Search Results (27)

The SARS-CoV-2 nucleocapsid protein (N protein) is critical in viral replication by undergoing liquid–liquid phase separation to seed the formation of a ribonucleoprotein (RNP) complex to drive viral genomic RNA (gRNA) translation and in suppressing both stress granules and processing bodies, which is postulated to increase uncoated gRNA availability. The N protein can also form biomolecular condensates with a broad range of host endogenous proteins including RNA binding proteins (RBPs). Amongst these RBPs are proteins that are associated with pathological, neuronal, and glial cytoplasmic inclusions across several adult-onset neurodegenerative disorders, including TAR DNA binding protein 43 kDa (TDP-43) which forms pathological inclusions in over 95% of amyotrophic lateral sclerosis cases. In this study, we demonstrate that the N protein can form biomolecular condensates with TDP-43 and that this is dependent on the N protein C-terminus domain (N-CTD) and the intrinsically disordered C-terminus domain of TDP-43. This process is markedly accelerated in the presence of RNA. In silico modeling suggests that the biomolecular condensate that forms in the presence of RNA is composed of an N protein quadriplex in which the intrinsically disordered TDP-43 C terminus domain is incorporated. Full article

The human immunodeficiency virus type 1 (HIV-1) capsid is a protein core formed by multiple copies of the viral capsid (CA) protein. Inside the capsid, HIV-1 harbours all the viral components required for replication, including the genomic RNA and viral enzymes reverse transcriptase (RT) and integrase (IN). Upon infection, the RT transforms the genomic RNA into a double-stranded DNA molecule that is subsequently integrated into the host chromosome by IN. For this to happen, the viral capsid must open and release the viral DNA, in a process known as uncoating. Capsid plays a key role during the initial stages of HIV-1 replication; therefore, its stability is intimately related to infection efficiency, and untimely uncoating results in reverse transcription defects. How and where uncoating takes place and its relationship with reverse transcription is not fully understood, but the recent development of novel biochemical and cellular approaches has provided unprecedented detail on these processes. In this review, we present the latest findings on the intricate link between capsid stability, reverse transcription and uncoating, the different models proposed over the years for capsid uncoating, and the role played by other cellular factors on these processes. Full article

Coxsackievirus A10 (CV-A10) is a prevailing causative agent of hand–foot–mouth disease, necessitating the isolation and adaptation of appropriate strains in cells allowed for human vaccine development. In this study, amino acid sequences of CV-A10 strains with different cell tropism on RD and Vero cells were compared. Various amino acids on the structural and non-structural proteins related to cell tropism were identified. The reverse genetic systems of several CV-A10 strains with RD⁺/Vero⁻ and RD⁺/Vero⁺ cell tropism were developed, and a set of CV-A10 recombinants were produced. The binding, entry, uncoating, and proliferation steps in the life cycle of these viruses were evaluated. P1 replacement of CV-A10 strains with different cell tropism revealed the pivotal role of the structural proteins in cell tropism. Further, seven amino acid substitutions in VP2 and VP1 were introduced to further investigate their roles played in cell tropism. These mutations cooperated in the growth of CV-A10 in Vero cells. Particularly, the valine to isoleucine mutation at the position VP1-236 (V1236I) was found to significantly restrict viral uncoating in Vero cells. Co-immunoprecipitation assays showed that the release of viral RNA from the KREMEN1 receptor-binding virions was restricted in r0195-V1236I compared with the parental strain r0195 (a RD⁺/Vero⁺ strain). Overall, this study highlights the dominant effect of structural proteins in CV-A10 adaption in Vero cells and the importance of V1236 in viral uncoating, providing a foundation for the mechanism study of CV-A10 cell tropism, and facilitating the development of vaccine candidates. Full article

Although the involvement of the ubiquitin-proteasome system (UPS) in several coronavirus-productive infections has been reported, whether the UPS is required for infectious bronchitis virus (IBV) and porcine epidemic diarrhea virus (PEDV) infections is unclear. In this study, the role of UPS in the IBV and PEDV life cycles was investigated. When the UPS was suppressed by pharmacological inhibition at the early infection stage, IBV and PEDV infectivity were severely impaired. Further study showed that inhibition of UPS did not change the internalization of virus particles; however, by using R18 and DiOC-labeled virus particles, we found that inhibition of UPS prevented the IBV and PEDV membrane fusion with late endosomes or lysosomes. In addition, proteasome inhibitors blocked the degradation of the incoming viral protein N, suggesting the uncoating process and genomic RNA release were suppressed. Subsequently, the initial translation of genomic RNA was blocked. Thus, UPS may target the virus-cellular membrane fusion to facilitate the release of incoming viruses from late endosomes or lysosomes, subsequently blocking the following virus uncoating, initial translation, and replication events. Similar to the observation of proteasome inhibitors, ubiquitin-activating enzyme E1 inhibitor PYR-41 also impaired the entry of IBV, enhanced the accumulation of ubiquitinated proteins, and depleted mono-ubiquitin. In all, this study reveals an important role of UPS in coronavirus entry by preventing membrane fusion and identifies UPS as a potential target for developing antiviral therapies for coronavirus. Full article

Rhinoviruses (RVs) are the major cause of common cold, a respiratory disease that generally takes a mild course. However, occasionally, RV infection can lead to serious complications in patients debilitated by other ailments, e.g., asthma. Colds are a huge socioeconomic burden as neither vaccines nor other treatments are available. The many existing drug candidates either stabilize the capsid or inhibit the viral RNA polymerase, the viral proteinases, or the functions of other non-structural viral proteins; however, none has been approved by the FDA. Focusing on the genomic RNA as a possible target for antivirals, we asked whether stabilizing RNA secondary structures might inhibit the viral replication cycle. These secondary structures include G-quadruplexes (GQs), which are guanine-rich sequence stretches forming planar guanine tetrads via Hoogsteen base pairing with two or more of them stacking on top of each other; a number of small molecular drug candidates increase the energy required for their unfolding. The propensity of G-quadruplex formation can be predicted with bioinformatics tools and is expressed as a GQ score. Synthetic RNA oligonucleotides derived from the RV-A2 genome with sequences corresponding to the highest and lowest GQ scores indeed exhibited characteristics of GQs. In vivo, the GQ-stabilizing compounds, pyridostatin and PhenDC3, interfered with viral uncoating in Na⁺ but not in K⁺-containing phosphate buffers. The thermostability studies and ultrastructural imaging of protein-free viral RNA cores suggest that Na⁺ keeps the encapsulated genome more open, allowing PDS and PhenDC3 to diffuse into the quasi-crystalline RNA and promote the formation and/or stabilization of GQs; the resulting conformational changes impair RNA unraveling and release from the virion. Preliminary reports have been published. Full article

Enterovirus A71, a non-enveloped single-stranded (+) RNA virus, enters host cells through three stages: attachment, endocytosis and uncoating. In recent years, receptors/co-receptors anchored on the host cell membrane and involved in this process have been continuously identified. Among these, hSCARB-2 was the first receptor revealed to specifically bind to a definite site of the EV-A71 viral capsid and plays an indispensable role during viral entry. It actually acts as the main receptor due to its ability to recognize all EV-A71 strains. In addition, PSGL-1 is the second EV-A71 receptor discovered. Unlike hSCARB-2, PSGL-1 binding is strain-specific; only 20% of EV-A71 strains isolated to date are able to recognize and bind it. Some other receptors, such as sialylated glycan, Anx 2, HS, HSP90, vimentin, nucleolin and fibronectin, were discovered successively and considered as “co-receptors” because, without hSCARB-2 or PSGL-1, they are not able to mediate entry. For cypA, prohibitin and hWARS, whether they belong to the category of receptors or of co-receptors still needs further investigation. In fact, they have shown to exhibit an hSCARB-2-independent entry. All this information has gradually enriched our knowledge of EV-A71’s early stages of infection. In addition to the availability of receptors/co-receptors for EV-A71 on host cells, the complex interaction between the virus and host proteins and various intracellular signaling pathways that are intricately connected to each other is critical for a successful EV-A71 invasion and for escaping the attack of the immune system. However, a lot remains unknown about the EV-A71 entry process. Nevertheless, researchers have been continuously interested in developing EV-A71 entry inhibitors, as this study area offers a large number of targets. To date, important progress has been made toward the development of several inhibitors targeting: receptors/co-receptors, including their soluble forms and chemically designed compounds; virus capsids, such as capsid inhibitors designed on the VP1 capsid; compounds potentially interfering with related signaling pathways, such as MAPK-, IFN- and ATR-inhibitors; and other strategies, such as siRNA and monoclonal antibodies targeting entry. The present review summarizes these latest studies, which are undoubtedly of great significance in developing a novel therapeutic approach against EV-A71. Full article

Hepatitis B virus (HBV) relies on the core protein (HBc) to establish productive infection, as defined by the formation of the covalently closed circularized DNA (cccDNA), as well as to carry out almost every step of the lifecycle following cccDNA formation. Multiple copies of HBc form an icosahedral capsid shell that encapsidates the viral pregenomic RNA (pgRNA) and facilitates the reverse transcription of pgRNA to a relaxed circular DNA (rcDNA) within the capsid. During infection, the complete HBV virion, which contains an outer envelope layer in addition to the internal nucleocapsid containing rcDNA, enters human hepatocytes via endocytosis and traffics through the endosomal compartments and the cytosol to deliver its rcDNA to the nucleus to produce cccDNA. In addition, progeny rcDNA, newly formed in cytoplasmic nucleocapsids, is also delivered to the nucleus in the same cell to form more cccDNA in a process called intracellular cccDNA amplification or recycling. Here, we focus on recent evidence demonstrating differential effects of HBc in affecting cccDNA formation during de novo infection vs. recycling, obtained using HBc mutations and small molecule inhibitors. These results implicate a critical role of HBc in determining HBV trafficking during infection, as well as in nucleocapsid disassembly (uncoating) to release rcDNA, events essential for cccDNA formation. HBc likely functions in these processes via interactions with host factors, which contributes critically to HBV host tropism. A better understanding of the roles of HBc in HBV entry, cccDNA formation, and host species tropism should accelerate ongoing efforts to target HBc and cccDNA for the development of an HBV cure and facilitate the establishment of convenient animal models for both basic research and drug development. Full article

It has been previously shown that amino acid polymorphisms in reovirus proteins μ2 and λ1 are associated with differing levels of interferon induction. In the present study, viruses carrying these polymorphisms in either or both proteins, were further studied. The two viral determinants exert a synergistic effect on the control of β-interferon induction at the protein and mRNA level, with a concomitant increase in RIG-I. In contrast, levels of phospho-Stat1 and interferon-stimulated genes are increased in singly substituted viruses but with no further increase when both substitutions were present. This suggests that the viral determinants are acting during initial events of viral recognition. Accordingly, difference between viruses was reduced when infection was performed with partially uncoated virions (ISVPs) and transfection of RNA recovered from early-infected cells recapitulates the differences between viruses harboring the different polymorphisms. Altogether, the data are consistent with a redundant or complementary role of μ2 and λ1, affecting either early disassembly or the nature of the viral RNA in the incoming viral particle. Proteins involved in viral RNA synthesis are thus involved in this likely critical aspect of the ability of different reovirus variants to infect various cell types, and to discriminate between parental and transformed/cancer cells. Full article

There is an immense healthcare challenge and financial pressure due to the COVID-19 pandemic caused by a newly identified human coronavirus, SARS-CoV-2. Effective COVID-19 prevention efforts in healthcare, home, and community settings highlight the need for rapid, efficient, and no-contact SARS-CoV-2 inactivation strategies. Here, we examined the photocatalytic and virucidal activity of the MACOMA™ TiO₂ photocatalytic film activated by an UVA-LED-12V-367 nm (MA-717836-1) lamp against the HCoV-OC43, a member of the beta coronaviruses family, like SARS-CoV-2, using quantitative RT-qPCR and virus infectivity assays. The UVA radiation-responsive TiO₂ film accelerated virus inactivation (decreased viral titer) compared to the uncoated glass surface when placed at a vertical distance of 1.2 feet (~14 inches) from virus samples for 10, 30, and 60 min. UVA-LED exposure for both 10 and 30 min effectively reduced the viral RNA copies and the infectious virus in samples on TiO₂-coated surfaces compared to the control surfaces. Importantly, a 60 min exposure of samples on the TiO₂ completely eliminated HCoV-OC43. These results confirmed that the MACOMA™ UVA/TiO₂-based disinfection system provides a rapid and complete surface inactivation of tested human coronavirus in a human-safe manner and has great potential for limiting the virus spread in poorly ventilated as well as high-traffic public places. Full article

Human coronavirus (HCoV) similar to other viruses rely on host cell machinery for both replication and to spread. The p97/VCP ATPase is associated with diverse pathways that may favor HCoV replication. In this study, we assessed the role of p97 and associated host responses in human lung cell line H1299 after HCoV-229E or HCoV-OC43 infection. Inhibition of p97 function by small molecule inhibitors shows antiviral activity, particularly at early stages of the virus life cycle, during virus uncoating and viral RNA replication. Importantly, p97 activity inhibition protects human cells against HCoV-induced cytopathic effects. The p97 knockdown also inhibits viral production in infected cells. Unbiased quantitative proteomics analyses reveal that HCoV-OC43 infection resulted in proteome changes enriched in cellular senescence and DNA repair during virus replication. Further analysis of protein changes between infected cells with control and p97 shRNA identifies cell cycle pathways for both HCoV-229E and HCoV-OC43 infection. Together, our data indicate a role for the essential host protein p97 in supporting HCoV replication, suggesting that p97 is a therapeutic target to treat HCoV infection. Full article

Viruses are obligate intracellular parasites that are dependent on host factors for their replication. One such host protein, p97 or the valosin-containing protein (VCP), is a highly conserved AAA ATPase that facilitates replication of diverse RNA- and DNA-containing viruses. The wide range of cellular functions attributed to this ATPase is consistent with its participation in multiple steps of the virus life cycle from entry and uncoating to viral egress. Studies of VCP/p97 interactions with viruses will provide important information about host processes and cell biology, but also viral strategies that take advantage of these host functions. The critical role of p97 in viral replication might be exploited as a target for development of pan-antiviral drugs that exceed the capability of virus-specific vaccines or therapeutics. Full article

Rhinoviruses (RVs) are the main cause of recurrent infections with rather mild symptoms characteristic of the common cold. Nevertheless, RVs give rise to enormous numbers of absences from work and school and may become life-threatening in particular settings. Vaccination is jeopardised by the large number of serotypes eliciting only poorly cross-neutralising antibodies. Conversely, antivirals developed over the years failed FDA approval because of a low efficacy and/or side effects. RV species A, B, and C are now included in the fifteen species of the genus Enteroviruses based upon the high similarity of their genome sequences. As a result of their comparably low pathogenicity, RVs have become a handy model for other, more dangerous members of this genus, e.g., poliovirus and enterovirus 71. We provide a short overview of viral proteins that are considered potential drug targets and their corresponding drug candidates. We briefly mention more recently identified cellular enzymes whose inhibition impacts on RVs and comment novel approaches to interfere with infection via aggregation, virus trapping, or preventing viral access to the cell receptor. Finally, we devote a large part of this article to adding the viral RNA genome to the list of potential drug targets by dwelling on its structure, folding, and the still debated way of its exit from the capsid. Finally, we discuss the recent finding that G-quadruplex stabilising compounds impact on RNA egress possibly via obfuscating the unravelling of stable secondary structural elements. Full article

Influenza is a zoonotic respiratory disease of major public health interest due to its pandemic potential, and a threat to animals and the human population. The influenza A virus genome consists of eight single-stranded RNA segments sequestered within a protein capsid and a lipid bilayer envelope. During host cell entry, cellular cues contribute to viral conformational changes that promote critical events such as fusion with late endosomes, capsid uncoating and viral genome release into the cytosol. In this focused review, we concisely describe the virus infection cycle and highlight the recent findings of host cell pathways and cytosolic proteins that assist influenza uncoating during host cell entry. Full article

The Human Immunodeficiency Virus type 1 (HIV-1) virion contains a conical shell, termed capsid, encasing the viral RNA genome. After cellular entry of the virion, the capsid is released and ensures the protection and delivery of the HIV-1 genome to the host nucleus for integration. The capsid relies on many virus–host factor interactions which are regulated spatiotemporally throughout the course of infection. In this paper, we will review the current understanding of the highly dynamic HIV-1 capsid–host interplay during the early stages of viral replication, namely intracellular capsid trafficking after viral fusion, nuclear import, uncoating, and integration of the viral genome into host chromatin. Conventional anti-retroviral therapies primarily target HIV-1 enzymes. Insights of capsid structure have resulted in a first-in-class, long-acting capsid-targeting inhibitor, GS-6207 (Lenacapavir). This inhibitor binds at the interface between capsid protein subunits, a site known to bind host factors, interferes with capsid nuclear import, HIV particle assembly, and ordered assembly. Our review will highlight capsid structure, the host factors that interact with capsid, and high-throughput screening techniques, specifically genomic and proteomic approaches, that have been and can be used to identify host factors that interact with capsid. Better structural and mechanistic insights into the capsid–host factor interactions will significantly inform the understanding of HIV-1 pathogenesis and the development of capsid-centric antiretroviral therapeutics. Full article

During the last two decades, progresses in bioimaging and the development of various strategies to fluorescently label the viral components opened a wide range of possibilities to visualize the early phase of Human Immunodeficiency Virus 1 (HIV-1) life cycle directly in infected cells. After fusion of the viral envelope with the cell membrane, the viral core is released into the cytoplasm and the viral RNA (vRNA) is retro-transcribed into DNA by the reverse transcriptase. During this process, the RNA-based viral complex transforms into a pre-integration complex (PIC), composed of the viral genomic DNA (vDNA) coated with viral and host cellular proteins. The protective capsid shell disassembles during a process called uncoating. The viral genome is transported into the cell nucleus and integrates into the host cell chromatin. Unlike biochemical approaches that provide global data about the whole population of viral particles, imaging techniques enable following individual viruses on a single particle level. In this context, quantitative microscopy has brought original data shedding light on the dynamics of the viral entry into the host cell, the cytoplasmic transport, the nuclear import, and the selection of the integration site. In parallel, multi-color imaging studies have elucidated the mechanism of action of host cell factors implicated in HIV-1 viral cycle progression. In this review, we describe the labeling strategies used for HIV-1 fluorescence imaging and report on the main advancements that imaging studies have brought in the understanding of the infection mechanisms from the viral entry into the host cell until the provirus integration step. Full article