Structural and Functional Properties of Viral Membrane Proteins
- 438 Downloads
Viruses have developed a large variety of transmembrane proteins to carry out their infectious cycles. Some of these proteins are simply anchored to membrane via transmembrane helices. Others, however, adopt more interesting structures to perform tasks such as mediating membrane fusion and forming ion-permeating channels. Due to the dynamic or plastic nature shown by many of the viral membrane proteins, structural and mechanistic understanding of these proteins has lagged behind their counterparts in prokaryotes and eukaryotes. This chapter provides an overview of the use of NMR spectroscopy to unveil the transmembrane and membrane-proximal regions of viral membrane proteins, as well as their interactions with potential therapeutics.
We will focus our discussion on two classes of transmembrane (TM) proteins encoded by viruses, viroporins (or viral channels) and membrane fusion proteins, as these proteins have been sought after as antiviral targets and they often exhibit peculiar structural features not seen in other membrane proteins. As implied by their name, the viroporin proteins can form channel-like structures in lipid bilayer that permeate ions or solutes. Now over a dozen viroporins from various sources have been characterized, covering a wide range of ion substrates including H+, K+, Na+, Ca2+, and Cl−, as well as larger substrates such as RNA. The exact function of ion channel activity in many viruses is not yet known, though their roles have been implicated in entry, virus assembly and virus release. The function of membrane fusion proteins is much better defined, that is, to enable viral entry by mediating virus-host membrane fusion. The viral fusion proteins have large extramembrane domains for receptor recognition and for grabbing onto the host cell membrane, but the function of their TM and membrane-proximal regions have been elusive. Functional mutagenesis studies have suggested that, at least in the cases of HIV-1 and influenza A viruses, the TM domains (TMDs) of fusion proteins are not merely membrane anchors, but play important roles in membrane fusion and viral infectivity. Apart from the channels and fusion proteins, some viruses have developed enzymatic domains anchored to the membrane, e.g., the polymerases of the hepatitis C virus and the neurominidase of the influenza viruses. In these cases, the TMDs are believed to only serve as membrane anchors and thus will not be discussed in this chapter.
6.1.1 Function of Viroporins
List of identified viroporin proteinsa
Name of virus
Names of viroporin
# of amino acid
Influenza A virus
Influenza B virus
Hepatitis C virus
Na+ over K+
Probably the best-defined roles of viroporins are that of the M2 proton channels of influenza A and B viruses, both of which involve equilibrating pH between compartments. One important role of the M2 is to equilibrate pH across the viral membrane during viral entry, which acidifies the virion interior immediately after endocytosis and facilitates RNA release [22, 23] (Fig. 6.1a). Another role of M2 is to equilibrate pH across the trans-Golgi membrane of the infected cells during viral maturation, which preserves the pre-fusion form of the hemagglutinin fusion protein through de-acidification of the Golgi lumen  (Fig. 6.1b). The roles of other cation-selective viroporins are less clear. One of the general roles proposed for viroporin is to depolarize either the ER or plasma membrane to facilitate membrane curvature formation during virus budding [25, 26] (Fig. 6.1c). In the case of HCV, for example, the p7-mediated channel activity has been reported to facilitate virus release [27, 28]. Another general role for viroporin is simply to cause cation leakage, which would induce cellular stress and programmed cell death  (Fig. 6.1d). In addition to ion permeation, several viroporins are known to participate in viral assembly through interaction with other proteins. For example, the influenza AM2 and BM2 proteins both have significant cytoplasmic domains that are believed to recruit matrix proteins M1 during virus assembly [30, 31, 32]. The HCV p7 protein has also been reported to interact with other non-structural proteins such as NS2, and this interaction appears to be crucial for the production of infectious HCV particles [33, 34].
6.1.2 Viral Membrane Fusion Proteins
Due to the essential roles of viral fusion proteins during infection, they have long been pursued as targets for antiviral intervention. For example, the approved small molecule drug Arbidol is used as a broad-spectrum inhibitor of influenza A and B virus, as well as hepatitis C virus [50, 51]. Unlike many other broad-spectrum antivirals, Arbidol has an established mechanism of action against the HAs in influenza A and B viruses that involves the inhibition of virus-mediated membrane fusion and thus viral entry . In addition, Enfuvirtide (T20, Fuzeon) is an approved peptide drug that blocks the HIV-1 entry ; it is derived from the C-terminal heptad repeat 2 region of the HIV-1 gp41 envelope glycoprotein, designated the C-peptide, that disrupts membrane fusion by competing with an intra-molecular interaction that is important for the refolding of gp41 during membrane fusion . Apart from being therapeutic targets, another important medical use of the fusion proteins is vaccine development. Viral fusion proteins are usually presented on the virion surface with high copy numbers, and thus are popular antigens for eliciting broad-spectrum neutralizing antibodies. For example, the native conformations of the HIV-1 envelope glycoprotein have being pursued intensely as immunogens for B-cell based vaccine development [54, 55].
6.2 Structural Methods for Investigating Viral Membrane Protein
For most single-pass TM proteins, the role of their TMDs beyond membrane anchoring is unresolved, partly because of the difficulty in structural studies: they are notorious for resisting crystallization and due to their small sizes, unfeasible for visualization by cryo-electron microscopy (EM). There have been only a few examples of crystal structures of the small TMDs, including the TMD of the AM2 [56, 57] and the more recent crystal structure of the TM helix dimer of the glycophorin A protein . As cryo-EM is rapidly approaching atomic resolution, it is becoming increasingly used to examine the structures of the much larger viral fusion proteins containing the TMD. Most notably, a recent cryo-EM study of the HIV-1 envelope glycoprotein (Env) including the TMD achieved a high resolution structure of the prefusion state of the HIV-1 Env . This study, however, showed that the TMD and the membrane-proximal regions of the Env are disordered, possibly due to incompatibility between the membrane-associated regions of the Env and the detergent used to solubilize the Env.
The purified hydrophobic peptides can be reconstituted in any membrane mimetic media, including detergent micelles, bicelles, and lipid nanodiscs. While nanodiscs are the closest mimic of native membrane, we find that the lipid/detergent bicelle system is a good compromise between having the capacity to provide a near lipid bilayer environment and generating good NMR spectra. Previous studies on the bicelle system with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) as lipid and (1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) as detergent have shown that when the molar ratio of lipid to detergent (q) > 0.5, the assembly reaches the ideal bicelle condition in which the lipid and detergents are well segregated [63, 64]. At q = 0.5, for example, the estimated diameter of the lipid bilayer region of the bicelle disc is ~45 Å according to the equation describing bicelle assembly [64, 65, 66], and this size is sufficiently large to accommodate small TMDs. Remarkably, even bicelles of such size could generate NMR spectra of sufficiently high quality to enable full-scale structure determination, as was demonstrated for the structure determination of the trimeric TMDs of the Fas death receptor  and HIV-1 Env . Here we provide an example of bicelle reconstitution from a previous study on the TMD of HIV-1 Env . The hydrophobic peptides (lyophilized) are first completely dissolved in strong organic solvent (e.g., hexafluoro-isopropanal) with calculated amount of DMPC. The solution is slowly dried to a thin film under nitrogen stream. The dried film is then dissolved in 8 M Urea containing calculated amount of DHPC. The denaturant is removed by dialysis, during which the DMPC: DHPC ratio is monitored by 1D NMR, and the loss of DHPC during dialysis is compensated by further addition of DHPC.
6.3 Viroporin Architectures and Mechanism
Structural studies of viroporins have been challenging because these small membrane proteins are typically dynamic and very hydrophobic. In the past 10 years, multiple biophysical techniques including solution NMR, solid-state NMR, X-ray crystallography, and EM have been used to gradually fill the structural gaps. Taking the influenza AM2 for example, there is now structural information in crystal and solution states, in lipid bilayer, under different pHs, and bound to different small molecules. These complementary structural data allow elucidation of the functional mechanism from different view angles.
6.3.1 The Influenza M2 Channels: Structural Solutions for Proton Conduction
The AM2 of influenza A and the BM2 of influenza B are 97- and 109-residues single-pass membrane proteins, respectively, that form homotetramers in membrane [72, 73, 74, 75, 76]. The sequences consist of three domains: an extracellular N-terminal domain, a transmembrane domain (TMD) and an intracellular C-terminal domain. These domains arrange into different structures. The only homologous sequence between the two proteins is the HxxxW sequence motif of the TMDs that is essential for channel activity. The TM region of the AM2 contains residues 24–46. The unstructured extracellular segment of AM2 is relatively conserved and has been sought after as a vaccine epitope [77, 78, 79, 80, 81]. BM2 has a similar sized TMD (residues 4–33), but a much larger C-terminal cytoplasmic domain [32, 82]. The BM2 cytoplasmic region also assembles into oligomers which are important for recruiting the matrix proteins to the cell surface during the viral assembly [30, 31, 83, 84].
6.3.2 The Funnel Architecture of the p7 Channel
Ion channels typically have two essential features: (1) pore elements that support selective ion dehydration; (2) a gate or constriction that prevents non-specific permeation, but can open in response to regulating factor such as pH, voltage, ligand, or the ion of selection itself [100, 101]. The channel interior of p7 has a number of strongly conserved residues that are likely candidates to serve the above functions. One suspect is Asn9, which forms a ring of carboxamide near the narrow end of the channel (Fig. 6.5b). Residue 9 is asparagine in all strains except being substituted with histidine in genotype 2 viruses. Formation of a ring of carboxylates or carboxamides has been a recurring theme in prokaryotic and eukaryotic channels that have selectivity for divalent cations. For examples, the CorA Mg2+ channel has a pentameric ring of asparagines , and the calcium release-activated calcium (CRAC) channel Orai has a hexameric ring of aspartic acids . These channels all have strong selectivity for divalent cations, although they can also conduct monovalent cations such as Na+ and K+. In addition to the Asn9 ring, residues near the hinge between H1 and H2 (Ser12, Asn16, Trp21) are in an arrangement that may also bind cations (Fig. 6.5b), although these residues have not yet been tested in functional assays.
In addition to what appears to be the cation selectivity ring near the narrow, N-terminal exit of the channel, the wider, C-terminal entrance of the channel is decorated with a conserved ring of arginines or lysines (Fig. 6.5b). Placement of a positively charged ring at the entrance was anticipated because it may repel cations. But an earlier study reported that a designed TM barrel with internal arginine-histidine dyads forms efficient cation selective channels  because the immobile arginines can recruit mobile anions, which in turn facilitate cations to diffuse through the pore. It is interesting to note that a highly basic region containing Arg155, Lys159 and Lys163 in the pore was also found in the CRAC Orai structure . One possible role of these basic residues in cation selective channels is binding and obstructing anions while allowing cations to diffuse into the pore. This mechanism would be consistent with the observation that replacing Arg35 with negatively charged aspartic acid largely abrogated conductance .
There are still many unanswered questions. Does the NMR structure, solved in the absence of Ca2+ and inhibitors, represent the open or closed state of the channel (if the two states exist)? How strong is the Ca2+ selectivity of p7? Or was p7 developed as a general, unspecific cation channel for the purpose of dissipating membrane potential? For example, another recently study reported p7 activity in dissipating proton gradients within cell membrane compartments . It is unclear what ion flux mediated by p7 plays a dominant role in the HCV life cycle. From a structural perspective, the funnel-like architecture is formed with multiple helical segments connected by hinges and short loops and we believe this flexibility can afford the dynamic opening and closing of the tip of the channel.
6.3.3 Drug Binding of Viroporins
Probably the most intriguing aspect of small molecule interaction with viroporin is the finding that the adamantane derivatives amantadine and rimantadine have inhibitory effect on multiple viroporins including the influenza AM2 and HCV p7. Amantadine (Symadine) or rimantadine (Flumadine) was the first licensed drug for treating influenza infections . In fact, the compound also played critical roles in the early days of functional characterization of the AM2 channel [107, 108, 109]. The BM2 channel is a functional and structural homolog of AM2 but is not sensitive to the adamantane family of drugs . Remarkably, the HCV p7 channel structure is completely different but showed detectable, though not strong, sensitivity to rimantadine [15, 110]. The mechanism of how amantadine/rimantadine inhibit the AM2 channel has been elusive for quite some time. Previous confusion came mainly from the multiple binding sites that have been observed experimentally. In a crystallographic study of the AM2 TMD (residues 24–46) in the presence of amantadine, the drug density was found inside the channel near residue Ser31, but at structural resolution of 3.5 Å, it was difficult to confirm the position of amantadine binding . At the same time, however, a solution NMR study of a longer version of AM2 (residues 18–60) showed that rimantadine binds to an external, lipid-facing pocket around residue Asp44 between adjacent TM helices .
In addition to blocking the AM2 channel, the amantadine and its derivatives have also been shown to pose some inhibitory effects on the p7 channel conductance [15, 113]. The physical binding sites of amantadine and rimantadine have been identified in p7 of genotype 5a using intermolecular NOE experiments . The NOE data revealed that amantadine or rimantadine binds to six equivalent hydrophobic pockets (due to the six-fold symmetry of the p7 channel) between the pore-forming and peripheral helices (Fig. 6.6b). In each site, Leu52, Leu53, and Leu56 from H3 and Val25, Val26, and Phe20 from H2 appear to form a hydrophobic pocket that wraps around the adamantane cage of the drug. The amino group of amantadine or rimantadine is facing the largely hydrophilic channel lumen. An important property of the drug binding site is that it consists of elements from different helical segments and from different monomers. As rationalized above, permeation of cations through the p7 channel may depend on opening the narrow end of the funnel, which in turn depends on the reorientation of the helical segments. The binding of adamantane derivatives to the pocket may inhibit channel activity allosterically by causing the channel to close. Indeed, an NMR relaxation dispersion study showed that residues at the H1-H2 hinge (Phe19) and the narrow end of the cavity (Val7, Leu8) experienced substantial chemical exchanges (kex~1000 ± 79 s−1 and ~10% excited state). This data is consistent with movements of the H1 helices that cause the tip of the funnel to open and close. More importantly, addition of rimantadine slowed down motion at the tip of the channel, as relaxation dispersion curve for Val7, which has significant chemical exchange in the apo state, is completely flat in the drug-bound state . The dispersion curve of Phe19 is also significantly flatter, and individual curve fit yielded kex value of 67 ± 182 s−1. Clearly, rimantadine binding makes the channel less dynamic. Therefore, the rimantadine may thus act as a “molecular wedge” that prevents the dynamic “breathing” of the channel required for ion conduction.
Comparing the amantadine or rimantadine binding mode of HCV p7 to that of influenza AM2 shows two fundamentally different mechanisms of drug inhibition. In the case of AM2, one drug binds to one channel. Drug binding inhibits proton transport by directly blocking the channel passage; it also prevents channel from opening. In the case of p7, amantadine and rimantadine are clearly too small to block the channel. They instead bind to six equivalent sites outside of the channel cavity, which can afford up to six drugs per channel. If rimantadine binding to this site is relevant to inhibition, as crudely suggested by previous functional mutagenesis, drug binding to these sites inhibits cation conduction with an allosteric mechanism, possibly by stabilizing the closed state of the channel.
Although the mechanisms of drug inhibition may be completely different, the structural bases that govern drug-binding affinity for AM2 and p7 are actually similar, and they involve hydrophobicity and size of the pocket, and position of the drug amino group (Fig. 6.6c). In the case of the AM2 tetramer, the drug adamantane cage fits snuggly in a hydrophobic pocket formed by eight methyl groups from Val27 and Ala30 (two from each subunit), while the drug amino group forms polar contact with the backbone oxygen of Ala30 and points to the polar region of the channel cavity. For the p7 channel, the adamantane cage is in contact with ten methyl groups and an aromatic group from the protein. These hydrophobic groups form a deep hydrophobic pocket that also matches closely the size of the adamantane cage. The amino group of amantadine or rimantadine points to the channel lumen; it is in position to form polar contacts with the electronegative groups such as backbone carbonyl of residues 15–17. Hence, having a greasy pocket for the adamantane cage and a nearby electronegative group to interact with the amino group may be a general requirement for amantadine or rimantadine binding.
6.4 The Transmembrane Domains of Viral Fusion Proteins
Previous functional mutagenesis studies have suggested that the TMDs of viral fusion proteins are not only limited to the function of membrane anchoring, but also involved in other functions such as membrane fusion or assembly of the fusion protein on the viral membrane. For example, sequence analysis of the HA from mutant viruses and site-specific mutagenesis of the fusion peptide identified a group of mutations in the N-terminal half of the influenza HA TMD severely affected membrane fusion (the hemifusion to pore formation) [115, 116, 117]. In the case of HIV-1, multiple lines of evidences suggest that the TMD of gp41 is not merely a membrane anchor, but plays critical roles in membrane fusion and viral infectivity [118, 119, 120, 121, 122]. The amino acid sequence of gp41 TMD is also highly interesting. There is a Gly rich motif in the TMD, which suggests some sort of oligomerization. Even more peculiar is the presence of a conserved arginine in the middle of the predicted TM region. Unlike the structural biology of viroporins, there is essentially no structural information of TMDs of viral fusion proteins except for that of the TMD of HIV-1 Env. Hence, we focus on the discussion on the trimeric membrane anchor of the TMD of the HIV-1 envelope spike.
6.4.1 HIV-1 Envelope Glycoprotein
HIV-1 envelope spike [Env; trimeric (gp160)3, cleaved to (gp120/gp41)3] is a type I membrane protein that fuses viral and host cell membranes to initiate viral infection . The gp120 and gp41 are the receptor recognition and membrane fusion proteins, respectively. Conformational changes in gp120 when triggered by binding to receptor (CD4) and co-receptor (e.g., CCR5 or CXCR4) lead to a cascade of refolding events in gp41 (similar to those illustrated in Fig. 6.2a), and ultimately to membrane fusion [39, 124, 125, 126]. The mature and functional Env spikes, (gp120/gp41)3, are the sole antigens on the virion surface and thus important candidates for vaccine development [127, 128]. The native prefusion conformation of HIV-1 Env is recognized by most broadly neutralizing antibodies (bnAbs) [129, 130, 131] and it is generally believed to have the potential to induce such antibody responses. Thus, the native conformation of Env spikes on the surface of virions is extremely important to immunogen design in B-cell based vaccine development.
One unusual feature is that the TMD trimer appears to be stabilized by two separate packing modes (Fig. 6.7c). The N-terminal half encompassing the GxxxG motif forms a coiled-coil trimer, whereas the C-terminal half is held together by a network of polar contacts, which we named the hydrophilic core. For the N-terminal coiled-coil, the helical wheel representation of the trimer clearly indicates packing of hydrophobic residues such as Ile and Val at the “a” and “d” positions of the heptad motif, forming a hydrophobic core. The GxxxG is a well-known motif that drives TMH dimerization [58, 140, 141]. In the classic example of the glycophorin A TMD dimer structure, the two glycines of one TMH allow close packing with the GxxxG face of another TMH, resulting in a very strong TMH dimeric complex [58, 140]. There has been no previous report, however, of the GxxxG involvement in TMH trimerization. In the coiled-coil region of the HIV-1 Env TMD, only G690 is involved in the trimer assembly, i.e., its small sidechain allows a close VDW contact with V689 of the adjacent TMH. The other glycine, G694, is on the periphery of the trimer facing outwards and its mutation to alanine or valine has essentially no effect on TMD trimerization, as well as Env functions . Therefore, the key difference in the structural role of the GxxxG motif between the glycophorin A TMD dimer and HIV-1 Env TMD trimer is that both the glycines are required for forming inter-monomer contacts in the dimer, while only one glycine is important for the trimeric assembly.
TM segments of many viral fusion proteins contain a GxxxG motif or “SmallxxxSmall” motifs (“Small” refers to residues with a small side chain, such as, glycine, alanine, serine or cysteine) [142, 143, 144], suggesting that oligomerization of their TMDs may be a common property. For example, recent biochemical evidence has shown that the TMDs of hepatitis C virus envelope glycoproteins E1 and E2 form stable dimers or trimers that are also resistant to SDS . No high-resolution structure of any TMD oligomer from other viral fusion proteins has been reported, due to technical challenges for structural studies of such constructs in the context of lipid bilayer. The NMR structure of the HIV-1 Env TMD may provide some clues for how other viral fusion proteins oligomerize in the membrane.
Another peculiar feature is the presence of three copies of arginine (R696) near the middle of the TMHs (Fig. 6.7b), suggesting three unbalanced charges in the hydrophobic core of the membrane if the Arg remains protonated. In the NMR structure, the tips of the long sidechains of these arginines are facing lipids and each of them is surrounded by three hydrophobic residues (L692, L695, and I697). It is also interesting to note that R696 occupies a “d” position in the coiled-coil, with its Cβ facing inwards to the trimer interface. Precise physical basis of how R696 is accommodated in the highly hydrophobic environment remains unclear, but the underlying mechanism must tolerate a Lys residue, which is present in some viral isolates at this position. It is interesting to mention that the NOE experiment for the R696 epsilon protons showed clear water NOE even with a short NOE mixing time of 60 ms, suggesting that the Arg is somehow hydrated in the membrane.
A positively-charged Arg or Lys in the TM segment of viral fusion protein is present in some related enveloped viruses, including simian immunodeficiency virus, caprine arthritis and encephalitis virus, equine infectious anemia virus, visna virus, and foamy virus, as well as in hepatitis C virus [145, 146, 147, 148, 149, 150], but absent in many others. It is therefore not a prerequisite for viral membrane fusion in general . Functional mutagenesis indicated that the R696A mutant of HIV-1 Env showed some defect in cell-cell fusion, but it could be fully compensated by high Env expression . Moreover, this mutant has wildtype viral infectivity. In vitro infectivity and cell-cell fusion do not, however, mimic all the conditions under which the virus moves from one cell to another in an infected individual, nor are those assays particularly sensitive to physiologically relevant kinetic parameters.
6.5 Future Perspective
The structures of viroporins discussed above are all substantially different from any of the channel structures found in prokaryotes and eukaryotes, and are thus clean structural targets for developing antiviral compounds. The available structures also suggest that viroporins generally adopt minimalist architecture and can possess structural features compatible with selective ion transport. The viral channels, however, may not be as functionally robust or specifically regulated as some of their counterparts in prokayrotes and eukaryotes, because viruses often do not need intricate regulation of channel activities during their infection cycles. Having minimalist structure also makes viroporins fragile and sensitive to the membrane environment. This is reflected by the conformational variations observed for the AM2 channel in different reconstitution media. Therefore, it remains important to examine these viroporins in more native environments, e.g., lipid bilayer and full-length proteins. As solid-state NMR continues to improve spectral resolution [87, 89], obtaining detailed structures of viroporins in liposomes should in principle be feasible in the near future. Alternatively, the ideal bicelle system can be explored with solution NMR to revisit some of the viroporin structures determined previously in detergent micelles. Future establishment of the NMR systems for viroporins under more native conditions would certainly provide versatile and effective platforms for investigating inhibitor binding.
Apart from the progress in structure determination, major challenges lie ahead in the functional aspects of viroporin research. The precise functional roles of many viroporins are still unclear. Moreover, due to the lack of robust single-channel recording setups suitable for viroporins, the ion conductance properties of most viroporins have not been fully characterized. Therefore, better definition of the functional roles and channel properties of viroporins will certainly draw greater enthusiasm for developing therapeutics that target this interesting family of membrane channels.
The example from HIV-1 Env suggests that TMDs of viral fusion proteins can adopt very interesting structures, and the distinct structural features certainly allude to their roles in viral fusion protein assembly and incorporation into the envelope, as well as in the process of membrane fusion. The NMR structural study of the HIV-1 gp41 TMD in bicelles with q = 0.5  demonstrates that the structures of most of the viral fusion protein TMDs can in principle be determined in essentially lipid bilayer environment using modern NMR techniques. Moreover, the combined use of ideal bicelles (q ≥ 0.5) and solvent paramagnetic relaxation enhancement measurements can be used to determine the membrane partition of the TMDs in the bilayer region of the bicelles . We believe the next major challenge lies in understanding the roles of the fusion protein TMDs in the fusion mechanism. In the cases of the flu HA and HIV gp41, for example, new experiments need to be designed to address questions such as, “Does the TMD interact with the fusion domain in the hemifusion stage in which the two domains are presumably in close proximity?” and “Does the TMD play a role in facilitating the conversion from the hemifusion to the pore formation?”
Understanding the structural properties and conformational stability of the TMD may also have far reaching implication to vaccine development and this has been recognized at least for HIV-1. As mentioned above, truncations in the CT of the Env could drastically alter the sensitivity of the Env ECD to the known trimer-specific bnAbs . The continuous structure from the N- to C- terminal ends of the Env TMD, as observed by NMR, suggests that the Env ECD can be structurally coupled via the TMD. Indeed, it has also been shown that the Env TMD can also modulate the antigenic structure of the ECD in a cell-cell fusion assay and a pseudovirus-based neutralization assay . The results show direct correlation that more disrupted TMD trimer led to less inhibition or neutralization by the trimer-specific bnAbs that target only the ECD. The results suggest that the stability of the TMD trimer is an important consideration when designing immunogens for HIV vaccines.
The HIV Env TMD is, however, not directly linked to the Env ECD. Another important segment known as the membrane-proximal external region (MPER) is the direct link that connects the TMD to the ECD, and thus it could in principle also affect the antigenic properties of the Env ECD. The MPER sequence is extremely conserved with five absolutely conserved tryptophans, suggesting that the MPER probably also has interesting structural features. The conformation of the MPER attached to the TMD trimer in a lipid bilayer environment is still unknown (Fig. 6.7d). Previous NMR studies of an isolated MPER peptide in detergent micelle suggested that the MPER is monomeric and folds into a kinked helix with many hydrophobic residues embedded in the micelles [151, 152]. This of course would have a rather negative implication for the MPER as a vaccine epitope because antibodies that bind to the MPER must dig it out of the lipids, which could be accompanied with polyreactivities. The Env TMD appears to be well structured and assembled strongly to trimers. Hence, the structure of the MPER when connected to the trimeric TMD and in the context of a lipid bilayer remains to be characterized.
- 145.West JT, Johnston PB, Dubay SR, Hunter E. Mutations within the putative membrane-spanning domain of the simian immunodeficiency virus transmembrane glycoprotein define the minimal requirements for fusion, incorporation, and infectivity. J Virol. 2001;75(20):9601–12.PubMedPubMedCentralCrossRefGoogle Scholar