Influenza defective interfering viral RNA is formed by internal deletion of genomic RNA.

The 3'- and 5'-terminal nucleotide sequences of the defective interfering (DI) RNAs present in a preparation of DI influenza virus were determined. It was found that all DI RNAs possessed identical terminal sequences for at least the first 13 nucleotides at the 5' end and at least the last 12 nucleotides at the 3' end. The sequence of the DI RNAs is (5')A-G-U-A-G-A-A-A-C-A-A-G-G-...-C-C-U-G-C-U-U-U-C-G-C-U-OH(3'). In addition, the same sequences were present at the 3' and 5' termini of the viral polymerase genes (P1, P2, and P3) from which these DI RNAs originate. These results indicate that DI RNAs of influenzing virus are formed by an internal deletion of the genomic RNA.     

Sequence of an oligonucleotide derived from the 3'' end of each of the four brome mosaic viral RNAs.

A 3'-terminal oligonucleotide fragment, 161 bases long, can be obtained from each of the four brome mosaic virus RNAs by means of nuclease digestion. Like the four intact brome mosaic virus RNAs, each fragment accepts tyrosine in a reaction catalyzed by wheat germ aminoacyl-tRNA synthetase. The complete nucleotide sequence of the RNA 4 fragment has been determined by use of standard radiochemical methods. Comparative data for the fragments from RNAs 1, 2, and 3 show that they have nearly the same sequence as the RNA 4 fragment. The eight bases adjacent to the 3' terminus of the RNA 4 fragment are identical in sequence to the eight terminal bases of tyrosine tRNA from Torula utilis and eleven interior bases are identical in sequence to eleven bases encompassing the anticodon region of tyrosine tRNA from Saccharomyces cerevisiae, T. utilis, and Escherichia coli. Nevertheless, reasonable base-pairing schemes yield, at best, a distorted cloverleaf secondary structure.     

Polyadenylic Acid at the 3′-Terminus of Poliovirus RNA

Poliovirus RNA that has been derivatized at the 3'-end with NaIO(4)-NaB(3)H(4) yields, after hydrolysis with alkali or RNase T2, predominantly labeled residues of modified adenosine; no labeled nucleoside derivative is produced by digestion with RNase A or RNase T1. The 3'-terminal bases of the RNA are, therefore,...ApA(OH). Hydrolyzates of poliovirus [(32)P]RNA, after exhaustive digestion with RNase T1 or RNase A, contain, besides internal oligonucleotides, polynucleotides resistant to further action of ribonucleases T1 and A, respectively; these polynucleotides were isolated by membrane-filter binding or ion-exchange chromatography. The sequence of the T1-resistant polynucleotide was determined to be (Ap)(n)A(OH), that of the RNase A-resistant polynucleotide was GpGp(Ap)(n)A(OH). The chain length (n) of the polyadenylic acid, as analyzed by different methods, averages 89 nucleotides. Gel electrophoresis revealed heterogeneity of the size of poly(A). Poliovirus RNA, when labeled in vitro at the 3'-end, contains [3'-(3)H]poly(A); when labeled in vivo with [(3)H]A, it contains [(3)H](Ap)(n)A(OH). The data establish that... YpGpGp(Ap)([unk])A(OH) is the 3'-terminal sequence of poliovirus RNA, Type 1 (Mahoney). Since this mammalian virus reproduces in the cell cytoplasm, these observations may modify prior interpretations of the function of polyadenylate ends on messenger RNAs.     

Stucture of the amino-acid accepting 3''-end of high-molecular-weight eggplant mosaic virus RNA.

The sequence of the first 59 nucleotides from the 3'-OH terminus of high-molecular-weight eggplant mosaic virus RNA has been determined by standard radio-chemical techniques. The fragment was identified among the products of partial T1 RNase digestion by making use of the reverse migration, at pH 2.5, of the 3'-OH terminal oligonucleotide. No abnormal bases were found. A model of secondary structure may be constructed for this fragment, which is known to fix valine in the presence of valyl-tRNA synthetase. Its relation to the structures of genuine tRNAs and to the 3'-OH termini of other viral RNAs that also accept amino acids is discussed.     

Segment-specific inverted repeats found adjacent to conserved terminal sequences in wound tumor virus genome and defective interfering RNAs.

Defective interfering (DI) RNAs are often associated with transmission-defective isolates of wound tumor virus (WTV), a plant virus member of the Reoviridae. We report here the cloning and characterization of WTV genome segment S5 [2613 base pairs (bp)] and three related DI RNAs (587-776 bp). Each DI RNA was generated by a simple internal deletion event that resulted in no sequence rearrangement at the deletion boundaries. Remarkably, although several DI RNAs have been in continuous passage for more than 20 years, their nucleotide sequences are identical to that of corresponding portions of segment S5 present in infrequently passaged, standard, transmission-competent virus. The positions of the deletion breakpoints indicate that the minimal sequence information required for replication and packaging of segment S5 resides within 319 bp from the 5' end of the (+)-strand and 205 bp from the 3' end of the (+)-strand. The terminal portions of segment S5 were found to contain a 9-bp inverted repeat immediately adjacent to the conserved terminal 5'-hexanucleotide and 3'-tetranucleotide sequences shared by all 12 WTV genome segments. The presence of a 6- to 9-nucleotide segment-specific inverted repeat immediately adjacent to the conserved terminal sequences was found to be a feature common to all WTV genome segments. These results reveal several basic principles that govern the replication and packaging of a segmented double-stranded RNA genome.     

Cells that express all five proteins of vesicular stomatitis virus from cloned cDNAs support replication, assembly, and budding of defective interfering particles.

An alternative approach to structure-function analysis of vesicular stomatitis virus (VSV) gene products and their interactions with one another during each phase of the viral life cycle is described. We showed previously by using the vaccinia virus-T7 RNA polymerase expression system that when cells expressing the nucleocapsid protein (N), the phosphoprotein (NS), and the large polymerase protein (L) of VSV were superinfected with defective interfering (DI) particles, rapid and efficient replication and amplification of (DI) particle RNA occurred. Here, we demonstrate that all five VSV proteins can be expressed simultaneously when cells are contransfected with plasmids containing the matrix protein (M) gene and the glycoprotein (G) gene of VSV in addition to plasmids containing the genes for the N, NS, and L proteins. When cells coexpressing all five VSV proteins were superinfected with DI particles, which because of their defectiveness are unable to express any viral proteins or to replicate, DI particle replication, assembly, and budding were observed and infectious DI particles were released into the culture fluids. Omission of either the M or G protein expression resulted in no DI particle budding. The vector-supported DI particles were similar in size and morphology to the authentic DI particles generated from cells coinfected with DI particles and helper VSV and their infectivity could be blocked by anti-VSV or anti-G antiserum. The successful replication, assembly, and budding of DI particles from cells expressing all five VSV proteins from cloned cDNAs provide a powerful approach for detailed structure-function analysis of the VSV gene products in each step of the replicative cycle of the virus.     

Sequence relationships among defective interfering influenza viral RNAs.

Each clone of ts-52 and ts+ WSN influenza virus, when serially passaged at high multiplicity, gives rise to defective interfering (DI) virus with a unique set of new RNA species. The new RNAs (DI RNA) from several DI viruses were examined by the technique of RNase T1 oligonucleotide two-dimensional electrophoresis. It was found that each DI RNA arises from a specific segment of standard viral RNA. All DI RNA studied arose from the viral polymerase genes (P1, P2, and P3). DI RNAs originating from the same polymerase gene were interrelated. Certain of these DI RNAs appeared to contain completely overlapping nucleotide sequences. Others contained both overlapping and nonoverlapping nucleotide sequences. The latter DI RNAs may be formed from the progenitor viral RNA segment by a mechanism other than a common initiation (or termination) point and a simple deletion from one end.     

Amino acid sequence of a mouse immunoglobulin mu chain.

The complete amino acid sequence of the mouse mu chain from the BALB/c myeloma tumor MOPC 104E is reported. The C mu region contains four consecutive homology regions of approximately 110 residues and a COOH-terminal region of 19 residues. A comparison of this mu chain from mouse with a complete mu sequence from human (Ou) and a partial mu chain sequence from dog (Moo) reveals a striking gradient of increasing homology from the NH2-terminal to the COOH-terminal portion of these mu chains, with the former being the least and the latter the most highly conserved. Four of the five sites of carbohydrate attachment appear to be at identical residue positions when the constant regions of the mouse and human mu chains are compared. The mu chain of MOPC 104E has a carbohydrate moiety attached in the second hypervariable region. This is particularly interesting in view of the fact that MOPC 104E binds alpha-(1 leads to 3)-dextran, a simple carbohydrate. The structural and functional constraints imposed by these comparative sequence analyses are discussed.     

Genetic analysis of Schizosaccharomyces pombe 7SL RNA: a structural motif that includes a conserved tetranucleotide loop is important for function.

We have studied the effects of mutations in a 6-base segment of Schizosaccharomyces pombe 7SL RNA, which lies within a 35-nucleotide domain whose sequence and secondary structure are conserved in RNAs from many divergent organisms, including the 7SL component of human signal recognition particle (SRP). Surprisingly, many changes in this region can be tolerated under normal growth conditions. An exception is the lethality of several mutations at positions 159 and 160, 2 nucleotides previously shown to be protected from RNase digestion by the 19-kDa canine SRP protein. Nucleotide 160 is, in addition, the most highly conserved base in a consensus sequence for the most common tetranucleotide loop in ribosomal RNAs. Mutations that are likely to affect the stability and/or conformation of the RNA give rise to a conditional phenotype: when osmolarity of the medium is raised, the RNAs become partially or completely defective in function at high temperature.     

Two small RNAs encoded by Epstein-Barr virus and complexed with protein are precipitated by antibodies from patients with systemic lupus erythematosus

Primate cells harboring the Epstein-Barr virus (EBV) genome synthesize large amounts of two small RNAs:EBER 1 and EBER 2 (EBV-encoded RNA). These RNAs are approximately 180 nucleotides long, possess 5' pppA termini, and lack poly(A). They have different T1 and pancreatic RNase digestion fingerprints. They are not found in normal B lymphocytes, in transformed B lymphocytes that lack EBV DNA, in T lymphocytes transformed by Herpesvirus ateles, or in a variety of other nonlymphoid mammalian cells. Hybridization analyses indicate that EBER 1 and EBER 2 are encoded by the EcoRI-J fragment of EBV (B95-8) DNA. In vivo both RNAs are associated with protein(s), allowing their specific precipitation by the systemic lupus erythematosus-associated antibody anti-La. The La antigen in uninfected mammalian cells consists of a heterogeneous class of small ribonucleoprotein particles, some of whose RNA components exhibit sequence homology with a highly repetitive, interspersed class of human DNA designated the Alu family. Possible functions for EBER 1 and EBER 2 in infection and cell transformation by EBV and their potential relationship to the pathogenesis of systemic lupus erythematosus are discussed.     

Primary structures of canine pancreatic lipase and phospholipase A2 messenger RNAs

cDNA clones coding for phospholipase A2 and lipase mRNA have been identified in a full-length cDNA library constructed from canine pancreatic poly (A) + mRNA. Phospholipase A2 mRNA contains 562 nucleotides and codes for a preproenzyme of 146 amino acids (Mr = 16,251) containing a 15 residue signal peptide (MetLysPheLeuValLeuAlaAlaLeuLeuThrValAlaAlaAla), a seven residue activation peptide (GluGlyGlyIleSerProArg), and a 124 residue mature enzyme, phospholipase A2 (79.2% homology with the porcine enzyme). The 5' nontranslated sequence contains a region where eight of nine bases show potential hybridization to the 3' end of 18S ribosomal RNA. Lipase mRNA contains 1,493 nucleotides and codes for a preenzyme with 467 amino acids (Mr = 51,489) which contains a 17 residue signal peptide (MetValSerIleTrpThrIleAlaLeuPheLeuLeuGlyAlaAlaLysAla) and a 450 residue mature enzyme, lipase (75.6% homology with porcine lipase). The 5' noncoding sequences for phospholipase A2 (28 bases) and lipase (34 bases) mRNAs both have an adenosine base three positions preceding the AUG initiation codon but otherwise demonstrate no homology.     

Recognition site of Escherichia coli B restriction enzyme on ?XsB1 and simian virus 40 DNAs: An interrupted sequence

Methyl groups placed on varphiXsB1 replicative form DNA by the Escherichia coli B modification enzyme are located in the overlap between fragments Mbo II-3 and Alu I-2, a 61-base-pair DNA segment. Mutations that led to loss of susceptibility to restriction by E. coli B occurred within this segment at three positions spanning 14 nucleotides. A sequence difference between varphiXsB1 and varphiXam3cs70, a varphiX174 strain not restricted by E. coli B, occurs at one of these positions. The site on simian virus 40 DNA methylated by the modification enzyme is located in the 115-base-pair overlap between fragments Hae III-I and Alu I-G. The sequences of these segments of varphiXsB1 and simian virus 40 DNA and two regions of phage f1 DNA recognized by the E. coli B restriction enzyme [Ravetch, J. V., Horiuchi, K. & Zinder, N. D. (1978) Proc. Natl. Acad. Sci. USA 75, 2266-2270] contain a homology of nine bases in the configuration:5'-T-G-A... 8N... T-G-C-T... 9N... T-N-N-T-3'.The sequence 5'-T-G-A... 8N... T-G-C-T-3' may constitute the restriction enzyme recognition site since it does not occur in varphiXam3cs70 DNA and occurs only once in simian virus 40 DNA, and since all observed mutations leading to loss of the site occur at one of the bases specified by this sequence. Analysis of the sequence of varphiXam3cs70 showed that if no other residues are recognized, all seven of these bases are essential for recognition and the interval between the two groups of specified bases must be precisely eight.     

Vesicular stomatitis virus glycoprotein is anchored in the viral membrane by a hydrophobic domain near the COOH terminus

We have determined the COOH-terminal and NH(2)-terminal amino acid sequences of the vesicular stomatitis virus (VSV) glycoprotein (G). A sequence of 122 COOH-terminal amino acids was deduced from the complete sequence of a cloned DNA insert carrying 470 nucleotides derived from the 3' end of the G mRNA. Evidence presented indicates that this portion of the polypeptide includes the domains of G that reside inside the virion and span the lipid bilayer of the virion. This seems clear because a partial amino acid sequence of a fragment of G that remains associated with the membrane of the virion after exhaustive proteolytic digestions can be located unambiguously in the predicted sequence. This predicted sequence contains an uninterrupted hydrophobic domain beginning 49 amino acids and ending 30 amino acids from the COOH terminus. This region presumably spans the lipid bilayer. The COOH-terminal portion of 29 amino acids contains a high proportion of basic residues and resides inside the virion. The COOH-terminal portion of the VSV G protein therefore resembles in structure that of glycophorin, an erythrocyte membrane protein well characterized previously. The configuration of G in the viral membrane demonstrated here is probably similar for other viral glycoproteins, although this has not been tested as directly in any other case. From the sequence of a DNA primer extended on the RNA genome from the adjacent M protein gene into the G protein gene, we have deduced an NH(2)-terminal G protein sequence of 53 amino acids, including the leader sequence of 16 amino acids. Our sequence confirms, extends, and corrects two partial amino acid sequences reported for this region previously.     

18S defective interfering RNA of Semliki Forest virus contains a triplicated linear repeat.

The nucleotide sequence of a nearly full-length cloned cDNA copy of an 18S defective interfering (DI) RNA of Semliki Forest virus has been determined. This corresponded to a major virus-specific cytoplasmic RNA species at the 11th undiluted passage of the virus in BHK cells. The 1652-nucleotide-long sequence consists of a unique 5'-terminal sequence followed by three tandem 484-nucleotide repeat units derived from the 5' two-thirds of the viral genome and a unique sequence of 106 nucleotides preceding the poly(A) of the 3' terminus. One of the tandem 484-nucleotide repeat units contains an extra segment of 60 nucleotides. Hybridization experiments showed that the cloned cDNA was colinear with an 18S DI RNA and that it contained an approximately 320-nucleotide-long segment colinear with the viral genomic RNA. Analysis of 18S DI RNA oligonucleotide fingerprints revealed that the molecule studied and the heterogeneous DI RNA population contain similar repeated sequences. The mechanism by which the DI RNAs are generated is not known, but it seems likely that multiple internal deletions and duplications are involved.     

Sequence of picornavirus RNAs containing a radioiodinated 5''-linked peptide reveals a conserved 5'' sequence.

Virion RNA (vRNA) from poliovirus type 1 (PV1), poliovirus type 2 (PV2), and coxsackie virus B1 (Cox B1) 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 nucleotides show 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.