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The main protease (Mpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of coronavirus disease (COVID-19), is an ideal target for pharmaceutical inhibition. Mpro is conserved among coronaviruses and distinct from human proteases. Viral replication depends on the cleavage of the viral polyprotein at multiple sites. We present crystal structures of SARS-CoV-2 Mpro bound to two viral substrate peptides. The structures show how Mpro recognizes distinct substrates and how subtle changes in substrate accommodation can drive large changes in catalytic efficiency. One peptide, constituting the junction between viral nonstructural proteins 8 and 9 (nsp8/9), has P1′ and P2′ residues that are unique among the SARS-CoV-2 Mpro cleavage sites but conserved among homologous junctions in coronaviruses. Mpro cleaves nsp8/9 inefficiently, and amino acid substitutions at P1′ or P2′ can enhance catalysis. Visualization of Mpro with intact substrates provides new templates for antiviral drug design and suggests that the coronavirus lifecycle selects for finely tuned substrate-dependent catalytic parameters.

Combination antiretroviral therapy has transformed HIV-1 infection, once a fatal illness, into a manageable chronic condition. Drug resistance, severe side effects and treatment noncompliance bring challenges to combination antiretroviral therapy implementation in clinical settings and indicate the need for additional molecular targets. Here, we have identified several small-molecule fusion inhibitors, guided by a neutralizing antibody, against an extensively studied vaccine target-the membrane proximal external region (MPER) of the HIV-1 envelope spike. These compounds specifically inhibit the HIV-1 envelope-mediated membrane fusion by blocking CD4-induced conformational changes. An NMR structure of one compound complexed with a trimeric MPER construct reveals that the compound partially inserts into a hydrophobic pocket formed exclusively by the MPER residues, thereby stabilizing its prefusion conformation. These results suggest that the MPER is a potential therapeutic target for developing fusion inhibitors and that strategies employing an antibody-guided search for novel therapeutics may be applied to other human diseases.

Env's transmembrane domain revealed HIV-1's envelope protein (Env) spans the viral membrane and grants the virus entry into host cells. Env is also the sole protein of HIV-1 that is targeted by antibodies, making it a key target for vaccine design. Dev et al. used nuclear magnetic resonance to determine an atomic-level structure of the membrane-spanning region of Env in a lipid bicelle. Env's transmembrane domain forms a well-ordered trimer, which includes a stabilizing C-terminal hydrophilic core. Disrupting this core alters the sensitivity of Env to broadly neutralizing antibodies, suggesting the potential importance of this region to vaccine design. Science , this issue p. 172

HIV-1 envelope glycoprotein gp41 undergoes large conformational changes to drive fusion of viral and target cell membranes, thereby exhibiting at least three distinct conformations during the viral entry process. Neutralizing antibodies against gp41 block HIV-1 infection by targeting its membrane proximal external region in a fusion-intermediate state. Here we report biochemical and structural evidence that non-neutralizing antibodies, capable of binding with high affinity to an immunodominant segment adjacent to the neutralizing epitopes in the membrane-proximal region, only recognize a gp41 conformation when membrane fusion is complete. We propose that these non-neutralizing antibodies are induced in HIV-1 infected patients by gp41 antigens in a triggered, postfusion form and contribute to production of ineffective humoral responses. These results have important implications for gp41-based vaccine design by rational strategies.

A piezoelectric flag is modeled as a membrane for energy harvesting purposes. The tension, pressure, piezoelectric material, and external force are introduced and employed in the model. In this model, the tension is caused by gravity, the piezoelectric material, and the fluid flow. The pressure acting on the flag consists of a non-circulatory and a circulatory component. Additionally, an external force is modeled to ensure that the pressure acting on the system does not dissipate. To model the system, Hamilton’s principle is employed to find differential equation of motion. In this study, the flag is vertically oriented. This is to ensure the flag does not droop, which would greatly complicate the effect of gravity. In studying the free response, it is found that the Bessel function of the first kind describes the flag. Lastly, Galerkin’s method is applied to the system. This allows for the deflection and the voltage produced by the flag to be found. It is found that the presented model reasonably predicts both the deflection and voltage of the piezoelectric flag.Copyright © 2015 by ASME

The trimeric HIV/SIV envelope glycoprotein, gp160, is cleaved to noncovalently associated fragments, gp120 and gp41. Binding of gp120 to viral receptors leads to large structural rearrangements in both fragments. The unliganded gp120 core has a disordered beta3-beta5 loop, which reconfigures upon CD4 binding into an ordered, extended strand. Molecular modeling suggests that residues in this loop may contact gp41. We show here that deletions in the beta3-beta5 loop of HIV-1 gp120 weaken the binding of CD4 and prevent formation of the epitope for monoclonal antibody (mAb) 17b (which recognizes the coreceptor site). Formation of an encounter complex with CD4 binding and interactions of gp120 with mAbs b12 and 2G12 are not affected by these deletions. Thus, deleting the beta3-beta5 loop blocks the gp120 conformational change and may offer a strategy for design of restrained immunogens. Moreover, mutations in the SIV beta3-beta5 loop lead to greater spontaneous dissociation of gp120 from cell-associated trimers. We suggest that the CD4-induced rearrangement of this loop releases structural constraints on gp41 and thus potentiates its fusion activity.

HIV-1 enters cells by membrane fusion, mediated by the trimeric viral envelope glycoprotein gp160, which is processed by a single proteolytic cleavage into stably associated gp120 and gp41. The gp120/gp41 trimer can be triggered to undergo an irreversible conformational change. Using a protein-based assay designed to mimic the gp41 conformational change, we screened for small molecules that prevent the formation of postfusion gp41. Several compounds were identified. One set of structurally related molecules inhibited formation of a postfusion-like assembly with an IC 50 of ≈5 μM. The compounds also inhibited envelope-mediated membrane fusion in both cell–cell fusion and viral infectivity assays. Thus, our screen identifies effective fusion inhibitors. Tested against a panel of envelope proteins from primary HIV-1 isolates, the compounds inhibited fusion across a broad range of clades, including both M and T tropic strains. They bind in a highly conserved, hydrophobic pocket on the inner core of the gp41 trimer, a region previously identified as a potential inhibitor site.

Mechanical characterization of thin samples is now routine due to the prominence of the Atomic Force Microscope. Advances in amplitude modulation techniques have allowed for accurate measurement of a sample’s elastic properties by interpreting the changes in the vibration of a cantilevered beam in intermittent contact. However, the nonlinearities associated with contact complicate attempts to find an accurate time-history for the beam. Furthermore, the inclusion of viscous effects, common to soft samples, puts an explicit solution even farther from reach. A numerical method is proposed that analyzes the time-history and frequency response of a microcantilever beam with a viscoelastic end-condition. The mathematics can be simplified by incorporating the viscoelastic end-condition into the equation of motion directly by modeling it as a distributed load. A forcing function can then be derived from the Standard Linear Solid model of viscoelasticity and implemented in the non-conservative work term of Hamilton’s principle. The Galerkin method can separate the resulting nonlinear equation of motion into time and space components. Performing a numerical analysis of the time factor equation provide the beam’s response over time. The results demonstrate the distinctive effects of viscoelasticity and periodic contact on the beam’s motion and provide the framework for the determination of viscous properties using dynamic techniques.

A flag is modeled as a membrane to investigate the two-dimensional characteristics of the vibration response to an uniform wind flow. Both the affecting tension and pressure functions for the wind flow with constant velocity are introduced and utilized in the modeling. In this case, the tension is caused by the weight of the flag. The pressure function is a function describing the pressure variations caused on the flag when in uniform flow. The pressure function is found by assuming that the air flow is relatively slow and that the flag is wide enough to minimize cross flow at the boundaries. An analysis of the downstream motion of the flag is necessary as well. Hamilton’s principle is employed to derive the partial differential equation of motion. The flag is oriented in the vertical direction to neglect the effect of the flag’s weight on the system’s response. Galerkin’s method is used to solve for the first four mode shapes of the system, and the system response is numerically solved. Simulations reveal a very reasonable model when the flag is modeled as a membrane.Copyright © 2014 by ASME

Prostate specific antigen (PSA, also known as human kallikrein 3) is an important diagnostic indicator of prostatic disease. PSA exhibits low protease activity (>10(4)-fold less than chymotrypsin) under the usual in vitro assay conditions. In addition, PSA does not react readily with prototypical serine protease inactivators. We expressed human PSA (rh-PSA) in Escherichia coli and have demonstrated that rh-PSA has properties similar to those of native PSA isolated from human seminal fluid. Both PSA and rh-PSA are >10(3)-fold more active in the presence of 1.3 M Na(2)SO(4). This activation is anion-dependent, following the Hofmeister series when normality is considered: SO(4)(2)(-) approximately citrate > Ac(-) > Cl(-) > Br(-) > I(-). The nature of the cation has little effect on salt activation. The rate of inactivation of rh-PSA by DFP is 30-fold faster in the presence of 0.9 M Na(2)SO(4), and the rate of inactivation by Suc-Ala-Ala-Pro-Phe-CK is >20-fold faster under these conditions. Azapeptides containing Phe or Tyr at position P(1) also inactivate rh-PSA in the presence of high salt concentrations. These compounds represent the first described inhibitors designed to utilize the substrate binding subsites of PSA. CD spectroscopy demonstrates that the conformation of rh-PSA changes in the presence of high salt concentrations. Analytical ultracentifugation and dynamic light scattering indicate that PSA remains monomeric under high-salt conditions. Interestingly, human prostatic fluid contains as much as 150 micro mol citrate/g wet weight, which suggests that salt concentrations may regulate PSA activity in vivo.

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.

Most antibodies induced by HIV-1 are ineffective at preventing initiation or spread of infection because they are either nonneutralizing or narrowly isolate-specific. Rare, “broadly neutralizing” antibodies have been detected that recognize relatively conserved regions on the envelope glycoprotein. Using stringently characterized, homogeneous preparations of trimeric HIV-1 envelope protein in relevant conformations, we have analyzed the molecular mechanism of neutralization by two of these antibodies, 2F5 and 4E10. We find that their epitopes, in the membrane-proximal segment of the envelope protein ectodomain, are exposed only on a form designed to mimic an intermediate state during viral entry. These results help explain the rarity of 2F5- and 4E10-like antibody responses and suggest a strategy for eliciting them.