A mutant retroviral receptor restricts virus superinfection interference and productive infection
© Liu and Eiden; licensee BioMed Central Ltd. 2012
Received: 2 March 2012
Accepted: 12 April 2012
Published: 12 June 2012
Both cell-free and cell-associated infection routes are important for retroviral dissemination. Regardless of the mechanism, the driving force of retroviral entry is the interaction between the viral envelope and its receptor. To date it remains unclear how decreased affinity of viruses for their receptors affects viral cell-free infection, cell-cell transmission, and spreading kinetics. We have previously characterized a mutant form of the amphotropic murine retrovirus receptor human phosphate transporter 2 (PiT2) wherein the single substitution of a glutamic acid for the lysine residue at position 522 of this receptor is sufficient to render it to function as a gibbon ape leukemia virus (GALV) receptor.
In this study we analyzed the binding affinity of the mutant receptor PiT2K522E and determined that it has a 1000 fold decreased GALV envelope binding affinity compared to the GALV wild type receptor. The decreased affinity does not restrict the initiation of cell-free GALV infection. The diminished binding affinity does, however, correlate with a decrease in the ability of GALV to spread in cells expressing this mutant receptor.
The reduced ability of GALV to subsequently spread among cells expressing PiT2K522E is likely resulted from reduced cell-cell transmission, the decreased ability of PiT2K522E-expressing cells to establish superinfection interference, and attendant cytopathic affects.
Both cell-free and cell-associated infections are important for retroviral dissemination. However, cell-associated viral infection is over a thousand fold more efficient in vivo . To enter cells, cell-free enveloped viruses bind to specific receptors on the target cell surface. They then penetrate the host cells by either direct fusion of viral and cellular lipid membranes or via an endocytotic pathway. Both entry pathways result in the release of the viral nucleocapsid into the cytoplasm [2–4]. Several mechanisms have been invoked for the transmission of virus from an infected cell to an uninfected cell. All involve the interaction of viral envelope in the membrane of the infected cell with the receptor on the uninfected target cell triggering a signal that causes cytoskeleton rearrangement. Several adhesion molecules are recruited to participate at the cell contact site to form a virological synapse and filopodial bridges [5–7].
The affinity thresholds that accompany the association of viral envelope proteins in the membrane of infected cells with their receptors required to trigger viral entry vary greatly. Influenza virus requires millimolar range affinity while human immunodeficiency virus (HIV) requires binding affinity in the nanomolar range . Higher affinities result in more efficient binding of a single viral particle to recruit several receptors thereby accelerating post-binding events that lead to membrane fusion and enhance efficiencies of viral entry. In cell-associated infection routes, envelope proteins are highly enriched on the infected cell interface. This enrichment allows more efficient recruitment of receptors and subsequent access to signaling proteins at levels that make cells more susceptible to viral replication. For example, this enrichment facilitates the utilization of actin-driven motion  and cellular proteins that interact with the host cell cytoskeleton  to support intracellular transport and membrane fusion events associated with viral entry. Decreased affinity between viral envelope and receptor has been reported to cause delayed viral replication kinetics and is linked to the failure to establish superinfection resistance, apoptosis, and induce syncytia formation [9–12]. However, it remains unclear how a decreased receptor affinity affects viral cell-associated infection and spreading kinetics.
As gammaretroviruses, amphotropic murine leukemia virus (A-MLV) and gibbon ape leukemia virus (GALV) have divergent host ranges and are not in the same interference class . The receptors for GALV and A-MLV encode distinct but related proteins originally designated GLVR1 and GLVR2 . Later, the GALV and A-MLV receptors were identified to function as type III inorganic phosphate transporters and renamed as PiT1 and PiT2 and are now referred to as SLC20A1 and SLC20A2 in accordance with their transporter classification. Herein, we use the PiT1 and PiT2 nomenclature for ease of cross-referencing. Previously, we reported that the PiT2 ortholog expressed on hamster E36 cells, HaPiT2, in contrast to the human form of the A-MLV receptor (PiT2), functions as a receptor not only for A-MLV, but also GALV . Based on comparison of the deduced amino acid sequences of the HaPiT2 and PiT2 proteins, it was eventually determined that the substitution of a single amino acid residue, glutamate (glutamic acid), for lysine residue at position 522 is sufficient to render PiT2 functional as a GALV receptor while retaining A-MLV receptor function. The titer of GALV enveloped retroviral vector is reduced 5 to 6 fold in cells expressing the mutant receptor PiT2K522E compared to those expressing the GALV receptor PiT1 [15, 16].
Although both PiT1 and PiT2K522E function to efficiently mediate transduction by GALV enveloped vectors, the ability of PiT2K522E to function as a receptor for replication-competent GALV has not been previously characterized. Surprisingly, PiT1 and PiT2K522E function very differently in their ability to bind GALV envelope, establish superinfection resistance and facilitate efficient GALV spread following exposure to cell-free virus.
Productive infection by GALV is severely restricted in cells expressing PiT2K522E compared to cells expressing PiT1
Even though CHOK1, like E36 cells, are derived from Chinese hamsters, these two cell types differ in their resistance to infection by GALV: CHOK1 cells are resistant to GALV [14, 18]. CHOK1 cells (Chinese hamster kidney cells) expressing either PiT1 or PiT2K522E employed in parallel studies showed similar results (data not shown).
PiT2K522E exhibits a markedly reduced ability to bind GALV compared to wild type PiT1
Nonspecific binding of murine leukemia virus (MLV) particles to cells has been previously reported [19–21]. To determine if the binding of GALV to MDTF cells expressing PiT2K522E is nonspecific, we examined GALV RBD binding to MDTF cells expressing PiT2 that are not susceptible to GALV infection. As shown in Figure 2B, binding of GALV RBD to MDTF cells expressing PiT2 was significantly lower than that achieved with MDTF cells expressing PiT2K522E. These results indicate that MDTF cells expressing PiT2K522E bind to GALV RBD at a substantially reduced affinity compared to MDTF cells expressing PiT1, but still at a level sufficient to facilitate GALV entry.
Cells expressing mutant receptor are not deficient in their ability to release infectious particles
Decreased GALV binding affinity to the mutant receptor leads to failure to establish superinfection resistance
GALV transmission from infected MDTFPiT1K522E to uninfected cells expressing PiT2K522E tagged with HA is not defective but is diminished
Summary of coculture experiments
GALV production cells
% recipient infected
MDTFPiT1 infected with GALV-GFP (90% GFP+)
MDTFPiT2K522E infected with GALV-GFP (35% GFP+)
Virological synapses were observed at a reduced frequency in GALV infected cells expressing PiT2K522E compared to those expressing PiT1
Syncytium formation is prominent in productively infected cells expressing PiT2K522E
Cell lines derived from Chinese hamster lung fibroblasts such as E36 cells are the only hamster cells susceptible to both GALV and A-MLV. They also express a PiT2 ortholog that functions as a GALV receptor . Cells expressing human PiT2 are not permissive for GALV infection nor does human PiT2 bind GALV RBD or GALV viral particles. Using chimeric E36 hamster PiT2 and human PiT2 proteins, we previously determined that a single residue difference at position 522 of PiT2 accounts for the ability of hamster PiT2 to facilitate GALV as well as A-MLV entry . This difference represents the presence of a glutamate residue in hamster PiT2 in place of the lysine residue present in PiT2. However, we have now determined that even though resistant cells such as murine MDTF cells expressing PiT2K522E, like MDTF cells expressing the human GALV receptor PiT1, are susceptible to GALV vectors, their ability to support infection by replication-competent GALV differ dramatically. In this report, we have attempted to determine what accounts for these differences.
The reduced affinity of the PiT2K522E mutant receptor to GALV compared to wild type PiT1 correlates with a reduced capacity for viral spread (Figures 1 and 2). We have shown that GALV infectious particles produced in cells expressing PiT2K522E are released into the supernatant at levels comparable to PiT1 expressing producer cells (Figure 3). This observation together with the dramatically reduced ability of PiT2K522E to bind GALV envelope suggests that infectious GALV particles are efficiently produced from cells bearing this mutant receptor and that the reduction in cell-to-cell transmission is not mediated by reduced virus production or particle stability. The weak affinity of GALV for target cells bearing PiT2K522E may limit virus spread due to a failure to recruit sufficient levels of receptor and induce conformation changes necessary to mediate viral uptake via viral synapse formation . The most efficient means of retrovirus spread following exposure to cell-free virus involves the transfer of viral particles from infected cells to uninfected target cells via viral synapses formed at the interface of these two types of cells [6, 7, 23–25]. GALV producer cells co-cultured with MDTFPiT1 efficiently triggered the formation of virological synapse mediating the apparent transfer of virus from one donor to, on average, three recipient cells in co-culture experiments. Similar experiments carried out with PiT2K522E expressing recipient cells showed a reduction in the ratio of donor to recipient cells (e.g., 1:1) and a reduction in synapse formation (Figure 7). Efficient cell-cell transmission is dependent on the formation of virological synapses or filopodia bridges that, in turn, depends on virus being efficiently maintained on the cell surface by receptors prior to extracellular release or retained in cells that fail to release mature viral particles . Cells expressing PiT2K522E form virological synapses or filopodia bridges less efficiently than cells expressing PiT1and this may account for the reduced levels of productively infected cells (Figure 1). We have shown previously, that cell-free GALV pseudotyped retroviral vectors transduce cells expressing PiT2K522E five times less efficiently than cells expressing PiT1 . The results presented here show that cell to cell transmission is also diminished in PiT2K522K infected cells indicating that reduced receptor binding affinity affects both GALV cell-free and cell to cell transmission.
Altered envelope receptor binding kinetics attributed to specific residue changes in the envelope has been shown to affect the cell fusion process and the spectrum of disease associated with ecotropic murine and feline retroviruses. It has been reported that adaptive changes in the envelope protein of gammaretroviruses that confer changes in receptor binding properties (compared to their prototypic equivalents) correlate with altered disease properties. A recent report characterized a feline leukemia virus subgroup A (FeLV A) isolate, FeLV-945, that causes an uncharacteristic disease and demonstrated that it has a significantly greater receptor binding capacity than the prototypic FeLV-A, FeLV61E . It was proposed that this enhanced receptor affinity might serve to increase the kinetics of virus spread and render target cells with reduced receptor numbers susceptible to infection in vivo thereby altering the disease status of infected cats. At the opposite end of the spectrum, a finding similar to the results presented here, has been reported by S.L. Murphy et al. . This group found a linkage of specific residue changes in the ecotropic MLV ENV (TR1.3) that lead to decreased receptor affinity, the loss of superinfection resistance, syncytium formation  and attendant pathology.
In this report, we demonstrate that a single virus, GALV, can exhibit distinct receptor affinity, superinfection interference properties, syncytium formation and viral spreading capacities depending on the receptor that is employed to enter cells. Thus the receptor ortholog or the envelope protein can mediate dramatic differences in receptor dependent properties with as little as a single residue difference as shown in this paper and previous studies . The cell lines expressing low affinity and high affinity GALV receptors (MDTFPiT2K522E and MDTFPiT1, respectively) employed in the analysis of viral infection and spread are useful tools in dissecting specific stages critical to cell-cell transmission. These cells should prove valuable for in vitro testing of reagents designed to prevent cell mediated viral spread.
Cell lines used in this study include Mus dunni tail fibroblasts MDTF , Chinese hamster ovary (CHOK1) cells, CCL61, (ATCC), and 293 T (formerly referred to as TSA cells) . All cells, with the exception of CHOK1, were maintained in Dulbecco’s modified Eagle’s medium with Glutamax (DMEM) (Invitrogen), supplemented with 10% fetal bovine serum, 100 units of penicillin/ml, and 100 ug/ml of streptomycin. CHOK1 cells were maintained in alpha minimal essential medium (MEM) supplemented with 10% fetal bovine serum, 100 units/ml, of penicillin and 100 ug/ml of streptomycin (Invitrogen). The GALV viral receptor PiT1, A-MLV viral receptor PiT2 and mutant receptor PiT2K522E all tagged with a hemagglutinin (HA) eptitope were generated as previously described . Vesicular stomatitis virus (VSV) G protein-enveloped retroviral vector pLNSX  expressing the appropriate receptors was used to transduce respective cell lines and G418 allowing selection of cells stably expressing these receptors.
The GALV-GZAP (generously provided by Christopher Logg, University of California, Los Angeles, CA,) is replication-competent ecotropic Moloney MLV backbone in which the GALV env gene replaces the MLV env gene. In addition an IRES driven GFP (IRES-GFP) is positioned between the 3′terminus of the env gene and untranslated region of 3′LTR . The GALV-GZAP variant, MSA2-GFP  contains an insertion of TCC at the MLV splice acceptor and was shown to replicate more efficiently than GALV-GZAP and was thus used as the GFP expressing GALV enveloped virus in this study. We further modified the MSA2-GFP plasmid removing the IRES-GFP fragment and this construct was used to produce GALV enveloped virus that does not express GFP. The GALV RBD fragment was inserted into the pcDNA3.1-V5-His B plasmid (Invitrogen). Fragments encoding envelope residues 1 to 219 of GALV RBD were amplified by PCR from the GALV envelope subclone  using primers containing EcoRI and XbaI at their 5′ and 3′ termini, respectively. The EcoRI and XbaI-digested PCR product was ligated into the pcDNA3.1-V5-His B plasmid. To construct a vector genome expressing fluorescent cherry red protein, RFP, the GFP gene in the retroviral vector, pRT43.2 GFP  was removed by digesting with PmlI (blunted) and NotI and this fragment was removed and replaced with RFP gene that was obtained from CMV-brainbow 1.1 M plasmid (Addgene, Inc.) cleaved by BglII (blunted) and NotI. All constructs generated by PCR were verified by nucleotide sequence analyses.
Production of replication-competent retroviruses, retroviral vectors and GALV RBD
The GALV enveloped replication-competent retroviruses (GALV-GFP and GALV), GALV enveloped retroviral vectors as well as GALV RBD were produced by calcium phosphate transfection of 293 T cells (Promega Corporation) as previously described . Supernatants were harvested 48 to 72 h post-transfection, then passed through a 0.45 mM Millipore (Millipore Corporation) and stored at −80 °C. GALV RBD purification was performed following manufacturer’s instruction (Invitrogen) with modifications. Supernatants that contain GALV RBD tagged with V5 and 6xHis epitope were concentrated greater than 10-fold using Amicon Ultra-15, centrifugal filters (Millipore Corporation), then mixed with 40 ml binding buffer (1x Hanks Balanced Salt Solution (HBSS) (Mediatech, Inc.) containing 10 mM imidazole (ACROS). Five ml samples were loaded onto the high-performance nickel-NTA (Ni-NTA) agarose column (Invitrogen), unbound proteins were removed with washing buffer (1x HBSS containing 20 mM imidazole) and bound GALV RBD was eluted with buffer (1x HBSS containing 250 mM imidazole) and collected. The concentrated GALV RBD was passed through Econo-Pac 10DG desalting column to remove imidazole (BioRad Life Science). The purified GALV RBD was quantified using the BCA protein assay kit (Pierce) and stored in aliquots at −80 °C.
Co-culture of GALV infected cells with target cells
MDTFPiT1 or MDTFPiT2K522E cells were exposed to replication-competent GALV-GFP viruses. One week after initial exposure, infected cells were mixed with an equivalent number of uninfected MDTFPiT1-HA or MDTFPiT2K522E-HA cells. Thirty hours after co-culture, HA-tagged receptors were detected on the cell surface by incubation of MDTF cells expressing receptors with monoclonal HA antibody HA.11 (Covance Inc.), followed by incubation with a RPE conjugated secondary antibody (Santa Cruz biotechnology) and then analyzed by flow cytometry.
Production of GALV-gag-YFP viruses and visualization of virological synapses
Approximately 2×105 293 T cells were exposed to replication-competent GALV viruses for one week, grown overnight on a 35 mm tissue culture dish with a 0.17 mm thick glass bottom, and then transfected with 0.2ug MLV-gag-YFP plasmid. MLV-gag-YFP consists of MLV gag protein fused in frame to YFP at the nucleotides encoding the residues PQ at present at the C-terminus of its gag nucleocapsid domain (Addgene Inc.). Sixteen hours later, these cells were incubated with 2×105 uninfected MDTFPiT1-HA, MDTFPiT2K522E-HA or MDTFPiT2-HA cells labeled with CMAC cell tracker blue (Invitrogen) according to manufacturer’s instructions. The de novo synthesized gag-YFP proteins in the infected 293 T cells were recruited and used in GALV assembly to produce GALV virions that contain YFP labeled gag at cell-cell contact site. Six hours after co-culture at 37 °C, cells were fixed with 2% paraformadehyde in PBS solution and visualized on an LSM510 inverted Meta confocal microscope (Carl Zeiss, Thornwood, NY) with a 63× 1.4 NA oil immersion objective. CMAC was excited with a 405 nm laser, YFP with the 514 nm line of a krypton/argon laser. YFP was imaged with a 530–600 nm bandpass filter, CMAC with a 420–500 nm bandpass filter.
Epics XL (Beckman Coulter, Fullerton, CA) and FACScan (Becton Dickinson, Franklin Lakes, NJ) flow cytometers were used to assess expression of GFP, RFP, HA and V5 tagged proteins in infected and transfected cells using a 525 nm or a 530 nm band pass emission filter. 20,000 cells from each sample were analyzed after trypsinization and suspension in HBSS. Propidium iodide (Sigma) was added to cell suspension at a concentration of 1ug/ml to exclude dead cells in FACS analysis.
We thank Karen B. Farrell for technical advice; Jim Nagle and Debbie Kauffmann at the NINDS Sequencing Facility (National Institutes of Health) for sequencing; Parisa Moghaddam-Taaheri for experimental help; Jon Marsh and Jill Russ for supervising FACS operation and Vincent Schram and Lynne Holtzclaw in the Microscropy & Imaging Core, NICHD, NIH for technical guidance in confocal microscopy, Phil McCoy in the NHLBI Flow Cytometry Core for technical support in cell cycle analysis, and Wenqin Xu for her thoughtful comments and editorial assistance. This work was supported by NIMH Intramural funding.
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