Structural-functional organization of the SV40 virus chromosome. II. Characteristics of viral genome containing deletion in the regulatory region

Structure of SV40 wild type virus genome differing from the well known 776 strain has been characterized. This particular strain (SV40) was used previously for viral chromatin analysis. We show here that SV40 strain differs from 776 strain by deletion in the beginning of the "late" region (enhancer). Restriction nucleases mapping and nucleotide sequencing through this region reveal the absence of one full-length copy of 72 bp repeat.     

Minimal transcriptional enhancer of simian virus 40 is a 74-base-pair sequence that has interacting domains.

We have assayed the ability of segments of the simian virus 40 (SV40) 72-base-pair (bp) repeat enhancer region to activate gene expression under the control of the SV40 early promoter and to compete for trans-acting enhancer-binding factors of limited availability in vivo in monkey CV-1 or human HeLa cells. The bacterial chloramphenicol acetyltransferase and the herpes simplex virus type 1 thymidine kinase genes were used as reporters in these assays. A 94-bp sequence located between SV40 nucleotides 179 and 272, including one copy of the 72-bp repeat, has been termed the minimal enhancer in previous studies. In the present study, we found that the 20-bp origin-proximal region located between nucleotides 179 and 198 was dispensable, since its removal caused only a slight reduction in enhancer activity. However, the deletion of another 4 bp up to nucleotide 202 abolished the enhancer activity. We propose that the minimal enhancer is a 74-bp sequence located between nucleotides 199 and 272, including 52 bp of one copy of the 72-bp repeat and a 22-bp adjacent sequence up to the PvuII site at 272. The nonamer 5'-AAGT/CATGCA-3', which we term the K core, occurred as a tandem duplication around the SphI site at nucleotide 200, and we found that this duplication was essential for enhancement and factor-binding activities. A heterologous core element (which we term the C core), 5'-GTGGA/TA/TA/TG-3', identified earlier (G. Khoury and P. Gruss, Cell 33:313-314, 1983; Weiher et al., Science 219:626-631, 1983) also occurred in duplicate, with one of the copies located within the 22-bp sequence near nucleotide 272 present outside the 72-bp repeat. We provide direct evidence that this 22-bp sequence augments enhancer activity considerably. We also found that in addition to the heterologous interaction occurring normally between the K and C cores within the minimal enhancer, certain homologous interactions were also permitted provided there was proper spacing between the elements.     

The SV40 72 base repair repeat has a striking effect on gene expression both in SV40 and other chimeric recombinants.

By introduction of recombinant plasmids into monkey CV1 cells, we have unambiguously demonstrated that sequences entirely within the 72 bp repeat, which is located upstream of the SV40 early region, are crucial for T-antigen expression in vivo. We have also shown that a DNA fragment containing the 72 bp repeat, inserted directly before chicken conalbumin or adenovirus-2 major late promoter sequences in chimeric plasmids where these promoters replace that of the SV40 early genes, caused a dramatic increase in the expression of T-antigen in vivo. This effect was independent of the orientation of the 72 bp repeat, but was sensitive to its location within the plasmid, when the 72 bp repeat was separated from the promoter sequences, T-antigen expression was reduced. Insertion of the 72 bp repeat into equivalent plasmids containing no known eukaryotic promoter sequences (plasmids which were not detectably expressed in vivo) gave rise to a measurable, but smaller level of expression. The stimulation of expression by the 72 bp repeat is cis-acting : it required covalent linkage to the recombinant. We discuss the possibility that the 72 bp repeat region in SV40 may act as a bi-directional entry site for RNA polymerase B such that promoter sequences linked to the repeat are more efficiently utilised.     

Specific interaction of cellular factors with the B enhancer of polyoma virus.

Specific interactions between proteins from mouse 3T6 cells and the enhancer sequence of polyoma virus were detected using the method of band shifting on polyacrylamide gels. Proteins eluted from 3T6 nuclei using a buffer containing 0.55 M NaCl, formed a stable complex with the B enhancer of polyoma virus. At least two different factors are involved in this interaction. The contact sites which were mapped on the DNA sequence using DNase I footprinting correspond to a GC-rich palindrome surrounded by two sequences homologous respectively to the immunoglobulin and to the immunoglobulin and SV40 enhancers. Moreover Bal31 deletion analysis confirmed that similar sequences are required for the formation of the complex. In spite of a common function and partial sequence homology among some enhancers, neither the polyoma A enhancer, the mouse immunoglobulin heavy chain gene enhancer, nor the origin-promoter-enhancer region of SV40 efficiently competed with the polyoma B enhancer for the binding of these molecules.     

Simian virus 40 DNA replication: functional organization of regulatory elements.

The efficiency of simian virus 40 (SV40) DNA replication is dependent on the structural organization of the regulatory region. The enhancing effect of the G + C-rich 21-base-pair (bp) repeats on SV40 DNA replication is position and dose dependent and to some extent orientation dependent. The inverted orientation is about 50% as effective as the normal orientation of the 21-bp repeat region. Movement of the 21-bp repeat region 180 or 370 bp upstream of the ori sequence abolishes its enhancing effect, whereas no replication is detected if the 21-bp repeat region is placed downstream of the ori sequence. The dose-dependent enhancement of the 21-bp repeat of SV40 DNA replication as first described in single transfection by Bergsma et al. (D. J. Bergsma, D. M. Olive, S. W. Hartzell, and K. N. Subramanian, Proc. Natl. Acad. Sci. USA 79:381-385, 1982) is dramatically amplified in mixed transfection. In the presence of the 21-bp repeat region, the 72-bp repeat region can enhance SV40 DNA replication. In the presence of the 21-bp repeats and a competitive environment, the 72-bp repeat region exhibits a cis-acting inhibitory effect on SV40 DNA replication.     

Nucleotide sequence analysis and enhancer function of long terminal repeats associated with an endogenous African green monkey retroviral DNA.

The nucleotide sequence and enhancer activity of the long terminal repeats (LTRs) associated with a cloned endogenous African green monkey (AGM) retroviral DNA designated as lambda-AGM-1 was studied. A unique feature of the endogenous AGM proviral LTRs was the presence of multiple copies of two types of directly repeating units in the U3 region: 16 8-base-pair (bp) repeats were present in the 5' LTR and 12 were present in the 3' LTR which were bound by a 6-bp perfect direct repeat; tandem duplication of a 32-bp sequence resulted in 3.5 copies in the 5' LTR and 2.5 copies in the 3' LTR. Nucleotide sequence homology was seen between the 8-bp direct repeats located in the AGM proviral LTRs and a 10-bp repeat unit of the deca-satellite present in AGM cellular DNA. The 32-bp repeats of the AGM proviral LTRs contained sequences which were related to the SV40 21-bp repeats and to the "core" of the SV40 72-bp enhancer element. Furthermore, the AGM provirus was distinct from known infectious retroviruses due to the presence of a primer-binding sequence complementary to the 3' terminus of mammalian tRNAGly. Functional analysis of the 3' LTR present in lambda-AGM-1 DNA by chloramphenicol acetyltransferase assay demonstrated enhancer activity associated with the 32-bp direct repeats. Sequences outside the 32-bp unit were necessary for full activator function, suggesting the presence of multiple enhancer domains in the AGM provirus.     

Pausing in simian virus 40 DNA replication by a sequence containing (dG-dA)27.(dT-dC)27.

A 200 bp sequence including a stretch of 54 base pairs of alternating guanosine and adenosine nucleotide residues [(dG-dA)27.(dT-dC)27] was cloned in the simian virus 40 (SV40) genome between the KpnI and HpaII sites. This sequence was discovered earlier as part of a region limiting the amplification of sequences adjacent to an integrated polyoma virus in a transformed rat cell line. The newly constructed DNA was transfected into African Green monkey kidney CV1 cells and the variant virus was isolated by plaque-purification. The insertion was stably maintained and the variant virus grew more slowly than the wild type, had lower titers and gave smaller plaques. In mixed infection experiments, the variant was found to be stable, though the wild type replicated more rapidly. Pulse labeling experiments indicated that the unusual inserted sequence acts as a pause site for fork progression during DNA replication, as evidenced by the rate of incorporation of radioactively labeled nucleotides into various regions of the SV40 genome. Statistical fit of the experimental curves with theoretically generated curves suggested the pause of fork progression to be about one minute.     

Transient expression of murine interferon-alpha genes in mouse and monkey cells

The coding regions of murine interferon-alpha (IFN-alpha) genes were combined with promoter and 3'-noncoding sequences from other eukaryotic genes. Transient expression of these fusion genes was achieved in monkey COS cells and in a mouse cell line (TOP cells) expressing polyoma virus (Py) large T antigen constitutively. The efficiency of the different expression plasmids was determined by measuring the amount of IFN secreted into the medium. Replacement of the 3'-noncoding region of an IFN-alpha gene by that of the rabbit beta-globin gene resulted in a fourfold higher IFN-alpha production. The SV40 early promoter and the Moloney murine leukemia virus (MoMLV) long terminal repeat (LTR) produced similar amounts of IFN-alpha in COS cells. However, a tandem combination of the SV40 enhancer/early promoter and the mouse metallothionein-I promoter appeared fivefold more active than the SV40 early promoter. In TOP cells the MoMLV LTR was found to be threefold more active than the Py early promoter.     

Specific interaction between enhancer-containing molecules and cellular components

Using an in vivo assay, we have obtained competition between several types of enhancer-containing molecules for cellular components that interact with them. The presence of these cellular factors is required for enhancer function. Specific competition involved enhancers and not other SV40 promoter elements such as the 21 bp repeats or TATA box. Point mutants within the 72 bp repeat of SV40 that were defective in enhancer function were unable to compete for the cellular components in the competition assay. Although heterologous enhancers compete in several types of cells for the same set of cellular molecules, the host cell preference of different enhancers is reflected in the competition assay. This result might explain the previously described host cell preference of enhancer elements.     

Assembly of transfected DNA into chromatin: structural changes in the origin-promoter-enhancer region upon replication

Chimeric SV40 DNA containing only the early region, or plasmid DNA harboring the origin-promoter-enhancer region of SV40, when introduced into CV-1 or Cos-1 monkey cells by DEAE-dextran mediated transfer are rapidly assembled in a typical chromatin structure revealed by the generation of a regular 190 bp repeat ladder after micrococcal nuclease digestion. DNA replication is not required for this assembly process. Chromatin-specific DNase I hypersensitive sites are observed in the enhancer region of these minichromosomes. The pattern of the sites differs between non-replicating and post-replicated chromatin. The latter is identical to that observed in the lytic cycle. The presence of large T antigen is not sufficient for the shift in the structure of the chromatin. These experiments suggest that replication can modulate protein-DNA interactions during viral infection or upon cell differentiation.