In vitro replication, packaging, and transcription of the segmented double-stranded RNA genome of bacteriophage phi 6: studies with procapsids assembled from plasmid-encoded proteins

Gottlieb, Paul; Strassman, J; Qiao, Xueying; Frucht, A. H.; Mindich, Leonard

doi:10.1128/jb.172.10.5774-5782.1990

Cited by 85 publications

(87 citation statements)

References 31 publications

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“…P124 procapsids lacking P7 were produced with cell line LM1115 and plasmid pLM574 (16). Procapsid Isolation-Cell cultures were grown overnight in minimal medium and induced with 1 mM isopropyl ␤-D-thiogalactopyranoside.…”

Section: Methodsmentioning

confidence: 99%

Initial Location of the RNA-dependent RNA Polymerase in the Bacteriophage Φ6 Procapsid Determined by Cryo-electron Microscopy

Sen

Heymann

Cheng

et al. 2008

Journal of Biological Chemistry

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The RNA-dependent RNA polymerases (RdRPs) of Cystoviridae bacteriophages, like those of eukaryotic viruses of the Reoviridae, function inside the inner capsid shell in both replication and transcription. In bacteriophage ⌽6, this inner shell is first assembled as an icosahedral procapsid with recessed 5-fold vertices that subsequently undergoes major structural changes during maturation. The tripartite genome is packaged as singlestranded RNA molecules via channels on the 5-fold vertices, and transcripts probably exit the mature capsid by the same route. The RdRP (protein P2) is assembled within the procapsid, and it was thought that it should be located on the 5-fold axes near the RNA entry and exit channels. To determine the initial location of the RdRP inside the procapsid of bacteriophage ⌽6, we performed cryo-electron microscopy of wild type and mutant procapsids and complemented these data with biochemical determinations of copy numbers. We observe ring-like densities on the 3-fold axes that are strong in a mutant that has ϳ10 copies of P2 per particle; faint in wild type, reflecting the lower copy number of ϳ3; and completely absent in a P2-null mutant. The dimensions and shapes of these densities match those of the known crystal structure of the P2 monomer. We propose that, during maturation, the P2 molecules rotate to occupy positions closer to adjacent 5-fold vertices where they conduct replication and transcription.

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Section: Methodsmentioning

confidence: 99%

Initial Location of the RNA-dependent RNA Polymerase in the Bacteriophage Φ6 Procapsid Determined by Cryo-electron Microscopy

Sen

Heymann

Cheng

et al. 2008

Journal of Biological Chemistry

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“…The procapsid-associated P4 sequentially translocates the three ssRNA genomic precursors into the particle interior (18 -20). The packaged RNA precursors are then replicated inside the viral particle into dsRNA genome segments (21). Although the procapsid-associated packaging is highly specific for the viral RNA, an unspecific, in vitro translocase activity was recently demonstrated for isolated P4 hexamers from bacteriophages 8 and 13 (13).…”

mentioning

confidence: 99%

Enzymatic Mechanism of RNA Translocation in Double-stranded RNA Bacteriophages

Lísal

Kainov

Bamford

et al. 2004

Journal of Biological Chemistry

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Many complex viruses acquire their genome by active packaging into a viral precursor particle called a procapsid. Packaging is performed by a viral portal complex, which couples ATP hydrolysis to translocation of nucleic acid into the procapsid. The packaging process has been studied for a variety of viruses, but the mechanism of the associated ATPase remains elusive. In this study, the mechanism of RNA translocation in doublestranded RNA bacteriophages is characterized using rapid kinetic analyses. The portal complex of bacteriophage 8 is a hexamer of protein P4, which exhibits nucleotide triphosphatase activity. The kinetics of ATP binding reveals a two-step process: an initial, fast, second-order association, followed by a slower, first-order phase. The slower phase exhibits a high activation energy and has been assigned to a conformational change. ATP binding becomes cooperative in the presence of RNA. Steady-state kinetics of ATP hydrolysis, which proceeds only in the presence of RNA, also exhibits cooperativity. On the other hand, ADP release is fast and RNA-independent. The steady-state rate of hydrolysis increases with the length of the RNA substrate indicating processive translocation. Raman spectroscopy reveals that RNA binds to P4 via the phosphate backbone. The ATP-induced conformational change affects the backbone of the bound RNA but leaves the protein secondary structure unchanged. This is consistent with a model in which cooperativity is induced by an RNA link between subunits of the hexamers and translocation is effected by an axial movement of the subunits relative to one another upon ATP binding.Genome encapsidation in many viruses is realized through energy-dependent packaging into a preformed capsid (procapsid). This requires molecular motors (portal complexes) converting chemical energy (ATP hydrolysis) into mechanical work (translocation). A wealth of structural and functional data has been obtained for portal complexes of dsDNA 1 bacteriophages (e.g. 29, , SPP1, P22, T4, and T-odd phages (1-6), but the molecular basis of the mechanochemical coupling is not understood. This is partly because of the complexity of portal complexes, in which the packaging ATPase is a multifunctional enzyme and associates transiently with the packaging machinery (i.e. the ATPase is a non-structural protein) (4). In particular, very little is known about the enzymatic action of these ATPases during packaging (7-9).The packaging system of dsRNA bacteriophages from the Cystoviridae family (phages 6-14 (10, 11)) is considerably simpler. The ssRNA translocation is fueled by a single structural protein P4. P4 forms stable hexamers (12, 13), which possess nonspecific nucleotide triphosphatase activity and resides at the 5-fold vertices of the procapsid (14, 15). The dodecahedral procapsid results from co-assembly of the major structural protein P1 with P4 hexamers and minor proteins P2 (RNA polymerase) and P7 (assembly and functional cofactor) (16,17).Cystoviruses have a tripartite dsRNA genome. The procapsid-associa...

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“…Purified 6 P1 is a monomer, but the equivalent protein from a related cystovirus, 8, forms tetramers in solution (25,43). Protein P2 is an RNAdependent RNA polymerase catalyzing genome replication and transcription (19,26,27). In dsRNA viruses of the family Reoviridae, such as orthoreovirus (52), rotavirus (29), rice dwarf virus (33), aquareovirus (11), and gram carp reovirus (10), the polymerase subunit localizes to the interior of the Tϭ1 shell, close to 5-fold axes of icosahedral symmetry, resulting in a maximal number of 12 polymerase subunits per particle.…”

mentioning

confidence: 99%

“…The resulting particles are competent for all enzymatic functions of the PC, including NTP hydrolysis-driven single-stranded RNA (ssRNA) packaging, replication of the packaged RNA molecules to the dsRNA form, and subsequent RNA transcription from the dsRNA templates (43). Incomplete PCs containing the main structural protein, P1, with one or two additional protein components, can be obtained using the recombinant expression system (14,17,19,38) or by omitting minor protein subunits from the self-assembly reaction mix (43). The 6 PC assembly pathway has been established based on kinetic studies of in vitro assembly reactions (43).…”

mentioning

confidence: 99%

Probing, by Self-Assembly, the Number of Potential Binding Sites for Minor Protein Subunits in the Procapsid of Double-Stranded RNA Bacteriophage φ6

Sun

Bamford

Poranen

2012

J Virol

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The double-stranded RNA bacteriophage 6 is an extensively studied prokaryotic model system for virus assembly. There are established in vitro assembly protocols available for the 6 system for obtaining infectious particles from purified protein and RNA constituents. The polymerase complex is a multifunctional nanomachine that replicates, transcribes, and translocates viral RNA molecules in a highly specific manner. The complex is composed of (i) the major structural protein (P1), forming a T‫1؍‬ icosahedral lattice with two protein subunits in the icosahedral asymmetric unit; (ii) the RNA-dependent RNA polymerase (P2); (iii) the hexameric packaging nucleoside triphosphatase (NTPase) (P4); and (iv) the assembly cofactor (P7). In this study, we analyzed several 6 virions and recombinant polymerase complexes to investigate the relative copy numbers of P2, P4, and P7, and we applied saturated concentrations of these proteins in the self-assembly system to probe their maximal numbers of binding sites in the P1 shell. Biochemical quantitation confirmed that the composition of the recombinant particles was similar to that of the virion cores. By including a high concentration of P2 or P7 in the self-assembly reaction mix, we observed that the numbers of these proteins in the resulting particles could be increased beyond those observed in the virion. Our results also suggest a previously unidentified P2-P7 dependency in the assembly reaction. Furthermore, it appeared that P4 must initially be incorporated at each, or a majority, of the 5-fold symmetry positions of the P1 shell for particle assembly. Although required for nucleation, excess P4 resulted in slower assembly kinetics.

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In vitro replication, packaging, and transcription of the segmented double-stranded RNA genome of bacteriophage phi 6: studies with procapsids assembled from plasmid-encoded proteins

Cited by 85 publications

References 31 publications

Initial Location of the RNA-dependent RNA Polymerase in the Bacteriophage Φ6 Procapsid Determined by Cryo-electron Microscopy

Initial Location of the RNA-dependent RNA Polymerase in the Bacteriophage Φ6 Procapsid Determined by Cryo-electron Microscopy

Enzymatic Mechanism of RNA Translocation in Double-stranded RNA Bacteriophages

Probing, by Self-Assembly, the Number of Potential Binding Sites for Minor Protein Subunits in the Procapsid of Double-Stranded RNA Bacteriophage φ6

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