Have NEC Coat, Will Travel

Abstract

Herpesviruses are unusual among enveloped viruses because they bud twice yet acquire a single envelope. Furthermore, unlike other DNA viruses that replicate in the nucleus, herpesviruses do not exit it by passing through the nuclear pores or by rupturing the nuclear envelope. Instead, herpesviruses have a complex mechanism of nuclear escape whereby nascent capsids bud at the inner nuclear membrane to form perinuclear virions that subsequently fuse with the outer nuclear membrane, releasing capsids into the cytosol. This makes them some of the very few known viruses that bud into the nuclear envelope. The envelope acquired during nuclear budding does not end up in the mature viral particle but instead allows the capsid to translocate from the nucleus into the cytosol. The viral nuclear egress complex (NEC) is a critical player in the nuclear egress, yet its function and mechanism have remained enigmatic. Recent studies have demonstrated that the NEC buds membranes without the help of other proteins by forming a honeycomb coat, which established the NEC as the first virally encoded budding machine that operates at the nuclear, as opposed to cytoplasmic, membrane. This review discusses our current understanding of the NEC budding mechanism, with the emphasis on studies that illuminated the structure of the NEC coat and its role in capsid budding during herpesvirus nuclear escape.

Introduction

Replication in eukaryotic host cells, which separate their compartments by multiple membranes, presents viruses with a number of challenges. During infection, viruses interact with cellular membranes in many ways. Viruses must breach host membranes to deliver their genomes inside the cells. Once infection is underway, host membranes are used as envelope sources during budding of enveloped viruses or for building replication compartments by some RNA viruses. To accomplish this, viruses have had to become experts in membrane manipulation, yet we are only beginning to understand the mechanisms by which these processes are accomplished.

Herpesviruses are double-stranded DNA, enveloped viruses that infect nearly all vertebrates, from mice to elephants, and even invertebrates such as oysters, scallops, and snails (Davison et al., 2009). The hallmark of herpesviruses is their ability to establish lifelong latent infections in the infected hosts from which they periodically reactivate. Reactivations result not only in a substantial disease burden but also in a high rate of new infections. Herpesviruses that infect mammals and birds belong to the family of Herpesviridae and are divided into three subfamilies, α-, β-, and γ-herpesviruses. Eight human herpesviruses, which contain members of all three subfamilies, are ubiquitous; yet, most infections are asymptomatic as the immune system controls the virus, a testament to the sophisticated mechanism of coexistence with the host. However, disruption of this coexistence results in viral reactivation and a range of ailments from skin lesions and ocular diseases to encephalitis, cancers, congenital infections, and disseminated disease in immunocompromised people, e.g., organ transplant recipients or AIDS patients. Understanding the mechanisms by which herpesviruses manipulate their hosts, including host membranes, is necessary to develop better ways to prevent and control infections.

Herpesviruses are challenging to study because of their inherent complexity: they encode nearly a hundred genes, many of which are unique; despite such large coding capacity, herpesviral proteins are nearly always multifunctional; and herpesviral processes often require multiple proteins where other viruses use only one. One of the most complicated stages in herpesviral replication is viral exit out of the cell, termed egress, which is coupled to viral morphogenesis.

During egress, herpesviruses have to get across several membranes at different cellular locations (Fig. 1). Herpesviruses are also enveloped, and while egressing the cell must gain a lipid envelope as well as other viral components. Enveloped viruses typically acquire their lipid envelope by budding at a cytoplasmic membrane, either the plasma membrane or other cellular membranes. Herpesviruses are unusual in that while they have a single-bilayer envelope, they bud twice. Only the second, and final, budding event at cytoplasmic membranes results in the formation and release of the mature infectious virus while the envelope acquired during the first budding event does not end up in the mature viral particle. The initial budding event is also unusual, because it occurs in the nucleus at the nuclear envelope and serves to allow the viral capsids to escape from the nucleus. Herpesviruses are dsDNA viruses, and their genomes are replicated and packaged into capsids inside the nucleus. Most traffic in and out of the nucleus, which is surrounded by the nuclear envelope, occurs through the nuclear pores. Herpesvirus capsids are too large to fit through the nuclear pores, and to exit the nucleus, capsids bud into the inner nuclear membrane (INM) forming immature viral particles in the perinuclear space (Fig. 1) (Johnson and Baines, 2011). This process is often referred to as the primary envelopment, to distinguish it from the secondary envelopment, which occurs in the cytosol. Perinuclear viral particles then fuse with the outer nuclear membrane (ONM) thereby releasing naked capsids into the cytoplasm in a process termed deenvelopment. As the result, capsids are translocated from the nucleus to the cytosol. Herpesviruses then bud again, this time into cytoplasmic membranes derived from Trans-Golgi Network or early endosomes (Hollinshead et al., 2012, Johnson and Baines, 2011, Owen et al., 2015) to be released from the cell by exocytosis (Hogue et al., 2014) (Fig. 1). Currently, there are only few examples for viruses that bud at the nuclear membrane. It has been reported that insect viruses use nuclear budding (Shen and Chen, 2012, Yuan et al., 2011), but of all known viruses that infect vertebrates, herpesviruses are unique in their nuclear exit strategy.

Efficient nuclear egress requires several viral and cellular proteins, but only two viral proteins are essential for the initial budding event at the INM (Bubeck et al., 2004, Chang and Roizman, 1993, Farina et al., 2005, Fuchs et al., 2002, Muranyi et al., 2002, Reynolds et al., 2001, Roller et al., 2000). These two conserved proteins, termed UL31 and UL34 in α-herpesviruses and known by other names in β- and γ-herpesviruses, form the nuclear egress complex (NEC). While the importance of the NEC in nuclear budding has been appreciated for a number of years, its specific function had remained enigmatic until very recently when the NEC was discovered to have an intrinsic ability to vesiculate membranes in vitro in the absence of any other proteins or chemical energy (Bigalke et al., 2014, Lorenz et al., 2015). The NEC drives membrane budding by oligomerizing on the membrane and forming a hexagonal scaffold, or a coat, inside the bud (Bigalke and Heldwein, 2015a, Bigalke et al., 2014, Hagen et al., 2015). The NEC is also capable of membrane scission, which makes it a virally encoded budding nanomachine that can operate independently of other viral or host factors. Furthermore, the crystal structures of NEC from several different herpesviruses (Bigalke and Heldwein, 2015b, Lye et al., 2015, Walzer et al., 2015, Zeev-Ben-Mordehai et al., 2015) have illuminated critical mechanistic and structural features of NEC-mediated budding. The NEC structures provide a three-dimensional roadmap to enable the dissection of its budding mechanism and the design of inhibitors to block it.

In this review, we describe the recent breakthroughs in our understanding of the NEC-mediated budding mechanism, with the emphasis on structures of the NEC heterodimer and the honeycomb lattice it forms in vitro and inside cells. These groundbreaking insights are transforming the way we think about how herpesviruses manipulate host membranes.