Stoichiometric packaging of the three genomic segments of double-stranded RNA bacteriophage Φ6

Qiao, Xueying; Qiao, Jian; Mindich, Leonard

doi:10.1073/pnas.94.8.4074

Cited by 58 publications

(57 citation statements)

References 36 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…A stable f6-eGFP carrier state cell line containing three copies of the eGFP segment and a single copy of the L kan segment, as determined by densitometry analysis, was selected for dsRNA production. Packaging of multiple copies of reduced size genome segments in f6 has been discussed previously (Mindich et al 1995;Qiao et al 1997;Poranen and Bamford 1999). Approximately 1.6 mg dsRNA/g wet cells were produced (z10% of the total RNA synthesized in the cell), both in batch cultures and in a 10 L fermenter, indicating a linear correlation between the dsRNA yield and the cell mass (Supplemental Table 1).…”

Section: Resultsmentioning

confidence: 79%

Large-scale production of dsRNA and siRNA pools for RNA interference utilizing bacteriophage ϕ6 RNA-dependent RNA polymerase

Aalto¹,

Sarin²,

Dijk³

et al. 2007

RNA

View full text Add to dashboard Cite

The discovery of RNA interference (RNAi) has revolutionized biological research and has a huge potential for therapy. Since small double-stranded RNAs (dsRNAs) are required for various RNAi applications, there is a need for cost-effective methods for producing large quantities of high-quality dsRNA. We present two novel, flexible virus-based systems for the efficient production of dsRNA: (1) an in vitro system utilizing the combination of T7 RNA polymerase and RNA-dependent RNA polymerase (RdRP) of bacteriophage f6 to generate dsRNA molecules of practically unlimited length, and (2) an in vivo RNA replication system based on carrier state bacterial cells containing the f6 polymerase complex to produce virtually unlimited amounts of dsRNA of up to 4.0 kb. We show that pools of small interfering RNAs (siRNAs) derived from dsRNA produced by these systems significantly decreased the expression of a transgene (eGFP) in HeLa cells and blocked endogenous pro-apoptotic BAX expression and subsequent cell death in cultured sympathetic neurons.

show abstract

Section: Resultsmentioning

confidence: 79%

Large-scale production of dsRNA and siRNA pools for RNA interference utilizing bacteriophage ϕ6 RNA-dependent RNA polymerase

Aalto¹,

Sarin²,

Dijk³

et al. 2007

RNA

View full text Add to dashboard Cite

show abstract

“…Second, the rate of encapsidation of deleterious mutants may provides a short-term advantage where (at least some) dominance by higher-fitness alleles allows greater overbe increased during co-infection. During virus reproduction empty virus capsids (protein shells that house all productivity of the virus population; Perrot et al (1991) similarly showed that diploidy can evolve despite the RNA) are generated, and replicated segments enter the capsid in an ordered fashion (Qiao et al 1997).…”

Section: Crow and Kimura 1965) That Sex Results In Acceleratedmentioning

confidence: 99%

Co-infection Weakens Selection Against Epistatic Mutations in RNA Viruses

et al. 2004

View full text Add to dashboard Cite

Co-infection may be beneficial in large populations of viruses because it permits sexual exchange between viruses that is useful in combating the mutational load. This advantage of sex should be especially substantial when mutations interact through negative epistasis. In contrast, co-infection may be detrimental because it allows virus complementation, where inferior genotypes profit from superior virus products available within the cell. The RNA bacteriophage φ6 features a genome divided into three segments. Co-infection by multiple φ6 genotypes produces hybrids containing reassorted mixtures of the parental segments. We imposed a mutational load on φ6 populations by mixing the wild-type virus with three single mutants, each harboring a deleterious mutation on a different one of the three virus segments. We then contrasted the speed at which these epistatic mutations were removed from virus populations in the presence and absence of co-infection. If sex is a stronger force, we predicted that the load should be purged faster in the presence of co-infection. In contrast, if complementation is more important we hypothesized that mutations would be eliminated faster in the absence of co-infection. We found that the load was purged faster in the absence of co-infection, which suggests that the disadvantages of complementation can outweigh the benefits of sex, even in the presence of negative epistasis. We discuss our results in light of virus disease management and the evolutionary advantage of haploidy in biological populations.

show abstract

“…Unless otherwise indicated, previously published 6 RNA packaging reaction conditions were used in all packaging experiments, namely, 50 mM Tris (pH 8.9), 80 mM ammonium acetate (NH 4 Ac), 5 mM MgCl 2 , 2 mM dithiothreitol (DTT), 0.1 mM EDTA, 6% polyethylene glycol 4000, 1 mM ATP, 1 U of rRNasin (Promega)/l, 0.5 to 1.1 g of each 32 P-labeled ssRNA segment in equimolar amounts (6), and freshly prepared procapsid preparations. Packaging reaction mixtures (25 l) were incubated at 30°C for 30 to 90 min.…”

Section: Methodsmentioning

confidence: 99%

Nonspecific Nucleoside Triphosphatase P4 of Double-Stranded RNA Bacteriophage φ6 Is Required for Single-Stranded RNA Packaging and Transcription

Pirttimaa

Paatero

Frilander

et al. 2002

J Virol

View full text Add to dashboard Cite

Bacteriophage 6 has a segmented double-stranded RNA genome. The genomic single-stranded RNA (ssRNA) precursors are packaged into a preformed protein capsid, the polymerase complex, composed of viral proteins P1, P2, P4, and P7. Packaging of the genomic precursors is an energy-dependent process requiring nucleoside triphosphates. Protein P4, a nonspecific nucleoside triphosphatase, has previously been suggested to be the prime candidate for the viral packaging engine, based on its location at the vertices of the viral capsid and its biochemical characteristics. In this study we were able to obtain stable polymerase complex particles that are completely devoid of P4. Such particles were not able to package ssRNA segments and did not display RNA polymerase (either minus-or plus-strand synthesis) activity. Surprisingly, a mutation in P4, S250Q, which reduced the level of P4 in the particles to about 10% of the wild-type level, did not affect RNA packaging activity or change the kinetics of packaging. Moreover, such particles displayed minus-strand synthesis activity. However, no plus-strand synthesis was observed, suggesting that P4 has a role in the plus-strand synthesis reaction also.

show abstract

Stoichiometric packaging of the three genomic segments of double-stranded RNA bacteriophage Φ6

Cited by 58 publications

References 36 publications

Large-scale production of dsRNA and siRNA pools for RNA interference utilizing bacteriophage ϕ6 RNA-dependent RNA polymerase

Large-scale production of dsRNA and siRNA pools for RNA interference utilizing bacteriophage ϕ6 RNA-dependent RNA polymerase

Co-infection Weakens Selection Against Epistatic Mutations in RNA Viruses

Nonspecific Nucleoside Triphosphatase P4 of Double-Stranded RNA Bacteriophage φ6 Is Required for Single-Stranded RNA Packaging and Transcription

Contact Info

Product

Resources

About