Structure of poliovirus replicative intermediate RNA: Electron microscope analysis of RNA cross-linked in vivo with psoralen derivative

The structure of the poliovirus replicative intermediate RNA was examined by electron microscopy after cross-linking in vivo with 4′-aminomethyl-4,5′,8-trimethylpsoralen. After purification from infected cells, undenatured RI² appeared as a double-stranded backbone of genome length, with an average of three (and occasionally up to eight) nascent, single-stranded tails. After denaturation, however, only single strands of heterogeneous length were visualized, indicating that the RI in the cell contains little or no duplex structure, and thus nascent chains are only transiently hydrogen-bonded to their template over short regions. The double-stranded backbone of undenatured RI, observed previously by others and in these experiments, is due to collapse of complementary chains during the deproteinization and purification procedures. The effectiveness of the in vivo cross-linking procedure was demonstrated by the complete inhibition of viral RNA synthesis in treated cells and by direct binding of [³H]AMT to RI molecules in vivo. Mature polio virions are impermeable to AMT; however, growth of virus in cells incubated with AMT in the dark resulted in normal yields of virus particles containing RNA genomes, whose infectivity could be subsequently photo-inactivated. The frequency of AMT-induced cross-linking was determined by analyses of double-stranded poliovirus RNA (RF). Cross-linking in vitro followed by spreading for electron microscopy under denaturing conditions yielded bubbled duplex structures with a minimum of one interstrand cross-link per 80 base-pairs. RF cross-linked in vivo also showed extensive cross-linking, decreased about fivefold from the in vitro cross-linked value. Thus, the failure to detect cross-linked RI under these conditions indicates that extensive base-pairing does not exist in vivo.     

An RNA hairpin at the extreme 5'' end of the poliovirus RNA genome modulates viral translation in human cells.

Several mutations were introduced into an infectious poliovirus cDNA clone by inserting different oligodeoxynucleotide linkers into preexisting DNA restriction endonuclease sites in the viral cDNA. Ten mutated DNAs were constructed whose lesions mapped in the 5' noncoding region or in the capsid coding region of the viral genome. Eight of these mutated cDNAs did not give rise to infectious virus upon transfection into human cells, one yielded virus with a wild-type phenotype, and one gave rise to a viral mutant with a small-plaque phenotype. This last mutant, designated 1-5NC-S21, bears a 6-nucleotide insertion in the loop of a stable RNA hairpin at the very 5' end of the viral genome. Detailed analysis of the biological properties of 1-5NC-S21 showed that the primary defect in mutant-infected cells is a fivefold decrease in translation relative to wild-type-infected cells. Transfection into HeLa cells of in vitro-synthesized RNA molecules bearing either the 5' noncoding region of 1-5NC-S21 or wild-type poliovirus upstream of a luciferase reporter gene showed that the mutated RNA hairpin was responsible for the observed decrease in viral translation in mutant-infected cells and conferred this defect to heterologous RNAs. These findings indicate that an RNA hairpin located at the extreme 5' end of the viral RNA and highly conserved among enteroviruses and rhinoviruses profoundly affects the translation efficiency of poliovirus RNA in infected cells.     

Characterization of Influenza Virus Ribonucleic Acid Duplex Produced by Annealing In Vitro

A ribonuclease-resistant ribonucleic acid (RNA) with a sedimentation coefficient of 12S was obtained by self-annealing influenza virus-specific RNA isolated from infected cells. It had the properties of double-stranded RNA. (i) Sedimentation behavior in sucrose gradient was independent of salt concentration. (ii) Thermal transition profile was sharp; the melting temperature is 83 C in 0.1 SSC (0.15 m NaCl plus 0.015 m sodium citrate) and 98 C in SSC. (iii) Buoyant density in cesium sulfate was 1.58 g/cm(3) compared to 1.64 g/cm(3) for single-stranded RNA. (iv) It gave rise to single-stranded RNA after denaturation. (v) The 12S RNA duplex contained both plus and minus strands of influenza virus. Labeled plus strands could be displaced by extraneous cold plus strands and extraneous (32)P-labeled plus strands could be incorporated into duplex after denaturation and reannealing.     

Separation and quantitation of intracellular forms of poliovirus RNA by agarose gel electrophoresis.

Intracellular poliovirus-specific RNA species can be measured directly by electrophoresis of total cytoplasmic nucleic acids through 1% agarose gels, resulting in the separation of single- and double-stranded forms of poliovirus RNA from each other and from HeLa cell 28S ribosomal RNA. Single-stranded RNA molecules differing by only 15% in length are resolved in this gel system. RNA species can be visualized as fluorescen bands appearing after staining of the gels with ethidium bromide and observation under ultraviolet illumination. The total amount of RNA can be determined by densitometric quantitation of the fluorescent response. In this way, the amount of poliovirus-specific RNA within the cytoplasm of HeLa cells infected for various times has been estimated. At 170-min postinfection, there are 0.67 X 10(5) molecules of single-stranded poliovirus RNA per cell and at 230 min, the amount has increased to 3.7 X 10(5) molecules/cell. Poliovirus double-strnaded RNA reaches a maximum of 0.7 X 10(5) molecules/cell at 330 min after infection.     

Intramolecular and intermolecular uridylylation by poliovirus RNA-dependent RNA polymerase

The 22-amino-acid protein VPg can be uridylylated in solution by purified poliovirus 3D polymerase in a template-dependent reaction thought to mimic primer formation during RNA amplification in infected cells. In the cell, the template used for the reaction is a hairpin RNA termed 2C-cre and, possibly, the poly(A) at the 3' end of the viral genome. Here, we identify several additional substrates for uridylylation by poliovirus 3D polymerase. In the presence of a 15-nucleotide (nt) RNA template, the poliovirus polymerase uridylylates other polymerase molecules in an intermolecular reaction that occurs in a single step, as judged by the chirality of the resulting phosphodiester linkage. Phosphate chirality experiments also showed that VPg uridylylation can occur by a single step; therefore, there is no obligatory uridylylated intermediate in the formation of uridylylated VPg. Other poliovirus proteins that could be uridylylated by 3D polymerase in solution were viral 3CD and 3AB proteins. Strong effects of both RNA and protein ligands on the efficiency and the specificity of the uridylylation reaction were observed: uridylylation of 3D polymerase and 3CD protein was stimulated by the addition of viral protein 3AB, and, when the template was poly(A) instead of the 15-nt RNA, the uridylylation of 3D polymerase itself became intramolecular instead of intermolecular. Finally, an antiuridine antibody identified uridylylated viral 3D polymerase and 3CD protein, as well as a 65- to 70-kDa host protein, in lysates of virus-infected human cells.     

Stable hairpin structure within the 5''-terminal 85 nucleotides of poliovirus RNA.

The primary sequence of a 5'-terminal fragment of poliovirus type 1 RNA, generated by digestion with RNase III, has been determined. This sequence reveals the presence of a stable hairpin structure beginning nine nucleotides from the terminally linked protein VPg. The sequence does not contain (i) the initiation codons AUG or GUG or (ii) the putative ribosome-binding sequence complementary to the 3' end of eucaryotic ribosomal 18S RNA. The stem-and-loop structure identified can be drawn in either plus or minus RNA strands. It is unclear to which strand functional significance (if any) can be assigned. It is possible that the hairpin structure is involved in ribosomal recognition and translation or in RNA synthesis by interacting with replicase molecules.     

Discontinuous DNA replication in mouse P-815 cells.

The length of newly synthesized DNA strands from mouse P-815 cells was analyzed after denaturation both by electrophoresis and by sedimentation in alkaline sucrose gradients. [3-H]-Thymidine pulses of 2-8 min at 37 degrees C predominantly label molecules of 20-60 S. With 30-s pulses at 25 degrees C, all the [3-H]thymidine appears in short DNA strands of 50-200 nucleotides. Thus, DNA strand elongation occurs discontinuously via Okazaki fragments at both the 5' end and the 3' end. In dodecylsulfate lysates, only 10% of the Okazaki fragments are found as single-stranded molecules. About 90% are resistant to hydrolysis by the single-strand-specific nuclease S-1 and band in isopycnic gradients at the buoyant density of double-stranded DNA. No evidence for ribonucleotides at the 5' end of Okazaki fragments was obtained either in isopycnic CsCl or Cs2SO4 gradients or after incubation with polynucleotide kinase and [gamma-32P]ATP.     

Polyadenylic acid on poliovirus RNA. II. poly(A) on intracellular RNAs.

The content, size, and mechanism of synthesis of 3'-terminal poly(A) on the various intracellular species of poliovirus RNA have been examined. All viral RNA species bound to poly(U) filters and contained RNase-resistant stretches of poly(A) which could be analyzed by electrophoresis in polyacrylamide gels. At 3 h after infection, the poly(A) on virion RNA, relicative intermediate RNA, polyribosomal RNA, and total cytoplasmic 35S RNA was heterogeneous in size with an average length of 75 nucleotides. By 6 h after infection many of the intracellular RNA's had poly(A) of over 150 nucleotides in length, but the poly(A) in virion RNA did not increase in size suggesting that the amount of poly(A) which can be encapsidated is limited. At all times, the double-stranded poliovirus RNA molecules had poly(A) of 150 to 200 nucleotides. Investigation of the kinetics of poly(A) appearance in the replicative intermediate and in finished 35S molecules indicated that poly(A) is the last portion of the 35S RNA to be synthesized; no nascent poly(A) could be detected in the replicative intermediate. Although this result indicates that poliovirus RNA is synthesized 5' leads to 3' like other RNA's, it also suggests that much of the poly(A) found in the replicative intermediate is an artifact possibly arising from the binding of finished 35S RNA molecules to the replicative intermediate during extraction. The addition of poly(A) to 35S RNA molecules was not sensitive to guanidene.     

Denaturation map of the circular mitochondrial genome of Neurospora crassa.

A denaturation map of mitochondrial DNA from the wild type strain 5256 of Neurospora crassa was constructed by computer analysis of the contour length distribution of single- and double-stranded regions of nineteen circular and three full length linear molecules after partial denaturation. The data suggest that mitochondrial DNA in this strain is a homogeneous population of a circular molecule of molecular weight 41 - 10(6) with an asymmetric distribution of AT-rich regions, and that linear molecules derive from this genome by random breaks during isolation.     

Secondary structure in heterogeneous nuclear RNA: involvement of regions from repeated DNA sites

Heterogeneous nuclear RNA was found to contain regions of secondary structure based on a relative resistance to nuclease treatment compared with mRNA or poliovirus RNA and a shift in density toward double-stranded RNA early in the course of nuclease digestion. The regions involved in this secondary structure are enriched for RNA segments transcribed from repeated sites in the DNA. Thus, to maximize hybridization to repetitive sites heterogeneous nuclear RNA molecules must be both denatured and fragmented. Some of the self-complementary regions in heterogeneous nuclear RNA are released by alkali denaturation and fragmentation below 1500 nucleotides but maximum release is not achieved until fragmentation below 500 nucleotides. These results indicate that these self-complementary regions (“loops” plus “stems”) are mainly below 500 nucleotides in length.     

Functional oligomerization of poliovirus RNA-dependent RNA polymerase.

Using a hairpin primer/template RNA derived from sequences present at the 3' end of the poliovirus genome, we investigated the RNA-binding and elongation activities of highly purified poliovirus 3D polymerase. We found that surprisingly high polymerase concentrations were required for efficient template utilization. Binding of template RNAs appeared to be the primary determinant of efficient utilization because binding and elongation activities correlated closely. Using a three-filter binding assay, polymerase binding to RNA was found to be highly cooperative with respect to polymerase concentration. At pH 5.5, where binding was most cooperative, a Hill coefficient of 5 was obtained, indicating that several polymerase molecules interact to retain the 110-nt RNA in a filter-bound complex. Chemical crosslinking with glutaraldehyde demonstrated physical polymerase-polymerase interactions, supporting the cooperative binding data. We propose a model in which poliovirus 3D polymerase functions both as a catalytic polymerase and as a cooperative single-stranded RNA-binding protein during RNA-dependent RNA synthesis.     

Molecular Weight of Poliovirus Ribonucleic Acid

Purified poliovirus single- and double-stranded ribonucleic acids (RNA) were examined by electron microscopy. The length of both molecules was found to be 2.37 mum. The uncorrected sedimentation coefficient for single-stranded RNA is 33S, as compared to 27S for the RNA of tobacco mosaic virus. It is calculated from these results that the molecular weight of the sodium form of poliovirus is 2.6 x 10(6) daltons.     

Accumulation of a 3'-terminal genome fragment in Japanese encephalitis virus-infected mammalian and mosquito cells

Japanese encephalitis virus (JEV) contains a single positive-strand RNA genome nearly 11 kb in length and is not formally thought to generate subgenomic RNA molecules during replication. Here, we report the abundant accumulation of a 3'-terminal 521- to 523-nucleotide (nt) genome fragment, representing a major portion of the 585-nt 3' untranslated region, in both mammalian (BHK-21) and mosquito (C6/36) cells infected with any of nine strains of JEV. In BHK-21 cells, the viral genome was detected as early as 24 h postinfection, the small RNA was detected as early as 28 h postinfection, and the small RNA was 0.25 to 1.5 times as abundant as the genome on a molar basis between 28 and 48 h postinfection. In C6/36 cells, the genome and small RNA were present 5 days postinfection and the small RNA was 1.25 to 5.14 times as abundant as the genome. The 3'-terminal 523-nt small RNA contains a 5'-proximal stable hairpin (nt 6 to 56) that may play a role in its formation and the conserved flavivirus 3'-cyclization motif (nt 413 to 420) and the 3'-terminal long stable hairpin structure (nt 440 to 523) that have postulated roles in genome replication. Abundant accumulation of the small RNA during viral replication in both mammalian and mosquito cells suggests that it may play a biological role, perhaps as a regulator of RNA synthesis.     

Defective interfering particles of poliovirus: mapping of the deletion and evidence that the deletions in the genomes of DI(1), (2) and (3) are located in the same region.

The deletions in RNAs of three defective interfering (DI) particles of poliovirus type 1 have been located and their approximate extent determined by three methods. (1) Digestion with RNase III of DI RNAs yields the same 3′-terminal fragments as digestion with RNase III of standard virus RNA. The longest 3′-terminal fragment has a molecular weight of 1.55 × 10⁶. This suggests that the deletions are located in the 5′-terminal half of the polio genome. (2) Fingerprints of RNase T₁-resistant oligonucleotides of all three DI RNAs are identical and lack four large oligonucleotides as compared to the fingerprints of standard virus, an observation suggesting that the deletions in all three DI RNAs are located in the same region of the viral genome. The deletion-specific oligonucleotides have also been shown to be within the 5′-terminal half of the viral genome by alkali fragmentation of the RNA and fingerprinting poly (A)-linked (3′-terminal) fragments of decreasing size. (3) Virion RNA of DI(2) particles was annealed with denatured double-stranded RNA (RF) of standard virus and the hybrid heteroduplex molecules examined in the electron microscope. A single loop, approximately 900 nucleotides long and 20% from one end of the molecules, was observed. Both the size and extent of individual deletions is somewhat variable in different heteroduplex molecules, an observation suggesting heterogeneity in the size of the deletion in RNA of the DI(2) population. Our data show that the DI RNAs of poliovirus contain an internal deletion in that region of the viral genome known to specify the capsid polypeptides. This result provides an explanation as to why poliovirus DI particles are unable to synthesize viral coat proteins.