Binding of ribosomes to the 5' leader sequence (N = 258) of RNA 3 from alfalfa mosaic virus.

RNA 3 of alfalfa mosaic virus (AlMV) contains information for two genes: near the 5' end an active gene coding for a 35 Kd protein and, near the 3' end, a silent gene coding for viral coat protein. We have determined a sequence of 318 nucleotides which contains the potential initiation codon for the 35 Kd protein at 258 nucleotides from the 5' end. This long leader sequence can form initiation complexes containing three 80 S ribosomes. A shorter species of RNA, corresponding to a molecule of RNA 3 lacking the cap and the first 154 nucleotides (RNA 3') has been isolated. The remaining leader sequence of 104 nucleotides in RNA 3' forms a single 80 S initiation complex with wheat germ ribosomes. The location of the regions of the leader sequence of RNA 3 involved in initiation complex formation with 80 S ribosomes is reported.     

Complete nucleotide sequence of genomic RNA of the potato M-virus

The complete nucleotide sequence of potato virus M genomic RNA has been determined to be 8534 nucleotides (with the exception of the poly(A) tail at the 3' end). The sequence contains six large open reading frames coding for proteins of mol. wt. 223206, 25438, 11893, 6793, 33906, and 12183 (in 5'----3' direction). According to its primary sequence analysis the 223K protein ORF codes for a virus RNA replicase. The in vitro translation product of 34K protein gene precipitates by the antisera against the RVM indicating that the 34K protein is the virus coat protein. The general aspects of carla- and potexvirus gene organization are discussed.     

Sequence of 1000 nucleotides at the 3'' end of tobacco mosaic virus RNA.

The sequence of 1000 nucleotides at the 3' end of tobacco mosaic virus RNA has been determined. The sequence contains the entire coat protein cistron as well as regions to its left and right. Sequence characterization was by conventional methods for use with uniformly 32P labeled RNA complemented by newer methods for in vitro 5' and 3' 32P end-labeling of RNA and its subsequent rapid analysis. The noncoding region separating the coat protein cistron from the 3' terminus is 204 residues long and may be folded into a clover-leaf-type secondary structure. The distribution of termination codons to the left of the coat protein cistron suggests that the end of the adjacent cistron is separated from the beginning of the coat protein cistron by only two nucleotides. The subgenomic viral coat protein mRNA was isolated from infected tissue and shown to be capped. The nontranslated sequence separating the cap from the AUG initiation codon is 9 residues long and thus overlaps a portion of the adjacent cistron on the genome RNA.     

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.     

Complete nucleotide sequence of alfalfa mosaic virus RNA 4.

Alfalfa mosaic virus RNA 4, the subgenomic messenger for viral coat protein, was partially digested with RNase T1 or RNase A and the sequence of a number of fragments was deduced by in vitro labeling with polynucleotide kinase and application of RNA sequencing techniques. From overlapping fragments, the complete primary sequence of the 881 nucleotides of RNA 4 was constructed: the coding region of 660 nucleotides (not including the initiation and termination codon) is flanked by a 5' noncoding region of 39 nucleotides and a 3' noncoding region of 182 nucleotides. The RNA sequencing data completely confirm the amino acid sequence of the coat protein as deduced by Van Beynum et al. (Fur.J. Biochem. 72, 63-78, 1977).     

Self-assembly of tobacco mosaic virus: the role of an intermediate aggregate in generating both specificity and speed

The tobacco mosaic virus (TMV) particle was the first macromolecular structure to be shown to self-assemble in vitro, allowing detailed studies of the mechanism. Nucleation of TMV self-assembly is by the binding of a specific stem-loop of the single-stranded viral RNA into the central hole of a two-ring sub-assembly of the coat protein, known as the 'disk'. Binding of the loop onto its specific binding site, between the two rings of the disk, leads to melting of the stem so more RNA is available to bind. The interaction of the RNA with the protein subunits in the disk cause this to dislocate into a proto-helix, rearranging the protein subunits in such a way that the axial gap between the rings at inner radii closes, entrapping the RNA. Assembly starts at an internal site on TMV RNA, about 1 kb from its 3'-terminus, and the elongation in the two directions is different. Elongation of the nucleated rods towards the 5'-terminus occurs on a 'travelling loop' of the RNA and, predominantly, still uses the disk sub-assembly of protein subunits, consequently incorporating approximately 100 further nucleotides as each disk is added, while elongation towards the 3'-terminus uses smaller protein aggregates and does not show this 'quantized' incorporation.     

Analysis of the genome structure of tobacco rattle virus strain PSG.

The sequence of the 3'-terminal 2077 nucleotides of genomic RNA 1 and the complete sequence of genomic RNA 2 of tobacco rattle virus (TRV, strain PSG) has been deduced. RNA 2 (1905 nucleotides) contains a single open reading frame for the viral coat protein (209 amino acids), flanked by 5'- and 3'-noncoding regions of 570 and 708 nucleotides, respectively. A subgenomic RNA (RNA 4) was found to lack the 5'-terminal 474 nucleotides of RNA 2 and is the putative messenger for coat protein. The deduced RNA 1 sequence contains the 3'-terminal part of a reading frame that probably corresponds to the TRV 170K protein and reading frames for a 29K protein and a 16K protein. Proteins encoded by the first two reading frames show significant amino acid sequence homology with corresponding proteins encoded by tobacco mosaic virus. Subgenomic RNAs 3 (1.6 kb) and 5 (0.7 kb) were identified as the putative messengers for the 29K and 16K proteins, respectively. At their 3'-termini all PSG-RNAs have an identical sequence of 497 nucleotides; at the 5'-termini homology is limited to 5 to 10 bases.     

Complete nucleotide sequences of the coat protein messenger RNAs of brome mosaic virus and cowpea chlorotic mottle virus

The nucleotide sequences of the subgenomic coat protein messengers (RNA4's) of two related bromoviruses, brome mosaic virus (BMV) and cowpea chlorotic mottle virus (CCMV), have been determined by direct RNA and CDNA sequencing without cloning. BMV RNA4 is 876 b long including a 5' noncoding region of nine nucleotides and a 3' noncoding region of 300 nucleotides. CCMV RNA 4 is 824 b long, including a 5' noncoding region of 10 nucleotides and a 3' noncoding region of 244 nucleotides. The encoded coat proteins are similar in length (188 amino acids for BMV and 189 amino acids for CCMV) and display about 70% homology in their amino acid sequences. Length difference between the two RNAs is due mostly to a single deletion, in CCMV with respect to BMV, of about 57 b immediately following the coding region. Allowing for this deletion the RNAs are indicate that mutations leading to divergence were constrained in the coding region primarily by the requirement of maintaining a favorable coat protein structure and in the 3' noncoding region primarily by the requirement of maintaining a favorable RNA spatial configuration.     

The nucleotide sequence at the 5'' end of foot and mouth disease virus RNA.

Foot and mouth disease virus RNA has been treated with RNase H in the presence of oligo (dG) specifically to digest the poly(C) tract which lies near the 5' end of the molecule (10). The short (S) fragment containing the 5' end of the RNA was separated from the remainder of the RNA (L fragment) by gel electrophoresis. RNA ligase mediated labelling of the 3' end of S fragment showed that the RNase H digestion gave rise to molecules that differed only in the number of cytidylic acid residues remaining at their 3' ends and did not leave the unique 3' end necessary for fast sequence analysis. As the 5' end of S fragment prepared form virus RNA is blocked by VPg, S fragment was prepared from virus specific messenger RNA which does not contain this protein. This RNA was labelled at the 5' end using polynucleotide kinase and the sequence of 70 nucleotides at the 5' end determined by partial enzyme digestion sequencing on polyacrylamide gels. Some of this sequence was confirmed from an analysis of the oligonucleotides derived by RNase T1 digestion of S fragment. The sequence obtained indicates that there is a stable hairpin loop at the 5' terminus of the RNA before an initiation codon 33 nucleotides from the 5' end. In addition, the RNase T1 analysis suggests that there are short repeated sequences in S fragment and that an eleven nucleotide inverted complementary repeat of a sequence near the 3' end of the RNA is present at the junction of S fragment and the poly(C) tract.     

Formation of ribosome-RNA initiation complexes with alfalfa mosaic virus RNA 4 and RNA 3.

RNA 4 of alfalfa mosaic virus (AMV) is a monocistronic messenger for the coat protein. We have determined the sequence of the 40 +/- 2 nucleotides in RNA 4 that were protected in the initiation complex formed with wheat germ 80 S ribosomes from digestion by T1 or pancreatic ribonucleases. The AUG coat protein initiation codon was near the middle of this protected region. We have found two ribosome-binding sites in RNA 3. The principal one, near the 5' end, is the initiation site for the major translation product, a 35,000 dalton protein. The second site binds ribosomes only weakly, at the beginning of the "silent" coat protein cistron, and is similar but not identical to the initiation site protection site is discussed.     

Location of the cistron of the tobacco mosaic virus coat protein.

Treatment of tobacco mosaic virus (TMV) RNA with T1 RNase under mild conditions cuts the RNA molecule into a large number of fragments, only a few of which may be specifically recognized by disks of TMV protein. It has been shown elsewhere that these specifically recognized RNA fragments are a part of the coat protein cistron, the portion coding for amino acids 95 to 129 of the coat protein. It is reported that different size classes of partially uncoated virus particles were prepared by limited reconstitution between TMV RNA and protein or by partial stripping of intact virus with DMSO. Both procedures produce nucleoprotein rods in which the 5'-terminal portion of the RNA is encapsidated and the 3'-terminal region is free. The free and the encapsidated portions of the RNA were each tested for the ability to give rise to the aforesaid specifically recognized fragments of the coat protein cistron upon partial T1 RNase digestion. It was found that only the 3'-terminal third of the virus particle need to be uncoated in order to expose the portion of the RNA molecule from which these fragments are derived. We conclude, therefore, that the coat protein cistron is situated upon the 3'-terminal third of the RNA chain, i.e. within 2000 nucleotides of the 3'-end.     

Nucleotide sequence of the 5'' noncoding region and part of the gag gene of mouse mammary tumor virus; identification of the 5'' splicing site for subgenomic mRNAs

We have determined the sequence of the first 1371 nucleotides at the 5' end of the genome of mouse mammary tumor virus using molecularly cloned proviral DNA of the GR virus strain. The most likely initiation codon used for the gag gene of mouse mammary tumor virus is the first one, located 312 nucleotides from the 5' end of the viral RNA. The 5' splicing site for the subgenomic mRNA's is located approximately 288 nucleotides downstream from the 5' end of the viral RNA. From the DNA sequence the amino acid sequence of the N-terminal half of the gag precursor protein, including p10 and p21, was deduced (353 amino acids).     

Nucleotide sequence of the 5' terminus of satellite tobacco necrosis virus ribonucleic acid.

Treatment of the RNA of satellite tobacco necrosis virus (STNV) with phosphomonoesterase followed by heat denaturation and treatment with polynucleotide kinase in the presence of [gamma-32P]ATP yields a STNV [5'-32P]RNA containing a homogeneous 5' terminus. Analyses of this STNV [5'-32P]RNA yield the sequence of the first 42 nucleotides from the 5'terminus of STNV RNA. This nucleotide sequence contains the translation initiation AUG codon starting at position 30 from the 5' terminus as indicated by match of subsequent nucleotides with the genetic code assignments for the N-terminal amino acids of STNV coat protein in the 5'-terminal sequence ppAGUAAAGACAGGAAACUU-UACUGACUAACAUGGCAAAACAAC. An interesting feature of this sequence is its potential to form a hairpin loop structure involving perfect Watson-Crick base pairing between the first seven nucleotides and nucleotides at positions 16--22.     

The complete nucleotide sequence of the potexvirus white clover mosaic virus.

The complete nucleotide sequence (5845 nucleotides) of the genomic RNA of the potexvirus white clover mosaic virus (WC1MV) has been determined from a set of overlapping cDNA clones. Forty of the most 5'-terminal nucleotides of WC1MV showed homology to the 5' sequences of other potexviruses. The genome contained five open reading frames which coded for proteins of Mr 147, 417, Mr 26,356, Mr 12,989, Mr 7,219 and Mr 20,684 (the coat protein). The Mr 147,417 protein had domains of amino acid sequence homology with putative polymerases of other RNA viruses. The Mr 26,356 and Mr 12,989 proteins had homology with proteins of the hordeivirus barley stripe mosaic virus RNA beta and the furovirus beet necrotic yellow vein virus (BNYVV) RNA-2. A portion of the Mr 26,356 protein was also conserved in the cylindrical inclusion proteins of two potyviruses. The Mr 7,219 protein had homology with the 25K putative fungal transmission factor of BNYVV RNA-3.     

Assembly of tobacco mosaic virus in vitro. Improved model for the elongation process by protein subunits.

The in vitro assembly reaction of tobacco mosaic virus (TMV), especially the elongation process of partially reconstituted RNA (PRR) by protein subunits, was observed by electron microscopy. After addition of TMV-protein subunits, the PRR appeared as rods with a clump at one end, believed to be a complex between added protein subunits and the RNA tail protruding from PRR. The subunits entrapped on the RNA tails in the forms of clumps were progressively incorporated into the growing rods on incubation, ending with the formation of completely reconstituted rods. The clumps were also observed after addition of cucumber green mottle mosaic virus (CGMMV) protein subunits to rods partially reconstituted from RNA and TMV-protein. In this case, the protein subunits, seen as clumps, did not become incorporated to form elongating rods. An improved model for the elongation of TMV rods is proposed. The elongation process is composed of two steps, with the first step being the interaction of protein subunits with the RNA tail protruding from the growing rod. Any protein having a specific binding site for TMV-rna, not limited to TMV-protein, will react in the first step. The second step is the incorporation of the protein on the RNA tail into a rod-shaped structure, with consequent elongation of the growing rod. It appears that only protein homologous with that in the partially reconstituted rods can partake in the second step.     

Nucleotide sequence of the gene encoding respiratory syncytial virus matrix protein.

The amino acid sequence of the matrix protein of the human respiratory syncytial virus (RS virus) was deduced from the sequence of a cDNA insert in a recombinant plasmid harboring an almost full-length copy of this gene. It specifically hybridized to a single 1,050-base mRNA from infected cells. The recombinant containing 944 base pairs of RS viral matrix protein gene sequence lacked five nucleotides corresponding to the 5' end of the mRNA. The nucleotide sequence of the 5' end of the mRNA was determined by the dideoxy sequencing method and found to be 5' NGGGC, wherein the C residue is one nucleotide upstream of the cloned viral sequence. The initiator ATG codon for the matrix protein is embedded in an AATATGG sequence similar to the canonical PXXATGG sequence present around functional eucaryotic translation initiation codons. There is no conserved sequence upstream of the polyadenylate tail, unlike vesicular stomatitis virus and Sendai virus, in which four nucleotides upstream of the polyadenylate tail are conserved in all genes. There is no equivalent of the eucaryotic polyadenylation signal AAUAAA upstream of the polyadenylate tail. The matrix protein of 28,717 daltons has 256 amino acids. It is relatively basic and moderately hydrophobic. There are two clusters of hydrophobic amino acid residues in the C-terminal third of the protein that could potentially interact with the membrane components of the infected cell. The matrix protein has no homology with the matrix proteins of other negative-strand RNA viruses, implying that RS virus has undergone extensive evolutionary divergence. A second open reading frame potentially encoding a protein of 75 amino acids and partially overlapping the C terminus of the matrix protein was also identified.     

Oligonucleotide binding to the coat protein disk of tobacco mosaic virus. Possible steps in the mechanism of assembly

Binding of the oligoribonucleotides AAG, AAGAAG and AAGAAGUUG to the disk aggregate of tobacco mosaic virus coat protein has been studied in solution under conditions favourable for virus assembly. The two longer oligomers bind strongly with Kd around 1 microM, approach complete saturation of binding sites and cause the formation of long, nicked helical rods resembling the virus. It is suggested that the binding of these oligomers, with sequences chosen from the assembly origin of the viral RNA, simulates the tobacco mosaic virus assembly process. No binding could be detected for AAG, indicating that chain length is a crucial determinant in the interaction. The binding of AAGAAG to coat protein crystals is very much weaker than that observed in solution, and the crystals crack at high oligomer concentrations. The corresponding oligodeoxyribonucleotide, d(AAGAAG), shows no binding to the protein in solution; the interaction is extremely specific for RNA.     

An uncapped RNA suggests a model for Caenorhabditis elegans polycistronic pre-mRNA processing

Polycistronic pre-mRNAs from Caenohabditis elegans operons are processed by internal cleavage and polyadenylation to create 3' ends of mature mRNAs. This is accompanied by trans-splicing with SL2 approximately 100 nucleotides downstream of the 3' end formation sites to create the 5' ends of downstream mRNAs. SL2 trans-splicing depends on a U-rich element (Ur), located approximately 70 nucleotides upstream of the trans-splice site in the intercistronic region (ICR), as well as a functional 3' end formation signal. Here we report the existence of a novel gene-length RNA, the Ur-RNA, starting just upstream of the Ur element. The expression of Ur-RNA is dependent on 3' end formation as well as on the presence of the Ur element, but does not require a trans-splice site. The Ur-RNA is not capped, and alteration of the location of the Ur element in either the 5' or 3' direction alters the location of the 5' end of the Ur-RNA. We propose that a 5' to 3' exonuclease degrades the precursor RNA following cleavage at the poly(A) site, stopping when it reaches the Ur element, presumably attributable to a bound protein. Part of the function of this protein can be performed by the MS2 coat protein. Recruitment of coat protein to the ICR in the absence of the Ur element results in accumulation of an RNA equivalent to Ur-RNA, and restores trans-splicing. Only SL1, however, is used. Therefore, coat protein is sufficient for blocking the exonuclease and thereby allowing formation of a substrate for trans-splicing, but it lacks the ability to recruit the SL2 snRNP. Our results also demonstrate that MS2 coat protein can be used as an in vivo block to an exonuclease, which should have utility in mRNA stability studies.