Niklaus H. Mueller

Varicella Zoster Virus Infection: Clinical Features, Molecular Pathogenesis of Disease, and Latency

Varicella zoster virus (VZV) is an exclusively human neurotropic alphaherpesvirus. Primary infection causes varicella (chickenpox), after which virus becomes latent in cranial nerve ganglia, dorsal root ganglia, and autonomic ganglia along the entire neuraxis. Years later, in association with a decline in cell-mediated immunity in elderly and immunocompromised individuals, VZV reactivates and causes a wide range of neurologic disease. This article discusses the clinical manifestations, treatment, and prevention of VZV infection and reactivation; pathogenesis of VZV infection; and current research focusing on VZV latency, reactivation, and animal models.

Clinical and molecular aspects of varicella zoster virus infection

A declining cell-mediated immunity to varicella zoster virus (VZV) with advancing age or immunosuppression results in virus reactivation from latently infected human ganglia anywhere along the neuraxis. Virus reactivation produces zoster, often followed by chronic pain (postherpetic neuralgia or PHN) as well as vasculopathy, myelopathy, retinal necrosis and cerebellitis. VZV reactivation also produces pain without rash (zoster sine herpete). Vaccination after age 60 reduces the incidence of shingles by 51%, PHN by 66% and the burden of illness by 61%. However, even if every healthy adult over age 60 years is vaccinated, there would still be about 500,000 zoster cases annually in the United States alone, about 200,000 of whom will experience PHN. Analyses of viral nucleic acid and gene expression in latently infected human ganglia and in an animal model of varicella latency in primates are serving to determine the mechanism(s) of VZV reactivation with the aim of preventing reactivation and the clinical sequelae.

Alpha-crystallin-mediated protection of lens cells against heat and oxidative stress-induced cell death.

In addition to their key role as structural lens proteins, α-crystallins also appear to confer protection against many eye diseases, including cataract, retinitis pigmentosa, and macular degeneration. Exogenous recombinant α-crystallin proteins were examined for their ability to prevent cell death induced by heat or oxidative stress in a human lens epithelial cell line (HLE-B3). Wild type αA- or αB-crystallin (WT-αA and WT-αB) and αA- or αB-crystallins, modified by the addition of a cell penetration peptide (CPP) designed to enhance the uptake of proteins into cells (gC-αB, TAT-αB, gC-αA), were produced by recombinant methods. In vitro chaperone-like assays were used to assay the ability of α-crystallins to protect client proteins from chemical or heat induced aggregation. In vivo viability assays were performed in HLE-B3 to determine whether pre-treatment with α-crystallins reduced death after exposure to oxidative or heat stress. Most of the five recombinant α-crystallin proteins tested conferred some in vitro protection from protein aggregation, with the greatest effect seen with WT-αB and gC-αB. All α-crystallins displayed significant protection to oxidative stress induced cell death, while only the αB-crystallins reduced cell death induced by thermal stress. Our findings indicate that the addition of the gC tag enhanced the protective effect of αB-crystallin against oxidative but not thermally-induced cell death. In conclusion, modifications that increase the uptake of α-crystallin proteins into cells, without destroying their chaperone-like activity and anti-apoptotic functions, create the potential to use these proteins therapeutically.

Cell Penetration Peptides for Enhanced Entry of αB-Crystallin into Lens Cells

PURPOSE The prevalence of cataract increases with age. Conversely, the abundance of native α-crystallin diminishes with age and cataract development. We hypothesize replenishing lens α-crystallin may delay or prevent cataract. Herein we investigated the ability of cell penetration peptides (CPP) to enhance entry of α-crystallins into lens-derived cells. METHODS Recombinant αB-crystallins were modified by the addition of CPPs. Candidate CPP were designed with reference to the HSV-1 glycoprotein C gene (gC) or the HIV-1 TAT peptide. αB-crystallins produced by fusing gC or TAT were over-expressed in E. coli. Purified proteins were subjected to size exclusion chromatography (SEC) to characterize oligomeric complexes (OC). Chaperone-like activity (CLA) was evaluated by measuring the ability of α-crystallins to suppress chemically-induced protein aggregation. To evaluate protein uptake, labeled α-crystallins were incubated with HLE B3 cells and monitored by fluorescence microscopy for 48 hours. RESULTS We examined the effects of the addition of CPP on the structure, CLA, and cell transduction properties of αB-crystallins. C-terminal CPP fused crystallins had poor solubility. In contrast, N-terminal tagged αB-crystallins were soluble. These modified αB-crystallins formed OC that were larger than wild-type based on SEC. Wild-type and gC tagged αB-crystallin displayed robust CLA. Subunit exchange was observed when gC-fused αB-crystallin was mixed with αA. In contrast to wild-type, modified α-crystallins accumulated in HLE B3 cells. CONCLUSIONS Addition of CPP improves the uptake of αB-crystallins into HLE B3 cells. No undesirable changes to the chaperone-like abilities of α-crystallins were observed in αB-crystallin modified by the addition of the gC-derived CPP.

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

ABSTRACT Varicella-zoster virus (VZV) open reading frame (ORF) 63 is abundantly transcribed in latently infected human 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.

Identification of phosphorylated residues on varicella-zoster virus immediate-early protein ORF63

Efficient replication of varicella-zoster virus (VZV) in cell culture requires expression of protein encoded by VZV open reading frame 63 (ORF63p). Two-dimensional gel analysis demonstrates that ORF63p is extensively modified. Mass spectroscopy analysis of ORF63p isolated from transiently transfected HEK 293 and stably transfected MeWo cells identified 10 phosphorylated residues. In VZV-infected MeWo cells, only six phosphorylated residues were detected. This report identifies phosphorylation of two previously uncharacterized residues (Ser5 and Ser31) in ORF63p extracted from cells infected with VZV or transfected with an ORF63p expression plasmid. Computational analysis of ORF63p for known kinase substrates did not identify Ser5 or Ser31 as candidate phosphorylation sites, suggesting that either atypical recognition sequences or novel cellular kinases are involved in ORF63p post-translational modification.

Generation of Mutant Murine Cytomegalovirus Strains from Overlapping Cosmid and Plasmid Clones

ABSTRACT We have developed a cosmid and plasmid system to generate mutant strains of murine cytomegalovirus (MCMV). The system is based on a series of seven overlapping cosmid clones that regenerate MCMV when cotransfected into mouse cells. The unaltered cosmids produce MCMV that is indistinguishable from wild-type MCMV based on restriction enzyme digest patterns of virus DNA and growth rates both in vitro and in vivo. Analysis of viral DNA from plaque-purified recombinant isolates taken from in vitro and in vivo stocks indicated that regeneration did not introduce novel mutations in the recombinant viral genomes. Isolation of specific genes and subsequent generation of specific mutant MCMVs was accomplished by replacement of cosmids with overlapping plasmid subclones. A new vector, PmeSUB, featuring a multiple cloning site and a stringent origin of replication, was constructed to make large subclones for use with smaller subclones containing the gene of interest. The utility of this system was demonstrated by the generation of two different mutant MCMVs from different combinations of overlapping plasmid subclones of one cosmid. The advantages of this system are that (i) target genes are maintained as small clones making them amenable to standard in vitro mutagenesis manipulations and that (ii) no reporter or selection genes are necessary to identify mutants.

Therapeutic potential of α-crystallin ☆

BACKGROUND The findings that α-crystallins are multi-functional proteins with diverse biological functions have generated considerable interest in understanding their role in health and disease. Recent studies have shown that chaperone peptides of α-crystallin could be delivered into cultured cells and in experimental animals with beneficial effects against protein aggregation, oxidation, inflammation and apoptosis. SCOPE OF REVIEW In this review, we will summarize the latest developments on the therapeutic potential of α-crystallins and their functional peptides. MAJOR CONCLUSIONS α-Crystallins and their functional peptides have shown significant favorable effects against several diseases. Their targeted delivery to tissues would be of great therapeutic benefit. However, α-crystallins can also function as disease-causing proteins. These seemingly contradictory functions must be carefully considered prior to their therapeutic use. GENERAL SIGNIFICANCE αA and αB-Crystallin are members of the small heat shock protein family. These proteins exhibit molecular chaperone and anti-apoptotic activities. The core crystallin domain within these proteins is largely responsible for these prosperities. Recent studies have identified peptides within the crystallin domain of both α- and αB-crystallins with remarkable chaperone and anti-apoptotic activities. Administration of α-crystallin or their functional peptides has shown substantial inhibition of pathologies in several diseases. However, α-crystallins have been shown to promote disease-causing pathways. These two sides of the proteins are discussed in this review. This article is part of a Special Issue entitled Crystallin Biochemistry in Health and Disease.

Temperature-dependent structural and functional properties of a mutant (F71L) αA-crystallin: molecular basis for early onset of age-related cataract.

alphaA crystallin and alphaB crystallin bind by molecular sieving(View interaction)

Recombinant Monoclonal Antibody Recognizes a Unique Epitope on Varicella-Zoster Virus Immediate-Early 63 Protein

ABSTRACT We previously constructed a recombinant monoclonal antibody (rec-MAb 63P4) that detects immediate-early protein IE63 encoded by varicella-zoster virus (VZV) in the cytoplasm of productively infected cells. Here, we used ORF63 truncation mutants to map the rec-MAb 63P4 binding epitope to amino acids 141 to 150 of VZV IE63, a region not shared with other widely used anti-IE63 antibodies, and found that the recombinant antibody does not bind to the simian IE63 counterpart.