Nuclear Viral RNA Research Articles

Fluorescent In Situ Detection of RNA-Protein Interactions in Intact Cells by RNA-PLA.

RNA-protein proximity ligation assay (RNA-PLA) enables the detection of specific RNA-protein interactions in fixed cells. In RNA-PLA, bridging and ligation of a circular DNA template occurs if the target RNA and protein are within 40 nanometers of each other. The resulting circular template is amplified by rolling circle amplification and abundantly recognized by fluorescent antisense DNA oligonucleotides. This strategy therefore enables localization of RNA-protein interactions in situ with high specificity and sensitivity. Here, we describe the use of RNA-PLA to detect interactions between a nuclear viral RNA and a host RNA-binding protein in Epstein-Barr virus (EBV)-infected B cells.

The Nuclear Matrix Protein SAFA Surveils Viral RNA and Facilitates Immunity by Activating Antiviral Enhancers and Super-enhancers

Pathogen pattern recognition receptors (PRRs) trigger innate immune responses to invading pathogens. All known PRRs for viral RNA have extranuclear localization. However, for many viruses, replication generates dsRNA in the nucleus. Here, we show that the nuclear matrix protein SAFA (also known as HnRNPU) functions as a nuclear viral dsRNA sensor for both DNA and RNA viruses. Upon recognition of viral dsRNA, SAFA oligomerizes and activates the enhancers of antiviral genes, including IFNB1. Moreover, SAFA is required for the activation of super-enhancers, which direct vigorous immune gene transcription to establish the antiviral state. Myeloid-specific SAFA-deficient mice were more susceptible to lethal HSV-1 and VSV infection, with decreased type I IFNs. Thus, SAFA functions as a nuclear viral RNA sensor and trans-activator to bridge innate sensing with chromatin remodeling and potentiate robust antiviral responses.

Nuclear-resident RIG-I senses viral replication inducing antiviral immunity

The nucleus represents a cellular compartment where the discrimination of self from non-self nucleic acids is vital. While emerging evidence establishes a nuclear non-self DNA sensing paradigm, the nuclear sensing of non-self RNA, such as that from nuclear-replicating RNA viruses, remains unexplored. Here, we report the identification of nuclear-resident RIG-I actively involved in nuclear viral RNA sensing. The nuclear RIG-I, along with its cytoplasmic counterpart, senses influenza A virus (IAV) nuclear replication leading to a cooperative induction of type I interferon response. Its activation signals through the canonical signaling axis and establishes an effective antiviral state restricting IAV replication. The exclusive signaling specificity conferred by nuclear RIG-I is reinforced by its inability to sense cytoplasmic-replicating Sendai virus and appreciable sensing of hepatitis B virus pregenomic RNA in the nucleus. These results refine the RNA sensing paradigm for nuclear-replicating viruses and reveal a previously unrecognized subcellular milieu for RIG-I-like receptor sensing.

A novel nuclear export activity in HIV-1 matrix protein required for viral replication.

An important aspect of the pathophysiology of human immunodeficiency virus type-1 (HIV-1) infection is the ability of the virus to replicate in non-dividing cells. HIV-1 matrix (MA), the amino-terminal domain of the Pr55 gag polyprotein (Pr55), bears a nuclear localization signal that promotes localization of the viral preintegration complex to the nucleus of non-dividing cells following virus entry. However, late during infection, MA, as part of Pr55, directs unspliced viral RNA to the plasma membrane, the site of virus assembly. How MA can mediate these two opposing targeting functions is not understood. Here we demonstrate that MA has a previously undescribed nuclear export activity. Although MA lacks the canonical leucine-rich nuclear export signal, nuclear export is mediated through the conserved Crm1p pathway and functions in both mammalian cells and yeast. A mutation that disrupts the MA nuclear export signal (MA-M4) mislocalizes Pr55 and genomic viral RNA to the nucleus, thereby severely impairing viral replication. Furthermore, we show that MA-M4 can act in a dominant-negative fashion to mislocalize genomic viral RNA even in the presence of wild-type MA. We conclude that the MA nuclear export signal is required to counteract the MA nuclear localization signal, thus ensuring the cytoplasmic availability of the components required for virion assembly.

Interaction of adenoviral E4 and E1b products in late gene expression

The adenovirus 294R protein of E4 ORF 6 forms a physical and functional complex with the 496R protein product of E1b. The E4 294R ORF 6 protein also functions in parallel with the E4 116R ORF 3 protein in viral late protein synthesis. We have examined the roles of these three proteins and the protein complex in viral late protein synthesis and late message metabolism, by comparing the phenotypes of E4 294R − , E4 116R −, and E1b 496R − mutants to those of a series of double mutants. Our data indicate that the 294R and 116R proteins act in parallel to permit the accumulation of normal levels of unprocessed late viral RNA in the nucleus of infected cells. Both 294R and 496R function in parallel with the 116R protein in viral nuclear RNA accumulation, but do so to different degrees, suggesting that 294R and 496R may have roles apart from the functional complex in mediating accumulation of viral messages in the nucleus, or that they have nonequivalent roles within the complex. Our results are also consistent with a role for the 496R/294R protein complex in mediating efficient transport of late messages from the nucleus to the cytoplasm and/or in maintaining the stability of those messages on reaching the cytoplasm, as suggested previously S. Pilder, M. Moore, J. Logan, and T. Shenk, 1986, Mol. Cell. Biol. 6, 470–476). Finally the 116R protein seems to act in parallel with the complex to permit normal viral DNA replication.

Alterations in early adenovirus transcription and mRNA abundance induced by translational inhibitors.

At least six viral genes are activated early after adenovirus 2 infection of HeLa cells. In an effort to define more clearly the effects of protein synthesis inhibition on early viral gene expression, the rate of transcription and the cytoplasmic abundance of viral mRNA from each transcription unit were studied in infected cells treated with translational inhibitors added at different times during infection and at concentrations known to inhibit 95 to 99% of protein synthesis. Tritiated uridine-labeled nuclear and cytoplasmic viral RNA molecules synthesized in infected HeLa cells in both the presence and absence of inhibitors were analyzed and compared by hybridization using specific DNA probes. Translational inhibitors (25 micrograms/ml of cycloheximide or 10 microM anisomycin) added 30 min to 1 h after infection stimulated the over-accumulation of cytoplasmic viral mRNA from 3- to 20-fold, early regions E1A, E2, and E4 being affected most. This overproduction of viral mRNA was found to result from enhanced transcription of the respective early genes. Treatment of cells with 100 microM anisomycin 1 h prior to and during infection did not prevent the accumulation of early viral mRNA molecules; however, total cellular RNA synthesis was drastically inhibited in 100 microM anisomycin-treated cells. These results support the hypothesis that a cellular factor may function in the regulation of early viral gene expression at a transcriptional level.

5′ termini of polyoma virus early region transcripts synthesized in vivo by wild-type virus and viable deletion mutants

The 5′ termini of polyoma virus early region transcripts synthesized during the productive infection of permissive mouse cells by wild-type or tsa virus, and those expressed in a variety of transformed rodent cell lines, have been mapped on the viral genome. Results were obtained using the S 1 nuclease and primer extension gel mapping procedures. Principal 5′ ends of cytoplasmic polyadenylated mRNAs in every instance mapped between nucleotides 145 and 156 (numbering according to Soeda et al., 1980) in the DNA sequence, 17 to 28 base-pairs before the translational initiation codon for early proteins and 26 to 37 base-pairs after a sequence agreeing with the TATA box consensus. Our data implied a minimum of two different termini with this region of the genome, at nucleotide 147 ± 2 and at 152 ± 2. The 5′ ends at 147 ± 2 were particularly common in the mRNA overproduced after thermal inactivation of the large T protein at late times during infection. The sequences determining the principal 5′ termini, unlike the analogous sequences in the closely related simian virus 40 (SV40), are distinct from those involved in the viral origin of DNA replication. A number of minor alternative 5′ termini of cytoplasmie mRNAs. located both before and after the principal 5′ ends, were also detected. Of those downstream from the principal termini, one at nucleotide 300 ± 2 was prominent. Although this apparent 5′ end is well within the early protein coding sequence, it occurs at a position 31 ± 2 base-pairs after a second TATA box. The several minor apparent 5′ termini mapping upstream of the principal termini occurred primarily in the vicinity of the highly conserved papovavirus DNA replication origin sequence. This sequence includes a third TATA box. Two of the minor 5′ termini, at 14 ± 2 and at 20 ± 2. were near the consensus distance from this TATA box, but the others mapped within or before it. Early region mRNA extracted at late times from the cytoplasm of cells infected with wild-type virus, or with tsa virus at the permissive temperature, was usually (but not invariably) enriched for RNA species with apparent 5′ termini mapping in the replication origin region, as well as for even longer RNAs. Such RNAs were correctly spliced and had the normal polyadenylated 3′ ends. They were very minor in the cytoplasmic mRNA overproduced after thermal inactivation of the large T protein at late times during infection, but the nuclear RNA from these cells comprised giant species with highly heterogeneous apparent 5′ ends, including predominantly those in the origin region and others further upstream. Nuclear viral RNA from most transformed cell lines lacked the giant species and had principal 5′ termini in the nt145 to 156 region. We consider two models to account for the presence of the longer mRNAs in the cytoplasm of infected cells at late times during infection. The first postulates a shift in transcriptional initiation sites to upstream positions because of the repressor action of the large T protein. The second, which we favour, proposes that the longer species occur because of inefficient transcriptional termination, which leads to transcription around the entire circular genome and consequently to the eventual accumulation of long cleavage products in the cytoplasm. We further studied the mRNAs expressed by three viable deletion mutants (Bendig et al., 1980) that lack all or part of the sequence determining the principal 5′ termini and the TATA box that precedes it. These efficiently synthesized early region mRNA with slightly or highly heterogeneous 5′ ends. Two of the mutants (dl-75 and dl-17) produced mRNAs with principal alternative 5′ ends located slightly before or after the deletions. The 5′ ends of these mutant mRNAs corresponded to those of very minor transcripts of wild-type templates. These results suggest that sequence information other than the TATA box has an important role in specifying the approximate position of mRNA 5′ ends.

Analysis of endogenous avian retrovirus DNA and RNA: Viral and cellular determinants of retrovirus gene expression

We have characterized endogenous avian retrovirus DNAs and RNAs in uninfected white leghorn chicken embryos. The embryos we studied include representations of four phenotypic classifications of chickens (gs − chf − , gs + chf + , gs − chf +, and V +), and contained the endogenous proviruses evl, 2, 3, 4, 5, 6, 8, 9, and 15. We confirmed the previously described phenotypes and restriction maps of evl, 3, 4, 5, 6, 8, and 15, and we confirmed the phenotype for ev9 and derived a restriction map for this locus. Evaluation of the structure of the individual proviruses and characterization of the nuclear and cytoplasmic viral RNA isolated from embryos of defined genotype yielded the following major conclusions. (i) Viral RNA could not be detected for ev4, ev5, ev8, or ev15. (ii) The amount of stable RNA produced from evl, ev3, ev6, or ev9 varied over a range of at least 100- to 300-fold and was at least 10- to 1000-fold lower than the amounts found in cells productively infected with exogenous avian retroviruses. (iii) A deletion in the provirus at ev3 can account for three abnormalities: the production of a viral protein that results from fusion of internal regions of the gag and pol genes, the abnormal structure of a subgenomic viral RNA, and failure of the abnormal RNA to appear in the cytoplasm. (iv) A deletion in the provirus at ev6 gives rise to at least two major anomalies: the initiation of transcription at an upstream cellular promoter, and the abnormal metabolism of an ev6 viral RNA. (v) ev9 has an ostensibly normal structure; nevertheless, the metabolism of an RNA arising from this locus is aberrant. We conclude that the expression of endogenous retrovirus genes in chickens is subject to control by both viral and cellular determinants.

Genome distribution of adenovirus total and self-complementary nuclear RNA at early times

The study of adenovirus types 2 and 5 viral nuclear RNAs present at early times during a productive infection was analyzed by hybridization of pulse-labeled RNA to restriction fragments of Ad2 and Ad5 DNA. The RNA studied was the total nuclear infected cell population and the double-stranded nuclear RNAs formed after self-annealing, RNase digestion, and selection by Sephadex chromatography. In addition, the viral RNAs were synthesized in the presence and absence of cycloheximide (to restrict the infection to the early phase) at 32° and 41° (to provide control conditions for the H5ts125 mutant defective in the initiation of viral DNA synthesis at the restrictive temperature). The results of these studies established that (1) transcription of the genome is not qualitatively, but is quantitatively, affected by the use of inhibitors of viral DNA synthesis, (2) all regions of the genome are abundantly transcribed including those regions that do not code for early viral mRNA, and (3) most of these non-early messenger RNAs (which include both late mRNA and anti-sense sequences) have been post-transcriptionally modified by polyadenylation. Additionally, liquid hybridization studies of early Ad2 viral nuclear RNA to separated strands of Ad2 Eco (R1-A) fragment DNA indicate that all of these sequences (both sense and anti-sense) are represented in the RNA population and that neither the l nor r strands are preferentially transcribed. All of these results show that self-complementary RNA transcription and the transcription of non-early mRNAs is an integral part of the productive infection of human KB cells by adenovirus 2 and 5. Some of the possible functions of these sequences are discussed.

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.

Efficiency of processing of viral RNA during the early and late phases of productive infection by polyoma virus.

The efficiency of processing of polyoma viral RNA and of its export from nucleus to cytoplasm was measured in primary mouse kidney cells by comparing the initial rates of incorporation of [3H]uridine into cytoplasmic and nuclear viral RNA. Appropriate methods of cell fractionation were chosen to maximize yields of cytoplasmic RNA and to minimize leakage of nuclear RNA. Incorporation of [3H]uridine into cellular 4S RNA in the cytoplasm was followed to monitor pool equilibration and maintenance of an excess of radioactive precursor throughout the experimental period. During the early phase of infection (9 to 11 h, in the presence of 5-fluorodeoxyuridine), viral RNA was rapidly and efficiently exported from nucleus to cytoplasm. Viral RNA appeared in the cytoplasm within 6 min of its synthesis, greater than half of the viral RNA synthesized in the nucleus was exported to the cytoplasm. In contrast, during the late phase of infection (28 to 30 h), viral RNA was exported more slowly, appearing in the cytoplasm 12 to 20 min after its synthesis, and much less efficiently-only 5% of late nuclear transcripts was exported. The poor efficiency of processing of late viral RNA may be, in part, a result of (i) the presence in nuclear transcripts of non-mRNA sequences which are removed during processing; (ii) the presence in nuclear transcripts of multiple copies of mRNA sequences, only one of which is incorporated into mature mRNA; and (iii) inefficient polyadenylation of viral nuclear RNA.

Characterization of intracellular viral RNA in interferon-treated cells chronically infected with murine leukemia virus.

We have recently found that Moloney murine leukemia virus assembles within cytoplasmic vacuoles of chronically infected NIH/3T3 cells rather than at their surface (submitted for publication). In the present study we found that if these cells were treated with interferon (IF) for 24 to 48 h the intracellular virus particles accumulated at a two- to threefold-higher level than that observed in untreated cells. Nevertheless, despite this accumulation, no difference between IF-treated and untreated cells was observed in the amount of the total cytoplasmic viral RNA or in its 35S or 21S species. When cellular virions were sedimented from the cytoplasmic fraction, a markedly higher amount of viral RNA was detected in the viral pellet of IF-treated cells than was detected in untreated cells, whereas the amount of viral RNA left in the virus-free cytoplasm of IF-treated cells was much lower than that in the untreated cells. Furthermore, the amount of the cytoplasmic polyriboadenylic acid-containing viral RNA was also remarkably higher in the IF-treated cells. Viral polyribosomes appeared to be fully functional in IF-treated cells, since no effect of IF on viral protein synthesis could be detected. Analysis of the nuclear viral RNA showed no difference between IF-treated and untreated cells after 24 h of IF treatment. Both contained a comparable amount of 35S viral RNA. However, at 48 h a significant accumulation of viral RNA was observed in the nucleus of the IF-treated cells as compared with the untreated cells, although in both cases only 35S species were evident. This accumulation appeared to activate a degradation process which destroyed nuclear viral RNA, since a dramatic shift toward smaller-sized molecules of viral RNA and a remarkable reduction in its amount were observed after 72 h of IF treatment.

Evidence for the role of double-helical structures in the maturation of simian virus-40 messenger RNA.

Simian Virus-40 infected BSC-1 cells were pretreated with glucosamine and briefly pulsed with [3H]-uridine. The labeling can be halted instantaneously by the addition of cold uridine and glucosamine. Under these pulse-chase conditions, the inhibitory effects of the intercalating agent proflavine on the processing of prelabeled nuclear RNA precursors were examined in vivo. Proflavine inhibits the cleavage of viral nuclear RNA precursors. However, turnover of the mature viral mRNAs in the cytoplasm is not inhibited. The effect of proflavine on processing is not a secondary consequence of its inhibition of protein synthesis. The data suggest that base-paired secondary structures in the primary transcripts are important processing signals in the generation of viral mRNA molecules.

RNA synthesis in isolated nuclei: Identification and comparison of adenovirus 2 encoded transcripts synthesized in vitro and in vivo

Nuclei isolated from HeLa cells 16 hours post infection by adenovirus serotype 2 synthesize high levels of viral specific RNA in vitro. This RNA shares many features in common with nuclear viral specific RNA synthesized in vivo. The size of the RNA is heterogeneous, although the bulk of the transcripts are 10 × 10 3 to 25 × 10 3 bases in length. The 5′ ends of transcripts synthesized in vitro that are colinear with the viral DNA map primarily at two co-ordinates: 16.5, the site of the major late promoter, and 19.5, the location of the first splice-point used in synthesis of late viral mRNA. Colinear transcripts with 5′ ends which map at sites corresponding to the locations of other splice-points are also labeled but are much less abundant. In particular, only low levels of fully spliced messenger RNA are produced after two hours of incubation in vitro. A fraction of the transcripts synthesized in vitro appears to extend all the way to the end of the genome. However, discrete RNA species with 3′ ends at 38.5, 50.0, 61.5 and 78.5 map units are also synthesized in vitro. Molecules with these co-ordinates are also detected in steady-state nuclear RNA in vivo. These four species are all accurately polyadenylated in vitro, although the efficiencies of polyadenylation vary: RNA molecules with 3′ ends at position 61.5 or 78.5 only accumulate in a poly(A) + form, while species terminated at co-ordinate 38.5 or 50.0 are either poly(A) + or poly(A) −. An identical distribution also exists in nuclear steady-state RNA synthesized in vivo. Further analysis of Ad2 † † Abbreviations used: Ad2, adenovirus serotype 2; kb, kilobase (10 3 bases); EGTA, ethylene glycol-bis (β-aminoethyl ether) N,N′-tetraacetie acid. -specific nuclear RNA, synthesized either in vitro or in vivo, reveals that large RNA molecules containing incompletely spliced tripartite leader segments (presumptive processing intermediates) are present in both poly(A) − and poly(A) + RNA. However, molecules with mature 5′ ends (presumably created by splicing of the tripartite leader segment onto main-body sequences) accumulate to detectable levels only as fully processed, polyadenylated mRNA.

Regulation of simian virus 40 early and late gene transcription without viral DNA replication

Primary cultures of African green monkey kidney cells were infected with the simian virus 40 temperature-sensitive mutant tsA58 at the nonpermissive temperature of 41 degrees C for 12 to 20 h. Under these conditions, a defective T antigen was produced and no viral DNA replication was detected. Viral transcription complexes were extracted from infected nuclei using Sarkosyl and the nascent chains of RNA elongated in vitro. Sixty to 70% of the viral RNA synthesized in vitro hybridized to late gene sequences. In contrast, 80 to 90% of the nuclear viral RNA labeled in vivo during a 15-min pulse with [3H]uridine hybridized to early gene sequences. This suggests that selective degradation of late gene transcripts occurs in vivo. The role of T antigen and viral DNA replication in regulation of simian virus 40 transcription is discussed.

Selective Degradation of Newly Synthesized Nonmessenger Simian Virus 40 Transcripts

By pretreating simian virus 40-infected BSC-1 cells with glucosamine, [(3)H]uridine labeling of both cellular and viral RNA can be halted instantaneously by addition of cold uridine. We have studied the fate of pulse-labeled viral RNA from cells at 45 h postinfection under these conditions. During a 5-min period of labeling, both the messenger and nonmessenger regions of the late strand were transcribed. After various chase periods, nuclear viral species which sediment at 19, 17.5, and 16S were observed. Nuclear viral RNA decays in a multiphasic manner. Of the material present at the beginning of the chase period, 50% was degraded rapidly with a half-life of 8 min (initial processing). This rapidly degraded material was that fraction of the late strand which did not give rise to stable late mRNA species. Forty percent was transported to the cytoplasm, and 10% remained in the nucleus as material which sedimented in the 2 to 4S region. These 2 to 4S viral RNAs had a half-life of 3 h, and hybridization studies suggest that they are in part coded for by the late-strand nonmessenger region and are derived from the initial nuclear processing step. Another part is coded for by the late-strand messenger region and may be generated by some subsequent nuclear cleavages of 19S RNA into 17.5 and 16S RNAs. Transport of nuclear viral RNA into the cytoplasm was detected after a 5-min pulse and a 7-min chase. The maximum amount of labeled viral RNA was accumulated in the cytoplasm after a 30-min to 1-h chase. At least two viral cytoplasmic species were observed. Kinetic data suggest that 19S RNA is transported directly from the nucleus. Whether cytoplasmic 16S is formed by cleavage of 19S RNA in the cytoplasm is not clear. The half-lives of cytoplasmic 19 and 16S RNAs can be approximated as 2 and 5 h, respectively.

Polyoma virus high molecular weight nuclear rna codes for capsid protein VP2 in vitro

RNA extracted from the nuclei of 3T6 mouse cells late after infection by pot yoma virus (Py) has been translated in vitro to give one predominant polypeptide, which was identified as virion protein VP2 by polyacrylamide gel electrophoresis, specific immunoprecipitation, and tryptic peptide analysis. The active nuclear RNA species sediment between 15 and 50 S on sucrose gradients after denaturation, and resediment true after further denaturation and recentrifugation on 50% formamide-sucrose gradients. The bulk of the active viral nuclear RNA does not bind to oligo(dT)-cellulose and synthesis of Py VP2 is inhibited by m 7Gp under conditions where translation of Py-cRNA and EMC RNA are not inhibited. These experiments suggest that large viral nuclear RNA molecules, which are probably capped but not polyadenylated, can serve as messengers for Py VP2.

Characterization of adenovirus RNA synthesized in the presence of an adenosine analog: Failure of poly(A) addition

Synthesis of adenovirus-specific RNA in the presence of toyocamycin, an adenosine analog, late in infection of HeLa S 3 cells has been investigated. The effect of this analog on nuclear metabolism has been examined because, under the appropriate conditions, there is an apparent accumulation of rapidly sedimenting nuclear viral RNA (HnRNA) and no new viral mRNA associates with polyribosomes. Under these conditions there was an approximate 10% substitution by toyocamycin for adenosine in viral HnRNA. A similar amount was incorporated into virus-associated RNA(s) but there was little effect on the synthesis, the size, or the appearance in the cytoplasm of this species of viral RNA. In the presence of the analog no polyadenylate-rich segments could be detected in nuclear viral RNA. Two 5′ termini containing the methylated components 7mG and m 6Am were recovered and constituted proportionately the same amount in selected RNA sequences whether or not synthesis had occurred in the presence of the adenosine analog. Relative to the recovery of 5′ termini, selectively extracted RNA synthesized in the presence of toyocamycin yielded nearly twofold less m 6Ap. Since rapid sedimentation of nuclear viral RNA implies incomplete processing of molecules, these results suggest that the incorporation of toyocamycin interferes with RNA metabolism because of its prevention of polyadenylation and/or reduction in methylation of internal adenosine residues. Our data also imply a sequence of events in which the introduction of at least some 5′ alterations and internal methylations can occur prior to and independent of poly(A) additions.

Synthesis and accumulation of polyadenylated and nonpolyadenylated virus-specific RNA in adenovirus-infected cells

In human adenovirus type 12-infected cells, nonpolyadenylated virus-specific RNA was synthesized at a higher rate than was polyadenylated RNA. A large proportion of the pulse-labeled nuclear viral RNA in all sizes was nonpolyadenylated. Both the polyadenylated and nonpolyadenylated viral RNA contained the same sequences as determined by competition-hybridization. More than 50% of the viral RNA accumulated in the nucleus was nonpolyadenylated. Thus, nuclear viral RNA lacking poly(A) was not degraded rapidly. Both polyadenylated and nonpolyadenylated viral RNA were transported to the cytoplasm. However, the nonpolyadenylated viral RNA was transported more quickly than the polyadenylated RNA. It was also found that the viral RNA that sedimented with polysomes in a sucrose gradient had a higher degree of polyadenylation than the total cytoplasmic viral RNA.

Strand-Specific Transcription of Polyoma Virus DNA Early in Productive Infection and in Transformed Cells

The DNA strand origin of nuclear and cytoplasmic polyoma-specific RNA in productively infected mouse cells and in a line of polyoma-transformed hamster cells was determined by hybridization of unlabeled RNA with radioactively labeled separated strands of polyoma DNA. Early in the productive cycle (10 h postinfection) nuclear viral RNA is complementary to only about 40% of the E strand of viral CNA. No RNA complementary to the L strand was detected even when the RNA was first self-annealed to enrich for possible minor species. Early cytoplasmic RNA is complementary to the same 40% of the E strand. Thus, only that part of the poloma genome which codes for early virual messenger RNA appears to be transcribed. Late in infection, nuclear viral RNA is complementary to most or all of the L strand and to at least 60% of the E strand. Late cytoplasmic viral RNA hybridizes to 40 to 45% of the E strand and 50 to 55% of the L strand. The transformed cell nuclear viral RNA is complementary to 60% of the E strand, whereas cytoplasmic RNA is complementary to 40% of the E strand and comprises the same polyoma-specific sequences as are found in RNA early in productive infection. No L strand transcripts could be detected. Thus, in the transformed cells and late in productive infection, viral RNA sequences in the cytoplasm are a specific subset of those in the nucleus.