Intracellular Assembly of Cyanophage Syn5 Proceeds through a Scaffold-Containing Procapsid

Abstract

Syn5 is a marine cyanophage that is propagated on the marine photosynthetic cyanobacterial strain Synechococcus sp. WH8109 under laboratory conditions. Cryoelectron images of this double-stranded DNA (dsDNA) phage reveal an icosahedral capsid with short tail appendages and a single novel hornlike structure at the vertex opposite the tail. Despite the major impact of cyanophages on life in the oceans, there is limited information on cyanophage intracellular assembly processes within their photosynthetic hosts. The one-step growth curve of Syn5 demonstrated a short cycle with an eclipse period of ϳ45 min, a latent phase of ϳ60 min, and a burst size of 20 to 30 particles per cell at 28°C. SDS-PAGE and Western blot analysis of cell lysates at different times after infection showed the synthesis of major virion proteins and their increase as the infection progressed. The scaffolding protein of Syn5, absent from virions, was identified in the lysates and expressed from the cloned gene. It migrated anomalously on SDS-PAGE, similar to the phage T7 scaffolding protein.Particles lacking DNA but containing the coat and scaffolding proteins were purified from Syn5-infected cells using CsCl centrifugation followed by sucrose gradient centrifugation. Electron microscopic images of the purified particles showed shells lacking condensed DNA but filled with protein density, presumably scaffolding protein. These findings suggest that the cyanophages form infectious virions through the initial assembly of scaffolding-containing procapsids, similar to the assembly pathways for the enteric dsDNA bacteriophages. Since cyanobacteria predate the enteric bacteria, this procapsid-mediated assembly pathway may have originated with the cyanophages.

A multitrophic model to quantify the effects of marine viruses on microbial food webs and ecosystem processes

“…The emergent ratios of organic elements have been shown to vary with latitude in the global oceans that may be driven by variation in the availability of inorganic nutrients (Martiny et al, 2013) Finally, there is a delay-the latent periodbetween infection and lysis, currently omitted from the model framework. Reported latent periods for marine viruses vary from less than an hour (Raytcheva et al, 2011) to dozens of hours (or more) (Suttle and Chan, 1993;Jacobsen et al, 1996;Lindell et al, 2007). Delays between infection and lysis can destabilize model predictions of population dynamics given a single virus-host pair (Levin et al, 1977;Smith and Thieme, 2012).…”

Polar freshwater cyanophage S-EIV1 represents a new widespread evolutionary lineage of phages

“…The detection of only six structural proteins for a phage of this size is undoubtedly an underestimation, especially given the complexity of the tail structure of S-EIV1. For example, SDS-PAGE analysis for cyanophage PaV-LD, which is about 80 nm in diameter and lacks a tail, resolved 13 structural proteins (Gao et al, 2012), while 14 structural proteins were resolved for Cyanophage Syn5 (Raytcheva et al, 2011). Ultimately, a mass spectrometry-based proteomics analysis would be a more sensitive approach for elucidating the structural proteins that make up the S-EIV1 virion.…”

Two Novel Proteins of Cyanophage Syn5 Compose Its Unusual Horn Structure

“…Growing host cells for plating (3 ϫ 10 8 to 4 ϫ 10 8 cells/ml) were diluted 2-fold into fresh artificial seawater (ASW) medium the day before the experiment and incubated overnight (8). Four serum dilutions (1:200, 1:2,000, 1:20,000, and 1:200,000) were prepared for each antibody-antiSyn5, anti-gp53, and anti-gp54.…”

“…Electron microscopy (EM) of Syn5 shows an icosahedral virion, with a head diameter of 60 nm and a short tail (7), which assembles through a procapsid intermediate lacking condensed DNA (8). The procapsid and a class of expanded capsids have been recently directly visualized within infected cells by cryo-electron tomography (cryo-ET) (9).…”

“…The smaller proteins (gp55, gp57, and gp58) are good candidates for the outer capsid knob proteins. The genes for three of these, gp53, gp54, and gp58, were cloned, the proteins were overexpressed and purified, and polyclonal antibodies were raised against them (8,12). These antibodies were used in immunogold labeling to analyze which virion structural components are composed of the novel proteins.…”