A pUL25 dimer interfaces the pseudorabies virus capsid and tegument

Abstract

Inside the virions of a-herpesviruses, tegument protein pUL25 anchors the tegument to capsid vertices through direct interactions with tegument proteins pUL17 and pUL36. In addition to promoting virion assembly, both pUL25 and pUL36 are critical for intracellular microtubule-dependent capsid transport. Despite these essential roles during infection, the stoichiometry and precise organization of pUL25 and pUL36 on the capsid surface remain controversial due to the insufficient resolution of existing reconstructions from cryo-electron microscopy (cryoEM). Here, we report a threedimensional (3D) icosahedral reconstruction of pseudorabies virus (PRV), a varicellovirus of the a-herpesvirinae subfamily, obtained by electron-counting cryoEM at 4.9 Å resolution. Our reconstruction resolves a dimer of pUL25 forming a capsidassociated tegument complex with pUL36 and pUL17 through a coiled coil helix bundle, thus correcting previous misinterpretations. A comparison between reconstructions of PRV and the g-herpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV) reinforces their similar architectures and establishes important subfamily differences in the capsidtegument interface.

Dissecting the herpesvirus architecture by targeted proteolysis

“…While we cannot rule out that there may be redundant tegument-capsid interactions that promote particle assembly, cryoEM reconstruction of PRV and HSV-1 virions do no attest to capsid interactions apart from pUL36 (6,7,70).…”

“…While this value could be coincidental, it is possible that 60 copies of the pUL25/ mCherry fusion are stably bound to capsids around the vertices, while a smaller number of pUL25 copies incorporate into virions by another means. We also note that a recent cryoEM reconstruction of PRV reveals that there are 10 copies of pUL25 per vertex, further indicating that fusing mCherry to pUL25 may reduce its copy number on capsids (6). Green-fluorescence intensity measurements were converted to copy numbers by normalizing the average intensities to the reported copy number for GFP-pUL36, which is 107.1 copies/virion ( Fig.…”

“…Virion production begins in the nucleus, where capsid assembly and genome encapsidation occurs, and concludes in the cytoplasm with the envelopment of the capsid-tegument complex (5). While the structure of the capsid is solved to 3.1 Å for HSV-1 and 4.9 Å for PRV (6)(7)(8), the majority of the tegument layer departs from the radial symmetry of the capsid shell and cannot be visualized in great detail by composite averaging methods such as cryo-electron microscopy (9). This technical limitation is overcome by the application of cryo-electron tomography, but this method has yet to achieve resolutions necessary to infer protein distributions within the herpesvirus virion (10,11).…”

“…Most of the inner tegument density is localized to capsid vertices, and to date, three tegument proteins have been proposed to contact the capsid directly: pUL16, pUL36, and pUL47 (16)(17)(18)(19). The best established capsid-tegument interaction is the pUL36 protein, which is directly anchored to the pUL25 capsid protein as part of a five-helix bundle around capsid vertices (6,7,18,(20)(21)(22).…”

DNA-Packing Portal and Capsid-Associated Tegument Complexes in the Tumor Herpesvirus KSHV

“…Of note, because the globular density accommodates only one copy of pORF19/pUL25 head domain (whereas the corresponding region in HSV-1 CATC accommodates two), we previously asserted-based on a 6-Å icosahedral reconstructionthat the KSHV CATC had a different stoichiometry than the HSV-1 CATC (Dai et al, 2014). But, as noted in a subsequent study demonstrating differences in globular head domain arrangement between KSHV and neurotropic alphaherpesviruses (Dai et al, 2014;Liu et al, 2017), and as our present sub-particle reconstructions reveal, the KSHV CATC does, in fact, bear the same stoichiometry/architecture as the HSV-1 CATC. These findings indicate that the second head domain of pORF19 is present but perhaps flexibly tethered elsewhere.…”