Purification and characterization of brome mosaic virus RNA-dependent RNA polymerase

Quadt, R.; Jaspars, E.M.J.

doi:10.1016/0042-6822(90)90393-6

Cited by 83 publications

(67 citation statements)

References 10 publications

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“…No products are observed when no RNA is added to the reaction mixture (Fig. 1, In contrast, the CMV RdRp does not exhibit tire strict template specifity that is observed with AIMV RdRp [9] and BMV RdRp [7]. CMV RdRp recognizes and utilizes a variety of viral RNAs as template for minusstrand synthesis leading to the formation of doublestranded RNAs corresponding to viral RNAs I, 2 and 3 and subgenomic RNAs (Fig.…”

Section: Resultsmentioning

confidence: 99%

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Characterization of cucumber mosaic virus RNA‐dependent RNA polymerase

Quadt¹,

Jaspars²

1991

FEBS Letters

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Section: Resultsmentioning

confidence: 99%

“…CMV RdRp can be purified by employing essentially the same method as for purification of BMV RdRp [7]. An 8914-fold increase in specific activity was obtained by solubilization of CMV RdRP followed by subsequent purification of the solubilized enzyme (Table Ill. The purified enzyme is completely dependent on exogenous template.…”

Section: Resultsmentioning

confidence: 99%

Characterization of cucumber mosaic virus RNA‐dependent RNA polymerase

Quadt¹,

Jaspars²

1991

FEBS Letters

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“…Surprisingly, tobacco mosaic virus RNA was used, at even lower efficiency, by plant-derived BMV RdRp but not by BMV RdRp extracted from yeast ( Fig. 1, lanes 8 and 4) [5][6][7][8]. Virion RNAs added as templates are indicated above each lane.…”

Section: Methodsmentioning

confidence: 99%

“…The resulting detergent extracts were used for RdRp assays. Preparation of highly purified RdRp from BMV-infected barley and RdRp activity assays were performed as described (5).…”

Section: Methodsmentioning

confidence: 99%

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

Quadt

Ishikawa

Janda

et al. 1995

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

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In this report we show that yeast expressing brome mosaic virus (BMV) replication proteins la and 2a and replicating a BMV RNA3 derivative can be extracted to yield a template-dependent BMV RNA-dependent RNA polymerase (RdRp) able to synthesize (-)-strand RNA from BMV (+)-strand RNA templates added in vitro. This virus-specific yeast-derived RdRp mirrored the template selectivity and other characteristics of RdRp from BMV-infected plants. Equivalent extracts from yeast expressing la and 2a but lacking RNA3 contained normal amounts of la and 2a but had no RdRp activity on BMV RNAs added in vitro. To determine which RNA3 sequences were required in vivo to yield RdRp activity, we tested deletions throughout RNA3, including the 5', 3', and intercistronic noncoding regions, which contain the cis-acting elements required for RNA3 replication in vivo. RdRp activity was obtained only from cells expressing la, 2a, and RNA3 derivatives retaining both 3' and intercistronic noncoding sequences. Strong correlation between extracted RdRp activity and BMV (-)-strand RNA accumulation in vivo was found for all RNA3 derivatives tested. Thus, extractable in vitro RdRp activity paralleled formation of a complex capable ofviral RNA synthesis in vivo. The results suggest that assembly of active RdRp requires not only viral proteins but also viral RNA, either to directly contribute some nontemplate function or to recruit essential host factors into the RdRp complex and that sequences at both the 3'-terminal initiation site and distant internal sites of RNA3 templates may participate in RdRp assembly and initiation of (-)-strand synthesis.Brome mosaic virus (BMV), a plant-infecting virus, is a representative member of the alphavirus-like superfamily of (+)-strand RNA viruses of animals and plants. The BMV genome consists of three separately encapsidated RNAs, designated RNA1-3. Monocistronic RNA1 and RNA2, respectively, encode proteins la (109 kDa) and 2a (94 kDa). la and 2a are essential mutually interacting components of the RNA-dependent RNA polymerase (RdRp) involved in BMV RNA replication (1, 2). This RdRp, whose functions have been studied in vivo and in vitro, is a complex of la, 2a, and host proteins (2-6). RNA3 contains two genes separated by an -250-nt intercistronic region. The 5'-proximal gene, encoding the 3a infection movement protein (7,8), is translated directly from RNA3. The 3'-proximal coat protein gene is expressed via a subgenomic mRNA, RNA4. The 3a and coat proteins are dispensable for RNA replication but are required for infection spread in plants (8).BMV-directed replication of RNA3 in vivo depends on cis-acting sequences in three regions of RNA3 (see Fig. 3A): the 3' and 5' noncoding regions (NCRs) and the intercistronic NCR (9). The last 160-200 nt of the 3' NCR contain signals for initiation of (-)-strand RNA synthesis in vitro and areThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U....

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“…CMV RdRp was purified from tobacco leaf tissues infected with a satellite-free preparation of CMV strain S using the procedure described previously [14], but where the DEAE-Biogel A column was substituted by a Q-Sepharose Fast Flow column (Pharmacia LKB Biotechnology Inc).…”

Section: Purification Of Rdrp /Rom Infected Tobaccomentioning

confidence: 99%

Replication of cucumber mosaic virus satellite RNA in vitro by an RNA‐dependent RNA polymerase from virus‐infected tobacco

Kaper

Jaspars

1991

FEBS Letters

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An RNA-dependent RNA polymerase purified from tobacco infected with cucumber mosaic virus catalyzes the synthesis of (-) and (+) strands of the viral satellite RNA, CARNA 5. but fails to replicate the satellite R.NA of peanut stunt virus (PSV). The enzyme replicates the genomie RNAs of the three principal cucumoviruses CMV. PSV and tomato aspermy virus (TAV) with varying efficiencies. The specificity with which CMV RdRp replicates differen', sequence-unrelated RNA templates suggests that the site of their recognition requires secondary or higher level structural organization.

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Purification and characterization of brome mosaic virus RNA-dependent RNA polymerase

Cited by 83 publications

References 10 publications

Characterization of cucumber mosaic virus RNA‐dependent RNA polymerase

Characterization of cucumber mosaic virus RNA‐dependent RNA polymerase

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

Replication of cucumber mosaic virus satellite RNA in vitro by an RNA‐dependent RNA polymerase from virus‐infected tobacco

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Purification and characterization of brome mosaic virus RNA-dependent RNA polymerase

Cited by 83 publications

References 10 publications

Characterization of cucumber mosaic virus RNA‐dependent RNA polymerase

Characterization of cucumber mosaic virus RNA‐dependent RNA polymerase

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

Replication of cucumber mosaic virus satellite RNA in vitro by an RNA&#x2010;dependent RNA polymerase from virus&#x2010;infected tobacco

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Replication of cucumber mosaic virus satellite RNA in vitro by an RNA‐dependent RNA polymerase from virus‐infected tobacco