A role for 3-O-sulfated heparan sulfate in promoting human cytomegalovirus infection in human iris cells.

Human cytomegalovirus (HCMV) has emerged as a clinically opportunistic pathogen that targets multiple types of ocular cells and tissues, including the iris region of the uveal tract during anterior uveitis. In this report, we used primary cultures of human iris stroma (HIS) cells derived from human eye donors to investigate HCMV entry. The following lines of evidence suggested the role of 3-O-sulfated heparan sulfate (3-OS HS) during HCMV-mediated entry and cell-to-cell fusion in HIS cells. First, 3-O-sulfotransferase-3 (3-OST-3) expression in HIS cells promoted HCMV internalization, while pretreatment of HIS cells with heparinase enzyme or with anti-3-OS HS (G2) peptide significantly reduced the HCMV-mediated formation of plaques/foci. Second, coculture of the HCMV-infected HIS cells with CHO-K1 cells expressing 3-OS HS significantly enhanced cell fusion. Finally, a similar trend of enhanced fusion was observed with cells expressing HCMV glycoproteins (gB, gO, and gH-gL) cocultured with 3-OS HS cells. Taken together, these results highlight the role of 3-OS HS during HCMV plaque formation and cell-to-cell fusion and identify a novel target for future therapeutic interventions.

Comprehensive analysis of herpes simplex virus 1 (HSV-1) entry mediated by zebrafish 3-O-Sulfotransferase isoforms: implications for the development of a zebrafish model of HSV-1 infection.

Binding of herpes simplex virus 1 (HSV-1) envelope glycoprotein D (gD) to the receptor 3-O-sulfated heparan sulfate (3-OS HS) mediates viral entry. 3-O-Sulfation of HS is catalyzed by the 3-O-sulfotransferase (3-OST) enzyme. Multiple isoforms of 3-OST are differentially expressed in tissues of zebrafish (ZF) embryos. Here, we performed a comprehensive analysis of the role of ZF 3-OST isoforms (3-OST-1, 3-OST-5, 3-OST-6, and 3-OST-7) in HSV-1 entry. We found that a group of 3-OST gene family isoforms (3-OST-2, -3, -4, and -6) with conserved catalytic and substrate-binding residues of the enzyme mediates HSV-1 entry and spread, while the other group (3-OST-1, -5, and -7) lacks these properties. These results demonstrate that HSV-1 entry can be recapitulated by certain ZF 3-OST enzymes, a significant step toward the establishment of a ZF model of HSV-1 infection and tissue-specific tropism.

Zebrafish 3-O-sulfotransferase-4 generated heparan sulfate mediates HSV-1 entry and spread.

Rare modification of heparan sulfate (HS) by glucosaminyl 3-O sulfotransferase (3-OST) isoforms generates an entry receptor for herpes simplex virus type-1 (HSV-1). In the zebrafish (ZF) model multiple 3-OST isoforms are differentially expressed. One such isoform is 3-OST-4 which is widely expressed in the central nervous system of ZF. In this report we characterize the role of ZF encoded 3-OST-4 isoform for HSV-1 entry. Expression of ZF 3-OST-4 into resistant Chinese hamster ovary (CHO-K1) cells promoted susceptibility to HSV-1 infection. This entry was 3-O sulfated HS (3-OS HS) dependent as pre-treatment of ZF 3-OST-4 cells with enzyme HS lyases (heparinase II/III) significantly reduced HSV-1 entry. Interestingly, co-expression of ZF 3-OST-4 along with ZF 3-OST-2 which is also expressed in brain rendered cells more susceptible to HSV-1 than 3-OST-4 alone. The role of ZF-3-OST-4 in the spread of HSV-1 was also evaluated as CHO-K1 cells that expressed HSV-1 glycoproteins fused with ZF 3-OST-4 expressing effector CHO-K1 cells. Finally, adding further evidence ZF 3-OST-4 mediated HSV-1 entry was inhibited by anti-3O HS G2 peptide. Taken together our results demonstrate a role for ZF 3-OST-4 in HSV-1 pathogenesis and support the use of ZF as a model to study it.

Susceptibility of human iris stromal cells to herpes simplex virus 1 entry.

Ocular herpes simplex virus 1 (HSV-1) infection can lead to multiple complications, including iritis, an inflammation of the iris. Here, we use human iris stroma cells as a novel in vitro model to demonstrate HSV-1 entry and the inflammatory mediators that can damage the iris. The upregulated cytokines observed in this study provide a new understanding of the intrinsic immune mechanisms that can contribute to the onset of iritis.

Contortrostatin, a homodimeric disintegrin isolated from snake venom inhibits herpes simplex virus entry and cell fusion.

BACKGROUND: Herpes simplex virus (HSV) causes significant health problems from periodical skin and corneal lesions to encephalitis. HSV entry provides a unique opportunity for therapeutic intervention. In this study, we evaluated contortrostatin (CN), an Arg-Gly-Asp motif containing disintegrin isolated from snake venom, as a novel therapeutic agent with ability to block HSV entry and related membrane fusion. METHODS: In vitro efficacy of CN against HSV was determined using an HSV type-1 (HSV-1) entry assay based on the measurement of ß-galactosidase reporter activity originating from the genome of a recombinant strain of HSV-1(KOS) gL86. HSV-1 glycoprotein-mediated cell-to-cell fusion was used to study the effect of CN on polykaryocyte formation. Primary as well as transformed cell lines were used for this study. RESULTS: Pre-treatment of Chinese hamster ovary (CHO-K1) cells expressing HSV-1 glycoprotein D receptors and primary cultures of human corneal fibroblasts (CF) with CN resulted in the inhibition of entry, cell-to-cell fusion, and polykaryocyte formation. Interestingly, a more pronounced anti-HSV-1 effect was observed in naturally susceptible CF than CHO-K1 cells. CONCLUSIONS: CN, a novel venom-derived peptide, exhibits the ability to block two key steps, entry and cell-to-cell fusion, in HSV infection. Showing strong promise for development as an anti-HSV agent, it also demonstrates better prophylactic efficacy in primary cells.

Role of heparan sulfate in sexually transmitted infections.

Cell surface heparan sulfate (HS), a polysaccharide composed of alternating uronic acid and glucosamine residues, represents a common link that many sexually transmitted infections (STIs) require for infection. Variable modifications within the monomeric units of HS chains together with their unique structural conformations generate heterogeneity, which expands the ability of HS to bind a diverse array of host and microbial proteins. Recent advances made in the field of glycobiology have critically enhanced our understanding of HS and its interactions with microbes and their significance in important human diseases. The role of HS has been elaborated for several STIs to include those caused by herpes simplex virus, human immunodeficiency virus, human papillomavirus, and Chlamydia. In addition, gonorrhea, syphilis, and yeast infections are also dependent on the presence of HS on human target cells. Critical steps such as pathogen adhesion or binding to host cells followed by internalization to enhance intracellular survival and possible spread to other cells are mediated by HS. In addition, HS guided cell signaling plays a role in the development of angiogenesis and inflammation associated with many STIs. Past and ongoing investigations are providing new push for the development of HS-mimetics and analogs as novel prevention strategies against many different STIs. This review article summarizes the significance of HS in STIs and describes how emerging new products that target HS can be used to control the spread of STIs.

Amyloid-ß42 alters apolipoprotein E solubility in brains of mice with five familial AD mutations.

Amyloid plaques composed of the 42 amino acid form of amyloid-ß peptide (Aß42) are a pathological hallmark of Alzheimer's disease (AD), but soluble and intraneuronal Aß42 are the more proximal causes of synaptic dysfunction and neurotoxicity. Apolipoprotein E (apoE) modulates this disease process, as inheritance of the ɛ4 allele of the apoE gene is the primary genetic risk factor for AD. To address the solubility of Aß42 and apoE, the 5xFAD-specific extraction profile for Aß42 was optimized, a protein extraction protocol was optimized in the presence of minimal to extensive Aß42 pathology. Sequential extractions with TBS, TBS+Triton X-100 (TBSX), and guanidine-HCl (GuHCl) or formic acid (FA) were used with tissue from young and old wild type or mice expressing 5 familial AD mutations (5xFAD), in disease-susceptible or -resistant brain regions. In older 5xFAD mice, the extraction of insoluble Aß42 and m-apoE protein was increased with FA compared to GuHCl. The 5 FAD mutations significantly increase production of Aß42, recapitulating AD-like pathology at a greatly accelerated rate. Consistent protein extraction and the specificity of extractions for soluble or membrane-associated proteins were demonstrated. Age-dependent increases in Aß42 were observed in all extraction fractions, particularly in the cortex and hippocampus. In both young and old 5xFAD mice, Aß42 is TBS- or GuHCl-soluble. While in WT mice m-apoE is TBSX-soluble, in 5xFAD mice m-apoE is TBS- or GuHCl-soluble. Thus, the 5xFAD-specific extraction profile of Aß42 paralleled that of m-apoE. As now characterized, this method identifies the extraction profile for disease relevant apoE and Aß in the brain, both normal or modified due to neuropathological processes.

Phosphorylation of the translation initiation factor eIF2alpha increases BACE1 levels and promotes amyloidogenesis.

beta-site APP cleaving enzyme-1 (BACE1), the rate-limiting enzyme for beta-amyloid (Abeta) production, is elevated in Alzheimer's disease (AD). Here, we show that energy deprivation induces phosphorylation of the translation initiation factor eIF2alpha (eIF2alpha-P), which increases the translation of BACE1. Salubrinal, an inhibitor of eIF2alpha-P phosphatase PP1c, directly increases BACE1 and elevates Abeta production in primary neurons. Preventing eIF2alpha phosphorylation by transfection with constitutively active PP1c regulatory subunit, dominant-negative eIF2alpha kinase PERK, or PERK inhibitor P58(IPK) blocks the energy-deprivation-induced BACE1 increase. Furthermore, chronic treatment of aged Tg2576 mice with energy inhibitors increases levels of eIF2alpha-P, BACE1, Abeta, and amyloid plaques. Importantly, eIF2alpha-P and BACE1 are elevated in aggressive plaque-forming 5XFAD transgenic mice, and BACE1, eIF2alpha-P, and amyloid load are correlated in humans with AD. These results strongly suggest that eIF2alpha phosphorylation increases BACE1 levels and causes Abeta overproduction, which could be an early, initiating molecular mechanism in sporadic AD.

Beta-site amyloid precursor protein cleaving enzyme 1 levels become elevated in neurons around amyloid plaques: implications for Alzheimer's disease pathogenesis.

Beta-site amyloid precursor protein cleaving enzyme 1 (BACE1) (beta-secretase) initiates generation of beta-amyloid (Abeta), which plays an early role in Alzheimer's disease (AD). BACE1 levels are increased in postmortem AD brain, suggesting BACE1 elevation promotes Abeta production and AD. Alternatively, the BACE1 increase may be an epiphenomenon of late-stage AD. To distinguish between these possibilities, we analyzed BACE1 elevation using a highly specific BACE1 antibody, BACE-Cat1, made in BACE1-/- mice, which mount a robust anti-BACE1 immune response. Previous BACE1 immunohistochemical studies lack consistent results because typical BACE1 antibodies produce nonspecific background, but BACE-Cat1 immunolabels BACE1 only. BACE1 elevation was recapitulated in two amyloid precursor protein (APP) transgenic mouse lines. 5XFAD mice form amyloid plaques at young ages and exhibit neuron loss. In contrast, Tg2576 form plaques at a more advanced age and do not show cell death. These two mouse lines allow differentiation between early Abeta-induced events and late phenomena related to neuron death. BACE1 levels became elevated in parallel with amyloid burden in each APP transgenic, starting early in 5XFAD and late in Tg2576. The increase in BACE1 protein occurred without any change in BACE1 mRNA level, indicating a posttranscriptional mechanism. In APP transgenic and AD brains, high BACE1 levels were observed in an annulus around Abeta42-positive plaque cores and colocalized with neuronal proteins. These results demonstrate that amyloid plaques induce BACE1 in surrounding neurons at early stages of pathology before neuron death occurs. We conclude that BACE1 elevation is most likely triggered by the amyloid pathway and may drive a positive-feedback loop in AD.

Intraneuronal beta-amyloid aggregates, neurodegeneration, and neuron loss in transgenic mice with five familial Alzheimer's disease mutations: potential factors in amyloid plaque formation.

Mutations in the genes for amyloid precursor protein (APP) and presenilins (PS1, PS2) increase production of beta-amyloid 42 (Abeta42) and cause familial Alzheimer's disease (FAD). Transgenic mice that express FAD mutant APP and PS1 overproduce Abeta42 and exhibit amyloid plaque pathology similar to that found in AD, but most transgenic models develop plaques slowly. To accelerate plaque development and investigate the effects of very high cerebral Abeta42 levels, we generated APP/PS1 double transgenic mice that coexpress five FAD mutations (5XFAD mice) and additively increase Abeta42 production. 5XFAD mice generate Abeta42 almost exclusively and rapidly accumulate massive cerebral Abeta42 levels. Amyloid deposition (and gliosis) begins at 2 months and reaches a very large burden, especially in subiculum and deep cortical layers. Intraneuronal Abeta42 accumulates in 5XFAD brain starting at 1.5 months of age (before plaques form), is aggregated (as determined by thioflavin S staining), and occurs within neuron soma and neurites. Some amyloid deposits originate within morphologically abnormal neuron soma that contain intraneuronal Abeta. Synaptic markers synaptophysin, syntaxin, and postsynaptic density-95 decrease with age in 5XFAD brain, and large pyramidal neurons in cortical layer 5 and subiculum are lost. In addition, levels of the activation subunit of cyclin-dependent kinase 5, p25, are elevated significantly at 9 months in 5XFAD brain, although an upward trend is observed by 3 months of age, before significant neurodegeneration or neuron loss. Finally, 5XFAD mice have impaired memory in the Y-maze. Thus, 5XFAD mice rapidly recapitulate major features of AD amyloid pathology and may be useful models of intraneuronal Abeta42-induced neurodegeneration and amyloid plaque formation.