Dissecting the Herpesvirus Architecture by Targeted Proteolysis

Abstract

Herpesvirus particles have a complex architecture consisting of an icosahedral capsid that is surrounded by a lipid envelope. Connecting these two components is a layer of tegument that consists of various amounts of 20 or more proteins. The arrangement of proteins within the tegument cannot easily be assessed and instead is inferred from tegument interactions identified in reductionist models. To better understand the tegument architecture, we have developed an approach to probe capsid-tegument interactions of extracellular viral particles by encoding tobacco etch virus (TEV) protease sites in viral structural proteins, along with distinct fluorescent tags in capsid and tegument components. In this study, TEV sites were engineered within the pUL36 large tegument protein, a critical structural element that is anchored directly on the capsid surface. Purified pseudorabies virus extracellular particles were permeabilized, and TEV protease was added to selectively cleave the exposed pUL36 backbone. Interactions with the capsid were assessed by monitoring the fate of the fluorescent signals following cleavage. Although several regions of pUL36 are proposed to bind capsids, pUL36 was found stably anchored to the capsid exclusively at its carboxyl terminus. Two additional tegument proteins, pUL37 and pUS3, were tethered to the capsid via pUL36, whereas the pUL16, pUL47, pUL48, and pUL49 tegument proteins were not stably bound to the capsid. Neuroinvasive alphaherpesviruses produce diseases of clinical and economic significance in humans and veterinary animals but are predominantly associated with less serious recurrent disease. Like all viruses, herpesviruses assemble a metastable particle that selectively dismantles during initial infection. This process is made more complex by the presence of a tegument layer that resides between the capsid surface and envelope. Components of the tegument are essential for particle assembly and also serve as critical effectors that promote infection upon entry into cells. How this dynamic network of protein interactions is arranged within virions is largely unknown. We present a molecular approach to dissect the tegument, and with it we begin to tease apart the protein interactions that underlie this complex layer of the virion architecture.

The Herpes Simplex Virus 1 Deamidase Enhances Propagation but Is Dispensable for Retrograde Axonal Transport into the Nervous System

“…The pUL37 tegument protein sustains dynein-based microtubule transport by restraining opposing plus-end motion (22). Consistent with these roles, pUL36 and pUL37 remain attached to capsids upon entry into cells (2,3,5,27), with pUL36 bound directly to the capsid surface (7,9,28,29) and pUL37 bound to pUL36 (10,11,30), such that pUL36 tethers pUL37 to the capsid (13). Understanding how the pUL36 and pUL37 tegument proteins mediate neuroinvasion is foundational to developing vectors for neural gene delivery and vaccination.…”

The herpes simplex virus type I deamidase enhances propagation but is dispensable for retrograde axonal transport into the nervous system

“…The pUL37 tegument protein sustains dynein-based microtubule transport by restraining opposing plus-end motion (22). Consistent with these roles, pUL36 and pUL37 remain attached to capsids upon entry into cells (2, 3, 5, 26), with pUL36 bound directly to the capsid surface (7, 9, 27, 28) and pUL37 bound to pUL36 (10, 11, 29) such that pUL36 tethers pUL37 to the capsid (13). Understanding how the pUL36 and pUL37 tegument proteins mediate neuroinvasion is foundational to developing vectors for neural gene delivery and vaccination.…”

“…However, at least three tegument proteins, pUL36, pUL37, and pUS3, remain capsid bound (2-5). The pUL36 tegument protein directly binds to the pUL25 component of the capsid surface (6-9), and tethers pUL37, pUS3, as well as the host dynein/dynactin microtubule motor, to the capsid (10-13). Each of the capsid-bound tegument proteins has a distinct enzymatic activity: pUL36 houses a deubiquitinase in its amino terminus (14, 15), pUL37 houses a deamidase in its carboxyl terminus (16), and pUS3 is a serine-threonine protein kinase (17).…”

Tegument proteins of Epstein-Barr virus: Diverse functions, complex networks, and oncogenesis

“…EBV BPLF1 is linked to BBRF2 [ 40 ], BOLF1 associates with BKRF4 ( Fig. 1 ) [ 41 ], and HSV-1 UL36 binds to UL48 [ 42 ] and US3 [ 43 ] ( Fig. 2 ).…”