HTLV-1 structural proteins.

HTLV-1 structural proteins do not appear to ensure virus transmission as efficiently as most other retrovirus structural proteins do, whereas all other retroviruses can be transmitted via either free virions or cell-to-cell contacts, infection by HTLV-1 by free virions is very inefficient, and effective infection requires the presence of HTLV-1 infected cells. This characteristic feature of HTLV-1 provides a unique tool which can be used to analyse retrovirus cellular transmission in the absence of simultaneous cell-free infection. Here we summarise what is known about HTLV-1 structural proteins and identify the questions about these proteins which remain to be answered.

Identification of two intracellular mechanisms leading to reduced expression of oncoretrovirus envelope glycoproteins at the cell surface.

All retrovirus glycoproteins have a cytoplasmic domain that plays several roles in virus replication. We have determined whether and how the cytoplasmic domains of oncoretrovirus glycoproteins modulate their intracellular trafficking, by using chimeric proteins that combined the alpha-chain of the interleukin-2 receptor with the glycoprotein cytoplasmic domains of five oncoretroviruses: human T-cell leukemia virus type 1 (HTLV-1), Rous sarcoma virus (RSV), bovine leukemia virus (BLV), murine leukemia virus (MuLV), and Mason-Pfizer monkey virus (MPMV). All of these proteins were synthesized and matured in the same way as a control protein with no retrovirus cytoplasmic domain. However, the amounts of all chimeric proteins at the cell surface were smaller than that of the control protein. The protein appearing at and leaving the cell surface and endocytosis were measured in stable transfectants expressing the chimera. We identified two groups of proteins which followed distinct intracellular pathways. Group 1 included chimeric proteins that reached the cell surface normally but were rapidly endocytosed afterwards. This group included the chimeric proteins with HTLV-1, RSV, and BLV cytoplasmic domains. Group 2 included chimeric proteins that were not detected at the cell surface, despite normal intracellular concentrations, and were accumulated in the Golgi complex. This group included the chimeric proteins with MuLV and MPMV cytoplasmic domains. Finally, we verified that the MuLV envelope glycoproteins behaved in the same way as the corresponding chimeras. These results indicate that retroviruses have evolved two distinct mechanisms to ensure a similar biological feature: low concentrations of their glycoproteins at the cell surface.

Interaction of CD82 tetraspanin proteins with HTLV-1 envelope glycoproteins inhibits cell-to-cell fusion and virus transmission.

The entry of retroviruses into their target cell involves interactions between the virus envelope glycoproteins and their cellular receptors, as well as accessory ligand-receptor interactions involving adhesion molecules that can also participate in fusion. We have studied the contribution of CD82 proteins to the transmission of the human T-cell leukemia virus type 1 (HTLV-1), which is greatly dependent on cell-to-cell contacts. CD82 proteins belong to a class of cell surface molecules, the tetraspanins, that can act as molecular facilitators in cellular adhesion processes. The coexpression of CD82 proteins with HTLV-1 envelope glycoproteins resulted in marked inhibition of syncytium formation, whereas CD82 proteins had no effect on syncytium formation induced by human immunodeficiency virus type 1 (HIV-1) envelope proteins. The presence of CD82 proteins also inhibited cell-to-cell transmission of HTLV-1. Coimmunoprecipitation and cocapping experiments showed that CD82 associates with HTLV-1 envelope glycoproteins, both within the cell and at the cell surface. Finally, whereas the intracellular maturation of HTLV-1 glycoproteins was not modified by the presence of CD82 proteins, HTLV-1 protein coproduction delayed the intracellular maturation of CD82 proteins. There thus seems to be a reciprocal interaction between virus and cell proteins, and the cellular proteins involved in adhesion modulate retrovirus transmission both positively, as shown in other systems, and negatively, as shown here.

The Y-S-L-I tyrosine-based motif in the cytoplasmic domain of the human T-cell leukemia virus type 1 envelope is essential for cell-to-cell transmission.

The human T-cell leukemia virus type 1 (HTLV-1) transmembrane glycoprotein has a 24-amino-acid cytoplasmic domain whose function in the viral life cycle is poorly understood. We introduced premature-stop mutations and 18 single-amino-acid substitutions into this domain and studied their effects on cell-to-cell transmission of the virus. The results show that the cytoplasmic domain is absolutely required for cell-to-cell transmission of HTLV-1, through amino acids which cluster in a Y-S-L-I tyrosine-based motif. The transmission defect in two motif mutants did not result from a defect in glycoprotein incorporation or fusion. It appears that the Y-S-L-I tyrosine-based motif of the HTLV-1 glycoprotein cytoplasmic domain has multiple functions, including involvement in virus transmission at a postfusion step.

Early assembly step of a retroviral envelope glycoprotein: analysis using a dominant negative assay.

As for most integral membrane proteins, the intracellular transport of retroviral envelope glycoproteins depends on proper folding and oligomeric assembly in the ER. In this study, we considered the hypothesis that a panel of 22 transport-defective mutants of the human T cell leukemia virus type 1 envelope glycoprotein might be defective in ER assembly. Upon cell cotransfection with wild-type envelope, however, the vast majority of these transport-defective mutants (21 of 22) exerted a specific trans-dominant negative effect. This effect was due to random dimerization of the mutated and wild-type glycoproteins that prevented the intracellular transport of the latter. This unexpected result suggests that association of glycoprotein monomers precedes the completion of folding. The only mutation that impaired this early assembly was located at the NH2 terminus of the protein. COOH-terminally truncated, soluble forms of the glycoprotein were also trans-dominant negative provided that their NH2 terminus was intact. The leucine zipper-like domain, although involved in oligomerization of the envelope glycoproteins at the cell surface, did not contribute to their intracellular assembly. We propose that, at a step subsequent to translation, but preceding complete folding of the monomers, glycoproteins assemble via their NH2-terminal domains, which, in turn, permits their cooperative folding.

Interactions of the cytoplasmic domains of human and simian retroviral transmembrane proteins with components of the clathrin adaptor complexes modulate intracellular and cell surface expression of envelope glycoproteins.

The cytoplasmic domains of the transmembrane (TM) envelope proteins (TM-CDs) of most retroviruses have a Tyr-based motif, YXXO, in their membrane-proximal regions. This signal is involved in the trafficking and endocytosis of membrane receptors via clathrin-associated AP-1 and AP-2 adaptor complexes. We have used CD8-TM-CD chimeras to investigate the role of the Tyr-based motif of human immunodeficiency virus type 1 (HIV-1), simian immunodeficiency virus (SIV), and human T-leukemia virus type 1 (HTLV-1) TM-CDs in the cell surface expression of the envelope glycoprotein. Flow cytometry and confocal microscopy studies showed that this motif is a major determinant of the cell surface expression of the CD8-HTLV chimera. The YXXO motif also plays a key role in subcellular distribution of the envelope of lentiviruses HIV-1 and SIV. However, these viruses, which encode TM proteins with a long cytoplasmic domain, have additional determinants distal to the YXXO motif that participate in regulating cell surface expression. We have also used the yeast two-hybrid system and in vitro binding assays to demonstrate that all three retroviral YXXO motifs interact with the micro1 and micro2 subunits of AP complexes and that the C-terminal regions of HIV-1 and SIV TM proteins interact with the beta2 adaptin subunit. The TM-CDs of HTLV-1, HIV-1, and SIV also interact with the whole AP complexes. These results clearly demonstrate that the cell surface expression of retroviral envelope glycoproteins is governed by interactions with adaptor complexes. The YXXO-based signal is the major determinant of this interaction for the HTLV-1 TM, which contains a short cytoplasmic domain, whereas the lentiviruses HIV-1 and SIV have additional determinants distal to this signal that are also involved.

Analysis of functional conservation in the surface and transmembrane glycoprotein subunits of human T-cell leukemia virus type 1 (HTLV-1) and HTLV-2.

Human T-cell leukemia virus types 1 and 2 (HTLV-1 and HTLV-2) are closely related retroviruses with nucleotide sequences that are 65% identical. To determine whether their envelope glycoproteins function similarly and to define the molecular determinants of HTLV-2 envelope-mediated functions, we have used pseudotyped viruses and have introduced mutations into regions of the HTLV-2 glycoproteins homologous to those known to be important for HTLV-1 glycoprotein functions. The envelopes of the two viruses could be exchanged with no loss of infectivity, suggesting that the glycoproteins function in broadly similar ways. However, comparative analysis of the HTLV-1 and HTLV-2 glycoproteins showed subtle differences in the structure-function relationships of the two surface glycoprotein (SU) subunits, even though they recognize the same receptor. Indeed, mutations introduced at equivalent positions in the two SU glycoproteins resulted in different phenotypes in the two viruses. The scenario is the opposite for the transmembrane glycoprotein (TM) subunits, in which the functional domains of the two viruses are strictly conserved, confirming the involvement of the TM ectodomain in postfusion events required for full infectivity of the HTLVs. Thus, although they recognize the same receptor, the HTLV-1 and HTLV-2 SU subunits have slightly different ways of transducing the conformational information that primes a common fusion mechanism effected by similar TM subunits.

The ectodomain of the human T-cell leukemia virus type 1 TM glycoprotein is involved in postfusion events.

To examine the contribution of the transmembrane envelope glycoprotein (TM) to the infectivity of the human T-cell leukemia virus type 1 (HTLV-1), single amino acid substitutions were introduced throughout its ectodomain. The mutated envelopes were tested for intracellular maturation and for functions, including ability to elicit syncytium formation and ability to mediate cell-to-cell transmission of the virus. Three major phenotypes, defining three functionally distinct regions, were identified. (i) Mutations causing defects in intracellular maturation of the envelope precursor are mostly distributed in the central portion of the TM ectodomain, containing the immunosuppressive peptide. This region, which includes vicinal cysteines thought to form an intramolecular disulfide bridge, is probably essential for correct folding of the protein. (ii) Mutations resulting in reduced syncytium-forming ability despite correct intracellular maturation are clustered in the amino-terminal part of the TM ectodomain, within the leucine zipper-like motif. Similar motifs with a propensity to form coiled-coil structures have been implicated in the fusion process driven by other viral envelope proteins, and HTLV-1 may thus conform to this general rule for viral fusion. (iii) Mutants with increased syncytium-forming ability define a region immediately amino-terminal to the membrane-spanning domain. Surprisingly, these mutants exhibited severe defects in infectivity, despite competence for fusion. Existence of this phenotype indicates that capacity for cell-to-cell fusion is not sufficient to ensure viral entry, even in cell-to-cell transmission. The ectodomain of the TM glycoprotein thus may be involved in postfusion events required for full infectivity of HTLV-1, which perhaps represents a unique feature of this poorly infectious retrovirus.

Two distinct oncornaviruses harbor an intracytoplasmic tyrosine-based basolateral targeting signal in their viral envelope glycoprotein.

It has been clearly established that the budding of the human immunodeficiency virus (HIV-1), a lentivirus, occurs specifically through the basolateral membrane in polarized epithelial cells. More recently, the signal was assigned to a tyrosine-based motif located in the intracytoplasmic domain of the envelope glycoprotein, as previously observed on various other viral and cellular basolateral proteins. In the present study, expression of human T-cell leukemia virus type 1 (HTLV-1) or Moloney murine leukemia virus envelope glycoproteins was used for trans-complementation of an envelope-negative HIV-1. This demonstrated the potential of oncornaviral retrovirus envelope glycoproteins to confer polarized basolateral budding in epithelial Madin-Darby canine kidney cells (MDCK cells). Site-directed mutagenesis confirmed the importance of a common motif encompassing at least one crucial membrane-proximal intracytoplasmic tyrosine residue. The conservation of a similar basolateral maturation signal in different retroviruses further supports its importance in the biology of this group of viruses.

A novel human T-leukemia virus type 1 cell-to-cell transmission assay permits definition of SU glycoprotein amino acids important for infectivity.

Human T-leukemia virus type 1 (HTLV-1) envelope glycoproteins play a major role in viral transmission, which in the case of this virus occurs almost exclusively via cell-to-cell contact. Until very recently, the lack of an HTLV-1 infectivity assay precluded the determination of the HTLV-1 protein domains required for infectivity. Here, we describe an assay which allows the quantitative evaluation of HTLV-1 cell-to-cell transmission in a single round of infection. Using this assay, we demonstrate that in this system, cell-to-cell transmission is at least 100 times more efficient than transmission with free viral particles. We have examined 46 surface (SU) glycoprotein mutants in order to define the amino acids of the HTLV-1 SU glycoprotein required for full infectivity. We demonstrate that these amino acids are distributed along the entire length of the SU glycoprotein, including the N-terminus and C-terminus regions, which have not been previously defined as being important for HTLV-1 glycoprotein function. For most of the mutated glycoproteins, the capacity to mediate cell-to-cell transmission is correlated with the ability to induce formation of syncytia. This result indicates that the fusion capacity is the main factor responsible for infectivity mediated by the HTLV-1 SU envelope glycoprotein, as is the case for other retroviral glycoproteins. However, other factors must also intervene, since two of the mutated glycoproteins were correctly fusogenic but could not mediate cell-to-cell transmission. Existence of this phenotype shows that capacity for fusion is not sufficient to confer infectivity, even in cell-to-cell transmission, and could suggest that postfusion events involve the SU.

The HTLV-I envelope glycoproteins: structure and functions.

The human T-cell lymphotropic virus type I (HTLV-I) envelope has a structural organization shared by all retroviral envelopes, which contain two mature viral glycoproteins deriving from a common precursor: an external surface protein (SU), associated with a transmembrane protein (TM) responsible for anchoring the SU-TM complex at the cell surface or in the viral envelope. Our understanding of the tertiary structure of these proteins is extremely poor. The intracellular maturation follows the normal cellular secretory pathway, resulting in expression of the mature glycoproteins at the cell surface. The five potential N-glycosylation sites are glycosylated. Most mutations artificially introduced into the glycoproteins result in loss of function, mostly due to abnormal intracellular maturation. This probably indicates a very compact structure of these proteins, where the entire structure is involved in correct conformation. Studies using neutralizing antibodies or mutagenesis have defined functional domains in the SU protein, which is responsible for receptor binding. These domains occur throughout the SU glycoprotein. Sequence analysis of the HTLV-I TM predicts a structure, and probably functions, similar to other retrovirus TMs: involvement of this glycoprotein in the different oligomerization steps leading to a fusogenic SU-TM complex and in the fusion process itself. These features remain to be proven, and it is not yet understood why the free HTLV-I viral particle is not infectious.

Identification of functional regions in the human T-cell leukemia virus type I SU glycoprotein.

Single conservative and nonconservative amino acid substitutions were introduced into the gp45 external envelope protein (SU) of human T-cell leukemia virus type I (HTLV-I). The mutated amino acids were those identified as being conserved in HTLV-I, HTLV-II, and simian T-cell leukemia virus type I (but not in bovine leukemia virus). The mutated envelopes were tested for intracellular maturation and for function. Mutants with three major phenotypes could be defined: (i) 9 mutants with a wild-type phenotype, which included most of the conservative amino acid changes (five of seven) distributed throughout the SU protein; (ii) 8 mutants with affected intracellular maturation, 6 of which define a region in the central part of the SU protein essential for correct folding of the protein; and (iii) 13 mutants with normal intracellular maturation but impaired syncytium formation. These mutations likely affect the receptor binding step or postbinding events required for fusion. Five of these mutations are located between amino acids 75 and 101 of the SU protein, in the amino-terminal third of the molecule. The other mutations involve positions 170, 181, 195, 197, 208, 233, and 286, suggesting that two other domains, one central and one carboxy terminal, are involved in HTLV-I envelope functions.

The open reading frame I (ORF I)/ORF II part of the human T-cell leukemia virus type I X region is dispensable for p40tax, p27rex, or envelope expression.

The X region of the human T-cell leukemia virus type I contains the second coding exon of the tax and rex regulatory proteins (open reading frame IV [ORF IV] and ORF III, respectively), as well as coding regions for more recently described proteins, p30II (or the tof protein) and p13II in ORF II and the putative rof protein and p12I in ORF I. Deletions and transcomplementation experiments showed that expression of the envelope, as well as that of the tax and rex proteins, was independent of the proteins encoded in the ORF I/ORF II region. Furthermore, p30II and p12I proteins could not replace the rex protein in a rex-dependent envelope or Gag protein expression system.