Bacteriophage ϕ6 Nucleocapsid Surface Protein 8 Interacts with Virus-Specific Membrane Vesicles Containing Major Envelope Protein 9

Abstract

Enveloped double-stranded RNA (dsRNA) bacterial virus Pseudomonas phage ϕ6 has been developed into an advanced assembly system where purified virion proteins and genome segments self-assemble into infectious viral particles, inferring the assembly pathway. The most intriguing step is the membrane assembly occurring inside the bacterial cell. Here, we demonstrate that the middle virion shell, made of protein 8, associates with the expanded viral core particle and the virus-specific membrane vesicle.

TEXT

Pseudomonas phage ϕ6 is an enveloped double-stranded RNA (dsRNA) virus of the family Cystoviridae (22, 32). The ϕ6 genome, which consists of three dsRNA segments (S, M, and L), is encapsidated within a nucleocapsid (NC) surrounded by a lipid membrane envelope derived from the plasma membrane of its host, the Gram-negative plant-pathogenic bacterium Pseudomonas syringae (1, 3, 10, 14, 31). The NC surface consists of protein 8 (P8) arranged in a T=13 icosahedral lattice with P4 at the vertices (3, 5, 10). The internal polymerase complex (PC) is composed of four protein species (P1, P2, P4, and P7) encoded by the L-segment (1, 13, 16, 17, 23). These proteins self-assemble into empty PCs that sequentially package the genomic precursor RNAs, which are converted into dsRNA by the internally located RNA polymerase (1, 6–8, 24, 25, 28), causing a dramatic expansion of the particle (3, 19). The dsRNA-filled PC particles transcribe genomic dsRNA templates for synthesis of late proteins encoded by the S- and M-segments (26). According to the established model of the ϕ6 assembly pathway, P8 is subsequently assembled onto dsRNA-filled PCs (1, 20, 21, 24) and the newly formed NC acquires a virus-specific lipid envelope from the plasma membrane in a process assisted by the viral nonstructural protein 12 (1). P9, the major viral envelope-associated membrane protein, and the assembly factor P12 are the minimal components required to induce the formation of virus-specific vesicles (1, 12, 30). In this study, we have set up a recombinant expression system (Fig. 1) to further characterize the interactions between P8, the PC, and the viral envelope.

Fig 1.

Construction of pMH expression plasmids. The schematic figure depicts the genes expressed by ϕ6 L-segment-based vectors pMH2, pMH9, and pMH10 (A), as well as ϕ6 S-segment-based vectors pMH4, pMH7, and pMH8 (B). The gene numbers correspond to those of the proteins. The ϕ6 L-segment is derived from pLM687 (18), and the ϕ6 S-segment from pLM659 (9). The restriction enzyme sites indicated were used for cloning (pMH2 and pMH4) or truncation of the insert (pMH7, pMH8, pMH9, and pMH10). Transcription of correct-length mRNA from pMH4, pMH7, and pMH8 is ensured by a 3′-terminal transcription terminator (T7 term.). Synthesis of the lytic enzyme P5 is prevented by a 4× stop codon insertion in gene 5 in the pMH4 and pMH7 vectors. Constructs based on plasmid pGZ119EH (15) contain a chloramphenicol resistance gene, whereas pLM659-based vectors carry an ampicillin resistance gene.

Production of P8 requires additional ϕ6 proteins.

Previously, P8 has only been obtained through sequential disassembly of purified virions (2, 21). We set out to acquire P8 by coexpression of its gene with other ϕ6 genes. Constructs derived from cDNA copies of the ϕ6 S- and L-segments (Fig. 1) were expressed in Escherichia coli JM109 cells for 16 h at room temperature in L-broth (1% [wt/vol] Bacto tryptone, 0.5% [wt/vol] Bacto yeast extract, 0.5% [wt/vol] NaCl) supplemented with the relevant antibiotics following induction with 1 mM isopropyl β-d-1-thiogalactopyranoside. The pelleted cells were disrupted with a French pressure cell, and the protein content was analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) (27). Recombinant P8 was recovered only when synthesized in concert with other ϕ6 proteins (Fig. 2); the recovery was most prominent when the gene was coexpressed with those encoding PC proteins P9 and P12 (Fig. 2, lane 5), as determined by Western blotting using a P8-specific polyclonal antibody and chemiluminescent detection (27).

Fig 2.

Western blot detection of recombinant P8 from E. coli lysates. The plus signs indicate that the corresponding gene was included in the DNA construct(s) expressed in E. coli JM109 cells. P8 obtained from purified bacteriophage ϕ6 virions (2) is included as a control (lane 1).

P8 associates with P9-specific lipid vesicles.

Previous in vitro assembly studies have shown that purified P8 readily reassembles onto both in vitro-constituted and virion-derived PCs (20, 21, 24). In the absence of other viral components, virion-derived P8 forms aberrant shell-like structures (4, 21). To further analyze P8 interactions, the supernatant derived from the lysate of E. coli BL21(DE3) synthesizing P1-P2-P4-P7-P8-P9-P12-P14 proteins (lysate centrifuged at 9,300 × g for 4 min) was subjected to rate zonal centrifugation (5 to 20% sucrose gradient in 20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10 mM MgCl₂ in a Sorvall TH641 rotor at 27,000 rpm, corresponding to approximately 90,000 × g, for 110 min at 15°C). After centrifugation, the gradient was fractionated and the protein content was analyzed by SDS-PAGE. Interestingly, it appeared that some P8 cosedimented with P9 (Fig. 3A). This slowly sedimenting P8-P9 particle and a faster P8-containing particle (Fig. 3A) were collected and subjected to equilibrium flotation centrifugation (Sorvall TH641 rotor at 40,000 rpm for 62 h at 4°C; as described in reference 14) to separate lipid-protein vesicles from proteinaceous viral particles. The gradients were fractionated, and the samples were analyzed by SDS-PAGE (Fig. 3B and C). Samples for transmission electron cryomicroscopy were vitrified (3) and transferred to a Gatan 626 cryostage. Micrographs were recorded on an FEI Tecnai F20 electron microscope operating at 200 kV (nominal magnification, ×50,000). Equilibrium flotation centrifugation of the slowly sedimenting material showed a significant excess of P8 and P9 in the lipid-containing low-density fractions, whereas PC proteins were less prominent (Fig. 3B). Cryoelectron microscopy confirmed the presence of membrane vesicles containing aberrant shell-like structures apparently consisting of P8 (Fig. 3D, panel I). Equilibrium flotation centrifugation of the faster-sedimenting material revealed expanded PCs with a P8 shell enclosed in membrane vesicles, similar to virions (Fig. 3C and D, panel II) (11). The pellet was highly enriched with PCs in their typical unexpanded conformation (Fig. 3C and D, panel III).

Fig 3.

P8 associates with P9-specific lipid vesicles. (A to C) Results of denaturing 15% polyacrylamide gel electrophoresis following rate zonal centrifugation of P1-P2-P4-P7-P8-P9-P12-P14 proteins derived from gene expression in E. coli BL21(DE3) (A) and equilibrium flotation centrifugation of slow-sedimenting (B) and fast-sedimenting (C) material. The positions of specific fractions following centrifugation are depicted in the schematic drawings (top). Purified ϕ6 (2) is included as an electrophoresis marker (lane ϕ6), and the relevant proteins are labeled. The P8-containing fractions (indicated as I to III) in panels B and C were further analyzed by cryoelectron microscopy (D, I to III). The schematic drawings depict the composition of particles representative for panels I to III. The scale bar in panel III is equivalent to 100 nm (applies to panels I to III).

Pathway for nucleocapsid and envelope assembly.

Based on the above-described observations, P8 can be recovered when its gene is coexpressed with those encoding the PC components (Fig. 2) and it readily associates with ϕ6-specific lipid vesicles (Fig. 3). Previous studies have shown that synthesis of P8 is required for envelopment of the virion, but interaction between P8 and the virus-specific vesicles during ϕ6 infection has not been detected (1, 29, 30). Apparently, the P8-vesicle interaction observed in E. coli (Fig. 3) may be suppressed in the P. syringae host until P8 is assembled on the PC. Furthermore, assembly of the P8 shell in vitro has only been accomplished on dsRNA-filled expanded PCs (21). Accordingly, the empty compact form of the PC is not observed in the enveloped particles (Fig. 3). These observations suggest that the assembly of the NC is regulated both by interaction with the expanded P1 shell in the PC and by interaction with the P9-containing lipid vesicle. Therefore, P8 assembly requires expansion of the metastable PC, which can occur either spontaneously or through sequential packaging and replication of the genomic precursors. The expanded PC provides the scaffold around which P8 and ϕ6-specific lipid vesicles assemble.