Nucleotide sequence at the 5' extremity of tobacco-mosaic-virus RNA. 1. The noncoding region (nucleotides 1-68)

The sequence of the 5' noncoding region of tobacco mosaic virus RNA has been determined. The noncoding region is 68 nucleotides long and is unusual in that it contains no internal guanosine residues. The long T1 oligonucleotide containing the guanosine-free tract was isolated from a T1 ribonuclease digest of tobacco mosaic virus RNA and sequenced by labelling techniques in vitro using polynucleotide kinase. The guanosine-free tract is terminated by the first potential initiation codon in the RNA molecule and several lines of evidence suggest that this AUG triplet is operational in initiating viral protein synthesis (see following paper). The 5'-noncoding region cannot base-pair extensively with the 3'-terminal sequence of 18-S ribosomal RNA from rabbit reticulocytes.     

Structure of the genome of Moloney murine leukemia virus: a terminally redundant sequence

The genome of the Moloney strain of murine leukemia virus (Mo-MuLV) has been analyzed by digestion with ribonuclease T1 and separation of the digestion products by two-dimensional gel electrophoresis. Thirty large oligonucleotides isolated from such a fingerprint have been characterized. One of these oligonucleotides (number 21) was found to be present in twice the molar yield of the rest. The 30 oligonucleotides were mapped on the genome by determining their yields in various size classes of 3' terminal fragments of Mo-MuLV RNA. The physical map obtained in this way suggested that oligonucletoide 21 was present very near the 3' end of the geome as well as in another location near or at the 5' end. The genome structure suggested by these results was confirmed by analyzing oligonucleotides in Mo-Mulv RNA complementary to strong stop DNA, which is shown to be a copy of the 5' terminal 134 nucleotides of the MoMuLV genome. Some of the oligonucleotides in the RNA protected from RNAase digestion by hybridization to this DNA, including oligonucleotide 21, were present near both the 3' and 5' ends. Comparison of these with the nucleotide sequence of strong stop DNA shows that there is a terminal redundancy of 49-60 nucleotides in the Mo-MuLV genome RNA.     

The 5'' end group of tobacco mosaic virus RNA is m7G5'' ppp5'' Gp.

RNA extracted from CsC1-purified virions of tobacco mosaic virus is shown to give rise to an unusual nucleotide on digestion which RNAase T2, in addition to the four major nucleotides. This minor component has the electrophoretic characteristics of a phosphorylated end group, but is partially resistant to bacterial alkaline phosphatase. It is, however, a substrate for venom phosphodiesterase or nucleotide pyrophosphatase, yielding products which imply the structure m7G5'ppp5'Gp. TMV RNA, like many animal cellular and viral mRNAs recently examined, therefore has a 5' terminus blocked by a methylated nucleotide inverted with respect to the rest of the chain.     

Poliovirus RNA-Dependent RNA Polymerase Synthesizes Full-Length Copies of Poliovirion RNA, Cellular mRNA, and Several Plant Virus RNAs In Vitro

The poliovirus RNA-dependent RNA polymerase was active on synthetic homopolymeric RNA templates as well as on every natural RNA tested. The polymerase copied polyadenylate. oligouridylate [oligo(U)], polycytidylate . oligoinosinate, and polyinosinate. oligocytidylate templates to about the same extent. The observed activity on polyuridylate. oligoadenylate was about fourfold less. Full-length copies of both poliovirion RNA and a wide variety of other polyadenylated RNAs were synthesized by the polymerase in the presence of oligo(U). Polymerase elongation rates on poliovirion RNA and a heterologous RNA (squash mosaic virus RNA) were about the same. Changes in the Mg(2+) concentration affected the elongation rates on both RNAs to the same extent. With two non-polyadenylated RNAs (tobacco mosaic virus RNA and brome mosaic virus RNA3), the results were different. The purified polymerase synthesized a subgenomic-sized product RNA on brome mosaic virus RNA3 in the presence of oligo(U). This product RNA appeared to initiate on oligo(U) hybridized to an internal oligoadenylate sequence in brome mosaic virus RNA3. No oligo(U)-primed product was synthesized on tobacco mosaic virus RNA. When partially purified polymerase was used in place of the completely purified enzyme, some oligo(U)-independent activity was observed on the brome mosaic virus and tobacco mosaic virus RNAs. The size of the product RNA from these reactions suggested that at least some of the product RNA was full-sized and covalently linked to the template RNA. Thus, the polymerase was found to copy many different types of RNA and to make full-length copies of the RNAs tested.     

Nucleotide sequence of cucumber mosaic virus RNA. 1. Presence of a sequence complementary to part of the viral satellite RNA and homologies with other viral RNAs

The nucleotide sequence of the 3389 residues of RNA 1 (Mr 1.15 X 10(6) of the Q strain of cucumber mosaic virus (CMV) was determined, completing the primary structure of the CMV genome (8617 nucleotides). CMV RNA 1 was sequenced by the dideoxy-chain-termination method using M13 clones carrying RNA 1 sequences as well as synthetic oligonucleotide primers on RNA 1 as a template. At the 5' end of the RNA there are 97 noncoding residues between the cap structure and the first AUG (98-100), which is the start of a single long open-reading frame. This reading frame encodes a translation product of 991 amino acid residues (Mr 110791) and stops 319 nucleotide residues from the 3' end of RNA 1. In addition to the conserved 3' region present in all CMV RNAs (307 residues in RNA 1), RNAs 1 and 2 have highly homologous 5' leader sequences, a 12-nucleotide segment of which is also conserved in the corresponding RNAs of brome mosaic virus (BMV). CMV satellite RNA can form stable base pairs with a region of CMV RNAs 1 and 2 including this 12-nucleotide sequence, implying a regulatory function. This conserved sequence is part of a hairpin structure in RNAs 1 and 2 of CMV and BMV and in CMV satellite RNA. The entire translation products of RNA 1 of CMV and BMV could be aligned with significant homology. Less prominent homologies were found with alfalfa mosaic virus RNA 1 translation product and with tobacco mosaic virus Mr-126000 protein.     

RNA sequencing provides evidence for allelism of determinants of the N-, B- or NB-tropism of murine leukemia viruses.

Previous genetic and biochemical studies identified three large RNAase T1-resistant oligonucleotides, each associated with either the N-, B- or NB-tropism of murine C-type viruses of BALB/c origin. These oligonucleotides were shown to lie in the 5' third of the oligonucleotide maps of their respective viruses. We sequenced the three oligonucleotides and found that they share a 10 base sequence. Together these observations provide good evidence that the determinants of N-, B- or NB-tropism monitored by the three oligonucleotides are allelic. The oligonucleotides associated with N- and B-tropism differ in sequence at four of sixteen nucleotides, while the B- and NB-tropism-associated oligonucleotides differ in sequence by only one base out of sixteen. These results are consistent with the possibilities that B-tropic viruses may arise from N-tropic viruses by recombination, while NB-tropic viruses may arise from B-tropic virus by mutation. An unexplained finding was that a 10 base sequence present in the oligonucleotide associated with N-tropism is also found in the 3' third of the genomes of the N-, B- and NB-tropic viruses studied.     

The amino acid sequence of ribonuclease U2 from Ustilago sphaerogena.

1. RNAase (ribonuclease) U2, a purine-specific RNAase, was reduced, aminoethylated and hydrolysed with trypsin, chymotrypsin and thermolysin. On the basis of the analyses of the resulting peptides, the complete amino acid sequence of RNAase U2 was determined, 2. When the sequence was compared with the amino acid sequence of RNAase T1 (EC 3.1.4.8), the following regions were found to be similar in the two enzymes; Tyr-Pro-His-Gln-Tyr (38-42) in RNAase U2 and Tyr-Pro-His-Lys-Tyr (38-42) in RNAase T1, Glu-Phe-Pro-Leu-Val (61-65) in RNAase U2 and Glu-Trp-Pro-Ile-Leu (58-62) in RNAase T1, Asp-Arg-Val-Ile-Tyr-Gln (83-88) in RNAase U2 and Asp-Arg-Val-Phe-Asn (76-81) in RNAase T1 and Val-Thr-His-Thr-Gly-Ala (98-103) in RNAase U2 and Ile-Thr-His-Thr-Gly-Ala (90-95) in RNAase T1. All of the amino acid residues, histidine-40, glutamate-58, arginine-77 and histidine-92, which were found to play a crucial role in the biological activity of RNAase T1, were included in the regions cited here. 3. Detailed evidence for the amino acid sequence of the sequence of the proteins has been deposited as Supplementary Publication SUP 50041 (33 PAGES) AT THE British Library (Lending Division)(formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1975), 145, 5.     

Inhibition of dengue virus by novel, modified antisense oligonucleotides.

Five different target regions along the length of the dengue virus type 2 genome were compared for inhibition of the virus following intracellular injection of the cognate antisense oligonucleotides and their analogs. Unmodified phosphodiester oligonucleotides as well as the corresponding phosphorothioate oligonucleotides were ineffective in bringing about a significant inhibition of the virus. Novel modified phosphorothioate oligonucleotides in which the C-5 atoms of uridines and cytidines were replaced by propynyl groups caused a significant inhibition of the virus. Antisense oligonucleotide directed against the target region near the translation initiation site of dengue virus RNA was the most effective, followed by antisense oligonucleotide directed against a target in the 3' untranslated region of the virus RNA. It is suggested that the inhibitory effect of these novel modified oligonucleotides is due to their increased affinity for the target sequences and that they probably function via an RNase H cleavage of the oligonucleotide:RNA heteroduplex.     

T1 oligonucleotide maps of N-, B-, and B leads to NB-tropic murine leukemia viruses derived from BALB/c.

We previously described and characterized RNase T1 RNA fingerprints of an N-, a B-, and five B leads to NB-tropic murine leukemia viruses derived from BALB/c mice (Faller and Hopkins, J. Virol. 23:188-195, 1977, and J. Virol. 24:609-617, 1977). These viruses share the majority of their large RNase T1-resistant oligonucleotides, but each possesses some "unique" oligonucleotides relative to the others. We have ordered the large T1-resistant oligonucleotides of the N-, the B-, and one NB-tropic virus relative to the 3' end of their genomes to obtain oligonucleotide maps. These maps indicate that (i) the large T1 oligonucleotides shared by the N-, B-, and NB-tropic viruses probably occupy the same relative positions on their genomes; (ii) the 14 T1 oligonucleotides that differ between the N- and B-tropic viruses are derived from regions scattered along the genomes; and (iii) an oligonucleotide that is present in five NB-tropic viruses but not in their B-tropic virus progenitors lies toward the 5' end of the NB-tropic virus oligonucleotide map.     

The putative 15 S precursor of globin mRNA contains a poly (A) sequence

[3H] Uridine or [3H] adenosine pulse-labelled nuclear RNA was isolated from chicken immature red blood cells and separated on denaturing formamide sucrose gradients. RNA of each gradient fraction was hybridized with unlabelled globin DNA complementary to mRNA (cDNA) and subsequently digested by RNAase A and RNAase T1. The experiments revealed two RNA species with globin coding sequences sedimenting 9 S and approx. 15 S, the latter probably representing a precursor of 9 S globin mRNA. A poly (A) sequence was demonstrated in this RNA by two different approaches. Nuclear RNA pulse-labelled with [3H] uridine was fractionated by chromatography on poly (U)-Sepharose. Part of the 15 S precursor was found in the poly(A)-containing RNA. In the second approach 15 S RNA pulse-labelled with [3H]adenosine was hybridized with globin cDNA, incubated with RNAase A and RNAase T1 and subjected to chromatography on hydroxyapatite. The hybrids were isolated and after separation of the strands degraded with DNAase I, RNAase A and RNAase T1. By this procedure poly(A) sequences of approximately 100 nucleotides could be isolated from the 15 S RNA with globin coding sequences. The poly(A) sequence was completely degraded by RNAase T2.     

Studies on the sequence of the 3'-terminal region of turnip-yellow-mosaic-virus RNA.

A fragment representing the 3'-terminal 'tRNA-like' region of turnip yellow mosaic (TYM) virus RNA has been purified following incubation of intact TYM virus RNA with Escherichia coli 'RNase P'. This fragment, which is 112+3-nucleotides long has been completely digested with T1 RNase and pancreatic RNase and all the oligonucleotides present in such digests have been sequenced using 32P-end labelling techniques in vitro. The TYM virus RNA fragment is free of modified nucleosides and does not contain a G-U-U-C-R sequence. Using nuclease P1 from Penicillium citrinum, the sequence of 26 nucleotides from the 5' end and 16 nucleotides from the 3' end of this fragment has been deduced. The nucleotide sequence at the 5' end of the TYM virus RNA fragment indicates that this fragment includes the end of the TYM virus coat protein gene.     

Location and encapsidation of the coat protein cistron of tobacco mosaic virus. A bidirectional elongation of the nucleoprotein rod.

The coat protein cistron of tobacco mosaic virus has been located on the viral RNA starting between 975 and 1050 nucleotides from the 3'-hydroxyl end. This locates its 5' end close to the origin for virus assembly, where the first protein disk interacts with RNA. It also means that the coat protein mRNA must have a short 5'-untranslated tail and a long (over 500 nucleotides) 3' one. The recovery of characteristic oligonucleotides in nuclease-protected rods during the growth from RNA and a protein disk preparation shows that elongation of the nucleated rods proceeds independently in both directions though, on average, much more rapidly along the longer 5' tail than the shorter 3' tail. Protected RNA of length equal to that in the complete virion is first seen within 6 min, showing that the most rapidly elongated particles are substantially complete by this time.     

Genetic structure and transforming sequence of avian sarcoma virus UR2.

We have recently shown that a newly isolated avian sarcoma virus, UR2, is defective in replication and contains no sequences homologous to the src gene of Rous sarcoma virus. In this study, we analyzed the genetic structure and transforming sequence of UR2 by oligonucleotide fingerprinting. The sizes of the genomic RNAs of UR2 and its associated helper virus, UR2AV, were determined to be 24S and 35S, respectively, by sucrose gradient sedimentation. The molecular weight of the 24S UR2 genomic RNA was estimated to be 1.1 x 10(6), corresponding to 3,300 nucleotides, by gel electrophoresis under the native and denatured conditions. RNase T1 oligonucleotide mapping indicated that UR2 RNA contains seven unique oligonucleotides in the middle of the genome and shares eight 5'- and six 3'-terminal oligonucleotides with UR2AV RNA. From these data, we estimated that UR2 RNA contains a unique sequence of about 12 kilobases in the middle of the genome, and contains 1.4 and 0.7 kilobases of sequences shared with UR2AV RNA at the 5' and 3' ends, respectively. Partial sequence analysis of the UR2-specific oligonucleotides by RNase A digestion revealed that there are no homologous counterparts to these oligonucleotides in the RNAs of other avian sarcoma and acute leukemia viruses studied to date. UR2-transformed non-virus-producing cells contain a single 24S viral RNA which is most likely the message coding for the transforming protein of UR2. On the basis of the uniqueness of the transforming sequence, we concluded that UR2 is a new member of the defective avian sarcoma viruses.     

Circular dichroism and sedimentation studies on the reconstitution of tobacco mosaic virus

Reconstitution of tobacco mosaic virus from its constituents, the coat protein and RNA, was investigated by means of ultracentrifugation and circular dichroism measurement. Tobacco mosaic virus protein forms a 20S double-layer disc under conditions favorable for tobacco mosaic virus reconstitution. Dibromination of the tyrosine 139 residue of tobacco mosaic virus protein prevents formation of the 20S disc.Acidification of the tobacco mosaic virus protein solution causes 20S discs to polymerize into long helical rods. Changes in the CD spectra of tobacco mosaic virus protein in the near-ultraviolet region suggest that stacking of the aromatic sidechains of amino acid residues stabilizes the helical rod. The dibrominated tobacco mosaic virus protein also has the ability of rod elongation under acidic condition. CD studies reveal that assembly of tobacco mosaic virus particles from its constituents is stabilized by the stacking effect between the base residues of RNA and the aromatic residues of tobacco mosaic virus protein.Cucumber green mottle mosaic virus protein, which acts as a substituent for tobacco mosaic virus protein in tobacco mosaic virus reconstitution, was also investigated.