The Eukaryotic Linear Motif resource: 2022 release.

Almost twenty years after its initial release, the Eukaryotic Linear Motif (ELM) resource remains an invaluable source of information for the study of motif-mediated protein-protein interactions. ELM provides a comprehensive, regularly updated and well-organised repository of manually curated, experimentally validated short linear motifs (SLiMs). An increasing number of SLiM-mediated interactions are discovered each year and keeping the resource up-to-date continues to be a great challenge. In the current update, 30 novel motif classes have been added and five existing classes have undergone major revisions. The update includes 411 new motif instances mostly focused on cell-cycle regulation, control of the actin cytoskeleton, membrane remodelling and vesicle trafficking pathways, liquid-liquid phase separation and integrin signalling. Many of the newly annotated motif-mediated interactions are targets of pathogenic motif mimicry by viral, bacterial or eukaryotic pathogens, providing invaluable insights into the molecular mechanisms underlying infectious diseases. The current ELM release includes 317 motif classes incorporating 3934 individual motif instances manually curated from 3867 scientific publications. ELM is available at: http://elm.eu.org.

Rotavirus Spike Protein VP4 Mediates Viroplasm Assembly by Association to Actin Filaments.

Rotavirus (RV) viroplasms are cytosolic inclusions where both virus genome replication and primary steps of virus progeny assembly take place. A stabilized microtubule cytoskeleton and lipid droplets are required for the viroplasm formation, which involves several virus proteins. The viral spike protein VP4 has not previously been shown to have a direct role in viroplasm formation. However, it is involved with virus-cell attachment, endocytic internalization, and virion morphogenesis. Moreover, VP4 interacts with actin cytoskeleton components, mainly in processes involving virus entrance and egress, and thereby may have an indirect role in viroplasm formation. In this study, we used reverse genetics to construct a recombinant RV, rRV/VP4-BAP, that contains a biotin acceptor peptide (BAP) in the K145-G150 loop of the VP4 lectin domain, permitting live monitoring. The recombinant virus was replication competent but showed a reduced fitness. We demonstrate that rRV/VP4-BAP infection, as opposed to rRV/wt infection, did not lead to a reorganized actin cytoskeleton as viroplasms formed were insensitive to drugs that depolymerize actin and inhibit myosin. Moreover, wild-type (wt) VP4, but not VP4-BAP, appeared to associate with actin filaments. Similarly, VP4 in coexpression with NSP5 and NSP2 induced a significant increase in the number of viroplasm-like structures. Interestingly, a small peptide mimicking loop K145-G150 rescued the phenotype of rRV/VP4-BAP by increasing its ability to form viroplasms and hence improve virus progeny formation. Collectively, these results provide a direct link between VP4 and the actin cytoskeleton to catalyze viroplasm assembly. IMPORTANCE The spike protein VP4 participates in diverse steps of the rotavirus (RV) life cycle, including virus-cell attachment, internalization, modulation of endocytosis, virion morphogenesis, and virus egress. Using reverse genetics, we constructed for the first time a recombinant RV, rRV/VP4-BAP, harboring a heterologous peptide in the lectin domain (loop K145-G150) of VP4. The rRV/VP4-BAP was replication competent but with reduced fitness due to a defect in the ability to reorganize the actin cytoskeleton, which affected the efficiency of viroplasm assembly. This defect was rescued by adding a permeable small-peptide mimicking the wild-type VP4 loop K145-G150. In addition to revealing a new role of VP4, our findings suggest that rRV harboring an engineered VP4 could be used as a new dual vaccination platform providing immunity against RV and additional heterologous antigens.

Actin cytoskeleton remodeling primes RIG-I-like receptor activation.

The current dogma of RNA-mediated innate immunity is that sensing of immunostimulatory RNA ligands is sufficient for the activation of intracellular sensors and induction of interferon (IFN) responses. Here, we report that actin cytoskeleton disturbance primes RIG-I-like receptor (RLR) activation. Actin cytoskeleton rearrangement induced by virus infection or commonly used reagents to intracellularly deliver RNA triggers the relocalization of PPP1R12C, a regulatory subunit of the protein phosphatase-1 (PP1), from filamentous actin to cytoplasmic RLRs. This allows dephosphorylation-mediated RLR priming and, together with the RNA agonist, induces effective RLR downstream signaling. Genetic ablation of PPP1R12C impairs antiviral responses and enhances susceptibility to infection with several RNA viruses including SARS-CoV-2, influenza virus, picornavirus, and vesicular stomatitis virus. Our work identifies actin cytoskeleton disturbance as a priming signal for RLR-mediated innate immunity, which may open avenues for antiviral or adjuvant design.

The mammalian endocytic cytoskeleton.

Clathrin-mediated endocytosis (CME) is the major route through which cells internalise various substances and recycle membrane components. Via the coordinated action of many proteins, the membrane bends and invaginates to form a vesicle that buds off-along with its contents-into the cell. The contribution of the actin cytoskeleton to this highly dynamic process in mammalian cells is not well understood. Unlike in yeast, where there is a strict requirement for actin in CME, the significance of the actin cytoskeleton to mammalian CME is variable. However, a growing number of studies have established the actin cytoskeleton as a core component of mammalian CME, and our understanding of its contribution has been increasing at a rapid pace. In this review, we summarise the state-of-the-art regarding our understanding of the endocytic cytoskeleton, its physiological significance, and the questions that remain to be answered.

Virus interactions with the actin cytoskeleton-what we know and do not know about SARS-CoV-2.

The actin cytoskeleton and actin-dependent molecular and cellular events are responsible for the organization of eukaryotic cells and their functions. Viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), depend on host cell organelles and molecular components for cell entry and propagation. Thus, it is not surprising that they also interact at many levels with the actin cytoskeleton of the host. There have been many studies on how different viruses reconfigure and manipulate the actin cytoskeleton of the host during successive steps of their life cycle. However, we know relatively little about the interactions of SARS-CoV-2 with the actin cytoskeleton. Here, we describe how the actin cytoskeleton is involved in the strategies used by different viruses for entry, assembly, and egress from the host cell. We emphasize what is known and unknown about SARS-CoV-2 in this regard. This review should encourage further investigation of the interactions of SARS-CoV-2 with cellular components, which will eventually be helpful for developing novel antiviral therapies for mitigating the severity of COVID-19.

Effect of Ivermectin and Atorvastatin on Nuclear Localization of Importin Alpha and Drug Target Expression Profiling in Host Cells from Nasopharyngeal Swabs of SARS-CoV-2- Positive Patients.

Nuclear transport and vesicle trafficking are key cellular functions involved in the pathogenesis of RNA viruses. Among other pleiotropic effects on virus-infected host cells, ivermectin (IVM) inhibits nuclear transport mechanisms mediated by importins and atorvastatin (ATV) affects actin cytoskeleton-dependent trafficking controlled by Rho GTPases signaling. In this work, we first analyzed the response to infection in nasopharyngeal swabs from SARS-CoV-2-positive and -negative patients by assessing the gene expression of the respective host cell drug targets importins and Rho GTPases. COVID-19 patients showed alterations in KPNA3, KPNA5, KPNA7, KPNB1, RHOA, and CDC42 expression compared with non-COVID-19 patients. An in vitro model of infection with Poly(I:C), a synthetic analog of viral double-stranded RNA, triggered NF-κB activation, an effect that was halted by IVM and ATV treatment. Importin and Rho GTPases gene expression was also impaired by these drugs. Furthermore, through confocal microscopy, we analyzed the effects of IVM and ATV on nuclear to cytoplasmic importin α distribution, alone or in combination. Results showed a significant inhibition of importin α nuclear accumulation under IVM and ATV treatments. These findings confirm transcriptional alterations in importins and Rho GTPases upon SARS-CoV-2 infection and point to IVM and ATV as valid drugs to impair nuclear localization of importin α when used at clinically-relevant concentrations.

Cytoskeleton-a crucial key in host cell for coronavirus infection.

The emerging coronavirus (CoV) pandemic is threatening the public health all over the world. Cytoskeleton is an intricate network involved in controlling cell shape, cargo transport, signal transduction, and cell division. Infection biology studies have illuminated essential roles for cytoskeleton in mediating the outcome of host‒virus interactions. In this review, we discuss the dynamic interactions between actin filaments, microtubules, intermediate filaments, and CoVs. In one round of viral life cycle, CoVs surf along filopodia on the host membrane to the entry sites, utilize specific intermediate filament protein as co-receptor to enter target cells, hijack microtubules for transportation to replication and assembly sites, and promote actin filaments polymerization to provide forces for egress. During CoV infection, disruption of host cytoskeleton homeostasis and modification state is tightly connected to pathological processes, such as defective cytokinesis, demyelinating, cilia loss, and neuron necrosis. There are increasing mechanistic studies on cytoskeleton upon CoV infection, such as viral protein‒cytoskeleton interaction, changes in the expression and post-translation modification, related signaling pathways, and incorporation with other host factors. Collectively, these insights provide new concepts for fundamental virology and the control of CoV infection.