Mink cell focus-forming murine leukemia virus killing of mink cells involves apoptosis and superinfection.

Induction of apoptosis by different types of pathogenic retroviruses is an important step in disease development. We have observed that infection of thymic lymphocytes by the mink cell focus-forming murine leukemia virus (MCF MLV) during the preleukemic period resulted in an enhancement of apoptosis of these cells. To further study the ability of MCF MLVs to induce apoptosis and the role of this process in viral pathogenesis, we have developed an in vitro system of virus-induced apoptosis. MCF13 MLV infection of mink epithelial cells resulted in the production of cytopathic foci. In contrast, infection of mink cells with the 4070A amphotropic MLV did not produce any cytopathic effects. Staining of MCF13 MLV-infected cells with propidium iodide and annexin V-fluorescein isothiocyanate indicated that virus-induced cell death was due to apoptosis. At 6 days postinfection, the percentage of apoptotic MCF13 MLV-infected cells was 27% compared with 2 to 3% for mock- or amphotropic MLV-infected cells, representing a 9- to 14-fold difference. Assays for caspase-3 activation confirmed the detection by flow cytometry of apoptosis of MCF13 MLV-infected cells. Large amounts of unintegrated linear viral DNA were detectable by Southern blot analysis during the acute phase of infection, which indicated that MCF13 MLV is able to superinfect mink cells. Unintegrated viral DNA of only the linear form was detectable in thymic lymphocytes isolated from MCF13 MLV-inoculated mice during the preleukemic period. These results indicated that the ability of MCF13 MLV to induce apoptosis is correlated with its ability to superinfect cells and that this occurs as an early step in thymic lymphoma development.

Role of the LTR region between the enhancer and promoter in mink cell focus-forming murine leukemia virus pathogenesis.

Long terminal repeat (LTR) sequences are important determinants of mink cell focus-forming (MCF) murine leukemia virus pathogenesis. These sequences include the enhancer and sequences between the enhancer and promoter (DEN). In a previous study we showed that a virus missing the DEN region in its LTR was severely attenuated in its ability to induce thymic lymphoma. In this study we observed that a virus with an LTR consisting of DEN but no enhancer sequences was pathogenic. We compared the pathogenicity of this DEN virus with other LTR mutant MCF13 viruses that contained a single enhancer (1R) or a single enhancer plus DEN (1R + DEN). All LTR mutant viruses generated thymic lymphoma, however, at a much lower incidence and with a longer latency compared with wild-type (WT) MCF13 virus. DEN virus replication in the thymus was the lowest compared with the 1R and 1R + DEN viruses. Viral replication in a different thymic subpopulation could not explain the decreased pathogenicity of the LTR mutant viruses compared with WT virus. However, lower levels of mutant virus replication in the thymus compared with WT during the preleukemic period may contribute to the attenuation of pathogenicity. The phenotype of tumors induced by the mutant viruses was similar and differed from tumors induced by WT virus by the presence of CD3(-)CD4(-)CD8(-) cells. Analysis of LTR sequences of infectious virus rescued from tumors induced by the 1R and 1R + DEN viruses showed that amplification of enhancer sequences had occurred during tumor development. The lack of DEN virus expression by tumor cells led us to propose that DEN sequences may play a role at an early step in tumorigenesis.

Mink cell focus-forming murine leukemia virus infection induces apoptosis of thymic lymphocytes.

In a previous study we identified the subpopulations of thymus cells that were infected by the lymphomagenic MCF13 murine leukemia virus (MLV) (F. K. Yoshimura, T. Wang, and M. Cankovic, J. Virol. 73:4890-4898, 1999) and observed an effect on thymus size by virus infection. In this report we describe our results which demonstrate that MCF13 MLV infection of thymuses reduced the number of T lymphocytes in this organ. Histological examination showed diffuse lymphocyte depletion, which was most striking in the CD4(+) CD8(+) lymphocyte-enriched cortical zone. Consistent with this, flow cytometric analysis showed that the lymphocytes which were depleted were predominantly the immature CD3(-) CD4(+) CD8(+) and CD3(+) CD4(+) CD8(+) cells. A comparison of the percentages of live, apoptotic, and dead cells of the gp70(+) and gp70(-) thymic lymphocytes suggested that this effect on thymus cellularity is a result of virus infection. Studies of the survival of thymic T lymphocytes in culture showed that cells from MCF13 MLV-inoculated mice underwent greater apoptosis and death than cells from control animals. Assays for apoptosis included 7-amino-actinomycin D staining, DNA fragmentation, and cleavage of caspase-3 and poly(ADP-ribose) polymerase proenzymes. Our results suggest that apoptosis of thymic lymphocytes by virus infection is an important step in the early stages of MCF13 MLV tumorigenesis.

Sequences between the enhancer and promoter in the long terminal repeat affect murine leukemia virus pathogenicity and replication in the thymus.

We previously showed that the 93-bp region between the enhancer and promoter (named DEN for downstream of enhancer) of the long terminal repeat (LTR) of the MCF13 murine leukemia virus is an important determinant of the ability of this virus to induce thymic lymphoma. In this study we observed that DEN plays a role in the regulation of virus replication in the thymus during the preleukemic period. A NF-kappaB site in the DEN region partially contributes to the effect of DEN on both lymphomagenicity and virus replication. To further study the effects of DEN and the NF-kappaB site on viral pathogenicity during the preleukemic period, we examined replication of wild-type and mutant viruses with a deletion of the NF-kappaB site or the entire DEN region in the thymus. Thymic lymphocytes which were infected with wild-type and mutant viruses were predominantly the CD3(-) CD4(+) CD8(+) and CD3(+) CD4(+) CD8(+) cells. The increase in infection by wild-type virus and both mutant viruses of these two subpopulations during the preleukemic period ranged from 9- to 84-fold, depending upon the time point and virus. The major difference between the wild-type and both mutant viruses was the lower rate and lower level of mutant virus replication in these thymic subpopulations. Significant differences in replication between wild-type and both mutant viruses were seen in the CD3(-) CD4(+) CD8(+) and CD3(-) CD4(-) CD8(-) subpopulations, suggesting that these thymic cell types are important targets for viral transformation.

Spacing between the enhancer and promoter of the long terminal repeat of a murine leukaemia retrovirus is required for transcriptional activation in T cells.

In T cells, transcriptional activation by the long terminal repeat (LTR) of the mink cell focus-forming murine leukaemia virus requires some spacing between the enhancer and promoter. A large size-range of intervening sequences (11-93 bp) is able to activate transcription efficiently. Neither a specific nucleotide sequence nor stereospecific alignment of the spacer is important.

Identification of nucleotide sequences that regulate transcription of the MCF13 murine leukemia virus long terminal repeat in activated T cells.

The region downstream of the enhancer (DEN) of the long terminal repeat of the mink cell focus-forming murine leukemia virus is important for viral pathogenicity. Another important activity of DEN is its control of transcription in activated T cells, and we have determined that an NF-kappaB site is critical for this activity.

Intrapatient sequence variation of the gag gene of human immunodeficiency virus type 1 plasma virions.

Because certain regions of the gag gene, such as p24, are highly conserved among human immunodeficiency virus (HIV) isolates, many therapeutic strategies have been directed at gag gene targets. Although intrapatient variation of segments of gag have been determined, little is known about the variability of the full-length gag gene for HIV isolated from a single individual. To evaluate intrapatient full-length gag variability, we derived the nucleotide sequences of at least 10 cDNA gag clones of virion RNA isolated from plasma for each of four asymptomatic HIV type 1-infected patients with relatively high CD4+ T-cell counts (300 to 450 cells per mm3). Mean values of intrapatient gag nucleotide variation obtained by pairwise comparisons ranged from 0.55 to 2.86%. For three subjects, this value was equivalent to that reported for intrapatient full-length env variation. The greatest range of intrapatient mean nucleotide variation for individual protein-coding regions was observed for p7. We did not detect any G-to-A hypermutation, as A-to-G and G-to-A transitions occurred at similar frequencies, accounting for 29 and 25%, respectively, of the changes. Mean variation values and phylogenetic analysis suggested that the extent of nucleotide variation correlated with the length of viral infection. Furthermore, no distinct subpopulations of quasispecies were detectable within an individual. The predicted amino acid sequences indicated that there were no regions within a gag protein that were comprised of clustered changes.

Characterization of nuclear protein binding to a site in the long terminal repeat of a murine leukemia virus: comparison with the NFAT complex.

We previously identified a protein-binding site (MLPal) that is located downstream of the enhancer element in the long terminal repeat (LTR) of a mink cell focusing-forming (MCF) murine leukemia virus (F. K. Yoshimura, K. Diem, H. Chen, and J. Tupper, J. Virol. 67:2298-2304, 1993). We determined that the MLPal site regulates transcription specifically in T cells and affects the lymphomagenicity of the MCF isolate 13 murine leukemia virus with a single enhancer repeat in its LTR. In this report, we present evidence that two different proteins, a T-cell-specific protein and a ubiquitous protein, bind the MLPal site in a sequence-specific manner. By mutational analysis, we determined that the T-cell-specific and the ubiquitous proteins require different nucleotides in the MLPal sequence for DNA binding. By competitive electrophoretic mobility shift assays, we demonstrated that the T-cell-specific protein that binds MLPal is identical or similar to a protein from nonactivable T cells that interacts with the binding site of the nuclear factor of activated T cells (NFAT). Unlike the NFAT-binding site, however, the MLPal site does not bind proteins that are inducible by T-cell activation. We observed that the MLPal sequence is conserved in the LTRs of other mammalian retroviruses that cause T-cell diseases. Furthermore, the MLPal sequence is present in the transcriptional regulatory regions of cellular genes that either are expressed specifically in T cells or are commonly rearranged by provirus integration in thymic lymphomas. Thus, the MLPal-binding proteins may play a role in the transcriptional regulation not only of the MCF virus LTR but also of cellular genes involved in T-cell development.

Identification of a region of a murine leukemia virus long terminal repeat with novel transcriptional regulatory activities.

The 93-bp region downstream of the enhancer (DEN) in the long terminal repeat (LTR) of the mink cell focus-forming virus (MCF13) has been shown to be important for transcriptional activation and viral lymphomagenicity (J. C. Tupper, H. Chen, E. F. Hays, G. C. Bristol, and F. K. Yoshimura, J. Virol. 66:7080-7088, 1992). In this report, we have further explored the role of the DEN region in transcriptional activation. We observed that it has enhancer-like abilities as well as some unique LTR properties. Transcriptional activation by the DEN region involved interactions with enhancer sequences that were either synergistic or additive, depending on the cell type. The most intriguing property of the DEN region is its ability to induce transcription in activated T cells. This activity is unique for the LTR in that no other LTR region can do this. We also examined the role of the DEN region in retroviral lymphomagenesis. We cloned and sequenced proviral LTRs integrated upstream of the cellular c-myc gene from DNA obtained from thymic tumors induced by DEN region deletion mutant viruses in AKR mice. We determined that for transcriptional activation of the c-myc proto-oncogene, enhancer sequences can substitute for the DEN region. This study identifies the significance of non-enhancer sequences in the LTR for the oncogenesis of the MCF13 retrovirus.

A protein-binding site with dyad symmetry in the long terminal repeat of the MCF13 murine leukemia virus that contributes to transcriptional activity in T lymphocytes.

We have previously identified regions in the long terminal repeat (LTR) of the MCF13 murine leukemia virus (MLV) that contribute to transcriptional activity in different cell types. We have observed that enhancer sequences and a region that resides 3' of the enhancer make significant contributions to transcriptional activity in T lymphocytes (T. Hollon and F. K. Yoshimura, J. Virol. 63:3353-3361, 1989). In this report, we have focused on the region of the MCF13 LTR that is 3' of the enhancer to identify binding sites for proteins that may play a role in the regulation of transcription in T cells. By gel shift and DNA footprint analyses, we have identified a single protein-binding site (MLPal) that includes a nucleotide sequence with dyad symmetry. A synthetic double-stranded oligonucleotide corresponding to this protein-binding site formed a specific protein-DNA complex. Deletion of this protein-binding site from the wild-type LTR decreased transcriptional activity in T lymphocytes but not in fibroblasts as determined by a transient expression assay. The MLPal sequence by itself cannot augment transcription in T cells but is able to do so in conjunction with enhancer sequences.

Contributions to transcriptional activity and to viral leukemogenicity made by sequences within and downstream of the MCF13 murine leukemia virus enhancer.

We have identified nucleotide sequences that regulate transcription in both a cell-type-specific and general manner in the long terminal repeat of the MCF13 murine leukemia virus. Besides the enhancer element, we have observed that the region between the enhancer and promoter (DEN) has a profound effect on transcription in different cell types. This effect, however, was dependent on the copy number of enhancer repeats and was detectable in the presence of a single repeat. When two enhancer repeats were present, the effect of DEN on transcription was abrogated except in T cells. DEN also makes a significant contribution to the leukemogenic property of the MCF13 retrovirus. Its deletion from the MCF13 virus dramatically reduced the incidence of thymic lymphoma and increased the latency of disease in comparison with the wild-type virus. This effect was most marked when one rather than two enhancer repeats was present in the mutant viruses. We also observed that the removal of one repeat alone remarkably reduced leukemogenicity by the MCF13 virus. A newly identified protein-binding site (MLPal) located within DEN affects transcription only in T cells, and its deletion attenuates the ability of an MCF13 virus with a single enhancer repeat to induce thymic lymphoma. This observation suggests that the MLPal protein-binding site contributes to the effect of the DEN region on T-cell-specific transcription and viral leukemogenicity. This study identifies the importance of nonenhancer sequences in the long terminal repeat for the oncogenesis of the MCF13 retrovirus.

In vitro transfection of fresh thymocytes and T cells shows subset-specific expression of viral promoters.

We describe conditions under which exogenous DNA templates can be introduced for transient expression into primary murine T lymphocytes. T cells at various stages of development, including concanavalin A-activated splenic T cells, immature pre-T cells, and even small cortical thymocytes, could be successfully transfected. A variety of model DNA constructs were compared in which different viral promoter regions were used to drive expression of the chloramphenicol acetyltransferase (CAT) reporter gene. All showed enhanced expression in cells that had been acutely stimulated with the Ca2+ ionophore A23187 and phorbol ester as chemical proxies for T-cell receptor-mediated signals. In addition, splenocytes but not thymocytes required prior treatment with a mitogen and interleukin-2 in order to express these constructs, implying that even postmitotic thymocytes may be held in a quasiactivated state. A most striking result was the finding that the viral regulatory sequences in the Rous sarcoma virus long terminal repeat and the simian virus 40 early region were subject to sharply differential regulation, with a rank order that changed depending on the developmental stage of the T cells. The most immature thymic blasts and several lymphoma cell lines expressed the pRSV-Cat and pSV2-Cat constructs similarly, but cortical thymocytes exhibited a strong preference for pSV2-Cat. Splenic concanavalin A-stimulated blasts, on the other hand, slightly preferred pRSV-Cat, a tendency which became exaggerated in factor-dependent T-cell lines. The ratio of pRSV-Cat to pSV2-Cat expression varied according to cell type by as much as 500-fold. These results argue against a trivial linkage of promoter preference to cell cycle status but instead provide evidence that activation of T cells at distinct stages of differentiation results in the expression of different ensembles of nuclear regulatory proteins. In contrast to the simian virus 40 and Rous sarcoma virus promoter regions, the long terminal repeats of the retroviruses mink cell focus-forming virus and Akv were expressed well in all primary T-lineage cells. Thus, they represent excellent model promoters for engineering developmental stage-independent expression of exogenous genes in murine T cells.

Differential DNA binding of nuclear proteins to a long terminal repeat region of the MCF13 and Akv murine leukemia viruses.

Long terminal repeat (LTR) sequences of murine leukemia viruses (MLVs) have been demonstrated to be mainly responsible for the pathogenic differences in these retroviruses. A region of the LTR which is downstream of the enhancer elements has been shown to contribute both to enhancer activity as well as to disease specificity of MLVs. We have identified protein-DNA complexes generated by this region of a lymphomagenic MLV (MCF13) and one which is nonpathogenic (Akv). One protein-DNA complex we have observed for this region is unique to MCF13 DNA sequences. Detection of protein involved in this unique MCF13 complex in different cell lines revealed that it was ubiquitous.

A nonuniform electrical field electroporation chamber design.

We show an inexpensive design for an electroporation chamber which subjects electroporated cells to a nonuniform electrical field. Our design, which we call an electroporation cylinder, improved transfection efficiency over that of a uniform field design (electroporation cuvettes) by about sixfold when tested in five mouse cell lines with a transient gene expression assay. Electroporation cylinders subjected cells to electrical field strengths at least as powerful as those of electroporation cuvettes, as judged by comparing the percentages of cells killed by electroporation. Cylinder and cuvette designs were similar in their effect on the variability of transfection efficiency. Electroporation cylinders may be particularly useful when the optimal electrical field strength for a cell line is not known or is unattainable with a given power supply.

Variation in enzymatic transient gene expression assays.

We examined causes for high variability in data from enzymatic transient gene expression assays. Our results strongly suggest that variation in transfection efficiency is the major cause of data variation and can seriously compromise valid interpretation of data. We compared averaging data from multiple transfections and cotransfection of a second reporter gene as methods for correcting for variation in transfection efficiency. We found that transfection efficiency can be so highly variable that neither method necessarily overcomes the resulting bias in data. Depending upon the degree in variation in transfection efficiency, a combination of the two methods may be advisable. The need to normalize data for transfection efficiency is dependent upon the difference in strengths of promoters being tested and the relative variability of the transfection method used. We also show that the level of reporter gene expression between transfection experiments performed on different days can vary by more than 10-fold.

Mapping of functional regions of murine retrovirus long terminal repeat enhancers: enhancer domains interact and are not independent in their contributions to enhancer activity.

We have used deletion and recombinant long terminal repeat (LTR) mutants to examine enhancer activity differences between LTRs of the nonpathogenic Akv and the thymus lymphomagenic MCF13 murine retroviruses. Deletion mutant analysis revealed that major control regions for MCF13 and Akv LTR enhancer activity were similar but not identical. For both LTRs, major control regions were distinctly different in a murine T-cell and a fibroblast cell line. Recombinant enhancer analysis showed that LTRs could be divided into three regions capable of altering the level of enhancer activity through cooperative or antagonistic interaction. The contribution of each region to enhancer activity was dependent on its context with respect to the other regions. LTR enhancer function in different cell types appears to be the result of the interaction of enhancer modular elements.

Different activities of viral enhancer elements before and after stable integration of transfected DNAs.

Analysis of the RNA and DNA levels of a selectable gene linked to a murine retroviral enhancer demonstrated a correlation between RNA levels and tissue-specific enhancer activity during transient expression in T cells but not in stably transformed cell lines.

Murine leukemia virus long terminal repeat sequences can enhance gene activity in a cell-type-specific manner.

We tested the ability of sequences in the long terminal repeat (LTR) of a mink cell focus-forming (MCF) murine leukemia virus to function as an enhancer in a cell-type-specific manner. In a stable transformation assay, the MCF or Akv LTR and the simian virus 40 enhancer had similar activities in murine fibroblasts. In contrast, the MCF LTR had a significantly greater activity in murine T lymphoid cells than did either the simian virus 40 enhancer or the Akv LTR.

AKR thymic lymphomas involving mink cell focus-inducing murine leukemia viruses have a common region of provirus integration.

Newly acquired proviruses related to a mink cell focus-inducing murine leukemia virus were detected in low copy number in restriction endonuclease-digested DNAs from thymic lymphomas of AKR/J mice. These extra proviruses were not present in DNAs of either normal thymus or leukemic brain tissues. Extra tumor-specific DNA fragments generated by restriction endonucleases either were identical in size or fell into similar size classes, suggesting a common site(s) of provirus integration. Characterization of extra EcoRI DNA fragments for mink cell focus-inducing viral sequences revealed that all of them contained large terminal repeat sequences and that a significant number represented proviruses with deletions.

Identification of a DNA fragment from a molecularly cloned mink cell focus-inducing murine leukemia virus specific for xenotropic virus-related sequences.

Molecular clones of closed circular DNA molecules of a mink cell focus-inducing murine leukemia virus (MCF-13 MuLV) were generated. Closed circular DNA molecules isolated from a Hirt extraction of recently infected NIH/3T3 cells were inserted at their unique EcoRI site into lambda gtWES.lambda B. Restriction endonuclease analysis of inserts of two clones indicated that they represented intact MCF-13 MuLV genomes. One viral insert contained two large terminal repeat sequences, and the other contained only one. A 300-base-pair DNA fragment located in the envelope region of the MCF-13 MuLV genome was determined to be related to xenotropic MuLV sequences.