Late RNA Research Articles

Both early and late control sequences of SV40 and polyoma promote transcription of Escherichia coli gpt gene in transfected cells.

Eucaryotic expression vectors containing two selective markers, the herpes simplex 1 thymidine kinase gene (tk) and the Escherichia coli gpt gene (Eco gpt) coding for a xanthine-guanine phosphoribosyl-transferase ( XGPRT ) were constructed. These plasmids were used to transfect mouse Ltk- cells followed by selection of either tk+ or XGPRT + colonies. The transcription and maturation of the Eco gpt mRNA is dependent on the presence of a eucaryotic promoter sequence at its 5' end and on the presence of a viral intron and a poly(A) addition site at its 3' end ( Mulligan and Berg, 1980). Here, we report that both simian virus 40 (SV40) and polyoma early and late promoters permit the transcription of this gene integrated into the cellular genome. Polyoma DNA fragments lacking the TATA box, or both the TATA and CAAT boxes directing early transcription, efficiently promote Eco gpt expression. Furthermore, a fragment terminating approximately 300 nucleotides upstream of the initiation site of early RNA permits Eco gpt synthesis when the early strand is joined to the Eco gpt-coding strand. The SV40 early promoter is 2- to 3-fold more efficient than the controlling sequence of late transcription. These results strongly suggest that the switch from a predominance of early RNA to that of late RNA occurring after the onset of DNA replication is caused by the increase in the abundance of template and by the concomitant repression of early transcription by the T antigen. The presence of the tk gene in all the plasmids constructed permits the analysis of Eco gpt expression as a non-selected marker in tk+ clones selected for growth in HAT medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Epstein-Barr virus RNA. VIII. Viral RNA in permissively infected B95-8 cells.

More than 50 RNAs expressed by Epstein-Barr virus late in productive infection have been identified. B95-8-infected cells were induced to a relatively high level of permissive infection with the tumor promotor 12-O-tetradecanoylphorbol-13-acetate. Polyadenylated RNAs were extracted from the cell cytoplasm, separated by size on formaldehyde gels, transferred to nitrocellulose, and hybridized to labeled recombinant Epstein-Barr virus DNA fragments. Comparison of RNAs from induced cultures with RNAs from induced cultures also treated with phosphonoacetic acid to inhibit viral DNA synthesis identifies two RNA classes: a persistent early class of RNAs whose abundance is relatively resistant to viral DNA synthesis inhibition and a late class of RNAs whose abundance is relatively sensitive to viral DNA synthesis inhibition. The persistent early and late RNAs are not clustered but are intermixed and scattered through most of segments UL and US. The cytoplasmic polyadenylated RNAs expressed during latent infection were not detected in productively infected cells, indicating that different classes of viral RNA are associated with latent and productive infection. Non-polyadenylated small RNAs originally identified in cells latently infected with Epstein-Barr virus are expressed in greater abundance in productively infected cells and are part of the early RNA class.

Characterization of two RNA polymerase activities induced by mouse hepatitis virus.

RNA-dependent RNA polymerase activity was found in mouse hepatitis virus strain A59 (MHV-A59)-infected cells. The enzyme was induced in the infected cells and could not be detected in the MHV-A59 virion. Two peaks of RNA polymerase activity, one early and the other late in infection, were detected. These polymerase activities were in temporal sequence with early and late virus-specific RNA synthesis. Both of them were found to be associated with membrane fractions. There were significant differences in the enzymatic properties of the two polymerases. The early polymerase, but not the late polymerase, could be activated by potassium ions in the absence of magnesium ions and also had a lower optimum pH than the late polymerase. It was therefore probable that the enzymes represent two different species of RNA polymerase and perform different roles in virus-specific RNA synthesis. The effects of cycloheximide on MHV-specific RNA synthesis were determined. Continuous protein synthesis was required for both early and late RNA synthesis and might also be required for shutoff of early RNA synthesis.

Evidence for simian virus 40 late transcriptional control: mixed infections of wild-type simian virus 40 and a late leader deletion mutant exhibit trans effects on late viral RNA synthesis.

Mixed infections involving equal multiplicities of wild-type simian virus 40 and viable deletion mutant dl861 resulted in decreased cytoplasmic levels of wild-type-derived male mRNA, as well as very low to undetectable levels of mutant-derived late mRNA, as compared with individual infections. The dl861 deletion removes 16 to 25 base pairs from the late leader region. This deletion was shown to be the direct cause of the mixed-infection effect; replacement of the deletion with wild-type sequences restored normal levels of late mRNAs in mixed infections. Other viral functions, e.g., early gene expression and replication, were found to be unaffected by the dl861 deletion. Further examination of the mixed-infection effect showed that the levels of unspliced nuclear precursors of late mRNA, derived from both the mutant and wild-type genomes, were decreased or undetectable, in accord with the cytoplasmic results. Thus, the effect appears to be occurring at the transcriptional level. These data demonstrate a trans-acting effect on late transcription, which is detectable due to the presence of the dl861 mutant in the mixed infection. This finding is indicative of a diffusible factor which exerts a control on simian virus 40 late gene expression at the transcriptional level. A model for positive control of simian virus 40 late gene expression is presented.

Colinearity of RNAs with the vaccinia virus genome: anomalies with two complementary early and late RNAs result from a small deletion or rearrangement within the inverted terminal repetition.

The colinearity of RNA transcripts with the vaccinia virus genome was investigated. Cytoplasmic RNA from infected cells was annealed to a cloned DNA segment that extended from 9 to 15.6 kilobase pairs from the left end of the genome and contained approximately 800 base pairs of the inverted terminal repetition (ITR). Remaining unhybridized single strands of DNA were digested with nuclease S1, and the lengths of the protected DNA fragments were determined by agarose gel electrophoresis under neutral and alkaline conditions. Uniformly 32P-labeled cloned DNA insert, separated recombinant DNA strands, and smaller restriction fragments, as well as 3' and 5' end-labeled DNA, were employed to map five early RNAs and one late RNA. One of the early RNAs hybridized to sequences within the ITR, and the other four hybridized to sequences proximal to the ITR. The late RNA was initiated proximal to the ITR but extended into it. Interestingly, the 3' portion of this late RNA was complementary to the early RNA transcribed from the opposite strand of the ITR. From a comparison of the lengths of the protected DNA fragments on neutral and alkaline gels, all except the complementary early and late RNAs appeared to be colinear with the genome. Although the anomalous nuclease S1 data obtained with the latter RNAs mimicked splicing, they were shown by DNA-DNA hybridization to result from a small deletion or rearrangement within the ITR. Thus far, no true examples of spliced vaccinia virus RNAs have been found.

Translational control of early protein synthesis at the late stage of vaccinia virus infection

The cytoplasmic RNA obtained at 1 hr (early) or 6 hr (late) after infection of Ehrlich ascites tumor (EAT) cells with vaccinia virus stimulated to similar extent the incorporation of [ 35S]methionine into proteins when translated in a micrococcal nuclease-treated lysate obtained also from EAT cells. Comparison on SDS-polyacrylamide gels of the in vitro translation products at nonsaturating RNA concentrations with those obtained in vivo by pulse labeling the infected cells showed that essentially all the early in vivo labeled proteins were made upon translation of the early RNA. Analysis of the cells pulse labeled in vivo at the late time demonstrated the presence of late proteins and of only residual amounts of some early proteins. The in vitro translation products of the late RNA, however, not only contained the late proteins but also appreciable amounts of several early proteins. This observation suggested that some early mRNA species were still present at the late time after infection but not translated in vivo, at least not to the same relative extent. In parallel, hybridization competition experiments revealed that the late cytoplasmic RNA contained almost twice as much early sequences as did the early cytoplasmic RNA. These results suggest that at the late stage of vaccinia virus infection the synthesis of the early proteins is subjected to translational control.

Simian virus 40-rabbit beta-globin recombinants lacking late mRNA splice sites express cytoplasmic RNAs with altered structures.

Deletions were introduced at exon-intron boundaries in the late region of a simian virus 40-beta-globin cDNA recombinant to study the role of splicing in the formation of simian virus 40 late cytoplasmic RNAs. The recombinant was used as a wild type because it allowed characterization of mutant RNAs expressed from defective genomes in the presence of comparable RNAs contributed by the coinfecting helper virus. Removal of a 17-base pair segment at map position 0.76, which included a portion of the leader sequence implicated in the splicing of the major 16S mRNA, prevented expression of 16S-type mRNA. The same mutant accumulated cytoplasmic 19S-type mRNA, but the assortment of the 5' ends of these mRNAs differed from the assortment of the wild-type counterparts. Another mutant that lacks nucleotide sequences implicated in the splicing of the major 16S mRNA and one of the principal 19S-type RNAs accumulated a 16S-type mRNA with a previously undetected leader splice, and assortment of 19S mRNAs with new or normally underrepresented splices, and even a species of unspliced cytoplasmic 19S mRNA.

SP01 gene 27 is required for viral late transcription.

The SP01 mutant sus HA20 (gene 27) was found to be defective for synthesis of viral late RNA. It is known that gene 27 is also required for viral DNA replication. The SP01 gene 27 product resembles the T4 gene 45 product, which also has a dual role in viral DNA replication and late transcription.

Monocistronic and polycistronic bacteriophage T4 gene 23 messages.

We studied transcription of T4 late genes by in vitro translation of size-fractionated late RNA and by hybridization of T4 late RNA to plasmids containing identified T4 late genes. We identified mRNA species that coded for the late proteins gp10, gp18, gp21, gp22, gp23, and gp24. Functional mRNA's that coded for the early proteins gp32 and IPIII were also detected after fractionation of late RNA. As in preparations of early RNA, gene 32 message activity was present in two species of RNA, which had molecular weights of approximately 0.5 x 10(6) and 0.8 x 10(6), and IPIII message activity was present in multiple species of RNA. Gene 24 and gene 10 message activities migrated as single species that had approximate molecular weights of 0.5 x 10(6) and 1.2 x 10(6), respectively. mRNA activity for gp18 migrated heterogeneously. We detected multiple transcripts from gene 23 by in vitro translation and by hybridization of late RNA to plasmids containing genes 21 through 23. Both types of analysis indicated that the major gene 23 transcript had a molecular weight of 0.75 x 10(6). In addition, two gene 23 transcripts having molecular weights of about 1.0 x 10(6) and 1.3 x 10(6) were present; these RNA species also coded for gp21 and gp22. Physical linkage of transcripts from genes 21, 22, and 23 was demonstrated by hybridization.

Late messenger RNA production by viable simian virus 40 mutants with deletions in the leader region

We have analyzed the structure of simian virus 40 messenger RNA in cells infected with mutant viruses in which DNA encoding various segments of the late leader has been deleted. The results show that large amounts of stable unspliced mRNA may accumulate in the cytoplasm and that some unspliced RNA appears in polyribosomes. The locations of the prominent 5′ ends of mRNA are influenced by relatively remote downstream sequences and bear no fixed relationship to any sequence of the simian virus 40 DNA retained in the mutant virus. Sequences downstream from the leader template may play a role in determining the region in which transcription initiates preferentially. Splices may occur even when nucleotides further than seven residues downstream from the upstream end of the splice are altered, and alternative splices with one junction in common may be differently affected by deletions near the common splice junction.

Growth patterns of temperature-sensitive mutants of Western equine encephalitis virus in cultured Aedes albopictus (mosquito) cells.

Several temperature-sensitive (ts) mutants of Western equine encephalitis virus (WEEV) have been isolated previously from persistently infected cultures of mosquito cells and divided into three groups: early passage RNA- mutants, early passage RNA+ mutants and late passage RNA- mutants (Maeda et al., 1979(. The growth patterns of these groups, as well as of several ts mutants isolated after chemical mutagenesis and of wild-type (wt) WEEV, have been compared in BHK cells and in two strains of mosquito cells. The late passage ts mutants grew much better in mosquito cells than either the wt WEEV or the chemically induced mutants. When mosquito cells were co-infected with a late passage mutant (A125) and Wt WEEV, infectious virions of both parental types as well as phenotypically mixed particles were produced. Infection of mosquito cells with WEEV resulted in a slight suppression of host DNA and protein synthesis during the acute stage of the infection (the first 1 or 2 days). Virus growth in a line of cloned mosquito in which WEEV produced a cytopathic infection (c.p.e.) was analysed with the result that the viruses could be divided into two groups: one in which wt WEEV, chemically induced ts mutants and early passage RNA+ mutants all induced maximal c.p.e., and another in which late passage RNA- mutants and one early passage RNA- mutant induced very little c.p.e., but released much more infections virus into the culture fluid. Electron microscopy showed that in these cloned mosquito cells infected with a virus of the first group, large amounts of virus accumulated on or in the plasma membrane.

Human cytomegalovirus DNA: Restriction enzyme cleavage maps and map locations for immediate-early, early, and late RNAs

Complete physical maps for human cytomegalovirus (Davis strain) DNA were constructed from fragments produced by restriction endonucleases HindIII, XbaI, and EcoRI. The results showed that the human cytomegalovirus genome has a structural organization similar to that of the herpes simplex virus genome: a long (L) segment, comprising 82% of the genome, joined to a short (S) segment, comprising the remaining 18% of the genome. Permutations of the S and L segments produce four different molecular arrangements which are represented in equimolar proportions in virion DNA preparations. Heterogeneity in the size of the DNA was detected within fragments which map at one terminus of the S segment and also in fragments which map at one terminus of the L segment. The regions of the genome from which RNA synthesized at various times after infection originates was determined. Immediate-early RNA (RNA synthesized in the presence of cycloheximide) was restricted to a few regions on the genome but hybridized in abundance to a region which mapped between 0.686 and 0.733 units (within the L segment). Early RNA (synthesized in the absence of viral DNA synthesis) hybridized to most areas of the genome, but in particular abundance to a small region which mapped between 0 and 0.046 units (also within the L segment). RNA which accumulated during the late phase of infection was not characterized by particular abundances to any region of the genome.

DNA synthesis and template activity in a mutant of polyoma virus with altered expression of late viral proteins.

The 3049 strain of polyoma virus overproduces late RNA and proteins. The synthesis and accumulation of virus DNA was measured in cells infected with 3049 and a wild-type virus, lpS, to assess the possible role of gene dosage in this phenomenon. The rate of incorporation of [3H]thymidine into the DNA of 3049 and lpS virus was shown to be identical. The number of genome copies per cell, determined by DNA-DNA reassociation kinetics, was found to be similar in either whole cell or Hirt extracts of cells infected with the two viruses. Viral transcription complexes were isolated by Sarkosyl extraction, and the specific transcriptional activity was measured by incorporation of [3H]UTP. The incorporation of [3H]UTP and the DNA content of transcription complexes were indistinguishable. These results suggest that overproduction of late RNA (and protein) by the 3049 virus is not due to either an increased number of viral genomes per cell or an increased fraction of genomes involved in transcription. These and other data (unpublished) support the conclusion that the altered phenotype is due to a posttranscriptional mechanism.

Transcriptional map of bacteriophage N4: Location and polarity of N4 RNAs

The strands of the four bacteriophage N4 DNA fragments derived by HhaI endonuclease digestion have been separated on agarose gels, and the eight fragments obtained have been related to the two complementary strands of N4 DNA. We have used the HpaI restriction endonuclease fragments (Zivin et al., 1980) and single-stranded fragments of HhaI-digested N4 DNA as probes to determine the temporal appearance, localization and polarity of N4 transcripts. These experiments have allowed us to localize on the N4 genome the two previously characterized classes (early and middle RNAs) of N4 rifampicin-resistant RNAs (Vander Laan et al., 1977) and to detect a new class of N4 RNAs, which are synthesized late in infection. The synthesis of late N4 RNAs is rifampicin sensitive and, as we show in this paper, requires the activity of the Escherichia coli RNA polymerase.

Extension of the transcriptional and translational map of the left end of the vaccinia virus genome to 21 kilobase pairs

Physical, transcriptional, and translational maps of an EcoRI fragment located between 15,800 and 20,600 base pairs from the left end of the vaccinia virus genome were prepared. Major polypeptides with molecular weights of 14,000 (14K polypeptide), 32,000 and 38,000 were synthesized in a reticulocyte cell-free system programmed with immediate early RNA made in the presence of cycloheximide and selected by hybridization to lambda recombinant DNA containing the EcoRI fragment. With early RNA made in the presence of cytosine arabinoside, an inhibitor of DNA replication, the polypeptide pattern was similar except for quantitative differences in which less 38K polypeptide was detected as a translation product. With late RNA, isolated 6 h after infection without inhibitors, only traces of the early translation products were found and a new 40K polypeptide was detected. The size of the mRNA's for the 14K, 32K, and 38K polypeptides were determined to be approximately 760,880, and 1,150 nucleotides, respectively, by several independent procedures. Several large early RNAs not shown to code for any additional translation products were also detected. The size of the late message for the 40K polypeptide varied from 920 to 3,100 nucleotides. This heterogeneity appeared to be a general property of vaccinia virus late mRNA's. No evidence of RNA splicing was obtained by analysis of RNA-DNA hybrids after nuclease S1 treatment. Further analyses using separated recombinant DNA strands and restriction fragments indicated that all mRNA's were encoded by the leftward-reading DNA strand and at least two were overlapping. Since early and late mRNA's were encoded by the same DNA strand, the possibility of temporal regulation by transcriptional strand switching was eliminated. In conjunction with previous studies, a transcriptional map of the left 20,600 base pairs of the vaccinia virus genome was derived.

Transcription of the human cytomegalovirus genome in productively infected cells.

Nuclear and cytoplasmic RNAs, synthesized in cells productively infected with human cytomegalovirus (HCMV) were analysed at various times after infection by liquid and filter DNA-RNA hybridization. Results of these experiments have revealed that: (i) the fraction of the genome transcribed increased as infection progressed. In the nucleus, transcripts represented approx. 20% of the virus DNA sequences at both 2 and 4 h post-infection (p.i.) and 36% of the virus DNA at 40 h p.i; (ii) the increase in virus sequences among nuclear transcripts at late times was prevented by the DNA synthesis inhibitor, 2'-deoxyfluorouridine; (iii) early virus RNA transcripts were subset of those represented in late RNA; (iv) two classes of early RNA were identified by competition hybridization; (v) approx. 10% of the late nuclear transcripts were symmetrical. Results of filter hybridization at DNA excess indicated that virus-specific RNA represented 0.6% of RNA labelled from 0 to 2 h p.i., and 1.8% of RNA labelled from 28 to 30 h. Polyadenylated RNA isolated from cytoplasm represented 1.2% and 10% of labelled mRNA at 2 h and 30 h respectively. Our data show that during productive infection of human cells by HCMV, gene expression is under temporal, quantitative and post-transcriptional control.

Transcriptional and translational mapping of a 6.6-kilobase-pair DNA fragment containing the junction of the terminal repetition and unique sequence at the left end of the vaccinia virus genome.

The penultimate EcoRI fragments from the left and right ends of the vaccinia virus genomes were cloned in phage lambda. Heteroduplex analysis and comparison of restriction fragments indicated that the inverted terminal repetition extended 780 base pairs (bp) beyond the EcoRI site or about 9,800 bp from each end of the genome. Detailed physical, transcriptional, and translational maps of the 6,600-bp left penultimate EcoRI fragment were prepared so as to extend previous maps of the 9,000-bp terminal EcoRI fragment. Polypeptides with molecular weights of 6,000 (6K polypeptide), 13,000, 19,000, 21,000, and 60,000 were synthesized in a reticulocyte cell-free system programmed with immediate early RNA (made in the presence of cycloheximide) or early RNA (made in the presence of cytosine arabinoside) and selected by hybridization to immobilized recombinant DNA. A 22K polypeptide was detected as a translation product of late RNA that hybridized to this DNA fragment. A variety of biochemical procedures were used to size and map the mRNA's. Of the five messages that hybridized to this 6,600-bp EcoRI fragment, only the one for the 21K polypeptide was encoded within the inverted terminal repetition and hybridized to the rightward-reading DNA strand. (Three additional early polypeptides were encoded within the first 9,000 bp of the inverted terminal repetition.) The remaining early polypeptides were encoded within the unique portion of the penultimate EcoRI fragment and were transcribed from the leftward-reading strand. Additional high-molecular-weight early RNAs of unknown function were also detected; however, there was no evidence indicating that mature mRNA's were spliced.

Cell-free translation of early and late mRNAs selected by hybridization to cloned DNA fragments derived from the left 14 million to 72 million daltons of the vaccinia virus genome

A preliminary translational map of the left 14 million to 72 million daltons of the vaccinia virus genome was constructed. To facilitate this study, the EcoRI D fragment of HindIII C and the successive HindIII N, M, K, F, E, O, I, G, L, J, H, and D fragments were cloned in the plasmid pBR322. The recombinant DNAs were immobilized on nitrocellulose filters and used to select immediate early viral RNA made in the presence of cycloheximide, early viral RNA made in the presence of cytosine arabinoside, and late viral RNA made in the absence of inhibitors. The selected mRNAs were translated in the reticulocyte cell-free system and the [ 35S]methionine-labeled products were analyzed by polyacrylamide gel electrophoresis. Approximately 75 polypeptides were detected among the translation products of early mRNAs selected to this portion of the genome. Most of these polypeptides were also made with immediate early mRNAs. Specific early mRNAs were selected by hybridization to each HindIII fragment; however, no immediate early mRNA hybridized to HindIII L. When selected late mRNAs were translated, more than 40 distinct new polypeptides were detected and synthesis of early polypeptides was greatly diminished. Late mRNAs were clustered near the center of the genome, particularly in HindIII fragments G, L, J, H, and D. No late polypeptides were detected by translation of mRNA hybridizing to fragments C′, N, M, and K and few were detected using mRNA that hybridized to fragments F, E, and O.

The metabolism of SV40 RNA is associated with the cytoskeletal framework

We prepared the cytoskeletal framework of SV40-infected BSC-1 cells late after infection by extracting the cells with Triton X-100. The cytoskeletal framework obtained by this procedure carries more than 80% of the newly synthesized viral polyribosomes. Almost all the viral-specific cytoplasmic RNA is engaged in the formation of cytoskeletal-associated polyribosomes. The cytoskeletal framework harbors both the poly(A) + 19 S and 16 S late viral RNAs, as well as most of the poly(A) − viral RNA. The poly(A) − viral RNA sediments in sucrose gradients between 4 and 18 S representing heterogeneous species and comprises about 30–50% of the total virus-specific RNA in the cytoplasm. Based on pulse-chase experiments it is concluded that: (i) the poly(A) + viral RNA emerges from the nucleus and becomes associated with the cytoskeletal framework, (ii) the decay rate of the poly(A) + RNA species on the cytoskeletal framework is faster than that of the poly(A) − viral RNA, and (iii) both the poly(A) + and poly(A) − viral RNAs detach from the cytoskeletal framework and move to the soluble fraction.

Structure of polyoma virus late nuclear RNA

We have investigated the structure of polyoma virus-specific RNA in the nuclei of 3T6 cells late during productive infection, using standard RNA fractionation techniques and adaptations of the S 1 nuclease/gel electrophoresis mapping technique (Berk & Sharp, 1977). Giant, tandemly repeated, short-lived transcripts of the entire circular viral genome predominate; a proportion of these molecules is polyadenylated. The 5′ termini of these molecules are heterogeneous. The majority map in a 200-nucleotide region centred on 67 map units; a single 5′ end maps in the early region at 92.6 map units. The 3′ termini of polyadenylated viral nuclear RNA molecules map at the same position as those of the cytoplasmic messenger RNAs, 24.9 map units. The only defined 3′ end of non-polyadenylated molecules maps at 93.8 map units. Spliced molecules were detectable only in the polyadenylated nuclear RNA fraction. In general, the structure of the giant late viral nuclear RNA is consistent with its serving as precursor to the late viral messenger RNAs, which each possess 5′ leader sequences comprising a variable number of exact tandem repeats of a short sequence unit present only once in the viral DNA.