Live imaging of Yersinia translocon formation and immune recognition in host cells

Yersinia enterocolitica employs a type three secretion system (T3SS) to translocate immunosuppressive effector proteins into host cells. To this end, the T3SS assembles a translocon/pore complex composed of the translocator proteins YopB and YopD in host cell membranes serving as an entry port for the effectors. The translocon is formed in a Yersinia -containing pre-phagosomal compartment that is connected to the extracellular space. As the phagosome matures, the translocon and the membrane damage it causes are recognized by the cell-autonomous immune system. We infected cells in the presence of fluorophore-labeled ALFA-tag-binding nanobodies with a Y. enterocolitica strain expressing YopD labeled with an ALFA-tag. Thereby we could record the integration of YopD into translocons and its intracellular fate in living host cells. YopD was integrated into translocons around 2 min after uptake of the bacteria into a phosphatidylinositol-4,5-bisphosphate enriched pre-phagosomal compartment and remained there for 27 min on average. Damaging of the phagosomal membrane as visualized with recruitment of GFP-tagged galectin-3 occurred in the mean around 14 min after translocon formation. Shortly after recruitment of galectin-3, guanylate-binding protein 1 (GBP-1) was recruited to phagosomes, which was accompanied by a decrease in the signal intensity of translocons, suggesting their degradation. In sum, we were able for the first time to film the spatiotemporal dynamics of Yersinia T3SS translocon formation and degradation and its sensing by components of the cell-autonomous immune system.

Click chemistry and molecular modeling methods in computer-aided design and identification of potential HIV-1 inhibitors

An integrated approach including the click chemistry methodology, molecular docking, quantum mechanics, and molecular dynamics was used to perform the computer-aided design of potential HIV-1 inhibitors able to block the membrane- proximal external region (MPER) of HIV-1 gp41 that plays an important role in the fusion of the viral and host cell membranes. Evaluation of the binding efficiency of the designed compounds to the HIV-1 MPER peptide was performed using the methods of molecular modeling, resulting in nine chemical compounds that exhibit the high-affinity binding to this functionally important site of the trimeric “spike” of the viral envelope. The data obtained indicate that the identified compounds are promising for the development of novel antiviral drugs, HIV fusion inhibitors blocking the early stages of HIV infection.

SARS-CoV-2 variants impact RBD conformational dynamics and ACE2 accessibility

The coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has killed over 5 million people and is causing a devastating social and economic impact all over the world. The rise of new variants of concern (VOCs) represents a difficult challenge due to the loss vaccine and natural immunity, and increased transmissibility. All circulating VOCs contain mutations in the spike glycoprotein, which mediates fusion between the viral and host cell membranes, via its receptor binding domain (RBD) that binds to angiotensin-converting enzyme 2 (ACE2). In an attempt to understand the effect of RBD mutations in circulating VOCs, a lot of attention has been given to the RBD-ACE2 interaction. However, this type of analysis is limited, since it ignores more indirect effects, such as the conformational dynamics of the RBD itself. Observing that some VOCs mutations occur in residues that are not in direct contact with ACE2, we hypothesized that they could affect RBD conformational dynamics. To test this, we performed long atomistic (AA) molecular dynamics (MD) simulations to investigate the structural dynamics of wt RBD, and that of three circulating VOCs (alpha, beta, and delta). Our results show that in solution, wt RBD presents two distinct conformations: an open conformation where it is free to bind ACE2; and a closed conformation, where the RBM ridge blocks the binding surface. The alpha and beta variants significantly impact the open/closed equilibrium, shifting it towards the open conformation by roughly 20%. This shift likely increases ACE2 binding affinity. Simulations of the currently predominant delta variant RBD were extreme in this regard, in that a closed conformation was never observed. Instead, the system alternated between the before mentioned open conformation and an alternative reversed one, with a significantly changed orientation of the RBM ridge flanking the RBD. This alternate conformation could potentially provide a fitness advantage not only due to increased availability for ACE2 binding, but also by aiding antibody escape through epitope occlusion. These results support the hypothesis that VOCs, and particularly the delta variant, impact RBD conformational dynamics in a direction that simultaneously promotes efficient binding to ACE2 and antibody escape.

Design and Identification of Potential HIV-1 Entry Inhibitors Using In Silico Click Chemistry and Molecular Modeling Methods

An integrated approach including the click chemistry methodology, molecular docking, quantum mechanics, and molecular dynamics was used to computer-aided design of potential HIV-1 inhibitors able to block the membrane-proximal external region (MPER) of HIV-1 gp41, which plays an important role in the fusion of the viral and host cell membranes. Evaluation of the binding efficiency of the designed compounds to the HIV-1 MPER peptide was performed using the methods of molecular modeling, resulting in nine chemical compounds exhibiting high-affinity binding to this functionally important site of the trimeric “spike” of the viral envelope. The data obtained indicate that the identified compounds are promising for the development of novel antiviral drugs, HIV fusion inhibitors blocking the early stages of HIV infection.

Diversity of Coronavirus Receptors

Several recent surges in COVID-19 cases due to newly emerging variant strains of SARS-CoV-2 with greater transmissibility have highlighted the virus’s capability to directly modulate spike-ACE2 interactions and promote immune evasion by sterically masking the immunogenic epitopes. Recently, there have also been reports of the bidirectional transfer of coronavirus between different animal species and humans. The ability of coronavirus to infect and adapt to a wide range of hosts can be attributed to new variants that modify the molecular recognition profile of the spike protein (S protein). The receptor-binding domain of the spike protein specifically interacts with key host receptor molecules present on the host cell membranes to gain entry into the host and begin the infection cycle. In this review, we discuss the molecular, structural, and functional diversity associated with the coronavirus receptors across their different phylogenetic lineages and its relevance to various symptomatology in the rapid human-to-human infection in COVID-19 patients, tropism, and zoonosis. Despite this seeming diversity of host receptors, there may be some common underlying mechanisms that influence the host range, virus transmissibility, and pathogenicity. Understanding these mechanisms may be crucial in not only controlling the ongoing pandemic but also help in stopping the resurgence of such virus threats in the future.

Modification of the Spike Protein for Vaccines against Enveloped RNA Viruses

Abstract— Most vaccines work by inducing neutralizing antibodies that target the viral envelope. Enveloped RNA viruses have evolved mechanisms for surface glycoproteins to evade host immune responses, which exhibit substantial variability, even among different strains. Natural infection and vaccines using native forms of surface proteins may induce broadly neutralizing antibodies, yet with low and ineffective levels. Class I membrane-fusion proteins of enveloped RNA viruses, HIV-1, influenza A virus, SARS-CoV-2, yield a stable conformation (so-called “pre-fusion”) in providing fusion between viral and host cell membranes. Modified viral surface proteins that are based on these features induce neutralizing antibodies with activity available against a broad spectrum of circulating strains and make it possible to overcome the difficulties associated with escape/variability of viral antigen.

Cathelicidin and PMB neutralize endotoxins by multifactorial mechanisms including LPS interaction and targeting of host cell membranes

Antimicrobial peptides (AMPs) contribute to an effective protection against infections. The antibacterial function of AMPs depends on their interactions with microbial membranes and lipids, such as lipopolysaccharide (LPS; endotoxin). Hyperinflammation induced by endotoxin is a key factor in bacterial sepsis and many other human diseases. Here, we provide a comprehensive profile of peptide-mediated LPS neutralization by systematic analysis of the effects of a set of AMPs and the peptide antibiotic polymyxin B (PMB) on the physicochemistry of endotoxin, macrophage activation, and lethality in mice. Mechanistic studies revealed that the host defense peptide LL-32 and PMB each reduce LPS-mediated activation also via a direct interaction of the peptides with the host cell. As a biophysical basis, we demonstrate modifications of the structure of cholesterol-rich membrane domains and the association of glycosylphosphatidylinositol (GPI)-anchored proteins. Our discovery of a host cell–directed mechanism of immune control contributes an important aspect in the development and therapeutic use of AMPs.

Transient Excursions to Membrane Core as Determinants of Influenza Virus Fusion Peptide Activity

Fusion of viral and host cell membranes is a critical step in the life cycle of enveloped viruses. In the case of influenza virus, it is mediated by subunit 2 of hemagglutinin (HA) glycoprotein whose N-terminal fragments insert into the target membrane and initiate lipid exchange. These isolated fragments, known as fusion peptides (HAfp), already possess own fusogenic activity towards liposomes. Although they have long been studied with the hope to uncover the details of HA-mediated fusion, their actual mechanism of action remains elusive. Here, we use extensive molecular dynamics simulations combined with experimental studies of three HAfp variants to fully characterize their free energy landscape and interaction with lipid bilayer. In addition to customary assumed peptides localization at lipid–water interface, we characterize membrane-spanning configurations, which turn out to be metastable for active HAfps and unstable for the fusion inactive W14A mutant. We show that, while the degree of membrane perturbation by surface peptide configurations is relatively low and does not show any mutation-related differences, the effect of deeply inserted configurations is significant and correlates with insertion depth of the N-terminal amino group which is the highest for the wild type HAfp. Finally, we demonstrate the feasibility of spontaneous peptide transition to intramembrane location and the critical role of strictly conserved tryptofan residue 14 in this process.

Large fluctuations of the fusion intermediate help SARS-CoV-2 capture host cell membranes

Cell entry of SARS-CoV-2 is accomplished by the S2 subunit of the spike S protein on the virion surface by fusion of viral and host cell membranes. Fusion requires the prefusion S2 to transit to its potent, fusogenic form, the fusion intermediate (FI). However, the FI structure is unknown, detailed computational models of the FI are not available, and the mechanisms of fusion and entry remain unclear. Here, we constructed a full-length model of the CoV-2 FI by extrapolating from known CoV-2 pre- and postfusion structures. Atomistic and coarse-grained simulations showed the FI is a remarkably flexible mechanical assembly executing large orientational and extensional fluctuations due to three hinges in the C-terminal base. Fluctuations lead to a large fusion peptide exploration volume and may aid capture of the host cell target membrane and define the clock for fluctuation-triggered refolding and membrane fusion. This work suggests several novel potential drug targets.

Engineering protease-resistant peptides to inhibit human parainfluenza viral respiratory infection

ABSTRACTThe lower respiratory tract infections affecting children worldwide are in large part caused by the parainfluenza viruses (HPIVs), particularly HPIV3, along with human metapneumovirus and respiratory syncytial virus, enveloped negative-strand RNA viruses. There are no vaccines for these important human pathogens, and existing treatments have limited or no efficacy. Infection by HPIV is initiated by viral glycoprotein-mediated fusion between viral and host cell membranes. A viral fusion protein (F), once activated in proximity to a target cell, undergoes a series of conformational changes that first extend the trimer subunits to allow insertion of the hydrophobic domains into the target cell membrane, and then refold the trimer into a stable postfusion state, driving the merger of the viral and host cell membranes. Lipopeptides derived from the C-terminal heptad repeat (HRC) domain of HPIV3 F inhibit infection by interfering with the structural transitions of the trimeric F assembly. Clinical application of this strategy, however, requires improving the in vivo stability of antiviral peptides. We show that the HRC peptide backbone can be modified via partial replacement of α-amino acid residues with β-amino acid residues to generate α/β-peptides that retain antiviral activity but are poor protease substrates. Relative to a conventional α-lipopeptide, our best α/β-lipopeptide exhibits improved persistence in vivo and improved anti-HPIV3 antiviral activity in animals.