Herpes Simplex Virus 1 Entry Glycoproteins Form Complexes before and during Membrane Fusion.

Herpesviruses-ubiquitous pathogens that cause persistent infections-have some of the most complex cell entry mechanisms. Entry of the prototypical herpes simplex virus 1 (HSV-1) requires coordinated efforts of 4 glycoproteins, gB, gD, gH, and gL. The current model posits that the glycoproteins do not interact before receptor engagement and that binding of gD to its receptor causes a "cascade" of sequential pairwise interactions, first activating the gH/gL complex and subsequently activating gB, the viral fusogen. But how these glycoproteins interact remains unresolved. Here, using a quantitative split-luciferase approach, we show that pairwise HSV-1 glycoprotein complexes form before fusion, interact at a steady level throughout fusion, and do not depend on the presence of the cellular receptor. Based on our findings, we propose a revised "conformational cascade" model of HSV-1 entry. We hypothesize that all 4 glycoproteins assemble into a complex before fusion, with gH/gL positioned between gD and gB. Once gD binds to a cognate receptor, the proximity of the glycoproteins within this complex allows for efficient transmission of the activating signal from the receptor-activated gD to gH/gL to gB through sequential conformational changes, ultimately triggering the fusogenic refolding of gB. Our results also highlight previously unappreciated contributions of the transmembrane and cytoplasmic domains to glycoprotein interactions and fusion. Similar principles could be at play in other multicomponent viral entry systems, and the split-luciferase approach used here is a powerful tool for investigating protein-protein interactions in these and a variety of other systems. IMPORTANCE Herpes simplex virus 1 (HSV-1) infects the majority of humans for life and can cause diseases ranging from painful sores to deadly brain inflammation. No vaccines or curative treatments currently exist. HSV-1 infection of target cells requires coordinated efforts of four viral glycoproteins. But how these glycoproteins interact remains unclear. Using a quantitative protein interaction assay, we found that HSV-1 glycoproteins form receptor-independent complexes and interact at a steady level. We propose that the 4 proteins form a complex, which could facilitate transmission of the entry-triggering signal from the receptor-binding component to the membrane fusogen component through sequential conformational changes. Similar principles could be applicable across other multicomponent protein systems. A revised model of HSV-1 entry could facilitate the development of therapeutics targeting this process.

The nuclear egress complex of Epstein-Barr virus buds membranes through an oligomerization-driven mechanism.

During replication, herpesviral capsids are translocated from the nucleus into the cytoplasm by an unusual mechanism, termed nuclear egress, that involves capsid budding at the inner nuclear membrane. This process is mediated by the viral nuclear egress complex (NEC) that deforms the membrane around the capsid. Although the NEC is essential for capsid nuclear egress across all three subfamilies of the Herpesviridae, most studies to date have focused on the NEC homologs from alpha- and beta- but not gammaherpesviruses. Here, we report the crystal structure of the NEC from Epstein-Barr virus (EBV), a prototypical gammaherpesvirus. The structure resembles known structures of NEC homologs yet is conformationally dynamic. We also show that purified, recombinant EBV NEC buds synthetic membranes in vitro and forms membrane-bound coats of unknown geometry. However, unlike other NEC homologs, EBV NEC forms dimers in the crystals instead of hexamers. The dimeric interfaces observed in the EBV NEC crystals are similar to the hexameric interfaces observed in other NEC homologs. Moreover, mutations engineered to disrupt the dimeric interface reduce budding. Putting together these data, we propose that EBV NEC-mediated budding is driven by oligomerization into membrane-bound coats.

A surface pocket in the cytoplasmic domain of the herpes simplex virus fusogen gB controls membrane fusion.

Membrane fusion during the entry of herpesviruses is carried out by the viral fusogen gB that is activated by its partner protein gH in some manner. The fusogenic activity of gB is controlled by its cytoplasmic (or intraviral) domain (gBCTD) and, according to the current model, the gBCTD is a trimeric, inhibitory clamp that restrains gB in the prefusion conformation. But how the gBCTD clamp is released by gH is unclear. Here, we identified two new regulatory elements within gB and gH from the prototypical herpes simplex virus 1: a surface pocket within the gBCTD and residue V831 within the gH cytoplasmic tail. Mutagenesis and structural modeling suggest that gH V831 interacts with the gB pocket. The gB pocket is located above the interface between adjacent protomers, and we hypothesize that insertion of the gH V831 wedge into the pocket serves to push the protomers apart, which releases the inhibitory clamp. In this manner, gH activates the fusogenic activity of gB. Both gB and gH are conserved across all herpesviruses, and this activation mechanism could be used by other gB homologs. Our proposed mechanism emphasizes a central role for the cytoplasmic regions in regulating the activity of a viral fusogen.

Well Put Together-A Guide to Accessorizing with the Herpesvirus gH/gL Complexes.

Herpesviruses are enveloped, double-stranded DNA viruses that infect a variety of hosts across the animal kingdom. Nine of these establish lifelong infections in humans, for which there are no cures and few vaccine or treatment options. Like all enveloped viruses, herpesviruses enter cells by fusing their lipid envelopes with a host cell membrane. Uniquely, herpesviruses distribute the functions of receptor engagement and membrane fusion across a diverse cast of glycoproteins. Two glycoprotein complexes are conserved throughout the three herpesvirus subfamilies: the trimeric gB that functions as a membrane fusogen and the heterodimeric gH/gL, the role of which is less clearly defined. Here, we highlight the conserved and divergent functions of gH/gL across the three subfamilies of human herpesviruses by comparing its interactions with a broad range of accessory viral proteins, host cell receptors, and neutralizing or inhibitory antibodies. We propose that the intrinsic structural plasticity of gH/gL enables it to function as a signal integration machine that can accept diverse regulatory inputs and convert them into a "trigger" signal that activates the fusogenic ability of gB.

Contributions of the Four Essential Entry Glycoproteins to HSV-1 Tropism and the Selection of Entry Routes.

Herpes simplex viruses (HSV-1 and HSV-2) encode up to 16 envelope proteins, four of which are essential for entry. However, whether these four proteins alone are sufficient to dictate the broad cellular tropism of HSV-1 and the selection of different cell type-dependent entry routes is unknown. To begin addressing this, we previously pseudotyped vesicular stomatitis virus (VSV), lacking its native glycoprotein G, with only the four essential entry glycoproteins of HSV-1: gB, gH, gL, and gD. This novel VSVΔG-BHLD pseudotype recapitulated several important features of HSV-1 entry: the requirement for gB, gH, gL, gD, and a cellular receptor and sensitivity to anti-gB and anti-gH/gL neutralizing antibodies. However, due to the use of a single cell type in that study, the tropism of the VSVΔG-BHLD pseudotype was not investigated. Here, we show that the cellular tropism of the pseudotype is severely limited compared to that of wild-type HSV-1 and that its entry pathways differ from the native HSV-1 entry pathways. To test the hypothesis that other HSV-1 envelope proteins may contribute to HSV-1 tropism, we generated a derivative pseudotype containing the HSV-1 glycoprotein C (VSVΔG-BHLD-gC) and observed a gC-dependent increase in entry efficiency in two cell types. We propose that the pseudotyping platform developed here has the potential to uncover functional contributions of HSV-1 envelope proteins to entry in a gain-of-function manner.IMPORTANCE Herpes simplex viruses (HSV-1 and HSV-2) contain up to 16 different proteins in their envelopes. Four of these, glycoproteins gB, gD, gH, and gL, are termed essential with regard to entry, whereas the rest are typically referred to as nonessential based on the entry phenotypes of the respective single genetic deletions. However, the single-gene deletion approach, which relies on robust loss-of-function phenotypes, may be confounded by functional redundancies among the many HSV-1 envelope proteins. We have developed a pseudotyping platform in which the essential four entry glycoproteins are isolated from the rest, which can be added back individually for systematic gain-of-function entry experiments. Here, we show the utility of this platform for dissecting the contributions of HSV envelope proteins, both the essential four and the remaining dozen (using gC as an example), to HSV entry.

Virus-derived peptide inhibitors of the herpes simplex virus type 1 nuclear egress complex.

Herpesviruses infect a majority of the human population, establishing lifelong latent infections for which there is no cure. Periodic viral reactivation spreads infection to new hosts while causing various disease states particularly detrimental in the immunocompromised. Efficient viral replication, and ultimately the spread of infection, is dependent on the nuclear egress complex (NEC), a conserved viral heterodimer that helps translocate viral capsids from the nucleus to the cytoplasm where they mature into infectious virions. Here, we have identified peptides, derived from the capsid protein UL25, that are capable of inhibiting the membrane-budding activity of the NEC from herpes simplex virus type 1 in vitro. We show that the inhibitory ability of the peptides depends on their length and the propensity to form an α-helix but not on the exact amino acid sequence. Current therapeutics that target viral DNA replication machinery are rendered ineffective by drug resistance due to viral mutations. Our results establish a basis for the development of an alternative class of inhibitors against nuclear egress, an essential step in herpesvirus replication, potentially expanding the current repertoire of available therapeutics.

Conserved Outer Tegument Component UL11 from Herpes Simplex Virus 1 Is an Intrinsically Disordered, RNA-Binding Protein.

A distinguishing morphological feature of all herpesviruses is the multiprotein tegument layer located between the nucleocapsid and lipid envelope of the virion. Tegument proteins play multiple roles in viral replication, including viral assembly, but we do not yet understand their individual functions or how the tegument is assembled and organized. UL11, the smallest tegument protein, is important for several distinct processes in replication, including efficient virion morphogenesis and cell-cell spread. However, the mechanistic understanding of its role in these and other processes is limited in part by the scant knowledge of its biochemical and structural properties. Here, we report that UL11 from herpes simplex virus 1 (HSV-1) is an intrinsically disordered, conformationally dynamic protein that undergoes liquid-liquid phase separation (LLPS) in vitro Intrinsic disorder may underlie the ability of UL11 to exert multiple functions and bind multiple partners. Sequence analysis suggests that not only all UL11 homologs but also all HSV-1 tegument proteins contain intrinsically disordered regions of different lengths. The presence of intrinsic disorder, and potentially, the ability to form LLPS, may thus be a common feature of the tegument proteins. We hypothesize that tegument assembly may involve the formation of a biomolecular condensate, driven by the heterogeneous mixture of intrinsically disordered tegument proteins.IMPORTANCE Herpesvirus virions contain a unique tegument layer sandwiched between the capsid and lipid envelope and composed of multiple copies of about two dozen viral proteins. However, little is known about the structure of the tegument or how it is assembled. Here, we show that a conserved tegument protein UL11 from herpes simplex virus 1, a prototypical alphaherpesvirus, is an intrinsically disordered protein that undergoes liquid-liquid phase separation in vitro Through sequence analysis, we find intrinsically disordered regions of different lengths in all HSV-1 tegument proteins. We hypothesize that intrinsic disorder is a common characteristic of tegument proteins and propose a new model of tegument as a biomolecular condensate.

Expression, Purification, and Crystallization of HSV-1 Glycoproteins for Structure Determination.

Herpes simplex viruses utilize glycoproteins displayed on the viral envelope to perform a variety of functions in the viral infectious cycle. Structural and functional studies of these viral glycoproteins can benefit from biochemical, biophysical, and structural analysis of purified proteins. Here, we describe a general protocol for expression and purification of viral glycoproteins from insect cells based on those developed for the HSV-1 gB and HSV-2 gH/gL ectodomains as well as the protocol for crystallization of these glycoproteins. This protocol can be used for generating milligram amounts of wild-type (WT) or mutant gB and gH/gL ectodomains or can be adapted to produce purified ectodomains of glycoproteins from HSV or other herpesviruses for biochemical and structural studies.

Expression, Purification, and Crystallization of Full-Length HSV-1 gB for Structure Determination.

HSV glycoproteins play important roles in the viral life cycle, particularly viral cell entry. Here we describe the protocol for expression, purification, and crystallization of full-length HSV-1 glycoprotein B. The protocol provides a framework for incorporating transmembrane domain-stabilizing amphipols into the crystallization setup and can be adapted to isolate other complete HSV glycoproteins.

The dynamic nature of the conserved tegument protein UL37 of herpesviruses.

In all herpesviruses, the space between the capsid shell and the lipid envelope is occupied by the unique tegument layer composed of proteins that, in addition to structural roles, play many other roles in the viral replication. UL37 is a highly conserved tegument protein that has activities ranging from virion morphogenesis to directional capsid trafficking to manipulation of the host innate immune response and binds multiple partners. The N-terminal half of UL37 (UL37N) has a compact bean-shaped α-helical structure that contains a surface region essential for neuroinvasion. However, no biochemical or structural information is currently available for the C-terminal half of UL37 (UL37C) that mediates most of its interactions with multiple binding partners. Here, we show that the C-terminal half of UL37 from pseudorabies virus UL37C is a conformationally flexible monomer composed of an elongated folded core and an unstructured C-terminal tail. This elongated structure, along with that of its binding partner UL36, explains the nature of filamentous tegument structures bridging the capsid and the envelope. We propose that the dynamic nature of UL37 underlies its ability to perform diverse roles during viral replication.

Structural basis for membrane anchoring and fusion regulation of the herpes simplex virus fusogen gB.

Viral fusogens merge viral and cell membranes during cell penetration. Their ectodomains drive fusion by undergoing large-scale refolding, but little is known about the functionally important regions located within or near the membrane. Here we report the crystal structure of full-length glycoprotein B (gB), the fusogen from herpes simplex virus, complemented by electron spin resonance measurements. The membrane-proximal (MPR), transmembrane (TMD), and cytoplasmic (CTD) domains form a uniquely folded trimeric pedestal beneath the ectodomain, which balances dynamic flexibility with extensive, stabilizing membrane interactions. The postfusion conformation of the ectodomain suggests that the CTD likewise adopted the postfusion form. However, hyperfusogenic mutations, which destabilize the prefusion state of gB, target key interfaces and structural motifs that reinforce the observed CTD structure. Thus, a similar CTD structure must stabilize gB in its prefusion state. Our data suggest a model for how this dynamic, membrane-dependent 'clamp' controls the fusogenic refolding of gB.

The pUL37 tegument protein guides alpha-herpesvirus retrograde axonal transport to promote neuroinvasion.

A hallmark property of the neurotropic alpha-herpesvirinae is the dissemination of infection to sensory and autonomic ganglia of the peripheral nervous system following an initial exposure at mucosal surfaces. The peripheral ganglia serve as the latent virus reservoir and the source of recurrent infections such as cold sores (herpes simplex virus type I) and shingles (varicella zoster virus). However, the means by which these viruses routinely invade the nervous system is not fully understood. We report that an internal virion component, the pUL37 tegument protein, has a surface region that is an essential neuroinvasion effector. Mutation of this region rendered herpes simplex virus type 1 (HSV-1) and pseudorabies virus (PRV) incapable of spreading by retrograde axonal transport to peripheral ganglia both in culture and animals. By monitoring the axonal transport of individual viral particles by time-lapse fluorescence microscopy, the mutant viruses were determined to lack the characteristic sustained intracellular capsid motion along microtubules that normally traffics capsids to the neural soma. Consistent with the axonal transport deficit, the mutant viruses did not reach sites of latency in peripheral ganglia, and were avirulent. Despite this, viral propagation in peripheral tissues and in cultured epithelial cell lines remained robust. Selective elimination of retrograde delivery to the nervous system has long been sought after as a means to develop vaccines against these ubiquitous, and sometimes devastating viruses. In support of this potential, we find that HSV-1 and PRV mutated in the effector region of pUL37 evoked effective vaccination against subsequent nervous system challenges and encephalitic disease. These findings demonstrate that retrograde axonal transport of the herpesviruses occurs by a virus-directed mechanism that operates by coordinating opposing microtubule motors to favor sustained retrograde delivery of the virus to the peripheral ganglia. The ability to selectively eliminate the retrograde axonal transport mechanism from these viruses will be useful in trans-synaptic mapping studies of the mammalian nervous system, and affords a new vaccination paradigm for human and veterinary neurotropic herpesviruses.

Crystal Structure of the N-Terminal Half of the Traffic Controller UL37 from Herpes Simplex Virus 1.

Inner tegument protein UL37 is conserved among all three subfamilies of herpesviruses. Studies of UL37 homologs from two alphaherpesviruses, herpes simplex virus 1 (HSV-1) and pseudorabies virus (PRV), have suggested that UL37 plays an essential albeit poorly defined role in intracellular capsid trafficking. At the same time, HSV and PRV homologs cannot be swapped, which suggests that in addition to a conserved function, UL37 homologs also have divergent virus-specific functions. Accurate dissection of UL37 functions requires detailed maps in the form of atomic-resolution structures. Previously, we reported the crystal structure of the N-terminal half of UL37 (UL37N) from PRV. Here, we report the crystal structure of HSV-1 UL37N. Comparison of the two structures reveals that UL37 homologs differ in their overall shapes, distributions of surface charges, and locations of projecting loops. In contrast, the previously identified R2 surface region is structurally conserved. We propose that within the N-terminal half of UL37, functional conservation is centered within the R2 surface region, whereas divergent structural elements pinpoint regions mediating virus-specific functions and may engage different binding partners. Together, the two structures can now serve as templates for a structure-guided exploration of both conserved and virus-specific functions of UL37.IMPORTANCE The ability to move efficiently within host cell cytoplasm is essential for replication in all viruses. It is especially important in the neuroinvasive alphaherpesviruses, such as human herpes simplex virus 1 (HSV-1), HSV-2, and veterinarian pseudorabies virus (PRV), that infect the peripheral nervous system and have to travel long distances along axons. Capsid movement in these viruses is controlled by capsid-associated tegument proteins, yet their specific roles have not yet been defined. Systematic exploration of the roles of tegument proteins in capsid trafficking requires detailed navigational charts in the form of their three-dimensional structures. Here, we determined the crystal structure of the N-terminal half of a conserved tegument protein, UL37, from HSV-1. This structure, along with our previously reported structure of the UL37 homolog from PRV, provides a much needed 3-dimensional template for the dissection of both conserved and virus-specific functions of UL37 in intracellular capsid trafficking.

Characterization of Vesicular Stomatitis Virus Pseudotypes Bearing Essential Entry Glycoproteins gB, gD, gH, and gL of Herpes Simplex Virus 1.

Herpes simplex viruses (HSVs) are unusual in that unlike most enveloped viruses, they require at least four entry glycoproteins, gB, gD, gH, and gL, for entry into target cells in addition to a cellular receptor for gD. The dissection of the herpes simplex virus 1 (HSV-1) entry mechanism is complicated by the presence of more than a dozen proteins on the viral envelope. To investigate HSV-1 entry requirements in a simplified system, we generated vesicular stomatitis virus (VSV) virions pseudotyped with HSV-1 essential entry glycoproteins gB, gD, gH, and gL but lacking the native VSV fusogen G. These virions, referred to here as VSVΔG-BHLD virions, infected a cell line expressing a gD receptor, demonstrating for the first time that the four essential entry glycoproteins of HSV-1 are not only required but also sufficient for cell entry. To our knowledge, this is the first time the VSV pseudotyping system has been successfully extended beyond two proteins. Entry of pseudotyped virions required a gD receptor and was inhibited by HSV-1 specific anti-gB or anti-gH/gL neutralizing antibodies, which suggests that membrane fusion during the entry of the pseudotyped virions shares common requirements with the membrane fusion involved in HSV-1 entry and HSV-1-mediated syncytium formation. The HSV pseudotyping system established in this study presents a novel tool for systematic exploration of the HSV entry and membrane fusion mechanisms. IMPORTANCE: Herpes simplex viruses (HSVs) are human pathogens that can cause cold sores, genital herpes, and blindness. No vaccines or preventatives are available. HSV entry into cells-a prerequisite for a successful infection-is a complex process that involves multiple viral and host proteins and occurs by different routes. Detailed mechanistic knowledge of the HSV entry is important for understanding its pathogenesis and would benefit antiviral and vaccine development, yet the presence of more than a dozen proteins on the viral envelope complicates the dissection of the HSV entry mechanisms. In this study, we generated heterologous virions displaying the four essential entry proteins of HSV-1 and showed that they are capable of cell entry and, like HSV-1, require all four entry glycoproteins along with a gD receptor. This HSV pseudotyping system pioneered in this work opens doors for future systematic exploration of the herpesvirus entry mechanisms.

Novel Structure and Unexpected RNA-Binding Ability of the C-Terminal Domain of Herpes Simplex Virus 1 Tegument Protein UL21.

UNLABELLED: Proteins forming the tegument layers of herpesviral virions mediate many essential processes in the viral replication cycle, yet few have been characterized in detail. UL21 is one such multifunctional tegument protein and is conserved among alphaherpesviruses. While UL21 has been implicated in many processes in viral replication, ranging from nuclear egress to virion morphogenesis to cell-cell spread, its precise roles remain unclear. Here we report the 2.7-Å crystal structure of the C-terminal domain of herpes simplex virus 1 (HSV-1) UL21 (UL21C), which has a unique α-helical fold resembling a dragonfly. Analysis of evolutionary conservation patterns and surface electrostatics pinpointed four regions of potential functional importance on the surface of UL21C to be pursued by mutagenesis. In combination with the previously determined structure of the N-terminal domain of UL21, the structure of UL21C provides a 3-dimensional framework for targeted exploration of the multiple roles of UL21 in the replication and pathogenesis of alphaherpesviruses. Additionally, we describe an unanticipated ability of UL21 to bind RNA, which may hint at a yet unexplored function. IMPORTANCE: Due to the limited genomic coding capacity of viruses, viral proteins are often multifunctional, which makes them attractive antiviral targets. Such multifunctionality, however, complicates their study, which often involves constructing and characterizing null mutant viruses. Systematic exploration of these multifunctional proteins requires detailed road maps in the form of 3-dimensional structures. In this work, we determined the crystal structure of the C-terminal domain of UL21, a multifunctional tegument protein that is conserved among alphaherpesviruses. Structural analysis pinpointed surface areas of potential functional importance that provide a starting point for mutagenesis. In addition, the unexpected RNA-binding ability of UL21 may expand its functional repertoire. The structure of UL21C and the observation of its RNA-binding ability are the latest additions to the navigational chart that can guide the exploration of the multiple functions of UL21.

gH/gL supercomplexes at early stages of herpesvirus entry.

Membrane fusion during herpesvirus entry into host cells is a complex process where multiple glycoproteins interact to relay the triggering signal from a receptor-binding protein to the conserved fusogen gB through the conserved heterodimer gH/gL. Crystal structures of individual glycoproteins are available, yet high-order 'supercomplexes' have been elusive. Recent structures of complexes between gH/gL from human cytomegalovirus or Epstein-Barr virus and the receptor-binding proteins that form at early stages of herpesviral entry highlighted mechanisms that control tropism and revealed dynamic intermediate complexes containing gH/gL that may directly participate in membrane deformation and juxtaposition. Determining how the triggering signal reaches the fusogen gB represents the next frontier in structural biology of herpesvirus entry.

Herpesvirus gB: A Finely Tuned Fusion Machine.

Enveloped viruses employ a class of proteins known as fusogens to orchestrate the merger of their surrounding envelope and a target cell membrane. Most fusogens accomplish this task alone, by binding cellular receptors and subsequently catalyzing the membrane fusion process. Surprisingly, in herpesviruses, these functions are distributed among multiple proteins: the conserved fusogen gB, the conserved gH/gL heterodimer of poorly defined function, and various non-conserved receptor-binding proteins. We summarize what is currently known about gB from two closely related herpesviruses, HSV-1 and HSV-2, with emphasis on the structure of the largely uncharted membrane interacting regions of this fusogen. We propose that the unusual mechanism of herpesvirus fusion could be linked to the unique architecture of gB.

Crystal Structure of the Human Cytomegalovirus Glycoprotein B.

Human cytomegalovirus (HCMV), a dsDNA, enveloped virus, is a ubiquitous pathogen that establishes lifelong latent infections and caused disease in persons with compromised immune systems, e.g., organ transplant recipients or AIDS patients. HCMV is also a leading cause of congenital viral infections in newborns. Entry of HCMV into cells requires the conserved glycoprotein B (gB), thought to function as a fusogen and reported to bind signaling receptors. gB also elicits a strong immune response in humans and induces the production of neutralizing antibodies although most anti-gB Abs are non-neutralizing. Here, we report the crystal structure of the HCMV gB ectodomain determined to 3.6-Å resolution, which is the first atomic-level structure of any betaherpesvirus glycoprotein. The structure of HCMV gB resembles the postfusion structures of HSV-1 and EBV homologs, establishing it as a new member of the class III viral fusogens. Despite structural similarities, each gB has a unique domain arrangement, demonstrating structural plasticity of gB that may accommodate virus-specific functional requirements. The structure illustrates how extensive glycosylation of the gB ectodomain influences antibody recognition. Antigenic sites that elicit neutralizing antibodies are more heavily glycosylated than those that elicit non-neutralizing antibodies, which suggest that HCMV gB uses glycans to shield neutralizing epitopes while exposing non-neutralizing epitopes. This glycosylation pattern may have evolved to direct the immune response towards generation of non-neutralizing antibodies thus helping HCMV to avoid clearance. HCMV gB structure provides a starting point for elucidation of its antigenic and immunogenic properties and aid in the design of recombinant vaccines and monoclonal antibody therapies.

Crystal structure of the herpesvirus inner tegument protein UL37 supports its essential role in control of viral trafficking.

UNLABELLED: In cells infected with herpesviruses, two capsid-associated, or inner tegument, proteins, UL37 and UL36, control cytosolic trafficking of capsids by as yet poorly understood mechanisms. Here, we report the crystal structure of the N-terminal half of UL37 from pseudorabies virus, an alphaherpesvirus closely related to herpes simplex viruses and varicella-zoster virus. The structure--the first for any alphaherpesvirus inner tegument protein--reveals an elongated molecule of a complex architecture rich in helical bundles. To explore the function of the UL37 N terminus, we used the three-dimensional framework provided by the structure in combination with evolutionary trace analysis to pinpoint several surface-exposed regions of potential functional importance and test their importance using mutagenesis. This approach identified a novel functional region important for cell-cell spread. These results suggest a novel role for UL37 in intracellular virus trafficking that promotes spread of viral infection, a finding that expands the repertoire of UL37 functions. Supporting this, the N terminus of UL37 shares structural similarity with cellular multisubunit tethering complexes (MTCs), which control vesicular trafficking in eukaryotic cells by tethering transport vesicles to their destination membranes. Our results suggest that UL37 could be the first viral MTC mimic and provide a structural rationale for the importance of UL37 for viral trafficking. We propose that herpesviruses may have co-opted the MTC functionality of UL37 to bring capsids to cytoplasmic budding destinations and further on to cell junctions for spread to nearby cells. IMPORTANCE: To move within an infected cell, viruses encode genes for proteins that interact with host trafficking machinery. In cells infected with herpesviruses, two capsid-associated proteins control the cytosolic movement of capsids by as yet poorly understood mechanisms. Here, we report the crystal structure for the N-terminal half of one of these proteins, UL37. Structure-based mutagenesis revealed a novel function for UL37 in virus trafficking to cell junctions for cell-cell spread. The unexpected structural similarity to components of cellular multisubunit tethering complexes, which control vesicular traffic, suggests that UL37 could be the first viral MTC mimic and provides a structural basis for the importance of UL37 for virus trafficking.