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.

Targeted expression of HTLV-I envelope proteins in muscle by DNA immunization of mice.

We have compared two types of plasmids for DNA immunization against HTLV-I envelope glycoproteins. One type of plasmid contains the coding DNA of the complete envelope gene of HTLV-I under the control of the CMV promoter with (CMVenvLTR) or without (CMVenv) the tax/rex genes. The second type contains the coding DNA of the complete env gene of HTLV-I under the control of the human desmin muscle specific promoter (DesEnv). These plasmids were inoculated into mice and the humoral response was studied by flow cytometry, ELISA and neutralization assays. Inoculation of the DesEnv construct elicited a higher humoral response with better neutralization properties than the injection of CMVenvLTR or CMVenv plasmids. The choice of vectors will be important for the design of genetic HTLV-I vaccines.

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.

Identification of exposed epitopes on the envelope glycoproteins of human T-cell lymphotropic virus type I (HTLV-I)

Several lines of evidence underscore the important role of the humoral response specific for HTLV-I envelope protein in the protection against viral infection. One approach to producing efficient immunogens is to synthesize peptides corresponding to the primary amino-acid sequence of neutralizing epitopes found in the external sub-unit gp46. In this study, we have selected synthetic peptides overlapping the major linear neutralizing determinants described earlier and used them as immunogens in rabbits and mice. All rabbit polyclonal anti-sera raised against peptides recognized epitopes in a denaturated context as well as MAbs raised against the HB peptide (aa287-311). By contrast, synthetic peptides O (aa89-110), HH (aa190-209), T (aa190-212) and HB (aa287-311) have generated antibodies efficiently binding their epitopes in a native context, suggesting that these domains are well exposed both at the heterodimer and at the oligomer surface. None of the antibodies induced by synthetic peptides show in vitro neutralizing properties, even those with a good capacity to bind the native form of HTLV-I envelope proteins.

Induction of neutralizing antibodies against HTLV-I envelope proteins after combined genetic and protein immunizations in mice.

Direct DNA inoculation can induce both protective humoral and cellular responses against several viruses. The HTLV-I envelope glycoproteins are the major antigens recognized by sera of HTLV-I infected patients that generate neutralizing immune responses in vitro and in vivo. We compared immune responses elicited after a single inoculation of two plasmids encoding the complete HTLV-I envelope proteins followed or not by gp62 Baculovirus recombinant protein boosts in BALB/c mice. First, we observe that the coexpression of env and rex genes is not sufficient to raise a detectable specific humoral response after a single DNA inoculation. Protein boosts generated a high antibody response in mice primed with DNA expressing HTLV-I envelope proteins as compared to naive and negative control vector primed groups. This humoral response presented high neutralizing antibody titers. These results suggest that a single inoculation of DNA expressing HTLV-I env gene can stimulate memory B-cell clones that are able to respond effectively to subsequent encounters with HTLV-I envelope proteins and a specific cellular T helper cell response in mice.

Production and characterization of a monoclonal antibody directed against HTLV-1 p19: use in a specific capture enzyme immunoassay.

An enzyme immunoassay (EIA) was developed for detection of Human T-cell Leukemia Virus antigen in culture supernatants and cell lysates. The assay used a mouse monoclonal antibody against HTLV-I p19 major core protein as capture antibody. It has a sensitivity of 1 microgram/ml of HTLV-I protein, 250 pg/ml of purified recombinant p19 and detected p19 in an 10(-2) diluted supernatant of MT2 infected cell and in a 100 MT2 cells lysate (10(6) cells taken at day 7 of culture). The assay enable us to discriminate between HTLV-I and HTLV-II antigens and is reproducibly negative for supernatants and cell lysates of uninfected cells and of HIV-1 infected cells. The assay was found to be more specific and 10 times more sensitive than the reverse transcriptase (RT) assay, and the EIA test became positive three days earlier than RT assay for the HTLV-I cell lines supernatants.