Vesicular stomatitis virus defective interfering particle containing a muted internal leader RNA gene.

Keene, Jack D.; Chien, I.; Lazzarini, Robert A.

doi:10.1073/pnas.78.4.2090

Cited by 38 publications

(31 citation statements)

References 31 publications

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“…A defective interfering particle (DI-LT2) ofVSV that contains the leader RNA gene at an internal site 71 nucleotides away from the 3' end has recently been described (7). Although the complete leader sequence and the start of the N gene are present, neither the leader RNA nor any other mRNA transcript is synthesized in vitro by DI-LT2.…”

Section: Resultsmentioning

confidence: 99%

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Sequence-specific contacts between the RNA polymerase of vesicular stomatitis virus and the leader RNA gene.

Keene¹,

Thornton²,

Emerson³

1981

Proc. Natl. Acad. Sci. U.S.A.

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Methylation-protection studies with nucleocapsids from vesicular stomatitis virus indicate that the viral polymerase (L and NS proteins) contacts the genomic RNA template in the middle ofthe leader gene, 16-30 nucleotides from the 3' terminus. The data suggest that the NS protein binds to the sequence 20 30 3' G-G-U-A-A-U-A-A-U-A-G-U-A-A-U 5'and may function as an initiator protein for transcription. Because transcription starts at the 3' terminus ofVSV RNA, the promoter for the polymerase is presumed to reside in the leader gene. However, the binding sites ofthe L and NS proteins along the genome have not been determined. Mellon and Emerson (5) have shown by reconstitution of polymerase-template complexes that L protein will only bind to the template in combination with NS protein. NS, on the other hand, will bind alone to purified templates. Thus, the NS protein may direct the binding properties of the L protein to the template.We have exploited the finding of Gilbert and coworkers (6) that some proteins can alter the susceptibility of base residues to methylation by dimethyl sulfate at,their site of binding. Regions of specific protein-nucleic acid contact often show perturbations of the sequence pattern when probed with dimethylsulfate. We report that the N protein of VSV causes no significant perturbations of methylation of guanosine residues for more than 100 nucleotides from the 3' end of VSV RNA. This finding has allowed us to examine other VSV proteins (L and NS) for their ability to interact at specific sites along the nucleocapsid. We have isolated nucleocapsids from VSV that contain only N, NS, and perhaps some L protein and compared them to nucleocapsids from which L and NS have been removed. We conclude that the VSV polymerase or NS protein alone contacts an A+ U-rich sequence in the middle of the leader gene that may be analogous to the Hogness box located near the initiation sites ofother eukaryotic genes. We postulate that the NS protein may function as an initiator for transcription by mediating interaction between the L protein and the template. MATERIALS AND METHODSGrowth of Cells and Virus. The 'Mudd-Summers strain of VSV Indiana was propagated in BHK21 cells as described (7,8). Virus particles were purified by two isopycnic bandings and, in some instances, were subsequently isolated by rate velocity sedimentation in 10-40% sucrose gradients (9).Isolation of Viral Nucleocapsids. Purified virus particles were lysed in 0.72 M NaCl/1.87% Triton X-100/0.6 mM dithiothreitol/ 9.35% (vol/vol) glycerol/0.05 M Tris-HCl, 7.4 (HSS buffer) for 30-60 min on ice (10). Nucleocapsids containing N and NS proteins were prepared by centrifugation oflysed virus particles for 15 hr at 24,000 rpm in an SW 27 rotor through 15.2% Renografin (in HSS buffer) and onto a cushion of 76% Renografin as described (11). The band at the 15.2%/76% Renografin interface was removed, diluted with 12 ml of HSS buffer, and pelleted for 2 hr at 35,000 rpm in the SW 41 rotor onto 50 A.l of glycerol. Alternatively, nucleoc...

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Section: Resultsmentioning

confidence: 99%

“…The 'Mudd-Summers strain of VSV Indiana was propagated in BHK21 cells as described (7,8). Virus particles were purified by two isopycnic bandings and, in some instances, were subsequently isolated by rate velocity sedimentation in 10-40% sucrose gradients (9).…”

Section: Methodsmentioning

confidence: 99%

Sequence-specific contacts between the RNA polymerase of vesicular stomatitis virus and the leader RNA gene.

Keene¹,

Thornton²,

Emerson³

1981

Proc. Natl. Acad. Sci. U.S.A.

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“…In fact, as artificial as the double-promoter constructs presented here can be-in nature the replication promoters are normally found at the extremity of the templates-an old report describing a vesicular stomatitis virus defective interfering (DI) RNA relates to the double promoter and the imprint-ing concepts (21). An internal DI deletion (DI-LT 2 ) carrying, before the genomic 3Ј end, 70 nucleotides complementary to the 5Ј end of the vesicular stomatitis virus genome (in other words, carrying an AGP sequence in front of the normal GP sequence) was described.…”

Section: Discussionmentioning

confidence: 99%

Given the Opportunity, the Sendai Virus RNA-Dependent RNA Polymerase Could as Well Enter Its Template Internally

Vulliémoz

Roux

2002

J Virol

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The negative-stranded RNA viral genome is an RNA-protein complex of helicoidal symmetry, resistant to nonionic detergent and high salt, in which the RNA is protected from RNase digestion. The 15,384 nucleotides of the Sendai virus genome are bound to 2,564 subunits of the N protein, each interacting with six nucleotides so tightly that the bases are poorly accessible to soluble reagents. With such a uniform structure, the question of template recognition by the viral RNA polymerase has been raised. In a previous study, the N-phase context has been proposed to be crucial for this recognition, a notion referring to the importance of the position in which the nucleotides interact with the N protein. The N-phase context ruled out the role of the template 3-OH congruence, a feature resulting from the obedience to the rule of six that implies the precise interaction of the last six 3-OH nucleotides with the last N protein. The N-phase context then allows prediction of the recognition by the RNA polymerase of a replication promoter sequence even if internally positioned, a promoter which normally lies at the template extremity. In this study, with template minireplicons bearing tandem replication promoters separated by intervening sequences, we present data that indeed show that initiation of RNA synthesis takes place at the internal promoter. This internal initiation can best be interpreted as the result of the polymerase entering the template at the internal promoter. In this way, the data are consistent with the importance of the N-phase context in template recognition. Moreover, by introducing between the two promoters a stretch of 10 A residues which represent a barrier for RNA synthesis, we found that the ability of the RNA polymerase to cross this barrier depends on the type of replication promoter, strong or weak, that the RNA polymerase starts on, a sign that the RNA polymerase may be somehow imprinted in its activity by the nature of the promoter on which it starts synthesis. Viral RNA synthesis on negative-stranded RNA virus infection consists of two distinct processes: genome replication and transcription of the viral mRNAs. Replication initiates at the very 3Ј-OH end of the viral RNA and proceeds via the synthesis of its full complement, the antigenome, which in turn serves as the template to amplify the viral genome. The genomic promoter (GP) and antigenomic promoter (AGP) for replication of the genome and the antigenome, respectively, are positioned at the RNA 3Ј end of the genome and the antigenome, respectively (for general information, see reference 23). For Sendai virus, a member of the Paramyxoviridae family with a nonsegmented genome of 15,384 nucleotides, the replication promoters are within the first 96 nucleotides (34).While the GP and AGP have the same gross primary structure organization, only the first 12 nucleotides of the promoters are identical. With the possible exception of members of the Pneumovirus genus (e.g., respiratory syncytial virus), the replication promoters of all members of t...

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“…Isolation of the 3' Strand Probe of Defective-Interfering (DI) Stem RNA. RNA from purified LT2 defective particles (18) was prepared by lysis of the virus with 0.5% NaDodSO4 and phenol followed by purification of the 31S species on 10-30% sucrose/NaDodSO4 gradients (40,000 rpm for 3 MSL-54 is 53 or 54 bases long. This limitation in the-probing method was described previously (4).…”

Section: Methodsmentioning

confidence: 99%

A host protein (La) binds to a unique species of minus-sense leader RNA during replication of vesicular stomatitis virus.

Wilusz¹,

Kurilla²,

Keene³

1983

Proc. Natl. Acad. Sci. U.S.A.

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Baby hamster kidney cells infected with the minus-strand RNA virus vesicular stomatitis virus (VSV) were found to contain three small viral leader RNA species of the minus sense. The longest minus-strand leader RNA was 54 nucleotides long and was complexed with the host cell La protein that was immunoprecipitated by antisera from patients with systemic lupus ery-*thematosus. The La protein is normally found associated with RNA polymerase Ill transcripts in their unprocessed form. Shorter minus-strand leader RNA species of 45-48 nucleotides were more abundant but were not associated with the La protein. Unlike the plus-strand leader RNA of VSV, the minus-strand leader RNAs were not detected in the nucleus in any form. The minus-strand leader RNAs accumulated gradually throughout the infection and could not be found in association with the viral nucleocapsid protein. The sequence required for La protein binding on the 54-nucleotide-long minus-strand leader is similar to that at the 3' end of the La protein binding-plus-strand leader RNA and, thus, we propose a role for the La protein in the replication of VSV.Vesicular stomatitis virus (VSV) is a minus-strand RNA cytoplasmic virus that packages RNA polymerase in purified virions. The 42S genomic RNA serves as template for five monocistronic, capped, and polyadenylylated mRNAs. Viral RNA polymerase initiates transcription in vivo and in vitro at the 3' end of the genome and sequentially transcribes the genes in the order 3' N-NS-M-G-L 5' (1). In addition, the polymerase also synthesizes a leader RNA from the 3' terminus of the genome. This plus-sense leader RNA has been implicated in the shutoff of host macromolecular synthesis (2, 3) and is transported to the nucleus early in the infection (4). In addition, it may serve as a decision point. for the switch from viral transcription to replication (5). The mechanism by which this switch occurs is unknown. In the process of replication, the virus must synthesize a full-length 42S plus-sense complementary product from the genomic minus-sense RNA. The 42S plus strand in turn serves as template for two kinds of RNA products: full-length minus strands for packaging into progeny particles and short minusstrand leader RNAs. The minus-strand leader RNA may serve as an early decision point for synthesis of the genomic minus strand during replication (5).Several lines of evidence have suggested a role for host cell factors in VSV transcription and replication. Host range mutants of VSV have been isolated that have altered transcriptive properties (6, 7) and extracts from uninfected cells have been found to alter the quality and quantity of in vitro transcription (refs. 8 and 9; unpublished data). The nature of these factors is not understood and whether they interact with the RNA polymerase, the ribonucleoprotein (RNP) template, or the viral transcripts has not been determined. Virus-host interactions affecting transcription may be central to understanding VSV .replication and viral cytopathology.We have recently foun...

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Vesicular stomatitis virus defective interfering particle containing a muted internal leader RNA gene.

Cited by 38 publications

References 31 publications

Sequence-specific contacts between the RNA polymerase of vesicular stomatitis virus and the leader RNA gene.

Sequence-specific contacts between the RNA polymerase of vesicular stomatitis virus and the leader RNA gene.

Given the Opportunity, the Sendai Virus RNA-Dependent RNA Polymerase Could as Well Enter Its Template Internally

A host protein (La) binds to a unique species of minus-sense leader RNA during replication of vesicular stomatitis virus.

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