Structure of epsilon15 bacteriophage reveals genome organization and DNA packaging/injection apparatus

Abstract

Using single particle electron cryomicroscopy that does not impose icosahedral averaging, we determined the structure of the entire infectious Salmonella phage Epsilon15 1 , including both icosahedral and non-icosahedral components. At least three layers of condensed viral DNA were observed to pack in coaxial coils with local 25 Å hexagonal inter-strand spacing. At one of the fivefold vertices, a portal complex with twelve subunits replaces a capsid pentamer. A tail hub with six projecting trimeric tailspikes sits on the external face of the portal. Below the portal is a cylindrical protein core. An extended shaft of density fills the central channel of the protein core and the portal complex and appears to consist of about 90 nucleotides at the terminus of the packaged DNA poised for injection. Using an icosahedral symmetry imposed reconstruction, the fold of the capsid shell protein is seen to resemble the capsid protein fold of other tailed double-stranded DNA phages 2-5 and human herpesvirus 6 . These common structural features suggest a common evolutionary origin among these viruses. Double-stranded DNA (dsDNA) phages are vectors for gene transfer among enteric bacteria, including important human pathogens 7 . For all the well-studied tailed dsDNA phages, a preformed procapsid shell is assembled, and the DNA is pumped into the shell through a portal complex located at a single vertex 8 . The phage tails are also assembled at this vertex. The portal complex together with packaging enzymes have been shown to function as components of a very powerful molecular motor 9 , but it has not been possible to visualize the complex within the intact virion.The inability to visualize the packed DNA and the portal vertex in the virion reflects the difficulties in determining these structural features which lack icosahedral symmetry and are lost in any icosahedral averaging used in X-ray crystallography or electron cryomicroscopy (cryoEM). Using a cryoEM single particle reconstruction technique without symmetry imposition, we have been able to determine the structure of these critical features of Salmonella phage Epsilon15, some of which are unexpected.Epsilon15 is a short tailed dsDNA bacteriophage that infects Salmonella anatum. Its genome (NCBI accession number: NC_004775) contains 39,671 base pairs with 49 open reading frames ( Supplementary Fig. 1) among which six, coding for structural proteins, were resolved by SDS-PAGE and identified by tryptic-digest/mass spectrometry ( Supplementary Fig. 2).

Biological consequences of tightly bent DNA: The other life of a macromolecular celebrity

“…These studies combine data from thousands of particles to produce three-dimensional images of the capsid and genomic DNA, allowing the visualization of several layers of DNA loops within the capsid. [11][12][13] Models of Tightly-Packed Viral DNA…”

Crystal and cryoEM structural studies of a cell wall degrading enzyme in the bacteriophage φ29 tail

“…Electron micrographs were recorded at a magnification of 33,000 with a CM300 FEG microscope under low-dose conditions. Both mutant virus reconstructions assumed no symmetry and were based on a procedure as described (4,7,34). The N-and C-terminal domains of gp13 were initially fitted manually into the cryoEM density map by using the program Chimera and then optimized with the program EMfit, assuming 2-fold symmetry.…”

Ionic effects on viral DNA packaging and portal motor function in bacteriophage φ29

“…Recent cryo-electron microscopy 3D reconstructions of P22 and epsilon15 virions (15)(16)(17) indicate at least partial spooling of the DNA around the portal axis; however, similar reconstructions of 29 do not appear to indicate such coaxial organization, especially in the early stages of packaging (L. Comolli, A. Spakowitz, C. Siegerist, S.G., P.J.J., D.L.A., C. Bustamante, and K. Downing, unpublished manuscript). In addition, several molecular dynamic simulations of packaging do not observe spontaneous DNA spooling (4,(30)(31)(32)(33)(34).…”

“…Purohit et al made similar calculations for the case of several different viruses (capsid geometries) in which the DNA strands were assumed to be packed in a hexagonal lattice that forms an inverse spool coaxial to the portal channel (6, 7). Cryo-EM reconstructions of some phages suggest that the DNA is at least partially organized in a coaxial spool (13)(14)(15)(16)(17), although the degree of order toward the interior (away from the capsid wall) remains unclear. X-ray diffraction studies of phages have consistently revealed short-range interaxial DNA separations of 2-3 nm (1).…”