Deletion of U L 21 Causes a Delay in the Early Stages of the Herpes Simplex Virus 1 Replication Cycle

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 ...

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confidence: 99%

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

Metrick

Heldwein

2016

“…Consistent with this, single-amino-acid changes within the CTD of full-length UL16 also activate binding to UL11, although they do not stimulate binding to gE and VP22 (12,36,37). Moreover, UL16 binds directly to UL21, and although the sites of contact are unknown, this interaction also activates binding to UL11 but, again, not to gE (26,(45)(46)(47)(48)(49)(50). Thus, it seems that UL16 has at least two functional domains: an NTD that mediates binding to partners on the membrane and a CTD that regulates those interactions in a complicated manner (36).…”

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confidence: 68%

Domain Interaction Studies of Herpes Simplex Virus 1 Tegument Protein UL16 Reveal Its Interaction with Mitochondria

Chadha

Sarfo

Zhang

et al. 2017

The UL16 tegument protein of herpes simplex virus 1 (HSV-1) is conserved among all herpesviruses and plays many roles during replication. This protein has an N-terminal domain (NTD) that has been shown to bind to several viral proteins, including UL11, VP22, and glycoprotein E, and these interactions are negatively regulated by a C-terminal domain (CTD). Thus, in pairwise transfections, UL16 binding is enabled only when the CTD is absent or altered. Based on these results, we hypothesized that direct interactions occur between the NTD and the CTD. Here we report that the separated and coexpressed functional domains of UL16 are mutually responsive to each other in transfected cells and form complexes that are stable enough to be captured in coimmunoprecipitation assays. Moreover, we found that the CTD can associate with itself. To our surprise, the CTD was also found to contain a novel and intrinsic ability to localize to specific spots on mitochondria in transfected cells. Subsequent analyses of HSV-infected cells by immunogold electron microscopy and live-cell confocal imaging revealed a population of UL16 that does not merely accumulate on mitochondria but in fact makes dynamic contacts with these organelles in a time-dependent manner. These findings suggest that the domain interactions of UL16 serve to regulate not just the interaction of this tegument protein with its viral binding partners but also its interactions with mitochondria. The purpose of this novel interaction remains to be determined. IMPORTANCEThe HSV-1-encoded tegument protein UL16 is involved in multiple events of the virus replication cycle, ranging from virus assembly to cell-cell spread of the virus, and hence it can serve as an important drug target. Unfortunately, a lack of both structural and functional information limits our understanding of this protein. The discovery of domain interactions within UL16 and the novel ability of UL16 to interact with mitochondria in HSV-infected cells lays a foundational framework for future investigations aimed at deciphering the structure and function of not just UL16 of HSV-1 but also its homologs in other herpesviruses.KEYWORDS domains, HSV-1, herpes simplex virus, mitochondria, tegument, UL16 H erpes simplex virus 1 (HSV-1) contains a 373-amino-acid tegument protein, UL16 (Fig. 1), which is conserved among all families of herpesviruses (1-15). Encoded by the late-expressing 16th open reading frame in the unique long segment of the viral genome, UL16 initially localizes to both the nucleus and the cytoplasm of the infected cell but later accumulates in the cytoplasm (4, 16). There, a population of UL16 is found to be stably associated with an unknown partner on cytoplasmic capsids (16,17), where it plays a role in envelopment by providing bridging functions via its interaction with at least three viral membrane proteins: UL11, glycoprotein E (gE), and VP22 (12, 18-43). Thus, UL16-null mutants produce only 1/10 the number of wild-type virions (1, 12), and

“…Residues 38 to 46, 76 to 87, and 199 to 216 were not resolved despite being present in the crystals. S0 is the first residue of the ␤1 strand, and residues QEF of the StrepII tag form a 3 10 helix.…”

Section: Figmentioning

confidence: 99%

“…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).…”

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confidence: 99%

“…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).…”

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confidence: 99%

See 1 more Smart Citation

The Unusual Fold of Herpes Simplex Virus 1 UL21, a Multifunctional Tegument Protein

Metrick

Chadha

Heldwein

2015

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.