The 3'-terminal consensus sequence of rotavirus mRNA is the minimal promoter of negative-strand RNA synthesis

Wentz, Melissa J.; Patton, John T.; Ramig, Robert F.

doi:10.1128/jvi.70.11.7833-7841.1996

Cited by 54 publications

(26 citation statements)

References 33 publications

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“…Our results are the first to directly demonstrate that VP1 alone lacks replicase activity. Previous studies have shown that the only essential cis-acting signal required for minus-strand synthesis in vitro is formed by the last seven nucleotides of rotavirus mRNA (21,27). Given that of the three core proteins, only VP1 specifically interacts with the 3Ј end of viral mRNA, it is likely that VP1 recognizes and binds to the cis-acting replication signal.…”

Section: Discussionmentioning

confidence: 99%

“…These investigators included as a source of replicase activity in the system either virion-derived cores or baculovirus-expressed VP1-VP2-VP3 CLPs that had been disrupted ("opened") by incubation with hypotonic buffers. Subsequently, the system was used to define the locations of cisacting signals that promote minus-strand synthesis in rotavirus gene 8 and 9 mRNAs (21,27,28). A signal essential for replication was mapped to the extreme 3Ј ends of gene 8 and 9 mRNAs and, because of the highly conserved nature of its sequence, is probably a common feature of all 11 rotavirus mRNAs (21).…”

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confidence: 99%

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Rotavirus VP1 alone specifically binds to the 3' end of viral mRNA, but the interaction is not sufficient to initiate minus-strand synthesis

Patton

1996

J Virol

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Recent studies have shown that disrupted (open) rotavirus cores have an associated replicase activity which supports the synthesis of dsRNA from viral mRNA in a cell-free system (. 68:7030-7039, 1994). To determine which of the core proteins, VP1, VP2, or VP3, recognizes the template mRNA during RNA replication, SA11 open cores were incubated with 32 P-labeled RNA probes of viral and nonviral origin and the reaction mixtures were analyzed for the formation of RNA-protein complexes by gel mobility shift assay. In mixtures containing a probe representing the 3 end of SA11 gene 8 mRNA, two closely migrating RNA-protein complexes, designated s and f, were detected. The interaction between the RNA and protein of the s and f complexes was shown to be specific by competitive binding assay with tRNA and brome mosaic virus RNA. By electrophoretic analysis of RNAprotein complexes recovered from gels, VP1 was shown to be the only viral protein component of the complexes, thereby indicating that VP1 specifically recognizes the 3 end of gene 8 mRNA. Analysis of VP1 purified from open cores by glycerol gradient centrifugation verified that VP1 recognizes the 3 end of viral mRNA but also showed that in the absence of other viral proteins, VP1 lacks replicase activity. When reconstituted with VP2-rich portions of the gradient, VP1 stimulated levels of replicase activity severalfold. These data indicate that VP1 can bind to viral mRNA in the absence of any other viral proteins and suggest that VP2 must interact with the RNA-protein complex before VP1 gains replicase activity.

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Rotavirus VP1 alone specifically binds to the 3' end of viral mRNA, but the interaction is not sufficient to initiate minus-strand synthesis

Patton

1996

J Virol

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“…For the Reoviridae, it is not clear how genomic packaging works. It has been possible to effect minus-strand synthesis in vitro with disrupted core particles of rotavirus (8,66,83). The reaction is specific in that sequences at the 5Ј and 3Ј ends of the RNA are important in determining the extent of synthesis.…”

Section: Packaging In Other Systems With Dsrna Genomesmentioning

confidence: 99%

Precise Packaging of the Three Genomic Segments of the Double-Stranded-RNA Bacteriophage φ6

Mindich

1999

Microbiol Mol Biol Rev

108

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SUMMARY Bacteriophage φ6 has a genome of three segments of double-stranded RNA. Each virus particle contains one each of the three segments. Packaging is effected by the acquisition, in a serially dependent manner, of the plus strands of the genomic segments into empty procapsids. The empty procapsids are compressed in shape and expand during packaging. The packaging program involves discrete steps that are determined by the amount of RNA inside the procapsid. The steps involve the exposure and concealment of binding sites on the outer surface of the procapsid for the plus strands of the three genomic segments. The plus strand of segment S can be packaged alone, while packaging of the plus strand of segment M depends upon prior packaging of S. Packaging of the plus strand of L depends upon the prior packaging of M. Minus-strand synthesis begins when the particle has a full complement of plus strands. Plus-strand synthesis commences upon the completion of minus-strand synthesis. All of the reactions of packaging, minus-strand synthesis, and plus-strand synthesis can be accomplished in vitro with isolated procapsids. Live-virus constructions that are in accord with the model have been prepared. Mutant virus with changes in the packaging program have been isolated and analyzed.

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“…This is the first report of an infectious RNA virus recovered from an aquatic species. The prerequisite for the construction of infectious clones of IPNV was the identification of the precise 5Јand 3Ј-terminal sequences, which are crucial for replication of viral RNA (25,30). We determined the 5Ј-and 3Ј-noncoding sequences from both genomic segments of IPNV by poly(A) tailing and 5Ј RACE methods.…”

Section: Discussionmentioning

confidence: 99%

“…For example, 32 of 50 nucleotides at the 5Ј-noncoding region and 29 of 50 nucleotides at the 3Јnoncoding region between the two segments are conserved. These termini should contain sequences that are important in the packaging and replication of the IPNV genome, as demonstrated for other double-stranded RNA viruses, such as mammalian reoviruses and rotaviruses (17,25,30,34).…”

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confidence: 99%

Generation of Infectious Pancreatic Necrosis Virus from Cloned cDNA

Yao¹,

Vakharia²

1998

J. Virol.

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We developed a reverse genetics system for infectious pancreatic necrosis virus (IPNV), a prototype virus of theBirnaviridae family, with the use of plus-stranded RNA transcripts derived from cloned cDNA. Full-length cDNA clones of the IPNV genome that contained the entire coding and noncoding regions of RNA segments A and B were constructed. Segment A encodes a 106-kDa precursor protein which is cleaved to yield mature VP2, nonstructural protease, and VP3 proteins, whereas segment B encodes the RNA-dependent RNA polymerase VP1. Plus-sense RNA transcripts of both segments were prepared by in vitro transcription of linearized plasmids with T7 RNA polymerase. Transfection of chinook salmon embryo (CHSE) cells with combined transcripts of segments A and B generated infectious IPNV particles 10 days posttransfection. Furthermore, a transfectant virus containing a genetically tagged sequence was generated to confirm the feasibility of this system. The presence and specificity of the recovered virus were ascertained by immunofluorescence staining of infected CHSE cells with rabbit anti-IPNV serum and by nucleotide sequence analysis. In addition, 3′-terminal sequence analysis of RNA from the recovered virus showed that extraneous nucleotides synthesized at the 3′ end during in vitro transcription were precisely trimmed or excluded during replication, and hence these were not incorporated into the genome. An attempt was made to determine if RNA-dependent RNA polymerase of IPNV and infectious bursal disease virus (IBDV), another birnavirus, can support virus rescue in heterologous combinations. Thus, CHSE cells were transfected with transcripts derived from IPNV segment A and IBDV segment B and Vero cells were transfected with transcripts derived from IBDV segment A and IPNV segment B. In either case, no infectious IPNV or IBDV particles were generated even after a third passage in cell culture, suggesting that viral RNA-dependent RNA polymerase is species specific. However, the reverse genetics system for IPNV that we developed will greatly facilitate studies of viral replication and pathogenesis and the design of a new generation of live attenuated vaccines.

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The 3'-terminal consensus sequence of rotavirus mRNA is the minimal promoter of negative-strand RNA synthesis

Cited by 54 publications

References 33 publications

Rotavirus VP1 alone specifically binds to the 3' end of viral mRNA, but the interaction is not sufficient to initiate minus-strand synthesis

Rotavirus VP1 alone specifically binds to the 3' end of viral mRNA, but the interaction is not sufficient to initiate minus-strand synthesis

Precise Packaging of the Three Genomic Segments of the Double-Stranded-RNA Bacteriophage φ6

Generation of Infectious Pancreatic Necrosis Virus from Cloned cDNA

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