INTRODUCTION

Top Abstract Introduction Materials & Methods Results Discussion References

Entry of an enveloped virus into its host is determined by a specific interaction between receptors on the cell surface and envelope glycoproteins on the virion. For most retroviruses, this process is pH independent; therefore, it has been hypothesized that for these viruses, receptor binding serves to trigger a series of conformational changes on the envelope protein required for viral-host membrane fusion. Thus, the receptor not only is likely to provide a binding site for the viral glycoprotein but also may initiate the conformational changes thought to be required for entry.

Infection of subgroup A avian leukosis and sarcoma virus (ALSV-A) is mediated by the cellular receptor, Tva, on avian cells (4, 23). Tva is a small, relatively simple protein with two functional isoforms, Tva 950 and Tva 800, that are produced by differential splicing (4). Tva 950 is tethered by a typical membrane-spanning domain near the carboxyl terminus of the protein, whereas Tva 800 appears to be anchored by a glycosylphosphatidylinositol tail. Within the extracellular region of both isoforms, near the amino terminus of Tva is a 40-residue, cysteine-rich motif that is closely related to a sequence in the low-density lipoprotein receptor (LDLR) (4). In LDLR, this module is repeated seven times and constitutes the ligand binding region of the protein (11, 22). The 40-residue LDLR-like module from Tva forms the ASLV-A interaction domain. It is sufficient for viral receptor function (19), and mutations within this module affect ASLV-A receptor function and envelope binding (5, 24, 25). A peptide corresponding to the 19 carboxyl-terminal residues of the Tva module inhibits ASLV-A entry, leading to the suggestion that this region comprises the receptor's viral interaction domain (24).

Tva specifically binds to ALSV-A envelope protein (7, 13), induces conformational changes in EnvA (10, 14, 15), and when incorporated into viral particles can direct infection of ASLV-A envelope (EnvA)-expressing cells (2). Together these results strongly support a direct role for Tva in triggering ALSV-A envelope-mediated entry. Recently, a tryptophan residue (W48) near the carboxyl terminus of Tva's LDLR module was reported to be specifically involved in triggering the conformational changes of ALSV-A envelope during viral entry (25). Additionally, W48 was proposed to be part of a common retroviral receptor motif consisting of an aromatic and adjacent acidic residue found in Tva and receptors for ecotropic murine leukemia virus and human immunodeficiency virus (17, 24).

To define further the requirements for Tva receptor function, we produced deletion and substitution mutations throughout the 40-residue LDLR module of Tva and analyzed separately the effect of these changes on EnvA binding and ASLV-A entry. We found that efficient EnvA binding requires an intact LDLR module since the majority of substitution or deletion mutations analyzed severely impaired envelope binding. However, the amino-terminal 17 residues of Tva's LDLR module could be deleted without significant impact on viral receptor function, suggesting that while this region contributes to EnvA binding, determinants sufficient for ASLV-A entry localize to the carboxyl-terminal 23 residues of Tva's LDLR module. Because of the proposed importance of residues 46 to 48 (DEW), an extensive mutational analysis of these three residues was also performed. Our analysis of 27 substitution mutants in residues 46 to 48 of Tva does not support the proposed model for a common motif in retroviral receptors, nor did we find evidence for a specific involvement of W48 in post-EnvA binding events. The effect of the mutations in Tva are discussed in relation to the recently elucidated structure for a module from the human LDLR (12).

	MATERIALS AND METHODS

Top Abstract Introduction Materials & Methods Results Discussion References

Cells and viruses. Human embryonic kidney 293T cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% calf serum and 300 µg of Geneticin per ml. Quail QT6 cells were grown in medium 199 containing 10% tryptose phosphate broth, 5% fetal calf serum, and 1% chicken serum. HeLa cells were grown in a spinner flask in Joklik's modified medium with 10% calf serum.

DNA methodology. All Tva mutants were generated in the quail Tva 950 background. Tva substitution and deletion mutants were constructed by using two rounds of PCR as previously described (19).

Protein expression and Western blotting. Transfection and transient expression of Tva mutant DNAs in 293T and QT6 cells were done as described previously (4, 19). The Western blots were probed with an anti-Tva polyclonal antiserum as described previously (4).

Infection. Tva mutants were transiently expressed in 293T cells by CaPO₄ transfection. Twenty-four hours after transfection, the cells were split into six-well plates for infection and 100-mm-diameter plates for analysis of protein expression. To determine the level of susceptibility conferred by a particular Tva mutant, a series of 10-fold dilutions of RCAS(A)AP was used to infect the transfected cells. Alkaline phosphatase (AP)-positive cells were detected and enumerated 48 h posttransfection as described previously (19).

Envelope binding assay. The ability of Tva mutants to block soluble wt Tva (sTva) binding to envelope was measured by a blocking ELISA. In this assay, gD-EnvA-His₆ was captured onto wells coated with monoclonal antibody 1D3. The wells were washed, and 10 µl of cell lysate containing wt Tva or mutant proteins was allowed to bind for 1 h at 4°C. After washing, 5 ng of biotin-labeled sTva was added, and the mixture was incubated at 4°C for an additional hour. After washing, bound sTva was detected by using streptavidin-horseradish peroxidase and 2,2'-azinobis(3-ethylbenzothiazolinesulfonic acid) (ABTS). Plates were read at room temperature in a Molecular Dynamics ELISA reader at 405 nm. Percent inhibition was calculated by the formula % inhibition = 100  100(S/T), where S is the test sample and T the total binding (no competitor). All binding experiments were performed at least three times in triplicate except for the wt Tva titration curve, which was performed twice in triplicate.

Immunofluorescence detection of Tva surface expression. Tva mutants were transiently expressed in QT6 cells by CaPO₄-mediated transfection. Twenty-four hours posttransfection, expression of wt or mutant proteins was detected by immunofluorescence as previously described (18), using a polyclonal anti-Tva antibody at a 1:250 dilution (4).

	RESULTS

Top Abstract Introduction Materials & Methods Results Discussion References

Substitution of regions from other LDLR-like repeats in Tva. A 40-residue region of Tva which is highly homologous to the modules that comprise the human LDLR ligand binding domain is sufficient for ASLV-A receptor function (19). Figure 1A shows a cartoon of Tva's LDLR-like module based on the recently reported disulfide bonding patterns of human LDLR modules 1, 2, and 5 (8, 9, 12). Three pairs of disulfide bonds connecting cysteines 1 and 3, 2 and 5, and 4 and 6 of Tva create a looped structure. To begin analyzing viral receptor function of the Tva LDLR motif, a series of conservative mutations of quail Tva 950 (4) was made by substituting residues between adjacent cysteines with the corresponding residues from other LDLR family members (Fig. 1B, C1C2, C2C3, C3C4, C4C5, and C5C6). For example, the residues between C2 and C3 of Tva (SEPPGAHGE) were replaced with those from a LDLR-like repeat of human heparin sulfate proteoglycan (HSGH) to create mutant C2C3. This strategy was taken for two considerations. First, we hypothesized that since other LDLR repeat-containing proteins do not function as ALSV-A receptors, then residues that determine the specificity of the Tva LDLR motif are likely to be nonconserved. Substitution of the nonconserved residues might enable identification of residues important for Tva viral receptor function. Second, it seems likely that conserved residues also play a structural role; therefore, alteration of these residues could abrogate folding. Thus, we initially avoided changes in the conservative residues.

View larger version (17K): [in this window] [in a new window]   FIG. 1.   Tva LDLR module and effects of homolog substitutions and deletions on viral receptor function. (A) Cartoon of the Tva LDLR module. Amino acids of the Tva LDLR module are labeled, and the proposed three disulfide bonds based on human LDLR repeats 1, 2, and 5 are indicated. The highly conserved residues found in most LDLR modules are shaded. (B) Sequences of the Tva LDLR module homolog substitution and deletion mutants. The functions of these mutants as viral receptors as assayed by infection with RCAS(A)AP are shown at the right. Values represent positive AP-staining cells per milliliter of viral stock [RCAS(A)-AP vector] used.

View larger version (25K): [in this window] [in a new window]   FIG. 2.   Expression and EnvA binding properties of Tva LDLR module homolog substitution and deletion mutants. (A) Transient expression of Tva mutants in 293T cells. Cell lysates from 293T cells transiently transfected with Tva mutant plasmids were analyzed by SDS-PAGE and Western blot analysis using a polyclonal antiserum against Tva protein. 293T, mock-transfected cells. Sizes are indicated in kilodaltons. (B) EnvA binding activity of Tva mutants measured by blocking ELISA. Cell lysates (10 µl) from 293T cells transiently expressing the mutant Tva proteins were used in the blocking ELISA as described in Materials and Methods. Percent inhibition of labeled sTva binding was calculated as described in Materials and Methods. Percent inhibition for each sample is the average from three experiments, each performed in triplicate. Bars indicate standard deviation. (C) Relative envelope binding activities of Tva mutants C1C3, C3C5, and C45/C1C3 with increasing amount of cell lysates measured by blocking ELISA, expressed as percent inhibition. Percent inhibition for each sample is the average from three experiments, each performed in triplicate. Bars indicate standard deviation.

Deletion mutants in the LDLR motif of Tva. Next, we examined whether the entire LDLR motif of Tva was required for efficient viral receptor function by deletion analysis. The deletions in Tva were designed to maintain an even number of cysteine residues and thus the potential to form disulfide bonds. Mutant C1C3 contains a deletion of the first 17 residues including the first two cysteine residues of the LDLR motif, while C3C5 has a deletion of 13 residues including cysteines 3 and 4. An additional mutant, C45/C1C3, combines the substitution mutant C4C5 with C1C3 (Fig. 1B).

Effects of Tva mutations on envelope binding. To directly analyze the effects of the mutations in Tva on envelope binding, we used an ELISA-based receptor-envelope binding assay and tested the ability of each of the mutants to block the binding of labeled sTva to EnvA. In this blocking assay, lysates from cells expressing the Tva mutants were first incubated with EnvA that had been captured on the ELISA plate; then after washing, 5 ng of labeled sTva was added. Therefore, if a Tva mutant can form a stable complex with EnvA, then binding of biotin-labeled sTva should be blocked.

View larger version (12K): [in this window] [in a new window]   FIG. 3.   Analysis of EnvA binding by wt Tva by using a blocking ELISA. The ability of Tva in a cell lysate to bind EnvA was indirectly analyzed by determining the level of inhibition of labeled sTva binding. Increasing amounts of lysate from 293T cells expressing wt Tva were incubated with captured gD-EnvA before biotin-labeled sTva was added in the blocking ELISA protocol as described in Materials and Methods. Each experiment was performed in triplicate, and the standard deviation was less than 10% of the mean optical density reading.

Roles of residues D46, E47, and W48 in viral receptor function of Tva. Residues D46, E47, and W48 near the carboxyl terminus of the Tva LDLR motif were previously proposed to be the critical determinants of Tva receptor function (24), and W48 was postulated to be involved in postbinding entry events (25). Because of the proposed critical role of these residues, a random mutagenesis strategy was used to alter each of these residues. The resulting mutants were examined for effects on EnvA binding and receptor function. Twenty-seven individual substitution mutations in residues D46, E47, and W48 of Tva were generated as described in Materials and Methods. The mutant receptors were transiently expressed in 293T cells by transfection and analyzed for Tva expression, ASLV-A receptor function, and EnvA binding as described above.

D46. Seven substitutions for residue D46 were generated and included a basic (R), an aromatic (Y), three nonpolar (G, A, and I), and two polar (C and S) residues. All the mutants were expressed well in 293T cells compared to wt Tva (Fig. 4A).

View larger version (26K): [in this window] [in a new window]   FIG. 4.   Analysis of Asp46 mutants of Tva. (A) Expression of Tva Asp46 mutants in 293T cells analyzed by Western blotting with polyclonal serum to Tva. Sizes are indicated in kilodaltons. (B) Blocking ELISA (10 µl of each cell lysate) was performed to measure the EnvA binding activity of Tva Asp46 mutants, expressed as percent inhibition of labeled sTva binding. Percent inhibition for each sample is the average from three experiments, each performed in triplicate. Bars indicate standard deviation.

E47. Ten substitution mutants for E47 were generated and included two aromatic (Y and F), two basic (H and R), three nonpolar (A, I, and P), and three polar (T, S, and N) residues. All of these mutants were expressed in 293T cells at a level comparable to that of wt Tva (Fig. 5A). Like the D46 substitutions, most of the E47 mutants displayed little or no EnvA binding capability, inhibiting sTva binding by less than 10% when 10 µl of lysate was used (Fig. 5B). As discussed above, this result implies that these mutants are severely defective for EnvA binding compared to wt Tva. A single D47 mutant, E47F, retained detectable EnvA binding activity, inhibiting sTva binding by about 45% when 10 µl of lysate was used. These results suggest that maintenance of a glutamate at this position is important for high levels of EnvA binding activity.

View larger version (34K): [in this window] [in a new window]   FIG. 5.   Analysis of Glu47 mutants of Tva. (A) Expression of Tva Glu47 mutants in 293T cells analyzed by Western blotting with antiserum to Tva. Sizes are indicated in kilodaltons. (B) Blocking ELISA (10 µl of each cell lysate) was performed to assess the EnvA binding activity of Tva Glu47 mutants, expressed as percent inhibition. Percent inhibition for each sample is the average from three experiments, each performed in triplicate. Bars indicate standard deviation.

W48. Ten substitution mutants of W48, including two basic (R and H), one acidic (D), four nonpolar (I, V, A, and L), and three polar (S, N, and T) residues, were analyzed. The expression level and pattern of modification of these mutants were similar to that of wt Tva (Fig. 6A).

View larger version (32K): [in this window] [in a new window]   FIG. 6.   Analysis of Trp48 mutants of Tva. (A) Expression of Tva Trp48 mutants in 293T cells analyzed by Western blotting with antiserum to Tva. Sizes are indicated in kilodaltons. (B) Blocking ELISA (10 µl of each cell lysate) was performed to assess the EnvA binding activity of Tva Trp48 mutants, expressed as percent inhibition. Percent inhibition for each sample is the average from three independent experiments, each performed in triplicate. Bars indicate standard deviation.

Titration of selected DEW mutants in blocking ELISA. To further assess the EnvA binding activities of the DEW mutants, a selected group of these mutants was analyzed over a range of input receptor concentration. These include mutants with high levels of receptor function (D46G, E47F, E47A, W48L, and W48R) and functionally impaired receptors (D46A, E47P, W48H, and W48A). As seen previously, there was a sharp response to input wt Tva such that with 1 µl of lysate EnvA, binding was saturated. Similarly, the inhibition by E47F appears to be responsive to the input amount of Tva; however, the highest level of inhibition achieved by E47F is significantly lower than for that of wt Tva when 100 times less receptor protein is used. None of the other eight mutants tested (D46A, D46G, E47A, E47P, W48A, W48H, W48L, and W48R) displayed detectable binding activity when the maximum amount of lysate (10 µl) was used (Fig. 7). These results suggest that even the most active DEW substitution mutant, E47F, has over 100-fold-lower EnvA binding activity compared to wt Tva and that the other substitutions at these three residues have significantly greater than a 100-fold decrease in EnvA binding. Thus, these three residues are critical for efficient envelope binding.

View larger version (22K): [in this window] [in a new window]   FIG. 7.   Effect of titration of selected Tva DEW mutants on EnvA binding. Various amounts of lysates from 293T cells expressing DEW mutants of Tva (0.1 to 10 µl) were used in the blocking ELISA. Percent inhibition of sTva binding to captured EnvA for each sample is the average from three independent experiments, each performed in triplicate. Bars indicate standard deviation.

Surface expression of Tva mutants by immunofluorescence. We examined whether those Tva mutants that did not mediate efficient viral infection expressed Tva protein on the cell surface by immunofluorescence. Tva mutants C34/56, D46R, D46C, E47R, and E47P that either did not mediate viral infection or functioned extremely poorly as viral receptors in the infection assay (Fig. 1A and Table 1) were transiently expressed in QT6 cells. Cells were fixed with 95% ethanol-5% acetic acid either before (intracellular expression) or after (extracellular expression) a rabbit anti-Tva polyclonal antibody was incubated with the cells. Bound anti-Tva antibody was visualized by incubating fixed cells with a fluorescein isothiocyanate-conjugated secondary anti-rabbit antibody. Expression of both intracellular and extracellular wt Tva or Tva mutants was easily detected (not shown). Therefore, the defect in function for the mutants analyzed here was not caused by a lack of Tva surface expression, since previous experiments with wt Tva demonstrated that surface levels of Tva below the threshold of detection are sufficient for full receptor activity in transiently transfected cells (5) and avian fibroblasts (4).

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

The sequence requirements for ASLV-A envelope binding and viral receptor function of the LDLR module in Tva were examined by mutational analysis. Our results demonstrate that the sequences required for high-affinity binding to EnvA map throughout the 40-residue LDLR module in Tva. Deletion and substitution mutations in all regions of the Tva LDLR module affected EnvA binding. In contrast to the binding studies, ASLV-A infectivity assays demonstrate that the N-terminal 17 residues of the LDLR module are dispensable for viral entry (Fig. 1B), confirming a previous finding suggesting that the major determinants for infection localize to the carboxyl terminus of the cysteine-rich module (24). This N-terminal deletion and many substitution mutants in Tva exhibited greatly reduced EnvA binding activity, yet when transiently expressed, these receptors were functional for viral entry.

Receptor function was impaired for many mutants only when multiple regions of the LDLR module of Tva were altered. For example, a homolog substitution mutant affecting residues between cysteines 3 and 4 (C3C4) mediated infection approximately fivefold less efficiently than wt Tva. However, when the C3C4 mutant was combined with a mutant affecting residues between cysteines 5 and 6 (C5C6) which had no effect on function, the resulting mutant, C34/56, was unable to mediate viral infection. Since all of these receptor mutants are expressed on the cell surface, then the most straightforward interpretation of these findings is that discontinuous regions in Tva form the envelope binding site and that the homolog substitution mutations either affect the presentation of the binding site or, we believe more likely, alter critical residues within the binding site.

Previously, it was hypothesized that a 19-amino-acid peptide corresponding to the carboxyl-terminal end of the LDLR module, from cysteines 4 to 6 and including residues D46, E47, and W48, contained all of the structural and functional information of the Tva viral interaction site (24). While analysis of the N-terminal deletion described in this report is in agreement with this hypothesis, our results demonstrating effects on function and EnvA binding by mutations in other regions of Tva do not support this model (Fig. 1B, 2B, and 2C). Additionally, we have recently demonstrated that a single module from the human LDLR which contains residues D46, E47, and W48 and other elements proposed to be sufficient for receptor function (24) will not mediate infection or bind envelope until additional residues in the module are altered to conform to the Tva sequence (21). Interestingly, one region of Tva found to affect the function of the human LDLR module was the sequence between cysteines 1 and 3 of Tva. Thus, while these residues are dispensable for receptor activity in Tva, they can confer some level of function on an otherwise defective LDLR module or negatively affect an otherwise functional receptor as demonstrated by the C45/ C1C3 mutant.

Analysis of a smaller set of Tva substitution mutants previously suggested that maintaining an aromatic residue at position 48 of Tva was crucial for efficient receptor function (25). Similar important aromatic residues in the human immunodeficiency virus and murine leukemia virus receptors CD4 and MCAT suggested that a common functional motif in these receptors consisted of an aromatic residue and an adjacent charged amino acid (17, 24). In contrast, our results analyzing a larger set of substitutions for W48 do not find an absolute requirement for an aromatic or even a hydrophobic residue at this position of Tva for high levels of receptor function. Similarly, although residues 46 and 47 in Tva are crucial for high levels of EnvA binding activity, they can be replaced by nonacidic residues without a significant effect on viral receptor function. Previous results had suggested that the requirement for an acidic residue at position 46 was absolute, while similar to our results, the requirement at position 47 was less stringent (24). Thus, our data from analysis of numerous Tva mutants at the DEW residues in Tva are not consistent with the previous hypothesis of a common functional motif in different retroviral receptors.

Substitutions of tryptophan 48 in Tva have also been reported to impair viral receptor function but not EnvA binding, leading to the suggestion that W48 may be involved in a postbinding step such as triggering conformational changes in EnvA (25). In our studies, we found that all 10 substitutions at this position produced severely diminished EnvA binding. Our analysis included the W48A substitution which was previously reported to dissociate binding from receptor function but that we found to be defective for both EnvA binding and function. The reason for these discrepant results may reflect differences in the assays used to measure envelope binding. The ELISA that we used evaluates monovalent binding of soluble receptor to a captured envelope protein, whereas the fluorescence-activated cell sorting assay used previously to test Tva-EnvA binding measures the avidity of a bivalent EnvA immunoadhesin for surface-expressed Tva (25). The bivalent nature of the immunoadhesin binding to Tva on the cell surface could underestimate decreases in Tva's EnvA binding activity, especially if the effect of the mutation was on the dissociation rate of the receptor-envelope complex, since both EnvA molecules would have to dissociate simultaneously for the complex to detach from the cell surface. Similarly, mutant Tva proteins that do not associate stably with the viral envelope would not compete with wt Tva in the ELISA, possibly explaining the receptor mutants that function efficiently in infection but appear not to bind EnvA (D46G, W48L, W48I, etc.).

Recent structural analysis of module 5 from the human LDLR demonstrates that residues corresponding to D46 and E47 in Tva, as well as D36 and D40, are part of a calcium box involved in coordinating a calcium ion (6, 12). For module 5, coordination of Ca²⁺ is absolutely required for proper in vitro folding of the module (6). Furthermore, many of the point mutations in LDLR which abolish LDLR function and cause familial hypercholesterolemia replace one of these acidic residues (16). Given their conservation in Tva and all other LDLR-like modules, it seems likely that the corresponding residues of Tva also coordinate Ca²⁺ and thus are important for Tva protein folding. Therefore, it is surprising that our results and previous studies on Tva substitution mutants (24) do not display an absolute requirement for acidic amino acids at the D47 E48 positions for efficient viral receptor function. In addition, the C3C5 mutant is deleted of two other acidic residues that coordinate the calcium ion and also two residues whose backbone carbonyl groups are also involved in coordination (12), yet this mutant expresses a functional, albeit impaired, receptor. Taken together, these data indicate that Tva can fold and function as a viral receptor under conditions where other LDLR modules are misfolded. In support of this view, mutation of individual cysteine residues in LDLR severely affects protein folding whereas similar mutants in Tva's LDLR module have no effect on receptor function (5).

One possible explanation that reconciles our findings with the structure of the LDLR module is that with D46 or E47 mutations the majority of the Tva proteins are misfolded, but a fraction of the molecules adopt a native conformation suitable for binding EnvA. Folding of human LDLR module 5 in vitro suggests that a small percentage of the proteins exist in a native conformation even in mutants where residues in the calcium box are altered (6). Since extremely low surface levels of Tva are sufficient for full receptor function (24) and the Tva mutants are overexpressed in the infectivity assays used here, then it is likely that a small percentage of correctly folded Tva exists at the cell surface and that this correctly folded protein mediates the low levels of infection detected. This small amount of correctly folded Tva would not be readily detected in the EnvA binding assays. Thus, this model would account for the discrepant binding and infectivity seen with some of the Tva mutants.

An intriguing alternative hypothesis for the apparent lack of effect of calcium box mutations on Tva function is that the residues at positions 46 and 47 have different roles, and thus different requirements, when Tva is free or bound to EnvA. This might explain the ability of residues which cannot participate in Ca²⁺ coordination to substitute for these acidic amino acids. Perhaps EnvA binding releases the Ca²⁺ and allows residues 46 and 47 of the receptor (and possibly residues 36 and 40 also) to participate in the ligand binding interaction. This model would explain why EnvA and a number of other LDLR module ligands contain clustered basic residues required for receptor binding (20). In addition, the affinity of Tva for EnvA (~1 to 2 nM) is roughly 50 times that of the LDL module (~70 nM) for calcium (6), suggesting that EnvA can displace the bound Ca²⁺. Future experiments examining the EnvA-Tva complex for wt and mutant receptors will be required to determine if any of these models are correct.