A fluorescence sedimentation assay for dsDNA antibodies.

The Farr assay is a radioimmunoassay (RIA) for dsDNA antibodies, based on antibody precipitation using ammonium sulphate and quantification using radio-labelled dsDNA. The RIA-Farr assay offers outstanding clinical specificity and sensitivity for systemic lupus erythematosus (SLE) compared to other assays but does also present some disadvantages as it utilizes radioactive-labelled dsDNA and requires high levels of technical expertise for safe handling. Here, a new precipitation assay, 'Fluoro-Farr' assay, is described. This assay maintains a high sensitivity and specificity for SLE but is based on precipitation with polyethylene glycol (PEG) and fluorescence of EvaGreen intercalated in dsDNA as detection principle. As dsDNA antibodies are quantified using fluorescence, the disadvantages of working with radioactivity are eliminated. The Fluoro-Farr assay was developed and validated, and the diagnostic efficiency of the assay was evaluated by testing 57 sera from SLE patients and 60 healthy controls. The Fluoro-Farr assay revealed a diagnostic sensitivity of 68% at a diagnostic specificity of 95% (ROC AUC 0.91). Furthermore, the new Fluoro-Farr assay was compared to the RIA-Farr assay, and showed a correlation of the outcomes from the two assays, but the Fluoro-Farr assay did not outperform the RIA-Farr assay due to its outstanding clinical diagnostic efficiency (ROC AUC 0.99). In conclusion, the Fluoro-Farr assay presents a viable alternative to the traditional RIA-Farr assay; especially in laboratories without facilities to perform assays with radioactivity-based read-out. As the RIA-Farr assay, the Fluoro-Farr assay has the advantage of being a precipitation assay allowing antibody:dsDNA interaction in solution using native dsDNA.

Chemical and thermal unfolding of calreticulin.

Calreticulin is a soluble endoplasmic reticulum chaperone, which has a relatively low melting point due to its remarkable structure with a relatively high content of flexible structural elements. Using far ultraviolet circular dichroism (CD) spectroscopy and a fluorescent dye binding thermal shift assay, we have investigated the chemical and thermal stability of calreticulin. When the chemical stability of calreticulin was assessed, a midpoint for calreticulin unfolding was calculated to 3.0M urea using CD data at 222 nm. Using the fluorescent dye binding thermal shift assay, calreticulin was found to obtain a molten structure in urea concentrations between 1-1.5 M urea, and to unfold/aggregate at high and low pH values. The results demonstrated that the fluorescent dye binding assay could measure the thermal stability of calreticulin in aqueous buffers with results comparable to melting points obtained by other techniques.

Epstein-Barr virus early antigen diffuse (EBV-EA/D)-directed immunoglobulin A antibodies in systemic lupus erythematosus patients.

OBJECTIVE: We sought to determine whether the serological response towards lytic cycle antigens of Epstein-Barr virus (EBV) is altered in systemic lupus erythematosus (SLE) patients. METHOD: We used enzyme-linked immunosorbent assay (ELISA) to investigate the prevalence of EBV early antigen diffuse (EBV-EA/D) antibodies in sera from 60 patients with SLE, 40 with scleroderma (SSc), 20 with primary Sjögren's syndrome (pSS), 20 with rheumatoid arthritis (RA), 20 healthy controls, and also subjects with various circulating autoantibodies. Samples from patients were obtained from clinics specialized within the diseases in Denmark and Sweden and samples from healthy controls were obtained from volunteers. RESULTS: A significant elevated titre of immunoglobulin (Ig)A, IgG, and IgM EBV-EA/D antibodies was found in SLE patients compared to healthy controls, a finding not explained by immunosuppressive treatment or disease activity. The largest difference was observed for IgA EBV-EA/D antibodies (p = 0.0013) with a seropositive rate of 58% in SLE patients and 0% in healthy controls. RA and SSc patients and individuals seropositive for anti-Scl-70 were additionally found to have elevated titres of IgA EBV-EA/D antibodies (40%, p = 0.014; 60%, p = 0.015; and 38.5%, p = 0.045, respectively). However, the titres were generally lower than in SLE patients. CONCLUSION: Our findings support an association between EBV and SLE. The elevated titre of EBV-EA/D-directed IgA antibodies found in SLE patients could suggest reactivation of EBV in epithelial cells or reinfection of epithelial cells after reactivation in B cells, indicating lack of control of the latent infection.

Interaction of the chaperone calreticulin with proteins and peptides of different structural classes.

The interaction of calreticulin with native and denatured forms and polypeptides in proteolytic digests of proteins representing structural classes of all-alpha-helix (hemoglobin, serum albumin), all-beta-sheet (IgG) and alpha-helix + beta-sheets (lysozyme, ovalbumin) was investigated. The binding of calreticulin to denatured proteins was found to depend on conformation and structural class of the protein. No interaction was observed with the native proteins, whereas binding was seen for the denatured proteins, the order of interaction being lysozyme = IgG > ovalbumin >> hemoglobin = serum albumin. Moreover, the interaction between calreticulin and the heat-denatured proteins depended on the temperature and time used for denaturation and the degree of proteolytic fragmentation. Calreticulin bound well to peptides in proteolytic digests from protease K or chymotrypsin treatment of lysozyme, IgG and ovalbumin but weakly or not at all to peptides in proteolytic digests of hemoglobin and serum albumin. Synthetic peptides from lysozyme and ovalbumin confirmed binding to hydrophobic peptides from these proteins. These results show that calreticulin has the ability to interact with denatured and fragmented forms of proteins with a preference for beta-strand structure and hydrophobicity.

Interaction of calreticulin with amyloid beta peptide 1-42.

The interaction of calreticulin with amyloid beta (Abeta) was investigated using solid phase and solution binding assays. Calreticulin bound Abeta 1-42 in a time and concentration dependent fashion. The binding was optimal at pH 5 and was stimulated by Ca2+ and inhibited by Zn2+ at pH 7. Interaction took place through the hydrophobic C-terminus of Abeta 1-42 and the polypeptide binding site of calreticulin. The results are discussed in the light of a reported role of calreticulin as a cell surface scavenger receptor.

Interaction of calreticulin with CD40 ligand, TRAIL and Fas ligand.

The molecular chaperone calreticulin has been shown to bind C1q and mannan-binding lectin (MBL), which are constituents of the innate immune defence system. C1q and MBL do not share a large sequence identity but have a similar overall molecular architecture: an N-terminal triple-helical collagen-like domain and a C-terminal globular domain with ligand-binding properties. C1q is a hetero-trimer, while MBL is a homo-trimer, but due to the presence of N-terminal cysteines they both form higher order oligomers of trimers, which are the mature functional molecules. The same molecular architecture is utilized by many other functionally diverse molecules and in this work the interaction of calreticulin with C1q and structurally similar molecules was investigated. In addition to C1q and MBL, CD40 ligand (CD40L), tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) and Fas ligand (FasL) were found to bind calreticulin strongly. A low level or no binding was observed for adiponectin, tumour necrosis factor-alpha (TNF-alpha), CD30L, surfactant protein-A and -D and collagen VIII. The interaction with calreticulin required a conformational change in CD40L, TRAIL and FasL and showed the same characteristics as calreticulin's interaction with C1q and MBL: a time-dependent saturable binding to immobilized protein, which was initially sensitive to salt but gradually developed into a salt-insensitive interaction. Thus, the interaction requires a structural change in the interaction partner and leads to a conformational change in calreticulin itself. The implications of these results are that calreticulin may function as a general response modifier for a whole group of immunologically important proteins.

Oral EBV and KSHV infection in HIV.

The gamma herpesviruses, Kaposi's-sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV), are tightly associated with the development of AIDS-associated oral disease and malignancy during immune suppression. The objective of this investigation was to characterize oral infection and pathogenesis in healthy and immune-suppressed individuals. To characterize oral EBV and KSHV infection, we examined throat washings and oral epithelial cells from HIV-positive and HIV-negative individuals. Quantitative/real-time polymerase-chain-reaction (PCR) assays, transmission electronmicroscopy, immunostaining, and sequence analysis were used to identify viral infection. Virus was isolated from throat-wash samples and was used to infect epithelial and lymphoid cell lines. We detected EBV and KSHV in the oral cavity in healthy and immune-suppressed individuals. Viral strain analysis of KSHV K1 in multiple clones from the oral cavities of healthy persons and immunosuppressed patients detected several strains previously detected in KS lesions, with minor strain variation within individuals. Immunoelectron microscopy for multiple viral antigens detected consistent expression of viral proteins and oral epithelial specimens. In oral epithelial cells infected with wild-type KSHV in vitro, the K8.1 glycoprotein associated with lytic KSHV infection was detected in both primary and telomerase immortalized oral epithelial cultures by 24 hours post-infection. Virions were detected, subsequent to infection, by scanning electron microscopy. Oral epithelial cells were also infected in vitro with wild-type EBV originating from throat washes. Analysis of these data suggests that, like EBV, KSHV infection is present in the oropharynx of healthy individuals, is transmissible in vitro, and may be transmitted by saliva.

Human immunodeficiency virus type 1 IIIB selected for replication in vivo exhibits increased envelope glycoproteins in virions without alteration in coreceptor usage: separation of in vivo replication from macrophage tropism.

Analysis of viral replication and pathogenicity after in vivo selection of human immunodeficiency virus type 1 (HIV-1) attenuated in vitro will help to define the functions involved in replication and pathogenesis in vivo. Using the SCID-hu Thy/Liv mouse and human fetal thymus organ culture as in vivo models, we previously defined HIV-1 env determinants (HXB2/LW) which were reverted for replication in vivo (L. Su et al., Virology 227:46-52, 1997). In this study, we examined the replication of four highly related HIV-1 clones directly derived from Lai/IIIB or after selection in vivo to investigate the envelope gp120 determinants associated with replication in macrophages and in the thymus models in vivo. The LW/C clone derived from the IIIB-infected laboratory worker and HXB2/LW both efficiently infected monocyte-derived macrophages (MDM) and the human thymus. Although the laboratory worker (LW) isolates showed altered tropism from IIIB, they still predominantly used CXCR4 as coreceptors for infecting peripheral blood mononuclear cells, macrophages, and the thymus. Interestingly, a single amino acid mutation in the V3 loop associated with resistance to neutralizing antibodies was also essential for the replication activity of the LW virus in the thymus models but not for its activity in infecting MDM. The LW virions were equally sensitive to a CXCR4 antagonist. We further demonstrated that the LW HIV-1 isolate selected in vivo produced more infectious viral particles that contained higher levels of the Env protein gp120. Thus, selection of the laboratory-attenuated Lai/IIIB isolate in vivo leads to altered tropism but not coreceptor usage of the virus. The acquired replication activity in vivo is correlated with an early A-to-T mutation in the V3 loop and increased virion association of HIV-1 Env gp120, but it is genetically separable from the acquired replication activity in macrophages.

Separation of human immunodeficiency virus type 1 replication from nef-mediated pathogenesis in the human thymus.

Human immunodeficiency virus type 1 (HIV-1) is frequently attenuated after long-term culture in vitro. The attenuation process probably involves mutations of functions required for replication and pathogenicity in vivo. Analysis of attenuated HIV-1 for replication and pathogenicity in vivo will help to define these functions. In this study, we examined the pathogenicity of an attenuated HIV-1 isolate in a laboratory worker accidentally exposed to a laboratory-adapted HIV-1 isolate. Using heterochimeric SCID-hu Thy/Liv mice as an in vivo model, we previously defined HIV-1 env determinants (HXB/LW) that reverted to replicate in vivo (L. Su, H. Kaneshima, M. L. Bonyhadi, R. Lee, J. Auten, A. Wolf, B. Du, L. Rabin, B. H. Hahn, E. Terwilliger, and J. M. McCune, Virology 227:46-52, 1997). Here we further demonstrate that HIV-1 replication in vivo can be separated from its pathogenic activity, in that the HXB/LW virus replicated to high levels in SCID-hu Thy/Liv mice, with no significant thymocyte depletion. Restoration of the nef gene in the recombinant HXB/LW genome restored its pathogenic activity, with no significant effect on HIV-1 replication in the thymus. Our results suggest that in vitro-attenuated HIV-1 lacks determinants for pathogenicity as well as for replication in vivo. Our data indicate that (i) the replication defect can be recovered in vivo by mutations in the env gene, without an associated pathogenic phenotype, and (ii) nef can function in the HXB/LW clone as a pathogenic factor that does not enhance HIV-1 replication in the thymus. Furthermore, the HXB/LW virus may be used to study mechanisms of HIV-1 nef-mediated pathogenesis in vivo.

Functional interaction between the cytoplasmic leucine-zipper domain of HIV-1 gp41 and p115-RhoGEF.

The long cytoplasmic tail of the human immunodeficiency virus (HIV)-1 transmembrane protein gp41 (gp41C) is implicated in the replication and cytopathicity of HIV-1 [1]. Little is known about the specific functions of gp41C, however. HIV-1 or simian immunodeficiency virus (SIV) mutants with defective gp41C have cell-type- or species-dependent phenotypes [2] [3] [4] [5] [6]. Thus, host factors are implicated in mediating the functions of gp41C. We report here that gp41C interacted with the carboxy-terminal regulatory domain of p115-RhoGEF [7], a specific guanine nucleotide exchange factor (GEF) and activator of the RhoA GTPase, which regulates actin stress fiber formation, activation of serum response factor (SRF) and cell proliferation [8] [9]. We demonstrate that gp41C inhibited p115-mediated actin stress fiber formation and activation of SRF. An amphipathic helix region with a leucine-zipper motif in gp41C is involved in its interaction with p115. Mutations in gp41C leading to loss of interaction with p115 impaired HIV-1 replication in human T cells. These findings suggest that an important function of gp41C is to modulate the activity of p115-RhoGEF and they thus reveal a new potential anti-HIV-1 target.

Induction of MHC class I expression on immature thymocytes in HIV-1-infected SCID-hu Thy/Liv mice: evidence of indirect mechanisms.

The SCID-hu Thy/Liv mouse and human fetal thymic organ culture (HF-TOC) models have been used to explore the pathophysiologic mechanisms of HIV-1 infection in the thymus. We report here that HIV-1 infection of the SCID-hu Thy/Liv mouse leads to the induction of MHC class I (MHCI) expression on CD4+CD8+ (DP) thymocytes, which normally express low levels of MHCI. Induction of MHCI on DP thymocytes in HIV-1-infected Thy/Liv organs precedes their depletion and correlates with the pathogenic activity of the HIV-1 isolates. Both MHCI protein and mRNA are induced in thymocytes from HIV-1-infected Thy/Liv organs, indicating induction of MHCI gene expression. Indirect mechanisms are involved, because only a fraction (<10%) of the DP thymocytes were directly infected by HIV-1, although the majority of DP thymocytes are induced to express high levels of MHCI. We further demonstrate that IL-10 is induced in HIV-1-infected thymus organs. Similar HIV-1-mediated induction of MHCI expression was observed in HF-TOC assays. Exogenous IL-10 in HF-TOC induces MHCI expression on DP thymocytes. Therefore, HIV-1 infection of the thymus organ leads to induction of MHCI expression on immature thymocytes via indirect mechanisms involving IL-10. Overexpression of MHCI on DP thymocytes can interfere with thymocyte maturation and may contribute to HIV-1-induced thymocyte depletion.

Varicella-zoster virus Fc receptor component gI is phosphorylated on its endodomain by a cyclin-dependent kinase.

Varicella-zoster virus (VZV) glycoprotein gI is a type 1 transmembrane glycoprotein which is one component of the heterodimeric gE:gI Fc receptor complex. Like VZV gE, VZV gI was phosphorylated in both VZV-infected cells and gI-transfected cells. Preliminary studies demonstrated that a serine 343-proline 344 sequence located within the gI cytoplasmic tail was the most likely phosphorylation site. To determine which protein kinase catalyzed the gI phosphorylation event, we constructed a fusion protein, consisting of glutathione-S-transferase (GST) and the gI cytoplasmic tail, called GST-gI-wt. When this fusion protein was used as a substrate for gI phosphorylation in vitro, the results demonstrated that GST-gI-wt fusion protein was phosphorylated by a representative cyclin-dependent kinase (CDK) called P-TEFb, a homologue of CDK1 (cdc2). When serine 343 within the serine-proline phosphorylation site was replaced with an alanine residue, the level of phosphorylation of the gI fusion protein was greatly reduced. Subsequent experiments with individually immunoprecipitated mammalian CDKs revealed that the VZV gI fusion protein was phosphorylated best by CDK1, to a lesser degree by CDK2, and not at all by CDK6. Transient-transfection assays carried out in the presence of the specific CDK inhibitor roscovitine strongly supported the prior results by demonstrating a marked decrease in gI phosphorylation while gI protein expression was unaffected. Finally, the possibility that VZV gI contained a CDK phosphorylation site in its endodomain was of further interest because its partner, gE, contains a casein kinase II phosphorylation site in its endodomain; prior studies have established that CDK1 can phosphorylate casein kinase II.

Epitope mapping and tagging by recombination PCR mutagenesis.

We describe a rapid PCR method that directly inserts an epitope tag into an open reading frame (ORF) to facilitate protein detection. This project was performed within a varicella-zoster virus (VZV) system. In earlier work, we produced a monoclonal antibody (MAb 3B3) to one VZV ORF called gE. MAb 3B3 bound to its epitope under extreme denaturing conditions. To further characterize the epitope, we devised a technique that identified the epitope by its insertion into another protein of interest. The 3B3 epitope was mapped to 11 residues (residues 151-161; QRQYGDVFKGD) in the gE ectodomain by using the technique of recombination PCR. At the same time, the 3B3 epitope was inserted in-frame into another VZV protein for which no MAb was available. The end result, VZV gL3B3.11, was a unique construct possessing a 33-bp insertion that expresses gL-3B3 protein recognized by the MAb 3B3. The 3B3 epitope was verified to be both highly functional and stable. An important advantage of this recombination PCR method of epitope mapping and tagging is that the epitope sequence can be inserted anywhere along the nucleotide sequence of an ORF, regardless of existing restriction sites.

Multiple regulatory effects of varicella-zoster virus (VZV) gL on trafficking patterns and fusogenic properties of VZV gH.

Varicella-zoster virus (VZV) is an extremely cell-associated alphaherpesvirus; VZV infection is spread almost exclusively via cell membrane fusion. The envelope glycoprotein H (gH) is highly conserved among the herpesviruses. A virus-encoded chaperone, glycoprotein L (gL), associates with gH, and the gH:gL complex is required for gH maturation and membrane expression. We recently demonstrated that in the VZV system, the gH:gL complex facilitated cell membrane fusion and extensive polykaryon formation in transfected cells (K. M. Duus, C. Hatfield, and C. Grose, Virology 210:429-440, 1995). To further define the functions of the unusual VZV gL chaperone protein, we have performed a series of mutagenesis experiments with both gH and gL and analyzed the mutants by laser scanning confocal microscopy in a transfection-based fusion assay. We established the fact that immature gH exited the endoplasmic reticulum (ER) when coexpressed with either gE or gI and appeared on the cell surface in a patch pattern. A similar effect was observed on the cell surface with gH with a cytoplasmic tail mutagenized to closely resemble the vaccinia virus hemagglutinin cytoplasmic tail. Site-directed mutagenesis of the five gL cysteine residues demonstrated that four of five cysteines participated in the gL chaperone function required for proper maturation of gH. On the other hand, the same gL mutants facilitated transport of immature gH to the cell surface, where patching occurred. Studies of gL processing demonstrated that maturation did not require transport beyond the medial-Golgi; furthermore, gL was not detected in the outer cell membrane, nor was it secreted into the medium. Colocalization studies with 3,3'-dihexyloxa-cabocyanine iodide and N-(e-7-nitrobenz-2-oxa-1,3-diazol-4-yl-aminocaproyl)-D-erythro-sphingosine confirmed that gL was found primarily in the ER and cis/medial-Golgi when expressed alone. When all of these data were considered, they suggested a posttranslational gH:gL regulation model whereby the gL chaperone modulated gH expression via retrograde flow from the Golgi to the ER. In this schema, mature gL returns to the ER, where it escorts immature gH from the ER to the Golgi; thereafter, mature gH is transported from the trans-Golgi to the outer cell membrane, where it acts as a major fusogen.

Cell surface expression and fusion by the varicella-zoster virus gH:gL glycoprotein complex: analysis by laser scanning confocal microscopy.

Varicella-zoster virus (VZV) open reading frames 37 and 60 encode the glycoproteins gH (gpIII) and gL (gpVI), respectively. The property of gH:gL complex formation is highly conserved among the herpesviruses, even though the VZV gL component diverges greatly from other herpesvirus gL homologs. VZV gL by itself was processed to a mature product within the Golgi. To evaluate the structure:function relationships for VZV gH:gL complex formation, the VZV gL product was modified by site-directed mutagenesis of three cysteine residues. When the transfection products were examined by laser scanning confocal microscopy, expression of the wild-type gH:gL complex was clearly visualized by a uniform distribution of gH molecules across the cell surface. In contrast, transfection with wild-type gH:mutant gL led to a marked change in the trafficking pattern; gH was not processed in the Golgi and not detected at the cell surface. Likewise, replacement of the gL cysteine residues interfered with the fusogenic properties of the gH:gL complex. Whereas coexpression of wild-type VZV gH:gL caused extensive cell-to-cell fusion with polykaryocytosis, no cell fusion occurred following transfection with gH:mutant gL. Whether another VZV glycoprotein could substitute for VZV gL was investigated within the same transfection system, with the discovery that either VZV gE (gpI) or VZV gI (gpIV) facilitated the cell surface expression of VZV gH. The gH:gE or gH:gI interaction led to a capping or patching phenomenon never seen on the surface of a cell expressing gH:gL complexes; furthermore, cell-to-cell fusion was not observed. The fact that VZV gL, unlike other herpesviral glycoproteins, lacked a traditional signal sequence was investigated further by computer-assisted BlockSearch sequence analysis. The BlockSearch program assigned VZV gL to a family of proteins which lack a typical endoplasmic reticulum signal sequence but possess instead an endoplasmic reticulum targeting sequence. Since the latter sequence is common to many chaperone proteins, VZV gL most likely behaves in a similar manner.

Blackpatch, a neural degeneration mutation that interacts with the Notch locus in Drosophila.

We have identified a gene in Drosophila melanogaster that is involved in the development of the adult eye and optic lobe of the brain and that interacts with facet alleles at the Notch locus. We have named this locus Blackpatch (Bpt). Mutant alleles of Bpt produce a variety of abnormal phenotypes in the presence of facet alleles. These phenotypes include neural degeneration in the eye and in the optic lobe of the adult brain that begins 60 hr after pupariation and produces a dark, necrotic eye spot in the adult eye. Other phenotypes include recessive embryonic lethality, pharate adult lethality, and premature adult death. We have isolated and characterized 10 Bpt alleles, all of which yield the neural eye/brain degeneration phenotype in individuals who are also homozygous or hemizygous for facet mutations. Only some of the facet alleles interact with Bpt. Bpt mutations also interact with the split mutation but do not interact with other types of Notch mutation. Somatic mosaic analysis and imaginal disc transplantation experiments suggest that the optic lobe of the brain may be the focus of Bpt action. We conclude that the Notch and Bpt genes have important functions during the interaction between the retina and the optic lobe of the brain.