Nucleotide sequences from the 3''-ends of vesicular stomatitis virus mRNA''s as determined from cloned DNA.

Molecular clones of vesicular stomatitis virus mRNA's were used to determine the 3'-terminal sequences of mRNA's encoding the N and NS proteins. This new approach to VSV mRNA sequencing allowed the first comparison of 3'-terminal sequences. The sequences showed a tetranucleotide homology, UAUG, immediately preceding the polyadenylic acid. In addition, both mRNA's had an AU-rich region including the tetranucleotide AUAU at positions 16 to 19 nucleotides from the polyadenylic acid. A possible secondary structure between the 3' end of N mRNA and the 5' end of the adjacent NS mRNA is noted. These structural features may serve as signals for termination (or cleavage) and polyadenylation of vesicular stomatitis virus mRNA's. Neither mRNA had the polyadenylic acidproximal hexanucleotide, AAUAAA, found in eucaryotic cellular and viral mRNA's transcribed from nuclear DNA. The probable location of the translation termination codon for the NS protein is only six nucleotides from polyadenylic acid in NS mRNA.     

Complexity and polyadenylic acid content of visna virus 60-70S RNA.

The genomic complexity of visna virus was measured by quantitative analysis of 18 RNase T1-resistant oligonucleotides from 60-70S RNA. T1-resistant oligonucleotides were separated by two-dimensional polyacrylamide gel electrophoresis. Visna virus had a genomic complexity of 3.6 X 10(6) daltons, very close to the size of a single 30-40S RNA subunit. It was therefore concluded that the visna virus genome is largely polyploid. Visna virus 60-70S RNA polyadenylic acid segment was purified by T1 RNase digestion followed by oligodeoxythymidylic acid-cellulose column chromatography. It contained over 99% AMP and had a size of about 200 nucleotides. The binding capacities on oligodeoxythymidylic acid-cellulose of native 60-70S RNA and purified 30-40S RNA subunits were examined. It was concluded that two out of three intact subunits contain a polyadenylic acid segment.     

Analysis of the recombination event generating a vesicular stomatitis virus deletion defective interfering particle.

cDNA clones of different portions of the L cistron and 5'-terminal region of the vesicular stomatitis virus genome have been prepared and used to identify the exact site of the deletion in the defective interfering particle, DI-LT. The deletion extends from nucleotide 251 from the beginning of the L gene to a position 342 nucleotides from the end of the genome. The nucleotide sequences flanking the deletion site, as well as those at the ends of the deleted segment, did not contain any obvious vesicular stomatitis virus initiation or termination signals as had been found near the recombination sites in other defective interfering particle RNAs. The results best fit a model for the origin of this type of defective interfering particle in which the polymerase interrupts its synthesis and moves with its nascent daughter strand to a new position on the template and resumes synthesis there, further extending the nascent strand. Neither the interruption nor the resumption of synthesis appears to be in response to the template nucleotide sequence. The sequences of two partial L cistron clones also reveal open reading frames that code for amino acid sequences likely to be the amino and carboxy termini of the L protein.     

Nucleotide sequence of a cDNA clone encoding the entire glycoprotein from the New Jersey serotype of vesicular stomatitis virus.

The nucleotide sequence of the mRNA encoding the glycoprotein from the New Jersey serotype of vesicular stomatitis virus (VSV) was determined from a cDNA clone containing the entire coding region. The sequence of 12 5'-terminal noncoding nucleotides present in the mRNA but not in the cDNA clone was determined from a primer extended to the 5' terminus of the mRNA. The mRNA is 1,573 nucleotides long (excluding polyadenylic acid) and encodes a protein of 517 amino acids. Only six nucleotides occur between the translation termination codon and the polyadenylic acid. Short homologies between the untranslated termini of this mRNA and the mRNAs of the Indiana serotype were found. The predicted protein sequence was compared with that of the glycoprotein of the Indiana serotype of VSV and with the glycoprotein of rabies virus, using a computer program which determines optimal alignment. An amino acid identity of 50.9% was found for the two VSV serotypes. Approximately 20% identity was found between the rabies virus and VSV New Jersey glycoproteins. The positions and sizes of the transmembrane domains, the signal sequences, and the glycosylation sites are identical in both VSV serotypes. Two of five serine residues which were possible esterification sites for palmitate in the glycoprotein from the Indiana serotype are changed to glycine residues in the glycoprotein from the New Jersey serotype. Because the glycoprotein of the New Jersey serotype does not contain esterified palmitate, we suggest that one or both of these residues are the probable esterification sites in the glycoprotein from the Indiana serotype.     

Nucleotide sequence of a cDNA clone carrying the glycoprotein gene of infectious hematopoietic necrosis virus, a fish rhabdovirus.

The nucleotide sequence of the mRNA encoding the glycoprotein of infectious hematopoietic necrosis virus was determined from a cDNA clone containing the entire coding region. The G-protein cDNA is 1,609 nucleotides long (excluding the polyadenylic acid) and encodes a protein of 508 amino acids. The predicted amino acid sequence was compared with that of the glycoprotein of the Indiana and New Jersey serotypes of vesicular stomatitis virus and with the glycoprotein of rabies virus, using a computer program which determined optimal alignment. An amino acid identity of approximately 20% was found between infectious hematopoietic necrosis virus and the two vesicular stomatitis virus serotypes and between infectious hematopoietic necrosis virus and rabies virus. The positions and sizes of the signal sequence and transmembrane domain and the possible glycosylation sites were determined.     

Comparisons of nucleotide sequences in the genomes of the New Jersey and Indiana serotypes of vesicular stomatitis virus.

Nucleotide sequences of around 200 residues were determined adjacent to the 3' terminus of the genome RNA of vesicular stomatitis virus, New Jersey serotype, and adjacent to the 3'-terminal polyadenylic acid tract of the N protein mRNA of the same virus. These sequences were compared with the corresponding sequences previously determined for the Indiana serotype of vesicular stomatitis virus. The sequences obtained for the two strains were readily aligned, showing 70.8% homology overall. Examination of the sequences allowed identification of the translation initiation and termination codons for the N mRNA of each serotype. The deduced N-terminal and C-terminal amino acid sequences of the two N polypeptides were each similar, and most of the differences between them consisted of substitution by a clearly homologous amino acid. It was proposed that these nucleotide sequences, within limits imposed by their functions, comprise reasonably representative measures of the extent of sequence homology between the genomes of the two serotypes, and that this is higher than previously estimated, but with little exact homology over extended regions.     

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.     

Vesicular stomatitis virus mutant with altered polyadenylic acid polymerase activity in vitro.

In vitro RNA synthesis by purified virions of a stock of tsG16(I) was aberrant compared with that of wild-type (wt) vesicular stomatitis virus. RNA made in vitro by tsG16(I) contained a larger proportion of A residues in polyadenylic acid [poly(A)] tracts than did RNA synthesized by wt virus, tsG13(I), tsG21(II) or tsG41(IV). Experiments to determine whether the aberrant polyadenylation was correlated with the known thermolability of the tsG16(I) L protein were inconclusive. Total product RNA made by tsG16(I) was methylated to almost the same extent as wt RNA, contained the same major methylated 5' cap structure as wt RNA, and was translated as well in a reticulocyte cell-free system, yielding the same molecular weight proteins in similar ratios. Most polyadenylated [poly(A)+] RNA made by tsG16(I) was considerably larger than wt poly(A)+ RNA and richer in AMP:UMP residues; however, the protein-coding capacities of mutant and wt poly(A)+ RNAs were similar. This suggested that most mRNAs made in vitro by tsG16(I) might possess very long poly(A)+ tracts, and digestion of RNA by T1 RNase supported this. It appeared, therefore, that a virally coded component of vesicular stomatitis virus could affect polyadenylation. This could be the poly(A) polymerase itself, a protein involved in control of polyadenylation, or a protein which affects an event spatially and temporally connected with polyadenylation (such as initiation of the subsequent mRNA).     

In vitro RNA transcription by the New Jersey serotype of vesicular stomatitis virus. I. Characterization of the mRNA species.

The in vitro RNA synthesis by the virion-associated RNA polymerase of vesicular stomatitis virus (VSV), New Jersey serotype, was compared with that of the serologically distinct Indiana serotype of VSV. The New Jersey serotype of VSV synthesized five distinct mRNA species in vitro, three of which were smaller than the corresponding species synthesized by the Indiana serotype of VSV. These included the mRNA's coding for the G, M, and NS proteins. By hybridization experiments, virtually no sequence homology was detected between the mRNA's of the two serotypes. Despite this lack of overall homology, the 12 to 18S mRNA species of both serotype contained a common 5'-terminal hexanucleotide sequence, G(5')ppp(5')A-A-C-A-G. The signicance of this finding in light of specific interactions between the two serotypes of VSV in vivo is discussed.     

Nucleotide sequences of the mRNA''s encoding the vesicular stomatitis virus G and M proteins determined from cDNA clones containing the complete coding regions.

The complete nucleotide sequences of the vesicular stomatitis virus mRNA's encoding the glycoprotein (G) and the matrix protein (M) have been determined from cDNA clones that contain the complete coding sequences from each mRNA. The G protein mRNA is 1,665 nucleotides long, excluding polyadenylic acid, and encodes a protein of 511 amino acids including a signal peptide of 16 amino acids. G protein contains two large hydrophobic domains, one in the signal peptide and the other in the transmembrane segment near the COOH terminus. Two sites of glycosylation are predicted at amino acid residues 178 and 335. The close correspondence of the positions of these sites with the reported timing of the addition of the two oligosaccharides during synthesis of G suggests that glycosylation occurs as soon as the appropriate asparagine residues traverse the membrane of the rough endoplasmic reticulum. The mRNA encoding the vesicular stomatitis virus M protein is 831 nucleotides long, excluding polyadenylic acid, and encodes a protein of 229 amino acids. The predicted M protein sequence does not contain any long hydrophobic or nonpolar domains that might promote membrane association. The protein is rich in basic amino acids and contains a highly basic amino terminal domain. Details of construction of the nearly full-length cDNA clones are presented.     

Relationship Between Moloney Murine Leukemia and Sarcoma Virus RNAs: Purification and Hybridization Map of Complementary DNAs from Defined Regions of Moloney Murine Sarcoma Virus 124

Complementary DNAs (cDNA's) specific for various regions of the Moloney murine sarcoma virus (MSV) 124 RNA genome were prepared by cross-hybridization techniques. A cDNA specific for the first 1,000 nucleotides adjacent to the RNA 3' end (cDNA 3') was prepared and shown to also be complementary to the 3'-terminal 1,000 nucleotides of a related Moloney murine leukemia virus (MLV) genome. A cDNA complementary to the "MSV-specific" portion of the MSV 124 genome was prepared. This cDNA was shown not to anneal to Moloney MLV RNA and to anneal to a portion of the viral RNA of about 1,500 to 1,800 nucleotides in length, located 1,000 nucleotides from the 3' end of MSV RNA. A cDNA common to the genome of MSV and MLV was also obtained and shown to anneal to the 5'-terminal two-thirds, as well as to the 3'-terminal 1,000 nucleotides, of the MSV RNA genome. This cDNA also annealed to the RNA from MLV and mainly to the 5'-terminal half of the MLV genome. It is concluded that the 6-kilobase Moloney MSV 124 RNA genome has a sequence arrangement that includes (i) a 3' portion of about 1,000 nucleotides, which is also present at the 3' terminus of MLV; (ii) an MSV-specific region, not shared with MLV, which extends between 1,000 and 2,500 nucleotides from the 3' terminus; and (iii) a second "common" region, again shared with MLV, which extends from 2,500 nucleotides to the 5' terminus. This second common region appears to be located in the 5' half of the 10-kilobase MLV genome as well. Experiments in which a large excess of cold MLV cDNA was annealed to (3)H-labeled polyadenylic acid-containing fragments of MSV RNA gave results consistent with this arrangement of the MSV genome.     

Translation of vesicular stomatitis and Sindbis virus mRNAs in cell-free extracts of Aedes albopictus cells

We have developed a cell-free system from Aedes albopictus (mosquito) cells which is able to carry out endogenous protein synthesis and is stable to freezing and thawing. Successful preparation of extracts was found to depend on the presence of purified placental RNase inhibitor during cell breakage. Micrococcal nuclease-treated extracts translated exogenously added Sindbis 26S or vesicular stomatitis virus mRNA with a high degree of fidelity, demonstrating that initiation of protein synthesis had occurred. Evidence is presented showing that when cell fractions containing intracellular membranes were used to translate vesicular stomatitis virus mRNA, the G protein was glycosylated and inserted into microsomal vesicles. Additional studies indicate that initiation of protein synthesis in this system is dependent on a capped and methylated 5'-terminal structure in the mRNA.