Syncytium-Inhibiting Monoclonal Antibodies Produced against Human T-Cell Lymphotropic Virus Type 1-Infected Cells Recognize Class II Major Histocompatibility Complex Molecules and Block by Protein Crowding

Four new monoclonal antibodies (MAbs) that inhibit human T-cell lymphotropic virus type 1 (HTLV-1)-induced syncytium formation were produced by immunizing BALB/c mice with HTLV-1-infected MT2 cells. Immunoprecipitation studies and binding assays of transfected mouse cells showed that these MAbs recognize class II major histocompatibility complex (MHC) molecules. Previously produced anti-class II MHC antibodies also blocked HTLV-1-induced cell fusion. Coimmunoprecipitation and competitive MAb binding studies indicated that class II MHC molecules and HTLV-1 envelope glycoproteins are not associated in infected cells. Anti-MHC antibodies had no effect on human immunodeficiency virus type 1 (HIV-1) syncytium formation by cells coinfected with HIV-1 and HTLV-1, ruling out a generalized disruption of cell membrane function by the antibodies. High expression of MHC molecules suggested that steric effects of bound anti-MHC antibodies might explain their inhibition of HTLV-1 fusion. An anti-class I MHC antibody and a polyclonal antibody consisting of several nonblocking MAbs against other molecules bound to MT2 cells at levels similar to those of class II MHC antibodies, and they also blocked HTLV-1 syncytium formation. Dose-response experiments showed that inhibition of HTLV-1 syncytium formation correlated with levels of antibody bound to the surface of infected cells. The results show that HTLV-1 syncytium formation can be blocked by protein crowding or steric effects caused by large numbers of immunoglobulin molecules bound to the surface of infected cells and have implications for the structure of the cellular HTLV-1 receptor(s).Human T-cell lymphotropic virus type 1 (HTLV-1) is a type C retrovirus and the etiologic agent of adult T-cell leukemia (43, 56, 59) and HTLV-1-associated myelopathy or tropical spastic paraparesis (15, 17, 49, 61). Although HTLV-1 shows tropism primarily for T cells, it can infect a variety of cell types including cells from some nonhuman species (6, 9, 27, 46, 48, 60, 62). Infection by free HTLV-1 tends to be highly inefficient, and the virus appears to be transmitted primarily by the cell-to-cell route (37). The HTLV-1 envelope glycoprotein is synthesized as a 61-kDa precursor which is cleaved into surface (gp46) and transmembrane (gp21) proteins (40, 57). gp46 is thought to serve as the virus attachment protein, as does gp120 for human immunodeficiency virus (HIV) (40, 57). Although previous reports have identified host cell molecules which might potentially mediate virus binding (9, 14), the cellular receptor for HTLV-1 has not been definitively identified. A recent study in which affinity chromatography was carried out with a gp46 peptide has provided evidence that the heat shock protein HSC70 binds directly to gp46 and may serve as a virus receptor (47).gp21 contains an N-terminal hydrophobic fusion domain and likely serves as a fusion protein similar to HIV gp41 (12, 61). Like many other retroviruses, HTLV-1 can induce syncytium formation between infected cells and certain uninfected cell types (28, 39). However, there are no data to indicate that virus transmission or virus persistence in vivo depends on syncytium formation. It is thought that cell-cell fusion involves the same receptors and occurs in a manner similar to virus-cell fusion. For this reason, HTLV-1 syncytium assays have been used to screen for cell surface molecules that may serve as virus receptors (13, 14, 25, 29). Monoclonal antibodies (MAbs) against a number of membrane proteins including members of the tetraspanner family (30, 31) have been found to block syncytium formation. My colleagues and I recently reported that expression of the cell adhesion molecule vascular cell adhesion molecule 1 (VCAM-1) on uninfected cells can confer sensitivity to HTLV-1-mediated syncytium formation (25). In this previous study, we were not able to block HTLV-1 cell fusion with MAbs against the major VCAM-1 counterreceptor VLA-4 (25). Others have reported that MAbs to other adhesion molecules including intercellular adhesion molecule 3 (ICAM-3) also block HTLV-1 syncytium formation (29). We have demonstrated that adhesion molecules also facilitate HIV type 1 (HIV-1) infection and syncytium formation (16, 24). Thus, adhesion molecules may be important accessory molecules for retroviruses generally.Earlier studies on accessory molecules involved in HTLV-1 biology have been extended by immunizing mice with HTLV-1-infected cells and screening for MAbs that block VCAM-1-supported HTLV-1 syncytium formation. Four new MAbs that completely block HTLV-1-mediated cell fusion have been generated. The MAbs were all determined to be specific for class II major histocompatibility complex (MHC) molecules. These MAbs had no effect on syncytium formation induced by HIV-1. Studies on the mechanism by which the MAbs mediate this effect have revealed a novel mode of antibody blockade of virus-induced cell fusion: protein crowding at the infected cell surface resulting in steric blockade of critical receptor-ligand interactions.     

Identification of a Domain within the Human T-Cell Leukemia Virus Type 2 Envelope Required for Syncytium Induction and Replication

In vitro infection by human T-cell leukemia virus type 1 and 2 (HTLV-1 and HTLV-2) can result in syncytium formation, facilitating viral entry. Using cell lines that were susceptible to HTLV-2-mediated syncytium formation but were nonfusogenic with HTLV-1, we constructed chimeric envelopes between HTLV-1 and -2 and assayed for the ability to induce syncytia in BJAB cells and HeLa cells. We have identified a fusion domain composed of the first 64 amino acids at the amino terminus of the HTLV-2 transmembrane protein, p21, the retention of which was required for syncytium induction. Construction of replication-competent HTLV genomic clones allowed us to correlate the ability of HTLV-2 to induce syncytia with the ability to replicate in BJAB cells. Differences in the ability to induce syncytia were not due to differences in the levels of total or cell membrane-associated envelope or in the formation of multimers. Therefore, we have localized a fusion domain within the amino terminus of the transmembrane protein of HTLV-2 envelope that is necessary for syncytium induction and viral replication.Human T-cell leukemia virus types 1 and 2 (HTLV-1 and HTLV-2) are type C retroviruses that have been associated with a variety of human malignancies. HTLV-1 is the etiological agent of adult T-cell leukemia as well as a degenerative neurological disorder, HTLV-1-associated myelopathy/tropical spastic paraparesis (28, 40, 58, 60, 83). Recent reports have also implicated HTLV-1 infection with arthropathy (42, 65), polymyosis (23, 37), and uveitis (48, 49, 51). HTLV-2 has been associated with a rare form of atypical hairy cell leukemia (62, 63, 68) as well as some cases of neuropathy (33, 39). It is estimated that between 10 million and 20 million individuals worldwide are infected with HTLV, with an overall risk of 5% of disease progression in infected individuals (14). HTLV is endemic in southern Japan, the Caribbean Basin, and Central and South America. In the United States, recent reports have identified a high proportion of HTLV, especially HTLV-2, infection in intravenous-drug abusers (44, 61, 64).Cell-to-cell contact is considered critical for the in vivo and in vitro transmission of HTLV-1 and HTLV-2, as infection by cell-free HTLV virus is inefficient in vitro and in vivo. By analogy with other enveloped viruses, HTLV infection of susceptible cells is likely mediated by the envelope glycoprotein. Antibodies against HTLV envelope are protective against infection in vivo (71, 80), and multiple epitopes that elicit neutralizing antibodies have been identified throughout the protein (31, 34, 56). Initially synthesized as a precursor protein, gp61, HTLV envelope is subsequently modified by glycosylation and cleaved into two subunits, gp46 and p21. The external surface glycoprotein, gp46, is anchored to the cell surface by noncovalent association with the transmembrane envelope glycoprotein, p21. Interaction of envelope with the as yet unidentified cellular receptor leads to cell-to-cell fusion and can result in syncytium formation.We were interested in identifying the molecular determinants of HTLV involved in syncytium formation and viral entry. Our laboratory has several cell lines that are permissive to HTLV-2- but not HTLV-1-mediated cell fusion. Therefore, we constructed recombinants between the HTLV-1 and -2 envelope genes and assayed for the loss of syncytium induction in BJAB cells and HeLa cells. Loss of a 64-amino-acid (aa) domain located at the amino terminus of the HTLV-2 transmembrane protein, p21, correlated with a loss in the ability of the envelope chimera to induce cell fusion. When the chimeric envelopes were expressed in the context of replication-competent genomic clones, there was a good correlation between syncytium induction and the ability to replicate in permissive cells. Present within the identified fusion domain is a hydrophobic region and a heptad repeat resembling a leucine zipper. We examined the contribution of the fusion domain to the structural integrity of the HTLV-2 envelope by using a vaccinia virus expression system. None of the recombinants affected the synthesis, transport, or oligomer formation of the HTLV glycoprotein complex.     

The Wavelet-Based Cluster Analysis for Temporal Gene Expression Data

A variety of high-throughput methods have made it possible to generate detailed temporal expression data for a single gene or large numbers of genes. Common methods for analysis of these large data sets can be problematic. One challenge is the comparison of temporal expression data obtained from different growth conditions where the patterns of expression may be shifted in time. We propose the use of wavelet analysis to transform the data obtained under different growth conditions to permit comparison of expression patterns from experiments that have time shifts or delays. We demonstrate this approach using detailed temporal data for a single bacterial gene obtained under 72 different growth conditions. This general strategy can be applied in the analysis of data sets of thousands of genes under different conditions.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29]