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 ␣-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. IMPORTANCEDue 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. Due to the limited genomic coding capacity of viruses, viral proteins are often multifunctional. Although herpesviruses have relatively large double-stranded DNA (dsDNA) genomes, they encode a number of proteins that mediate more than one unrelated function. A notable example is proteins located within the tegument, a thick protein layer located between the nucleocapsid and the envelope of a herpesviral virion. Although tegument proteins play structural roles, many have additional functions unrelated to viral assembly. For example, while UL36 (1, 2) and UL37 (3, 4) are required for proper viral assembly, both proteins are also necessary for efficient capsid trafficking (5-7), and UL36 additionally harbors deubiquitinating activity in its N terminus (8). UL48 serves as a hub of tegumentation and is necessary for herpes simplex virus 1 (HSV-1) morphogenesis (9-11); it is also known as ...

Respiratory syncytial virus (RSV) is the leading infectious cause of severe respiratory disease in infants and a major cause of respiratory illness in the elderly. There remains an unmet vaccine need despite decades of research. Insufficient potency, homogeneity, and stability of previous RSV fusion protein (F) subunit vaccine candidates have hampered vaccine development. RSV F and related parainfluenza virus (PIV) F proteins are cleaved by furin during intracellular maturation, producing disulfide-linked F1 and F2 fragments. During cell entry, the cleaved Fs rearrange from prefusion trimers to postfusion trimers. Using RSV F constructs with mutated furin cleavage sites, we isolated an uncleaved RSV F ectodomain that is predominantly monomeric and requires specific cleavage between F1 and F2 for self-association and rearrangement into stable postfusion trimers. The uncleaved RSV F monomer is folded and homogenous and displays at least two key RSV-neutralizing epitopes shared between the prefusion and postfusion conformations. Unlike the cleaved trimer, the uncleaved monomer binds the prefusion-specific monoclonal antibody D25 and human neutralizing immunoglobulins that do not bind to postfusion F. These observations suggest that the uncleaved RSV F monomer has a prefusion-like conformation and is a potential prefusion subunit vaccine candidate. IMPORTANCERSV is the leading infectious cause of severe respiratory disease in infants and a major cause of respiratory illness in the elderly. Development of an RSV vaccine was stymied when a clinical trial using a formalin-inactivated RSV virus made disease, following RSV infection, more severe. Recent studies have defined the structures that the RSV F envelope glycoprotein adopts before and after virus entry (prefusion and postfusion conformations, respectively). Key neutralization epitopes of prefusion and postfusion RSV F have been identified, and a number of current vaccine development efforts are focused on generating easily produced subunit antigens that retain these epitopes. Here we show that a simple modification in the F ectodomain results in a homogeneous protein that retains critical prefusion neutralizing epitopes. These results improve our understanding of RSV F protein folding and structure and can guide further vaccine design efforts.

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.

bUL21 is a conserved protein in the tegument of alphaherpesviruses and has multiple important albeit poorly understood functions in viral replication and pathogenesis. To provide a roadmap for exploration of the multiple roles of UL21, we determined the crystal structure of its conserved N-terminal domain from herpes simplex virus 1 to 2.0-Å resolution, which revealed a novel sail-like protein fold. Evolutionarily conserved surface patches highlight residues of potential importance for future targeting by mutagenesis. Herpesviruses are double-stranded DNA (dsDNA), enveloped viruses that cause lifelong latent infections and ailments ranging in severity from skin lesions to blindness, encephalitis, and cancer (1, 2). A unique feature of all herpesviruses is a multiprotein tegument layer between the capsid and the envelope. Besides being necessary for viral assembly, tegument proteins also play critical roles at early stages of the viral replication cycle, in which some are released to regulate expression of viral (3) or cellular genes (4) while others remain bound to the capsid and mediate its trafficking (5-7). The involvement of tegument proteins at multiple stages during viral replication makes them attractive antiviral targets, yet few have been characterized in detail.Herpes simplex virus 1 (HSV-1) UL21 is a 535-amino-acid tegument protein that is conserved within the Alphaherpesvirus subfamily (8) and may have analogs among other herpesviruses. UL21 is important for replication in culture because UL21-null mutants of HSV-1 and pseudorabies virus (PRV) show reductions in titer and small plaques (9, 10), whereas HSV-2 cannot replicate without UL21 (8). A lack of UL21 also leads to defects in pathogenesis of PRV in mice and pigs (11)(12)(13)(14). UL21 is involved in secondary envelopment (15) and cell-cell spread of mature virions (16) through binding of the tegument proteins UL16 (15, 17) and UL11 (16,17). The UL21-UL16-UL11 heterotrimer binds the cytoplasmic domain of glycoprotein E (gE), a viral glycoprotein required for viral cell spread (18, 19) and cell-cell fusion of infected cells (20), and regulates these functions (16). UL21 may also have a role in cytosolic capsid transport through association with microtubules (21). In the absence of UL21, expression of viral genes is delayed, possibly due to lower mRNA levels (8, 10). Finally, capsids of UL21-null HSV-2 are unable to undergo nuclear egress (8), suggesting a potential nuclear function for UL21, which localizes not only to the cytoplasm but also to the nucleus (17, 21), specifically the nuclear rim (8, 9, 16).Although UL21 clearly plays multiple roles in the viral replication cycle, little is known about it due to the lack of sequence Citation Metrick CM, Chadha P, Heldwein EE. 2015. The unusual fold of herpes simplex virus 1 UL21, a multifunctional tegument protein.