Disassembly of the cystovirus ϕ6 envelope by montmorillonite clay

Abstract

Prior studies of clay-virus interactions have focused on the stability and infectivity of nonenveloped viruses, yielding contradictory results. We hypothesize that the surface charge distribution of the clay and virus envelope dictates how the components react and affect aggregation, viral stability, and infectivity. The bacteriophage Cystoviridae species φ6 used in this study is a good model for enveloped pathogens. The interaction between φ6 and montmorillonite (MMT) clay (the primary component of bentonite) is explored by transmission electron microscopy. The analyses show that MMT-φ6 mixtures undergo heteroaggregation, forming structures in which virtually all the virions are either sequestered between MMT platelet layers or attached to platelet edges. The virions swell and undergo disassembly resulting in partial or total envelope loss. Edge-attached viral envelopes distort to increase contact area with the positively charged platelet edges indicating that the virion surface is negatively charged. The nucleocapsid (NCs) remaining after envelope removal also exhibit distortion, in contrast to detergent-produced NCs which exhibit no distortion. This visually discernible disassembly is a mechanism for loss of infectivity previously unreported by studies of nonenveloped viruses. The MMT-mediated sequestration and disassembly result in reduced infectivity, suggesting that clays may reduce infectivity of enveloped pathogenic viruses in soils and sediments.

Keywords: Viral envelope disassembly; viral infectivity; virus adsorption; virus-montmorillonite heteroaggregation; ϕ6 nucleocapsid..

Figure 1

TEM micrographs of (A) φ6 and (B) NCs with trace amounts of MMT. φ6 and NC particles appear consistently round. Only two NCs are identifiable in (A) (labeled NC) in a field of 10² φ6 virions.

Figure 2

A typical TEM micrograph of MMT platelets of varying thickness and relative orientation showing lack of features on a size scale of viral particles. The edge view (arrow) is indicative of platelet stacking.

Figure 3

TEM micrograph showing a population of virions in two clay particles in differing states of disassembly associated with two MMT particles. A population of virions attached to platelet surface is labeled A. Particle labeled B is an edge-attached virion with a disassembled envelope. Dashed arrows indicate distorted or partially disassembled virions; solid arrows point to virions that have undergone total envelope removal leaving only the NCs. Particles labeled C (distorted φ6) and D (φ6 with envelope disassembled) are shown in respective insets.

Figure 4

TEM micrograph of assemblies of virions in varying states of disassembly. Dashed arrows indicate distorted or partially disassembled virions; solid arrows point to virions that have undergone total envelope removal leaving only the NCs. Elongated particle labeled A is a fused virion. Inset shows alternating tetrahedral and octahedral layers in the clay indicative of edge-on orientation. Note that several virions are congregated near platelet edges at upper left of micrograph.

Figure 5

(A) TEM micrograph showing two φ6 virions attached to MMT platelet edges. The virions are distorted resulting in increased contact with the platelet edge. Outlines of the virion envelopes and NCs are shown for particles labeled #1 and #2 in (B) and (C), respectively. Envelope distortions and partial disassembly is evident from the tendency toward ellipsoidal shape of the virions and NC and increased spacing between the NC and envelope.

Figure 6

TEM micrograph showing detergent-produced NCs aggregated with MMT. The majority of NC particles in the aggregate exhibit little or no distortion and remain spherical with a diameter of ˜57 nm.

Figure 7

SDS-PAGE chromatograph showing the four larger molecular-weight proteins (P1, P2, P3, and P4) of phage φ6. The lanes are as follows: (1) isolated φ6 virions; (2) MMT–φ6 virion pellet; (3) MMT-φ6 virion supernatant isolated from pellet preparation; (4) isolated NC; (5) MMT-NC pellet; and (6) MMT-NC supernatant isolated from NC pellet preparation.

Figure 8

φ6 infectivity as a function of the MMT-to-φ6 ratio.