High mutation rate of a spleen necrosis virus-based retrovirus vector.

Abstract

Spleen necrosis virus (SNV) is an avian retrovirus that efficiently infects some mammalian cells (e.g., dog and rat cells). We constructed an SNV-based vector, which contains less than 1 kilobase (kb) of the retrovirus sequence, and a number of derivatives containing selectable markers. We obtained high-titer virus stocks, over 106 transforming units per ml, with a vector whose genomic RNA consists of 1,850 bases (full-length SNV RNA is 7.7 kb). We also studied two vectors that both carry two genes which should be expressed from a single promoter, one gene from unspliced mRNA and the other gene from spliced mRNA. In one vector, both genes were efficiently expressed as expected. However, in the other vector, expression of the gene 3' to the splice acceptor was inhibited. When we selected for expression of the 3' gene is this latter case, we found that the resistant cells contained mutant proviruses in which the 3' gene could be expressed. Furthermore, we found that mutations were generated during a single round of virus replication (provirus to provirus) at a rate of approximately 0.5% mutations per cycle.Retroviruses are RNA viruses that replicate through a DNA intermediate. The DNA intermediate, the provirus, stably integrates into the chromosomal DNA of the host cell. Moreover, retroviruses can effectively infect most or all cells in a culture (37). Because of these properties, retroviral vectors represent an attractive system for stable introduction of exogenous genes into cells.Retroviral vectors that contain two expressed genes have been of particular interest because they can carry both a gene of interest and a selectable gene, enabling easy selection of cells that have been infected by vector-derived virus. There are two major ways such a vector can be constructed. One is to make a vector with an internal promoter so that one gene is expressed from the retrovirus long terminal repeat (LTR) promoter while the other gene is expressed from an internal promoter. The other method is to contruct a vector so that both genes are expressed from a single LTR promoter, one from unspliced retroviral RNA and the other from spliced retroviral RNA. Both types of vectors have been made (2, 4-6, 9, 10, 12, 15, 16, 20, 22, 26, 28-30, 32, 36, 40).We are interested in using spleen necrosis virus (SNV)-based retroviral splicing vectors to introduce different genes into avian cells, but we previously found that inserting different genes into reticuloendotheliosis virus (Rev) strain T (Rev-T) suppresses transformation by reducing the amount of spliced . Consequently, we thought that it would be prudent to study in detail the biological properties of retroviral vectors with spliced mRNA. We term these constructions splicing vectors. Moreover, because of the interest in using splicing vectors as vehicles in somatic therapy of human genetic diseases, a deeper understanding of their properties should be helpful in developing successful protocols for their use in this therapy. We constructed two SNV-based splicing vectors that each...

The activation of TC10, a Rho small GTPase, contributes to v-Rel-mediated transformation

“…The spleen necrosis virus (SNV)-based bicistronic retroviral vector pMJ612 was derived from pJD216neoHy (Dougherty and Temin, 1986) and contains two long terminal repeats (LTR), neomycin and hygromycin resistance genes, and a gene encoding the green fluorescence protein (GFP). The sequences between the SmaI site and the SalI site of pMJ612 encode the hygromycin resistance gene and were replaced with double-stranded adapters (5 0 -GCCACCATGCCCGGGG GAGGGCGGGCT-3 0 and 5 0 -CATTGTCGACTCACGTGA TCAAACAGCAGT-3 0 ) to facilitate the cloning of the ORFs of TC10, the gain-of-function mutant TC10Q76L, or the dominant-negative mutant TC10T32N.…”

“…Viral supernatant fluids were harvested 7-12 days after drug selection. The infectious units (IU) of the MJ612 virus were determined by infection of D17 cells and quantitation of neomycin-resistant colonies as described previously (Dougherty and Temin, 1986).…”

Somatic transgenesis using retroviral vectors in the chicken embryo

“…RSV-based vectors provide higher levels expression than many of the other vectors but are capable of infecting only specific bird species (Payne et al, 1992;Fekete and Cepko, 1993a). SNV-based viruses (Dougherty and Temin, 1986) exhibit a broad host range in both birds and mammals (including human) but poorly infect rodent cells (Mikawa et al, 1991;Koo et al, 1991). Thus, selection of appropriate vectors will depend on the host species and cell types being targeted.…”

“…In addition, replication-defective vectors can accept exogenous genes of ϳ7 kb, offering enough space for inserting a gene of interest together with a reporter gene to identify the cells expressing exogenous genes in a somatic transgenic model. In earlier attempts of dual gene expression from a replication-defective viral vector, a di-cistronic provirus was designed to generate two subgenomic messages by introducing a splicing site (Dougherty and Temin, 1986;Fig. 3B) or by a second promoter (Cepko, 1988; Fig.…”

“…3C). These vectors, however, often fail to transmit and express both genes simultaneously (Emerman and Temin, 1984;Dougherty and Temin, 1986;Nawrotzki et al, 1995), giving rise to a subpopulation that expresses a reporter but not a gene of interest and vice versa.…”