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. 1999 Dec;73(12):9803-9.
doi: 10.1128/JVI.73.12.9803-9809.1999.

Replication of Mus dunni endogenous retrovirus depends on promoter activation followed by enhancer multimerization

G Wolgamot  1 , A D Miller
Affiliations

Replication of Mus dunni endogenous retrovirus depends on promoter activation followed by enhancer multimerization

G Wolgamot et al. J Virol. 1999 Dec.

Abstract

Mus dunni endogenous virus (MDEV) is an apparently intact retrovirus that normally lies transcriptionally silent in cultured M. dunni cells, but the provirus can be activated by treatment of the cells with hydrocortisone or 5-iodo-2'-deoxyuridine. Sequence analysis of a molecular clone of the replicating virus revealed a simple retrovirus with a chimeric VL30/GALV-like structure. Interestingly, in the region of the long terminal repeat (LTR) that typically contains the retroviral transcription enhancers, we found over six 80-bp repeats with only a single mismatch, indicating that acquisition of the repeats was a recent event. Here we provide evidence for the following model of MDEV activation and replication. The MDEV provirus in M. dunni cells has a chimeric structure similar to that of the molecular clone but has only 1.15 copies of the 80-bp repeat sequence found in the molecular clone. Activating chemicals directly stimulate transcription from the LTR, allowing a low level of virus replication. Copying errors made during reverse transcription allow multimerization of the 80-bp enhancer region, resulting in viruses with higher transcriptional rates and improved fitness, but increased enhancer copy number is likely balanced by the natural instability of retroviral repeats and constraints imposed by virion packaging limits. The resultant population of replicating MDEV is widely heterogeneous, having from 2.15 to 13.15 enhancer repeats in the LTR. These results reveal a novel mechanism for regulation of transcription and replication of an endogenous retrovirus, in terms of both activation of the virus by the steroid hydrocortisone and the large number and variation in enhancer repeats observed.

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Figures

FIG. 1
FIG. 1
Elements with a chimeric VL30/GALV-like structure exist in the M. dunni genome. Samples of DNA from the indicated cell lines and from a pMDEV9 plasmid preparation were subjected to PCR using the primers shown, which flank the MDEV 3′ recombination breakpoint. The PCR products were subjected to Southern analysis using the indicated MfeI-BsaBI env fragment of pMDEV9 as a probe.
FIG. 2
FIG. 2
DNA sequences consistent with the presence of an MDEV provirus containing 1.15 LTR repeats in the M. dunni genome. Genomic DNA (10 μg) or pMDEV9 plasmid DNA (100 pg) was digested with XhoI or XhoI plus SalI and subjected to Southern analysis using the indicated SalI-XbaI fragment of pMDEV9 as a probe. Ethidium bromide staining of the gel prior to blotting indicated approximately equal loading of genomic DNA samples (data not shown). The two panels are from the same gel and blot, with irrelevant plasmid-containing central lanes removed.
FIG. 3
FIG. 3
MDEV populations have variable numbers of repeats. Genomic DNA samples (10 μg of each) from MDEV-infected (lanes 1 to 6) or MDEV-transfected (lanes 7 and 8) G355/LAPSN cells or pMDEV plasmid DNA (10 pg) (rightmost lane) were digested with AluI and subjected to Southern analysis using an 80-bp XbaI fragment cut from the pMDEV9 repeat region as a probe. The ethidium bromide stain of the gel prior to blotting indicated approximately equal loading of genomic DNA samples (not shown). See Results for descriptions of the samples analyzed.
FIG. 4
FIG. 4
The expanded MDEV LTR has greater promoter strength than the unexpanded LTR in G355 and dunni cells. Cells were seeded at 2 × 105 per 3.5-cm-diameter well of six-well plates on day 1. On day 2, the medium was replaced and 4 μg of test plasmid mixed with 1 μg of the β-Gal expression plasmid pEQ176 was introduced into the G355 cells by CaPO4 precipitation or into the dunni cells by using Lipofectamine (Gibco). The medium was replaced on day 3, and medium and cells were harvested separately on day 4 for SEAP and β-Gal assays. β-Gal activities were similar for all assays, indicating similar gene transfer efficiencies. Results shown are means from one experiment, with error bars indicating standard deviations. This experiment was repeated several times, and also with different culture media and substrate concentrations, with consistent results.
FIG. 5
FIG. 5
The MDEV LTR is responsive to HC and IdU in dunni cells. Each SEAP construct was mixed at a 20:1 ratio with the selectable plasmid pSV2neo and introduced into dunni cells by electroporation. Drug-resistant colonies were selected by using 0.7 mg of active G418 (Gibco) per ml, and at least 16 clones for each construct were combined to make a polyclonal population. For assay, cells were plated at 2 × 105 per 3.5-cm-diameter well of six-well dishes on day 1. On days 2 and 3, cells were fed with medium with or without 10−4 M HC (A) or medium with or without a saturating concentration of IdU (B). The saturated solution of IdU was made by incubating medium with an excess of IdU crystals at 37°C and filtering the medium to remove undissolved IdU. Solutions containing IdU and cells treated with IdU were kept in the dark, or under low light conditions when necessary for manipulation. On day 4, the medium samples were harvested and assayed for SEAP activity. Gene copy number was determined by Southern analysis using a SEAP cDNA probe followed by quantitation of band intensities by phosphorimaging. Band intensities were compared to that obtained from cells having a single integrated copy of a retrovirus vector containing the SEAP cDNA to determine copy number. SEAP activity is expressed as the amount of enzyme product (4-methylumbelliferone [MU]) produced per cell divided by the SEAP gene copy number per cell, or enzyme product per gene copy. Each experiment was performed in triplicate, and the data represent means ± standard deviations of from two to four experiments for each condition.
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