Roles of the Minor Capsid Protein P7 in the Assembly and Replication of Double-Stranded RNA Bacteriophage ϕ6

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.

“…6C). These results are consistent with previous observations (42,43) and confirm the role of P7 as an assembly cofactor.…”

Section: Biochemical Quantitation Of Relative Protein Amounts Insupporting

confidence: 93%

Section: Discussionmentioning

confidence: 99%

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confidence: 99%

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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

Poranen

2012

J Virol

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“…Such capsid organization in which asymmetric dimers of the major capsid protein form a Tϭ1 icosahedral lattice is common among dsRNA viruses but not observed in any other virus group (18). In addition to P4 and P1, the PC contains two minor subunits, the RNA-dependent RNA polymerase P2 (19,20) and the assembly cofactor P7 (1,21), both located at the interior of the PC shell close to the 3-fold axes of symmetry (22,23).…”

mentioning

confidence: 98%

Rescue of Maturation Off-Pathway Products in the Assembly of Pseudomonas Phage ϕ6

Sun

Pirttimaa

et al. 2013

J Virol

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Many complex viruses use an assembly pathway in which their genome is packaged into an empty procapsid which subsequently matures into its final expanded form. We utilized Pseudomonas phage 6, a well-established virus assembly model, to probe the plasticity of the procapsid maturation pathway. The 6 packaging nucleoside triphosphatase (NTPase), which powers sequential translocation of the three viral genomic single-stranded RNA molecules to the procapsid during capsid maturation, is part of the mature 6 virion but may spontaneously be dissociated from the procapsid shell. We demonstrate that the dissociation of NTPase subunits results in premature capsid expansion, which is detected as a change in the sedimentation velocity and as defects in RNA packaging and transcription activity. However, this dead-end conformation of the procapsids was rescued by the addition of purified NTPase hexamers, which efficiently associated on the NTPase-deficient particles and subsequently drove their contraction to the compact naive conformation. The resulting particles regained their biological and enzymatic activities, directing them into a productive maturation pathway. These observations imply that the maturation pathways of complex viruses may contain reversible steps that allow the rescue of the off-pathway conformation in an overall unidirectional virion assembly pathway. Furthermore, we provide direct experimental evidence that particles which have different physical properties (distinct sedimentation velocities and conformations) display different stages of the genome packaging program and show that the transcriptional activity of the 6 procapsids correlates with the number of associated NTPase subunits.

Assembly of Large Icosahedral Double-Stranded RNA Viruses

Poranen

Advances in Experimental Medicine and Biology

2011

Double-stranded RNA (dsRNA) viruses are a diverse group of viruses infecting hosts from bacteria to higher eukaryotes. Among the hosts are humans, domestic animals, and economically important plant species. Fine details of high-resolution virion structures have revealed common structural characteristics unique to these viruses including an internal icosahedral capsid built from 60 asymmetric dimers (120 monomers!) of the major coat protein. Here we focus mainly on the structures and assembly principles of large icosahedral dsRNA viruses belonging to the families of Cystoviridae and Reoviridae. It is obvious that there are a variety of assembly pathways utilized by different viruses starting from similar building blocks and reaching in all cases a similar capsid architecture. This is true even with closely related viruses indicating that the assembly pathway per se is not an indicator of relatedness and is achieved with minor changes in the interacting components.