Self-Assembly of a Viral Molecular Machine from Purified Protein and RNA Constituents

Poranen, Minna M.; Paatero, Anja O.; Tůma, Roman; Bamford, Dennis H.

doi:10.1016/s1097-2765(01)00228-3

Cited by 90 publications

(171 citation statements)

References 45 publications

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“…These observations set BTV apart from bacteriophages φ6 and φ8 of Cystoviridae family, in which the assembly takes place in the absence of RNA and its reconstitution was possible, expressing in vitro each viral protein in Escherichia coli. The RNA is then packaged a posteriori to the formation of procapsids (26)(27)(28).…”

Section: Discussionmentioning

confidence: 99%

In vitro reconstitution of Bluetongue virus infectious cores

Lourenco

Roy

2011

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

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Bluetongue virus (BTV) is a vector-borne, nonenveloped icosahedral particle that is organized in two capsids, an outer capsid of two proteins, VP2 and VP5, and an inner capsid (or core) composed of two major proteins, VP7 and VP3, in two layers. The VP3 layer (subcore) encloses viral transcription complex (VP1 polymerase, VP4 capping enzyme, VP6 helicase) and a 10-segmented double-stranded (dsRNA) genome. Although much is known about the BTV capsids, the order of the core assembly and the mechanism of genome packaging remain unclear. Here, we established a cell-free system to reconstitute subcore and core structures with the proteins and ssRNAs, demonstrating that reconstituted cores are infectious in insect cells. Furthermore, we showed that the BTV ssRNAs are essential to drive the assembly reaction and that there is a distinct order of internal protein recruitment during the assembly process. The in vitro engineering of infectious BTV cores is unique for any member of the Reoviridae and will facilitate future studies of RNA-protein interactions during BTV core assembly.assembly pathway | Orbivirus | engineered particle

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Section: Discussionmentioning

confidence: 99%

In vitro reconstitution of Bluetongue virus infectious cores

Lourenco

Roy

2011

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

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“…This type of capsid organization is common for dsRNA viruses but has not been observed in any other type of virus (41). 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).…”

mentioning

confidence: 99%

“…Bacteriophage 6, which belongs to the family Cystoviridae (2), is a well-studied prokaryotic model system for dsRNA virus assembly, with an established in vitro self-assembly system to produce infectious viral nucleocapsids from purified protein and nucleic acid constituents (43). The 6 virion core is composed of a dodecahedral polymerase complex enclosing the three genomic dsRNA segments (S, M, and L segments) (6,45).…”

mentioning

confidence: 99%

“…Furthermore, it has been determined (34) that a single recombinant PC preparation can be composed of particles containing 0 to 12 P4 hexamers, with the majority of particles having either one or five 5-fold symmetry positions occupied. Protein P7 is an assembly cofactor that accelerates PC assembly and is essential for the formation of infectious virions (42,43). P7 forms elongated dimers in solution (23); however, recent structural studies indicate that P7 subunits are arranged as monomers around the 3-fold axes of empty PCs (35).…”

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confidence: 99%

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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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“…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).…”

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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Self-Assembly of a Viral Molecular Machine from Purified Protein and RNA Constituents

Cited by 90 publications

References 45 publications

In vitro reconstitution of Bluetongue virus infectious cores

In vitro reconstitution of Bluetongue virus infectious cores

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

Enzymatic Mechanism of RNA Translocation in Double-stranded RNA Bacteriophages

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