Abs that arise in HCV-infected individuals in response to viral infection are anticipated to react with the truly native conformation of the viral envelope structure. Recently, several HMAbs have been identified that react with conformational epitopes within E2 (1
). Moreover, some of these HMAbs have been shown to have neutralization-of-binding (NOB) activity (1
) defined by their ability to neutralize binding of recombinant, truncated HCV-E2 to human cells (37
). Previously, we identified 10 HMAbs that bind to full-length HCV-E2 glycoproteins from genotypes 1a, 1b, 2a, and 2b. Nine of these Abs reacted with conformational epitopes, six of which were NOB positive based on their ability to block E2 binding to cells or to CD81-coated plates (24
). Additionally, two of the NOB-positive HMAbs inhibited binding of infectious HCV virus particles (genotype 1a) to CD81 immobilized on polystyrene beads (24
), suggesting that these two HMAbs recognize important conformational epitopes within E2.
Preliminary experiments using this panel of Abs indicated that CBH-2, one of the two NOB-positive HMAbs that inhibited binding of infectious virus particles, reacted selectively with E2 glycoprotein only when coexpressed with E1. To follow up on this observation, HEK 293 cells cultured in Dulbecco’s modified Eagle medium-10% fetal calf serum were transiently transfected (using the GenePORTER 2 transfection reagent; Gene Therapy System, San Diego, Calif.) with 10 μg of plasmid encoding different forms of HCV glycoproteins from genotype 1a, H strain: full-length E1 (E1; amino acids 171 to 383), truncated E2 (E2 661; amino acids 364 to 661), full-length E2 (E2; amino acids 364 to 746), E1 and E2 (E1E2; amino acids 171 to 746), and E1E2p7NS2 (amino acids 171 to 1026). Transfected cells were lysed in lysis buffer, 4% Triton X-100 (Sigma, St. Louis, Mo.), 100 mM Tris-HCl (pH 8.0), 1 mM EDTA, and Complete Mini protease inhibitor cocktail tablets (Roche Diagnostics, Mannheim, Germany), for 30 min on ice and were clarified by centrifugation at 20,000 × g
for 30 min at 4°C. Protein A-immobilized CBH-2 was incubated 2 h at 4°C with clarified cell lysates. Ab-antigen complexes were washed four times with phosphate-buffered saline containing 0.2% Triton X-100. CBH-2 immunoprecipitations and aliquots of each lysate were boiled for 3 min in reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer and were electrophoresed on 10% polyacrylamide gels (Invitrogen, Carlsbad, Calif.). Proteins were transferred to polyvinyl difluoride (Immobilon-P; Millipore, Bedford, Mass.) and were immunoblotted with an anti-E2 MAb (3/11 [19
]) and detected with a mouse horseradish peroxidase-conjugated anti-rat (human-adsorbed) Ab (BioSource, Camarrillo, Calif.). Detection was performed using enhanced chemiluminescence detection reagents (Amersham Pharmacia Biotech, Little Chalfont, United Kingdom).
Interestingly, CBH-2 did not precipitate (Fig. ) truncated E2661
(upper panel, lane 2) or full-length E2 (upper panel, lane 3) glycoprotein, although cell lysates contained E2 proteins as determined by Western blotting (WB) with the anti-E2 MAb (Fig. , upper panel, lanes 8 and 9). However, CBH-2 was able to precipitate E2 when coexpressed with E1 (Fig. , upper panel, lanes 4 and 5). The additional faint higher-molecular-weight band observed in lane 5 is probably the unprocessed E2-NS2 precursor. Negative controls included empty vector-transfected cell lysates (Fig. , upper panel, lanes 6 and 12) or lysates prepared from cells expressing E1 alone (Fig. , upper panel, lanes 1 and 7). The blots were subsequently stripped by incubating in stripping buffer (62.5 mM Tris-HCl [pH 6.7], 100 mM β-mercaptoethanol, and 2% SDS) for 30 min at 50°C and were reprobed with an anti-E1 MAb (A4 [15
]), which was used to confirm the presence of E1 in the immunoprecipitates and lysates. As previously observed with the conformation-dependent anti-E2 MAb, H53 (7
), CBH-2 was able to coprecipitate E1 with E2 when the HCV envelope glycoproteins were coexpressed (lower panel, lanes 4 and 5). The requirement of CBH-2 to bind E2 only when expressed in the presence of E1 suggests that this HMAb may be recognizing a conformation of E2 that closely resembles native E2 glycoproteins as they exist on virus particles.
FIG. 1. CBH-2 HMAb immunoprecipitates (IP) E2 only when coexpressed intracellularly with E1. HEK 293 cells transfected with plasmids encoding E1 (lane 1), truncated E2661 (lane 2), E2 (lane 3), E1E2 (lane 4), E1E2p7NS2 (lane 5), or an empty vector (lane 6) were (more ...)
Properly folded E1 and E2 glycoproteins interact to form noncovalently linked heterodimers (12
), which are believed to be the native, prebudding form of the virus (31
). Previous studies have shown that E1E2 heterodimers can be formed when HCV envelope proteins are expressed both in cis
and in trans
). In order to know whether CBH-2 recognizes these heterodimers, 293 cells were transfected with plasmids encoding E1 alone, E2 alone, or E1E2 (cis
) or were cotransfected with a plasmid expressing E1 and a plasmid expressing E2 (trans
). Following transfection, cells were lysed and intracellular HCV glycoproteins were immunoprecipitated with CBH-2, separated, and detected as detailed above (Fig. ). To control for postlysis heterodimer formation, lysates from cells transfected separately with E1 or E2 were mixed prior to immunoprecipitation with CBH-2 (Fig. , lane 5). Cell lysates contained similar amounts of E2 glycoprotein as determined by WB with 3/11 (Fig. , lanes 8 to 10). The cell lysates contained additional diffuse bands in the upper part of the gel that likely represent aggregates (Fig. , lanes 8 to 10). Again, CBH-2 failed to precipitate E2 molecules when expressed in the absence of E1 (Fig. , upper panel, lane 2). In contrast, coexpression of E1 and E2, either in cis
(E1E2) or in trans
(E1 plus E2), resulted in efficient binding of E2 by the HMAb (upper panel, lanes 3 and 4). Proper folding and formation of E1E2 complexes did not appear to occur postlysis, as the Ab did not precipitate an appropriately sized band when incubated with a mixture of E1 and E2 lysates (Fig. , upper panel, lane 5). The blot was subsequently stripped and reprobed with A4, which was used to confirm the presence of E1 in the immunoprecipitates. As previously observed with the conformation-dependent anti-E2 MAb H53 (6
), CBH-2 was able to coprecipitate E1 with E2 when the HCV envelope glycoproteins were cotranslationally expressed (lower panel, lanes 3 and 4) but not when they were mixed postlysis (Fig. , lower panel, lane 5). Taken together, these results demonstrate that CBH-2 binds only to E2 glycoproteins that are complexed with E1, suggesting that this HMAb recognizes a prebudding, possibly native form of E2 envelope protein.
FIG. 2. Cotranslational expression of E1 and E2 is required for immunoprecipitation of E2 by CBH-2. HEK 293 cells were transfected with plasmids expressing E1 (lane 1), E2 (lane 2), E1E2 (lane 4), and empty vector (lane 6) or were cotransfected with a plasmid (more ...)
CBH-2 requires cotranslational expression of E1 to efficiently bind to E2 glycoproteins. This result was surprising, since this HMAb was originally identified by assays that required its binding to full-length E2 expressed in the absence of E1 (24
). The assays used previously detected reactivity with immobilized E2, whereas we used immobilized Abs to precipitate the envelope proteins from a soluble cell lysate. Under such conditions, it is likely that only high-affinity interactions (>108
/mol) could be detected (25
). It is also possible that the binding of E2 on solid phase may mimic the interaction with E1 to create the CBH-2 epitope.
The effect of HCV envelope proteins on folding of each other has been addressed by several studies. E1 has been shown to fold improperly, remaining in the reduced state, in the absence of E2 (30
). Insertion of alanine substitutions within the transmembrane domains of E1 and E2 disrupted E1E2 heterodimer formation and decreased the amount of properly folded E1 (32
). Similarly, glycosylation of E1 was dramatically improved when it was coexpressed with E2, indicating that glycosylation is also dependent on the presence of polypeptide sequences downstream of E1 (14
). These results indicate that E2 possesses a chaperone-like function to facilitate proper folding of E1 (for review, see reference 31
). In contrast to E1, E2 expressed in the absence of E1 was shown to fold properly (30
). Yet, coexpression of E1 either in cis
or in trans
was required for stable association of E2 with the ER membrane, suggesting that interaction between the hydrophobic transmembrane domains of these proteins is required for efficient ER membrane insertion and complex formation (6
). The requirement of CBH-2 to bind E2 only when coexpressed with E1 suggests once more that the presence of E1 may also influence the folding of E2, such that the epitope recognized by CBH-2 is formed only when E1 and E2 are in a complex.
The unique ability of CBH-2 to react with E1E2 heterodimers is seen only in the context of genotype 1a. Recently, Triyatni et al. have shown that the ability of CBH-2 to bind HCV-like particles (HCV-LPs) is restricted to genotype 1a (H77 strain); CBH-2 did not react with HCV-LPs from genotype 1b (J strain) (38
). In that study other conformation-dependent mouse MAbs, H2 and H53, also showed preferential binding to the HCV-LP 1a. The interaction of HCV-LP 1b with Abs was broader, exhibiting reactivity with Abs to both conformational and to linear epitopes (38