Chapter Three - Have NEC Coat, Will Travel: Structural Basis of Membrane Budding During Nuclear Egress in Herpesviruses
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.
Section snippets
The NEC Is Composed of UL31 and UL34
The NEC is a heterodimer (Bigalke et al., 2014) composed of UL31 and UL34 (Liang and Baines, 2005, Lotzerich et al., 2006, Roller et al., 2010) that is located at the INM (Gonnella et al., 2005, Lotzerich et al., 2006, Reynolds et al., 2001, Sam et al., 2009) and face the nucleoplasm (Reynolds et al., 2001). UL31 and UL34 genes are conserved among all herpesviruses but are known by other names in β-herpesviruses (CMV: UL53 and UL50) and γ-herpesviruses (EBV: BFLF2 and BFRF1). HSV-1 UL34 is a
The Overall Architecture of the NEC
Recently, five structures of NEC from three different herpesviruses, two α-herpesviruses HSV-1 and PRV and a β-herpesvirus HCMV, were determined (PDB IDs as follows: HSV-1: 4ZXS (2.8 Å resolution) (Bigalke and Heldwein, 2015b); PRV: 4Z3U (2.8 Å resolution) (Bigalke and Heldwein, 2015b) and 5E8C (2.9 Å resolution) (Zeev-Ben-Mordehai et al., 2015); and HCMV: 5DOB (2.5 Å resolution) (Lye et al., 2015) and 5D5N (2.4 Å resolution) (Walzer et al., 2015)). The overall NEC fold is very similar in all
The NEC Assembles into a Hexagonal Lattice In Vitro
One mechanism of membrane deformation used by proteins is forming an ordered array or coat (Zimmerberg and Kozlov, 2006). Consistent with its ability to bud membranes, NEC assembles into honeycomb-like hexagonal lattices on membranes both in vitro (Bigalke et al., 2014) and in vivo (Hagen et al., 2015). The ability of NEC to oligomerize was first observed in vitro with the purified recombinant HSV-1 NEC lacking the UL34 TM region (aka soluble NEC) (Bigalke et al., 2014). Soluble NEC is a
NEC Lattice Curvature
The hexagonal crystal lattice formed by the NEC is flat, whereas the honeycomb coats are spherical. While flat arrays permit strictly symmetrical hexagonal packing works, formation of a curved array, such as a coat, requires distortions, or defects, in hexagonal packing. While models of purely hexagonal NEC coats have been proposed (Bigalke et al., 2014, Hagen et al., 2015), their veracity is yet unclear. A spherical particle characterized by hexagonal symmetry is typically achieved through a
Regulation of NEC Lattice Assembly
The NEC has a robust membrane vesiculation activity both in vitro (Bigalke et al., 2014, Lorenz et al., 2015) and in cells stably expressing the UL31 and UL34 (Desai et al., 2012, Klupp et al., 2007, Luitweiler et al., 2013). But during infection, empty perinuclear vesicles are rarely observed (Hagen et al., 2015, Klupp et al., 2011). Therefore, in infected cells, the intrinsic budding ability of the NEC is likely regulated to avoid nonproductive budding. Given that NEC oligomerization is the
NEC and Capsid Deenvelopment at the ONM
Capsid budding at the INM, which leads to the formation of the perinuclear viral particles, is an essential step in herpesvirus nuclear egress. But, nuclear egress is not completed until the membrane of the perinuclear viral particle fuses with the ONM, and the capsids are released into the cytosol. This final stage of nuclear egress is termed deenvelopment. At its core, deenvelopment is membrane fusion, but relatively little is known about the mechanism of this process, and even the nature of
Summary
Herpesviral capsids are translocated from the nucleus into the cytoplasm by an unusual mechanism—termed nuclear egress—whereby capsids bud at the INM (primary envelopment), and the resulting primary virions fuse with the ONM (deenvelopment). The conserved NEC, located at the INM, is essential for nuclear egress. Recent studies have established the NEC as a virally encoded budding machine and illuminated the mechanism of NEC-mediated membrane budding that is driven by the formation of a
Acknowledgments
This work on nuclear egress in the Heldwein lab was supported by the NIH grants 1R21AI097573, 1R01GM111795, and the Burroughs Wellcome Fund. J.M.B. is a recipient of a postdoctoral fellowship from the Deutsche Forschungsgemeinschaft GZ: BI 1658/1-1.
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