Entry of alphaherpesviruses mediated by poliovirus receptor-related protein 1 and poliovirus receptor.

A human member of the immunoglobulin superfamily was shown to mediate entry of several alphaherpesviruses, including herpes simplex viruses (HSV) 1 and 2, porcine pseudorabies virus (PRV), and bovine herpesvirus 1 (BHV-1). This membrane glycoprotein is poliovirus receptor-related protein 1 (Prr1), designated here as HveC. Incubation of HSV-1 with a secreted form of HveC inhibited subsequent infection of a variety of cell lines, suggesting that HveC interacts directly with the virus. Poliovirus receptor (Pvr) itself mediated entry of PRV and BHV-1 but not of the HSV strains tested. HveC was expressed in human cells of epithelial and neuronal origin; it is the prime candidate for the coreceptor that allows both HSV-1 and HSV-2 to infect epithelial cells on mucosal surfaces and spread to cells of the nervous system.

Combining experimental information from crystal and solution studies: joint X-ray and NMR refinement.

Joint refinement of macromolecules against crystallographic and nuclear magnetic resonance (NMR) observations is presented as a way of combining experimental information from the two methods. The model of interleukin-1 beta derived by the joint x-ray and NMR refinement is shown to be consistent with the experimental observations of both methods and to have crystallographic R value and geometrical parameters that are of the same quality as or better than those of models obtained by conventional crystallographic studies. The few NMR observations that are violated by the model serve as an indicator for genuine differences between the crystal and solution structures. The joint x-ray-NMR refinement can resolve structural ambiguities encountered in studies of multidomain proteins, in which low- to medium-resolution diffraction data can be complemented by higher resolution NMR data obtained for the individual domains.

Use of a glucocorticoid-inducible promoter for expression of herpes simplex virus type 1 glycoprotein gC1, a cytotoxic protein in mammalian cells.

Abundant expression of herpes simplex virus type 1 glycoprotein gC (gC1) in transfected mammalian cells has not previously been achieved, possibly because gC1 protein is toxic to cells. To approach this problem, the gC1 coding sequence was placed under the control of the weak but inducible glucocorticoid-responsive promoter from the mouse mammary tumor virus (MMTV) long terminal repeat (LTR). As controls to evaluate for gC1 cytotoxicity, the MMTV LTR promoter was used to express glycoprotein gD1, and a strong, constitutive promoter from the Moloney murine sarcoma virus LTR was used to express gC1. L cells were transfected with these constructs, and a clone expressing gC1 from the inducible MMTV LTR promoter was analyzed. In the absence of glucocorticoid (dexamethasone) stimulation, only a low level of gC1 mRNA expression was detected; after overnight stimulation with dexamethasone, transcription increased approximately 200-fold. Abundant gC1 protein that was functionally active in that it bound complement component C3b, was produced. From passages 5 through 26 (70 cell population doublings), the gC1-producing clone became less responsive to overnight dexamethasone stimulation. The block to gC1 expression occurred at the level of transcription and was associated with hypermethylation of the MMTV LTR DNA. Treatment of the clone with 5-aza-2'-deoxycytidine partially reversed the block in gC1 protein production. Late-passage cells assumed a gC1-negative phenotype that appeared to offer a selective growth advantage, which suggested that gC1 was cytotoxic. Several findings support this view: (i) some cells expressing gC1 after overnight stimulation with dexamethasone assumed bizarre, syncytial shapes; (ii) continuous stimulation with dexamethasone for 5 weeks resulted in death of most cells; (iii) cells transfected with gC1 under the control of the strong Moloney murine sarcoma virus promoter assumed bizarre shapes, and stable gC1-expressing clones could not be established; and (iv) cells induced to express gD1 retained a normal appearance after overnight stimulation or 15 weeks of continuous stimulation with dexamethasone. The inducible MMTV LTR promoter is useful for expressing gC1 and may have applications for expressing other cytotoxic proteins.

Phosphocholine binding immunoglobulin Fab McPC603. An X-ray diffraction study at 2.7 A.

The crystal structure of the Fab of McPC603, a phosphocholine-binding mouse myeloma protein, has been refined at 2.7 A resolution by a combination of restrained least-squares refinement and molecular modeling. The overall structure remains as previously reported, with an elbow bend angle between the variable and constant modules of 133 degrees. Some adjustments have been made in the structure of the loops as a result of the refinement. The hypervariable loops are all visible in the electron density map with the exception of three residues in the first hypervariable loop of the light chain. A sulfate ion occupies the site of binding of the phosphate moiety of phosphocholine.

Structure and refinement at 1.8 A resolution of the aspartic proteinase from Rhizopus chinensis.

The structure of rhizopuspepsin (EC 3.4.23.6), the aspartic proteinase from Rhizopus chinensis, has been refined to a crystallographic R-factor of 0.143 at 1.8 A resolution. The positions of 2417 protein atoms have been determined with a root-mean-square (r.m.s.) error of 0.12 A. In the final model, the r.m.s. deviation from ideality for bond distances is 0.010 A, and for angle distances it is 0.034 A. During the course of the refinement, a calcium ion and 373 water molecules, of which 17 are internal, have been located. The active aspartate residues, Asp35 and Asp218, are involved in similar hydrogen-bonding interactions with neighboring residues and with several water molecules. One water molecule is located between the two carboxyl groups of the catalytic aspartate residues in a tightly hydrogen-bonded position. The refinement resulted in an unambiguous interpretation of the highly mobile "flap", a beta-hairpin loop region that projects over the binding pocket. Large solvent channels are formed when the molecules pack in the crystal, exposing the binding pocket and making it easily accessible. Intermolecular contacts involve mainly solvent molecules and a few protein atoms. The three-dimensional structure of rhizopuspepsin closely resembles other aspartic proteinase structures. A detailed comparison with the structure of penicillopepsin showed striking similarities as well as subtle differences in the active site geometry and molecular packing.

Antibody Fab assembly: the interface residues between CH1 and CL.

The effective assembly of an antibody molecule requires the proper association of the light and heavy chains, namely the tight, canonical association of VH with VL, and of CH1 with CL. In this paper the interaction of CH1 is examined by looking at the degree of conservation of residues in the interface between CH1 and CL, where CH1 can belong to any of the heavy chain classes, and CL can be either lambda or kappa. The three-dimensional structures of four antibody Fabs have been examined to see which are the significant interacting residues and to see whether they also correspond to the conserved residues in the different classes. It was found that there are a few hydrophobic residues buried in the interface which make numerous contacts with residues of the other chain and which remain invariant, or else are highly conserved. Around the periphery of the interface there are numerous interacting residues that have appreciable variability. Within the interface there is a cavity, the function of which may be to permit some changes in the central interface residues while still preserving the same relative orientation of CH1 and CL.

Localization of rabbit kappa light chain allotypic determinants.

The amino acid sequences of the constant regions of rabbit kappa light chains (C kappa) are remarkably divergent. The K1 allotypes differ at 47 of 106 positions; the K1 and K2 isotypes differ at three additional positions. Variability and structural dissimilarity plots reveal that most of these differences occur in clusters. Major hydrophilic areas are also found near some of these clusters. The structures of rabbit C kappa are modeled using the known alpha-carbon backbone structure of the Fab fragment of mouse myeloma protein McPC603. The effect of sequence variations upon the hypothetical three-dimensional structures was assessed and immunogenic determinants predicted and located. It was found that predicted determinants were external and located in or near loops. Two clusters of potentially interacting regions were predicted. Within each there could be several "topographical" and overlapping sets of epitopes that are recognized by different antibody-combining sites. One of the predicted immunogenic sites clearly interacts with the CH1 domain of the heavy chain. A heavy-chain dependent serological determinant has been correlated with amino acid differences in this region (kappa chain positions 121 and 124).

The three HveA receptor ligands, gD, LT-alpha and LIGHT bind to distinct sites on HveA.

The herpes virus entry mediator A (HveA), a member of the tumor necrosis factor receptor (TNFR) superfamily, interacts with three different protein ligands; lymphotoxin-alpha (LT-alpha) and LIGHT (LIGHT stands for lymphotoxin homolog, which exhibits inducible expression and competes with HSV glycoprotein D for HveA and is expressed on T-lymphocytes) from the host and the herpes simplex virus (HSV) surface glycoprotein gD. It has been reported that the gD binding site on HveA is located within the receptor's two N-terminal CRP domains, and that gD and LIGHT compete for their binding to HveA. However, whether these ligands interact with the same or different sites on the receptor is unclear. We analyzed and compared the sites of interaction between HveA and its TNF ligands, by using two recombinant forms of the receptor, comprising the full-receptor ectodomain (HveA (200t)) and its two first CRP domains (HveA (120t)), as well as several monoclonal antibodies recognizing HveA. Two HveA peptide ligands (BP-1 and BP-2) that differentially inhibit binding of soluble gD and LT-alpha to the receptor were also used to demonstrate that gD, LIGHT and LT-alpha bind to distinct sites on the receptor. Our results suggest that binding of a ligand to HveA may alter the conformation of this receptor, thereby affecting its interaction with its other ligands.

The structural and genetic basis for expression of normal and latent VHa allotypes of the rabbit.

The immunoglobulin heavy chain variable regions of the rabbit are unusual in having genetically controlled, serologically detectable alternative forms, the VHa allotypes, as well as minor VH allotypes of the x, y and w groups. New insights into the probable structural basis for the VHa allotypes have come from re-examination of earlier protein sequence data in the light of newly deduced protein sequences derived from sequencing cloned cDNAs and genomic DNAs encoding VH regions. Here we review this sequence information, and define the allotype-correlated differences at seven positions in framework region 1 and 10 positions in framework region 3 that may lead to the serologically detectable allotypic determinants (allotopes). Most alternative amino acids at allotype-correlated positions can be derived from each other by single-base changes. Thus somatic mutations and/or gene conversion-like events must be considered along with other serological and genetic explanations for various reported observations of the production of latent VHa allotypes. The proximity of rabbit VH genes (approximately 3 kb apart) might enhance the likelihood of conversion-like events in both germline and somatic cells.

"Painless" spinal epidural hematoma during anticoagulant therapy.

Severe back or radicular pain has previously been considered as the earliest and most prominent symptom in spinal epidural hematoma. In the patient described here, a "painless" spinal epidural hematoma developed during anticoagulant therapy. The absence of pain delayed recognition of the lesion and institution of therapy. The absence of pain is distinctively rare, but should not delay appropriate diagnostic procedures when other signs suggest the presence of a spinal epidural hematoma.

Prevention of herpes keratitis by monoclonal antibodies specific for discontinuous and continuous epitopes on glycoprotein D.

Seven monoclonal antibodies (mAb) specific for defined discontinuous and continuous epitopes on glycoprotein D of herpes simplex virus type 1 (HSV-1) were surveyed for their capacity to protect against virus-induced corneal disease in a murine ocular infection model. A known amount of purified mAb was transferred passively to BALB/c mice 24 hr after topical infection with HSV-1 on their scarified corneas. At high doses (50-136 micrograms), all seven mAbs protected against the development of persistent necrotizing stromal keratitis. Significant protection was also observed at low doses (20 micrograms) with two mAbs to discontinuous epitopes and two mAbs to continuous epitopes. Selected high-dose mAbs also were able to reduce the severity of blepharitis. These results indicated that at least seven different antigenic sites on glycoprotein D can serve as targets for effective antibody therapy in the murine model of HSV-1 ocular infection.

Functional T cell recognition of synthetic peptides corresponding to continuous antibody epitopes of herpes simplex virus type 1 glycoprotein D.

Four synthetic peptides which correspond to continuous antibody epitopes of herpes simplex virus (HSV) type 1 glycoprotein D (gD) within amino acid residues 1-23 (8-23), 268-287 and 340-356 were evaluated for in vitro stimulating activity on HSV-primed murine T lymphocytes. All peptides stimulated lymphoproliferative responses and interleukin 2 (IL2) production from draining lymph node (LN) cell populations taken 5 days after footpad immunization with live HSV. Similar responses were elicited from splenic memory T cells only if these T cells were restimulated with HSV in vitro and rested prior to peptide stimulation. Furthermore, peptide stimulated memory T cell populations released soluble factor(s) into the culture supernates which modulated the induced lymphoproliferative and cytotoxic T lymphocyte (CTL) activities of HSV-stimulated, HSV-immune splenocytes (indicator cultures). Memory T cell supernates suppressed lymphoproliferation of indicator cultures, while CTL activity of indicator cultures was either enhanced or suppressed, depending on the peptide and concentration. In contrast, supernates generated by peptide stimulation of draining LN cells had no effect on CTL activity of indicator cultures. However, the lymphoproliferative responses were augmented with three of the four peptides at the highest concentration of peptides tested. Our experiments indicate T helper (Th) and T suppressor (Ts) lymphocyte recognition of four synthetic peptides which encompass continuous antibody epitopes of HSV gD. Immunization with one of these peptides (1-23) induces virus neutralizing antibodies and protection against lethal viral challenge. Th lymphocyte recognition of this peptide in particular, together with its observed function in the induction of protection against HSV infection, indicates that this peptide is a promising candidate as a synthetic vaccine against HSV infection.

Thymosin-induced differentiation of murine thymocytes in allogeneic mixed lymphocyte cultures.

Calf thymosin is shown to enhance the one-way MLR of CBA thymocytes cultured with allogeneic mitomycin-C- treated C57BL/J6 spleen cells. Thymosin does not enhance the one-way MLR of CBA thymocytes cultured with syngeneic mitomucin-C-treated spleen cells. Based on this finding we present a relatively simple, rapid and quantitative in vitro microculture hioassay for inducers of T-cell differentiation and propose that thymosin treatment, when accompanied by antigen presentation, induces the two-step maturational sequence of pre-T yields T1 yields T2.

Expression and Purification of Secreted Forms of HSV Glycoproteins from Baculovirus-Infected Insect Cells.

Herpes simplex virus (HSV) remains a major human pathogen worldwide (25 causing cold sores, eye and genital infections, blindness, encephalitis, and neonatal infections. Most adults have antibodies against the oral form of the virus HSV-1 (9), and a significant number are infected with the genital form, HSV-2. Both serotypes establish lifelong latent infections and reactivate periodically to produce recurrent disease (25). After infection, virus-encoded glycoproteins are expressed on all cellular membranes and are major targets of the host's immune response. The virion envelope contains 10 glycoproteins that are important for infection and pathogenesis of HSV-1 and HSV-2. Because HSV contains so many glycoproteins, sorting out their functions in virus entry remains a difficult task. Our approach has focused on establishing structure-function relationships of the individual glycoproteins with particular emphasis on gC and gD. After many years of studying the properties of these proteins in HSV-infected and plasmid-transfected mammalian cells, we have now begun to overexpress the proteins using a baculovirus expression system.

Specific herpes simplex virus antigen in human gingiva.

Herpes simplex virus type 1 (HSV-1) specific antigens were found to be present in the sulcular epithelial cells of patients that were undergoing periodontal treatment. Four out of 14 cases were positive to specific HSV-1 antigen, as demonstrated by the indirect immunofluorescent assay. No nuclear staining was seen in any epithelial cells. These observations seem to indicate that the viral genome resides in the sulcular epithelial cells of the gingiva and is possibly localized in the stratum granulosum and spinosum.

A variant of toxic shock syndrome. Clinical, microbiologic, and autopsy findings in a fatal case.

A 16-year-old boy had toxic shock syndrome (TSS); Staphylococcus aureus bacteremia developed 11 hours prior to his death, which was three days after onset of the illness. The isoelectric focusing pattern of the staphylococcal isolate differed from both non-TSS and classic TSS S aureus isolates. Anatomic findings suggest three pathogenetic mechanisms: (1) immune complex-associated pulmonary microangiitis and vasculitis in the skin and skeletal muscle; (2) parenchymal cell "microvesicular" fatty metamorphosis in the liver, myocardium, renal tubules, and pancreas, and (3) pancarditis.

Regulation of immune balance by thymosin: potential role in the development of suppressor T-cells.

Studies in a variety of animal and human models indicate that thymosin plays a role in the differentiation of a number of T-cell subpopulations. The hypothesis presented is that a normal immune balance depends heavily upon the presence of thymosin-activated suppressor or regulator T-cells. A major thrust in our present research program is to determine whether or not the various disorders discussed here are causally related to abnormal thymosin production by the thymus gland. We are also assessing in animal models the potential value of thymsin in the treatment of specific autoimmune diseases. This information may yield new insights for the management of autoimmune type disorders such as SLE. Results from clinical trials to date suggest that thymosin will have a role in boosting the immune responses of patients with specific thymic malfunctions and may indeed exert an influence via the production of suppressor or regulator T-cells.