We have examined the role of transmembrane domains TM1 and TM3 in HBsAg particle formation. The assembly of native HBsAg virus-like particles depends on the first two TM domains of HBsAg (5
). However, using a strategy of domain swapping, we found that the entire TM1 domain of HBsAg could be replaced by the TM domain of HIV gp41 without disrupting particle assembly. Similarly, particles formed when the TM3 domain of HBsAg was replaced by the TM domain of gp41, or when both TM1 and TM3 domains were replaced by TM of gp41. Despite little or no direct sequence homology, one functional TM domain could replace another, suggesting that TM function was more important than sequence homology. HBsAg hybrid particles provide a novel way to express membrane-associated viral proteins, including transmembrane proteins, on a lipid surface. For antigenic determinants like the MPER of HIV gp41, this approach could enhance antibody binding by increasing valency and by presenting MPER in its natural milieu on a lipid surface.
The topology of particle formation is shown in Fig. , based on the membrane-spanning model of Stirk et al. (42
). The constructs can be divided into those where MPER was expressed at the exposed domains of HBsAg (TM12, TM32F, and TM34) and those where the transmembrane domain of the insert replaced a TM domain of HBsAg (constructs TM16 and TM20 and the double inserts). The first group was expected to form particles, since the TM domains were unaffected by the insert. However, the ability of the second group to form particles despite substitution for one or both of the TM1 and TM3 domains of HBsAg suggests that the inserted TM domain of gp41 also contributes to particle formation. The fact that the gp41 TM shared little or no sequence homology with HBsAg TM1 or TM3 suggests that other functional transmembrane domains could also permit particle assembly.
The assembly and folding of HBsAg-MPER chimeric particles is consistent with the two-stage model proposed for membrane proteins (15
). In the first stage, each transmembrane domain functions independently as it partitions into the lipid bilayer and forms a hydrophobic alpha helix. In the second stage, the TM domains organize through side-to-side interactions to generate functional proteins, and these can form disulfide bonds to neighboring HBsAg proteins to form dimers. The dimers oligomerize and eventually assemble into 22-nm particles. The ability to swap transmembrane domains as a complete unit (as in TM16), but not partial domains (TM14), agrees with the two-stage model, since an entire TM domain is the functional unit. Gp41 is an evolved transmembrane protein whose TM domain can partition into lipid (step 1) and self associate to form trimers (step 2). In addition, the current model of HBsAg particle formation suggests that two steps in subviral particle morphogenesis occur in distinct cellular compartments (17
In the second stage, the inserted TM domain of gp41 must interact favorably with the three remaining TM domains of HBsAg to allow protein folding and particle assembly. Unlike some enveloped viruses, such as HIV, which readily accept membrane proteins of the host cell (1
) or other unrelated viruses (47
), HBsAg particles rarely take up foreign proteins. HBsAg can form mixed particles when coexpressed with surface antigen of the closely related woodchuck hepatitis virus (WCsAg) but not duck hepatitis virus (16
). In addition, an HBsAg/WCsAg protein chimera formed particles when transmembrane domains TM1 and TM2 of WCsAg were replaced with the corresponding domains of HBsAg (38
). In these chimeras, the TM1 domain of HBsAg shared 12/21 amino acids with the WCsAg sequence it replaced (Table and ). The TM2 domains were even more conserved, sharing 19/22 amino acids between the two proteins, and this may allow both TM2 domains to function interchangeably.
TM1 domains of HBsAg chimeras capable of assemblya
TM3 domains of HBsAg chimeras capable of assemblya
In the current study, we held membrane-spanning domains 2 and 4 of HBsAg constant while replacing domain TM1 or TM3 of HBsAg with the TM domain of gp41. Although the gp41 TM domain shared only 2/21 amino acids with TM1 of HBsAg (Table ), it could replace TM1 and still form particles, as in construct TM16. HIV envelope gp41 readily forms pseudotypes with other membrane proteins, and this may explain its favorable interactions with the remaining HBsAg domains TM2, TM3, and TM4 during particle formation. Similarly, although the transmembrane domain of gp41 shared only 2/21 amino acids with the TM3 domain of HBsAg (Table ), construct TM20 formed particles, suggesting cooperative interactions with the remaining HBsAg domains TM1, TM2, and TM4 in particle formation. Once the first TM domain of gp41 is substituted into HBsAg particles (TM16 or TM20), it may be easier to accept the second TM substitution, as in TM16 + 20 particles. In this hybrid, two TM domains of gp41 may interact with each other, as they do in HIV envelope trimers. We have not substituted for domains TM2 or TM4, and TM2 may be essential for particle assembly.
Similar results have been reported recently for HBsAg chimeras containing the ectodomains and TM domains of envelope proteins E1 and E2 of hepatitis C virus (11
). These authors replaced TM1 of HBsAg with the TM domains of the HCV envelope proteins E1 and E2. Despite differences in nearly all amino acids of the TM domain (Tables and ), chimeric HBsAg-HCV proteins could participate in particle assembly. Unlike our TM constructs, particle formation depended on coexpressing HBsAg-E1 or HBsAg-E2 with native HBsAg. Difficulty in assembling HBsAg-HCV particles by themselves may reflect the size of the inserts, which included the entire E1 and E2 ectodomains and were much larger (54 and 85 kDa) than our HBsAg-MPER constructs (27 kDa).
The MPER determinant is an important target of HIV neutralizing antibodies (4
). Its sequence is conserved among diverse HIV isolates, and human monoclonal antibodies to MPER can neutralize a broad spectrum of HIV isolates from North America and western Europe (4
). MPER-specific neutralizing antibodies have been detected occasionally in infected humans, but they have never been elicited by immunization (27
). Similarly, in this study, despite immunizing rabbits with monovalent, divalent, or pentavalent HBsAg-MPER particles, we have not reliably elicited antibodies to MPER. Although an MPER immunogen is highly desirable, it presents a number of challenges. First, MPER is weakly immunogenic (34
). Many successful vaccines depend on particle formation to enhance vaccine potency. These include inactivated viral particles, such as inactivated polio vaccine (48
) and rabies (35
), and recombinant proteins that form virus-like particles, such as HBsAg (9
) and human papillomavirus (HPV) (18
). A number of experimental vaccines have gained potency by linking antigens with HBsAg to form virus-like particles, including vaccines against malaria (43
) and hepatitis C virus (30
). Similarly, we have made HBsAg-MPER particles to take advantage of the effect of multivalency on vaccine potency.
Second, the MPER determinant may require expression on a lipid surface (31
). By providing the lipid component, HBsAg-MPER particles could improve antibody quality by eliciting antibodies specific for MPER, as found on the viral surface. Third, it is located in a dynamic part of gp41, so neutralizing antibodies may need to bind a conformation of MPER that appears only transiently during viral entry (27
). This form of MPER might not be found on particles, and further modifications to the MPER insert would be required to produce this epitope. Finally, MPER may cross-react with certain host proteins (46
), and the immune response would need to be targeted selectively to avoid these potentially self-reactive epitopes.
HBsAg is a self-assembling membrane protein that presents viral antigens in the form of virus-like particles. We have probed its particle-forming activity by substituting an entire heterologous TM sequence for domains TM1 and TM3 of HBsAg. Particle formation depended on a functional TM domain rather than a conserved TM sequence. The particles displayed foreign envelope or transmembrane antigens, such as the HIV MPER determinant, anchored to a lipid surface. Antigenicity was enhanced by linking MPER to the amino end of the carrier, anchoring it through its own TM domain, and increasing its valency. Similarly modified HBsAg particles could provide a flexible platform for vaccine development against a variety of enveloped viruses.