HAstV-1 virions suppress serum hemolytic complement activity and display similar kinetics to CVF.
To investigate whether HAstV-1 virions affect serum complement activity, we utilized a standard hemolytic complement assay. In this procedure, sheep RBCs are sensitized with antibody and incubated with 2% NHS, and hemolytic complement activity is measured (Fig. , NHS, black bar). As a positive control for the inhibition of hemolysis, we utilized CVF. CVF is a structural and functional homolog to C3b that acts as a C3/C5 convertase to rapidly deplete C3 and the terminal complement cascade components, thus depleting functional serum complement activity (Fig. , NHS + CVF) (30
). As increasing amounts of infected cell lysate containing HAstV-1 virions were added to NHS, a dose-dependent response was seen, with 70 μl of the virus-containing lysate (corresponding to 2.41 × 108
genome copies) inhibiting lysis to approximately the same extent as 1 μg (4.27 × 1012
molecules) of CVF (Fig. , shaded bars). At 85 μl of virus-containing lysate (2.92 × 108
genome copies), inhibition of hemolysis surpassed that of CVF (P
= 0.0143). To demonstrate that the inhibition of hemolytic complement activity was due to HAstV-1 virions, NHS was incubated with equivalent amounts of cell lysate from uninfected cells to show that this activity was virus specific (Fig. , white bars). In addition, 7.64 × 1010
particles of sucrose gradient-purified FHV, a heterologous icosahedral RNA virus that infects insects (15
), were found not to inhibit hemolysis (RBC lysis, 107% ± 6.57%; n
FIG. 2. HAstV-1 virions suppress complement activity in a hemolytic complement assay, with similar kinetics to those of CVF. (A) Antibody-sensitized sheep RBCs were incubated with 20 μl NHS alone or in the presence of 1 μg CVF (black bars), along (more ...)
To characterize the kinetics of activity of the virions on NHS in the hemolytic complement assay, a time course comparing HAstV-1 virions and CVF was conducted. Infected cell lysates containing HAstV-1 particles (2.92 × 108 genome copies) and 1 μg CVF were incubated for 0 to 60 min in the presence of 2% NHS. At increasing time intervals, aliquots were removed and exposed to sensitized RBCs. Both CVF and the virus showed similar kinetics in the inhibition of hemolysis (Fig. ). While CVF displayed a more pronounced suppression of hemolytic activity at earlier time points, at the 60-min time point the virus demonstrated a stronger inhibitory effect, of 93%, than the 75% suppression for CVF (P = 0.0001).
Other HAstV serotypes suppress hemolytic complement activity.
To determine whether HAstV serotypes other than type 1 exhibit the same effects on complement activity, equivalent amounts of cell lysates infected with serotypes 1 to 4 were analyzed in the hemolytic complement assay, with all four serotypes demonstrating nearly identical levels of hemolysis inhibition (Table ). These findings suggest that the complement-suppressing effect reported here is a conserved property of the HAstVs.
Hemolytic assay of HAstV serotypes 1 to 4
Soluble HAstV-1 CP suppresses hemolytic complement activity.
While HAstV-infected cell lysates demonstrated suppression of hemolytic complement activity, one cannot rule out the possibility that a newly induced/overproduced cellular protein or viral nonstructural protein was responsible for the observed complement suppression activity. To address this contingency, we isolated soluble HAstV-1 CP produced from a recombinant baculovirus. While small numbers of intact virus-like particles were visualized in infected IPLB-Sf21 cells by thin-section electron microscopy, most of the CP was in the form of aggregates (data not shown). To produce soluble CP, we utilized a four-step purification procedure in which CP aggregates were initially isolated in crude form by extraction of cells with 2% NP-40 followed by 0.5 mg of DNase I per ml (22
). After high-speed pelleting, the CP-containing pellet was resuspended in dissociation buffer with freezing overnight to solubilize the aggregated CP (25
) and then purified further by sucrose gradient ultracentrifugation in the same buffer. During centrifugation, the majority of the CP was found to peak in fractions 6 to 8, along with some CP degradation products, as analyzed by SDS-PAGE followed by Coomassie blue staining (Fig. ) and immunoblotting using antibody to HAstV-1 particles (2
) (Fig. ).
FIG. 3. SDS-PAGE and immunoblot analysis of fractions obtained during the sucrose gradient ultracentrifugation step of HAstV-1 CP purification. The first 18 fractions of the gradient were run in SDS-PAGE gels and stained with Coomassie blue (A) or subjected to (more ...)
SDS-PAGE analysis of the CP-containing fractions at each step of the purification procedure was monitored by Coomassie blue staining (Fig. ) and immunoblotting (Fig. ). Densitometric scanning of the Coomassie blue-stained gel demonstrated that CP constituted >94% of the protein present in the peak fraction (in multiple preparations). A small portion of this signal was due to a CP-specific degradation product that migrated slightly below the full-length 87-kDa band. When gradient-purified CP was boiled in the presence of β-mercaptoethanol and run in a 7.5% SDS-PAGE gel, CP was detected at 87 kDa, as expected (Fig. ). However, CP that was not boiled gave rise to a band that migrated just above the 250-kDa molecular size standard (Fig. ). The size of this band is consistent with a trimer of CP molecules, which would be predicted to run at approximately 261 kDa (87 kDa × 3), and possibly represents an assembly intermediate. However, fully assembled virus-like particles were not detected under these dissociation conditions by negative-stain electron microscopy (data not shown). Consistent with our findings, HAstV serotype 8 CP was recently reported to form trimers in translation experiments performed in vitro (19
). We also subjected the purified CP to digestion with trypsin, which is normally required to cleave the viral capsid into proteins of 34, 29, and 26 kDa, as resolved by SDS-PAGE (1
). Trypsin cleavage of the CP resulted in digestion products of <25 kDa, further demonstrating that the soluble CP was structurally different from intact virus-like particles (data not shown).
FIG. 4. Analysis of HAstV-1 CP purification, oligomerization state, and activity in the hemolytic complement assay. Coomassie blue staining (A) and immunoblot analysis (B) of the CP-containing fraction were performed at each stage of the purification procedure. (more ...)
To ascertain whether soluble CP displays complement-suppressing activity, as demonstrated for authentic virus, we again utilized the hemolytic complement assay (Fig. ). As expected, NHS alone and NHS plus purified BSA protein demonstrated equivalent amounts of RBC lysis, whereas NHS plus CVF inhibited lysis significantly. As increasing amounts of CP were added to NHS, a dose-dependent response in hemolysis demonstrated decreased complement activity, as seen with HAstV-1-infected cell lysates. A total of 1.5 μg CP (3.51 × 1012
molecules of trimer) suppressed hemolysis to a similar degree as did 1 μg (4.27 × 1012
molecules) of CVF (see Fig. ), suggesting that CP possesses potent complement-suppressing activity. Equivalent amounts of sucrose gradient buffer alone or sucrose gradient buffer from a recombinant baculovirus expressing a soluble, heterologous viral protein (the RNA polymerase of FHV) (15
) that was subjected to an identical purification procedure to that used for HAstV-1 CP demonstrated no effect on hemolytic complement activity (data not shown).
FIG. 7. HAstV-1 CP inhibits iC3b formation, terminal complement cascade activation, and C4d formation. Reaction mixtures containing NHS were preincubated alone, with 5.4 μg CP, or with 1 μg CVF at 37°C for 3 h and then aliquoted. CVF is (more ...) HAstV-1 CP specifically suppresses the classical pathway.
To determine if CP suppresses complement activation of the classical or alternative pathway, sera depleted of complement factors specific for each pathway were utilized in the hemolytic complement assay. To evaluate suppression of classical pathway activation by CP, NHS and fBD serum were compared. Factor B is essential for alternative pathway activation, and utilizing fBD serum allows the specific testing of classical pathway activation. Both NHS and fBD serum demonstrated a dose-dependent suppression of hemolysis by CP (Fig. ). However, CP was considerably more effective at suppressing fBD serum than NHS. For 3.6 μg of CP, hemolysis was suppressed 16% for fBD serum, compared with 55% for NHS (P = 0.0159). Increased inhibition when the classical pathway was isolated (fBD serum) compared with that when activation could occur by any or all pathways (NHS) suggested that CP may more strongly suppress activation of the classical pathway than the alternative pathway. As with CP, infected cell lysates containing HAstV-1 particles (2.92 × 108 genome copies) also inhibited hemolysis in fBD serum (Fig. ). It should be noted that fBD serum retains the same capacity for hemolysis as NHS because C3 and C5 to C9 are intact. It is possible that the lectin pathway may also be activated under these conditions, but it is currently unknown whether astrovirus capsids are glycosylated.
FIG. 5. HAstV-1 CP and virions strongly suppress classical pathway activity. (A) Antibody-sensitized sheep RBCs were incubated with NHS (white bars) or fBD serum (shaded bars) in the presence of the indicated amounts of CP. (B) Antibody-sensitized sheep RBCs (more ...)
We tested the effects of CP and virus on the alternative pathway as well. As demonstrated in Fig. , in an assay to test for alternative pathway activation (i.e., the rabbit RBCs were not sensitized with antibody and only the alternative pathway may activate in Mg-EGTA-GVBS buffer), NHS lysed rabbit RBCs as expected. When 6.3 μg of CP was added to NHS, there was only a modest effect on the suppression of hemolysis (Fig. , NHS + CP). To confirm that activation was limited to the alternative pathway, C2D serum was utilized in place of NHS. By depleting NHS of C2 in this assay, the classical and lectin pathways are blocked and any RBC lysis is due exclusively to alternative pathway activity. As with NHS, C2D serum in the presence of CP suppressed hemolysis approximately 25% compared to that with C2D serum alone (Fig. , C2D and C2D + CP). The same results were obtained when HAstV-1-infected cell lysates (2.92 × 108 genome copies) were tested for alternative pathway activity (Fig. ). In both of these alternative pathway activation assays, CP appeared to only partially block activation, suggesting that HAstV-1 CP suppresses hemolytic complement activity via the classical pathway more than via the alternative pathway.
FIG. 6. HAstV-1 CP and virions have a modest effect on alternative pathway activity. NHS or C2D serum was incubated with rabbit RBCs in Mg-EGTA-GVBS buffer alone or in the presence of 6.3 μg CP (A) or HAstV-1 virions (30 μl of cell lysate, corresponding (more ...) HAstV-1 CP inhibits iC3b, C5b-9 membrane attack complex, and C4d formation.
While CP was found to suppress complement in hemolytic assays, it was unclear whether this effect was due to activation and depletion of serum complement components, as occurs with CVF, or as the result of inhibition of activation. To test these competing hypotheses, aliquots of NHS alone, NHS plus CP, or NHS plus CVF were incubated for 3 h at 37°C, and the amount of iC3b produced was measured by immunoblotting and an iC3b-specific ELISA. Upon activation of the three pathways of the complement system, C3 is cleaved to C3a and C3b. Some C3b is further cleaved to the inactive form iC3b by soluble complement regulators in NHS (factor I plus factor H). Utilizing an immunoblot with an antibody specific for C3 fragments, two bands were present for the uncleaved C3 control, namely, C3-alpha (114 kDa) and C3-beta (75 kDa) (Fig. , lane C3). Cleavage of C3b to iC3b results in two cleavage products from the alpha chain, of 68 kDa (α′1) and 42 kDa (α′2), while the C3-beta chain remains invariant (Fig. , lane iC3b). NHS alone produced only a small amount of the 42-kDa iC3b product after a 3-h incubation, consistent with low-level, spontaneous activation of C3 (Fig. , lane NHS). In the CP-containing sample, very little of the 42-kDa iC3b product was observed (Fig. , lane NHS + CP lane). In contrast, CVF depleted the C3-alpha chain, generating large amounts of the 42-kDa iC3b band (Fig. , lane NHS + CVF). To more precisely quantify the amount of iC3b generated, duplicate aliquots of these samples were measured by iC3b-specific ELISA. Consistent with the immunoblot data, CP suppressed iC3b formation to a greater extent than did NHS alone (P = 0.0314), whereas NHS plus CVF generated significant amounts of iC3b, consistent with the known actions of CVF to activate and deplete the complement system (Fig. ). Thus, even though HAstV-1 virions displayed similar kinetics to those of CVF (Fig. ), these data show that the CP of this virus does not cause complement activation resulting in cleavage of C3, suggesting that suppression of serum complement activation occurs through an inhibitory mechanism.
To measure the effect of CP inhibition of serum complement on the terminal complement cascade, aliquots of the samples used above were assayed for formation of the membrane attack complex, utilizing an ELISA specific for SC5b-9 complex formation. As shown in Fig. , for NHS alone, a modest amount of SC5b-9 complex was detected, whereas virtually no complex was present in NHS treated with CP (P = 0.0286), suggesting no terminal complement cascade activation. In CVF-treated serum, large amounts of SC5b-9 complex were generated, as expected for relentless activation and depletion.
The ability of CP to inhibit iC3b and SC5b-9 formation, coupled with its strong suppression of the classical pathway (Fig. ), led us to investigate if CP suppressed the complement system at the level of C4, the second component of the classical pathway, after C1. Upon activation of the C1 complex, C4 is cleaved and then C2 is cleaved to form the classical pathway C3 convertase (31
). An ELISA that detects a specific by-product of C4 activation (C4d) demonstrated that in the presence of CP, serum generated very low levels of C4d, in contrast to that in the presence of NHS alone (P
= 0.0286) (Fig. ), suggesting an inhibition of spontaneous classical pathway activation. The classical pathway activator heat-aggregated IgG greatly increased C4d generation, yet when CP was added simultaneously with heat-aggregated IgG to NHS, C4d formation was greatly inhibited (P
= 0.0286) (Fig. ). These results show that HAstV-1 CP can inhibit classical pathway activation by a potent classical activator, heat-aggregated IgG, before C4 cleavage can occur, suggesting that CP is a strong classical pathway inhibitor and that inhibition likely occurs at C1.
HAstV-1 CP binds to the A chain of C1q.
CP inhibition of the classical pathway led us to speculate that CP interacts with one of the classical pathway factors (C1 complex, C4, or C2). To ascertain whether CP binds to specific complement factors, we utilized a modified virus overlay protein binding assay approach (3
). To this end, C1 complex and highly purified C2, C3, and C4 were mixed with SDS sample buffer lacking reducing agents and loaded onto a 7.5% SDS-PAGE gel without being boiled. Following electrophoresis, proteins were transferred to nitrocellulose, blocked, and then probed with or without purified CP in blocking solution for 1 h, after which the blots were washed. CP binding was then detected with antiserum to HAstV-1 (2
) followed by a labeled secondary antibody. As shown in Fig. specific band of approximately 59 kDa, consistent with a dimer of C1 chains, was present in the C1 lane, while no binding was detected for C2 to C4. In the absence of the CP probe, no signal was detected for C1 (Fig. ). The fact that C1 is a multimolecular complex of C1q, C1r, and C1s along with the fact that the gels were run under nonreducing conditions without boiling the samples made it difficult to determine exactly which component of C1 interacts with the viral CP. To address this issue, all three of the highly purified constituents of the C1 complex (C1q, C1r, and C1s) were boiled, reduced, loaded onto 12% SDS-PAGE gels, transferred to nitrocellulose, and probed with or without CP as described above. The blot probed with CP showed a band of ~34 kDa in the C1 and C1q lanes, whereas there was no signal for BSA, C1r, or C1s (Fig. ). As expected, no signal was detected in the duplicate blot that did not receive the CP probe (Fig. ). The blots were stripped and reprobed with antisera to C1q, C1r, and C1s to reveal the individual protein constituents of C1 (Fig. ). C1q is composed of six subunits, each of which contains three polypeptide chains, i.e., A, B, and C. As shown in the C1 and C1q lanes, all three C1q chains react with C1q antisera, and under reducing conditions, chain C runs at 27.5 kDa, chain B runs at 31.6 kDa, and chain A runs at 34.8 kDa (23
). CP was found to overlay precisely with the 34-kDa band corresponding to the A chain of C1q in both the C1 and C1q lanes. We believe that the band detected by CP at approximately 59 kDa in the nonreduced C1 lanes (Fig. ) corresponds to disulfide-bonded A-B C1q dimers.
FIG. 8. HAstV-1 CP binds to the A chain of C1q. (A) The indicated complement factors were loaded onto a 7.5% SDS-PAGE gel in the absence of boiling or reduction. Proteins were then transferred to nitrocellulose, blocked, and probed with CP for 1 h. (B) (more ...) Exogenous C1 can reconstitute hemolytic activity and C3 activation of CP-treated NHS.
Based on the overlay blot data, CP appears to interact with the A chain of C1q. If C1 interacts with CP, then additional exogenous C1 should reconstitute hemolytic complement activity. Thus, enough CP was added to NHS for 1 h to inhibit lysis approximately 50% (Fig. ). Exogenous C1 (10 μg) was then added to CP-treated NHS, and hemolytic activity was fully restored, from 56% to 97% (P = 0.0286). Reconstitution of hemolytic activity did not occur when BSA was substituted for C1 or when additional C1 was added to CVF-treated NHS, suggesting that CP reversibly inhibits complement activation via C1.
FIG. 9. Exogenous C1 reconstitutes hemolytic activity and C3 activation for CP-treated NHS. (A) NHS was incubated alone, with 6.3 μg CP, or with 1 μg CVF for 1 h at 37°C. Heat-inactivated NHS (HI NHS) was used as an additional control. (more ...)
To confirm the reversal of CP inhibition of complement activity by C1, the deposition of C3 on zymosan was measured (Fig. ). Zymosan particles activate complement and serve as a target for C3 fragment binding. Duplicate aliquots of NHS were incubated with CP or CVF for 1 h at 37°C. After the incubation, C1 was added to the indicated samples, followed by the addition of zymosan to all samples. After 10 min at 37°C, the samples were treated with methylamine to cleave off bound C3 fragments, which were measured in a C3-specific ELISA. NHS alone bound substantial amounts of C3 fragments on zymosan, as expected, whereas CP-treated NHS bound much lower levels of C3 fragments (Fig. ). The addition of exogenous C1 to CP-treated NHS increased C3 fragment binding, again showing the ability of excess C1 to reconstitute complement activity. As expected, the addition of excess C1 to CVF-treated NHS did not significantly increase C3 deposition. Taken together, these experiments suggest that CP inhibits complement activation at C1 and that the inhibition of the complement activation cascade is reversible.