Role for the αV Integrin Subunit in Varicella-Zoster Virus-Mediated Fusion and Infection

Abstract

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 ␣V 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-␣V 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 ␣V 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 ␣V subunit knockdown cells. Thus, the ␣V integrin subunit is important for VZV gB/gH-gL fusion and infection. IMPORTANCE Varicella-zoster virus (VZV) is a highly infectious pathogen that causes chickenpox and shingles. A common complication ofshingles 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. V aricella-zoster virus (VZV) is an alphaherpesvirus and a hostspecific human pathogen that causes the diseases varicella and herpes zoster, commonly known as chickenpox and shingles (1). Prior to the approval of attenuated vaccines by the Food and Drug Administration, varicella was endemic in the United States population and it was estimated that one in three individuals would develop zoster in their lifetime (1, 2). The program for universal varicella vaccination of children in the United States has proven to be successful in preventing disease by reducing varicella incidence by 57% to 90% (3). The zoster vaccine has been effective in reducing the zoster incidence by 51.3% (4). Individuals afflicted with zoster risk developing p...

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

“…To determine the role of the gBcyt lysine cluster in VZV gB/gH-gL-mediated cell-cell fusion, gB mutants were generated and evaluated in the stable reporter fusion assay (SRFA), a quantitative cell-cell fusion assay (15). Lysines can be positively charged at physiological pH and are susceptible to posttranslational modifications (PTMs), including ubiquitination and acetylation (29,30).…”

“…CHO-DSP1 cells were propagated using F-12K nutrient mixture with Kaighn's modification (Invitrogen) supplemented with 10% fetal bovine serum (FBS; Invitrogen) and penicillin (100 U/ml; Invitrogen) and maintained under puromycin selection (8 g/ml; Invitrogen) (15). MeWo (human melanoma cells) (HTB-65; ATCC) and Mel-DSP2 cells were propagated in minimal essential medium (Invitrogen) supplemented with 10% FBS, nonessential amino acids (100 M; Omega Scientific), and antibiotics (penicillin, 100 U/ml; streptomycin, 100 g/ml; Invitrogen), with the latter cell line maintained under puromycin selection (5 g/ml) (15). The parental Oka strain of VZV (pOka) was derived from a self-excisable BAC (pOka-DX) and used throughout the study (45).…”

“…Stable reporter fusion assay of VZV gB/gH-gL-mediated fusion. Quantification of fusion events was performed as previously described (15,47). Briefly, CHO-DSP1 cells were transfected with equimolar amounts of pcDNA3.1-gL, pME18s-gH-TL, and pCAGGs-gB plasmids (gifts from Tadahiro Suenaga and Hisashi Arase, Osaka University, Osaka, Japan) (13) or pCAGGs vector expressing mutant gB using Lipofectamine 2000 (Invitrogen).…”

“…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). In contrast, other human herpesviruses require additional viral accessory proteins for membrane fusion, including glycoprotein D (gD) for herpes simplex virus 1 (HSV-1) and gp42 for Epstein-Barr virus (EBV) for specific cell types (16,17).…”

“…However, the role of the lysine cluster in the context of herpesvirus infection has not been previously examined. To characterize the VZV lysine cluster, mutant gB constructs were generated with alanine or arginine substitutions of the lysine cluster and evaluated in the recently developed gB/gH-gL stable reporter fusion assay (SRFA) (15). The role of the lysine cluster in VZV syncytium formation, replication kinetics, and spread was examined by transferring the mutations into recombinant VZV.…”