The objective of this study was to determine the location of protein P7, the RNA packaging factor, in the procapsid of the φ6 cystovirus. A comparison of cryo-electron microscopy high-resolution single particle reconstructions of the φ6 complete unexpanded procapsid, the protein P2-minus procapsid (P2 is the RNA directed RNA-polymerase), and the P7-minus procapsid, show that prior to RNA packaging the P7 protein is located near the three-fold axis of symmetry. Difference maps highlight the precise position of P7 and demonstrate that in P7-minus particles the P2 proteins are less localized with reduced densities at the three-fold axes. We propose that P7 performs the mechanical function of stabilizing P2 on the inner protein P1 shell which ensures that entering viral single-stranded RNA is replicated.

Cryo-electron microscopy projection image analysis and tomography is used to describe the overall architecture of influenza B/Lee/40. Algebraic reconstruction techniques with utilization of volume elements (blobs) are employed to reconstruct tomograms of this pleomorphic virus and distinguish viral surface spikes. The purpose of this research is to examine the architecture of influenza type B virions by cryo-electron tomography and projection image analysis. The aims are to explore the degree of ribonucleoprotein disorder in irregular shaped virions; and to quantify the number and distribution of glycoprotein surface spikes (hemagglutinin and neuraminidase) on influenza B. Projection image analysis of virion morphology shows that the majority (∼83%) of virions are spherical with an average diameter of 134±19 nm. The aspherical virions are larger (average diameter = 155±47 nm), exhibit disruption of the ribonucleoproteins, and show a partial loss of surface protein spikes. A count of glycoprotein spikes indicates that a typical 130 nm diameter type B virion contains ∼460 surface spikes. Configuration of the ribonucleoproteins and surface glycoprotein spikes are visualized in tomogram reconstructions and EM densities visualize extensions of the spikes into the matrix. The importance of the viral matrix in organization of virus structure through interaction with the ribonucleoproteins and the anchoring of the glycoprotein spikes to the matrix is demonstrated.

Cryo-electron tomography and subtomogram averaging are utilized to determine that the bacteriophage ϕ12, a member of the Cystoviridae family, contains surface complexes that are: toroidal in shape; composed of six globular domains with six-fold symmetry; and have a discrete density connecting them to the virus membrane-envelope surface. The lack of a hexameric spike in a reassortant of ϕ12 demonstrates that the gene for the hexameric spike is located in ϕ12’s medium length genome segment, likely to the P3 open reading frames which are the proteins involved in viral-host cell attachment. Based on this, and on protein mass estimates derived from the obtained averaged structure, it is suggested that each of the globular domains is most likely composed of a total of four copies of P3a and/or P3c proteins. Our findings may have implications in the study of the evolution of the cystovirus species in regard to their host specificity.

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.

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