scholarly journals Inhibition of Varicella-Zoster Virus by Penciclovir in Cell Culture and Mechanism of Action

1996 ◽  
Vol 7 (2) ◽  
pp. 71-78 ◽  
Author(s):  
T. H. Bacon ◽  
J. Gilbart ◽  
B. A. Howard ◽  
R. Standring-Cox
Keyword(s):  
Dna Synthesis ◽  
Varicella Zoster Virus ◽  
Clinical Isolates ◽  
Continuous Treatment ◽  
Lung Fibroblasts ◽  
Human Embryonic Lung ◽  
Viral Dna ◽  
Plaque Reduction Assay ◽  
Varicella Zoster ◽  
Infected Cells

The effect of penciclovir (BRL 39123) on the replication of varicella-zoster virus (VZV) in human embryonic lung fibroblasts (MRC-5 cells) was similar to aciclovir when the compounds were present continuously. However, when the compounds were withdrawn the antiviral activity of penciclovir was maintained more effectively than that of aciclovir. In the plaque reduction assay, median 50% effective concentrations (EC50s) were 3.8 μg ml−1 for penciclovir and 4.2 μg ml−1 for aciclovir ( n = 29 clinical isolates). Similarly, penciclovir and aciclovir were equally effective in reducing the numbers of VZV-infected MRC-5 cells and in reducing VZV DNA synthesis within infected cells following continuous treatment. Within VZV-infected cells (S)-penciclovir-triphosphate was formed from penciclovir with >95% enantiomeric purity, and the concentration of penciclovir-triphosphate was 360-fold greater than aciclovir-triphosphate immediately after treatment. This phosphorylation ratio compensates for the lower affinity of VZV DNA polymerase for penciclovir-triphosphate compared with aciclovir-triphosphate (Kis = 7.5 μM and 0.2 μM, respectively). When VZV-infected cultures were treated for 3 days, followed by withdrawal of the compound, inhibition of viral DNA synthesis by penciclovir was maintained for 24 h, whereas viral DNA synthesis resumed more readily after removal of aciclovir. Furthermore, following 8 h daily pulse treatment for 5 days, penciclovir was significantly more active than aciclovir in reducing VZV DNA synthesis ( p = 0.006, n = 10 clinical isolates). The long intracellular half-life of penciclovir-triphosphate (9.1 h) compared with that of aciclovir-triphosphate (0.8 h) accounts for the sustained inhibition of virus replication by penciclovir. This property may contribute to the clinical efficacy of famciclovir, the oral form of penciclovir.

scholarly journals Varicella-Zoster Virus Immediate-Early Protein 62 Blocks Interferon Regulatory Factor 3 (IRF3) Phosphorylation at Key Serine Residues: a Novel Mechanism of IRF3 Inhibition among Herpesviruses

2010 ◽  
Vol 84 (18) ◽  
pp. 9240-9253 ◽  
Author(s):  
Nandini Sen ◽  
Marvin Sommer ◽  
Xibing Che ◽  
Kris White ◽  
William T. Ruyechan ◽  
...  
Keyword(s):  
Varicella Zoster Virus ◽  
Immediate Early ◽  
Lung Fibroblasts ◽  
Poly I:C ◽  
Interferon Response ◽  
Luciferase Reporter ◽  
Type I ◽  
Human Embryonic Lung ◽  
Varicella Zoster ◽  
Early Protein

ABSTRACT Varicella-zoster virus (VZV) is an alphaherpesvirus that is restricted to humans. VZV infection of differentiated cells within the host and establishment of latency likely require evasion of innate immunity and limited secretion of antiviral cytokines. Since interferons (IFNs) severely limit VZV replication, we examined the ability of VZV to modulate the induction of the type I IFN response in primary human embryonic lung fibroblasts (HELF). IFN-β production was not detected, and transcription of two interferon response factor 3 (IRF3)-dependent interferon-stimulated genes (ISGs), ISG54 and ISG56, in response to poly(I:C) stimulation was downregulated in VZV-infected HELF. Inhibition of IRF3 function did not require VZV replication; the viral immediate-early protein 62 (IE62) alone was sufficient to produce this effect. IE62 blocked TBK1-mediated IFN-β secretion and IRF3 function, as shown in an IFN-stimulated response element (ISRE)-luciferase reporter assay. However, IRF3 function was preserved if constitutively active IRF3 (IRF3-5D) was expressed in VZV-infected or IE62-transfected cells, indicating that VZV interferes with IRF3 phosphorylation. IE62-mediated inhibition was mapped to blocking phosphorylation of at least three serine residues on IRF3. However, IE62 binding to TBK1 or IRF3 was not detected and IE62 did not perturb TBK1-IRF3 complex formation. IE62-mediated inhibition of IRF3 function was maintained even if IE62 transactivator activity was disrupted. Thus, IE62 has two critical but discrete roles following VZV entry: to induce expression of VZV genes and to disarm the IFN-dependent antiviral defense through a novel mechanism that prevents IRF3 phosphorylation.

scholarly journals A Novel Human Skin Tissue Model To Study Varicella-Zoster Virus and Human Cytomegalovirus

2020 ◽  
Vol 94 (22) ◽  
Author(s):  
Megan G. Lloyd ◽  
Nicholas A. Smith ◽  
Michael Tighe ◽  
Kelsey L. Travis ◽  
Dongmei Liu ◽  
...  
Keyword(s):  
Varicella Zoster Virus ◽  
Human Skin ◽  
Mouse Models ◽  
Human Cytomegalovirus ◽  
Fetal Tissue ◽  
Target Cells ◽  
Varicella Zoster ◽  
Adult Human ◽  
Infected Cells ◽  
Adult Human Skin

ABSTRACT The herpesviruses varicella-zoster virus (VZV) and human cytomegalovirus (HCMV) are endemic to humans. VZV causes varicella (chicken pox) and herpes zoster (shingles), while HCMV causes serious disease in immunocompromised patients and neonates. More effective, less toxic antivirals are needed, necessitating better models to study these viruses and evaluate antivirals. Previously, VZV and HCMV models used fetal tissue; here, we developed an adult human skin model to study VZV and HCMV in culture and in vivo. While VZV is known to grow in skin, it was unknown whether skin could support an HCMV infection. We used TB40/E HCMV and POka VZV strains to evaluate virus tropism in skin organ culture (SOC) and skin xenograft mouse models. Adult human skin from reduction mammoplasties was prepared for culture on NetWells or mouse implantation. In SOC, VZV infected the epidermis and HCMV infected the dermis. Specifically, HCMV infected fibroblasts, endothelial cells, and hematopoietic cells, with some infected cells able to transfer infection. VZV and HCMV mouse models were developed by subcutaneous transplantation of skin into SCID/beige or athymic nude mice at 2 independent sites. Viruses were inoculated directly into one xenograft, and widespread infection was observed for VZV and HCMV. Notably, we detected VZV- and HCMV-infected cells in the contralateral, uninoculated xenografts, suggesting dissemination from infected xenografts occurred. For the first time, we showed HCMV successfully grows in adult human skin, as does VZV. Thus, this novel system may provide a much-needed preclinical small-animal model for HCMV and VZV and, potentially, other human-restricted viruses. IMPORTANCE Varicella-zoster virus and human cytomegalovirus infect a majority of the global population. While they often cause mild disease, serious illness and complications can arise. Unfortunately, there are few effective drugs to treat these viruses, and many are toxic. To complicate this, these viruses are restricted to replication in human cells and tissues, making them difficult to study in traditional animal models. Current models rely heavily on fetal tissues, can be prohibitively expensive, and are often complicated to generate. While fetal tissue models provide helpful insights, it is necessary to study human viruses in human tissue systems to fully understand these viruses and adequately evaluate novel antivirals. Adult human skin is an appropriate model for these viruses because many target cells are present, including basal keratinocytes, fibroblasts, dendritic cells, and lymphocytes. Skin models, in culture and xenografts in immunodeficient mice, have potential for research on viral pathogenesis, tissue tropism, dissemination, and therapy.

scholarly journals Antiherpesvirus Activities of (1′S,2′R)-9-{[1′,2′-Bis(hydroxymethyl)cycloprop-1′-yl]methyl}guanine (A-5021) in Cell Culture

1998 ◽  
Vol 42 (7) ◽  
pp. 1666-1670 ◽  
Author(s):  
Satoshi Iwayama ◽  
Nobukazu Ono ◽  
Yuko Ohmura ◽  
Katsuya Suzuki ◽  
Miho Aoki ◽  
...  
Keyword(s):  
Antiviral Agent ◽  
Inhibitory Effect ◽  
Clinical Isolates ◽  
Human Embryonic Lung ◽  
Growth Inhibition Assay ◽  
Varicella Zoster ◽  
Hel Cells ◽  
Simplex Virus ◽  
Short Time ◽  
Hsv 1

ABSTRACT Antiherpetic activity of (1′S,2′R)-9-{[1′,2′-bis(hydroxymethyl)cycloprop-1′-yl]methyl}guanine (A-5021) was compared with those of acyclovir (ACV) and penciclovir (PCV) in cell cultures. In a plaque reduction assay using a selection of human cells, A-5021 showed the most potent activity in all cells. Against clinical isolates of herpes simplex virus type 1 (HSV-1,n = 5) and type 2 (HSV-2, n = 6), mean 50% inhibitory concentrations (IC50s) for A-5021 were 0.013 and 0.15 μg/ml, respectively, in MRC-5 cells. Corresponding IC50s for ACV were 0.22 and 0.30 μg/ml, and those for PCV were 0.84 and 1.5 μg/ml, respectively. Against clinical isolates of varicella-zoster virus (VZV, n = 5), mean IC50s for A-5021, ACV, and PCV were 0.77, 5.2, and 14 μg/ml, respectively, in human embryonic lung (HEL) cells. A-5021 showed considerably more prolonged antiviral activity than ACV when infected cells were treated for a short time. The selectivity index, the ratio of 50% cytotoxic concentration to IC50, of A-5021 was superior to those of ACV and PCV for HSV-1 and almost comparable for HSV-2 and VZV. In a growth inhibition assay of murine granulocyte-macrophage progenitor cells, A-5021 showed the least inhibitory effect of the three compounds. These results show that A-5021 is a potent and selective antiviral agent against HSV-1, HSV-2, and VZV.

scholarly journals Exocytosis of Varicella-Zoster Virus Virions Involves a Convergence of Endosomal and Autophagy Pathways

2016 ◽  
Vol 90 (19) ◽  
pp. 8673-8685 ◽  
Author(s):  
Erin M. Buckingham ◽  
Keith W. Jarosinski ◽  
Wallen Jackson ◽  
John E. Carpenter ◽  
Charles Grose
Keyword(s):  
Varicella Zoster Virus ◽  
Endocytic Pathway ◽  
Autophagic Flux ◽  
Varicella Zoster ◽  
Viral Particles ◽  
Cytoplasmic Vesicles ◽  
Infected Cells ◽  
Autophagy Pathway ◽  
Simplex Virus ◽  
2D And 3D

ABSTRACTVaricella-zoster virus (VZV) is an extremely cell-associated herpesvirus with limited egress of viral particles. The induction of autophagy in VZV-infected monolayers is easily detectable; inhibition of autophagy leads to decreased VZV glycoprotein biosynthesis and diminished viral titers. To explain how autophagic flux could exert a proviral effect on the VZV infectious cycle, we postulated that the VZV exocytosis pathway following secondary envelopment may converge with the autophagy pathway. This hypothesis depended on known similarities between VZV gE and autophagy-related (Atg) Atg9/Atg16L1 trafficking pathways. Investigations were carried out with highly purified fractions of VZV virions. When the virion fraction was tested for the presence of autophagy and endosomal proteins, microtubule-associated protein 1 light chain (MAP1LC3B) and Ras-like GTPase 11 (Rab11) were detected. By two-dimensional (2D) and 3D imaging after immunolabeling, both proteins also colocalized with VZV gE in a proportion of cytoplasmic vesicles. When purified VZV virions were enumerated after immunoelectron microscopy, gold beads were detected on viruses following incubation with antibodies to VZV gE (∼100%), Rab11 (50%), and LC3B (30%). Examination of numerous electron micrographs demonstrated that enveloped virions were housed in single-membraned vesicles; viral particles were not observed in autophagosomes. Taken together, our data suggested that some viral particles after secondary envelopment accumulated in a heterogeneous population of single-membraned vesicular compartments, which were decorated with components from both the endocytic pathway (Rab11) and the autophagy pathway (LC3B). The latter cytoplasmic viral vesicles resembled an amphisome.IMPORTANCEVZV infection leads to increased autophagic flux, while inhibition of autophagy leads to a marked reduction in virus spread. In this investigation of the proviral role of autophagy, we found evidence for an intersection of viral exocytosis and autophagy pathways. Specifically, both LC3-II and Rab11 proteins copurified with some infectious VZV particles. The results suggested that a subpopulation of VZV particles were carried to the cell surface in single-walled vesicles with attributes of an amphisome, an organelle formed from the fusion of an endosome and an autophagosome. Our results also addressed the interpretation of autophagy/xenophagy results with mutated herpes simplex virus lacking its ICP34.5 neurovirulence gene (HSVΔ34.5). The VZV genome lacks an ICP34.5 ortholog, yet we found no evidence of VZV particles housed in a double-membraned autophagosome. In other words, xenophagy, a degradative process documented after infection with HSVΔ34.5, was not observed in VZV-infected cells.

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