Human T-cell leukemia virus type 1 envelope-mediated syncytium formation can be activated in resistant Mammalian cell lines by a carboxy-terminal truncation of the envelope cytoplasmic domain - PubMed (original) (raw)

Human T-cell leukemia virus type 1 envelope-mediated syncytium formation can be activated in resistant Mammalian cell lines by a carboxy-terminal truncation of the envelope cytoplasmic domain

Felix J Kim et al. J Virol. 2003 Jan.

Abstract

Human T-cell leukemia virus (HTLV) envelope (Env) glycoproteins induce fusion, leading to rampant syncytium formation in a broad range of cell lines. Here, we identified murine, hamster, canine, and porcine cell lines that are resistant to HTLV-1 Env-induced syncytium formation. This resistance was not due to the absence of functional receptors for HTLV Env, as these cells were susceptible to infection with HTLV Env-pseudotyped virions. As murine leukemia virus (MLV) Env and HTLV Env present close structural homologies (F. J. Kim, I. Seiliez, C. Denesvre, D. Lavillette, F. L. Cosset, and M. Sitbon, J. Biol. Chem. 275:23417-23420, 2000), and because activation of syncytium formation by MLV Env generally requires cleavage of the R peptide in the cytoplasmic domain of the Env transmembrane (TM) component, we assessed whether truncation of the cytoplasmic domain of HTLV Env would alleviate this resistance. Indeed, in all resistant cell lines, truncation of the last 8 amino acids of the HTLV Env cytoplasmic domain (HdC8) was sufficient to overcome resistance to HTLV Env-induced syncytium formation. Furthermore, HdC8-mediated cell-to-cell infection titers varied according to the target cell lines and could be significantly higher than that observed with HTLV Env on HeLa cells. These data indicate that a determinant located within the 8 carboxy-terminal cytoplasmic amino acids of TM plays a distinct role in HTLV Env-mediated cell-to-cell infection and syncytium formation.

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Figures

FIG. 1.

FIG. 1.

Distinct susceptibility of NIH 3T3 and NIH 3T3(TK−) cells to HTLV Env-induced syncytium formation. (A) NIH 3T3 and NIH 3T3(TK−) cells were tested for their susceptibility to HTLV-1 Env (H)-induced syncytium formation. Cells were transfected with up to 6 μg of envelope expression vector. As controls, both cell lines were transfected with vectors encoding either the parental, full-length Friend MLV Env (F) or the fusogenic Friend MLV Env lacking the R peptide (FΔR). Syncytia are observed as dark plaques. (B) NIH 3T3(TK−) cells transfected with the HTLV-1 Env, NIH 3T3(TK−)+H, were tested for their ability to trigger syncytium formation with overlaid HeLa (left panel) and NIH 3T3(TK−) target cells (right panel).

FIG. 2.

FIG. 2.

Truncation of HTLV-1 Env TM induces syncytium formation in NIH 3T3(TK−) cells. (A) Schematic representation of HTLV Env. The SU and TM subunits are indicated. The HTLV-1 Env signal peptide, fusion peptide, and TM membrane anchor are shown as dotted boxes, from the amino terminus to the carboxy terminus, respectively. Amino acid residues of the membrane anchor (anchor) and cytoplasmic domain (CD) are shown, and the position of the last carboxy-terminal residue of each mutant is indicated with an arrowhead. Amino acid residue numbering starts from the first signal peptide methionine of the HTLV-1 Env precursor. (B) Parental HTLV-1 Env (H) and truncation mutants lacking the 8 (HdC8) or 16 (HdC16) carboxy-terminal amino acids of the TM were tested for their ability to form syncytia after transfection in NIH 3T3 or NIH 3T3(TK−) cells. Open arrows in the panels on the right point to syncytia.

FIG. 3.

FIG. 3.

Expression of HTLV-1 Env TM truncation mutants and their incorporation into virions. Equal amounts of cell extracts were obtained from 2 × 106 to 5 × 106 293T cells cotransfected with 8 μg of an MLV-based lacZ reporter gene vector, 4 μg of the MLV p/CLGag-Pol expression vector, and 4 μg of either an HTLV Env-derived expression vector (H, HdC8, HdC16, or HΔC) or a control vector (Mock). The different HTLV Env TM mutants are diagrammed in Fig. 2. Cell extracts were immunoblotted with the 5a anti-HTLV TM monoclonal antibody (3) (left panel). Pelleted virions were obtained from supernatants of 293T transfectants after concentration by ultracentrifugation on a 20% sucrose cushion. Resulting pellets were immunoblotted with either the 5a anti-HTLV TM monoclonal antibody (anti-TM, center panel) or the 1C11 anti-HTLV SU monoclonal antibody (anti-SU, right panel). Respective levels of virion expression were controlled by immunoblotting with the R187 anti-MLV capsid monoclonal antibody (a kind gift of B. Chesebro) (anti-CA, center panel). The positions of the HTLV-1 Env precursors (Pr Env), mature TM, and mature SU envelope proteins are indicated.

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References

    1. Audit, M., J. Dejardin, B. Hohl, C. Sidobre, T. J. Hope, M. Mougel, and M. Sitbon. 1999. Introduction of a cis-acting mutation in the capsid-coding gene of Moloney murine leukemia virus extends its leukemogenic properties. J. Virol. 73:10472-10479. - PMC - PubMed
    1. Brody, B. A., and E. Hunter. 1994. Postassembly cleavage of a retroviral glycoprotein cytoplasmic domain removes a necessary incorporation signal and activates fusion activity. J. Virol. 68:4620-4627. - PMC - PubMed
    1. Carrington, C. V., N. Paul, J. Cordell, and T. F. Schulz. 1996. Probing the conformation of the human T-lymphotropic virus I envelope protein complex with monoclonal antibodies. J. Gen. Virol. 77:2025-2029. - PubMed
    1. Daenke, S., and S. Booth. 2000. Molecular mechanisms affecting HTLV type 1-dependent fusion at the cell membrane: implications for inhibiting viral transmission. AIDS Res. Hum. Retroviruses. 16:1731-1736. - PubMed
    1. Daenke, S., S. A. McCracken, and S. Booth. 1999. Human T-cell leukaemia/lymphoma virus type 1 syncytium formation is regulated in a cell-specific manner by ICAM-1, ICAM-3 and VCAM-1 and can be inhibited by antibodies to integrin beta2 or beta7. J. Gen. Virol. 80:1429-1436. - PubMed

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