The viral transmembrane superfamily: possible divergence of Arenavirus and Filovirus glycoproteins from a common RNA virus ancestor.

BACKGROUND: Recent studies of viral entry proteins from influenza, measles, human immunodeficiency virus, type 1 (HIV-1), and Ebola virus have shown, first with molecular modeling, and then X-ray crystallographic or other biophysical studies, that these disparate viruses share a coiled-coil type of entry protein. RESULTS: Structural models of the transmembrane glycoproteins (GP-2) of the Arenaviruses, lymphochoriomeningitis virus (LCMV) and Lassa fever virus, are presented, based on consistent structural propensities despite variation in the amino acid sequence. The principal features of the model, a hydrophobic amino terminus, and two antiparallel helices separated by a glycosylated, antigenic apex, are common to a number of otherwise disparate families of enveloped RNA viruses. Within the first amphipathic helix, demonstrable by circular dichroism of a peptide fragment, there is a highly conserved heptad repeat pattern proposed to mediate multimerization by coiled-coil interactions. The amino terminal 18 amino acids are 28% identical and 50% highly similar to the corresponding region of Ebola, a member of the Filovirus family. Within the second, charged helix just prior to membrane insertion there is also high similarity over the central 18 amino acids in corresponding regions of Lassa and Ebola, which may be further related to the similar region of HIV-1 defining a potent antiviral peptide analogue. CONCLUSIONS: These findings indicate a common pattern of structure and function among viral transmembrane fusion proteins from a number of virus families. Such a pattern may define a viral transmembrane superfamily that evolved from a common precursor eons ago.

Membrane interface-interacting sequences within the ectodomain of the human immunodeficiency virus type 1 envelope glycoprotein: putative role during viral fusion.

We have identified a region within the ectodomain of the fusogenic human immunodeficiency virus type 1 (HIV-1) gp41, different from the fusion peptide, that interacts strongly with membranes. This conserved sequence, which immediately precedes the transmembrane anchor, is not highly hydrophobic according to the Kyte-Doolittle hydropathy prediction algorithm, yet it shows a high tendency to partition into the membrane interface, as revealed by the Wimley-White interfacial hydrophobicity scale. We have investigated here the membrane effects induced by NH(2)-DKWASLWNWFNITNWLWYIK-CONH(2) (HIV(c)), the membrane interface-partitioning region at the C terminus of the gp41 ectodomain, in comparison to those caused by NH(2)-AVGIGALFLGFLGAAGSTMGARS-CONH(2) (HIV(n)), the fusion peptide at the N terminus of the subunit. Both HIV(c) and HIV(n) were seen to induce membrane fusion and permeabilization, although lower doses of HIV(c) were required for comparable effects to be detected. Experiments in which equimolar mixtures of HIV(c) and HIV(n) were used indicated that both peptides may act in a cooperative way. Peptide-membrane and peptide-peptide interactions underlying those effects were further confirmed by analyzing the changes in fluorescence of peptide Trp residues. Replacement of the first three Trp residues by Ala, known to render a defective gp41 phenotype unable to mediate both cell-cell fusion and virus entry, also abrogated the HIV(c) ability to induce membrane fusion or form complexes with HIV(n) but not its ability to associate with vesicles. Hydropathy analysis indicated that the presence of two membrane-partitioning stretches separated by a collapsible intervening sequence is a common structural motif among other viral envelope proteins. Moreover, sequences with membrane surface-residing residues preceding the transmembrane anchor appeared to be a common feature in viral fusion proteins of several virus families. According to our experimental results, such a feature might be related to their fusogenic function.

Viral induction, transmission and apoptosis among cells infected by a Human Intracisternal A-type retrovirus.

Sjogren's Syndrome, a systemic autoimmune disease, is characterized by lymphocytic infiltration of the salivary or lacrimal glands, producing xerostomia or xerophthalmia. Although definitive proof of viral etiology has not been established, a cell line containing viral particles termed Human Intracisternal A-type Particles (HIAP) resulted from co-culture with patient lip biopsies. We stimulated these chronically infected cells with phorbol myristate acetate (PMA) in an effort to enhance production of viral particles for further characterization. We report that the virus present in the HIAP cell line can be induced to become lytic when subjected to PMA and that there is a difference in the effects of PMA on H9 and HIAP cell groups, with apparent protection from apoptosis due to PMA being exerted by viral presence. Delayed apoptosis may prolong exposure of the foreign/self complex, thus enhancing an autoimmune response. Polyacrylamide gel electrophoresis (PAGE) revealed the presence of new peptides in pellets of supernatants of PMA-stimulated HIAP cells, with prominent bands at 55 and 43 kDa, and several fainter ones. HIAP infection was transferred by cell-free filtered supernatants from stimulated cells to H9 cells, which became identical to parent HIAP cells by PAGE and fluorescence activated cell sorter.

Reactivity of sera from systemic lupus erythematosus and Sjögren's syndrome patients with peptides derived from human immunodeficiency virus p24 capsid antigen.

We have previously demonstrated that about one-third of patients with either Sjögren's syndrome (SS) or systemic lupus erythematosus (SLE) react to human immunodeficiency virus (HIV) p24 core protein antigen without any evidence of exposure to, or infection with, HIV itself. Herein, we further characterize the specificity of this reaction using enzyme-linked immunosorbent assay to peptides representing fragments of p24. Characteristic epitope-specific profiles were seen for SS and SLE patients. SS patients had significantly increased responses to peptides F (p24 amino acids 69 to 86) and H (amino acids 101 to 111) and diminished reactivity to peptides A (amino acids 1 to 16) and P (amino acids 214 to 228). SLE patients had increased reactivity to peptides E (amino acids 61 to 76), H, and P. Utilization of peptide P hyporeactivity as the criterion to select for SS patients results in a screen that is moderately sensitive (64%) and specific (79.3%). Adding hyperreactivity to one other peptide (F or H) as an additional criterion yields an expected decrease in sensitivity (to 41%) while increasing specificity (to 93.1%). All sera-reactive peptides from regions of known structure of HIV p24 were located in the apex of the p24 molecule. Thus, the specificity of the peptide reactivities described here indicates a specific pattern of a nonrandom cross-reactivity between HIV type 1 p24 and autoimmune sera which may be partially syndrome specific. The future focus of our work will be to optimize assays of the peptide as diagnostic tools.

A general model for the surface glycoproteins of HIV and other retroviruses.

A hypothetical model of the surface (SU) glycoproteins of human immunodeficiency virus (HIV) and other retroviruses is proposed. The model is based on repetition of a limited number of sequence motifs conserved within the virus family; similarities in biological, immunological, or genetic properties; as well as the tendency for regions of dissimilar sequence to share protein structures predicted by computer algorithms. It is proposed that the protein consists of three structural and functional domains interspersed by relatively conserved interdomain regions. For each retrovirus, these amino-terminal, central, and carboxy-terminal domains may play different roles in binding, postbinding events, and the immune response to viral infection.

PCR amplification of HIV-1 proteinase sequences directly from lab isolates allows determination of five conserved domains.

HIV-1 replication requires limited proteolysis of gag and gag-pol encoded precursor proteins by a specific viral proteinase (PR). Sequences of 20 different HIV-1 strains were compared in order to determine regions of conservation and variability within the PR gene. Viral strains included: (a) five new ones derived from New Orleans patient isolates, (b) four established ones grown in our laboratory, (c) eight, whose sequences were published in the Los Alamos Data Base (1990), (d) one Ugandan, and (e) two Brazilian isolates. In the first two groups, HIV proviral DNA extracted from infected lymphocytes was grown in tissue culture and directly amplified by PCR using specific primers flanking the PR gene. Amplified DNA was directly sequenced using a modified di-deoxy sequencing procedure. Sequence data showed a 25% variation among the 20 different HIV strains studied at the amino acid level, including 8% nonconservative changes and 17% conservative changes. Moreover, five noncontiguous regions were able to be delineated in which the PR showed no amino acid changes. These areas included amino acids (I) 1-9 (amino terminal sequence); (II) 21-32 (sequence around the active site); (III) 47-56 (top of the flap); (IV) 78-88; and (V) 94-99 (carboxy terminal sequence). Our results are consistent with those obtained from X-ray crystallography studies as well as single site mutational analysis.

Intrahepatic assembly of very low density lipoproteins: immunologic characterization of apolipoprotein B in lipoproteins and hepatic membrane fractions and its intracellular distribution.

Monoclonal antibodies, prepared against rat apoB, were used to examine apoB structure in serum lipoproteins and characterize the forms and localization of apoB in liver membrane fractions and cultured hepatocytes. Of the several antibodies obtained, four, having separate epitopes, were characterized. Western blot analysis showed that three (DB11, F4, and LB14) antibodies recognized both apoBL and apoBS. One antibody (HB41) recognized only apoBL. This antibody showed unusual properties. Competition ELISA assays showed that the epitope recognized by HB41 was more effectively expressed on low density lipoproteins (LDL) compared to very low density lipoproteins (VLDL). In addition, treatment of lipoproteins with detergents and sulfhydryl reducing agents also increased the expression of the HB41 epitope. Since HB41 has been found to inhibit LDL binding to hepatocyte receptors, these data indicate that the HB41 epitope is located on the carboxy-terminal side of the apoBS junction (probably within the LDL receptor binding domain). Western blotting hepatic microsomal subfractions showed that in the rough and smooth microsomes, HB41 recognized only apoBL, while in the Golgi it recognized both apoBL and a protein having a molecular weight slightly smaller. In contrast, Western blotting with a polyclonal antibody known to recognize both apoBL and apoBS showed that, in rough and smooth microsomes, proteins in addition to apoBL and apoBS having molecular weights between 120,000 and 30,000 were recognized. These proteins, likely to be proteolytic fragments of apoB, were barely detectable in the Golgi. Additional biosynthetic studies show that the [35S]methionine-labeled proteins smaller than apoB were immunoprecipitated from the rough microsome subfraction. Pulse-chase experiments show that these are produced with the same kinetics as full-size apoBL and apoBS, indicating that they are not incomplete nascent chains. Finally, immunofluorescence microscopy was used to determine the localization of monoclonal epitopes. ApoB monoclonal antibodies that recognized exclusively apoBL (HB41) and apoBL and apoBS (DB11) produced an immunofluorescence pattern characteristic of the endoplasmic reticulum, but not the Golgi. These data suggest that, in cultured rat hepatocytes, the majority of both molecular weight forms of apoB are localized in the endoplasmic reticulum, the initial site of VLDL assembly. The additional finding that proteolytic fragments of apoB are enriched in the microsomal fraction suggests that if the proteolysis occurs during subcellular fractionation, immature apoB is susceptible to proteolysis.(ABSTRACT TRUNCATED AT 400 WORDS)

A general model for the transmembrane proteins of HIV and other retroviruses.

A hypothetical model of the transmembrane (TM) protein of human immunodeficiency virus (HIV) is proposed that is derived from the known structure of the influenza TM protein HA2. This model is consistent with computer algorithms of predicted protein structure and with known properties of TM proteins determined by sequence homology, site-directed mutations, peptide analogs, immunochemistry, or other biologic means. It is applicable to a wide variety of retroviral TM proteins differing widely in overall molecular weight.

Monoclonal antibodies to rat C apolipoproteins: production and characterization of a unique antibody whose binding to apoC-I is inhibited by nonionic detergents.

Four monoclonal antibodies to rat apo (apolipoproteins) C were produced. Three of the monoclonals reacted to apoC-I and one to apoC-III. The IgG monoclonals LRB 21 and LRB 45 recognized a spatially close together or identical apoC-I epitope. The monoclonal LRB 80, however, recognized an epitope that is close to, but distinct from, that recognized by LRB 45 and LRB 21. The antibody LRB 45 recognized an apoC-I epitope that is specific for rat apoC-I, and the antibody did not cross-react with dog or human lipoproteins. Rat apoC-I could be detected in all lipoprotein density fractions, but not in the d greater than 1.21 g/ml fraction. Freezing and thawing of serum did not alter the antibody antigen binding. However, lipolysis of whole serum resulted in a 30% increase in antigenic epitope expression. Antibody antigen reaction could be inhibited by subcritical micellar concentration of nonionic detergents. The inhibition was specific but could be partially reversed if lipid-containing serum was used as a dilution buffer. On feeding animals a diet of olive oil and cholesterol for 2 weeks, apoC-I levels decreased.

Identification of a mouse monoclonal antibody, LHLP-1, specific for human Lp(a).

Heretofore, immunologic reagents used to define and quantify human Lp(a) have been polyclonal in origin and therefore heterogeneous in antigenic specificity. We report here the isolation of a mouse monoclonal antibody, LHLP-1, monospecific for Lp(a). The antigen reactive with LHLP-1 was expressed in both lipoprotein Lp(a) as well as apolipoprotein Lp(a) delipidated by SDS treatment; however, disulfide reduction of apolipoprotein Lp(a) inhibited LHLP-1 reactivity. The antigen reactive with LHLP-1 on Lp(a), therefore, appears not to require lipid for expression of its conformationally dependent (disulfide-inhibitable) epitope. Antigen reactivity was virtually absent in the apoB and other proteins contained in very low density, low density, and high density lipoprotein particles. Immunologic quantification of Lp(a) in individual serum samples with a rabbit reference antiserum or LHLP-1 showed good correlation. We conclude that the monoclonal antibody LHLP-1 identifies an antigen unique to Lp(a) and that this antibody may therefore be useful in the further characterization and measurement of human Lp(a).

Production and characterization of a monoclonal antibody to dog hepatic lipase.

Partially purified dog hepatic lipase was used as antigen to produce monoclonal antibodies in mice. In addition to enzyme-linked immunosorbent assay (ELISA), a reliable and efficient procedure for screening antibodies reacting to hepatic lipase has been developed. A method to distinguish antibodies directing to active site or non-active site epitopes has also been described. We obtained three positive clones that survived after subcloning and expansion. All three monoclonal antibodies possess gamma one (gamma 1) heavy chains and kappa (kappa) light chains. Specificity of monoclonal antibody LDHL No. 537 to dog hepatic lipase was demonstrated by passing post-heparin plasma through its immunoaffinity column. Only dog hepatic lipase was removed by LDHL No. 537 from post-heparin plasma. The immunoaffinity chromatography also demonstrated the co-existence of three enzyme activities (mono- and triacylglycerol lipase and phospholipase A1) on the dog hepatic lipase molecule. The subunit weight of dog hepatic lipase has been estimated at 57500 +/- 600 (n=3) by using immunoaffinity chromatography and the combination of immunoprecipitation and autoradiography methods.

A quantitative assay for cytolysis induced by Newcastle disease virus.

We report here an assay for quantifying virus-induced lysis, in the absence of antibody and complement, produced within 2 hr after adsorption. This technique makes use of 51CrO4 release from cell monolayers pre-incubated overnight with the isotope. The release of 51Cr is specific for virus-induced lysis and is suppressible by 0.001 M-Ca2+. This assay clearly distinguishes between wild-type Chinese hamster ovary (CHO) cells, clone K and fusion-resistant mutant (CHO-15B), which was found to be resistant to virus-induced cytolysis. The stability of the association of isotope with monolayers of this cell type under the labelling conditions described makes this technique applicable to the study of the cytolytic effects of virus infection.

Insensitivity of a ricin-resistant mutant of Chinese hamster ovary cells to fusion induced by Newcastle disease virus.

The role of membrane components in the interaction of cells with Newcastle disease virus (NDV) was studied using a ricin-resistant mutant of Chinese hamster ovary cells (CHO-15B), in which there is a deficiency in distal saccharides at the plasma membrane. Compared to the parental wild type, the mutant was shown to be 4- to 10-fold less sensitive to either fusion from without or fusion from within induced by NDV. The mutant and wild type were nearly indistinguishable with respect to other interactions with NDV. Viral attachment was investigated with 125I-labeled NDV and found to be comparable in both lines. Functionally equivalent amounts of hemagglutinin were produced, as measured by the fraction of cells positive for hemadsorption, or by the number of erythrocytes adsorbed per cell. No significant differences in the morphogenesis or yield of progeny virus were seen. The ability of the mutant to produce a fusion factor was measured by transfer of infected cells to uninfected monolayers. Infected CHO-15B cells were capable of inducing fusion normally in the indicator wild-type monolayers, but were incapable of inducing fusion in mutant monolayers. These results suggest that the insensitivity of the CHO-15B mutants to fusion may be due to inhibition of an early virus-cell interaction subsequent to viral attachment, whereas other events in infection appear to be unaffected by the cell surface mutation.

Identification of receptors for animal viruses.

Present knowledge of cell surface receptors for animal viruses is reviewed. The methods used for enumeration and identification of receptors are critically examined with respect to particular advantages and disadvantages. Specific controls and alternative interpretations are suggested in connection with the reactions of lectins which block viral attachment to cells. Currently available information for each group of animal viruses is summarized in order to define the extent to which the corresponding receptors have been identified. It is concluded that the full range of virus-receptor interactions has not yet been explored even for those viruses of which there is the most detailed knowledge. For some groups, moreover, the receptor is totally uncharacterized. Six areas in which future investigative effort might be productive are identified, including the isolation of membrane components and the immunochemical definition of viral receptors.