Phenotypic complementation of the SV40 tsA mutant defect in viral DNA synthesis following microinjection of SV40 T antigen.

African green monkey cells (CV-1P) were microinjected with highly purified SV40 T antigen using protein-loaded red cell ghosts and polyethylene glycol as fusagen. The microinjected cells were infected with a temperature-sensitive mutant of SV40 (tsA209) which is defective in the initiation of viral DNA synthesis. Using in situ hybridization as an assay method, we found that PEG-microinjection of both partially and highly purified T antigen resulted in an increase in the amount of viral DNA sequences in the monolayer. Moreover, 3H-thymidine-labeled and unlabeled Hirt supernatant from microinjected, tsA209-injected cells contained significantly more SV40 DNA than comparable extracts from sham-injected, tsA209-infected or uninfected cells, which were tested in parallel. Thus the introduction of highly purified, "large" SV40 T antigen led to phenotypic complementation of the tsA defect in viral DNA synthesis.     

Regulation of gene amplification and expression in cells that constitutively express a temperature sensitive SV40 T-antigen.

Simian cells have been transformed with SV40 origin-defective recombinant plasmids containing the tsA209 T-antigen gene. These plasmids contain deletions of either 5 or 52 nucleotides that include the BglI site at the SV40 ori, are defective for replication in COS-1 cells but retain a functional SV40 early promoter. Two cell lines transformed with these plasmids, U4 and S7, and their respective clonal derivatives E5 and F11, contain the tsA209 T-antigen gene integrated into the cell DNA and express T-antigen as detected by immunoprecipitation and immunofluorescence. These cells behave as ts-COS cells, since they complement in a temperature dependent manner the replication of an SV40 derived recombinant plasmid. When transfected with recombinant plasmids containing the chloramphenicol acetyl transferase (CAT) gene cloned into SV40 replicons, ts-COS cells were able to regulate the induction of the CAT activity by temperature. The ratios of CAT activity observed at permissive versus restrictive temperature were in the range of 20-400. Thus, these ts-COS cells are useful systems for the regulated expression of cloned genes in simian cells.     

Saccharomyces cerevisiae mRNA populations of different intrinsic stability in unstressed and heat shocked cells display almost constant m7GpppA:m7GpppG 5'-cap structure ratios

The half-lives of mRNAs in yeast vary from about 1 to over 100 min. While mRNA stabilities must strongly influence overall gene expression in this organism, very little is known about how they are determined. Labellings of yeast cells were conducted to investigate whether the 5'-cap structures of yeast mRNAs might influence their stability. Variation of the pulse-labelling period from 7.5 min to 120 min did not have any major influence on the relative labelling of m7GpppA (A cap) and m7GpppG (G cap) in total polyadenylated RNA. Whether an mRNA has the A cap or the G cap does not therefore have a marked effect on its stability. During the heat shock response the relative labelling of A caps to G caps in total polyadenylated RNA also does not fluctuate appreciably. This indicates that cap structure alone does not determine the destabilisation of non-heat shock mRNAs and stabilisation of heat shock mRNAs during this stress response.     

Initiation of simian virus 40 DNA replication in vitro: identification of RNA-primed nascent DNA chains.

Cell-free extracts of simian virus 40 (SV40)-infected CV-1 cells can initiate large tumor antigen dependent bidirectional replication in circular DNA molecules containing a functional SV40 origin of replication (ori). To determine whether or not DNA replication under these conditions involves RNA-primed DNA synthesis, replication was carried out in the presence of 5-mercuri-deoxycytidine triphosphate to label nascent DNA chains. Newly synthesized mercurated DNA was isolated by its affinity for thiol-agarose, and the 5'-ends of the isolated chains were radiolabeled to allow identification of RNA primers. At least 50% of the isolated chains contained 4 to 7 ribonucleotides covalently linked to their 5'-end; 80% of the oligoribonucleotides began with adenosine and 19% began with guanosine. About 60% of the nascent DNA chains annealed to the SV40 ori region, and about 80% of these chains were synthesized in the same direction as early mRNA. These results are consistent with the properties of SV40 DNA replication in vivo and support a model for initiation of SV40 DNA replication in which DNA primase initiates DNA synthesis on that strand of ori that encodes early mRNA.     

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.     

Interaction of a limited set of proteins with different mRNAs and protection of 5''-caps against pyrophosphatase digestion in initiation complexes.

A variety of 5'-3H-methyl-labeled, oxidized viral mRNAs were used as probes for detecting in wheat germ initiation complexes proteins that interact with, and can be cross-linked to, the 5'-cap structure. A limited and reproducible set of specific proteins was obtained with the different mRNAs. The binding of these proteins to the 5'-end of mRNA apparently results in protection against nucleotide pyrophosphatase digestion of the cap even in initiation complexes in which the 5'-end is susceptible to pancreatic RNase digestion. Cross-linked proteins from mammalian initiation complexes comigrated with several of the subunits of similarly treated eIF-3. A model for cap binding protein interaction with mRNA cap during initiation of translation is suggested.     

Ultraviolet-irradiated simian virus 40 activates a mutator function in rat cells under conditions preventing viral DNA replication

The UV-irradiated temperature-sensitive early SV40 mutant tsA209 is able to activate at the nonpermissive temperature the expression of mutator and recovery functions in rat cells. Unirradiated SV40 activates these functions only to a low extent. The expression of these mutator and recovery functions in SV40-infected cells was detected using the single-stranded DNA parvovirus H-1 as a probe. Because early SV40 mutants are defective in the initiation of viral DNA synthesis at the nonpermissive temperature, these results suggest that replication of UV-damaged DNA is not a prerequisite for the activation of mutator and recovery functions in mammalian cells. The expression of the mutator function is dose-dependent, i.e., the absolute number of UV-irradiated SV40 virions introduced per cell determines its level. Implications for the interpretation of mutation induction curves in the progeny of UV-irradiated SV40 in permissive host cells are discussed.     

Multiple methylated cap sequences in adenovirus type 2 early mRNA.

The methylated constituents of early adenovirus 2 mRNA were studied. RNA was isolated from polyribosomes of cells double labeled with [methyl-3H]methionine and 32PO4 from 2 to 7 g postinfection in the presence of cycloheximide. Cycloheximide ensures that methylation and processing are performed by preexisting host cell enzymes. RNA was fractionated into polyadenylic [poly(A)]+ and poly(A)- molecules using poly(U)-Sepharose, and undergraded virus-specific RNA was isolated by hybridization to viral DNA in 50% formamide at 37 degrees C. Viral mRNA was digested with RNase T2 and chromatographed on DEAE-Sephadex in 7 M urea. Two 3H-labeled RNase T2-resistant oligonucleotide fractions with charges between -5 and -6 were obtained, consistent with two classes of 5' terminal methyl "cap" structures, m7G(5')ppp(5')NmpNp (cap 1) and m7G(5')ppp(5')NmNmpNp (cap 2) (Nm is a ribose 2'-O-methylation). The putative cap 1 contains all the methylated constituents of cap 1 plus Cm. The molar ratios of m7G to 2'-O-methylnucleosides is about 1.0 for cap 1 and 0.5 for cap 2, consistent with the proposed cap structures. Most significant, compositional analysis indicates four different cap 1 structures and at least three different cap 2 structures. Thus there is a minimum of seven early viral mRNA species with different cap structures, unless each type of mRNA can have more than one 5' terminus. In addition to methylated caps, early mRNA contains internal base methylations, exclusively as m6A, as shown by analyses of the mononucleotide (-2 charge) fraction. m6A was present in the ratio of 1 mol of m6Ap per 450 nucleotides. Thus viral mRNA molecules contain two to three internal m6A residues per methyl cap, since there is on the average 1 cap per 1,250 nucleotides.     

Synthesis of Leishmania cap-4 intermediates, cap-2 and cap-3

Synthesis of Leishmania mRNA 5'-cap analogs, m(7)Gpppm(2)(6)AmpAm (cap-2), and m(7)Gpppm(2)(6)AmpAmpCm (cap-3) is reported. Binding affinities of those cap analogs for LeishIF4E proteins were determined using fluorescence spectroscopy. Cap-3 showed similar affinity to LeishIF4Es compared to the mature trypanosomatids cap structure (cap-4).     

Blocked and Methylated 5'-Terminal Cap Structures of Rat Brain Messenger Ribonucleic Acids

Abstract: The presence and identity of 5'-terminal cap structures in rat brain polysomal mRNA were investigated by radiolabeling the mRNA by periodate oxidation and [³H]sodium borohydride reduction or by β -elimination of 5'-terminal nucleoside and incorporation of ³²P in the presence of polynucleotide kinase. The labeled mRNAs were digested with nucleases and the cap structures were isolated and identified by chromatographic and electrophoretic procedures. The results showed that rat brain mRNAs contained cap 1 and cap 2 structures and no caps of the zero type. The proportion of cap 2 was higher than that of cap 1. Both caps had 7-methylguanosine (m⁷G) as the 5'-terminal nucleoside, which was linked to the next nucleoside by an inverted triphosphate bridge, as in other eukaryotic mRNAs. The most prominent nucleoside in the 5'-penultimate position was 6-methyl-2'- O -methyiadenosine [m⁶A(m)] followed by 2'- O -methyladenosine [A(m)], which together contributed to nearly 70% of both cap 1 and cap 2 structures. 2'- O -Methylguanosine [G(m)] accounted for approximately 18%, the rest being made up of 2'- O -methyl-cytidine [C(m)] and 2'- O -methyluridine [U(m)].     

Simian virus 40 gene A regulation of cellular DNA synthesis. I. In permissive cells.

The kinetics of host cellular DNA stimulation by simian virus 40 (SV40) tsA58 infection was studied by flow microfluorometry and autoradiography in two types of productively infected monkey kidney cells (AGMK, secondary passage, and the TC-7 cell line). Prior to infection, the cell populations were maintained predominantly in G0-G1 hase of the cell cycle by low (0.25%) serum concentration. Infection of TC-7 or AGMK cells by wild-type SV40, viable deletion mutant dl890, or by SV40 tsA58 at 33 degrees C induced cells through S phase after which they were blocked with a 4N DNA content in the G2 phase. The infection of TC-7 cells by tsA58 at 41 degrees C, which was a nonpermissive temperature for viral DNA replication, induced a round of cell DNA synthesis in approximately 30% of the cell population. These cells proceeded through S phase but then re-entered the G1 resting state. In contrast, infection of AGMK cells by tsA58 at 41 degrees C induced DNA synthesis in approximately 50% of the cells, but this population remained blocked in the G2 phase. These results indicate that the mitogenic effect of the A gene product upon cellular DNA is more heat resistant than its regulating activity on viral DNA synthesis and that the extent of induction of cell DNA synthesis by the A gene product may be influenced by the host cell.     

Recombination Between Endogenous and Exogenous Simian Virus 40 Genes. I. Rescue of a Simian Virus 40 Temperature-Sensitive Mutant by Passage in Permissive Transformed Monkey Lines

Passage of the simian virus 40 (SV40) temperature-sensitive (ts) mutant tsD202 at the permissive temperature in each of three permissive lines of SV40-transformed monkey CV1 cells resulted in the emergence of temperature-insensitive virus, which plated like wild-type SV40 at the restrictive temperature on normal CV1 cells. In independent experiments, the amount of temperature-insensitive virus that appeared after passage on transformed cells was from 10(3)- to 10(6)-fold greater than the amount of ts-revertant virus that appeared after an equal number of passages in nontransformed CV1 cells. The virus rescued by passage on transformed cells bred true upon sequential plaque purification, plated on normal CV1 cells with single-hit kinetics at the restrictive temperature, and displayed no selective growth advantage on transformed cells compared to non-transformed cells. Hence, the reversion of the ts phenotype is neither due to complementation effects nor to the selection of preexisting revertants, which grow better on transformed cells. In the accompanying article (T. Vogel et al., J. Virol. 24:541-550, 1977), we present biochemical evidence that the rescue of tsD202 mediated by passage on transformed cells is due to recombination with the resident SV40 genome. Parallel experiments in which tsA, tsB, and tsC SV40 mutants were passaged in each of the three permissive lines of SV40-transformed monkey cells resulted in either only borderline levels of rescue (tsA mutants) or no detectable rescue (tsB and tsC mutants). Evidence is presented that the resident SV40 genome of the transformed monkey lines is itself a late ts mutant, and we suggest that this accounts for the lack of detectable rescue of the tsB and tsC mutants. We furthermore suggest that the borderline level of rescue observed with two tsA mutants is related to a previous finding (Y. Gluzman et al., J. Virol. 22:256-266, 1977) which indicated that the resident SV40 genome of the permissive transformed monkey cells is defective in the function required for initiation of viral DNA synthesis.     

Role of mRNA 5'-terminal caps in translational dormancy of Physarum polycephalum

Translational regulation of protein synthesis accompanies sclerotization in Physarum polycephalum. Plasmodial and sclerotial poly(A)+ RNA were translated in a message-dependent wheat germ lysate in the presence of the cap analogue 7-methylguanosine-triphosphate to determine whether 5' structural alterations in mRNA accompany translational repression. The translation of plasmodial and sclerotial poly(A)+ RNA was reduced to identical levels suggesting that both RNA populations are capped. The 5'-termini of plasmodial and sclerotial poly(A)+ RNA were identified as m7G5'ppp5'Cm. Alterations in the 5'-cap of mRNA during sclerotization do not appear to be responsible for translational dormancy.     

Timing of ultraviolet light induced mutagenesis in simian virus 40 relative to viral DNA replication in monkey cells

Summary Simian virus 40 (SV40) was used to probe ultraviolet light (UV) — induced mutation in mammalian cells. Viral mutations were scored as reversions of early and late temperature-sensitive (ts) mutants to the wild-type (WT) phenotype. When virus was exposed to moderate or high UV doses, WT revertants were obtained at a frequency related to the square of the dose from two early (tsA) and one late (tsBC) mutant grown at the restrictive temperature. The reversions generated in the progeny of UV-irradiated early mutants presumably arose before the onset of viral DNA replication because, at the non-permissive temperature, tsA mutants are unable to express the functions responsible for the initiation of viral DNA synthesis. Moreover, the early mutant tsA209 underwent similar levels of induced reversion at the permissive and restrictive temperatures, suggesting that the pre-replicative mutational pathway might predominate for moderately and heavily irradiated virus, even under conditions where DNA synthesis can be initiated. The analysis of bursts from revertant plaques produced at the restrictive temperature was consistent with this interpretation. Although the mechanism of pre-replicative mutagenesis is not known, it is likely to be mediated by cellular activities owing to the low genetic complexity of the virus.     

Simian Virus 40 Mutants with Deletions at the 3′ End of the Early Region Are Defective in Adenovirus Helper Function

Coinfection of monkey cells with simian virus 40 (SV40) and adenovirus type 2 (Ad2) increased the Ad2 yield 1,000-fold over that obtained by Ad2 infection alone of monkey cells (A. S. Rabson, G. T. O'Conor, I. K. Berezesky, and F. J. Paul, Proc. Soc. Exp. Biol. Med. 116:187-190, 1964). The ability of viable mutants of SV40 that contain deletions at various sites in the viral DNA to enhance Ad2 growth in monkey cells was examined. Only those mutants with deletions near the 3' end of the early region were deficient in providing this helper function. Mutants dl1265, lacking 39 base pairs at map position 0.18, and dl1263, lacking 33 base pairs at map position 0.20 (H. van Heuverswyn, C. Cole, P. Berg, and W. Fiers, J. Virol. 30:936-941, 1979), were approximately 4 and 30% as effective as wild-type SV40, respectively. The extent of enhancement of Ad2 yield depended on the multiplicity of infection by SV40, but not by Ad2 (at a multiplicity of infection of 相似文献   

SV40 large tumor antigen can regulate some cellular transcripts in a positive fashion

Eleven cDNA clones identified from a cDNA library prepared from the mRNA fraction of SV40 transformed cells detected, by hybridization, higher levels of cellular mRNA in SV40-transformed cells than in nontransformed cells. Three of these cDNA clones detected levels of cellular mRNA that were more than 100-fold greater in SV40tsA transformed cell lines grown at the permissive temperature than in those grown at the nonpermissive temperature. Northern blot hybridizations confirmed these results and in some cases detected RNA species of multiple sizes that were regulated in a temperature-dependent fashion in SV40tsA transformed cell lines. Infection of 3T3 cells with SV40 stimulated the levels of RNAs complementary to these cDNA clones. The results demonstrate that the SV40 large T antigen can regulate the steady state levels of some cellular RNA species.