Formation of brome mosaic virus RNA-dependent RNA polymerase in yeast requires coexpression of viral proteins and viral RNA.

Quadt, R.; Ishikawa, Masayuki; Janda, Michael; Ahlquist, Paul

doi:10.1073/pnas.92.11.4892

Cited by 93 publications

(100 citation statements)

References 24 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This result parallels the in vivo BMV RNA synthesis pattern and suggests that synthesis of negative-strand RNA in this system depends on the replication enhancer element that is necessary for negative-strand RNA synthesis, which is absent in RNA 4 but present in the intercistronic region of RNA 3 (6). This finding is in contrast to the detergent-solubilized BMV RNA polymerase isolated from infected cells, which efficiently synthesizes negative-strand RNA 4 (29).…”

Section: Replication Of Bmv and Tcv Rnas In Bylcontrasting

confidence: 53%

Replication of plant RNA virus genomes in a cell-free extract of evacuolated plant protoplasts

Komoda

Naito

Ishikawa

2004

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

109

107

View full text Add to dashboard Cite

The replication of eukaryotic positive-strand RNA virus genomes occurs through a complex process involving multiple viral and host proteins and intracellular membranes. Here we report a cell-free system that reproduces this process in vitro. This system uses a membrane-containing extract of uninfected plant protoplasts from which the vacuoles had been removed by Percoll gradient centrifugation. We demonstrate that the system supported translation, negative-strand RNA synthesis, genomic RNA replication, and subgenomic RNA transcription of tomato mosaic virus and two other plant positive-strand RNA viruses. The RNA synthesis, which depended on translation of the genomic RNA, produced virus-related RNA species similar to those that are generated in vivo. This system will aid in the elucidation of the mechanisms of genome replication in these viruses.

show abstract

Section: Replication Of Bmv and Tcv Rnas In Bylcontrasting

confidence: 53%

Replication of plant RNA virus genomes in a cell-free extract of evacuolated plant protoplasts

Komoda

Naito

Ishikawa

2004

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

109

107

View full text Add to dashboard Cite

show abstract

“…4), and these effects are tightly linked to selecting RNA3 templates for replication and simultaneously inhibiting RNA3 translation (2,19). Loss of the RE and these 1a-mediated events inhibits RNA3 negative-strand synthesis and thus RNA replication approximately 100-fold (19,36,37). Similar mechanisms linking selection of viral RNAs for replication to translation inhibition are found in poliovirus and bacteriophage Q␤ (3) and may be common or universal among positive-strand viruses to avoid ribosome-polymerase collisions.…”

Section: Discussionmentioning

confidence: 99%

“…Exchanges between BMV and a related bromovirus show that 1a and the RE are the trans and cis determinants of template selectivity in RNA3 replication (13,35). The RE is a crucial, 100-fold enhancer of RNA3 negative-strand synthesis (36) and thus of RNA3 replication (19,37). RE mutations have parallel inhibitory or, in a few cases, stimulatory effects on 1a-induced RNA3 stabilization and (1aϩ2a)-dependent RNA3 replication (17).…”

Section: Lsm1pmentioning

confidence: 99%

Identification and characterization of a host protein required for efficient template selection in viral RNA replication

Díez

Ishikawa

Kaido

et al. 2000

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

130

126

View full text Add to dashboard Cite

P ositive-strand RNA viruses cause numerous human, animal, and plant diseases including encephalitis, hemorrhagic fevers, and hepatitis. Hepatitis C virus, e.g., chronically infects hundreds of millions of people, leading to progressive liver damage and cancer. Such positive-strand RNA viruses encapsidate messenger-sense RNA genomes, replicate through negative-strand RNA intermediates with no natural DNA phase, and share fundamental similarities in RNA replication. At the beginning of infection, the viral genomic RNA is first translated, yielding RNA replication factors that must recruit the genomic RNA out of translation for 3Ј to 5Ј copying by viral polymerase (1-3). The resulting RNA replication complexes are associated with intracellular membranes, although the nature and functions of this association are poorly understood (4-6).Genetic and biochemical results suggest that RNA virus replication involves unidentified host factors (7-10). Identifying and characterizing the relevant host factors is thus an important frontier for understanding and controlling viral replication as well as host range and pathology and should also illuminate important host cell pathways. To facilitate such studies, our laboratory identified two higher eukaryotic viruses able to replicate in the genetically tractable yeast Saccharomyces cerevisiae (11,12). One of these, brome mosaic virus (BMV), is a member of the alphavirus-like superfamily of human-, animal-, and plant-infecting positive-strand RNA viruses (13).The BMV genome is divided among three 5Ј capped RNAs (Fig. 1A). RNA1 and RNA2 encode replication factors 1a and 2a, which share conserved domains with all members of alphavirus-like superfamily. 1a contains domains implicated in RNA helicase and RNA capping functions (14, 15), and 2a contains an RNA-dependent RNA polymerase domain (13). 1a and 2a colocalize in an endoplasmic reticulum-associated replication complex that is the site of BMV-specific RNA synthesis (4, 5). RNA3 encodes cell-to-cell movement and coat proteins that direct systemic infection in the natural plant hosts of BMV but are dispensable for RNA replication. Coat protein is not translated from RNA3 but only from a subgenomic mRNA, RNA4 (11, 16). Expression of the coat gene or genes replacing it thus requires RNA3 replication to produce negative-strand RNA3, followed by subgenomic mRNA synthesis (Fig. 1 A). Like RNAs of hepatitis C virus and many other RNA viruses, BMV RNAs lack the 3Ј poly(A) of cellular mRNAs. Instead, the BMV RNAs have a tRNA-like structure that interacts with multiple tRNA-specific cellular enzymes (17,18).In yeast expressing 1a and 2a, RNA3 or RNA3 derivatives introduced by transfection or in vivo transcription are replicated and synthesize subgenomic RNA4 (Fig. 1 A). This yeast system reproduces all known features of BMV RNA replication in natural plant hosts, including localization to the endoplasmic reticulum; dependence on 1a, 2a, and the same cis-acting RNA signals; similar ratios of positive to negative-strand RNA; and other featu...

show abstract

“…4A). The ribozyme was derived from hepatitis ␦ virus (HDV) ribozyme, and has been shown previously to function in yeast (Quadt et al 1995).…”

Section: Tethered Pab1p Stabilizes Mrnas That Do Not Receive a Tailmentioning

confidence: 99%

“…4), the natural MFA2 3Ј UTR in the MFA2/MS2 reporter was deleted and replaced with the HDV ribozyme. This construct was generated by inserting the Acc65I-SmaI fragment of pMM2-1 into the SnaB1-StuI site of pB2RQ39 (Quadt et al 1995) creating pMM2-6.…”

Section: Plasmid Constructionmentioning

confidence: 99%

mRNA stabilization by poly(A) binding protein is independent of poly(A) and requires translation

1998

View full text Add to dashboard Cite

Translation and mRNA stability are enhanced by the presence of a poly(A) tail. In vivo, the tail interacts with a conserved polypeptide, poly(A) binding protein (Pab1p). To examine Pab1p function in vivo, we have tethered Pab1p to the 3 UTR of reporter mRNAs by fusing it to MS2 coat protein and placing MS2 binding sites in the 3 UTR of the reporter. This strategy allows us to uncouple Pab1p function from its RNA binding activity. We show that mRNAs that lack a poly(A) tail in vivo are stabilized by Pab1p, and that the portions of Pab1p required for stabilization are genetically distinct from those required for poly(A) binding. In addition, stabilization by Pab1p requires ongoing translation of the mRNA. We conclude that the primary, or sole, function of poly(A) with respect to mRNA stability is simply to bring Pab1p to the mRNA, and that mRNA stabilization is an intrinsic property of Pab1p. The approach we describe may be useful in identifying and assaying 3 UTR regulatory proteins, as it uncouples analysis of function from RNA binding.

show abstract

Formation of brome mosaic virus RNA-dependent RNA polymerase in yeast requires coexpression of viral proteins and viral RNA.

Cited by 93 publications

References 24 publications

Replication of plant RNA virus genomes in a cell-free extract of evacuolated plant protoplasts

Replication of plant RNA virus genomes in a cell-free extract of evacuolated plant protoplasts

Identification and characterization of a host protein required for efficient template selection in viral RNA replication

mRNA stabilization by poly(A) binding protein is independent of poly(A) and requires translation

Contact Info

Product

Resources

About