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

Abstract

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.

Electrostatic Interactions Drive the Self-Assembly and the Transcription Activity of the Pseudomonas Phage ϕ6 Procapsid

“…described previously (21). The P4-deficient particles harbor approximately one P4 hexamer and display an expanded conformation and a slower sedimentation rate than the compact wild-type PC (19,21). At 100 mM NaCl, approximately 7 P4 hexamers were assembled on the P4-deficient particles, and the particle sedimentation behavior resembled that of the wild type ( Fig.…”

“…The positions of double-stranded (L, M, and S) and single-stranded (m and s) RNA molecules are indicated on the left. mixture results in their association on the P4-deficient particles with a concomitant rescue of the transcription activity (21). Consequently, the observed reduction in transcription activity (Fig.…”

“…The potentials range from ϩ256 mV (blue) to Ϫ256 mV (red). described previously (21). The P4-deficient particles harbor approximately one P4 hexamer and display an expanded conformation and a slower sedimentation rate than the compact wild-type PC (19,21).…”

Backtracking behavior in viral RNA-dependent RNA polymerase provides the basis for a second initiation site

“…A link can be made between the observed force dependence of backtracking and the behavior of P2 within the Φ6 bacteriophage. Within the bacteriophage, the hexameric NTPase (denoted P4) operates as a conduit for the plus-strand RNA release ( 41 – 43 ). By analogy with the role of the applied force in our magnetic tweezers experiments, we speculate that P4 may, by limiting the excursions of the plus-strand RNA, prevent P2 from becoming trapped in a backtracked state.…”

Single-molecule measurements of viral ssRNA packaging

“…In this case, the in vitro packaging efficiency at single-molecule resolution would be considerably decreased. Also, particles that have less P4 (on average only one P4 hexamer) are expanded and do not recognize the s + segment for packaging (Sun et al 2013), further decreasing the chances of successful experiments.…”