A Self-Excisable Infectious Bacterial Artificial Chromosome Clone of Varicella-Zoster Virus Allows Analysis of the Essential Tegument Protein Encoded by
            <i>ORF9</i>

Varicella-zoster virus (VZV) is an exclusively human, neurotropic alphaherpesvirus. Primary infection typically produces chickenpox (varicella), after which virus becomes latent in cranial nerve ganglia, dorsal root ganglia, and autonomic ganglia along the entire neuraxis (16). Decades later, virus reactivation results in shingles (zoster), frequently complicated by chronic pain (postherpetic neuralgia) as well as stroke (VZV vasculopathy), paralysis and incontinence (VZV myelopathy), and blindness (VZV progressive outer retinal necrosis).Knowledge of the full extent of VZV gene transcription and translation during latency is a prerequisite to hypotheses concerning how virus establishes latency and reactivates. The exact extent of VZV transcription in latently infected ganglia is unknown. Sequence analysis of the 3Ј terminus and its polyadenylated tracts in cDNA synthesized from RNA extracted from latently infected human ganglia identified transcripts mapping to VZV genes 21, 29, 62, 63, and 66 (8, 11), of which VZV gene 63 transcripts were the most prevalent and abundant (12). In situ hybridization (ISH) also detected expression of VZV genes 4, 18, 21, 29, 40, 62, and 63 (13, 16, 17). Using multiplex reverse transcription (RT)-PCR and the GenomeLab genetic analysis system (GeXPS), we detected as few as 20 copies of all 68 predicted VZV open reading frames (ORFs) in VZV-infected cells in tissue culture (33). Herein, we applied the same strategy and technology to analyze the extent of VZV transcription in latently infected human ganglia.

Section: Discussionmentioning

confidence: 99%

Varicella-Zoster Virus Transcriptome in Latently Infected Human Ganglia

Nagel

Choe

Traktinskiy

et al. 2011

“…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). The recombinant virus pOka-TK-RFP (pOka-rTK) was generated in a previous study (46).…”

Section: Methodsmentioning

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

Self Cite

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

“…The 293T (CRL-3216; ATCC) cells were propagated using Dulbecco's modified Eagle's medium (DMEM) with 4.5 g/liter glucose, L-glutamine, and sodium pyruvate (Invitrogen) supplemented with 10% FBS and penicillin. The parental Oka strain of VZV (pOka) (gift from Klaus Osterrieder, Freie Universität, Berlin, Germany) was derived from a self-excisable bacterial artificial chromosome (BAC) and used throughout the study (28).…”

Section: Cells and Virusesmentioning

confidence: 99%

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

Yang

Arvin

Oliver

2016

Self Cite

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...