Bacteriophage φ6--structure investigated by fluorescence Stokes shift spectroscopy.

The Stokes shift of tryptophan (Trp) fluorescence from layers of the lipid-containing bacteriophage ϕ6 are compared to determine the relative effect of the layers on virus hydrophobicity. In the inner most layer, the empty procapsid (PC) which contains 80% – 90% of the virion Trp residues, λmax = 339.8 nm. The PC emission is substantially more red-shifted than the other ϕ6 layers and nearer to that of the Pseudomonad host cell than the other ϕ6 layers. The Trp emission from the nucleocapsid (NC) with λmax = 337.4 nm, is blue shifted by 2.4 nm relative to the PC although the number of Trp in the NC is identical to the PC. This shift represents an increase in Trp hydrophobicity, likely a requirement for the maintenance of A-form dsRNA. Fluorescence from the completely assembled virion indicates it is in a considerably more hydrophobic environment with λmax = 330.9 nm. Density measurements show that the water content in the NC does not changed during envelope assembly, therefore the blue-shifted ϕ6 emission suggests that the envelope changes the PC environment, probably via the P8 layer. This change in hydrophobicity likely arises from charge redistribution or envelope-induced structural changes in the PC proteins. Introduction The bacteriophage ϕ6 is a member of Cystoviridae, a family of viruses that are related to reoviruses. The cystoviruses contain a genome composed of three segments of doubled-stranded RNA (dsRNA) and are structurally analogous to reoviruses in that they have a multiple layered architecture. ϕ6 has a three-layer structure, shown schematically in Fig. 1. The inner core of the virus consists of four proteins designated P1, P2, P4 and P7, which together form the procapsid (PC). The PC has the capability of packaging three segments of viral message RNA that is subsequently replicated to dsRNA. The three dsRNA segments are named according to their size: small (S), medium (M) and large (L). Of the four proteins that comprise the PC, P1 is the major structural protein that is believed to alter its conformation during the RNA packaging reactions (1). The atomic structure of P1 has not been established, however studies of the isolated P1 from ϕ8 suggest that it is tetrameric. P1 from ϕ6 has only been purified in small amounts as a monomer but in vitro assembly assays show a fourth-order concentration dependence, further suggesting it is tetrameric (2, 3). The remaining three procapsid proteins, P2, P4, and P7 together constitute the RNA packaging/replication portal and the atomic structure of each has been determined (4–6). P2 is the RNA-directed RNA polymerase (RdRP) a monomeric protein that replicates and transcribes the dsRNA viral genome (7). P4 is a hexameric nucleotide-triphosphorylase that packages each viral mRNA segment. P4 is located at the five-fold axis of symmetry of the PC creating a symmetry mismatch by imposing its six-fold symmetry (8). The position of the P7 dimer within the assembled PC has not been confirmed but data from single particle reconstructions of the ϕ12 virus particle tentatively identified electron densities at the five-fold axis and surrounding the P4 hexamer as being P7. Fig. 1 A) Schematic of ϕ6 and inner layers: B) ϕ6−P3; C) NC; and D) PC. The cystovirus replicative mechanism starts with assembly of the empty PC. This is followed by the specific selection and packaging of three segments of viral mRNA within the PC (7, 9–11). The dodecahedral-shaped PC contains the RNA packaging and replication apparatus at each of 12 portals constituting multiple polymerase elements (PX). The PX consists of P2, P4 and, based on cryo-electron tomography of the related ϕ12 virus, P7 (12). The dsRNA segments are packaged in a specific size order and replicated to the double-stranded format utilizing RdRP only after packaging is complete. The RdRPs are located at each of the 12 portals and appear to undergo a position shift from the three-fold axis to the five-fold axis of the PX after PC expansion (12, 13). The PC undergoes a specific and sequential expansion during RNA packaging that selectively exposes RNA binding sites for each of the three RNA segments (1, 4, 14). The genomic RNA interacts with the internal structure of the PC and may modify P1, the main structural component. Mutations in the genome RNA positive stranded segments that alter packaging order have resulted in selection of P1 mutations that alter the overall charge distribution of the PC (15, 16). After packaging and RNA replication, a matrix composed of 200 trimeric copies of protein P8, is assembled around the filled PC. The P8 matrix is an open structure which can allow passage of water, cations and other smaller molecules (1). The PC, P8 and genome together form the nucleocapsid (NC). A lipid-bilayer-membrane envelope, derived from the Pseudomonas host-cell, is assembled around the NC. The mechanism of viral envelope assembly is not well understood. It involves the non-structural P12 protein which is not present in the completely assembled virion. The envelope contains four membrane proteins, P6, P9, P10 and P13, within a lipid bilayer. Based on cryo-electron microscopy of the related ϕ12 virus, protein P5 is likely positioned between the NC and the envelope (12, 17). P3 proteins form external spikes on the envelope whose function are to bind to the host cell receptor. In ϕ6, the three discrete layers can be selectively removed to reveal the subviral elements (17, 18). Alternatively, recombinant RNA packaging-competent procapsid structures can be assembled (7, 19). In the work presented here, we employ fluorescence Stokes shift spectroscopy of each subviral layer to investigate the hydrophobic and hydrophilic qualities of the layers. The extent of hydrophobicity has implications for the internal environment of the virus in regard to packaged viral genomic RNA and viral replication. Trp fluorescence has been employed extensively to study proteins (20). It is well known that the Stokes shift and quantum efficiency of tryptophan (Trp) fluorescence are dependent on the local environment and protein structure (21, 22) with the emission being more red-shifted in a hydrophilic environment. Of the aromatic amino acids, Trp is both the predominant fluorophore (20, 23) and strongest absorber (20) and Trp is the predominant fluorophore in viruses. However, there have been very few reports on the use of Stokes shift spectroscopy to study proteins in phage. Kitchell et al. investigated Trp accessibility in bacteriophage f2 and observed that the blue-shifted Trp fluorescence indicated a hydrophobic Trp environment (24). Kneale and Van Resandt studied the DNA binding protein in bacteriophage Pf1 using spectral shifts and time-resolved measurements of Trp fluorescence (25). Urbaneja et al. investigated nucleic acid interactions in phage ϕ29 by Trp fluorescence (26). More recently, Alimova et al. used Trp Stokes shift fluorescence to study the infection processes of cystoviruses ϕ6 and ϕ12 (27, 28). In Alimova 2004, it was observed that the ϕ6 Trp emission exhibited a decrease in hydrophobicity shortly after infection. In the work present here, we compare the Trp emission for the different ϕ6 layers to examine the differences in hydrophobicity of the layers and its implication for protein interactions during viral assembly. To the best of our knowledge, this is the first time Stokes shift fluorescence spectroscopy has been used to probe the multi-layers of any viral system.