Functional characterization of the N termini of murine leukemia virus envelope proteins

Abstract

The function of the N terminus of the murine leukemia virus (MuLV) surface (SU) protein was examined. A series of five chimeric envelope proteins (Env) were generated in which the N terminus of amphotropic 4070A was replaced by equivalent sequences from ecotropic Moloney MuLV (M-MuLV). Viral titers of these chimeras indicate that exchange with homologous sequences could be tolerated, up to V17eco/T15ampho (crossover III). Constructs encoding the first 28 amino acids (aa) of ecotropic M-MuLV resulted in Env expression and binding to the receptor; however, the virus titer was reduced 5- to 45-fold, indicating a postbinding block. Additional exchange beyond the first 28 aa of ecotropic MuLV Env resulted in defective protein expression. These N-terminal chimeras were also introduced into the AE4 chimeric Env backbone containing the amphotropic receptor binding domain joined at the hinge region to the ecotropic SU C terminus. In this backbone, introduction of the first 17 aa of the ecotropic Env protein significantly increased the titer compared to that of its parental chimera AE4, implying a functional coordination between the N terminus of SU and the C terminus of the SU and/or transmembrane proteins. These data functionally dissect the N-terminal sequence of the MuLV Env protein and identify differential effects on receptor-mediated entry.

FIG. 1

Generation of chimeric MuLV Env N terminus. Ecotropic envelope protein sequences were introduced to replace the equivalent amino acids of the amphotropic Env N terminus. Five chimeric ecotropic-amphotropic (EA) junctions at the N terminus were generated. They were examined in amphotropic 4070A and AE4 backbones, yielding five EA chimeras and five EAE chimeras. The constructs were numbered according to the location of junction points, starting from the N terminus. The alignment of ecotropic and amphotropic N termini is shown in the middle, and the N-linked glycosylation sites are boxed. The sequence of each chimeric N terminus is shown. The ecotropic sequence is underlined. The two boxes at the top illustrate the SU protein; the ecotropic sequence is shaded. The general features of the SU protein are indicated in the open box of the amphotropic part: VRA, VRB, the hinge region (H), and the PRR.

FIG. 2

Infectivity with N-terminal chimeric envelope proteins. Viruses bearing chimeric Env proteins were examined for their abilities to infect canine D17 cells. Viral supernatants were collected from TELCeB6 cells transiently transfected with the env constructs indicated, diluted with media, and incubated with D17 cells in the presence of Polybrene. Shown are titers of N-terminal chimeras in an amphotropic 4070A Env backbone (A) and equivalent chimeras in an AE4 backbone (B). Titers are presented as LacZ infectious units per milliliter of viral supernatant. The average from a triplicate experiment is shown with the error bar indicating standard deviation. The experiment was performed five times, and a representative set is shown.

FIG. 3

Surface expression of chimeric envelope proteins on TELCeB6 cells. TELCeB6 cells were transiently transfected with vectors expressing chimeric Env proteins. The names of individual constructs are in the right upper corner of each panel. Each gray line represents the level of fluorescence of mock-transfected cells. The expression of each construct is shown by a black line. The x axis is the fluorescence intensity detected for cell surface-associated Env protein. The y axis represents the cell number counted.

FIG. 4

Western blot analysis of virus-associated SU proteins. Env proteins were expressed and metabolically labeled in D17 gag-pol cells. Viruses were isolated, and virus-associated SU proteins were analyzed by Western blotting as described in Materials and Methods. Proteins were separated on SDS–10% polyacrylamide gels and probed with anti-SU antibody 80S-019. The individual constructs are indicated above each lane. Mock transfection served as an Env⁻ control for the D17 gag-pol cells.

FIG. 5

Binding of chimeric envelope proteins on target cells. Viral supernatants were collected from D17 gag-pol cells transiently transfected with chimeric env expression vectors, and the binding was tested on 293T cells. The level of binding was analyzed by flow cytometry as described for the surface expression assay. The basal level of fluorescence determined for each mock-transfected cell supernatant for binding is shown by a gray line. Binding intensities of each Env construct are shown by black lines. The mean channel number of the binding is shown at the upper right corner of each panel.

FIG. 6

N-terminal sequences on a molecular model of the amphotropic Env RBD. The proposed RBD structure was generated using the X look program, which is based on the X-ray crystal structure of the Friend MuLV RBD (22). VRA and VRB are shown in cyan and blue, respectively, to illustrate the spatial correlation with the N terminus. The sequence before crossover I (A1-H5) is not within the structure. Y8 and N9 within crossover II are shown in red. V10 to V14, between junctions II and III, are green. The region between III and IV (T15-T26) is orange. Sequences beyond crossover IV and before crossover V are yellow. Amino acids between junction III and junction IV form a loop adjacent to VRA. The amino acids that could mediate contacts between VRA and this loop are labeled.