Inhibition of cell-free human T-cell leukemia virus type 1 infection at a postbinding step by the synthetic peptide derived from an ectodomain of the gp21 transmembrane glycoprotein

Abstract

To investigate the roles of human T-cell leukemia virus type 1 (HTLV-1) envelope (Env) proteins gp46 and gp21 in the early steps of infection, the effects of the 23 synthetic peptides covering the entire Env proteins on transmission of cell-free HTLV-1 were examined by PCR and by the plaque assay using a pseudotype of vesicular stomatis virus (VSV) bearing the Env of HTLV-1 [VSV(HTLV-1)]. The synthetic peptide corresponding to amino acids 400 to 429 of the gp21 Env protein (gp21 peptide 400-429, Cys-Arg-Phe-Pro-Asn-Ile-Thr-Asn-Ser-His-Val-Pro-Ile-Leu-Gln-Glu-Arg-P ro-Pro-Leu-Glu-Asn-Arg-Val-Leu-Thr-Gly-Trp-Gly-Leu) strongly inhibited infection of cell-free HTLV-1. By using the mutant peptide, Asn407, Ser408, and Leu413, -419, -424, and -429 were confirmed to be important amino acids for neutralizing activity of the gp21 peptide 400-429. Addition of this peptide before or during adsorption of HTLV-1 at 4 degrees C did not affect its entry. However, HTLV-1 infection was inhibited about 60% when the gp21 peptide 400-429 was added even 30 min after adsorption of HTLV-1 to cells, indicating that the amino acid sequence 400 to 429 on the gp21 Env protein plays an important role at the postbinding step of HTLV-1 infection. In contrast, a monoclonal antibody reported to recognize the gp46 191-196 peptide inhibited the infection of HTLV-1 at the binding step.

FIG. 1

Detection of reverse-transcribed HTLV-1 RNA by PCR and effects of the HTLV-1 Env synthetic peptides on transmission of cell-free HTLV-1. (A) MOLT-4 cells were infected with serially diluted cell-free HTLV-1, lysed after incubation for 20 h, and examined by PCR amplification using the pXO 7302-7326 and pXO 7504-7481 primers (lanes 1 to 3). MOLT-4 cells were infected with cell-free HTLV-1 treated with HTLV-1-seronegative human serum (normal human serum [NHS]) or with HTLV-1-seropositive human serum (α-HTLV-1) for 1 h at 37°C. Lane 6, mock-infected control. PCR amplification was performed without template DNA (lane 7). (B) MOLT-4 cells were infected with HTLV-1 for 1 h in the presence of the Env gp46 or gp21 synthetic peptides at 10 μM. Amino acid positions of Env (the initiation codon of Met is 1) are shown above each lane. The amino acid sequences of the peptides are listed in Table 1. β-Globin DNA was amplified as a control to confirm the efficiency of the amplification of each sample.

FIG. 2

Dose-dependent and specific inhibition of cell-free HTLV-1 infection by the gp21 peptide 400-429. (A) MOLT-4 cells were infected with HTLV-1 in the presence of the gp21 peptide 400-429 at 0 to 10 μM. Formation of HTLV-1 DNA in the MOLT-4 cells was detected by PCR as described for Fig. 1. (B) Relative intensities of pX DNA were determined by densitometry: band intensities of pX DNA were divided by those of β-globin bands. (C) Inhibitory effect of the gp21 peptide 400-429 on plating of the VSV(HTLV-1) pseudotype. The VSV pseudotype infection was carried out in the presence of the gp21 peptide 400-429 (circles) or the gp21 peptide 458-488 (squares). About 100 plaques were formed in the absence of the peptides. Each datum point is the mean for two independent experiments. (D) Effect of the gp21 peptide 400-429 on transmission of cell-free HIV-1. MOLT-4 cells were infected with serially diluted cell-free HIV-1 (IIIB strain) (lanes 1 to 3), lysed 20 h later, and examined by PCR using the primers specific for the HIV-1 gag region. Cell-free HIV-1 was inoculated into MOLT-4 cells in the absence (lane 4) or presence (lane 5) of the gp21 peptide (Pep.) 400-429 (10 μM). Lane 6, MOLT-4 cells treated with neutralizing MAb against CD4 receptor (α-CD4) prior to infection. Lane 7, mock-infected control. β-Globin DNA was amplified as a control.

FIG. 3

Effects of mutations in the gp21 peptide 400-429 on inhibitory activity. (A) Amino acid sequences of the mutant peptides. (B) Effects of the mutant peptides on the transmission of cell-free HTLV-1. MOLT-4 cells were infected with either undiluted (odd-number lanes) or diluted (even-number lanes) cell-free HTLV-1 in the presence of the peptides at 10 μM. Cell lysates were examined by PCR as described for Fig. 1. β-Globin DNA was amplified as a control.

FIG. 4

Effects of the gp21 peptide 400-429 and a neutralizing MAb against gp46 on the early stage of HTLV-1 infection. (A) Schematic representation of the experiments (Exp.) investigating HTLV-1 infection at the binding step or the postbinding step. Precooled MOLT-4 cells were inoculated with cell-free HTLV-1 at 4°C for 1 h (Exp. 2, binding step), washed, and resuspended in fresh culture medium. Then, the incubation temperature of the cells was shifted to 37°C for 20 h (Exp. 3, postbinding step). The reverse-transcribed HTLV-1 RNA in MOLT-4 cells was detected by nested PCR. (B) Inhibition of the postbinding step of HTLV-1 infection by the gp21 peptide 400-429. To detect the formation of HTLV-1 DNA, serially diluted first PCR products were used as templates for nested PCR amplification using the pXI 7341-7360 and pXI 7460-7441 primers (lanes 1 to 4). The temperatures at the time of addition of the gp21 peptide (pep.) 400-429 are indicated in panel A as hatched bars. Fiftyfold-diluted first PCR products were used as templates for nested PCR. The 120 bp of pX DNA in lanes 6 to 8 corresponds to Exp. 1 to 3 in panel A, respectively. (C) Inhibition of transmission of cell-free HTLV-1 at the binding step by anti-gp46 neutralizing MAb LAT-27. HTLV-1 was incubated with LAT-27 MAb (30 μg/ml) or control MAb against mouse recombinant IL-2 (30 μg/ml) for 1 h at 37°C prior to infection. Then, experiments were done as described above. Lane 1, no-MAb control. MAbs were added to MOLT-4 cells either at 4°C (lanes 2 and 4) or at 37°C (lanes 3 and 5). β-Globin DNA was amplified as a control. (D) Effect of the timing of the addition of the gp21 peptide 400-429 after adsorption of HTLV-1 to MOLT-4 cells on the transmission of cell-free HTLV-1. After adsorption of HTLV-1 to MOLT-4 cells at 4°C, the gp21 peptide 400-429 was added at the indicated times and the cells were cultured for 20 h in the presence of the peptide. Formation of HTLV-1 DNA in MOLT-4 cells was detected by nested PCR (120 bp of pX). β-Globin DNA was amplified as a control. Relative intensities of pX DNA bands were calculated by densitometry. Inhibition by the no-peptide control was assigned a value of 0%.

FIG. 5

Schematic representation of the HTLV-1 Env glycoprotein gp21 and the amino acid sequence of the gp21 peptide 400-429. The fusion peptide domain (FP), extracellular domain (EC), transmembrane domain (TM), and intracellular domain (IC) are indicated. The positions of the leucine zipper-like region (LZR) (amino acids 340 to 392) and the immunodominant region (IDR) (amino acids 377 to 402) are also indicated. The N-glycosylation site of HTLV-1 Env gp21 (at position 404) (closed circle) is underlined.