Enhancer-deficient amphotropic murine leukemia virus and recombinants with heterologous transcription elements can be efficiently amplified and detected in Mus dunni fibroblasts.

Amphotropic murine leukemia virus (MLV) replicates in cells from various mammalian species, including humans, and is a potential contaminant in MLV vector preparations for human gene transfer studies. Mus dunni fibroblasts are routinely used for amplification and detection of contaminating virus. We have recently characterized an amphotropic MLV mutant lacking the 75-bp viral enhancer elements and spontaneous MLV-(RCMV) recombinants that have acquired cytomegalovirus (CMV) transcription elements. Both of these viruses replicate in specific human cell types. To test whether the formation of such viruses can be detected and controlled with current routine procedures, we have analyzed the replication of these amphotropic MLV mutants in Mus dunni fibroblasts. We find that M. dunni cells are permissive for enhancer-deficient and CMV promoter-recombinant MLV from several human cell lines. Thus, M. dunni fibroblasts are suitable for the amplification and subsequent detection of enhancer-deficient and enhancer-recombinant MLV in vector preparations.

Replication of enhancer-deficient amphotropic murine leukemia virus in human cells.

Amphotropic murine leukemia virus (MLV) replicates in cells from various mammalian species, including humans, and is a potential contaminant in MLV vector preparations for human gene transfer studies. The generation of replication-competent virus is considered less likely with vectors that delete the viral transcription elements. This conclusion is based on data obtained in rodents, where MLV replication depends on the expression of viral genes under the control of 75-bp enhancer elements in the long terminal repeat. We demonstrate here that in some human cells replication of amphotropic MLV is possible in the absence of these enhancer elements. Replication of the enhancer-deficient virus MLV-(MOA)Delta E is observed in selected human sarcoma and B lymphoma lines and proceeds at a lower rate than that of the intact virus. No insertion of a foreign promoter or enhancer into the long terminal repeat was detected. Our data suggest the presence of a secondary enhancer element within the MLV provirus that can in selected human cells mediate virus transcription and replication in the absence of the 75-bp U3 enhancers.

Amphotropic murine leukemia virus replication in human mammary epithelial cells and the formation of cytomegalovirus-promoter recombinants.

Amphotropic murine leukemia virus (MLV) can replicate in human cells and is a potential contaminant in vector preparations for human gene transfer studies. We have recently shown that replication of amphotropic MLV in specific human sarcoma and lymphoma lines is possible in the absence of the viral 75-bp transcription enhancer elements. Here, we have tested the replication of an amphotropic MLV, MLV-(MOA), and an enhancer-deficient mutant of this virus in human breast carcinoma-derived cell lines. The proviral expression plasmids use a cytomegalovirus (CMV) promoter for the initial transcription of virus RNA. We found that all cells analyzed are permissive for replication of MLV-(MOA). Enhancer-deficient virus is unable to replicate. However, in two lines the replication defect can be rescued by the spontaneous insertion of a CMV promoter and enhancer into the U3 region. This recombinant virus MLV-(RCMV) replicates with kinetics similar to that of MLV-(MOA) but is restricted to specific cell lines. The potential formation of RCMV recombinants during MLV vector preparation must be considered.

Mouse mammary tumor virus superantigen expression is reduced by glucocorticoid treatment.

Expression of mouse mammary tumor virus (MMTV)-encoded superantigens in B lymphocytes are required for viral transmission and pathogenesis. Due to problems with detection and quantification of the superantigen protein, most reports about the mechanism of superantigen expression from the viral sag gene rely on the quantitative analysis of putative sag mRNAs. The description of multiple promoters as a source of putative sag mRNA has complicated the situation even further. All conclusions about the level of superantigen protein expression based on these data remain circumstantial. To test the effect of the glucocorticoid hormone dexamethasone on the total superantigen expression from an infectious MMTV provirus we used a quantitative assay that is based on a superantigen-luciferase fusion protein. MMTV gene expression from the major promoter in the 5' long terminal repeat (LTR) is strongly induced in the presence of glucocorticoid hormones. We now demonstrate that, in the presence of dexamethasone, sag gene expression is reduced despite increased transcription from the MMTV 5' LTR and increased amounts of putative sag mRNA initiated at the LTR promoter. These data show that the expression of the MMTV sag gene does not correlate with the activity of the major LTR promoter and thus differs from all other MMTV genes.

The mouse mammary tumor virus transcription enhancers for hematopoietic progenitor and mammary gland cells share functional elements.

Expression of mouse mammary tumor virus (MMTV)-encoded superantigens in B lymphocytes is required for viral transmission and pathogenesis. We have previously established a critical role of an enhancer element within the long terminal repeat (LTR) for MMTV sag gene expression in B-lymphoid progenitor cells. We now demonstrate enhancer activity of this element in a promyelocytic progenitor cell line. We also map the position of the enhancer within the U3 region of the MMTV LTR and show that the progenitor cell enhancer shares functional elements with a previously described mammary gland-specific enhancer.

Mouse mammary tumor virus superantigen expression in B cells is regulated by a central enhancer within the pol gene.

Expression of mouse mammary tumor virus (MMTV)-encoded superantigens in B lymphocytes is required for viral transmission and pathogenesis. The mechanism of superantigen expression from the viral sag gene in B cells is largely unknown, due to problems with detection and quantification of these low-abundance proteins. We have established a sensitive superantigen-luciferase reporter assay to study the expression and regulation of the MMTV sag gene in B-cell lymphomas. The regulatory elements for retroviral gene expression are generally located in the 5' long terminal repeat (LTR) of the provirus. However, we found that neither promoters nor enhancers in the MMTV 5' LTR play a significant role in superantigen expression in these cells. Instead, the essential regulatory regions are located in the pol and env genes of MMTV. We report here that maximal sag expression in B-cell lines depends on an enhancer within the viral pol gene which can be localized to a minimal 183-bp region. Regulation of sag gene expression differs between B-cell lymphomas and pro-B cells, where an enhancer within the viral LTRs is involved. Thus, MMTV sag expression during B-cell development is achieved through the use of two separate enhancer elements.

Genetics of intracisternal-A-particle-related envelope-encoding proviral elements in mice.

Intracisternal-A-particle-related envelope-encoding (IAPE) proviral elements in the mouse genome encode and express an envelope-like protein that may allow transmission of IAPEs as infectious agents. To test IAPE mobility and potential transmission in mice, we have analyzed the distribution of IAPE elements in the genomes of Mus spretus and Mus musculus inbred strains and wild-caught animals. Potential full-length (IAPE-A) proviral elements are present as repetitive copies in DNA from male but not female animals of M. musculus inbred strains and Mus musculus castaneus. Analysis of IAPE-cellular junction fragments indicates that fixation of most IAPEs in the germ line occurred in M. musculus and M. spretus after speciation but before M. musculus inbred strains were derived.

Stimulation of mouse mammary tumor virus superantigen expression by an intragenic enhancer.

The mechanisms regulating expression of mouse mammary tumor virus (MMTV)-encoded superantigens from the viral sag gene are largely unknown, due to problems with detection and quantification of these low-abundance proteins. To study the expression and regulation of the MMTV sag gene, we have developed a sensitive and quantitative reporter gene assay based on a recombinant superantigen-human placental alkaline phosphatase fusion protein. High sag-reporter expression in Ba/F3, an early B-lymphoid cell line, depends on enhancers in either of the viral long terminal repeats (LTRs) and is largely independent of promoters in the 5' LTR. The same enhancer region is also required for general expression of MMTV genes from the 5' LTR. The enhancer was mapped to a 548-bp fragment of the MMTV LTR lying within sag and shown to be sufficient to stimulate expression from a heterologous simian virus 40 promoter. No enhancer activity of the MMTV LTR was observed in XC sarcoma cells, which are permissive for MMTV. Our results demonstrate a major role for this enhancer in MMTV gene expression in early B-lymphoid cells.

Expression of intracisternal A-particle-related retroviral element-encoded envelope proteins detected in cell lines.

Intracisternal A-particle (IAP) retrotransposons of rodents express gag and pol proteins for assembly of intracellular viruslike particles but lack an env gene. The recently described IAP-related family of retroviral elements contains a reading frame with close resemblance to retroviral env genes (IAPEs) (F. U. Reuss and H. C. Schaller, J. Virol. 65:5702-5709, 1991). I now report the analysis of cellular IAPE mRNAs and detection of IAPE env proteins. IAPE elements are transcribed in cell lines NH15-CA2 and AtT20. Four major transcripts of 4.2, 3.9, 2.8, and 1.3 kb are detected and characterized by probes specific for defined regions of the cloned IAPE-1 cDNA. The 2.8-kb mRNA is shown to lack gag and pol genes but comprises an env gene and U3 region, as expected for a subgenomic env mRNA. Polymerase chain reaction amplification and cloning of such mRNAs confirmed the absence of gag and pol genes 5' from the env gene and implicates env mRNA generation by a splicing event. A polyclonal anti-IAPE env antiserum, raised against a bacterial IAPE-env fusion protein, specifically detects N-glycosylated env proteins of 91 kDa or less in cell lines positive for IAPE mRNA. IAPE env proteins of different sizes represent independent translation products. After inhibition of N-glycosylation, env proteins in the size predicted from the env gene sequence or smaller are present. These results provide evidence that putative IAPE env proteins are synthesized in vivo. Envelope protein expression by an IAP-related retroviral element identifies IAPEs as a possible missing link between IAP retrotransposons and retroviruses.

cDNA sequence and genomic characterization of intracisternal A-particle-related retroviral elements containing an envelope gene.

Intracisternal A-particle retrotransposons (IAPs) are retroviruslike elements that are defective in envelope protein synthesis and exist without an extracellular stage. We have isolated a novel class of cDNAs that are related to known IAP elements in the nucleotide and deduced protein sequence of gag and pol genes but also contain a previously unidentified reading frame between the pol gene and putative U3 region. Analysis of the deduced protein sequence reveals features of the putative protein that are characteristic of retroviral envelope proteins. The isolated cDNAs represent transcripts of multiple retroid elements in the mouse genome that were termed IAPE (intracisternal A-particle-related elements coding for envelope). IAPE env genes exist in approximately 200 copies per haploid genome as integral parts of the majority of these retroid elements. Four major IAPE subgroups could be distinguished after EcoRI digestion of genomic DNA.