Simian varicella virus reactivation in cynomolgus monkeys

Varicella-zoster virus (VZV) open reading frame (ORF) 63 is abundantly transcribed in latently infectedhuman ganglia and encodes a 278-amino-acid protein, IE63, with immediate-early kinetics. IE63 is expressed in the cytoplasm of neurons during VZV latency and in both the cytoplasm and the nucleus during productive infection; however, the mechanism(s) involved in IE63 nuclear import and retention has remained unclear. We constructed and identified a recombinant monoclonal antibody to detect a posttranslationally modified form of IE63. Analysis of a series of IE63 truncation and substitution mutants showed that amino acids 186 to 195 are required for antibody binding. Synthetic peptides corresponding to this region identified IE63 S186 as a target for casein kinase II phosphorylation. In addition, acidic charges supplied by E194 and E195 were required for antibody binding. Immunofluorescence analysis of VZV-infected MeWo cells using the recombinant monoclonal antibody detected IE63 exclusively in the nuclei of infected cells, indicating that casein kinase II phosphorylation of S186 occurs in the nucleus and possibly identifying an initial molecular event operative in VZV reactivation.Primary infection with the human varicella-zoster virus (VZV) typically causes varicella (chickenpox), after which the virus becomes latent in ganglionic neurons along the entire neuraxis. Reactivation decades later usually produces zoster (shingles) (15). During latency, transcripts mapping to VZV open reading frames (ORFs) 62, 63, and 66 and proteins corresponding to ORFs 21, 29, 62, 63, and 66 have been detected in human ganglia obtained at autopsy (7,9,12,16,19,20,25,26,36). Quantitative PCR has shown that ORF 63 is the most prevalent and abundant VZV transcript detected during latency (8).VZV ORF 63, which is located within the terminal and internal repeat regions of the virus genome and is present as two copies, encodes the immediate-early (IE) protein IE63. This 278-amino-acid protein is present in the cytoplasm during latency but is located predominantly in the nucleus during productive infection (13,26).During productive infection in tissue culture cells, VZV IE63 is phosphorylated by casein kinase I (CKI), casein kinase II (CKII), and cyclin-dependent kinase 1 (CDK1) (2,4,17,35,37). In vitro phosphorylation assays have shown that IE63 is phosphorylated by CKII, CDK1, and a protein kinase encoded by ORF 47 (2,4,17,22). Sequence analysis has suggested 19 potential phosphorylation sites (2, 4). IE63 amino acid S224 is phosphorylated by CDK1 (17), while S165, S173, and S185 are phosphorylated in vitro by serine/threonine kinase, as indicated by alanine substitution mutation of IE63 (2).Previous studies have shown that phosphorylation of IE63 is required for efficient VZV replication in cell culture (2, 6).Using an antibody that recognizes a posttranslationally modified (PTM) form of IE63, we found that IE63 S186 is phosphorylated by CKII and that phosphorylated S186 is present only in the nuclei of VZV-infected cells. MATERIALS...

Section: Discussionmentioning

confidence: 99%

Phosphorylation of the Nuclear Form of Varicella-Zoster Virus Immediate-Early Protein 63 by Casein Kinase II at Serine 186

Mueller

Graf²,

Orlicky³

et al. 2009

“…SVV (a deltaherpesvirus strain) isolated from a naturally infected monkey (Erythrocebus patas) was propagated in Vero (African green monkey kidney) cells, and a virus stock was prepared as described previously (9). SVV-seronegative rhesus macaques (monkeys HB62, HI83, HF39, HC44, HA95, II49, and IK10) were inoculated intrabronchially with 10 4 PFU of wild-type SVV (monkeys HB62, HI83, HA95, II49, and IK10) or SVV expressing enhanced green fluorescent protein (SVV-EGFP) (monkeys HF39 and HC44) as described previously (4,10). Earlier, we demonstrated that SVV-EGFP is pathogenic in African green monkeys and that SVV-EGFP is mildly attenuated compared to wild-type SVV (11,12).…”

Section: Methodsmentioning

confidence: 99%

“…All immunosuppressive treatments were performed as described previously (4). Five months after primary infection, monkeys HB62, HI83, HF39, and HC44 were transported by van (2-h round trip) from the Tulane National Primate Research Center in Covington, LA, to the Tulane University Cancer Center in New Orleans, LA, anesthetized, and exposed to a single dose of 200-cGy total body X-irradiation and then started on daily oral treatment with 500 g (80 g/kg of body weight/day) of tacrolimus (Prograf) and 5 mg (2 mg/kg/ day) of prednisone until they were euthanized.…”

Section: Methodsmentioning

confidence: 99%

“…Simian varicella virus (SVV) infection of nonhuman primates has served as a good model with which to study the pathogenesis of VZV because of the pathological, immunological, and virological similarities of SVV to VZV (1,2). VZV reactivation is increased in patients receiving chemotherapy or after X-irradiation (3); similarly, SVV reactivates in latently infected African green monkeys (AGM) and cynomolgus monkeys (CM) after immunosuppression and environmental stress (4). In rhesus macaques, intrabronchial inoculation with SVV produces varicella, followed by the establishment of latency (5) and virus reactivation after X-irradiation (6,7).…”

Section: Importancementioning

confidence: 99%

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Simian Varicella Virus Is Present in Macrophages, Dendritic Cells, and T Cells in Lymph Nodes of Rhesus Macaques after Experimental Reactivation

Traina‐Dorge

Doyle-Meyers

Sanford

et al. 2015

Self Cite

Like varicella-zoster virus (VZV), simian varicella virus (SVV) reactivates to produce zoster. In the present study, 5 rhesus macaques were inoculated intrabronchially with SVV, and 5 months later, 4 monkeys were immunosuppressed; 1 monkey was not immunosuppressed but was subjected to the stress of transportation. In 4 monkeys, a zoster rash developed 7 to 12 weeks after immunosuppression, and a rash also developed in the monkey that was not immunosuppressed. Analysis at 24 to 48 h after zoster revealed SVV antigen in the lung alveolar wall, in ganglionic neurons and nonneuronal cells, and in skin and in lymph nodes. In skin, SVV was found primarily in sweat glands. In lymph nodes, the SVV antigen colocalized mostly with macrophages, dendritic cells, and, to a lesser extent, T cells. The presence of SVV in lymph nodes, as verified by quantitative PCR detection of SVV DNA, might reflect the sequestration of virus by macrophages and dendritic cells in lymph nodes or the presentation of viral antigens to T cells to initiate an immune response against SVV, or both. IMPORTANCEVZV causes varicella (chickenpox), becomes latent in ganglia, and reactivates to produce zoster and multiple other serious neurological disorders. SVV in nonhuman primates has proved to be a useful model in which the pathogenesis of the virus parallels the pathogenesis of VZV in humans. Here, we show that SVV antigens are present in sweat glands in skin and in macrophages and dendritic cells in lymph nodes after SVV reactivation in monkeys, raising the possibility that macrophages and dendritic cells in lymph nodes serve as antigen-presenting cells to activate T cell responses against SVV after reactivation. P rimary varicella-zoster virus (VZV) infection produces chickenpox (varicella), after which the virus becomes latent in ganglionic neurons along the entire neuraxis. As VZV-specific T cell immunity declines with advancing age, VZV reactivates to produce zoster, which may be complicated by multiple neurological and ocular disorders. Simian varicella virus (SVV) infection of nonhuman primates has served as a good model with which to study the pathogenesis of VZV because of the pathological, immunological, and virological similarities of SVV to VZV (1, 2). VZV reactivation is increased in patients receiving chemotherapy or after X-irradiation (3); similarly, SVV reactivates in latently infected African green monkeys (AGM) and cynomolgus monkeys (CM) after immunosuppression and environmental stress (4). In rhesus macaques, intrabronchial inoculation with SVV produces varicella, followed by the establishment of latency (5) and virus reactivation after X-irradiation (6, 7). At the time of SVV reactivation in CM, expression of CXCL10 (a chemokine which recruits activated T cells and NK cells) correlates with transient T cell infiltration in ganglia (8). In the study described here, we determined if a combination of irradiation and treatment with prednisone and tacrolimus induces reactivation of SVV in latently infected rhesus macaques to s...

“…Indeed, immunization of Erythrocebus patas with VZV can protect the animals from SVV challenge, demonstrating the antigenic relatedness of the two viruses (30). SVV can also reactivate in animals exposed to social/environmental stress such as transportation or introduction of new animals into an existing group (31)(32)(33)(34) as well as in animals subjected to immune system-suppressive treatments (35)(36)(37)(38). Thus, infection of nonhuman primates (NHP) with SVV provides a translational model with which to gain insight into the anti-VZV T cell response.…”

mentioning

confidence: 99%

Genome-Wide Analysis of T Cell Responses during Acute and Latent Simian Varicella Virus Infections in Rhesus Macaques

Haberthur

Kraft

Arnold

et al. 2013

f Varicella zoster virus (VZV) is the etiological agent of varicella (chickenpox) and herpes zoster (HZ [shingles]). Clinical observations suggest that VZV-specific T cell immunity plays a more critical role than humoral immunity in the prevention of VZV reactivation and development of herpes zoster. Although numerous studies have characterized T cell responses directed against select VZV open reading frames (ORFs), a comprehensive analysis of the T cell response to the entire VZV genome has not yet been conducted. We have recently shown that intrabronchial inoculation of young rhesus macaques with simian varicella virus (SVV), a homolog of VZV, recapitulates the hallmarks of acute and latent VZV infection in humans. In this study, we characterized the specificity of T cell responses during acute and latent SVV infection. Animals generated a robust and broad T cell response directed against both structural and nonstructural viral proteins during acute infection in bronchoalveolar lavage (BAL) fluid and peripheral blood. During latency, T cell responses were detected only in the BAL fluid and were lower and more restricted than those observed during acute infection. Interestingly, we identified a small set of ORFs that were immunogenic during both acute and latent infection in the BAL fluid. Given the close genome relatedness of SVV and VZV, our studies highlight immunogenic ORFs that may be further investigated as potential components of novel VZV vaccines that specifically boost T cell immunity.