Cellular mobile genetic elements in the regulatory region of the pneumotropic mouse polyomavirus genome: structure and function in viral gene expression and DNA replication

Abstract

DNA from the murine pneumotropic virus was extracted from virus in lung tissue of infected mice, and the regulatory region of the genome was amplified by PCR. The regulatory region of individual plasmid cloned DNA molecules appeared to have heterogeneous enhancer segments, whereas the protein-coding part of the genome had a uniform length. Nucleotide sequence analysis revealed that the majority of the DNA molecules had a structure differing from the standard type. A 220-bp insertion at nucleotide position 142 with a concomitant deletion of nucleotides 143 to 148 was prominent. There were two variants of the 220-bp insertion, differing at two nucleotide positions at one of the termini. Other DNA molecules had complete or partial deletions of these structures and surrounding sequences in the viral enhancer. However, the end of the insertion at nucleotide 142 was frequently preserved. The viral early and late promoter activity of the variant regulatory regions was tested in a luciferase reporter assay by using transfected NIH 3T3 cells. In relation to the standard-type DNA, all variants, including a G272T mutant, had much stronger late promoters. In contrast, the early promoter activity was influenced in a positive or negative direction by individual mutations. Also, the activity of the viral origin of DNA replication was affected by the sequence variation of the regulatory region, although the effects were smaller than for the late promoter. Analysis by Southern blotting and quantification using dot blots showed that approximately 10(3) copies of material related to the 220-bp insert in murine pneumotropic virus DNA was present in mouse and human DNA but not in Escherichia coli DNA. Moreover, analysis by PCR indicated that there were multiple copies in the mouse genome of sequences that were identical or closely related to the 220-bp viral DNA segment. These data together with the nucleotide sequence analysis suggest that the 220-bp insertion is related to a transposable element of a novel type.

FIG. 1.

Analysis of MPtV genome structure. (A) The regulatory region of MPtV DNA extracted from a crude virus preparation (VS) was amplified by PCR by using primers complementary to the region immediately adjacent to protein coding sequences. As a control DNA from the plasmid pstMPtV (VP) was used. The amplification products were separated by agarose gel electrophoresis. (B) Southern blot analysis of XbaI-digested DNA from lung extracts (VS) and from pstMPtV (VP). Annealing was done with ³²P-labeled MPtV DNA isolated from the recombinant plasmid pstMPtV, which was used as a probe. The mobility of DNA size markers is indicated to the left. kb, kilobase pairs.

FIG. 2.

Organization of the stMPtV regulatory region and the structure of variant genomes isolated from MPtV in mouse lung tissue. The regulatory region of the viral genome was amplified by PCR and ligated to pGL2-basic DNA. Recombinant plasmids were cloned, and the nucleotide sequence of the regulatory region was determined. The data were related to the published nucleotide sequence of MPtV DNA (31), here called standard type (stMPtV). (A) Short arrows indicate large T antigen recognition pentanucleotides (GPuGGC) in the sense of the early (E→) and late (←L) DNA strands, respectively. The hatched box shows the position of the putative viral replication origin. Abbreviations: inA and inB, insertion type A and type B, respectively (numbers refer to the nucleotide positions of the 220-bp segments); dl, deletion; dp, duplication; G272T, G-to-T base change at nt 272. (B) Predicted recognition sites of DNA-binding transcription factors (40, 47).

FIG. 3.

Southern blot analysis of MPtV insert-related sequences in cellular DNA. High-molecular-weight DNA was purified from human HeLa cells (lane 1); from mouse NIH 3T3 (lane 2), UAE (lane 4), and FM3A cells (lane 5); and from E. coli JM109 cells (lane 3). (A) Two micrograms of the DNA preparations was subjected to agarose gel electrophoresis and then stained with ethidium bromide. (B) Ten micrograms of the DNA preparations was digested with the restriction endonuclease BglII. The resulting fragments were resolved by agarose gel electrophoresis and then stained with ethidium bromide. (C) The resolved DNA fragments were subjected to Southern blot analysis with ³²P-labeled DNA of the 220-bp MPtV insert as a probe. As a positive control, 5 ng of pstMPtV linearized by BglII cleavage was used (+). The positions of size markers (in kilobase pairs) are shown on the right.

FIG. 4.

Estimation of copy number of MPtV insert-related material in genomic DNA. The same DNA preparations and radioactive probe were used as in the experiment described in the Fig. 3 legend. Radioactivity annealing to dot blotted material was quantified in a PhosphorImager. Copy numbers were estimated in relation to the signals obtained with purified pstMPtV DNA.

FIG. 5.

Agarose gel electrophoresis of PCR products from amplification of MPtV-related sequences in mouse DNA. DNA was extracted from the spleen of a C3H mouse and then purified. PCR amplification was done for 35 cycles in the presence or absence (−) of template DNA by using a primer pair that would generate a 179-bp product with the inA or inB insert of MPtV DNA as a template. After amplification, DNA was resolved by electrophoresis in a 1.5% agarose gel in the presence of 0.5 μg of ethidium bromide per ml. DNA was visualized in UV light. The final concentration of MgCl₂ in the PCR is indicated. The electrophoretic mobility of size markers is displayed to the left.

FIG. 6.

Nucleotide sequences of putative transposon in MPtV DNA and the integration site. The complete nucleotide sequence of the 220-bp inA insertion in the MPtV regulatory region is shown in panel A. The integration site in stMPtV, as well as the junctions between standard-type DNA and selected insertions, is shown in panel B. In the standard-type nucleotide sequence, thin vertical lines indicate the insertion site and dashed horizontal lines indicate a mirror symmetry of the pentanucleotide TGAATA. In the MPtV variant sequences, inserted segments are shaded and the TGAATA deletion is indicated. In inB DNA, the arrows show terminal direct repeats.