The first step of infection of host cells by HIV-1 is the binding of the envelope (Env) glycoprotein, which is expressed on the surface of the virus, to cellular receptors. Env is a metastable assembly of three heterodimeric complexes that form a trimer. Writing in Nature, Lu et al.1 present thought-provoking data that relaunch the search for the native conformational states of the Env trimer.
Before binding to cellular receptors, the Env trimer is in a closed (also referred to as native) conformational state. This conformation masks certain domains that are essential for the virus to enter cells, but that would otherwise be targeted by Env-specific neutralizing antibodies generated by the host’s immune system in response to HIV-1 infection. Interaction with cellular receptors — first CD4, then a co-receptor — triggers conformational changes that open up the Env trimer and pave the way for the fusion of viral- and host-cell membranes2 (Fig. 1). Defining the diverse structures of the Env trimer is key to understanding the host’s neutralizing-antibody response and to developing vaccines based on Env-derived immunogens — the proteins that stimulate the production of protective antibodies.
Despite being shielded from attacks by most neutralizing antibodies, the native trimer is potently targeted by broadly neutralizing antibodies (bnAbs). These antibodies have exceptional neutralization breadth; that is, they are active against a wide spectrum of HIV-1 subtypes and variants that emerge when the gene that encodes Env mutates. These antibodies have been investigated in HIV-vaccine development, but so far no vaccine has been able to promote a bnAb response in animals or humans2–4. The search is on for Env-derived immunogens that can successfully trigger bnAb activity. Resolving the structure of the native Env trimer is key, because this is the entity that is present before the virus binds to and enters cells. This might also be the immunogen that triggers bnAb production in people infected with HIV-1.
The inherent flexibility of the Env trimer, which transitions between conformations even when it is not bound to a cellular receptor, limits the options for structural analysis, because dynamic processes and metastable states cannot be captured by crystallization and cryo-electron microscopy. Our knowledge, therefore, rests on data obtained from analyses of Env proteins that have been artificially stabilized, such as engineered mutant Env proteins or complexes between Env and antibodies2–4. Two distinct conformations of the Env trimer — one closed, and one open and bound to the CD4 receptor — have been defined by structural analyses.
Structures known as SOSIP trimers comprise one type of engineered stabilized Env trimer that has been investigated for immunization, and in structural studies3. The capacity of antibodies to bind SOSIP trimers correlates with the extent of their neutralization breadth3,5. This indicates that these trimers represent a native, neutralization-relevant conformation of the Env protein, which makes them promising candidate immunogens for vaccines.
A technique called single-molecule fluorescence resonance energy transfer (smFRET) imaging provides information on the proximity of specifically labelled protein regions, which changes with the protein’s conformational state. This technique does not allow visualization of the Env protein’s overall structure, but tracks the opening and closing of the Env trimer. Three Env states can be distinguished before fusion of the virus with host cells: state 1, an unbound state that shows the highest degree of closeness; state 2, a slightly more open, intermediate state; and state 3, an open, CD4-bound state6 (Fig. 1). Lu and colleagues used smFRET on stabilized Env variants investigated in structural analyses to understand which of the smFRET states matches the established models of Env-trimer structure. They also analysed the wild-type Env that is expressed on virus particles.
Through a series of technically challenging measurements, the authors show that the stabilized Env trimers predominantly adopt the intermediate state 2 conformation, and not the supposedly more closed state 1 adopted by wild-type Env. Four bnAbs isolated from cows vaccinated with a state 2 SOSIP Env immunogen7 also targeted the state 2 conformation of Env. This finding suggests that antibodies induced by vaccination might preferentially recognize the conformational state of whichever Env immunogen is used in the vaccine. By contrast, and astonishingly, most HIV-1 bnAbs isolated from HIV-1-infected people that were assessed in this study targeted the state 1 conformation.
Given that the Env immunogens currently under investigation for vaccine development are in a state 2 conformation2–4, these observations call scientists to action. Which of the unbound conformations of Env, state 1 or state 2, is the better target for neutralizing the virus? Which state triggers bnAb responses in a natural infection? And which state provides the best immunogen for vaccination? Defining the structure of the elusive state 1 and its functional properties is essential to answer these questions. Furthermore, researchers need to identify specific inhibitors for distinct Env states, because they will indicate which conformations are neutralization-relevant targets.
As intriguing as the observations by Lu and colleagues are, much remains to be done to understand the implications fully. A conformation that predominates on native virus particles might not be a good immunogen, despite its physiological relevance, if it cannot be engineered to be stable. Furthermore, any differences between states 1 and 2 might be too small to induce different bnAb responses. Only a head-to-head comparison of vaccines using immunogens based on both Env conformations will address this point. Although state 1 immunogens are not available, upcoming vaccine trials with state 2 immunogens will bring relevant data to this debate.
Defining the relative importance of the different conformational states of the Env trimer of HIV-1 before its fusion with host cells resembles the task Dutch artist M. C. Escher carved out for viewers in his 1938 optical illusion Three Birds (see go.nature.com/2upmu3f). Much like choosing the most dominant colour of bird in his fluttering flock, selecting the most relevant Env conformation among the transitioning pre-CD4-bound states of the trimer depends on the context in which they are viewed. Although the differences between states 1 and 2 might be subtle, defining these states structurally and functionally is essential to inform the HIV-vaccine field. We can only be sure that the bird in the hand is indeed worth two in the bush if we seek them all out.
Nature 568, 321-322 (2019)