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. 2003 Apr;77(7):3993-4003.
doi: 10.1128/jvi.77.7.3993-4003.2003.

Distinct mechanisms of neutralization by monoclonal antibodies specific for sites in the N-terminal or C-terminal domain of murine leukemia virus SU

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Distinct mechanisms of neutralization by monoclonal antibodies specific for sites in the N-terminal or C-terminal domain of murine leukemia virus SU

Michael Dominic Burkhart et al. J Virol. 2003 Apr.

Abstract

The epitope specificities and functional activities of monoclonal antibodies (MAbs) specific for the murine leukemia virus (MuLV) SU envelope protein subunit were determined. Neutralizing antibodies were directed towards two distinct sites in MuLV SU: one overlapping the major receptor-binding pocket in the N-terminal domain and the other involving a region that includes the most C-terminal disulfide-bonded loop. Two other groups of MAbs, reactive with distinct sites in the N-terminal domain or in the proline-rich region (PRR), did not neutralize MuLV infectivity. Only the neutralizing MAbs specific for the receptor-binding pocket were able to block binding of purified SU and MuLV virions to cells expressing the ecotropic MuLV receptor, mCAT-1. Whereas the neutralizing MAbs specific for the C-terminal domain did not interfere with the SU-mCAT-1 interaction, they efficiently inhibited cell-to-cell fusion mediated by MuLV Env, indicating that they interfered with a postattachment event necessary for fusion. The C-terminal domain MAbs displayed the highest neutralization titers and binding activities. However, the nonneutralizing PRR-specific MAbs bound to intact virions with affinities similar to those of the neutralizing receptor-binding pocket-specific MAbs, indicating that epitope exposure, while necessary, is not sufficient for viral neutralization by MAbs. These results identify two separate neutralization domains in MuLV SU and suggest a role for the C-terminal domain in a postattachment step necessary for viral fusion.

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Figures

FIG. 1.
FIG. 1.
Mapping of the 35/56 and 83A25 epitopes. (A) Black rectangles indicate sequences from Friend clone 57 MuLV SU, and white rectangles indicate sequences from AKR.623 MuLV SU. Residue numbers (derived from the Friend sequence) are indicated above the bars. Only the SU sequence is shown. NIH 3T3 cells infected with MuLVs bearing these chimeras were assayed for 35/56 and 83A25 reactivity by immunofluorescence. +, positive reaction between antibody and infected cells; −, no reaction. (B) Sequence alignment for various MuLV SUs from residues 390 to 425. The primary amino acid sequence for AKR.623 is shown at the top. A dash indicates conservation; polymorphisms are indicated by the corresponding residue letter. Reactivity with 35/56 or 83A25, based on data taken from references and , is indicated on the right. An N-linked glycosylation site present in some sequences is underlined.
FIG. 2.
FIG. 2.
Antigens used for ELISAs to map SU-specific MAbs. (A) Black rectangles represent sequences derived from MuLV SU, and the letter H represents a six-histidine affinity tag. Amino acid numbers are given above the bars, and single-amino-acid differences from the Friend clone 57 sequence are indicated. The Friend SU bearing an insert between residues 243 and 244 has been described previously (24); viruses bearing this mutation are infectious. (B) Two synthetic peptides based on the Friend clone 57 MuLV SU PRR are shown: a 20-mer, 226-245, and a 25-mer, 229-253, which is shaded. The sequences are aligned, and the corresponding residue numbers of Friend SU are indicated. A schematic of Friend SU below the peptides shows the domain organization of SU. C-term., C-terminal domain.
FIG. 3.
FIG. 3.
Neutralization of MuLV by SU-specific MAbs. Neutralization assays for the indicated MAbs were performed with MuLV-luciferase-encoding virions pseudotyped with Friend clone 57 (A) or Friend/AKR 328/445 chimeric Env (B). Values are averages of triplicate samples.
FIG. 4.
FIG. 4.
Inhibition of MuLV SU binding to mCAT-1 by MAbs. (A) Purified Friend/AKR 328/445 chimeric SU labeled with 125I was incubated with the MAbs at the indicated concentrations for 1 h at 37°C and then assayed for the ability to bind 293.mCAT cells. The results are presented as percent inhibition of the binding level in the absence of MAb. Values are averages of triplicate samples. (B) MuLV pseudotyped with the Friend/AKR 393/426 chimeric Env was incubated with 293 or 293.mCAT cells (top panel) for 1 h at 37°C, and virion binding was detected by FACS. The MuLV particles were pretreated with 59C9, 5145a, or polyclonal anti-SU serum (PC) (middle panel) or 83A25 or 35/56 (bottom panel) for 1 h at 37°C and then assayed for binding as described above. The MAbs were used at 20 μg/ml, and the serum was used at a 1:100 dilution.
FIG. 5.
FIG. 5.
Inhibition of syncytium formation by MAbs. (A) Viral supernatant from NIH 3T3 cells infected with MuLV bearing the Friend/AKR 393/426 Env was incubated with or without MAbs for 1 h at 37°C. The supernatant was then used to overlay XC cells, and syncytium formation was analyzed after a 2.5-h incubation at 37°C. The MAbs were used at 15 μg/ml. (B) The indicated MAbs were titrated for their ability to inhibit syncytium formation as described in panel A. After fixation and staining of syncytia, fused nuclei were counted in four equal areas for each data point, and this number was used to calculate a percent inhibition relative to no antibody treatment.
FIG. 6.
FIG. 6.
Precipitation of intact virions with Env-specific MAbs. Culture supernatants from NIH 3T3 cells infected with MuLV bearing the chimeric Friend/AKR 393/426 Env labeled with [35S]methionine and cysteine were immunoprecipitated in the presence (+) or absence (−) of NP-40 with the MAbs (20 μg/ml) or polyclonal sera indicated. Precipitates were analyzed by SDS-PAGE and autoradiography. TM refers to the transmembrane subunit of Env, p15E, and CA refers to the MuLV capsid protein, p30. The presence of a CA band is indicative of binding of the SU-specific MAbs to intact virions.

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References

    1. Albritton, L. M., L. Tseng, D. Scadden, and J. M. Cunningham. 1989. A putative murine ecotropic retrovirus receptor gene encodes a multiple membrane-spanning protein and confers susceptibility to virus infection. Cell 57:659-666. - PubMed
    1. Bae, Y., S. M. Kingsman, and A. J. Kingsman. 1997. Functional dissection of the Moloney murine leukemia virus envelope protein gp70. J. Virol. 71:2092-2099. - PMC - PubMed
    1. Barnett, A. L., R. A. Davey, and J. M. Cunningham. 2001. Modular organization of the Friend murine leukemia virus envelope protein underlies the mechanism of infection. Proc. Natl. Acad. Sci. USA 98:4113-4118. - PMC - PubMed
    1. Burkhart, M. D. 2002. A study of retroviral neutralization by monoclonal antibodies specific for the murine leukemia virus envelope subunit SU. Ph.D. dissertation. New York University, New York, N.Y.
    1. Burton, D., E. Saphire, and P. Parren. 2001. A model for neutralization of viruses based on antibody coating of the virion surface, p. 109-143. In D. Burton (ed.), Antibodies in viral infection. Springer-Verlag, Berlin, Germany. - PubMed

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