Bachelor of Science, University of California Berkeley (2005)
Doctor of Philosophy, University of California Berkeley (2011)
The highly conserved herpesvirus glycoprotein complex gB/gH-gL mediates membrane fusion during virion entry and cell-cell fusion. Varicella-zoster virus (VZV) characteristically forms multinucleated cells, or syncytia, during the infection of human tissues, but little is known about this process. The cytoplasmic domain of VZV gB (gBcyt) has been implicated in cell-cell fusion regulation because a gB[Y881F] substitution causes hyperfusion. gBcyt regulation is necessary for VZV pathogenesis, as the hyperfusogenic mutant gB[Y881F] is severely attenuated in human skin xenografts. In this study, gBcyt-regulated fusion was investigated by comparing melanoma cells infected with wild-type-like VZV or hyperfusogenic mutants. The gB[Y881F] mutant exhibited dramatically accelerated syncytium formation in melanoma cells caused by fusion of infected cells with many uninfected cells, increased cytoskeleton reorganization, and rapid displacement of nuclei to dense central structures compared to pOka using live-cell confocal microscopy. VZV and human transcriptomes were concurrently investigated using whole transcriptome sequencing (RNA-seq) to identify viral and cellular responses induced when gBcyt regulation was disrupted by the gB[Y881F] substitution. The expression of four vital VZV genes, ORF61 and the genes for glycoproteins gC, gE, and gI, was significantly reduced at 36 h postinfection for the hyperfusogenic mutants. Importantly, hierarchical clustering demonstrated an association of differential gene expression with dysregulated gBcyt-mediated fusion. A subset of Ras GTPase genes linked to membrane remodeling were upregulated in cells infected with the hyperfusogenic mutants. These data implicate gBcyt in the regulation of gB fusion function that, if unmodulated, triggers cellular processes leading to hyperfusion that attenuates VZV infection.The highly infectious, human-restricted pathogen varicella-zoster virus (VZV) causes chickenpox and shingles. Postherpetic neuralgia (PHN) is a common complication of shingles that manifests as prolonged excruciating pain, which has proven difficult to treat. The formation of fused multinucleated cells in ganglia might be associated with this condition. An effective vaccine against VZV is available but not recommended for immunocompromised individuals, highlighting the need for new therapies. This study investigated the viral and cellular responses to hyperfusion, a condition where the usual constraints of cell membranes are overcome and cells form multinucleated cells. This process hinders VZV and is regulated by a viral glycoprotein, gB. A combination of live-cell imaging and next-generation genomics revealed an alteration in viral and cellular responses during hyperfusion that was caused by the loss of gB regulation. These studies reveal mechanisms central to VZV pathogenesis, potentially leading to improved therapies.
View details for PubMedID 27795423
The conserved glycoproteins gB and gH-gL are essential for herpesvirus entry and cell-cell fusion induced syncytium formation, a characteristic of varicella-zoster 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 tyrosine-based 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 (-1.5 log10), and smaller plaques than with the parental Oka (pOka) strain. In contrast, arginine substitutions resulted in syncytia with only 2-fold more nuclei, a -0.5-log10 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.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.
View details for PubMedID 27795427
Varicella-zoster virus (VZV) is an alphaherpesvirus that causes varicella and herpes zoster. Membrane fusion is essential for VZV entry and the distinctive syncytium formation in VZV-infected skin and neuronal tissue. Herpesvirus fusion is mediated by a complex of glycoproteins gB and gH-gL, which are necessary and sufficient for VZV to induce membrane fusion. However, the cellular requirements of fusion are poorly understood. Integrins have been implicated to facilitate entry of several human herpesviruses, but their role in VZV entry has not yet been explored. To determine the involvement of integrins in VZV fusion, a quantitative cell-cell fusion assay was developed using a VZV-permissive melanoma cell line. The cells constitutively expressed a reporter protein and short hairpin RNAs (shRNAs) to knock down the expression of integrin subunits shown to be expressed in these cells by RNA sequencing. The αV integrin subunit was identified as mediating VZV gB/gH-gL fusion, as its knockdown by shRNAs reduced fusion levels to 60% of that of control cells. A comparable reduction in fusion levels was observed when an anti-αV antibody specific to its extracellular domain was tested in the fusion assay, confirming that the domain was important for VZV fusion. In addition, reduced spread was observed in αV knockdown cells infected with the VZV pOka strain relative to that of the control cells. This was demonstrated by reductions in plaque size, replication kinetics, and virion entry in the αV subunit knockdown cells. Thus, the αV integrin subunit is important for VZV gB/gH-gL fusion and infection.Varicella-zoster virus (VZV) is a highly infectious pathogen that causes chickenpox and shingles. A common complication of shingles is the excruciating condition called postherpetic neuralgia, which has proven difficult to treat. While a vaccine is now available, it is not recommended for immunocompromised individuals and its efficacy decreases with the recipient's age. These limitations highlight the need for new therapies. This study examines the role of integrins in membrane fusion mediated by VZV glycoproteins gB and gH-gL, a required process for VZV infection. This knowledge will further the understanding of VZV entry and provide insight into the development of better therapies.
View details for DOI 10.1128/JVI.00792-16
View details for PubMedID 27279620
Varicella-zoster virus (VZV), of the family Alphaherpesvirinae, causes varicella in children and young adults, potentially leading to herpes zoster later in life on reactivation from latency. The conserved herpesvirus glycoprotein gB and the heterodimer gHgL mediate virion envelope fusion with cell membranes during virus entry. Naturally occurring neutralizing antibodies against herpesviruses target these entry proteins. To determine the molecular basis for VZV neutralization, crystal structures of gHgL were determined in complex with fragments of antigen binding (Fabs) from two human monoclonal antibodies, IgG-94 and IgG-RC, isolated from seropositive subjects. These structures reveal that the antibodies target the same site, composed of residues from both gH and gL, distinct from two other neutralizing epitopes identified by negative-stain electron microscopy and mutational analysis. Inhibition of gB/gHgL-mediated membrane fusion and structural comparisons with herpesvirus homologs suggest that the IgG-RC/94 epitope is in proximity to the site on VZV gHgL that activates gB. Immunization studies proved that the anti-gHgL IgG-RC/94 epitope is a critical target for antibodies that neutralize VZV. Thus, the gHgL/Fab structures delineate a site of herpesvirus vulnerability targeted by natural immunity.
View details for DOI 10.1073/pnas.1501176112
View details for Web of Science ID 000354390600062
View details for PubMedID 25918416