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 varying amounts of twenty 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 within 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 in situ 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. IMPORTANCE: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, begin to tease apart the protein interactions that underlie this complex layer of the virion architecture.

Navigating the Cytoplasm: Delivery of the Alphaherpesvirus Genome to the Nucleus

“…This is consistent with symmetrydependent cryo-electron microscopy reconstructions of the capsid that have failed to resolve pUL36 apart from its carboxyl terminus (Dai and Zhou, 2018;Wang et al, 2018). In addition to pUL37, pUL36 tethers the pUS3 protein kinase to the capsid (Daniel et al, 2018) and links to the remainder of the tegument through an interaction with the pUL48 tegument protein (Ko et al, 2010;Svobodova et al, 2012). The pUL36 interaction with pUL48 appears complex, with pUL48 possibly undergoing a substantial conformational change as a result of binding to pUL36 (Svobodova et al, 2012).…”

“…Other tegument proteins are also reported to bind capsid proteins, including pUL21 (de Wind et al, 1992;Takakuwa et al, 2001), pUL37 (Lee et al, 2008;Uetz et al, 2006), and pUL47 (Scholtes et al, 2010), but it is not clear that these tegument proteins directly bind to assembled capsids. In fact, multiple lines of evidence argue against a direct interaction between pUL37 and capsids, with pUL37 instead tethered to capsids by pUL36 (Cardone et al, 2012;Daniel et al, 2018;Fuchs et al, 2004;Ko et al, 2010;Sandbaumhuter et al, 2013). Nevertheless, there is indirect evidence of additional capsidtegument interactions, including that virions can form to some degree in the absence of the pUL36-pUL25 interaction (Coller et al, 2007;Schipke et al, 2012), and that the link between pUL36 and the remainder of the tegument is also dispensable for virion formation (Fuchs et al, 2002;Svobodova et al, 2012).…”

“…In addition to these activities, capsid-retained pUL36 is also essential because it tethers pUL37 to the PECC (Daniel et al, 2018).…”