Involvement of the C-terminal disulfide-bonded loop of murine leukemia virus SU protein in a postbinding step critical for viral entry

Abstract

A role for the C-terminal domain (CTD) of murine leukemia virus (MuLV) Env protein in viral fusion was indicated by the potent inhibition of MuLV-induced fusion, but not receptor binding, by two rat monoclonal antibodies (MAbs) specific for epitopes in the CTD. Although these two MAbs, 35/56 and 83A25, have very different patterns of reactivity with viral isolates, determinants of both epitopes were mapped to the last C-terminal disulfide-bonded loop of SU (loop 10), and residues in this loop responsible for the different specificities of these MAbs were identified. Both MAbs reacted with a minor fraction of a truncated SU fragment terminating four residues after loop 10, indicating that while the deleted C-terminal residues were not part of these epitopes, they promoted their formation. Neither MAb recognized the loop 10 region expressed in isolated form, suggesting that these epitopes were not completely localized within loop 10 but required additional sequences located N terminal to the loop. Direct support for a role for loop 10 in fusion was provided by the demonstration that Env mutants containing an extra serine or threonine residue between the second and third positions of the loop were highly attenuated for infectivity and defective in fusion assays, despite wild-type levels of expression, processing, and receptor binding. Other mutations at positions 1 to 3 of loop 10 inhibited processing of the gPr80 precursor protein or led to increased shedding of SU, suggesting that loop 10 also affects Env folding and the stability of the interaction between SU and TM.

FIG. 1.

(Top) Diagram of the Friend/AKR (393-426) chimeric SU (7). Friend MuLV clone 57 sequences (shaded areas) and AKR.623 sequences (white areas) are indicated. (Bottom) Sequence alignment of residues 393 to 426 of Friend MuLV and AKR MuLV SU, corresponding to the AKR fragment exchanged into Friend MuLV clone 57 to generate the Fr/AKR (393-426) chimera. Conserved residues are indicated by dashes, while polymorphic residues are listed.

FIG. 2.

Analysis of immunoreactivity of SU fragments containing loop 10. A. Representations of constructs used for this analysis. Construct 1 is the fragment expressing isolated AKR loop 10 together with six N-terminal and four C-terminal flanking residues (residues 397 to 420), surrounded by HA and myc epitope tags followed by a C-terminal PDGRF TM domain tag. Construct 2 is secreted Friend/AKR (393-426) SU (SU-sec) generated by inserting a stop codon after the codon for residue 445 at the C terminus of SU. Construct 3 is the secreted fragment of chimeric Friend/AKR (328-420) SU generated by inserting a stop codon after the codon for residue 420. B. Western blot analysis of immunoreactivity of isolated loop 10 (construct 1 in panel A). (Left) Cells expressing this construct were lysed, immunoprecipitated with antibody to the N-terminal HA tag (αHA), separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) both before and after reduction and probed with antibody to the C-terminal myc epitope tag. The larger band in the reduced sample is a minor contaminant that may represent an alternatively processed form of the fusion protein. (−)DTT and (+)DTT, absence and presence of dithiothreitol, respectively. (Right) The same protein was immunoprecipitated with anti-myc antibody and probed after blotting under nonreducing conditions with anti-HA antibody (αHA) and MAbs 83A25 and 35/56. C. Analysis of expression of conformational epitopes in secreted SU (construct 2 in panel A) and a secreted SU fragment truncated after loop 10 at residue E420 (construct 3 in panel A). Cell supernatants containing labeled full-length (SU-sec) or truncated (E420-stop) proteins were immunoprecipitated two times sequentially with the indicated MAbs at 15 μg/ml (lanes 1 and 2), and any remaining Env protein was then detected by precipitation with polyclonal anti-gp70 serum (lanes PC). Immunoprecipitated proteins were analyzed by SDS-PAGE and detected by autoradiography.

FIG. 3.

Titration of infectivity and SU and CA levels of wild-type and loop 10 mutant MuLV pseudotypes. (A) Dilutions of MuLV/luciferase pseudotypes bearing the indicated SUs were used to infect NIH 3T3 target cells and assayed for luciferase expression at 2 (left panel) or 3 days postinfection (right panel). Black columns, 1:2 dilution; grey columns, 1:6 dilution. All samples were assayed in triplicate, and infectivity is expressed as relative light unit (RLU) output. (B) Transfected 293 cells used to produce the MuLV/luciferase pseudotypes analyzed in Fig. 2A were labeled with [³⁵S]cysteine, and the secreted virions were pelleted by centrifugation and assayed for SU and CA content by precipitating with polyclonal sera specific for these viral proteins, separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and visualized by autoradiography. (C) Pulse-chase analysis of wild-type and defective Fr/AKR (393-426) Env loop 10 mutants. 293 cells transfected with plasmids expressing the indicated env genes were pulse-labeled at 24 h with [³⁵S]cysteine for 30 min and chased for the indicated time points in hours, and cell lysates were precipitated with anti-SU polyclonal serum followed by SDS-PAGE. Pre, Env precursor.

FIG. 4.

A. Analysis of binding and fusion activity of MuLV pseudotypes containing Friend MuLV clone 57 (Friend 57) wild-type, Fr/AKR(NLV), and Fr/AKR(NLV)S405+S Env proteins. (Left panels) Pseudotyped particles were incubated at the indicated serial dilutions with 293.mCAT cells, and the extent of binding of virions was quantitated by flow cytometry. (Right panels) XC cells were overlaid with the indicated MuLV pseudotypes, and syncytium formation was analyzed by microscopy (photographed at a magnification of ×400). B. Measurement of receptor binding and envelope expression of the highly defective S405T+T loop 10 mutant. (Left) Flow cytometric measurement of binding of MuLV pseudotypes bearing Fr/AKR(NLV) wild-type and Fr/AKR(NLV) S405T+T mutant Env proteins to 293.mCAT cells. Particles without any Env proteins [(−) Env] were included as controls. (Right) Analysis of virion protein content for mutant S405T+T Env and wild-type Fr/AKR(NLV) pseudotypes. Particles in supernatant medium were pelleted through 20% sucrose, lysed, and analyzed for SU and CA content by Western blotting. Fivefold serial dilutions of the samples were probed by polyclonal anti-SU serum and anti-CA serum.

FIG. 5.

Denaturation-induced isomerization of the intersubunit disulfide bond. [³⁵S]cysteine-labeled NLV virions with (+) and without (−) pretreatment with MAb 83A25 (left gels) and NLV S504T+S virus (right gels) were exposed to 1% NP-40 or 2 M urea with (+) and without (−) prior treatment with NEM. All samples were treated with NEM prior to immunoprecipitation with polyclonal anti-SU serum and analyzed by nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The film exposure time shown for the NLV S504T+S samples was four times longer than for the NLV samples to adjust for lower incorporation of [³⁵S]cysteine.