An Ocular Protein Triad Can Classify Four Complex Retinal Diseases.

Retinal diseases generally are vision-threatening conditions that warrant appropriate clinical decision-making which currently solely dependents upon extensive clinical screening by specialized ophthalmologists. In the era where molecular assessment has improved dramatically, we aimed at the identification of biomarkers in 175 ocular fluids to classify four archetypical ocular conditions affecting the retina (age-related macular degeneration, idiopathic non-infectious uveitis, primary vitreoretinal lymphoma, and rhegmatogenous retinal detachment) with one single test. Unsupervised clustering of ocular proteins revealed a classification strikingly similar to the clinical phenotypes of each disease group studied. We developed and independently validated a parsimonious model based merely on three proteins; interleukin (IL)-10, IL-21, and angiotensin converting enzyme (ACE) that could correctly classify patients with an overall accuracy, sensitivity and specificity of respectively, 86.7%, 79.4% and 92.5%. Here, we provide proof-of-concept for molecular profiling as a diagnostic aid for ophthalmologists in the care for patients with retinal conditions.

Molecular and Serological Intraocular Fluid Analysis of Coxiella burnetii-seropositive Patients with Concurrent Idiopathic Uveitis.

PURPOSE: Previous studies have suggested a link between Q fever and uveitis. We determined whether Coxiella burnetii causes intraocular infection in C. burnetii-seropositive patients with idiopathic uveitis. METHODS: From a retrospective observational case series, paired aqueous humor and serum samples from 10 C. burnetii-seropositive patients with idiopathic uveitis were examined for intraocular antibody production by using the Goldmann-Witmer coefficient and by polymerase chain reaction (PCR). RESULTS: Although intraocular IgG against C. burnetii was detected, no intraocular antibody production was observed (low Goldmann Wittmer coefficients). All PCR results were negative. CONCLUSIONS: Uveitis due to an intraocular infection with C. burnetii is unlikely.

Viral causes of unexplained anterior uveitis in Thailand.

AIMS: To assess the possible role of virus infection in patients with unexplained anterior uveitis (AU). METHODS: Intraocular fluid and plasma samples of 30 HIV-negative AU patients who were unresponsive or poorly responsive to topical steroid therapy were analyzed for nucleic acid of cytomegalovirus (CMV), herpes simplex virus (HSV), and varicella zoster virus (VZV) by real-time polymerase chain reaction (PCR) and for intraocular antibodies against these viruses by Goldmann-Witmer coefficient (GWC) analysis. Of these 30 cases, 21 were tested for rubella virus by GWC analysis, 16 of which also had PCR assessment of aqueous for rubella virus. RESULTS: Viral uveitis determined by either real-time PCR and/or GWC was documented in 20 out of 30 patients (67%). Of 30 paired samples tested by both methods for HSV, CMV, and VZV, 15 showed positive results (CMV (10), HSV (4), and VZV (1)). Real-time PCR was positive in 8/15 (53%), whereas GWC was positive in 10/15 (67%). Out of 10 CMV-positive patients, four had endotheliitis, two had Posner-Schlossman syndrome, and one Fuchs heterochromic uveitis syndrome (FHUS). Five out of 21 (24%) samples tested by GWC for Rubella virus were positive, three of which exhibited clinical features of FHUS. CONCLUSIONS: Our results indicate that CMV is a major cause of AU in Thailand and show that FHUS can be caused by both CMV and Rubella virus.

[Relationship between rubella virus and Fuchs heterochromic uveitis; 2 patients]. / Samenhang tussen rubellavirus en fuchs-heterochromie-uveïtis; 2 patiënten.

Two otherwise healthy men, aged 26 and 29 years, were diagnosed with Fuchs heterochromic uveitis (FHU) on the basis of the presence of iris heterochromia or iris atrophy, stellate corneal precipitates, and/or cataract. Microbiological investigation of aqueous humour demonstrated intraocular antibody production against rubella virus, but not against Toxoplasma gondii, herpes simplex virus or varicella zoster virus. Microbial nucleic acid detection was negative for all pathogens. Some time later, both patients underwent cataract surgery, which improved their vision considerably. FHU is a chronic, generally unilateral iridocyclitis, accompanied by the above-mentioned ophthalmologic manifestations in the absence of systemic disease. Little is known about the pathogenesis ofFHU, but recent publications have provided evidence for the possible involvement of the rubella virus.

Luxury at a cost? Recombinant mouse hepatitis viruses expressing the accessory hemagglutinin esterase protein display reduced fitness in vitro.

Group 2 coronaviruses encode an accessory envelope glycoprotein species, the hemagglutinin esterase (HE), which possesses sialate-O-acetylesterase activity and which, presumably, promotes virus spread and entry in vivo by facilitating reversible virion attachment to O-acetylated sialic acids. While HE may provide a strong selective advantage during natural infection, many laboratory strains of mouse hepatitis virus (MHV) fail to produce the protein. Apparently, their HE genes were inactivated during cell culture adaptation. For this report, we have studied the molecular basis of this phenomenon. By using targeted RNA recombination, we generated isogenic recombinant MHVs which differ exclusively in their expression of HE and produce either the wild-type protein (HE+), an enzymatically inactive HE protein (HE0), or no HE at all. HE expression or the lack thereof did not lead to gross differences in in vitro growth properties. Yet the expression of HE was rapidly lost during serial cell culture passaging. Competition experiments with mixed infections revealed that this was not due to the enzymatic activity: MHVs expressing HE+ or HE0 propagated with equal efficiencies. During the propagation of recombinant MHV-HE+, two types of spontaneous mutants accumulated. One produced an anchorless HE, while the other had a Gly-to-Trp substitution at the predicted C-terminal residue of the HE signal peptide. Neither mutant incorporated HE into virion particles, suggesting that wild-type HE reduces the in vitro propagation efficiency, either at the assembly stage or at a postassembly level. Our findings demonstrate that the expression of "luxury" proteins may come at a fitness penalty. Apparently, under natural conditions the costs of maintaining HE are outweighed by the benefits.

Structure-function analysis of the feline herpesvirus virulence factors gE and gI.

The biosynthesis and function of the gE-gI complex of feline herpesvirus (FHV) was studied by heterologous expression of the cloned genes in the vaccinia virus-based vTF7-3 expression system and by analysis of FHV recombinants. This work has led to the identification of domains in gI involved in complex formation with gE, and to a model for the disulfide-bonded structure of gI. The effects of mutations in gI on gE-gI-dependent cell-to-cell transmission are discussed.

Complementation of a gl-deficient feline herpesvirus recombinant by allotopic expression of truncated gl derivatives.

The alphaherpesvirus glycoproteins gE and gI form a hetero-oligomeric complex involved in cell-to-cell transmission. The gI-deficient recombinant feline herpesvirus (FHV), FHVdeltagI-LZ, produces plaques that are only 15% the size of those of wild-type FHV. Here, we have complemented FHV(delta)gI-LZ allotopically by expressing intact gI and C-terminally truncated gI derivatives from the thymidine kinase locus. The effect on gE-gI-mediated cell-to-cell spread was assessed by plaque assay employing computer-assisted image analysis (software available at http://www.androclus.vet.uu.nl/spotter/spotter.htm+ ++). Allotopic complementation with intact gI fully restored plaque size. Deletion of the C-terminal 11 residues of gI did not affect cell-to-cell spread, whereas deletion of the complete cytoplasmic tail reduced plaque size by only 35%. Mutants expressing gI166, roughly corresponding to the N-terminal half of the ectodomain, displayed a small-plaque phenotype. Nevertheless, their plaques were reproducibly larger than those of matched gI-deficient controls, indicating that the gE-gI166 hetero-oligomer, though crippled, is still able to mediate cell-to-cell spread. Our data demonstrate that plaque analysis provides a reliable and convenient tool to measure and quantitate gE-gI function in vitro.

The disulfide-bonded structure of feline herpesvirus glycoprotein I.

Alphaherpesvirus glycoproteins E and I (gE and gI, respectively) assemble into a hetero-oligomeric complex which promotes cell-to-cell transmission, a determining factor of virulence. Focusing on gI of feline herpesvirus (FHV), we examined the role of disulfide bonds during its biosynthesis, its interaction with gE, and gE-gI-mediated spread of the infection in vitro. The protein's disulfide linkage pattern was determined by single and pairwise substitutions for the four conserved cysteine residues in the ectodomain. The resulting mutants were coexpressed with gE in the vaccinia virus-based vTF7-3 system, and the formation and endoplasmic reticulum (ER)-to-Golgi transport of the hetero-oligomeric complex were monitored. The results were corroborated biochemically by performing an endoproteinase Lys-C digestion of a [35S]Cys-labeled secretory recombinant form of gI followed by tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the peptides under reducing and nonreducing conditions. We found that (i) gI derivatives lacking Cys79 (C1) and/or Cys223 (C4) still assemble with gE into transport-competent complexes, (ii) mutant proteins lacking Cys91 (C2) and/or Cys102 (C3) bind to gE but are retained in the ER, (iii) radiolabeled endoproteinase Lys-C-generated peptide species containing C1 and C4 are linked through disulfide bonds, and (iv) peptides containing both C2 and C3 are not disulfide linked to any other peptide. From these findings emerges a model in which C1 and C4 as well as C2 and C3 form intramolecular disulfide bridges. Since the cysteines in the ectodomain have been conserved during alphaherpesvirus divergence, we postulate that the model applies for all gI proteins. Analysis of an FHV recombinant with a C1-->S substitution confirmed that the C1-C4 disulfide bond is not essential for the formation of a transport-competent gE-gI complex. The mutation affected the posttranslational modification of gI and caused a slight cold-sensitivity defect in the assembly or the intracellular transport of the gE-gI complex but did not affect plaque size. Thus, C1 and the C1-C4 bond are not essential for gE-gI-mediated cell-to-cell spread, at least not in vitro.

Structure-function analysis of the gE-gI complex of feline herpesvirus: mapping of gI domains required for gE-gI interaction, intracellular transport, and cell-to-cell spread.

Alphaherpesvirus glycoproteins gE and gI form a noncovalently associated hetero-oligomeric complex, which is involved in cell-to-cell spread. In the absence of gI, feline herpesvirus (FHV) gE is transport incompetent and fully retained in the endoplasmic reticulum. Here, we assess the effect of progressive C-terminal truncations of FHV gI on the biosynthesis, intracellular transport, and function of the gE-gI complex. The truncated gI proteins were coexpressed with gE in the vaccinia virus-based vTF7-3 expression system. The results were corroborated and extended by studying FHV recombinants expressing truncated gI derivatives. The following conclusions can be drawn. (i) Deletion of the cytoplasmic tail, the transmembrane region plus the C-terminal half of the ectodomain of gI, does not affect intracellular transport of gE. Apparently, the N-terminal 166 residues of gI constitute a domain involved in gE-gI interaction. (ii) A region mediating stable association with gE is located within the N-terminal 93 residues of gI. (iii) The cytoplasmic domain of gI is not essential for gE-gI-mediated cell-to-cell transmission of FHV, as judged from plaque morphology. Deletion of the cytoplasmic tail of gI reduced plaque size by only 35%. (iv) Recombinants expressing the N-terminal 166 residues of gI display a small-plaque phenotype but produce larger plaques than recombinants with a disrupted gI gene. Thus, a complex consisting of gE and the N-terminal half of the gI ectodomain may retain residual biological activity. The implications of these findings for gE-gI interaction and function are discussed.

Biosynthesis of glycoproteins E and I of feline herpesvirus: gE-gI interaction is required for intracellular transport.

The biosynthesis of glycoproteins E and I of feline herpesvirus was studied by using the vaccinia virus vTF7-3 expression system. gE and gI were synthesized as N-glycosylated, endoglycosidase H (EndoH)-sensitive precursors with Mrs of 83,000 and 67,000, respectively. When coexpressed, gE and gI formed sodium dodecyl sulfate-sensitive hetero-oligomeric complexes that were readily transported from the endoplasmic reticulum (ER). Concomitantly, the glycoproteins acquired extensive posttranslational modifications, including O glycosylation, leading to an increase in their apparent molecular weights to 95,000 and 80,000 to 100,000 for gE and gI, respectively. In the absence of gE, most gI remained EndoH sensitive. Only a minor population became EndoH resistant, but these molecules were processed aberrantly as indicated by their Mrs (100,000 to 120,000). By immunofluorescence microscopy, gI was detected primarily in the ER but also at the plasma membrane. gE, when expressed by itself, remained EndoH sensitive and was found only in the ER and the nuclear envelope. These results were corroborated by studying the biosynthesis of gE in feline herpesvirus (FHV)-infected cells. In cells infected with wild-type FHV, gE acquired the same co- and posttranslational modifications as during vTF7-3-driven expression. However, an FHV mutant lacking gI failed to produce mature gE. We conclude that gE is retained in the ER, presumably by associating with molecular chaperones, and becomes transport competent only when in a complex with gI.