Electron Microscopy of In-Plaque Phage T3 Assembly: Proposed Analogs of Neurodegenerative Disease Triggers

Abstract

Increased knowledge of virus assembly-generated particles is needed for understanding both virus assembly and host responses to virus infection. Here, we use a phage T3 model and perform electron microscopy (EM) of thin sections (EM-TS) of gel-supported T3 plaques formed at 30 °C. After uranyl acetate/lead staining, we observe intracellular black particles, some with a difficult-to-see capsid. Some black particles (called LBPs) are larger than phage particles. The LBP frequency is increased by including proflavine, a DNA packaging inhibitor, in the growth medium and increasing plaque-forming temperature to 37 °C. Acidic phosphotungstate-precipitate (A-PTA) staining causes LBP substitution by black rings (BRs) that have the size and shape expected of hyper-expanded capsid containers for LBP DNA. BRs are less frequent in liquid cultures, suggesting that hyper-expanded capsids evolved primarily for in-gel (e.g., in-biofilm) propagation. BR-specific A-PTA staining and other observations are explained by α-sheet intense structure of the major subunit of hyper-expanded capsids. We hypothesize that herpes virus triggering of neurodegenerative disease occurs via in-gel propagation-promoted (1) generation of α-sheet intense viral capsids and, in response, (2) host production of α-sheet intense, capsid-interactive, innate immunity amyloid protein that becomes toxic. We propose developing viruses that are therapeutic via detoxifying interaction with this innate immunity protein.

In-Gel Isolation and Characterization of Large (and Other) Phages

“…In-plaque EM-TS is effective for non-tailed dsDNA phages [48] and podophage T3 [30]. Figure 3 shows effectiveness for myophage G. Several G capsids (C in Figure 3a), some associated with condensed DNA (C-D) are in this image.…”

“…Furthermore, the gel-breaking pressure exerted by elongating bacterial cells results in counter pressure on cells. This counter pressure is in the range of pressures that cause change in state of the bacterial cytoplasm [26], a change that, in turn, might explain an observed [30] altering of phage T3 infection in-gel.…”

“…Perhaps, the importance of plaque-supporting gel P E has been obscured by the fact that bacteria propagate within agar gels as concentrated as at least 1%. However, we now know that the bacteria push gel fibers aside even when propagating in 0.6% agarose gels [30]. Phages cannot do this.…”

A Protein Assembly Hypothesis for Population-Specific Decrease in Dementia with Time

“…More recently, yet additional data suggest that the details of this innate immune reaction are the following [11,12]. 1The structural basis is a (self-complementary) sheetlike protein structure, originally discovered by Pauling and Corey [13], and later called α-sheet [14][15][16].…”

“…This rotation places all α-carboxyl groups (negatively charged at neutral pH) on one edge of the sheet and all α-amino groups (positively charged at neutral pH) on the opposite edge. Based on observation and characterization of DNA packaging-generated hyper-expanded capsids (details, below), α-sheet is proposed to be a major conformation of virus capsid proteins during virus assembly [11,12]. However, in healthy human cells, α-sheet is present only in stretches of 4-6 amino acids [14,15].…”