Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the current coronavirus disease 2019 (COVID-19) pandemic. A major virulence factor of SARS-CoVs is the ...nonstructural protein 1 (Nsp1), which suppresses host gene expression by ribosome association. Here, we show that Nsp1 from SARS-CoV-2 binds to the 40
ribosomal subunit, resulting in shutdown of messenger RNA (mRNA) translation both in vitro and in cells. Structural analysis by cryo-electron microscopy of in vitro-reconstituted Nsp1-40
and various native Nsp1-40
and -80
complexes revealed that the Nsp1 C terminus binds to and obstructs the mRNA entry tunnel. Thereby, Nsp1 effectively blocks retinoic acid-inducible gene I-dependent innate immune responses that would otherwise facilitate clearance of the infection. Thus, the structural characterization of the inhibitory mechanism of Nsp1 may aid structure-based drug design against SARS-CoV-2.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) evades most innate immune responses but may still be vulnerable to some. Here, we systematically analyze the impact of SARS-CoV-2 proteins ...on interferon (IFN) responses and autophagy. We show that SARS-CoV-2 proteins synergize to counteract anti-viral immune responses. For example, Nsp14 targets the type I IFN receptor for lysosomal degradation, ORF3a prevents fusion of autophagosomes and lysosomes, and ORF7a interferes with autophagosome acidification. Most activities are evolutionarily conserved. However, SARS-CoV-2 Nsp15 antagonizes IFN signaling less efficiently than the orthologs of closely related RaTG13-CoV and SARS-CoV-1. Overall, SARS-CoV-2 proteins counteract autophagy and type I IFN more efficiently than type II or III IFN signaling, and infection experiments confirm potent inhibition by IFN-γ and -λ1. Our results define the repertoire and selected mechanisms of SARS-CoV-2 innate immune antagonists but also reveal vulnerability to type II and III IFN that may help to develop safe and effective anti-viral approaches.
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•Numerous SARS-CoV-2 proteins synergize to suppress immune sensing and signaling•Nsp14 targets IFNAR1 for lysosomal degradation•ORF3a and ORF7a block autophagy by different mechanisms•Synergistic treatment with IFN-γ and -λ1 is highly effective against SARS-CoV-2
Hayn et al. analyze the impact of individual SARS-CoV-2 proteins on virus sensing, interferon signaling, and autophagy. They define the repertoire of viral antagonists of innate immune defenses, determine selected underlying mechanisms, and identify remaining vulnerabilities of SARS-CoV-2.
Zika virus (ZIKV) causes severe birth defects and can be transmitted via sexual intercourse. Semen from ZIKV-infected individuals contains high viral loads and may therefore serve as an important ...vector for virus transmission. Here we analyze the effect of semen on ZIKV infection of cells and tissues derived from the anogenital region. ZIKV replicates in all analyzed cell lines, primary cells, and endometrial or vaginal tissues. However, in the presence of semen, infection by ZIKV and other flaviviruses is potently inhibited. We show that semen prevents ZIKV attachment to target cells, and that an extracellular vesicle preparation from semen is responsible for this anti-ZIKV activity. Our findings suggest that ZIKV transmission is limited by semen. As such, semen appears to serve as a protector against sexual ZIKV transmission, despite the availability of highly susceptible cells in the anogenital tract and high viral loads in this bodily fluid.
We present a protocol for analyzing the impact of SARS-CoV-2 proteins in interferon signaling using luciferase reporter assays. Here, the induction of defined promoters can be quantitatively assessed ...with high sensitivity and broad linear range. The results are similar to those obtained using qPCR to measure endogenous mRNA induction. The assay requires stringent normalization and confirmation of the results in more physiological settings. The protocol is adaptable for other viruses and other innate immune stimuli.
For complete details on the use and execution of this protocol, please refer to Hayn et al. (2021).
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•Measure the impact of viral proteins on innate immune activation•Rapid, cost-effective, and reliable procedure that can be adapted to other viruses•Flexible system can be adjusted for different types of immune stimuli
We present a protocol for analyzing the impact of SARS-CoV-2 proteins in interferon signaling using luciferase reporter assays. Here, the induction of defined promoters can be quantitatively assessed with high sensitivity and broad linear range. The results are similar to those obtained using qPCR to measure endogenous mRNA induction. The assay requires stringent normalization and confirmation of the results in more physiological settings. The protocol is adaptable for other viruses and other innate immune stimuli.
Peptide nanofibrils (PNFs) are considered a novel and highly efficient class of retroviral transduction enhancers due to their ability to efficiently bind virus particles and promote their attachment ...to the membrane of target cells. In this study, the virus‐binding properties of enhancing factor C (EF‐C) PNFs are used to develop a method for concentrating and isolating HIV‐1 particles without the need for centrifugation. Upon incubation in a virus‐laden sample, PNF‐coated magnetic beads facilitate magnetic separation, effectively depleting virus particles from the solution and consequently reducing its infectious nature. Remarkably, the isolated virus particles maintain their infectivity and exhibit an enhanced ability to infect target cells due to the complexation with EF‐C PNFs. This enhancement is pivotal for further virus propagation in cell cultures or detailed analysis. The presented method addresses the urgent need for more rapid, sensitive detection, and purification technologies for viral agents. It provides a tool to efficiently capture, isolate, and concentrate HIV‐1 particles. This may significantly improve the sensitivity of existing diagnostic tools and analytical tests.
Peptide nanofibrils of the enhancing factor C peptide are applied to capture HIV‐1 particles and increase their attachment to cells. This makes them excellent retroviral transduction enhancers. Using magnetic beads and the biotin‐streptavidin interaction for peptide nanofibril immobilization, this approach can be used to efficiently capture and isolate viruses from virus‐laden samples. Figure created with BioRender.com.
Endogenous Peptide Inhibitors of HIV Entry Harms, Mirja; Hayn, Manuel; Zech, Fabian ...
Advances in experimental medicine and biology,
2022, Letnik:
1366
Journal Article
Recenzirano
The discovery of the G-protein coupled-receptor (GPCR) CXCR4 as a major coreceptor of HIV-1 entry about three decades ago explained why the chemokine SDF-1/CXCL12 inhibits specific viral strains. The ...knowledge that RANTES, MlP-1α, and MlP-1β specifically inhibit other primary HIV-1 strains allowed the rapid discovery of CCR5 as second major viral coreceptor and explained why individuals with deletions in CCR5 are protected against sexual HIV-1 transmission. Here, we provide an update on endogenous ligands of GPCRs that act as endogenous inhibitors of HIV-1, HIV-2, and simian immunodeficiency virus (SIV) entry. In addition, we summarize the development of optimized derivatives of endogenous GPCR ligands and their perspectives as antiviral agents and beyond. Finally, we provide examples for other endogenous peptides that may contribute to our innate immune defense against HIV-1 and other viral pathogens and offer prospects for preventive or therapeutic development.
Amyloid‐like peptide nanofibrils (PNFs) are abundant in nature providing rich bioactivities and playing both functional and pathological roles. The structural features responsible for their unique ...bioactivities are, however, still elusive. Supramolecular nanostructures are notoriously challenging to optimize, as sequence changes affect self‐assembly, fibril morphologies, and biorecognition. Herein, the first sequence optimization of PNFs, derived from the peptide enhancing factor‐C (EF‐C, QCKIKQIINMWQ), for enhanced retroviral gene transduction via a multiparameter and a multiscale approach is reported. Retroviral gene transfer is the method of choice for the stable delivery of genetic information into cells offering great perspectives for the treatment of genetic disorders. Single fibril imaging, zeta potential, vibrational spectroscopy, and quantitative retroviral transduction assays provide the structure parameters responsible for PNF assembly, fibrils morphology, secondary and quaternary structure, and PNF‐virus‐cell interactions. Optimized peptide sequences such as the 7‐mer, CKFKFQF, have been obtained quantitatively forming supramolecular nanofibrils with high intermolecular β‐sheet content that efficiently bind virions and attach to cellular membranes revealing efficient retroviral gene transfer.
Enhancing factor‐C (EF‐C), a fragment of the human immunodeficiency virus‐1 glycoprotein, is previously shown to self‐assemble into nanofibrils and enhance viral transduction. Herein, a peptide library based on EF‐C is synthesized through multiparameter and multiscale optimization. It is evaluated for fibril formation, secondary/quaternary structure, virion‐fibril‐cell interaction, and critical parameters for enhanced transduction can be determined in a structure–activity fashion.
GPR15 is a G protein-coupled receptor (GPCR) proposed to play a role in mucosal immunity that also serves as a major entry cofactor for HIV-2 and simian immunodeficiency virus (SIV). To discover ...novel endogenous GPR15 ligands, we screened a hemofiltrate (HF)-derived peptide library for inhibitors of GPR15-mediated SIV infection. Our approach identified a C-terminal fragment of cystatin C (CysC95-146) that specifically inhibits GPR15-dependent HIV-1, HIV-2, and SIV infection. In contrast, GPR15L, the chemokine ligand of GPR15, failed to inhibit virus infection. We found that cystatin C fragments preventing GPR15-mediated viral entry do not interfere with GPR15L signaling and are generated by proteases activated at sites of inflammation. The antiretroviral activity of CysC95-146 was confirmed in primary CD4
T cells and is conserved in simian hosts of SIV infection. Thus, we identified a potent endogenous inhibitor of GPR15-mediated HIV and SIV infection that does not interfere with the physiological function of this GPCR.
Retroviral gene transfer is the method of choice for the stable introduction of genetic material into the cellular genome. However, efficient gene transfer is often limited by low transduction rates ...of the viral vectors. We have recently described a 12-mer peptide, termed EF-C, that forms amyloid-like peptide nanofibrils (PNF), strongly increasing viral transduction efficiencies. These nanofibrils are polycationic and bind negatively charged membranes of virions and cells, thereby overcoming charge repulsions and resulting in increased rates of virion attachment and gene transfer. EF-C PNF enhance vector transduction more efficiently than other soluble additives and offer prospects for clinical applications. However, while the transduction-enhancing activity of PNF has been well-characterized, the exact mechanism and the kinetics underlying infection enhancement as well as the cellular fate of the fibrils are hardly explored. This is partially due to the fact that current labeling techniques for PNF rely on amyloid probes that cause high background staining or lose signal intensities after cellular uptake. Here, we sought to generate EF-C PNF covalently coupled with fluorescent labels. To achieve such covalent bioconjugates, the free amino groups of the EF-C peptide were coupled to the ATTO 495 or 647N NHS ester dyes. When small amounts of the labeled peptides were mixed with a 100- to 10 000-fold excess of the native peptide, PNF formed that were morphologically indistinguishable from those derived from the unlabeled peptide. The fluorescence of the fibrils could be readily detected using fluorescence spectroscopy, microscopy, and flow cytometry. In addition, labeled and nonlabeled fibrils captured viral particles and increased retroviral transduction with similar efficacy. These covalently fluorescence-labeled PNF are valuable tools with which to elucidate the mechanism(s) underlying transduction attachment and the fate of the fibrils in cells, tissues, and animal models.
Eliminating latently infected cells is a highly challenging, indispensable step toward the cure for HIV/AIDS. Hypothesis put forward herein is that the unique HIV protease cut site (Phe‐Pro) can be ...reconstructed using a potent inhibitor of tubulin polymerization, monomethyl auristatin F (MMAF), which features Phe at its C‐terminus. This presents opportunities to design prodrugs that are specifically activated by the HIV protease. To this end, a series of MMAF derivatives is synthesized and evaluated in cell culture using latently HIV‐infected cells. The cytotoxicity of compounds is indeed enhanced upon latency reversal by up to 11‐fold. As a result, in a mixed cell population, nanomolar concentrations of the lead compounds depletes predominantly the HIV‐infected cells and in doing so markedly enriches the pool of the uninfected cells. Affinity of the lead compounds to the viral protease is validated computationally and experimentally but despite expectations, the mechanism of action of the synthesized toxins is shown to be independent from the enzymatic activity of the HIV protease.
Medicinal agents toward the elimination of HIV infected cells via the “shock and kill” strategy are designed. Lead compound exhibits an 11‐fold higher toxicity in cells upon HIV latency reversal. Dosed in vitro at nanomolar concentrations, the lead compound preferentially eliminates the virus‐positive cells and significantly enriches the pool of cells with the virus‐negative cells.
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