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 A 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
). 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. B, 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.
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, (more ...)
The receptor mutants (C1C2, C2C3, C3C4, C4C5, and C5C6) were transiently expressed in 293T cells, and the proteins were detected by Western blot analysis using a polyclonal antiserum against Tva. As shown in Fig. A, all five homolog substitution mutants were expressed well in 293T cells. The pattern of numerous bands seen for the mutants and wt Tva is characteristic for Tva and probably reflects differential modifications by N- and O-glycosylation.
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 (more ...)
Function of the mutant receptors was assessed by infection of transfected 293T cells with a recombinant ALSV-A vector carrying an AP reporter gene [RCAS(A)AP]. Because ALSV does not replicate in mammalian cells, this is a single-step infection assay and thus the number of AP-positive cells reflects the function of the receptor. The results are summarized in Fig. B. Infection of 293T cells expressing wt Tva and four Tva mutants (C1C2, C2C3, C4C5, and C5C6) resulted in very similar numbers of AP-positive cells. Therefore, it appears that these homolog substitution mutants do not significantly affect Tva viral receptor function. One exception is mutant C3C4, which in multiple independent experiments consistently functioned roughly fivefold less efficiently than wt Tva, indicating that this mutant is slightly defective.
Two possible alternative explanations for why the homolog substitutions did not affect Tva function were that the mutated residues are not involved in viral entry and that the mutated residues contribute to the envelope interaction site but individual alteration does not abrogate ASLV-A receptor function. To address the latter possibility, the substitution mutants C3C4, C4C5, and C5C6 were combined to create three mutants (C34/45; C34/56, and C45/56; [Fig. B]). Because of the deletion mutations described below, we concentrated our analysis on the nonconserved residues from C28 to the end of the LDLR motif. The C34/45, C34/56, and C45/56 proteins were expressed well in 293T cells and displayed the numerous bands characteristic of Tva; however, the pattern of modification for each mutant varies somewhat (Fig. A). The effect of the mutations on receptor function was again measured by assaying susceptibility to RCAS(A)AP. Combining the substitutions between the third and fourth or fourth and fifth cysteines (C34/45) had no significant effect on Tva function (Fig. B). In contrast, mutant C34/56 was unable to mediate viral infection in numerous experiments. Mutant C45/56 was also significantly impaired for receptor function, displaying roughly a 50-fold decrease compared to wt Tva. From these results, it appears that the nine nonconserved amino acids between the third cysteine (residue 28) and the end of the LDLR module of Tva are significant determinants of receptor function; however, their importance is manifested only when multiple residues are altered. This finding implies that the EnvA interaction site in Tva is composed of residues from discontinuous regions in the carboxyl-terminal half of the LDLR motif and suggests that numerous interactions contribute to receptor function.
Mutant C34/45 functioned similarly to wt Tva, yet one of the homolog substitution parents (C3C4) was slightly impaired in its ability to mediate infection. This discrepancy appears to be due to the fact that the C34/45 mutant has a wt leucine at residue 34, which was corrected during the overlap PCR protocol used for mutagenesis, while the parent C3C4 has an alanine at this position. Similar to data presented previously (24
), this result suggests that L34 plays a role in EnvA recognition. In support of this conclusion, analysis of human LDLR repeat 4-Tva chimeras also identifies an important role for L34 in ASLV-A receptor function (21
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 ΔC1C3 contains a deletion of the first 17 residues including the first two cysteine residues of the LDLR motif, while ΔC3C5 has a deletion of 13 residues including cysteines 3 and 4. An additional mutant, C45/ΔC1C3, combines the substitution mutant C4C5 with ΔC1C3 (Fig. B).
Unlike the substitution mutants described above, the level of expression of the Tva deletion mutants after transient transfection of 293T cells was much lower than that of wt Tva (Fig. A). It is possible that these mutant proteins are not detected well by the anti-Tva polyclonal antibody used. However, because the deletions are predicted to affect normal disulfide bond formation, it seems more likely that the deletions affected the expression of Tva. The ability of these mutants to mediate viral entry was examined by using RCAS(A)AP viruses to infect 293T cells transiently expressing the mutant proteins (Fig. B). Mutant ΔC1C3 was only slightly impaired as a viral receptor, displaying approximately 50% of wt Tva receptor activity in numerous experiments. The deletion encompassing the central region of the LDLR motif (ΔC3C5) had a more dramatic effect, reducing receptor function approximately 20-fold compared to wt Tva.
A mutant, C45/ΔC1C3, combining the amino-terminal deletion and the C4C5 homolog substitution decreased the receptor function of Tva more than 100-fold. Similar to the combined homolog substitution mutants discussed above, this result supports the hypothesis that the EnvA interaction domain of Tva is comprised of dispersed residues in the LDLR module encompassing both the C1C3 region and the C4C5 region. Alternatively, it is possible that the deleted and mutated regions are not directly involved in binding EnvA but affect folding of the module and thus presentation of the virus interaction site. Finally, although the three deletion mutants were expressed to significantly lower levels than wt Tva, it seems unlikely that decreased expression accounts for the defective receptor function since expression of wt Tva in transiently transfected cells at levels significantly below those of the deletion mutants resulted in full receptor function (5
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.
To determine the conditions for the binding inhibition experiments, lysates of transiently transfected 293T cells expressing wt Tva were used at various concentrations, and the range of inhibition was measured. We found that 10 μl of wt Tva lysate (Fig. A, wt Tva lane) could completely block labeled sTva binding to captured EnvA. Furthermore, as little as 0.1 μl of wt Tva lysate could block roughly 70% of sTva binding (Fig. ). Based on these results, 5 ng of biotin-labeled sTva and 10 μl of each cell lysate were used to assess the relative EnvA binding capabilities of the Tva mutants. Using this amount of the mutant Tvas in the binding assay ensures that even low levels of binding capability should be detectable since the amount of receptor added is 100 times greater than the level of wt protein required for significant inhibition.
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 (more ...)
Tva mutants C1C2, C2C3, and C4C5 inhibited sTva binding as efficiently as wt Tva (greater than 90% inhibition with 10 μl of lysate [Fig. B]), suggesting that these receptors display relatively normal EnvA binding activity. Mutant C3C4 appears to be partially impaired for EnvA binding since it inhibited sTva binding by approximately 65%. Mutants C5C6 and C34/45 displayed approximately 40% inhibition, while mutant ΔC1C3 inhibited binding by only 20%. None of these mutants with detectable EnvA binding were significantly impaired for receptor function (compare Fig. B and B). In contrast, mutants C34/56, C45/56, ΔC3C5, and C45/ΔC1C3 did not block sTva binding, suggesting that they do not stably associate with EnvA. These mutants, except C34/56, displayed some receptor function.
Since mutants ΔC1C3, ΔC3C5, and C45/ΔC1C3 appear to express much lower levels of receptor protein than either wt Tva or the other mutants, the effect of adding increasing amounts of the lysates containing these mutant proteins to the blocking assay was examined. For mutant ΔC1C3, there was a dose response to added lysate such that when 60 μl was added, sTva binding was inhibited by nearly 60%. In contrast, mutants ΔC3C5 and C45/ΔC1C3 displayed less than 20% inhibition when 60 μl of cell lysate was added (Fig. C). From these results, it appears that the deletion mutant ΔC1C3 retains significant EnvA binding activity; however, mutants ΔC3C5 and C45/ΔC1C3 have severely impaired envelope binding activities.
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.
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. A).
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 (more ...)
The binding affinity of each of the D46 mutants was analyzed by using the blocking ELISA with 10 μl of lysate as described above (Fig. B). EnvA binding was undetectable for most D46 mutants, suggesting that they do not stably associate with EnvA. However, one mutant, D46G, displayed a very low level of EnvA binding activity. These results demonstrate that there is little or no inhibition by any of the D46 mutants at levels of receptor protein more than 100-fold higher than that required for significant inhibition by wt Tva (compare Fig. and B). Therefore, conservation of aspartic acid at this position appears to be critical for efficient EnvA binding by Tva.
The effect of the D46 mutations on Tva receptor function was assayed by using RCAS(A)AP as described above. Consistent with the EnvA binding results, most substitutions for residue D46 were functionally impaired (Table ). Substitution of residue 46 with arginine, cysteine, or tyrosine reduced viral receptor function of Tva at least 1,000-fold, while alanine, isoleucine, and serine at same position affected viral infection 10- to 100-fold. However, mutation of residue 46 to glycine only slightly decreased receptor function despite the fact that this substitution appears to significantly reduce EnvA binding. Thus, while D46 appears to be important for receptor function, maintaining an acidic residue at this position is not essential for function.
TABLE 1 Effects of mutations on Tva viral receptorfunction 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. A). 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. B). 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.
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 (more ...)
The effects of the E47 mutants on virus receptor function did not correlate with the binding data. Consistent with the binding results, substitutions of glutamate at this position with four residues (H, Y, R, and P) either greatly reduced or abolished the viral receptor function of Tva (Table ). Substitution by threonine reduced receptor function about 10-fold. In contrast, substitution of E47 by four residues (A, S, N, and I) either had no effect or caused only a two- to fourfold decrease in receptor function. Mutant E47F had the least effect on envelope binding and had no effect on Tva’s ability to mediate viral infection. From these results, it is evident that Tva receptor function does not require an acidic residue at this position and that it can be replaced with residues having quite divergent characteristics.
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. A).
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 (more ...)
Like the mutations at D46 and E47, all 10 substitutions at W48 greatly decreased Tva’s EnvA binding activity. The level of inhibition of sTva binding by these mutants was consistently less than 10% in blocking ELISA (Fig. B). Thus, efficient EnvA binding appears to require a tryptophan residue at this position.
In contrast, a number of the W48 substitutions functioned efficiently as ALSV-A receptors (Table ). Substitutions of residue W48 with three nonpolar residues (leucine, isoleucine, or valine) only marginally affected viral infection. Interestingly, W48 can be replaced with a basic residue (R) with only a threefold effect on receptor function. Substitutions with other charged or polar residues (H, T, D, N, and S) greatly decreased the viral infection, with the number of infected cells ranging from 200- to 1,000-fold lower than with wt Tva (Table ). Finally, alanine was not well tolerated and decreased receptor function by 3 logs. From these data, it appears that hydrophobic residues and a bulky charged amino acid are functionally tolerated at this position whereas less bulky charged and nonpolar residues are not; however, as for the D46 and E47 positions, there is no strict amino acid requirement at this position.
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. ). 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.
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 (more ...) 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. A and Table ) 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