States of phage T3/T7 capsids: buoyant density centrifugation and cryo-EM

Abstract

Mature double-stranded DNA bacteriophages have capsids with symmetrical shells that typically resist disruption, as they must to survive in the wild. However, flexibility and associated dynamism assist function. We describe biochemistry-oriented procedures used to find previously obscure flexibility for capsids of the related phages, T3 and T7. The primary procedures are hydration-based buoyant density ultracentrifugation and purified particle-based cryo-electron microscopy (cryo-EM). We review the buoyant density centrifugation in detail. The mature, stable T3/T7 capsid is a shell flexibility-derived conversion product of an initially assembled procapsid (capsid I). During DNA packaging, capsid I expands and loses a scaffolding protein to form capsid II. The following are observations made with capsid II. (1) The in vivo DNA packaging of wild type T3 generates capsid II that has a slight (1.4%), cryo-EM-detected hyper-expansion relative to the mature phage capsid. (2) DNA packaging in some altered conditions generates more extensive hyper-expansion of capsid II, initially detected by hydration-based preparative buoyant density centrifugation in Nycodenz density gradients. (3) Capsid contraction sometimes occurs, e.g., during quantized leakage of DNA from mature T3 capsids without a tail.

Characterization of a Novel Tanay Virus Isolated From Anopheles sinensis Mosquitoes in Yunnan, China

“…This can be due to: (1) we did not choose good timing and angle of the slice or; (2) perhaps the tail is packaged when it is budding from the virus or it was a; (3) limitation of the used microscope. The recently developed cryo-EM, that has less artifact-prone alternative to thin-section, might be the best instrument of observation in this case (Serwer et al, 2018). Besides, the TANAV virus projection-like structure is comparable to the injection needle of phages (Guan et al, 2019), hence future studies should investigate whether the structure plays a role in virus binding to the host cell receptors, increasing its adsorption during virus entry.…”

Single-particle virology

“…Most of our knowledge has come from the high-resolution structures provided by electron microscopy studies of the past decades, which enabled us to predict the functions of different parts of viral nanomachines. In the recent past, cryo-electron microscopy has been providing an ever-increasing detail about the structure of viruses, lending clues to the infection mechanisms (Guo et al 2014 ; Kaelber et al 2017 ; Pham et al 2015 ; Serwer et al 2018 ; Shingler et al 2013 ; Wrapp et al 2020 ). Besides imaging methods, bulk assays relying on molecular biological techniques have provided key elements to understanding the steps of viral infection.…”

Nanomedicine and Phage Capsids

“…The DNA remaining packaged is obtained by (1) DNase I-digestion of external DNA; and (2) expulsion from the capsid of DNA remaining packaged [ 26 ]. This leakage-quantization phenomenon is best explained by quantized gp10 shell contraction that evolved via selection for control of the rate of infection-initiating DNA injection [ 26 , 33 ].…”