Functional analysis of the role of the A + T-rich region and upstream flanking sequences in simian virus 40 DNA replication.

One boundary of the minimal origin of replication of simian virus 40 DNA lies within the A + T-rich region. Deletion of only a few bases into the adenine-thymine (AT) stretch results in a DNA template which is defective for replication both in vivo and in vitro (B. Stillman, R. D. Gerard, R. A. Guggenheimer, and Y. Gluzman, EMBO J. 4:2933-2939, 1985). In the present study, such deletion mutations have been reconstructed into a simian virus 40 genome containing an intact early promoter-enhancer region. The resulting mutants synthesized wild-type levels of T antigen, but were defective for replication and would not form plaques on CV-1 monkey cells. Replication-competent phenotypic revertants were selected after transfection of large quantities of the replication-defective viral DNAs into CV-1 cells. DNA sequence analysis showed that most of these revertants contained insertions or point mutations which partially regenerate the length of the AT stretch. These genotypic alterations were shown to be responsible for the revertant phenotype by replication analysis in vivo of subcloned revertant origin fragments. In general, our results emphasize the importance of the AT region to simian virus 40 origin function. However, one revertant retained the altered AT region but deleted six nucleotides upstream. Experiments using this mutant indicate that the 21-base-pair repeats identified as part of the early transcriptional promoter may compensate for defects in simian virus 40 DNA replication in vivo caused by mutations in the A + T-rich region when positioned at an appropriate distance from the core origin.

Rescue of functional replication origins from embedded configurations in a plasmid carrying the adenovirus genome.

A variant of the adenovirus type 5 genome which lacks EcoRI sites has been cloned in a bacterial plasmid after the addition of EcoRI oligonucleotide linkers to its ends. Closed circular forms of the recombinant viral genome were not infectious upon their introduction into permissive eucaryotic cells. The linear genome released by digestion of the 39-kilobase recombinant plasmid (pXAd) with EcoRI produced infectious virus at about 5% of the level of wild-type controls. The viruses which arose were indistinguishable from the parental strain, and the normal termini of the viral genome had been restored. Marker rescue experiments demonstrate that provision of a DNA fragment with a normal viral end improves infectivity. When a small fragment carrying a wild-type left end (the 0 to 2.6% ClaI-B fragment) was ligated to ClaI-linearized pXAd, virus was produced with efficiencies comparable to a similar reconstitution of the two ClaI fragments of the wild-type genome. These viruses stably carry the left-end fragment at both ends, leaving the normal right end embedded in 950 base pairs of DNA. The embedded right origin is inactive. The consensus of the analyses reported here is that a free end is a necessary configuration for the sequences which make up the adenovirus origin of replication.

Construction of a helper-free recombinant adenovirus that expresses polyomavirus large T antigen.

Adenovirus-polyomavirus recombinant viruses were constructed in vitro by inserting a hybrid transcription unit composed of the adenovirus type 2 major late promoter and the early coding region of polyomavirus into the adenovirus type 5 vector Ad5 delta E1/dl309. The vector lacks the E1a and E1b transcription units and contains a unique restriction endonuclease cleavage site in their place. The polyomavirus genomic insert contained a small deletion which precluded the synthesis of functional small and middle T antigen but allowed for the synthesis of large T antigen. One recombinant virus, Ad5PyR39, which contained the hybrid transcription unit in the opposite transcriptional orientation from the overall direction of late-gene transcription, was studied in detail. Ad5PyR39 replicated efficiently without a helper virus in human 293 cells and expressed hybrid mRNAs of the expected size and composition that were translated to yield large T antigen. The large T antigen synthesized in 293 cells was the same size as that produced in mouse 3T6 cells lytically infected with polyomavirus, and this protein bound efficiently and specifically to the large-T-antigen-binding sites in polyomavirus DNA. Moreover, the large T antigen encoded by the recombinant virus proved capable of catalyzing the replication in mouse 3T6 cells of a plasmid containing the polyomavirus origin for DNA replication. Comparison of the amount of large T antigen produced in 3T6 cells infected with polyomavirus with that in 293 cells infected with Ad5PyR39, under optimal conditions for each system, revealed at least a fivefold greater yield of the protein on a per cell basis in the latter system compared with the former. Ad5PyR39 should prove to be useful to isolate large quantities of functional polyomavirus large T antigen for structural and biochemical studies.

Properties of the DNA-binding domain of the simian virus 40 large T antigen.

T antigen (Tag) from simian virus 40 binds specifically to two distinct sites in the viral origin of replication and to single-stranded DNA. Analysis of the protein domain responsible for these activities revealed the following. (i) The C-terminal boundary of the origin-specific and single-strand-specific DNA-binding domain is at or near amino acid 246; furthermore, the maximum of these DNA-binding activities coincides with a narrow C-terminal boundary, spanning 4 amino acids (246 to 249) and declines sharply in proteins with C termini which differ by a few (4 to 10) amino acids; (ii) a polypeptide spanning residues 132 to 246 of Tag is an independent domain responsible for origin-specific DNA binding and presumably for single-stranded DNA binding; and (iii) a comparison of identical N-terminal fragments of Tag purified from mammalian and bacterial cells revealed differential specificity and levels of activity between the two sources of protein. A role for posttranslational modification (phosphorylation) in controlling the DNA-binding activity of Tag is discussed.

Efficient transformation of human fibroblasts by adenovirus-simian virus 40 recombinants.

The origin-defective simian virus 40 (SV40) mutant 6-1 has been useful in transforming human cells (Small et al., Nature [London] 296:671-672, 1982; Nagata et al., Nature [London] 306:597-599, 1983). However, the low efficiency of transformation achieved by DNA transfection is a major drawback of the system. To increase the efficiency of SV40-induced transformation of human fibroblasts, we used recombinant adenovirus-SV40 virions which contain a complete SV40 early region including either a wild-type or defective (6-1) origin of replication. The SV40 DNA was cloned into the adenovirus vector in place of early region 1. Cell lines transformed by viruses containing a functional origin of replication produced free SV40 DNA. These cell lines were subcloned, and some of the subclones lost the ability to produce free viral DNA. Subclones that failed to produce free viral DNA were found to possess a mutated T antigen. Cell lines transformed by viruses containing origin-defective SV40 mutants did not produce any free DNA. Because of the high efficiency of transformation, we suggest that the origin-defective chimeric virus is a convenient system for establishing SV40-transformed cell lines from any human cell type that is susceptible to infection by adenovirus type 5.

New host cell system for regulated simian virus 40 DNA replication.

Transformed monkey cell lines (CMT and BMT) that inducible express simian virus 40 (SV40) T antigen from the metallothionein promoter have been isolated and characterized. Immunoprecipitation of pulse-labeled T antigen demonstrates a 5- to 12-fold increase in the rate of synthesis on addition of heavy-metal inducers to the culture medium. Radioimmunoassay of cell extracts indicates the accumulation of three- to fourfold more total T antigen after 2 days of induction by comparison with uninduced controls. A direct correlation was found between the level of T-antigen synthesis and the extent of SV40 DNA replication in inducible cells. Inducible BMT cells expressing a low basal level of T antigen were efficiently transformed by a vector carrying the neomycin resistance marker and an SV40 origin of replication. These vector sequences were maintained in an episomal form in most G418-resistant cell lines examined and persisted even in the absence of biochemical selection. Extensive rearrangements were observed only if the vector contained bacterial plasmid sequences. Expression of a protein product under the control of the SV40 late promoter in such vectors was increased after heavy-metal-dependent amplification of the template. These results demonstrate the ability of BMT cells to maintain a cloned eucaryotic gene in an amplifiable episomal state.

Replication and supercoiling of simian virus 40 DNA in cell extracts from human cells.

Soluble extracts prepared from the nucleus and cytoplasm of human 293 cells are capable of efficient replication and supercoiling of added DNA templates that contain the origin of simian virus 40 replication. Extracts prepared from human HeLa cells are less active than similarly prepared extracts from 293 cells for initiation and elongation of nascent DNA strands. DNA synthesis is dependent on addition of purified simian virus 40 tumor (T) antigen, which is isolated by immunoaffinity chromatography of extracts from cells infected with an adenovirus modified to produce large quantities of this protein. In the presence of T antigen and the cytoplasmic extract, replication initiates at the origin and continues bidirectionally. Initiation is completely dependent on functional origin sequences; a plasmid DNA containing an origin mutation known to affect DNA replication in vivo fails to replicate in vitro. Multiple rounds of DNA synthesis occur, as shown by the appearance of heavy-heavy, bromodeoxyuridine-labeled DNA products. The products of this reaction are resolved, but are relaxed, covalently closed DNA circles. Addition of a nuclear extract during DNA synthesis promotes the negative supercoiling of the replicated DNA molecules.

Simian virus 40 large T-antigen point mutants that are defective in viral DNA replication but competent in oncogenic transformation.

The large T antigen of simian virus 40 (SV40) is a multifunctional protein that is essential in both the virus lytic cycle and the oncogenic transformation of cells by SV40. To investigate the role of the numerous biochemical and physiological activities of T antigen in the lytic and transformation processes, we have studied DNA replication-deficient, transformation-competent large T-antigen mutants. Here we describe the genetic and biochemical analyses of two such mutants, C2/SV40 and C11/SV40. The mutants were isolated by rescuing the integrated SV40 DNA from C2 and C11 cells (CV-1 cell lines transformed with UV-irradiated SV40). The mutant viral early regions were cloned into the plasmid vector pK1 to generate pC2 and pC11. The mutations that are responsible for the deficiency in viral DNA replication were localized by marker rescue. Subsequent DNA sequencing revealed point mutations that predict amino acid substitutions in the carboxyl third of the protein in both mutants. The pC2 mutation predicts the change of Lys----Arg at amino acid 516. pC11 has two mutations, one predicting a change of Pro----Ser at residue 522, and another predicting a Pro----Arg change at amino acid 549. The two C11 mutations were separated from each other to form two distinct viral genomes in pC11A and pC11B. pC2, pC11, pC11A, and pC11B are able to transform both primary and established rodent cell cultures. The C11 and C11A T antigens are defective in ATPase activity, suggesting that wild-type levels of ATPase activity are not necessary for the oncogenic transformation of cells by T antigen.

DNA-binding properties of simian virus 40 T-antigen mutants defective in viral DNA replication.

Three simian virus 40 (SV40)-transformed monkey cell lines, C2, C6, and C11, producing T-antigen variants that are unable to initiate viral DNA replication, were analyzed with respect to their affinity for regulatory sequences at the viral origin of replication. C2 and C11 T antigens both bound specifically to sequences at sites 1 and 2 at the viral origin region, whereas C6 T antigen showed no specific affinity for any viral DNA sequences under all conditions tested. Viral DNA sequences encoding the C6 T antigen have recently been cloned out of C6 cells and used to transform an established rat cell line. T antigen from several cloned C6-SV40-transformed rat lines failed to bind specifically to the origin. C6 DNA contains three mutations: two located close to the amino terminus of T antigen at amino acid positions 30 and 51 and a third located internally at amino acid position 153. Two recombinant SV40 DNA mutants were prepared containing either the amino-terminal mutations at positions 30 and 51 (C6-1) or the internally located mutation at position 153 (C6-2) and used to transform Rat 2 cells. Whereas T antigen from C6-2-transformed cells lacked any specific affinity for these sequences. Therefore, the single mutation at amino acid position 153 (Asn leads to Thr) is sufficient to abolish the origin-binding property of T antigen. A T antigen-specific monoclonal antibody, PAb 100, which had been previously shown to immunoprecipitate an immunologically distinct origin-binding subclass of T antigen, recognized wild-type or C6-1 antigens, but failed to react with C6 or C6-2 T antigens. These results indicate that viral replication function comprises properties of T antigen that exist in addition to its ability to bind specifically to the SV40 regulatory sequences. Furthermore, it is concluded from these data that specific viral origin binding is not a necessary feature of the transforming function of T antigen.

Preliminary X-ray crystallographic analysis of a modified basic fibroblast growth factor.

Human basic fibroblast growth factor (hbFGF) has been modified, with Ala3 and Ser5 substituted by glutamic acid, and the purified recombinant protein has been crystallized. The crystals are triclinic (space group P1) with unit cell parameters a = 31.0 A, b = 33.6 A, c = 34.7 A, alpha = 88 degrees, beta = 85 degrees, gamma = 76 degrees, and they diffract to at least 2 A.

Immortalization of virus-free human placental cells that express tissue-specific functions.

Human pregnancy-specific glycoproteins (PSGs) are a family of closely related placental proteins that, together with the carcinoembryonic antigen members, comprise a subfamily within the immunoglobulin superfamily. To facilitate study of the control of PSG expression, we immortalized human placental cell lines with adenovirus-origin-minus (ori-)-simian virus-40 (SV40) recombinant viruses containing either wild-type or temperature-sensitive (ts) A mutants of SV40. Cells transformed with the SV40 tsA chimera (HP-A1 and HP-A2), but not the SV40 wild-type chimera (HP-W1), were temperature sensitive for transformation. All three cell lines expressed trophoblast-specific genes, including PSG and the alpha- and beta-subunits of hCG. Human CG alpha expression was greatly stimulated by (Bu)2cAMP in all three cell lines; shifting HP-A1 and HP-A2 cells to the nonpermissive temperature (39.5 C) further increased hCG alpha expression. At both 33 C (permissive temperature) and 39.5 C, the transformed placental cells expressed PSG mRNAs of 2.2 and 1.7 kilobases; expression was greatly stimulated by sodium butyrate. In the absence of an inducer, the three placental lines synthesized a PSG of 64 kilodaltons (kDa). In the presence of butyrate, they synthesized PSGs of 72, 64, and 54 kDa, similar to the placental PSGs. However, in placenta the predominant species is the 72-kDa product. At 39.5 C, butyrate selectively increased synthesis of the 72-kDa PSG in HP-A1 and HP-A2 cells. To characterize PSG promoter activity, we constructed chloramphenicol acetyltransferase (CAT) fusion genes containing -809 to -44 basepairs up-stream of the translational start site of the PSG6 gene. Using transient expression assays, we demonstrated that the -809/-44 region of the PSG6 gene contained cis-acting sequences that can direct CAT expression in human placental cells. Sodium butyrate, which stimulates PSG expression, greatly increased CAT activity, indicating that butyrate-induced PSG expression is regulated primarily at the level of gene transcription.

Preferential integration of the Ad5/SV40 hybrid virus at the highly recombinogenic human chromosomal site 1p36.

Human fibroblasts transformed with an adenovirus-5/simian virus 40 recombinant construct (Ad5/SV40) were analyzed to determine the chromosomal site(s) of virus integration. This was firstly done by in situ hybridization using metaphase and prometaphase chromosomes and 125I-labeled Ad5 DNA. Out of seven transformed cell lines (six of clonal origin and one uncloned), six were proven to have integrated the viral genome at the short- or the long-subtelomeric regions of autosome 1, two regions known to include chromosomal modification sites induced by acute infection with Ad12. Characterization of the integration sites was carried out by restriction analysis. Transformed cell lines with the same major chromosomal integration site were found to have the viral genome inserted in restriction fragments of different size, indicating that viral integration has occurred at different sites within a relatively small chromosomal region. Molecular studies carried out on one of the transformed cell lines (H13.1) gave an independent confirmation of the viral integration at the subterminal region of autosome 1 short arm. Nucleotide sequencing at this cellular-viral junction has shown that the virus has integrated within tandemly repeated Alu-like elements and that the cellular flanking sequences have several homologies with variable number of tandem repeats core sequences. Many possible open reading frames were identified in the DNA segment adjacent to the Alu-like elements.

Glycylcyclines. 1. A new generation of potent antibacterial agents through modification of 9-aminotetracyclines.

This report describes the discovery of a new generation of tetracycline antibacterial agents, the "glycylcyclines". These agents are notable for their activity against a broad spectrum of tetracycline-susceptible and -resistant Gram-negative and Gram-positive aerobic and anaerobic bacteria possessing various classes of tetracycline-resistant determinants [tet B (efflux), tet M (ribosomal protection)]. The design and synthesis of a number of 7-substituted 9-substituted-amido 6-demethyl-6-deoxytetracyclines are described.

Structure/activity relationships in basic FGF.

Although the FGFs have been subject to extensive biological studies, only limited progress has been made so far in determining the critical elements of structure-activity relationships in the FGFs. Among the recognized structural elements with potential to affect the biological activity of FGFs are the cysteine residues, and the heparin- and receptor-binding domains. These features have been studied using a variety of experimental approaches, but the available data are inconclusive. For example, ambiguity regarding the presence of a disulfide structure in FGFs was not resolved until the availability of x-ray crystal structure data. Furthermore, the functionally important heparin- and receptor-binding domains have been poorly characterized, with some interpretations being controversial. In this report, we describe a novel fragment of basic FGF (bFGF) with high biological activity [Ser78,96-bFGF(70-153)]. This fragment was generated by pronase treatment of heparin-bound recombinant Glu3,5Ser78,96-bFGF mutant and is active in vitro at an ED50 of about 100 ng/ml. The structure of the fragment and the manner by which it was generated provide additional insight into important aspects of structure-activity relationships in FGFs. Specifically, we conclude that (a) the cysteines in our bFGF mutant do not form a disulfide bond, (b) the high-affinity heparin binding of bFGF critically depends on an intact 3-dimensional structure of the growth factor rather than on specific heparin-binding sequence domains, and (c) the bFGF sequence between residues 70 and 122 is important for high biological activity.

[Inhibitory analysis of DNA polymerase of herpes simplex type 1]. / Ingibitornyi analiz DNK-polimerazy virusa gerpesa prostogo tipa 1.

The inhibitory potency of new analogs of nucleoside 5'-triphosphates modified at the sugar residue and or alpha-phosphate against herpes simplex virus type 1 DNA polymerase has been evaluated in a cell-free system containing M13mp10 phage DNA and a synthetic primer. Triphosphates of new acyclic nucleosides [1-(5-hydroxy-2-cis-pentenyl)nucleosides] were the most effective inhibitors among 15 types of nucleoside 5'-triphosphates under investigation, being threefold less active than acyclovirtriphosphate. 5'-Phosphonylmethyl-2'-deoxythymidine beta, gamma-diphosphate proved to be a poor substrate for DNA polymerase. Compounds with other modifications at alpha-phosphate were inactive. Constants of hydrolysis rate of acyclonucleosides incorporated into the 3' end of primer were determined.

SV40-transformed simian cells support the replication of early SV40 mutants.

CV-1, an established line of simian cells permissive for lytic growth of SV40, were transformed by an origin-defective mutant of SV40 which codes for wild-type T antigen. Three transformed lines (COS-1, -3, -7) were established and found to contain T antigen; retain complete permissiveness for lytic growth of SV40; support the replication of tsA209 virus at 40 degrees C; and support the replication of pure populations of SV40 mutants with deletions in the early region. One of the lines (COS-1) contains a single integrated copy of the complete early region of SV40 DNA. These cells are possible hosts for the propagation of pure populations of recombinant SV40 viruses.

Duplications of a mutated simian virus 40 enhancer restore its activity.

Enhancers are cis-acting control elements which can stimulate at a distance the activity of a variety of eukaryotic promoters. First identified as a repeated 72 base pair (bp) sequence upstream of the simian virus 40 (SV40) early gene promoter, enhancers have since been shown to be associated with numerous other viral and cellular genes. Although there are no strong homologies between the sequences of different enhancers, a number of short and degenerate consensus sequences have been identified, including the 'core' element GTGGA/TA/TA/TG and stretches of alternating purines and pyrimidines which may have the potential to form left-handed Z DNA. To study the functional significance of two alternating purine and pyrimidine sequences in the SV40 enhancer, we have introduced various combinations of point mutations into a modified SV40 enhancer which contained only one copy of the 72 bp element (W.H., Y.G., A. Nordheim and A. Rich, unpublished results); one of these combinations impaired both the activity of the enhancer and growth of SV40. We describe here the structure of 18 revertants of this mutant and suggest that in each of the 18 revertants, the defects of the original mutant have been overcome by simple tandem duplications in the enhancer region, all of which include the 'core' element.

A herpesvirus genetic element which affects translation in the absence of the viral GADD34 function.

Novel suppressor variants of conditionally lethal HSV-1 gamma34.5 deletion mutants have been isolated which exhibit restored ability to grow on neoplastic neuronal cells. Deletion of the viral gamma34.5 genes, whose products share functional similarity with the cellular GADD34 gene, renders the virus non-neurovirulent and imposes a block to viral replication in neuronal cells. Protein synthesis ceases at late times post-infection and the translation initiation factor eIF2alpha is phosphorylated by the cellular PKR kinase [Chou et al. (1990) Science, 252, 1262-1266; (1995) Proc. Natl Acad. Sci. USA, 92, 10516-10520]. The suppressor mutants have overcome the translational block imposed by PKR. Multiple, independent isolates all contain rearrangements within a 595 bp element in the HSV-1 genome where the unique short component joins the terminal repeats. This alteration, which affects the production of the viral mRNA and protein from the Us11 and Us12 genes, is both necessary and sufficient to confer the suppressor phenotype on gamma34.5 mutant viruses. HSV-1 thus encodes a specific element which inhibits ongoing protein synthesis in the absence of the viral GADD34-like function. Since this inhibition involves the accumulation of phosphorylated eIF2alpha, the element identified by the suppressor mutations may be a discrete PKR activator. Activation of the PKR kinase thus does not proceed through a general, cellular 'antiviral' sensing mechanism. Instead, the virus deliberately activates PKR and encodes a separate function which selectively prevents the phosphorylation of at least one PKR target, eIF2alpha. The nature of this potential activator element, and how analogous cellular elements could affect PKR pathways which affect growth arrest and differentiation are discussed.

Genetic and biochemical analysis of transformation-competent, replication-defective simian virus 40 large T antigen mutants.

To study the role of the biochemical and physiological activities of simian virus 40 (SV40) large T antigen in the lytic and transformation processes, we have analyzed DNA replication-defective, transformation-competent T-antigen mutants. Here we describe two such mutants, C8/SV40 and T22/SV40, and also summarize the properties of all of the mutants in this collection. C8/SV40 and T22/SV40 were isolated from C8 and T22 cells (simian cell lines transformed with UV-irradiated SV40). Early regions encoding the defective T antigens were cloned into a plasmid vector to generate pC8 and pT22. The mutations responsible for the defects in viral DNA replication were localized by marker rescue, and subsequent DNA sequencing revealed missense and one nonsense mutation. The T22 mutation predicts a change of histidine to glutamine at residue 203. C8 has two mutations, one predicts lysine224 to glutamamic acid and the other changes the codon for glutamic acid660 to a stop codon; therefore, C8 T antigen lacks the 49 carboxy-terminal amino acids. pC8A and pC8B were constructed to contain the C8 mutations separately. Plasmids pT22, pC8, pC8A, and pC8B were able to transform primary rodent cell cultures. T22 T antigen is defective in binding to the SV40 origin. C8B (49-amino-acid truncation) is a host-range mutant defective in a late function in CV-1 but not BSC cells. Analysis of T antigens in mutant SV40-transformed mouse cells suggests that the replicative function of T antigen is important in generating SV40 DNA rearrangements that allow the expression of "100K" variant T antigens in the transformants.