Incorporation of Three Endocytosed Varicella-Zoster Virus Glycoproteins, gE, gH, and gB, into the Virion Envelope

Herpes simplex viruses (HSVs) are prevalent human pathogens that establish latency in human neuronal cells and efficiently evade the immune system. It has been a major medical challenge to eradicate them and, despite intensive efforts, an effective vaccine is not available. We previously showed that upon infection of antigen-presenting cells, HSV type 1 (HSV-1) rapidly and efficiently downregulates the major histocompatibility complex class I-like antigen-presenting molecule, CD1d, and potently inhibits its recognition by CD1d-restricted natural killer T (NKT) cells. It suppresses CD1d expression primarily by inhibiting its recycling to the cell surface after endocytosis. We identify here the viral glycoprotein B (gB) as the predominant CD1d-interacting protein. gB initiates the interaction with CD1d in the endoplasmic reticulum and stably associates with it throughout CD1d trafficking. However, an additional HSV-1 component, the serine-threonine kinase US3, is required for optimal CD1d downregulation. US3 expression in infected cells leads to gB enrichment in the trans-Golgi network (TGN) and enhances the relocalization of both gB and CD1d to this compartment, suggesting that following internalization CD1d is translocated from the endocytic pathway to the TGN by its association with gB. Importantly, both US3 and gB are required for efficient inhibition of CD1d antigen presentation and NKT cell activation. In summary, our results suggest that HSV-1 uses gB and US3 to rapidly inhibit NKT cell function in the initial antiviral response.

Section: Discussionmentioning

confidence: 99%

Herpes Simplex Virus 1 Glycoprotein B and US3 Collaborate To Inhibit CD1d Antigen Presentation and NKT Cell Function

Rao

Pham

Kulkarni

et al. 2011

“…The three glycoproteins are also present on the alphaherpesvirus particles and are required for fusion of the virion envelope and cell membrane during entry (11,12). Transient expression of VZV gB and gH-gL has been shown to be necessary and sufficient to induce membrane fusion using in vitro cell-cell fusion assays (13)(14)(15).…”

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confidence: 99%

“…After translation, gB is exported from the endoplasmic reticulum (ER), processed in the Golgi apparatus, trafficked to the cell surface, endocytosed, and then returned to the trans-Golgi network (TGN) for insertion into the viral envelope (12,19,20). The protein forms a trimer, with each 931-amino-acid (aa) monomer having an ectodomain, transmembrane domain, and a 124-aa cytoplasmic domain (21,22).…”

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confidence: 99%

The Glycoprotein B Cytoplasmic Domain Lysine Cluster Is Critical for Varicella-Zoster Virus Cell-Cell Fusion Regulation and Infection

Yang

Arvin

Oliver

2017

The conserved glycoproteins gB and gH-gL are essential for herpesvirus entry and cell-cell fusion induced syncytium formation, a characteristic of varicellazoster virus (VZV) pathology in skin and sensory ganglia. VZV syncytium formation, which has been implicated in the painful condition of postherpetic neuralgia, is regulated by the cytoplasmic domains of gB (gBcyt) via an immunoreceptor tyrosinebased inhibition motif (ITIM) and gH (gHcyt). A lysine cluster (K894, K897, K898, and K900) in the VZV gBcyt was identified by sequence alignment to be conserved among alphaherpesviruses, suggesting a functional role. Alanine and arginine substitutions were used to determine if the positive charge and susceptibility to posttranslational modifications of these lysines contributed to gB/gH-gL cell-cell fusion. Critically, the positive charge of the lysine residues was necessary for fusion regulation, as alanine substitutions induced a 440% increase in fusion compared to that of the wild-type gBcyt while arginine substitutions had wild-type-like fusion levels in an in vitro gB/gH-gL cell fusion assay. Consistent with these results, the alanine substitutions in the viral genome caused exaggerated syncytium formation, reduced VZV titers (Ϫ1.5 log 10 ), and smaller plaques than with the parental Oka (pOka) strain. In contrast, arginine substitutions resulted in syncytia with only 2-fold more nuclei, a Ϫ0.5-log 10 reduction in titers, and pOka-like plaques. VZV mutants with both an ITIM mutation and either alanine or arginine substitutions had reduced titers and small plaques but differed in syncytium morphology. Thus, effective VZV propagation is dependent on cell-cell fusion regulation by the conserved gBcyt lysine cluster, in addition to the gBcyt ITIM and the gHcyt.IMPORTANCE Varicella-zoster virus (VZV) is a ubiquitous pathogen that causes chickenpox and shingles. Individuals afflicted with shingles risk developing the painful condition of postherpetic neuralgia (PHN), which has been difficult to treat because the underlying cause is not well understood. Additional therapies are needed, as the current vaccine is not recommended for immunocompromised individuals and its efficacy decreases with the age of the recipient. VZV is known to induce the formation of multinuclear cells in neuronal tissue, which has been proposed to be a factor contributing to PHN. This study examines the role of a lysine cluster in the cytoplasmic domain of the VZV fusion protein, gB, in the formation of VZV induced multinuclear cells and in virus replication kinetics and spread. The findings further elucidate how VZV self-regulates multinuclear cell formation and may provide insight into the development of new PHN therapies.KEYWORDS cell-cell fusion, glycoproteins, human herpesviruses, varicella-zoster virus Citation Yang E, Arvin AM, Oliver SL. 2017. The glycoprotein B cytoplasmic domain lysine cluster is critical for varicella-zoster virus cellcell fusion regulation and infection. J Virol

“…For a large number of envelope glycoproteins, however, it has been shown that they follow an internalization pathway after having reached the plasma membrane and accumulate in interior compartments of the cell, such as the TGN or vesicles derived from this cellular structure (1,8,9,9,17,30,42,43). A wellstudied member of this group of proteins is glycoprotein B (gB), which has been shown for a number of herpesviruses to be internalized from the plasma membrane and incorporated into the virion (4,14,21,45,57).…”

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confidence: 99%

Optimal Replication of Human Cytomegalovirus Correlates with Endocytosis of Glycoprotein gpUL132

Kropff¹,

Koedel

Britt

et al. 2010

Envelopment of a herpesvirus particle is a complex process of which much is still to be learned. We previously identified the glycoprotein gpUL132 of human cytomegalovirus (HCMV) as an envelope component of the virion. In its carboxy-terminal portion, gpUL132 contains at least four motifs for sorting of transmembrane proteins to endosomes; among them are one dileucine-based signal and three tyrosine-based signals of the YXXØ and NPXY (where X stands for any amino acid, and Ø stands for any bulky hydrophobic amino acid) types. To investigate the role of each of these trafficking signals in intracellular localization and viral replication, we constructed a panel of expression plasmids and recombinant viruses in which the signals were rendered nonfunctional by mutagenesis. In transfected cells wild-type gpUL132 was mainly associated with the trans-Golgi network. Consecutive mutation of the trafficking signals resulted in increasing fractions of the protein localized at the cell surface, with gpUL132 mutated in all four trafficking motifs predominantly associated with the plasma membrane. Concomitant with increased surface expression, endocytosis of mutant gpUL132 was reduced, with a gpUL132 expressing all four motifs in mutated form being almost completely impaired in endocytosis. The replication of recombinant viruses harboring mutations in single trafficking motifs was comparable to replication of wild-type virus. In contrast, viruses containing mutations in three or four of the trafficking signals showed pronounced deficits in replication with a reduction of approximately 100-fold. Moreover, recombinant viruses expressing gpUL132 with three or four trafficking motifs mutated failed to incorporate the mutant protein into the virus particle. These results demonstrate a role of endocytosis of an HCMV envelope glycoprotein for incorporation into the virion and optimal virus replication.Morphogenesis of herpesviruses is a complex multistep process which requires the assembly of an infectious viral particle containing Ͼ70 proteins. The proteins are organized into distinct regions in the particle, including the capsid containing viral DNA, the tegument, and the envelope of the virion, suggesting an ordered addition of viral proteins during assembly.Most data in the literature favor a model in which the final virion is assembled by multiple budding and fusion events of the nascent particle. According to this model, genome-containing capsids acquire a first envelope by budding at the inner nuclear membrane in a step termed primary envelopment (34).