Late Messenger RNA Research Articles

Assessing the detection of human papillomavirus late mRNA in liquid base cytology samples for risk stratification of cervical disease

Molecular human papillomavirus (HPV) testing is an important and developing tool for cervical disease management. However there is a requirement to develop new HPV tests that can differentiate between clinically significant and benign, clinically insignificant infection. Evidence would indicate that clinically significant infection is linked to an abortive HPV replication cycle. In particular the later stages of the replication cycle (i.e., production of late messenger (m) RNAs and proteins) appear compromised. Compared to current DNA-based tests which indicate only presence or absence of virus, detecting virus mRNAs by reverse transcriptase PCR (RT-PCR) may give a more refined insight into viral activity and by implication, clinical relevance. A novel quantitative (q)RT-PCR assay was developed for the detection of mRNAs produced late in the viral replication cycle. Initially this was validated on HPV-containing cell lines before being applied to a panel of 223 clinical cervical samples representing the cervical disease spectrum (normal to high grade). Samples were also tested by a commercial assay which detects expression of early HPV E6/E7 oncoprotein mRNAs. Late mRNAs were found in samples associated with no, low and high grade disease and did not risk-stratify HPV infection. The data reveal hidden complexities within the virus replication cycle and associated lesion development. This suggests that future mRNA tests for cervical disease may require quantitative detection of specific novel viral mRNAs. J. Med. Virol. 86:627–633, 2014. © 2013 Wiley Periodicals, Inc.

Human Polyomavirus JC (JCV) Infection of Human B Lymphocytes: A Possible Mechanism for JCV Transmigration across the Blood‐Brain Barrier

It has been suggested that JC virus (JCV) might travel to the central nervous system in infected B cells. Moreover, recent data suggest the presence of JCV in bone marrow plasma cells. However, the evidence for infection and replication of JCV in B cells is unclear. To address this question, we infected Epstein-Barr virus-transformed B cells with JCV and found that the viral genome decreased >1000-fold from days 0 to 20 after infection, which concurred with the absence of viral early and late messenger RNA transcripts and proteins. However, immunofluorescent images of B cells infected with fluorescein isothiocyanate-conjugated JCV demonstrated that JCV enters the B cells, and DNase protection assay confirmed the presence of intact JCV virions inside the B cells. Moreover, JCV-infected B cells were able to transmit infection to naive glial cells. These data confirm that JCV nonproductively infects B cells and possibly uses them as a vehicle for transmigration across the blood-brain barrier.

Transcription Initiation Sites of Prototype and Variant JC Virus Early and Late Messenger RNAs

The locations of the authentic viral transcription initiation sites utilized during the course of a JCV(Mad1) infection of primary human fetal glial (PHFG) cells have been identified using the S1 nuclease and primer extension techniques. Early viral mRNA start sites were located 20 to 30 nucleotides (positions 89-92 and 5115-5125) downstream from the two TATA elements present within the promoter-enhancer region of the JCV(Mad1) strain. Absence of the distal TATA element, as seen in the JCV(Mad4) promoter-enhancer, resulted in the alteration of the position and intensity of these sites. Several start sites were observed for late viral mRNAs; two sites were located 25 and 35 nucleotides (positions 191-192 and 200-203) downstream from a potential surrogate TATA signal. To determine whether the presence of constitutively expressed JCV large T protein would alter the pattern of transcription initiation, RNA isolated from JCV(Mad1)-infected POJ cells was analyzed. Although the positions of the early and late viral mRNA start sites were similar to those observed in JCV(Mad1)-infected PHFG cells, they were detected earlier in the course of the infection.

Novel second-site suppression of a cold-sensitive defect in phage P22 procapsid assembly

The DNA packaging portal of the phage P22 procapsid is formed of 12 molecules of the 90,000 dalton gene 1 protein. The assembly of this dodecameric complex at a unique capsid vertex requires scaffolding subunits. The mechanism that ensures the location of the 12-fold symmetrical portal at only one of the 12 5-fold vertices of an icosahedral virus capsid presents a unique assembly problem, which, in some viruses, is solved by the portal also acting as initiator of procapsid assembly. Phage P22 procapsids, however, are formed in the absence of the portal protein. The 1 − csH137 mutation prevents the incorporation of the portal protein into procapsids. In a mixed infection with cs + phage, the mutant subunits are able to form functional portals, suggesting that the cold-sensitivity does not affect portal-portal interactions, but affects the interaction of portal subunits with some other molecular species involved in the initiation of portal assembly. Interestingly, the cs defect is suppressed by temperature-sensitive folding mutations at four sites in the P22 tailspike gene 9. The suppression is allelespecific; other tailspike tsf mutations fail to suppress the cs defect. Translation through a suppressor site is required for suppression. This observation is unexpected, since analysis of nonsense mutations in this gene indicates that it is not required for procapsid assembly. Examination of the nucleic acid sequences in the neighborhood of each of the suppressor sites shows significant sequence similarity with the scaffolding gene translational initiation region on the late message. This supports a previously proposed model, in which procapsid assembly is normally initiated in a region on the late messenger RNA that includes the gene 8 start site. By this model, the suppressor mutations may be acting through protein-RNA interactions, changing sequences that identify alternative or competing sites at which the mutant portal subunits may be organized for assembly into the differentiated vertex of the phage capsid.

Bacteriophage P2 late promoters: II. Comparison of the four late promoter sequences

The late genes of bacteriophage P2 are clustered into four transcription units. We have reported the transcription initiation sites for two of the late messenger RNAs, encoding genes QP and ONMLKRS. We have now located the 5′ ends of the two remaining late mRNAs. The first gene in the VJHG transcription unit has been located by DNA sequence determination of the single nucleotide change in a V amber mutant. Location of the first gene in the FETUD transcription unit has been inferred from the DNA sequence. The 5′ ends of the mRNAs for these two transcription units were located by protection of end-labeled restriction fragments in RNA-DNA hybrids from digestion with nuclease S 1. Similar protection of hybrids using RNA that had been 5′ end-labeled with [ α- 32P]GTP and guanylyl transferase confirmed that these 5′ termini resulted from initiation of transcription. The DNA sequences preceding the P2 late transcription starts are different from the Escherichia coli promoter consensus sequences at −10 and −35, consistent with the apparent requirement for phage-encoded proteins in the regulation of P2 late gene expression. The four P2 late promoters do share sequence homologies in the −10 and −35 regions, however, and several additional homologies further upstream. P2 late gene expression also appears to involve negative regulation by a product of the ONMLKRS gene cluster. When cells are infected with P2 polar O amber mutants, a marked increase in the levels of proteins encoded by the other three gene clusters is observed. This increase is reflected in the amounts of late mRNAs, suggesting that RNA synthesis is normally repressed or that late mRNAs are more labile in the presence of a gene product from the ONMLKRS transcription unit. Satellite phage P4 induces P2 late gene expression without the usual requirement for P2 DNA replication. The 5′ ends of the P2 late mRNAs are the same during P4 transactivation as during normal P2 late gene expression. Thus, the regulation of P2 late gene expression by P4 does not involve altered promoter selection.

Simian virus 40-infected, interferon-treated cells contain 2‘,5‘-oligoadenylates which do not activate cleavage of RNA.

2',5'-oligoadenylates can be assayed sensitively in cell extracts by use of an antiserum having maximum specificity for any compound containing the moiety -pA2'pA2'pA-. These compounds reached high concentrations (25-2000 nM) in monkey CV-1 cells after infection with simian virus 40 (SV40) and treatment with human leukocyte interferon. The levels were highest late in infection and increased in parallel with the accumulation of SV40 late messenger RNAs. Alone, neither interferon nor SV40 caused the 2',5'-oligoadenylate concentrations to increase above the levels present in untreated CV-1 cells, 3 nM or less. Analyses by high performance liquid chromatography revealed little or no (p)pp(A2'p)2A or (p)pp(A2'p)3A, and the extracts showed only very low activity in functional assays with ppp(A2'p)nA-dependent nucleases, equivalent to 3 nM ppp(A2'p)3A or less. Some of the 2',5'-oligoadenylates eluted in the positions of the nonphosphorylated "cores," (A2'p)nA, and a substantial fraction was found in several peaks intermediate between ppp(A2'p)3A and cores. The positions of most of these peaks did not change when digestion with alkaline phosphatase was performed before chromatography, indicating that most of the 2',5'-oligoadenylates lack exposed phosphate groups. In contrast to the effects of infection with SV40, addition of poly(I) X poly(C) to interferon-treated CV-1 cells led to accumulation of high levels (up to 3000 nM) of 2',5'-oligoadenylate-5'-di- or triphosphates capable of activating the ppp(A2'p)nA-dependent ribonuclease.

Proteins encoded near the adenovirus late messenger RNA leader segments

Small fragments of adenovirus 2 DNA cloned into the single-strand phage M13 were used to select adenoviral messenger RNAs transcribed from the R-strand between map positions 16 and 30. Cell-free translation of these mRNAs produced proteins of 13.5K, 13.6K, and 11.5K, respectively encoded between the first and second segments of the tripartite major late leader, within the “i”-leader segment, and immediately preceding the third leader segment. Partial sequence analysis of the 13.6K protein is consistent with the hypothesis that it is encoded within the Header segment.

Alternative splicing pathways exist in the formation of adenoviral late messenger RNAs

Total rapidly labeled RNA was extracted from nuclei of HeLa cells late after infection with adenovirus type 2. Most of this nuclear RNA is transcribed from the major late transcription unit (16·2 to 100·0 map units (m.u.)). To study the cleavage reactions that are involved in RNA splicing, we used the S 1 nuclease mapping technique with HinIII B (16·8 to 31·5 m.u.) and XhoI F (15·5 to 22·4 m.u.) restriction fragments as DNA probes. The S 1 mapping data showed that both intron 1 (16·3 to 19·1 m.u.) and intron 2 (19·4 to 26·2 m.u.) can be excised in more than one step. Kinetic labeling and pulse-chase experiments demonstrated that certain cleavage sites in the RNA are used within three minutes after the start of transcription, while other cleavages occur only after a significant time-lag. The use of 5,6-dichloro-1-β- d-ribofuranosylbenzimidazole enabled us to label the nuclear RNA exclusively from the 5′ end. Such an approach showed that the first cleavages are introduced in the nascent RNA before the RNA polymerase has passed more than 2000 nucleotides beyond the cleavage site. The chronology of the appearance of processing intermediates that results from cleavage of the RNA indicates that preferentially intron 1 is removed before intron 2. This is in agreement with the finding that leader 1 is ligated to leader 2 before ligation of leader 2 to leader 3 takes place. However, we have found that alternative splicing pathways exist in the excision of introns 1 and 2, demonstrating that cleavage in intron 2 may occur before intron 1 is attacked by the splicing enzymes.

RNA synthesis in isolated nuclei: Processing of adenovirus serotype 2 late messenger RNA precursors

The mechanisms of formation of the 3′ ends of the adenovirus serotype 2 late transcripts have been investigated. RNA samples synthesized in isolated nuclei were studied by pulse-chase analysis followed by nuclease S 1 mapping. The results suggest that the 3′ ends of different precursor RNAs derived from a primary transcript may be formed by different mechanisms. The 3′ ends of the abundant messenger RNAs for the virion protein hexon and the 100K M r protein (the L3 and L4 families) are formed by cleavage of larger precursors. The cleavages appear to be endonucleolytic and do not require ongoing transcription. However, cleavage is tightly coupled with the subsequent polyadenylation of the RNA 3′ end: no non-polyadenylated 3′ end could be detected. In contrast, late RNAs with 3′ ends located at 38.5 map units (the L1 family) are not created by this mechanism. Although RNA species with 3′ ends at these positions are synthesized during a pulse label and, in fact, throughout an entire incubation in vitro, their synthesis depends on ongoing transcription. At best, only very low levels of splicing can be detected in the isolated nuclei. The implications of these results are discussed.

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.

Visualization of the major late r strand transcription unit of adenovirus serotype 2

Using chromatin spreading for electron microscopy, we have visualized the major late r strand transcription unit of adenovirus serotype 2 in a transcriptionally active state. Adenovirus 2 genomes were identified in the dispersed nuclear contents of infected HeLa cells by their discrete length (8 to 12 μm), relatively smooth deoxyribonucleoprotein configuration, and occurrence as both Y-form and linear form replicative intermediates. At 16 hours after infection, near the time of maximal synthesis of late messenger RNA, any one genome was likely to exhibit either several or no nascent transcripts. The percentage of adenovirus 2 genomes utilized as transcriptional templates appeared low and was not limited to non-replicating molecules. Analysis of individual transcribing viral genomes revealed an average of 3.9 transcripts per adenovirus 2 genome (ranging between 1 and 15; n = 51) and, in general, single gradients of fibril length. Linear regression analyses on 20 genomes with four or more transcripts yielded an average extrapolated promoter site at adenovirus map unit 16.6, very near the previously determined promoter for the late r strand transcription unit of 16.4. Both individual genomes and a constructed “composite genome” exhibited increasing gradients of fibril length from the promoter to the distal end of the genome, suggesting that only one large transcription unit was active, and that transcription was completed before discernible RNA cleavage events. Viral transcripts exhibited a high degree of ribonucleoprotein packaging and a secondary structure suggesting intramolecular interactions. The low transcript density on the viral genomes was similar to that seen on host heterogeneous nuclear RNA transcription units. A calculation is presented that indicates that the observed frequency of transcription is sufficient to account for late viral mRNA production.

Construction of an SV40-derived cloning vector

A new reiterated variant of simian virus 40 (SV40; dl1142) has been constructed. It should be useful for the purpose of cloning foreign pieces of DNA in SV40 virions. Up to 80% of the SV40 genome has been made available for substitution with foreign DNA and the vector contains a number of unique (single-cut) restriction sites which will facilitate cloning. The 5' and 3' regions of both the SV40 early and late messenger RNAs are included in the vector. Prokaryotic DNA has been successfully cloned in the early region of the vector. The transcriptional properties of the recombinant have been studied, and it was found that both the vector and insert DNA are transcribed, mainly as non-adenylated RNA.

Translational discrimination against bacteriophage T7 gene 0·3 messenger RNA

Based on genetic manipulation of T7 late messenger RNA levels in vivo, we previously hypothesized that wild-type T7 infection of Escherichia coli develops in mRNA excess and that there is translational discrimination against T7 gene 0·3 mRNA (Strome & Young, 1978). The results presented here support our hypothesis. The discrimination against 0·3 mRNA translation observed in vivo can be mimicked in a cell-free system by increasing the concentration of T7 RNA beyond the level needed to saturate the translational machinery or by translating T7 RNA with a low concentration of ribosomes. This discrimination can be overcome by adding ribosomes to the cell-free system (increasing the ribosome to mRNA ratio) or by slowing the rate of polypeptide chain elongation. In addition 0·3 mRNA activity as well as a substantial fraction of T7 late mRNA activity is found to be shifted off of polysomes late in T7 infection. Our results are indicative of a low initiation rate constant for 0·3 mRNA compared to T7 late mRNAs.

Determination and analysis of adenovirus-2 DNA sequences which may include signals for late messenger RNA processing

Nucleotide sequences have been determined for regions of the adenovirus-2 genome from which the three principal leaders of late messenger RNA are transcribed. We have also sequenced the region which surrounds a fourth leader only on the fiber mRNA and the region of the genome immediately preceding the translated portion. These sequences were examined directly for repeated features and were also used to draw secondary structure models. Our study of these sequences has revealed specific repeated primary and secondary structural features at both the 5′ and 3′ ends of each of the conserved RNA segments present in adenovirus-2 late mRNA transcripts.

Histone gene expression during sea urchin embryogenesis: Isolation and characterization of early and late messenger RNAs of Strongylocentrotus purpuratus by gene-specific hybridization and template activity

We have examined the molecular mechanisms responsible for the shifts in histone protein phenotype during embryogenesis in the sea urchin Strongylocentrotus purpuratus. The H1, H2A, and H2B classes of histone synthesized at the earliest stages of cleavage are heterogeneous: These proteins are replaced at late embryogenesis by a different set of histone-like polypeptides, some of which are also heterogeneous. The H3 and H4 histones appear to be homogeneous classes and remain constant. We have isolated from both early and late embryos the individual messenger RNAs coding for each of the multiple protein subtypes. The RNAs were isolated by hybridization to cloned DNA segments coding for a single histone protein or by elution from polyacrylamide gels. Each RNA was then analyzed and identified by its mobility on polyacrylamide gels and by its template activity in the wheat germ cell-free protein synthesizing system. The mRNAs for each of the five early histone protein classes are heterogeneous in size and differ from the late stage templates. The late mRNAs consist of at least 11 separable types coding for the 5 classes of histones. Each of the 11 has been separated and identified. The late stage proteins were shown to be authentic histones since many of their templates hybridize with histone coding DNA. The early and late stage mRNAs are transcribed from different sets of histone genes since (1) late stage H1 and H2A mRNAs fail to hybridize to cloned early stage histone genes under ideal conditions for detecting homologous early stage hybrids, (2) late stage H2B, H3, and H4 RNA/DNA hybrids melt at 14, 11, and 11°C lower, respectively, than do homologous RNA/DNA hybrids, and (3) purified late stage mRNAs direct the synthesis of the variant histone proteins which are synthesized only during later stages. The time course of synthesis of the late stage mRNAs suggests that they appear many hours before the late histone proteins can be detected, possibly as early as fertilization. In addition, early mRNAs are synthesized in small quantities as late as 40 hr after fertilization, during gastrulation. Thus, the major modulations of histone gene expression are neither abrupt nor an absolute on-off switch, and may represent only a gradual and relative repression of early gene expression. Two histones are detected only transiently during early cleavage. The mRNA for one of them, a subtype of H2A, can be detected in the cytoplasm for as long as 40 hr after fertilization. However, template activity for the other, a subtype of H2B, can be detected only at the blastula stage. Thus, the histone genes represent a complex multigene family that is developmentally modulated.

Nucleotide sequence at the 5' terminus of adenovirus 2 late messenger RNA.

One major late promoter is transcriptionally active for the majority of adenovirus 2 late RNA synthesis. Nucleotide sequences proximal to this promoter are spliced onto the 5' termini of all messenger RNAs generated from it. We report here the complete nucleotide sequence of the capped 5-'terminal undecanucleotide common to these late mRNAs. The sequence m7G5'ppp5'm6AmCUCUCUUCCGp shows striking similarities to the 5'-terminal sequences of the eukaryotic alpha- and beta-globin mRNAs and contains the sequence 5'CUUCCG3' complementary to a six-base sequence near the 3' terminus of most eukaryotic 18 S ribosomal RNAs. On the basis of this sequence and the finding that this same sequence is present at the 5' end of adenovirus 2 late nuclear RNA precursor, it should now be possible to locate the major late promotor within the adenoviral genomic DNA sequence.

Nucleotide Sequence of the Simian Virus 40 Hind II + III Restriction Fragment J and the Total Amino Acid Sequence of the Major Structural Protein VP1

The HindII + III restriction fragment J (Hind-J) represents 4.58% of the simian virus 40 genome. The information present in Hind-J is expressed as part of the major, late 16-S messenger RNA, which codes for the structural protein VP1. The nucleotide sequence of the 240-base-pairs-long Hind fragment J has been determined by analysis of each oligonucleotide from both strands resulting from T1 or pancreatic RNase digestion of RNA transcribed from the DNA and from RNase digestion of ribo-substituted DNA. Large oligonucleotide blocks which could be constructed mainly on the basis of complementarity were subsequently ordered by partial chemical degradation of terminally labeled DNA. This direct DNA sequencing approach also completely confirmed the results obtained by both aforementioned RNase degradation methods. In the strand with the same polarity as the late mRNA, triplets corresponding to termination codons are present in two of the three reading frames. The one open reading frame connects in phase with the open reading frame of the neighboring HindII/ III fragments K, F and G, which have been published previously and which together with Hind-J span the total VP1 gene. Some features of the primary nucleotide sequence of this VP1 gene and the derived VP1 amino acid sequence are discussed.

RNA structures near poly(A) of adenovirus-2 late messenger RNAs

RNA sequences near poly(A) of adenovirus-2 late messenger RNAs have been mapped within the Ad2 genome and compared by T 1 ribonuclease fingerprinting. Polyadenylated viral mRNAs transcribed 14 to 17 hours post-infection were prepared highly labeled with 32P, and then fractionated by hybridization to restriction fragments of Ad2 DNA or by gel electrophoresis. The 3′-terminal regions of purified mRNAs were isolated by partially degrading the mRNA with alkali or T 1 ribonuclease to an average length of ~100 to 200 residues, and selecting fragments with poly(A) by poly(U)-Sepharose chromatography. Six different 3′-terminal structures, all derived from rightward transcripts, were identified. The map positions of these structures were initially deduced by establishing the fingerprints of sequences near poly(A) of mRNAs hybridized to DNA restriction fragments with known map co-ordinates. In four cases the DNA templates for the termini were located more precisely by identifying short restriction fragments of known map position which encode, in vitro, large T 1 oligo-nucleotides found in vivo near poly(A). Five 3′ termini map within restriction fragments Sma1 I/ HinIII J (co-ordinates 37.3 to 40.5), Sma1 D/ Kpn D (47.4 to 52.6), EcoRI B (58.5 to 70.7), EcoRI D (75.9 to 83.4), and Sma1 C/ Eco R1 C (89.7 to 91.9). These 3′ termini fall within the boundaries of the major Ad2 late transcription unit (co-ordinates ~16 to 100; Evans et al., 1977). The sixth terminus maps outside this region in Sma1 M (11.2 to 11.6) and thus is transcribed from a second transcription unit which also functions late in infection. Each of the six 3′ termini yielded large T 1 oligonucleotides which were specific to the particular 3′-terminal region, rather than common to different structures. The hexanucleotide sequence A-A-U-A-A-A, which is located near poly(A) of several previously studied eukaryotic messengers (Proudfoot & Brownlee, 1976), is present in at least two of the Ad2 3′-terminal structures, and potentially in all six. The RNA sequences surrounding the hexanucleotide are not identical in the different viral cases, although these mRNAs are transcribed from a single DNA genome. The location of Ad2 mRNA 3′-terminal sequences within the viral genome is discussed in terms of the Ad2 late mRNA map and a hypothesis for the role of 3′ terminus formation in Ad2 mRNA synthesis.

The Genome of Simian Virus 40

The nucleotide sequence of SV40 DNA was determined, and the sequence was correlated with known genes of the virus and with the structure of viral messenger RNA's. There is a limited overlap of the coding regions for structural proteins and a complex pattern of leader sequences at the 5' end of late messenger RNA. The sequence of the early region is consistent with recent proposals that the large early polypeptide of SV40 is encoded in noncontinguous segments of DNA.

Overlapping of the VP 2-VP 3 gene and the VP 1 gene in the SV40 genome

The nucleotide sequence of the SV40 Hind E fragment has been determined mainly by the partial chemical degradation procedure of Maxam and Gilbert (1977). The sequence of the strand with the same polarity as the late messenger RNA shows only one open reading frame for translation. Considering that VP 3 corresponds to the carboxyl terminal part of VP 2, and considering various evidence which indicates that the SV40 Hind E segment is part of the VP 2-VP 3 gene, we conclude that the open reading frame mentioned above codes for part of the amino acid sequence of VP 2-VP 3. It continues clockwise in Hind K, where it terminates with a UAA signal. The latter is located 110 nucleotides beyond the initiation signal for the major structural protein VP 1 ( Fiers et al., 1975; Van de Voorde et al., 1976). Hence this small overlapping region of the genome codes for the synthesis of three different proteins in two different reading frames. The deduced amino acid sequence covers a major part of the VP 3 polypeptide, and the amino acid composition is in good agreement with published values ( Greenaway and Levine, 1973).