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The human T-cell leukemia virus type 1 (HTLV-1) is a human retrovirus that is the causal agent of adult T-cell leukemia and HTLV-1-associated myelopathy or tropical spastic paraparesis. The principal target cell in vivo is the CD4 ϩ T lymphocyte, whereas the in vitro tropism spans a broad spectrum of permissive cell types. Despite a considerable effort by a number of laboratories, the cellular receptor(s) required for HTLV-1 entry into permissive cells remains elusive. Clues to the breadth of receptor expression have come from a number of studies, including analyses of syncytium formation between permissive and HTLV-infected or envelope glycoprotein (Env)-expressing cells (22,26,33) and infection of cells by 11,20,25,42,44) and by human immunodeficiency virus type 1 (HIV-1) pseudotyped with HTLV-1 Env (36, 37). Cell surface binding of HTLV-1 virions and more recently soluble, recombinant HTLV-1 surface envelope-Fc fusion proteins (16-18) has allowed a more precise analysis of cell types expressing putative receptors. It has been proposed that HTLV-1 and HTLV-2 share a receptor encoded by a gene on chromosome 17 (34, 35), although this result is now somewhat controversial (16, 36). The majority of studies aimed at characterizing receptors have been based on monoclonal antibody (MAb) inhibition of viral fusion in syncytium formation assays. In this way a number of candidate structures have been implicated in HTLV-1 infection, including vascular cell adhesion molecule and other adhesion molecules (5, 10, 38), the heat shock protein Hsc70 (31), and tetraspanin C33 (15). However, the binding pattern of soluble HTLV-1 Env and the in vitro tropism of HIV-1 pseudotyped with HTLV-1 Env are not compatible with a central role for these candidate receptors (16,17,37).Proteoglycans are a group of proteins that carry sulfated polysaccharide side chains, called glycosaminoglycans (GAGs), consisting of repeating disaccharide units (1). One of their many functions is to trap soluble mediators such as cytokines and chemokines onto the solid phase via electrostatic interactions, establishing a concentration gradient for migrating leukocytes (1). A variety of microorganisms bind the GAG chains of proteoglycans, and many appear to use this association as a way to attach to target cells. Thus herpes simplex virus types 1 and 2, HIV-1, vaccinia virus, pseudorabies virus, dengue virus, and others bind highly sulfated GAG forms termed heparan sulfate proteoglycans (HSPGs), whereas others use the less densely sulfated chondroitin or dermatan sulfate (39, 41).Here we show that HTLV-1 gp46 binds to HSPGs on cells permissive for HTLV-1 fusion and infection, that the expression of HSPGs on target cells enhances infection by HTLV-1 Env-pseudotyped viral particles, and that the soluble polyanion dextran sulfate (DexS) inhibits HTLV-1 syncytium formation and infection in HSPG-expressing cells. On this basis we propose that HTLV-1 may have evolved to use HSPGs as accessory receptors. MATERIALS AND METHODSRecombinant proteins, enzymes, antibodies...

The (RRE). The RRE adopts an extensive secondary structure and binds Rev both in vitro and in vivo (1,5,7,(11)(12)(13). It has been suggested that in the absence of Rev, HIV-1 splice donor and acceptor sequences function as important determinants of nuclear retention for viral mRNAs (14). It was proposed that binding of Rev to the RRE actively displaces splicing complexes from nascent transcripts, allowing export of the incompletely spliced mRNAs to the cytoplasm (14).Alternatively, binding of Rev to the RRE may direct viral transcripts through a nuclear export pathway, effectively removing these intron-containing mRNAs from the activity of the splicing machinery (8).While splice signals have been implicated as mediators of nuclear retention, nuclear sequestration and Rev responsiveness have also been observed for HIV-1-derived transcripts that lack functional splice sites (6-8, 15, 16); these observations challenge the notion that splice signals are the primary determinants of nuclear sequestration. Furthermore, a number of cis-acting sequences derived from various regions of HIV-1 have been shown to reduce expression of chimeric reporter genes (16)(17)(18)(19), suggesting that such sequences may be responsible for the nuclear sequestration of HIV-1 mRNAs. These elements designated as cis-acting repressive sequences (CRS), or instability sequences, have been mapped to the gag, pol, and env regions of the HIV-1 genome. However, chimeric transcripts containing these negative elements retain some splice signal information, albeit nonfunctional, and consequently a role for splice signals in the nuclear retention mechanism cannot be ruled out.Expression of the HIV-1 envelope glycoprotein in transfected Drosophila melanogaster cells is also dependent upon coexpression of Rev, and this pattern of regulation directly reflects the Rev-mediated trans-activation observed in mammalian cells (20). Here we employ stably transfected Drosophila cells as a model system in which to examine the cis-acting sequences responsible for the nuclear retention of HIV-1 envelope mRNAs. We demonstrate that HIV-1 splice donor and acceptor sites are not required for nuclear retention of envelope-derived mRNAs or for Rev responsiveness. Furthermore, we provide evidence that a region of env that overlaps the RRE acts as a primary determinant of nuclear retention and restricted cytoplasmic accumulation for envelope mRNAs.

Recent evidence indicates that primary clinical isolates of human immunodeficiency virus type 1 (HIV-1) require significantly more soluble CD4 (sCD4) to block infection than the prototypic laboratory strain HTLV-IIIB. The currently accepted explanation for these observations is that the envelope glycoproteins from primary clinical isolates possess lower affinities for CD4 than laboratory strains. This observation has far reaching implications for the clinical effectiveness of sCD4. To test whether the resistance of clinical isolates to sCD4 neutralization correlates with low-affinity binding to gpl20, we have compared gpl20 glycoproteins derived from the clinical isolates HIV-1 JR-CSF and JR-FL with those derived from the prototypic strain HIV-1 BH10 in quantitative sCD4 binding studies. Surprisingly, our results demonstrate that gpl20 derived from HIV-1 JR-CSF and JR-FL possess sCD4 binding affinities of equal or greater magnitude than gpl20 derived from HIV-i BH10. Thus primary clinical HIV-1 isolates can and do possess gpl20 with high affinity for CD4, and sensitivity to neutralization by sCD4 is dependent upon factors other than the intrinsic affinity of gpl20 for CD4.

The envelope glycoproteins of human T-cell leukemia virus type 1 (HTLV-1) perform functions that are crucial for virus entry into cells. The surface glycoprotein (SU) is responsible for viral recognition of, and binding to, target cells through its interaction with an unknown cell surface receptor. To facilitate molecular analysis of the receptor-binding properties of SU and to characterize the cellular receptor employed by HTLV-1, we have expressed a recombinant SU fused to the Fc domain of human immunoglobulin G. Here, we demonstrate that this novel SU-immunoadhesin retains both the biochemical properties of Fc and the receptorbinding specificity of the HTLV-1 SU. We use this SU-immunoadhesin to demonstrate, by direct cell surface binding assays, that the receptor used by HTLV-1 has been conserved through vertebrate evolution. Moreover, using murine-human somatic cell hybrids we provide data that do not support the previously assigned location for the HTLV-1 receptor on human chromosome 17. Most importantly, we show that many cell lines that are resistant to HTLV-1 envelope-mediated infection and syncytium formation express functional receptors that are recognized by the HTLV-1 SU. Based on our results, we suggest that for some HTLV-1-resistant cell lines the block to viral entry occurs at a late post-receptor-binding step of the entry process. Our findings will be of value in developing new strategies to identify the cellular receptor used by HTLV-1.Human T-cell leukemia virus (HTLV-1) is the etiologic agent of a rare but aggressive adult T-cell leukemia-lymphoma and a progressive demyelinating disease known as HTLV-1-associated myelopathy or tropical spastic paraparesis. HTLV-1 is endemic in Southern Japan, West Africa, Central and South America, and the Caribbean basin. Although uncommon in Europeans, HTLV-1 infections have been reported among indigenous and immigrant European populations and are prevalent among intravenous-drug users both in Europe and in the United States (reviewed in references 1, 4, 21, and 48). HTLV-1 primarily infects and immortalizes human CD4 ϩ T cells in vivo, but in in vitro coculture systems HTLV-1 infection, viral replication, and virally induced syncytium formation can be supported by a variety of primate and nonprimate cell types (1,4,30,45,46,49).The promiscuous pattern of tropism observed for HTLV-1 in vitro has generated considerable interest in the molecular events that promote viral entry into cells, and a number of informative studies have highlighted the crucial role played by the viral envelope glycoproteins in the entry process (31-36, 44). The envelope is expressed as a 68-kDa precursor that is posttranslationally cleaved by a cellular protease to yield the 46-kDa surface glycoprotein (gp46, SU) and a 21-kDa transmembrane glycoprotein (gp21, TM) (4,8,31,36). The gp46 surface glycoprotein remains associated with the transmembrane glycoprotein by noncovalent interactions following precursor cleavage, and this envelope complex is retained on the surfaces of virions or i...