeptides was only approximately 4 X 10(-7) M. The half-lives of the alpha, gamma, and delta subunits at 37 degrees C were all found to be quite short (approximately 43 min), while the half-life of the beta subunit was found to be even shorter (approximately 12 min). The unique half-life of the beta subunit suggests that it might perform a key regulatory role in the process of AChR subunit assembly. One stable fibroblast cell line was established by transfection that expressed beta, gamma, and delta subunits simultaneously. When this cell line was infected with a retroviral alpha recombinant, fully functional cell surface AChRs were produced. The successful expression of this pentameric protein complex combining transfection and infection techniques demonstrates one strategy for stably introducing the genes of a heterologous multisubunit protein complex into cells.

Analysis of recombination in mammalian cells using SV40 and SV40-derived vectors

Viruses and viral vectors have played a crucial role in our understanding of the pathways of homologous and non-homologous recombination in mitotically dividing mammalian cells. In particular, they have allowed the confirmation of the preponderance of non-homologous over homologous recombination events and led to schemes for the selection and isolation of homologous recombination products. These studies have allowed an examination of the properties of reciprocal and non-reciprocal homologous recombination events extrachromosomally, in the chromosome and between plasmids and chromosomes. They suggest that it is feasible now to direct DNA segments to predetermined chromosomal locations by homologous recombination.

Identification of immunoglobulin heavy chain binding protein as glucose-regulated protein 78 on the basis of amino acid sequence, immunological cross-reactivity, and functional activity

Immunoglobulin heavy chain binding protein (BiP) associates transiently with various proteins destined for the secretory pathway. To investigate the relationship between BiP and the 78K (K = 10(3) Mr) glucose-regulated protein (GRP78), we have determined a partial amino acid sequence of purified mouse BiP and isolated and sequenced a full-length cDNA clone encoding mouse GRP78. The 26 amino-terminal residues of the mature BiP protein are identical to a sequence of amino acids located near the start of the open reading frame encoding GRP78. A polyclonal antiserum raised against mouse GRP78 protein expressed in bacteria from the cloned GRP78 cDNA could immunoprecipitate complexes consisting of BiP and unfolded forms of immunoglobulin heavy chains. Furthermore, a monoclonal antibody raised against mouse BiP immunoprecipitated mouse GRP78 expressed in monkey CV-1 cells from an SV40-GRP78 recombinant vector. Finally, like the endogenous BiP of simian cells, mouse GRP78 associated with malfolded, non-glycosylated forms of influenza hemagglutinin (HA) when GRP78 and HA were co-expressed from SV40 vectors in CV-1 cells. These studies confirm that BiP is identical to GRP78. Comparison of the nucleic acid and deduced amino acid sequence of mouse GRP78 with those of other rodent and human GRP78s revealed an extremely high degree of sequence identity. BiP/GRP78 is closely related (approximately 60% identity) to the cytoplasmic 70K heat-shock proteins. Surprisingly, the carboxy-terminal 29 amino acids of BiP/GRP78, which are not conserved in HSP70 proteins, are almost identical in sequence to the steroidogenesis activator peptide found in the cytoplasm of rat Leydig tumor cells. Possible relationships between these polypeptides are discussed.

The expression of the Escherichia coli uvrA gene in human cells

Cells cultured from xeroderma pigmentosum (XP) patients are defective in excision repair of damaged DNA specifically at the incision step. In Escherichia coli this step is mediated by the UvrA, UvrB and UvrC gene products. Our goal is to express each of these genes in XP cells, singly or in combination, and to determine the most suitable conditions for generating faithful E. coli protein copies in functional concentrations and properly localized for the eventual repair of damaged chromosomal DNA or DNA which is introduced exogenously. The E. coli gpt gene in pSV2 gpt is used as a selection marker for uvr gene transfection into XP cells. The uvr genes were cloned into composite pBR322, SV40 and gpt vectors in which each E. coli gene is flanked by individual SV40 regulatory elements. SV40-transformed XP-A cells were transfected with pSV2 uvrASV2 gpt, gpt + colonies were selected, and cell lines esttablished. Several times were examined in detail. Cell lines 714 and 1511 contain uvrA together with flanking SV40 regulatory elements integrated intact in genomic DNA and express UvrA protein as well as a 95 000-dalton UvrA-related protein. The expression of uvrA was found to be 50–100-fold lower than the expression of gpt. Attempts were made to assay the mammalian UvrA protein for functionality, but endogenous activities interfered with assays for each of the UvrA protein's three activities. The peptide maps derived from partial proteolysis of the “mammalian” UvrA protein are identical to the E. coli UvrA protein. The sub-cellular location of UvrA protein in uvrA + XP cells was investigated by fractionation of cell extracts in which an indirect immunofluorescence method revealed its location as being largely extra-nuclear. Two uvrA + cell lines were examined for their UV_resistant phenotye and not unexpectedly were found not to be reverted to a state of repair proficiency.

Heterologous transmembrane and cytoplasmic domains direct functional chimeric influenza virus hemagglutinins into the endocytic pathway.

Chimeric genes were created by fusing DNA sequences encoding the ectodomain of the influenza virus hemagglutinin (HA) to DNA coding for the transmembrane and cytoplasmic domains of either the G glycoprotein of vesicular stomatitis virus or the gC glycoprotein of Herpes simplex virus 1. CV-1 cells infected with SV40 vectors carrying the recombinant genes expressed large amounts of the chimeric proteins, HAG or HAgC on their surfaces. Although the ectodomains of HAG and HAgC differed in their immunological properties from that of HA, the chimeras displayed the biological functions characteristic of the wild-type protein. Both HAG and HAgC bound erythrocytes as efficiently as HA did and, after brief exposure to an acidic environment, induced the fusion of erythrocyte and CV-1 cell membranes. However, the behavior of HAG and HAgC at the cell surface differed from that of HA in several important respects. HAG and HAgC were observed to collect in coated pits whereas wild-type HA was excluded from those structures. In the presence of chloroquine, which inhibits the exit of receptors from endosomes, HAG and HAgC accumulated in intracellular vesicles. By contrast, chloroquine had no effect on the location of wild-type HA. HAG and HAgC labeled at the cell surface exhibited a temperature-dependent acquisition of resistance to extracellular protease at a rate similar to the rates of internalization observed for many cell surface receptors. HA acquired resistance to protease at a rate at least 20-fold slower. We conclude that HAG and HAgC are efficiently routed into the endocytic pathway and HA is not. However, like HA, HAG was degraded slowly, raising the possibility that HAG recycles to the plasma membrane.

Sialic acid is incorporated into influenza hemagglutinin glycoproteins in the absence of viral neuraminidase

We have analyzed the pronase-derived glycopeptides of the hemagglutinin glycoproteins expressed from SV40 vectors carrying cloned cDNA copies of the HA gene and of HA isolated from influenza virions (A/Jap/305/57). The glycopeptides derived from the HA glycoprotein obtained from cloned genes were heterogeneous, ranging in size from 3800 to 2800 dallons. Upon treatment with neuraminidase, sialic acid was released from the glycopeptides and their size was reduced to 2900–2400 daltons. However, under the same conditions, no sialic acid was detected in the virion HA. The presence of sialic acid was confirmed by monosaccharide analysis of the HA glycoprotein derived from products of cloned genes. These results support the idea that during replication of influenza virus, the viral neuraminidase cleaves sialic acid from the HA glycoprotein in infected cells.

Sequences involved in the regulated expression of the human interferon-beta1 gene in recombinant SV40 DNA vectors replicating in monkey cells.

The human genomic EcoRI fragment of 1.83 kb containing the interferon (IFN) gene IFN-beta1 with 285 nucleotides of 5'-flanking sequences was transfected into monkey kidney CV-1 cells as part of an SV40-pML2 vector. Induction of the monkey cells to produce IFN led to a rapid accumulation of IFN-beta1 RNA whose 5' ends were identical to the IFN-beta1 mRNA of human fibroblasts. This induction occurred with all recombinants tested. Expression from the SV40 late promoter was also seen in non-induced cells. We conclude that the regulation of the IFN-beta1 gene is retained in the replicating episomal SV40 vectors with high copy number, even when the gene is being transcribed from an external promoter. When the 5'-flanking sequences were deleted to leave only 40 bp before the presumed cap site of the IFN-beta1 gene, inducible formation of IFN-RNA with authentic 5' ends could still be demonstrated. However, inducibility and expression depended on the position of the deleted IFN-beta1 gene in the vector. We conclude that the sequences around the TATAA box and cap site on the IFN gene are involved in the regulation of its expression. Regulated short-term expression of the human IFN-beta1 gene in SV40 vectors provides a defined system in which the structures required to maintain the regulation and the influence of known external transcription signals can be examined.