Analysis of two monoclonal antibodies reactive with envelope proteins of murine retroviruses: one pan specific antibody and one specific for Moloney leukemia virus

Abstract

Many monoclonal antibodies (MAbs) reactive with various proteins of murine leukemia viruses (MuLVs) have been developed. In this report two additional MAbs with differing and unusual specificities are described. MAb 573 is reactive with the envelope protein of all MuLVs tested including viruses in the ecotropic, xenotropic, polytropic and amphotropic classes. Notably, MAb 573 is one of only two reported MAbs that react with the envelope protein of amphotropic MuLVs. This MAb appears to recognize a conformational epitope within the envelope protein, as it reacts strongly with live virus and live infected cells, but does not react with formalin-fixed or alcohol-fixed infected cells or denatured viral envelope protein in immunoblots. In contrast, Mab 538 reacts only with an epitope unique to the envelope protein of the Moloney (Mo-) strain of MuLV, a prototypic ecotropic MuLV that is the basis for many retroviral tools used in molecular biology. MAb 538 can react with live cells and viruses, or detergent denatured or fixed envelope protein. The derivation of these antibodies as well as their characterization with regard to their isotype, range of reactivity with different MuLVs and utility in different immunological procedures are described in this study.

Keywords: Amphotropic MuLV; Env protein; Moloney MuLV; Monoclonal antibodies; Retroviruses.

Figure 1

Recombinant polytropic MuLV SU proteins and their reactivity to MAb 538. The bar diagrams depict the amino acid sequences of several polytropic MuLV SU proteins derived by recombination with Mo-MuLV (Alamgir, Owens et al., 2005). Open (white) regions of the bars represent Mo-MuLV-derived regions while closed (black) regions represent regions derived from endogenous polytropic MuLV sequences. Numbers above the diagrams indicate the positions of amino acids numbered from the beginning of the processed SU protein. The sequence at the bottom of the figure depicts the proline rich region (PRR) of the Mo-MuLV SU protein with conserved and variable regions indicated. The reactivity of each of the SU proteins to MAb 538 is indicated.

Figure 2

Detection of cell surface expression of MuLV SU proteins by MAb 573 and MAb 538 by flow cytometry. (A). 3T3 cells chronically infected with the xenotropic MuLV, CasE no.1, the amphotropic MuLV, 4070A, the ecotropic MuLV, Fr-MuLV or the polytropic MuLV, M-RV 7A, were treated with MAb 573 and a secondary FITC goat Ab directed at mouse Ig (white) or with the secondary FITC antibody alone (grey) and analyzed by FACS. (B). 3T3 cells chronically infected with Mo-MuLV were treated with MAb 538 and the secondary goat FITC Ab (white) or with the secondary antibody alone (grey).

Figure 3

Detection of the Mo-MuLV SU protein by immunoblotting using MAb 538. Virions released from cells producing the Mo-MuLV-derived polytropic recombinant virus MCF 383-5S, Mo-MuLV or Fr-MuLV were purified by isopycnic gradient centrifugation, and equivalent levels of virion protein were electrophoresed on 10% polyacrylamide gels, transferred to Immobilon-FL PVDF membranes (Millipore) and subsequently developed using MAb 538 and goat anti-mouse DyLight 680 (Thermo Scientific).

Figure 4

Neutralization of MuLVs by MAbs 573 and 538. 0.5 ml of virus stocks of Mo-MuLV or Fr-MuLV adjusted to ~6000 focus-forming units/ml were mixed with 0.5 ml dilutions of hybridoma supernatant tissue culture medium containing MAb 573 (A) (16.7 ug IgM/ml) or Mab 538 (15 ug IgM/ml) (B), and incubated for 30 minutes at 37^oC. The treated MuLV stocks were subsequently assessed for infectivity on NIH 3T3 cells using a focal immunofluorescence assay (Sitbon et al., 1985).

Figure 5

Detection of cell-surface env protein on amphotropic 1504A-infected cells. The reactivity of Mus dunni cells chronically infected with the amphotropic MuLV 1504A with MAb 83A25 or MAb 573 was compared by indirect membrane immunofluorescence assays using trypsinized virus-infected cells (Chesebro et al., 1981).