Roles of the Interhexamer Contact Site for Hexagonal Lattice Formation of the Herpes Simplex Virus 1 Nuclear Egress Complex in Viral Primary Envelopment and Replication

Abstract

During the nuclear export of nascent nucleocapsids of herpes simplex virus 1 (HSV-1), the nucleocapsids acquire a primary envelope by budding through the inner nuclear membrane into the perinuclear space between the inner and outer nuclear membranes. This unique budding process, termed primary envelopment, is initiated by the nuclear egress complex (NEC), composed of the HSV-1 UL31 and UL34 proteins. Earlier biochemical approaches have shown that the NEC has an intrinsic ability to vesiculate membranes through the formation of a hexagonal lattice structure. The significance of intrahexamer interactions of the NEC in the primary envelopment of HSV-1-infected cells has been reported. In contrast, the contribution of lattice formation of the NEC hexamer to primary envelopment in HSV-1-infected cells remains to be elucidated. Therefore, we constructed and characterized a recombinant HSV-1 strain carrying an amino acid substitution in a UL31 residue that is an interhexamer contact site for the lattice formation of the NEC hexamer. This mutation was reported to destabilize the interhexamer interactions of the HSV-1 NEC. Here, we demonstrate that the mutation causes the aberrant accumulation of nucleocapsids in the nucleus and reduces viral replication in Vero and HeLa cells. Thus, the ability of HSV-1 to form the hexagonal lattice structure of the NEC was linked to an increase in primary envelopment and viral replication. Our results suggest that the lattice formation of the NEC hexamer has an important role in HSV-1 replication by regulating primary envelopment. IMPORTANCE The scaffolding proteins of several envelope viruses required for virion assembly form high-order lattice structures. However, information on the significance of their lattice formation in infected cells is limited. Herpesviruses acquire envelopes twice during their viral replication. The first envelop acquisition (primary envelopment) is one of the steps in the vesicle-mediated nucleocytoplasmic transport of nascent nucleocapsids, which is unique in biology. HSV-1 NEC, thought to be conserved in all members of the Herpesviridae family, is critical for primary envelopment and was shown to form a hexagonal lattice structure. Here, we investigated the significance of the interhexamer contact site for hexagonal lattice formation of the NEC in HSV-1-infected cells and present evidence suggesting that the lattice formation of the NEC hexamer has an important role in HSV-1 replication by regulating primary envelopment. Our results provide insights into the mechanisms of the envelopment of herpesviruses and other envelope viruses.

Structural basis for capsid recruitment and coat formation during HSV-1 nuclear egress

“…Additionally, HSV-1 NEC formed a hexagonal lattice of the same dimensions in crystals ( Bigalke and Heldwein, 2015 ). The high-resolution crystal structure of the hexagonal NEC lattice revealed interactions at the lattice interfaces ( Bigalke and Heldwein, 2015 ), and subsequent work confirmed that mutations that disrupt oligomeric interfaces reduce budding in vitro ( Bigalke and Heldwein, 2015 ; Bigalke et al, 2014 ) and in vivo ( Arii et al, 2019 ; Roller et al, 2010 ). Collectively, these findings established the NEC as a viral budding machine that generates negative membrane curvature by oligomerizing into a hexagonal coat on the surface of the membrane.…”

Structural basis for capsid recruitment and coat formation during HSV-1 nuclear egress

“…Additionally, HSV-1 NEC formed a hexagonal lattice of the same dimensions in crystals (Bigalke and Heldwein 2015). The high-resolution crystal structure of the hexagonal NEC lattice revealed interactions at the lattice interfaces (Bigalke and Heldwein 2015), and subsequent work confirmed that mutations that disrupt oligomeric interfaces reduce budding in vitro (Bigalke et al 2014, Bigalke and Heldwein 2015) and in vivo (Roller et al 2010, Arii et al 2019). Collectively, these findings established the NEC as a viral budding machine that generates negative membrane curvature by oligomerizing into a hexagonal coat on the surface of the membrane.…”

Host and Viral Factors Involved in Nuclear Egress of Herpes Simplex Virus 1

“…These findings suggest that the NEC has an intrinsic ability to deform membranes. Disruption of the membrane-coupled hexagonal NEC lattice by mutation of the inter- or intrahexagonal contact site impairs vesiculation both in vitro and in infected cells [ 4 , 21 , 49 , 63 , 65 ]. Thus, it is likely that formation of the NEC lattice drives vesiculation [ 4 , 21 , 63 ].…”