The receptors for gibbon ape leukemia virus and amphotropic murine leukemia virus are not downregulated in productively infected cells

Abstract

Background: Over the last several decades it has been noted, using a variety of different methods, that cells infected by a specific gammaretrovirus are resistant to infection by other retroviruses that employ the same receptor; a phenomenon termed receptor interference. Receptor masking is thought to provide an earlier means of blocking superinfection, whereas receptor down regulation is generally considered to occur in chronically infected cells.

Results: We used replication-competent GFP-expressing viruses containing either an amphotropic murine leukemia virus (A-MLV) or the gibbon ape leukemia virus (GALV) envelope. We also constructed similar viruses containing fluorescence-labeled Gag proteins for the detection of viral particles. Using this repertoire of reagents together with a wide range of antibodies, we were able to determine the presence and availability of viral receptors, and detect viral envelope proteins and particles presence on the cell surface of chronically infected cells.

Conclusions: A-MLV or GALV receptors remain on the surface of chronically infected cells and are detectable by respective antibodies, indicating that these receptors are not downregulated in these infected cells as previously proposed. We were also able to detect viral envelope proteins on the infected cell surface and infected cells are unable to bind soluble A-MLV or GALV envelopes indicating that receptor binding sites are masked by endogenously expressed A-MLV or GALV viral envelope. However, receptor masking does not completely prevent A-MLV or GALV superinfection.

Figure 1

A schematic representation of the viruses used in this study. A-MLV-GFP and GALV-GFP are replication-competent MoMLV in which the MLV envelope (env) gene has been replaced with either A-MLV [14] or GALV env [14,15]. Both viruses contain an IRES-GFP cassette between the env gene and 3'LTR. In addition, GALV-GFP also contains an insertion of TCC just upstream of the splice acceptor (SA) resulting in a virus with enhanced infection and replication properties [15]. GALV-GFP-C11D8 is identical to GALV-GFP except that the C11D8 epitope tag (QVMTITPPQAMGPNLVLP) that derives from the amino acid terminus of the FeLV-B proline rich region (PRR) was introduced into the GALV PRR [37]. The relative position of PRR within SU and transmembrane (TM ) subunits of GALV envelope protein is shown. GALV-Gag tomato red was generated by replacing GFP of GALV-GFP with Gag fused in frame to fluorescent tomato red gene in the GALV-GFP plasmid. The retroviral vector plasmid, pRT43.2 βgal contains a CMV immediate early enhancer/promoter in the 5' LTR as well as a β-galactosidase reporter gene.

Figure 2

Representative flow cytometric analyses carried out on control uninfected and GALV-GFP-C11D8 infected MDTF cells expressing the HA-tagged GALV receptor SLC20A1 cells. The cells were stained with monoclonal antibodies against V5, HA and C11D8 epitopes as well as R-phyoerythrin conjugated goat anti-mouse isotope specific secondary antibodies. In histograms, solid purple represents control groups; blue lines represent uninfected MDTFSLC20A1-HA cells; red lines represent MDTFSLC20A1-HA cells infected with GALV-GFP-C11D8 viruses. The relative amounts of cell surface detected V5-tagged GALV RBD (A), HA-tagged SLC20A1 (B) GALV envelope tagged with C11D8 epitope (C) and V5-tagged A-MLV RBD (D) are shown on the x-axis. In these experiments, we employed MDTF or CHOK1 cells as negative controls (data not shown). The experiment was performed for three independent times with similar results.

Figure 3

FACS analysis of SLC20A1-HA expression and GALV (C11D8) envelope associated with the surface of MDTFSLC20A1-HA cells chronically infected (one month-post exposure) with GALV-GFP-C11D8 is shown in histograms. The level of SLC20A1-HA expression (A) and the relative amount of GALV envelope glycoprotein (C11D8) bound to the cells (B) on the surface of MDTFSLC20A1-HA cells uninfected or chronically infected with GALV-GFP-C11D8 viruses. We employed MDTF cells as negative controls for receptor detection and viral infection (data not shown). The experiment was performed three independent times, and images are from one representative experiment.

Figure 4

SLC20A2-HA expression, A-MLV envelope and soluble A-MLV or GALV RBD bound to the surface of CHOK1 cells expressing SLC20A2-HA or MDBK cells expressing SLC20A1-HA cells chronically infected with A-MLV-GFP (one month after infection) or uninfected control cells was assayed by FACS and displayed in histograms. The cells were stained with primary antibodies specifically against A-MLV (83A25) and HA and V5 epitopes. The corresponding secondary antibodies used are species and isotope specific and conjugated with R-phyoerythrin. Solid purple lines represent control groups; blue lines represent uninfected cells; red lines represent cells infected with A-MLV-GFP. V5 epitope tagged A-MLV RBD bound to CHOK1 expressing SLC20A2-HA cells (A) or to MDBK expressing SLC20A2-HA cells (B) and V5 epitope tagged GALV RBD bound to MDBK cells expressing SLC20A2-HA (C), The expression level of HA-tagged SLC20A2 on the surface of CHOK1 expressing SLC20A2-HA cells (D), or the relative amounts of A-MLV envelope bound to CHOK1 expressing SLC20A2-HA cells (E) are shown on the x-axis. The experiment was performed three times, and images are from one representative experiment.

Figure 5

SLC20A1 protein physically interacts with GALV envelope protein. MDTFSLC20A1-HA cells incubated with V5 tagged GALV RBD were lysed, co-immunoprecipitated with agarose beads covalently linked to V5 antibody and subjected to western blots probed with antibody to V5 or HA (A). Western blots of the cells cross-linked with BS3 and then immunoprecipitated with agarose beads covalently linked to V5 antibody and subjected to western blot analysis using antibody to V5 or HA as a probe (B).

Figure 6

GALV enveloped retroviral vectors expressing cherry red protein superinfecting MDTFSLC20A1-HA cells productively infected with GALV (GALV-GFP-C11D8) for one week (A) or one month (B). After 48 hours, the cells were harvested for FACs analysis and the densitograms (top panel) and the quadrant statistics are presented in the table at the bottom. The experiment was performed three independent times, and the representative analysis is presented.

Figure 7

Immunofluorescence confocal microscopy of superinfection of GALV infected cells. MDTFSLC20A1-HA cells infected with GALV-GFP-C11D8 for one month and then exposed to GALV enveloped vector expressing cherry red fluorescent protein. After 48 hours, cells were fixed and stained by C11D8 monoclonal antibody and dylight conjugated goat anti-mouse IgG antibody.

Figure 8

Nonspecific attachment of GALV to chronically infected MDTFSLC20A1-HA cells. MDTFSLC20A1-HA cells uninfected or chronically infected with GALV were adsorbed with fluorescently labeled GALV viruses (GALV-Gag tomato red). Images were taken at 63x magnification on a LSM510 invert Meta confocal microscope. The arrows point to the tomato labeled GALV particles. The images are representatives of three independent experiments.