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

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.     

A short nucleotide sequence required for regulation of HIS4 by the general control system of yeast

We have made deletions of the HIS4 5' noncoding region in vitro and inserted these deletions into the yeast genome by transformation. Deletions that extend from -588 to -235 have no detectable effects on either promoter or regulatory functions. Deletions that extend to -138 affect promoter function, but are still regulated by the general control of amino acid biosynthesis. A deletion that extends to -136 cannot derepress HIS4 mRNA in response to the general control. This deletion removes all copies of the sequence 5'-TGACTC-3', which appears at positions -194, -182 and -138 in strains without the deletion. The importance of at least one copy of this repeat for regulation of HIS4 is shown by the reappearance of this sequence in revertants of the -136 deletion that have regained the regulatory response. The fact that deletion of this sequence leads to the inability to derepress suggests that HIS4 is under positive control.     

Selection of mouse neuroblastoma cell-specific polyoma virus mutants with stage differentiative advantages of replication.

Two mouse neuroblastoma cell lines were analyzed for their permissivity for polyoma virus growth. One (N18) is fully permissive for polyoma replication, the other (41A3) shows limited permissivity and the viral genome persists, without noticeable cell death. Virus persistence does not seem to alter the cells' ability to differentiate in vitro and leads to selection of viral mutants altered in the untranscribed regulatory region of the genome. The mutant types obtained appear to be related to the degree of host cell differentiation. Nucleotide sequence analysis of the restriction fragment covering the regulatory region shows that duplications are present in all mutants, while deletions in the non-duplicated segment are only present in mutants selected from less differentiated cells. These alterations involve both domains of the regulatory region that are considered to be essential for DNA replication and for enhancer activity. Mixed infections with polyoma wild type show that the selected mutants have cis-advantage in replication in neuroblastoma cells and not in 3T6 cells. Mutants carrying the deletion in the non-duplicated segment of the enhancer show a selective advantage in replication over the undeleted one in mixed infection. This advantage is much stronger in neuroblastoma cells in suspension (less-differentiated stage) than in monolayer cells (more-differentiated stage). An interpretation of the overall structure of the regulatory enhancer region, based on the observed differences between the mutants selected at different stages of differentiation in neuroblastoma and previously described mutants selected in undifferentiated cells, is discussed.     

Mapping genetic elements of Epstein-Barr virus that facilitate extrachromosomal persistence of Epstein-Barr virus-derived plasmids in human cells.

The Epstein-Barr virus (EBV) genome becomes established as a multicopy plasmid in the nucleus of infected B lymphocytes. A cis-acting DNA sequence previously described within the BamHI-C fragment of the EBV genome (J. Yates, N. Warren, D. Reisman, and B. Sugden, Proc. Natl. Acad. Sci. USA 81:3806-3810, 1984) allows stable extrachromosomal plasmid maintenance in latently infected cells, but not in EBV-negative cells. In agreement with the findings of Yates et al., deletion analysis permitted the assignment of this function to a 2,208-base-pair region (nucleotides 7315 to 9517 of the B95-8 strain of EBV) of the BamHI-C fragment that contained a striking repetitive sequence and an extended region of dyad symmetry. A recombinant vector, p410+, was constructed which carried the BamHI-K fragment (nucleotides 107565 to 112625 of the B95-8 strain, encoding the EBV-associated nuclear antigen EBNA-1), the cis-acting sequence from the BamHI-C fragment, and a dominant selectable marker gene encoding G-418 resistance in animal cells. After being transfected into HeLa cells, this plasmid persisted extrachromosomally at a low copy number, with no detectable rearrangements or deletions. Two mutations in the BamHI-K-derived portion of p410+, a large in-frame deletion and a linker insertion frameshift mutation, both of which alter the carboxy-terminal portion of EBNA-1, destroyed the ability of the plasmid to persist extrachromosomally in HeLa cells. A small in-frame deletion and linker insertion mutation in the region encoding the carboxy-terminal portion of EBNA-1, which replaced 19 amino acid codons with 2, had no effect on the maintenance of p410+ in HeLa cells. These observations indicate that EBNA-1, in combination with a cis-acting sequence in the BamHI-C fragment, is in part responsible for extrachromosomal EBV-derived plasmid maintenance in HeLa cells. Two additional activities have been localized to the BamHI-C DNA fragment: (i) a DNA sequence that could functionally substitute for the simian virus 40 enhancer and promoter elements controlling the expression of G-418 resistance and (ii) a DNA sequence which, although not sufficient to allow extrachromosomal plasmid maintenance, enhanced the frequency of transformation to G-418 resistance in EBV-positive (but not EBV-negative) cells. These findings suggest that the BamHI-C fragment contains a lymphoid-specific or EBV-inducible promoter or enhancer element or both.     

A splicing enhancer in the E4 coding region of human papillomavirus type 16 is required for early mRNA splicing and polyadenylation as well as inhibition of premature late gene expression

Successful inhibition of human papillomavirus type 16 (HPV-16) late gene expression early in the life cycle is essential for persistence of infection, the highest risk factor for cervical cancer. Our study aimed to locate regulatory RNA elements in the early region of HPV-16 that influence late gene expression. For this purpose, subgenomic HPV-16 expression plasmids under control of the strong human cytomegalovirus immediate early promoter were used. An exonic splicing enhancer that firmly supported the use of the E4 3' splice site at position 3358 in the early region of the HPV-16 genome was identified. The enhancer was mapped to a 65-nucleotide AC-rich sequence located approximately 100 nucleotides downstream of the position 3358 3' splice site. Deletion of the enhancer caused loss of both splicing at the upstream position 3358 3' splice site and polyadenylation at the early polyadenylation signal, pAE. Direct splicing occurred at the competing L1 3' splice site at position 5639 in the late region. Optimization of the position 3358 3' splice site restored splicing to that site and polyadenylation at pAE. Additionally, a sequence of 40 nucleotides with a negative effect on late mRNA production was located immediately downstream of the enhancer. As the E4 3' splice site is employed by both early and late mRNAs, the enhancer constitutes a key regulator of temporal HPV-16 gene expression, which is required for early mRNA production as well as for the inhibition of premature late gene expression.     

A polyoma-based episomal vector efficiently expresses exogenous genes in mouse embryonic stem cells.

We describe the ability of novel episomally maintained vectors to efficiently promote gene expression in embryonic stem (ES) cells as well as in established mouse cell lines. Extrachromosomal maintenance of our vectors is based on the presence of polyoma virus DNA sequences, including the origin of replication harboring a mutant enhancer (PyF101), and a modified version of the polyoma early region (LT20) encoding the large T antigen only. Reporter gene expression from such extrachromosomally replicating vectors was approximately 10-fold higher than expression from replication-incompetent control plasmids. After transfection of different ES cell lines, the polyoma virus-derived plasmid variant pMGD20neo (7.2 kb) was maintained episomally in 16% of the G418-resistant clones. No chromosomal integration of pMGD20neo vector DNA was detected in ES cells that contained episomal vector DNA even after long term passage. The vector's replication ability was not altered after insertion of up to 10 kb hprt gene fragments. Besides undifferentiated ES cells, the polyoma-based vectors were also maintained extrachromosomally in differentiating ES cells and embryoid bodies as well as in established mouse cell lines.