Although the m7G5'ppp5'N(m)pNp "capping" group has been found on virtually all known mammalian mRNAs (1), poliovirus polyribosomal RNA has pUp at its 5'-end (2-4). Because poliovirion RNA will direct viral protein synthesis in a cell-free system (5), it was thought to be identical to polyribosomal poliovirus RNA. Instead, we found that less than 10% of the virion molecules contained a pUp 5'-end (2).Lee et al. (6) recently presented evidence that a protein might be linked to the 5'-terminus of poliovirion RNA. We also have obtained evidence for a 5'-terminal protein in virion RNA. When total RNase digests of 32P-labeled virion RNA were examined by paper ionophoresis at pH 3.5, some labeled material was found moving toward the cathode, the direction opposite that of pure nucleotides. This material had the properties of a protein-pUp 5'-terminus of the RNA, a structure also suggested by Lee et al. (6). We have further found that cellulose acetate electrophoresis of a RNase T1 digest of virion RNA separates a protein-linked oligonucleotide. We show here that it consists of the structure protein-pU-U-A-A-A-A-C-A-G, which appears to be the 5'-terminus of poliovirion RNA

Poliovirus RNA purified from virus-specific polyribosomes does not contain m7G in a 5'-5'-pyrophosphate linkage at its 5'-end. The only potential 5'-end found in ribonuclease digests of this RNA is pUp, which is present in a yield of 1 mole/mole of poliovirus RNA. We conclude that a 5'-terminal m7G is not required for translation of at least one RNA species in animal cells.Recent evidence indicates that messenger RNA molecules of eukaryotic cells and their viruses have an unusual 5'-terminal structure of the general form m7G(5')ppp(5')Nm-N(m)-. . . This structure has been found on viral mRNAs synthesized by virion RNA polymerases of viruses containing double-and single-stranded RNA genomes (1, 2) as well as double-stranded DNA genomes (3). Viral mRNAs synthesized in virus-infected cells also contain this structure (4), and in the case of vesicular stomatitis virus mRNAs as many as 5 base and ribose methylations may occur near the 5'-end (5, 6). Animal cell mRNAs also contain a variety of "m7G-blocked" 5'-terminal structures (7,8). It has been proposed that the 5'-terminal m7G is required for translation of viral RNAs, since reovirus and vesicular stomatitis virus mRNAs lacking this nucleotide are translated poorly in a cell-free system derived from wheat embryo (9). It has also been suggested that this nucleotide may be required for translation of all mRNAs in eukaryotic cells, since its chemical removal from globin mRNA prevents translation (10).Virions of poliovirus contain a single-stranded RNA, the major 5'-end of which has been reported to be pAp (11); this RNA can be completely translated in a cell-free system (12). To examine whether the 5'-end of the viral mRNA active in infected cells might have the m7G blocking group, we have analyzed the structure of the 5'-terminus of poliovirus polyribosomal RNA prepared from infected HeLa cells. MATERIALS AND METHODSPreparation of Poliovirus RNA. The growth of HeLa cells in suspension using Joklik's modified minimal essential medium plus 7% horse serum and their infection by type 1 poliovirus have been described (13). For the preparation of 32P-labeled virus-specific RNA, 4 X 108 HeLa cells were harvested and washed three times with phosphate-free minimum essential medium buffered at pH 7.2 with 25 mM Hepes (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) and 10 mM Tes [N-tris(hydroxymethyl)methylarinoethanesulfonic acid]. The cells were resuspended at approximately 4 X 107 cells per ml in phosphate-free medium containing sufficient poliovirus to give a multiplicity of infection of 20. After room temperature adsorption for 30 min, the cells were diluted to 4 X 106/ml in phosphate-free medium plus 5% dialyzed horse serum (100 ml final volume). At 15 min post-infection, the culture was treated with 5 ,g/ml of actinomycin D and at 30 min post-infection, 600W Ci/ml of carrier-free 32p were added. The course of the infection was monitored by ['4C]uridine uptake into RNA in a portion of the culture that did not receive 32P.At 210 min post-infection, the cell...

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Because the ribonucleoprotein forms of the segments of the Uukuniemi virus genome have previously been characterized as circular, we examined the isolated RNAs by electron microscopy under conditions of increasing denaturation. After spreading under moderately denaturing conditions (50 or 60% formamide), 50 to 70% of the molecules were circular. Increasing the formamide concentration to 70 and 85% decreased the number of circular forms, and only linear forms were observed after incubation of the RNA at 60 degrees C for 15 min in 99% formamide. When spread from 4 M urea-80% formamide--another condition known to denature RNA--only 5 to 30% circular molecules were observed. Pretreatment of the RNA with 0.5 M glyoxal at 37 degrees C for 15 min prior to spreading from 50% formamide gave less than 5% cirucular forms. Length measurement of the molecules showed that they were not significantly degraded by any of the methods employed. The circular molecules were destroyed by treatment with pancreatic RNase, but were unaffected by DNase or proteinase K treatment. After complete denaturation of the RNA, the circles could be reformed under reannealing conditions. We conclude that the three size classes of RNA that comprise the Uukuniemi virus genome are circular molecules probably maintained in that form by base pairing between inverted complementary sequences at the 3' and 5' ends of linear molecules.

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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.

Virion RNA (vRNA) from poliovirus type 1 (PV1), poliovirus type 2 (PV2), and coxsackie virus Bi (Cox Bi) were treated with proteinase K to remove all but a small peptide of the covalently attached 5' genome-linked virion protein (VPg) The peptide on these RNA molecules was then treated with Bolton-Hunter 125I reagent, which iodinates primary amine groups, in order to obtain specific 5'-terminal radioactive labeling. Sequences of 125I-labeled vRNAs were determined by using a set of base-specific RNases and a partial alkaline hydrolysis "ladder." The first 20 positions of these RNAs show a remarkable conservation of sequence. The initial 10 nucleotides are identical in PV1, PV2, and Cox B1, with the sequence VPg-pU-U-A-A-A-A-C-A-G-C-. The next 10 nucleotide s ow a one-base difference between PV1 and PV2 and 50% homology between PV1 and Cox B1. This conserved 5' region may provide a recognition site for interaction between the viral mRNA and the host translation system. The translation of picornavirus mRNA within the infected host cell is unusual because these viral RNAs are not capped and methylated at the 5' terminus (1-4). In fact, the virion RNA (vRNA) of several picornaviruses containsa small genome-linked protein (VPg) attached covalently to the 5' terminus via a bond between a phosphate and a tyrosine residue (5-10). Polyribosome-associated vRNA of poliovirus type 1 (PV1) does not contain VPg but instead terminates with pUp (1-3). The presence of VPg on PV1 has been used to select a RNase Ti-resistant oligonucleotide and therefore to obtain the 5'-terminal sequence (VPg)-pU-U-A-A-A-A-C-A-G (6).We have specifically labeled the 5' terminus of vRNAs by iodination (using Bolton-Hunter 125I reagent*) of a proteinase K-resistant core of VPg attached to the RNA molecule (11) and have used these molecules to obtain sequence information. We report here a comparison of the 5'-terminal sequence of PV1 (Mahoney), poliovirus type 2 (PV2), and coxsackie virus Bi (Cox Bi). We find a remarkable conservation of sequence within the initial 20 nucleotides. Because picornavirus infection results in a specific inhibition of host protein synthesis and a preferential translation of viral proteins, this sequence suggests that a common 5' structure in the viral mRNA may be required for ribosomal recognition. MATERIALS AND METHODSGrowth of Cells and Viruses. HeLa S3 cells were grown in suspension culture as described (12). The growth and purification of PV1 in these-cells was by methods published elsewhere (13). PV2 and Cox Bi were obtained from J. Holland (University of California, San Diego). Virus was grown in HeLa cells as described for PV1. PV2 and Cox B1 were purified by lysing infected cells with 0.01 M NaCl/0.01 M Tris1HCI, pH 7.4, containing 1% Nonidet P-40 and pelleting virus from the cytoplasm in the absence of sodium dodecyl sulfate (NaDod-S04). Virus was purified by isopycnic banding in CsCl.Purification and Proteinase K Treatment of vRNA. Intact vRNA from each virus was obtained by acid/NaDodSO4 lysis (14) ...

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