Cryo-EM structure of SARS-CoV-2 postfusion spike in membrane.

The entry of SARS-CoV-2 into host cells depends on the refolding of the virus-encoded spike protein from a prefusion conformation, which is metastable after cleavage, to a lower-energy stable postfusion conformation1,2. This transition overcomes kinetic barriers for fusion of viral and target cell membranes3,4. Here we report a cryogenic electron microscopy (cryo-EM) structure of the intact postfusion spike in a lipid bilayer that represents the single-membrane product of the fusion reaction. The structure provides structural definition of the functionally critical membrane-interacting segments, including the fusion peptide and transmembrane anchor. The internal fusion peptide forms a hairpin-like wedge that spans almost the entire lipid bilayer and the transmembrane segment wraps around the fusion peptide at the last stage of membrane fusion. These results advance our understanding of the spike protein in a membrane environment and may guide development of intervention strategies.

Antibody-mediated SARS-CoV-2 entry in cultured cells.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters host cells by first engaging its cellular receptor angiotensin converting enzyme 2 (ACE2) to induce conformational changes in the virus-encoded spike protein and fusion between the viral and target cell membranes. Here, we report that certain monoclonal neutralizing antibodies against distinct epitopic regions of the receptor-binding domain of the spike can replace ACE2 to serve as a receptor and efficiently support membrane fusion and viral infectivity in vitro. These receptor-like antibodies can function in the form of a complex of their soluble immunoglobulin G with Fc-gamma receptor I, a chimera of their antigen-binding fragment with the transmembrane domain of ACE2 or a membrane-bound B cell receptor, indicating that ACE2 and its specific interaction with the spike protein are dispensable for SARS-CoV-2 entry. These results suggest that antibody responses against SARS-CoV-2 may help expand the viral tropism to otherwise nonpermissive cell types with potential implications for viral transmission and pathogenesis.

Structural basis of coreceptor recognition by HIV-1 envelope spike.

HIV-1 envelope glycoprotein (Env), which consists of trimeric (gp160)3 cleaved to (gp120 and gp41)3, interacts with the primary receptor CD4 and a coreceptor (such as chemokine receptor CCR5) to fuse viral and target-cell membranes. The gp120-coreceptor interaction has previously been proposed as the most crucial trigger for unleashing the fusogenic potential of gp41. Here we report a cryo-electron microscopy structure of a full-length gp120 in complex with soluble CD4 and unmodified human CCR5, at 3.9 Å resolution. The V3 loop of gp120 inserts into the chemokine-binding pocket formed by seven transmembrane helices of CCR5, and the N terminus of CCR5 contacts the CD4-induced bridging sheet of gp120. CCR5 induces no obvious allosteric changes in gp120 that can propagate to gp41; it does bring the Env trimer close to the target membrane. The N terminus of gp120, which is gripped by gp41 in the pre-fusion or CD4-bound Env, flips back in the CCR5-bound conformation and may irreversibly destabilize gp41 to initiate fusion. The coreceptor probably functions by stabilizing and anchoring the CD4-induced conformation of Env near the cell membrane. These results advance our understanding of HIV-1 entry into host cells and may guide the development of vaccines and therapeutic agents.

ACH2.0/E, the Consolidated Theory of Conventional and Unconventional Alzheimer's Disease: Origins, Progression, and Therapeutic Strategies.

The centrality of amyloid-beta (Aß) is an indisputable tenet of Alzheimer's disease (AD). It was initially indicated by the detection (1991) of a mutation within Aß protein precursor (AßPP) segregating with the disease, which served as a basis for the long-standing Amyloid Cascade Hypothesis (ACH) theory of AD. In the intervening three decades, this notion was affirmed and substantiated by the discovery of numerous AD-causing and AD-protective mutations with all, without an exception, affecting the structure, production, and intraneuronal degradation of Aß. The ACH postulated that the disease is caused and driven by extracellular Aß. When it became clear that this is not the case, and the ACH was largely discredited, a new theory of AD, dubbed ACH2.0 to re-emphasize the centrality of Aß, was formulated. In the ACH2.0, AD is caused by physiologically accumulated intraneuronal Aß (iAß) derived from AßPP. Upon reaching the critical threshold, it triggers activation of the autonomous AßPP-independent iAß generation pathway; its output is retained intraneuronally and drives the AD pathology. The bridge between iAß derived from AßPP and that generated independently of AßPP is the neuronal integrated stress response (ISR) elicited by the former. The ISR severely suppresses cellular protein synthesis; concurrently, it activates the production of a small subset of proteins, which apparently includes components necessary for operation of the AßPP-independent iAß generation pathway that are absent under regular circumstances. The above sequence of events defines "conventional" AD, which is both caused and driven by differentially derived iAß. Since the ISR can be elicited by a multitude of stressors, the logic of the ACH2.0 mandates that another class of AD, referred to as "unconventional", has to occur. Unconventional AD is defined as a disease where a stressor distinct from AßPP-derived iAß elicits the neuronal ISR. Thus, the essence of both, conventional and unconventional, forms of AD is one and the same, namely autonomous, self-sustainable, AßPP-independent production of iAß. What distinguishes them is the manner of activation of this pathway, i.e., the mode of causation of the disease. In unconventional AD, processes occurring at locations as distant from and seemingly as unrelated to the brain as, say, the knee can potentially trigger the disease. The present study asserts that these processes include traumatic brain injury (TBI), chronic traumatic encephalopathy, viral and bacterial infections, and a wide array of inflammatory conditions. It considers the pathways which are common to all these occurrences and culminate in the elicitation of the neuronal ISR, analyzes the dynamics of conventional versus unconventional AD, shows how the former can morph into the latter, explains how a single TBI can hasten the occurrence of AD and why it takes multiple TBIs to trigger the disease, and proposes the appropriate therapeutic strategies. It posits that yet another class of unconventional AD may occur where the autonomous AßPP-independent iAß production pathway is initiated by an ISR-unrelated activator, and consolidates the above notions in a theory of AD, designated ACH2.0/E (for expanded ACH2.0), which incorporates the ACH2.0 as its special case and retains the centrality of iAß produced independently of AßPP as the driving agent of the disease.

On the Inadequacy of the Current Transgenic Animal Models of Alzheimer's Disease: The Path Forward.

For at least two reasons, the current transgenic animal models of Alzheimer's disease (AD) appear to be patently inadequate. They may be useful in many respects, the AD models; however, they are not. First, they are incapable of developing the full spectrum of the AD pathology. Second, they respond spectacularly well to drugs that are completely ineffective in the treatment of symptomatic AD. These observations indicate that both the transgenic animal models and the drugs faithfully reflect the theory that guided the design and development of both, the amyloid cascade hypothesis (ACH), and that both are inadequate because their underlying theory is. This conclusion necessitated the formulation of a new, all-encompassing theory of conventional AD-the ACH2.0. The two principal attributes of the ACH2.0 are the following. One, in conventional AD, the agent that causes the disease and drives its pathology is the intraneuronal amyloid-ß (iAß) produced in two distinctly different pathways. Two, following the commencement of AD, the bulk of Aß is generated independently of Aß protein precursor (AßPP) and is retained inside the neuron as iAß. Within the framework of the ACH2.0, AßPP-derived iAß accumulates physiologically in a lifelong process. It cannot reach levels required to support the progression of AD; it does, however, cause the disease. Indeed, conventional AD occurs if and when the levels of AßPP-derived iAß cross the critical threshold, elicit the neuronal integrated stress response (ISR), and trigger the activation of the AßPP-independent iAß generation pathway; the disease commences only when this pathway is operational. The iAß produced in this pathway reaches levels sufficient to drive the AD pathology; it also propagates its own production and thus sustains the activity of the pathway and perpetuates its operation. The present study analyzes the reason underlying the evident inadequacy of the current transgenic animal models of AD. It concludes that they model, in fact, not Alzheimer's disease but rather the effects of the neuronal ISR sustained by AßPP-derived iAß, that this is due to the lack of the operational AßPP-independent iAß production pathway, and that this mechanism must be incorporated into any successful AD model faithfully emulating the disease. The study dissects the plausible molecular mechanisms of the AßPP-independent iAß production and the pathways leading to their activation, and introduces the concept of conventional versus unconventional Alzheimer's disease. It also proposes the path forward, posits the principles of design of productive transgenic animal models of the disease, and describes the molecular details of their construction.

The Amyloid Cascade Hypothesis 2.0 for Alzheimer's Disease and Aging-Associated Cognitive Decline: From Molecular Basis to Effective Therapy.

With the long-standing amyloid cascade hypothesis (ACH) largely discredited, there is an acute need for a new all-encompassing interpretation of Alzheimer's disease (AD). Whereas such a recently proposed theory of AD is designated ACH2.0, its commonality with the ACH is limited to the recognition of the centrality of amyloid-ß (Aß) in the disease, necessitated by the observation that all AD-causing mutations affect, in one way or another, Aß. Yet, even this narrow commonality is superficial since AD-causing Aß of the ACH differs distinctly from that specified in the ACH2.0: Whereas in the former, the disease is caused by secreted extracellular Aß, in the latter, it is triggered by Aß-protein-precursor (AßPP)-derived intraneuronal Aß (iAß) and driven by iAß generated independently of AßPP. The ACH2.0 envisions AD as a two-stage disorder. The first, asymptomatic stage is a decades-long accumulation of AßPP-derived iAß, which occurs via internalization of secreted Aß and through intracellular retention of a fraction of Aß produced by AßPP proteolysis. When AßPP-derived iAß reaches critical levels, it activates a self-perpetuating AßPP-independent production of iAß that drives the second, devastating AD stage, a cascade that includes tau pathology and culminates in neuronal loss. The present study analyzes the dynamics of iAß accumulation in health and disease and concludes that it is the prime factor driving both AD and aging-associated cognitive decline (AACD). It discusses mechanisms potentially involved in AßPP-independent generation of iAß, provides mechanistic interpretations for all principal aspects of AD and AACD including the protective effect of the Icelandic AßPP mutation, the early onset of FAD and the sequential manifestation of AD pathology in defined regions of the affected brain, and explains why current mouse AD models are neither adequate nor suitable. It posits that while drugs affecting the accumulation of AßPP-derived iAß can be effective only protectively for AD, the targeted degradation of iAß is the best therapeutic strategy for both prevention and effective treatment of AD and AACD. It also proposes potential iAß-degrading drugs.

Next Generation Therapeutic Strategy for Treatment and Prevention of Alzheimer's Disease and Aging-Associated Cognitive Decline: Transient, Once-in-a-Lifetime-Only Depletion of Intraneuronal Aß (iAß) by Its Targeted Degradation via Augmentation of Intra-iAß-Cleaving Activities of BACE1 and/or BACE2.

Although the long-standing Amyloid Cascade Hypothesis (ACH) has been largely discredited, its main attribute, the centrality of amyloid-beta (Aß) in Alzheimer's disease (AD), remains the cornerstone of any potential interpretation of the disease: All known AD-causing mutations, without a single exception, affect, in one way or another, Aß. The ACH2.0, a recently introduced theory of AD, preserves this attribute but otherwise differs fundamentally from the ACH. It posits that AD is a two-stage disorder where both stages are driven by intraneuronal (rather than extracellular) Aß (iAß) albeit of two distinctly different origins. The first asymptomatic stage is the decades-long accumulation of Aß protein precursor (AßPP)-derived iAß to the critical threshold. This triggers the activation of the self-sustaining AßPP-independent iAß production pathway and the commencement of the second, symptomatic AD stage. Importantly, Aß produced independently of AßPP is retained intraneuronally. It drives the AD pathology and perpetuates the operation of the pathway; continuous cycles of the iAß-stimulated propagation of its own AßPP-independent production constitute an engine that drives AD, the AD Engine. It appears that the dynamics of AßPP-derived iAß accumulation is the determining factor that either drives Aging-Associated Cognitive Decline (AACD) and triggers AD or confers the resistance to both. Within the ACH2.0 framework, the ACH-based drugs, designed to lower levels of extracellular Aß, could be applicable in the prevention of AD and treatment of AACD because they reduce the rate of accumulation of AßPP-derived iAß. The present study analyzes their utility and concludes that it is severely limited. Indeed, their short-term employment is ineffective, their long-term engagement is highly problematic, their implementation at the symptomatic stages of AD is futile, and their evaluation in conventional clinical trials for the prevention of AD is impractical at best, impossible at worst, and misleading in between. In contrast, the ACH2.0-guided Next Generation Therapeutic Strategy for the treatment and prevention of both AD and AACD, namely the depletion of iAß via its transient, short-duration, targeted degradation by the novel ACH2.0-based drugs, has none of the shortcomings of the ACH-based drugs. It is potentially highly effective, easily evaluable in clinical trials, and opens up the possibility of once-in-a-lifetime-only therapeutic intervention for prevention and treatment of both conditions. It also identifies two plausible ACH2.0-based drugs: activators of physiologically occurring intra-iAß-cleaving capabilities of BACE1 and/or BACE2.

Structure of the membrane proximal external region of HIV-1 envelope glycoprotein.

The membrane-proximal external region (MPER) of the HIV-1 envelope glycoprotein (Env) bears epitopes of broadly neutralizing antibodies (bnAbs) from infected individuals; it is thus a potential vaccine target. We report an NMR structure of the MPER and its adjacent transmembrane domain in bicelles that mimic a lipid-bilayer membrane. The MPER lies largely outside the lipid bilayer. It folds into a threefold cluster, stabilized mainly by conserved hydrophobic residues and potentially by interaction with phospholipid headgroups. Antigenic analysis and comparison with published images from electron cryotomography of HIV-1 Env on the virion surface suggest that the structure may represent a prefusion conformation of the MPER, distinct from the fusion-intermediate state targeted by several well-studied bnAbs. Very slow bnAb binding indicates that infrequent fluctuations of the MPER structure give these antibodies occasional access to alternative conformations of MPER epitopes. Mutations in the MPER not only impede membrane fusion but also influence presentation of bnAb epitopes in other regions. These results suggest strategies for developing MPER-based vaccine candidates.

Antigenicity-defined conformations of an extremely neutralization-resistant HIV-1 envelope spike.

The extraordinary genetic diversity of the HIV-1 envelope spike [Env; trimeric (gp160)3, cleaved to (gp120/gp41)3] poses challenges for vaccine development. Envs of different clinical isolates exhibit different sensitivities to antibody-mediated neutralization. Envs of difficult-to-neutralize viruses are thought to be more stable and conformationally homogeneous trimers than those of easy-to-neutralize viruses, thereby providing more effective concealment of conserved, functionally critical sites. In this study we have characterized the antigenic properties of an Env derived from one of the most neutralization-resistant HIV-1 isolates, CH120.6. Sequence variation at neutralizing epitopes does not fully account for its exceptional resistance to antibodies. The full-length, membrane-bound CH120.6 Env is indeed stable and conformationally homogeneous. Its antigenicity correlates closely with its neutralization sensitivity, and major changes in antigenicity upon CD4 engagement appear to be restricted to the coreceptor site. The CH120.6 gp140 trimer, the soluble and uncleaved ectodomain of (gp160)3, retains many antigenic properties of the intact Env, consistent with a conformation close to that of Env spikes on a virion, whereas its monomeric gp120 exposes many nonneutralizing or strain-specific epitopes. Thus, trimer organization and stability are important determinants not only for occluding many epitopes but also for conferring resistance to neutralization by all but a small set of antibodies. Env preparations derived from neutralization-resistant viruses may induce irrelevant antibody responses less frequently than do other Envs and may be excellent templates for developing soluble immunogens.

Stable, uncleaved HIV-1 envelope glycoprotein gp140 forms a tightly folded trimer with a native-like structure.

The HIV-1 envelope spike [trimeric (gp160)3, cleaved to (gp120/gp41)3] is the mediator of viral entry and the principal target of humoral immune response to the virus. Production of a recombinant preparation that represents the functional spike poses a challenge for vaccine development, because the (gp120/gp41)3 complex is prone to dissociation. We have reported previously that stable HIV-1 gp140 trimers, the uncleaved ectodomains of (gp160)3, have nearly all of the antigenic properties expected for native viral spikes. Because of recent claims that uncleaved gp140 proteins may adopt a nonnative structure with three gp120 moieties "dangling" from a trimeric gp41 ectodomain in its postfusion conformation, we have inserted a long, flexible linker between gp120 and gp41 in our stable gp140 trimers to assess their stability and to analyze their conformation in solution. The modified trimer has biochemical and antigenic properties virtually identical to those of its unmodified counterpart. Both forms bind a single CD4 per trimer, suggesting that the trimeric conformation occludes two of the three CD4 sites even when a flexible linker has relieved the covalent constraint between gp120 and gp41. In contrast, an artificial trimer containing three gp120s flexibly tethered to a trimerization tag binds three CD4s and has antigenicity nearly identical to that of monomeric gp120. Moreover, the gp41 part of both modified and unmodified gp140 trimers has a structure very different from that of postfusion gp41. These results show that uncleaved gp140 trimers from suitable isolates have compact, native-like structures and support their use as candidate vaccine immunogens.

Mechanism of HIV-1 neutralization by antibodies targeting a membrane-proximal region of gp41.

Induction of broadly neutralizing antibodies (bNAbs) is an important goal for HIV-1 vaccine development. Two autoreactive bNAbs, 2F5 and 4E10, recognize a conserved region on the HIV-1 envelope glycoprotein gp41 adjacent to the viral membrane known as the membrane-proximal external region (MPER). They block viral infection by targeting a fusion-intermediate conformation of gp41, assisted by an additional interaction with the viral membrane. Another MPER-specific antibody, 10E8, has recently been reported to neutralize HIV-1 with potency and breadth much greater than those of 2F5 or 4E10, but it appeared not to bind phospholipids and might target the untriggered envelope spikes, raising the hope that the MPER could be harnessed for vaccine design without major immunological concerns. Here, we show by three independent approaches that 10E8 indeed binds lipid bilayers through two hydrophobic residues in its CDR H3 (third heavy-chain complementarity-determining region). Its weak affinity for membranes in general and preference for cholesterol-rich membranes may account for its great neutralization potency, as it is less likely than other MPER-specific antibodies to bind cellular membranes nonspecifically. 10E8 binds with high affinity to a construct mimicking the fusion intermediate of gp41 but fails to recognize the envelope trimers representing the untriggered conformation. Moreover, we can improve the potency of 4E10 without affecting its binding to gp41 by a modification of its lipid-interacting CDR H3. These results reveal a general mechanism of HIV-1 neutralization by MPER-specific antibodies that involves interactions with viral lipids.

The Amyloid Cascade Hypothesis 2.0: Generalization of the Concept.

Recently, we proposed the Amyloid Cascade Hypothesis 2.0 (ACH2.0), a reformulation of the ACH. In the former, in contrast to the latter, Alzheimer's disease (AD) is driven by intraneuronal amyloid-ß (iAß) and occurs in two stages. In the first, relatively benign stage, Aß protein precursor (AßPP)-derived iAß activates, upon reaching a critical threshold, the AßPP-independent iAß-generating pathway, triggering a devastating second stage resulting in neuronal death. While the ACH2.0 remains aligned with the ACH premise that Aß is toxic, the toxicity is exerted because of intra- rather than extracellular Aß. In this framework, a once-in-a-lifetime-only iAß depletion treatment via transient activation of BACE1 and/or BACE2 (exploiting their Aß-cleaving activities) or by any means appears to be the best therapeutic strategy for AD. Whereas the notion of differentially derived iAß being the principal moving force at both AD stages is both plausible and elegant, a possibility remains that the second AD stage is enabled by an AßPP-derived iAß-activated self-sustaining mechanism producing a yet undefined deleterious "substance X" (sX) which anchors the second AD stage. The present study generalizes the ACH2.0 by incorporating this possibility and shows that, in this scenario, the iAß depletion therapy may be ineffective at symptomatic AD stages but fully retains its preventive potential for both AD and the aging-associated cognitive decline, which is defined in the ACH2.0 framework as the extended first stage of AD.

Effect of Lecanemab in Early Alzheimer's Disease: Mechanistic Interpretation in the Amyloid Cascade Hypothesis 2.0 Perspective.

In clinical trials, lecanemab and donanemab showed statistically significant yet marginal slowdown of Alzheimer's disease (AD)-associated cognitive decline. This could be due to their sub-optimal design and/or deployment; alternatively, their limited efficiency could be intrinsic. Distinguishing between the two is of great importance considering the acute need of efficient AD therapy and tremendous resources being invested in its pursuit. The present study analyzes the mode of operation of lecanemab and donanemab within the framework of recently proposed Amyloid Cascade Hypothesis 2.0 and concludes that the second possibility is correct. It suggests that substantial improvement of the efficiency of these drugs in symptomatic AD is unlikely and proposes the alternative therapeutic strategy.

Principles of Design of Clinical Trials for Prevention and Treatment of Alzheimer's Disease and Aging-Associated Cognitive Decline in the ACH2.0 Perspective: Potential Outcomes, Challenges, and Solutions.

With the Amyloid Cascade Hypothesis (ACH) largely discredited, the ACH2.0 theory of Alzheimer's disease (AD) has been recently introduced. Within the framework of the ACH2.0, AD is triggered by amyloid-ß protein precursor (AßPP)-derived intraneuronal Aß (iAß) and is driven by iAß produced in the AßPP-independent pathway and retained intraneuronally. In this paradigm, the depletion of extracellular Aß or suppression of Aß production by AßPP proteolysis, the two sources of AßPP-derived iAß, would be futile in symptomatic AD, due to its reliance on iAß generated independently of AßPP, but effective in preventing AD and treating Aging-Associated Cognitive Decline (AACD) driven, in the ACH2.0 framework, by AßPP-derived iAß. The observed effect of lecanemab and donanemab, interpreted in the ACH2.0 perspective, supports this notion and mandates AD-preventive clinical trials. Such trials are currently in progress. They are likely, however, to fail or to yield deceptive results if conducted conventionally. The present study considers concepts of design of clinical trials of lecanemab, donanemab, or any other drug, targeting the influx of AßPP-derived iAß, in prevention of AD and treatment of AACD. It analyzes possible outcomes and explains why selection of high-risk asymptomatic participants seems reasonable but is not. It argues that outcomes of such AD preventive trials could be grossly misleading, discusses inevitable potential problems, and proposes feasible solutions. It advocates the initial evaluation of this type of drugs in clinical trials for treatment of AACD. Whereas AD protective trials of these drugs are potentially of an impractical length, AACD clinical trials are expected to yield unequivocal results within a relatively short duration. Moreover, success of the latter, in addition to its intrinsic value, would constitute a proof of concept for the former. Furthermore, this study introduces concepts of the active versus passive iAß depletion, contends that targeted degradation of iAß is the best therapeutic strategy for both prevention and treatment of AD and AACD, proposes potential iAß-degrading drugs, and describes their feasible and unambiguous evaluation in clinical trials.

Structural and functional characteristics of the SARS-CoV-2 Omicron subvariant BA.2 spike protein.

The Omicron subvariant BA.2 has become the dominant circulating strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in many countries. Here, we have characterized structural, functional and antigenic properties of the full-length BA.2 spike (S) protein and compared replication of the authentic virus in cell culture and an animal model with previously prevalent variants. BA.2 S can fuse membranes slightly more efficiently than Omicron BA.1, but still less efficiently than other previous variants. Both BA.1 and BA.2 viruses replicated substantially faster in animal lungs than the early G614 (B.1) strain in the absence of pre-existing immunity, possibly explaining the increased transmissibility despite their functionally compromised spikes. As in BA.1, mutations in the BA.2 S remodel its antigenic surfaces, leading to strong resistance to neutralizing antibodies. These results suggest that both immune evasion and replicative advantage may contribute to the heightened transmissibility of the Omicron subvariants.

Role of HIV membrane in neutralization by two broadly neutralizing antibodies.

Induction of effective antibody responses against HIV-1 infection remains an elusive goal for vaccine development. Progress may require in-depth understanding of the molecular mechanisms of neutralization by monoclonal antibodies. We have analyzed the molecular actions of two rare, broadly neutralizing, human monoclonal antibodies, 4E10 and 2F5, which target the transiently exposed epitopes in the membrane proximal external region (MPER) of HIV-1 gp41 envelope during viral entry. Both have long CDR H3 loops with a hydrophobic surface facing away from the peptide epitope. We find that the hydrophobic residues of 4E10 mediate a reversible attachment to the viral membrane and that they are essential for neutralization, but not for interaction with gp41. We propose that these antibodies associate with the viral membrane in a required first step and are thereby poised to capture the transient gp41 fusion intermediate. These results bear directly on strategies for rational design of HIV-1 envelope immunogens.

The Amyloid Cascade Hypothesis 2.0: On the Possibility of Once-in-a-Lifetime-Only Treatment for Prevention of Alzheimer's Disease and for Its Potential Cure at Symptomatic Stages.

We posit that Alzheimer's disease (AD) is driven by amyloid-ß (Aß) generated in the amyloid-ß protein precursor (AßPP) independent pathway activated by AßPP-derived Aß accumulated intraneuronally in a life-long process. This interpretation constitutes the Amyloid Cascade Hypothesis 2.0 (ACH2.0). It defines a tandem intraneuronal-Aß (iAß)-anchored cascade occurrence: intraneuronally-accumulated, AßPP-derived iAß triggers relatively benign cascade that activates the AßPP-independent iAß-generating pathway, which, in turn, initiates the second, devastating cascade that includes tau pathology and leads to neuronal loss. The entire output of the AßPP-independent iAß-generating pathway is retained intraneuronally and perpetuates the pathway's operation. This process constitutes a self-propagating, autonomous engine that drives AD and ultimately kills its host cells. Once activated, the AD Engine is self-reliant and independent from Aß production in the AßPP proteolytic pathway; operation of the former renders the latter irrelevant to the progression of AD by relegating its iAß contribution to insignificant, and brands its manipulation for therapeutic purposes, such as BACE (beta-site AßPP-cleaving enzyme) inhibition, as futile. In the proposed AD paradigm, the only valid direct therapeutic strategy is targeting the engine's components, and the most effective feasible approach appears to be the activation of BACE1 and/or of its homolog BACE2, with the aim of exploiting their Aß-cleaving activities. Such treatment would collapse the iAß population and 'reset' its levels below those required for the operation of the AD Engine. Any sufficiently selective iAß-depleting treatment would be equally effective. Remarkably, this approach opens the possibility of a short-duration, once-in-a-lifetime-only or very infrequent, preventive or curative therapy for AD; this therapy would be also effective for prevention and treatment of the 'common' pervasive aging-associated cognitive decline. The ACH2.0 clarifies all ACH-unresolved inconsistencies, explains the widespread 'resilience to AD' phenomenon, predicts occurrences of a category of AD-afflicted individuals without excessive Aß plaque load and of a novel type of familial insusceptibility to AD; it also predicts the lifespan-dependent inevitability of AD in humans if untreated preventively. The article details strategy and methodology to generate an adequate AD model and validate the hypothesis; the proposed AD model may also serve as a research and drug development platform.

Cryo-EM structure of SARS-CoV-2 postfusion spike in membrane.

Entry of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) into host cells depends on refolding of the virus-encoded spike protein from a prefusion conformation, metastable after cleavage, to a lower energy, stable postfusion conformation. This transition overcomes kinetic barriers for fusion of viral and target cell membranes. We report here a cryo-EM structure of the intact postfusion spike in a lipid bilayer that represents single-membrane product of the fusion reaction. The structure provides structural definition of the functionally critical membraneinteracting segments, including the fusion peptide and transmembrane anchor. The internal fusion peptide forms a hairpin-like wedge that spans almost the entire lipid bilayer and the transmembrane segment wraps around the fusion peptide at the last stage of membrane fusion. These results advance our understanding of the spike protein in a membrane environment and may guide development of intervention strategies.

Structural and functional impact by SARS-CoV-2 Omicron spike mutations.

The Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), bearing an unusually high number of mutations, has become a dominant strain in many countries within several weeks. We report here structural, functional, and antigenic properties of its full-length spike (S) protein with a native sequence in comparison with those of previously prevalent variants. Omicron S requires a substantially higher level of host receptor ACE2 for efficient membrane fusion than other variants, possibly explaining its unexpected cellular tropism. Mutations not only remodel the antigenic structure of the N-terminal domain of the S protein but also alter the surface of the receptor-binding domain in a way not seen in other variants, consistent with its remarkable resistance to neutralizing antibodies. These results suggest that Omicron S has acquired an extraordinary ability to evade host immunity by excessive mutations, which also compromise its fusogenic capability.

Structural and functional characteristics of SARS-CoV-2 Omicron subvariant BA.2 spike.

The Omicron subvariant BA.2 has become the dominant circulating strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in many countries. We have characterized structural, functional and antigenic properties of the full-length BA.2 spike (S) protein and compared replication of the authentic virus in cell culture and animal model with previously prevalent variants. BA.2 S can fuse membranes more efficiently than Omicron BA.1, mainly due to lack of a BA.1-specific mutation that may retard the receptor engagement, but still less efficiently than other variants. Both BA.1 and BA.2 viruses replicated substantially faster in animal lungs than the early G614 (B.1) strain in the absence of pre-existing immunity, possibly explaining the increased transmissibility despite their functionally compromised spikes. As in BA.1, mutations in the BA.2 S remodel its antigenic surfaces leading to strong resistance to neutralizing antibodies. These results suggest that both immune evasion and replicative advantage may contribute to the heightened transmissibility for the Omicron subvariants.