Overview of the SARS-CoV-2 nucleocapsid protein.

Since the emergence of SARS-CoV in 2003, researchers worldwide have been toiling away at deciphering this virus's biological intricacies. In line with other known coronaviruses, the nucleocapsid (N) protein is an important structural component of SARS-CoV. As a result, much emphasis has been placed on characterizing this protein. Independent research conducted by a variety of laboratories has clearly demonstrated the primary function of this protein, which is to encapsidate the viral genome. Furthermore, various accounts indicate that this particular protein disrupts diverse intracellular pathways. Such observations imply its vital role in regulating the virus as well. The opening segment of this review will expound upon these distinct characteristics succinctly exhibited by the N protein. Additionally, it has been suggested that the N protein possesses diagnostic and vaccine capabilities when dealing with SARS-CoV. In light of this fact, we will be reviewing some recent headway in the use cases for N protein toward clinical purposes within this article's concluding segments. This forward movement pertains to both developments of COVID-19-oriented therapeutic targets as well as diagnostic measures. The strides made by medical researchers offer encouragement, knowing they are heading toward a brighter future combating global pandemic situations such as these.

Three Distinct Reporter Systems of Hepatitis E Virus and Their Utility as Drug Screening Platforms.

The hepatitis E virus (HEV) is increasingly acknowledged as the primary cause of acute hepatitis. While most HEV infections are self-limiting, cases of chronic infection and fulminant hepatitis necessitate the administration of anti-HEV medications. However, there is a lack of specific antiviral drugs designed for HEV, and the currently available drug (ribavirin) has been associated with significant adverse effects. The development of innovative antiviral drugs involves targeting distinct steps within the viral life cycle: the early step (attachment and internalization), middle step (translation and RNA replication), and late step (virus particle formation and virion release). We recently established three HEV reporter systems, each covering one or two of these steps. Using these reporter systems, we identified various potential drug candidates that target different steps of the HEV life cycle. Through rigorous in vitro testing using our robust cell culture system with the genotype 3 HEV strain (JE03-1760F/P10), we confirmed the efficacy of these drugs, when used alone or in combination with existing anti-HEV drugs. This underscores their significance in the quest for an effective anti-HEV treatment. In the present review, we discuss the development of the three reporter systems, their applications in drug screening, and their potential to advance our understanding of the incompletely elucidated HEV life cycle.

Spying on SARS-CoV-2 with Fluorescent Tags and Protease Reporters.

The SARS-CoV-2 coronavirus has caused worldwide disruption through the COVID-19 pandemic, providing a sobering reminder of the profound impact viruses can have on human well-being. Understanding virus life cycles and interactions with host cells lays the groundwork for exploring therapeutic strategies against virus-related diseases. Fluorescence microscopy plays a vital role in virus imaging, offering high spatiotemporal resolution, sensitivity, and spectroscopic versatility. In this opinion piece, we first highlight two recent techniques, SunTag and StayGold, for the in situ imaging of viral RNA translation and viral assembly. Next, we discuss a new class of genetically encoded fluorogenic protease reporters, such as FlipGFP, which can be customized to monitor SARS-CoV-2's main (Mpro) or papain-like (PLpro) protease activity. These assays have proven effective in identifying potential antivirals through high-throughput screening, making fluorogenic viral protease reporters a promising platform for viral disease diagnostics and therapeutics.

A roadmap for developing Venezuelan equine encephalitis virus (VEEV) vaccines: Lessons from the past, strategies for the future.

Venezuelan equine encephalitis (VEE) is a zoonotic infectious disease caused by the Venezuelan equine encephalitis virus (VEEV), which can lead to severe central nervous system infections in both humans and animals. At present, the medical community does not possess a viable means of addressing VEE, rendering the prevention of the virus a matter of paramount importance. Regarding the prevention and control of VEEV, the implementation of a vaccination program has been recognized as the most efficient strategy. Nevertheless, there are currently no licensed vaccines or drugs available for human use against VEEV. This imperative has led to a surge of interest in vaccine research, with VEEV being a prime focus for researchers in the field. In this paper, we initially present a comprehensive overview of the current taxonomic classification of VEEV and the cellular infection mechanism of the virus. Subsequently, we provide a detailed introduction of the prominent VEEV vaccine types presently available, including inactivated vaccines, live attenuated vaccines, nucleic acid, and virus-like particle vaccines. Moreover, we emphasize the challenges that current VEEV vaccine development faces and suggest urgent measures that must be taken to overcome these obstacles. Notably, based on our latest research, we propose the feasibility of incorporation codon usage bias strategies to create the novel VEEV vaccine. Finally, we prose several areas that future VEEV vaccine development should focus on. Our objective is to encourage collaboration between the medical and veterinary communities, expedite the translation of existing vaccines from laboratory to clinical applications, while also preparing for future outbreaks of new VEEV variants.

IFIT3 mediated the type I interferon antiviral response by targeting Senecavirus A entry, assembly and release pathways.

Senecavirus A (SVA) is a newly emerging etiological agent of vesicular disease associated with sow abortion and acute piglet death, causing devastating economic consequences to global pig industry. IFN-induced protein with tetratricopeptide repeats (IFIT) genes are versatile in combating a variety of viruses, but the detailed mechanisms-of-action against SVA is unexplored. Transcriptomic analysis and immunoblot revealed high abundance of IFIT transcripts and proteins following SVA infection, initially implying the correlation between IFITs and SVA. Type I IFNs restricted SVA replication accompanied with substantial elevation of IFIT expression, potentializing IFITs as anti-SVA effectors downstream of IFN signaling. Gain-of-function assay demonstrated that IFIT3 rather than IFIT1/2 potently inhibited SVA replication, which was consistently verified by SVA strain SVV CH-FJ-2017 by TCID50 titration and an eGFP-tagged recombinant SVA using fluorescent microscopy. Afterwards, IFIT3 disrupted SVA life cycle at the early stage of virus binding and internalization, and at the late stage of virus assembly and release, as quantified by copy number and transmission electron microscopy, respectively. Directly transfecting SVA infectious RNA in IFIT3-overexpressed cells bypassed its antiviral activity, further suggesting that IFIT3 targeted viral life cycle beyond RNA replication. Further investigations showed that IFIT3 overexpression was incapable of regulating host immune responses against pathogens. Those results identified IFIT3 as a potent inhibitor of SVA and implicated IFIT3 pathway in the regulation of virus entry/assembly. In short, IFIT3 exerted significant inhibitory effects on the replication and spread of SVA, and played different functions in the life cycle of SVA.

Functional diversity: update of the posttranslational modification of Epstein-Barr virus coding proteins.

Epstein-Barr virus (EBV), a human oncogenic herpesvirus with a typical life cycle consisting of latent phase and lytic phase, is associated with many human diseases. EBV can express a variety of proteins that enable the virus to affect host cell processes and evade host immunity. Additionally, these proteins provide a basis for the maintenance of viral infection, contribute to the formation of tumors, and influence the occurrence and development of related diseases. Posttranslational modifications (PTMs) are chemical modifications of proteins after translation and are very important to guarantee the proper biological functions of these proteins. Studies in the past have intensely investigated PTMs of EBV-encoded proteins. EBV regulates the progression of the latent phase and lytic phase by affecting the PTMs of its encoded proteins, which are critical for the development of EBV-associated human diseases. In this review, we summarize the PTMs of EBV-encoded proteins that have been discovered and studied thus far with focus on their effects on the viral life cycle.

COVID-19 Treatment-Current Status, Advances, and Gap.

COVID-19, which emerged in December 2019, was declared a global pandemic by the World Health Organization (WHO) in March 2020. The disease was caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It has caused millions of deaths worldwide and caused social and economic disruption. While clinical trials on therapeutic drugs are going on in an Accelerating COVID-19 Therapeutic Interventions and Vaccines (ACTIV) public-private partnership collaboration, current therapeutic approaches and options to counter COVID-19 remain few. Therapeutic drugs include the FDA-approved antiviral drugs, Remdesivir, and an immune modulator, Baricitinib. Hence, therapeutic approaches and alternatives for COVID-19 treatment need to be broadened. This paper discusses efforts in approaches to find treatment for COVID-19, such as inhibiting viral entry and disrupting the virus life cycle, and highlights the gap that needs to be filled in these approaches.

Palmitic Amide Triggers Virus Life Cycle via Enhancing Host Energy Metabolism.

Viruses contribute to the mortality of organisms, consequentially altering biological species composition of an ecosystem and having a threat on human health. As the most famous model for the initiation of virus infection, the Hershey-Chase experiment has revealed that on infection, the bacteriophage genomic DNA is injected into its host bacterium, while the viral capsid is left on the outer membrane of host cell. However, little is known about the injection of any other materials into the cytoplasm of host cells along with genomic DNA to trigger the virus life cycle. In this study, the results showed that palmitic amide packaged in the virions of GVE2, a bacteriophage infecting deep-sea hydrothermal vent thermophile Geobacillus sp. E263, promoted virus infection. Palmitic amide was interacted with acetate kinase to increase its enzymatic activity, thus enhancing the acetate-mediated energy metabolism. Furthermore, palmitic amide promoted tricarboxylic acid cycle (TCA cycle) to support virus infection. These data indicated that palmitic amide, packaged in the virions, might serve as a second messenger at the initiation step of virus infection by enhancing the host energy metabolism. Therefore our study revealed a novel mechanism for the initiation of the virus life cycle.

The Life of SARS-CoV-2 Inside Cells: Replication-Transcription Complex Assembly and Function.

The persistence of the coronavirus disease 2019 (COVID-19) pandemic has resulted in increasingly disruptive impacts, and it has become the most devastating challenge to global health in a century. The rapid emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants challenges the currently available therapeutics for clinical application. Nonstructural proteins (also known as replicase proteins) with versatile biological functions play central roles in viral replication and transcription inside the host cells, and they are the most conserved target proteins among the SARS-CoV-2 variants. Specifically, they constitute the replication-transcription complexes (RTCs) dominating the synthesis of viral RNA. Knowledge of themolecular mechanisms of nonstructural proteins and their assembly into RTCs will benefit the development of antivirals targeting them against existing or potentially emerging variants. In this review, we summarize current knowledge of the structures and functions of coronavirus nonstructural proteins as well as the assembly and functions of RTCs in the life cycle of the virus.

Multiple Regions Drive Hepatitis Delta Virus Proliferation and Are Therapeutic Targets.

Hepatitis Delta Virus (HDV) is the smallest mammalian single-stranded RNA virus. It requires host cells and hepatitis B virus (HBV) to complete its unique life cycle. The present review summarizes the specific regions on hepatitis D antigen (HDAg) and hepatitis B surface antigen (HBsAg) that drive HDV to utilize host cell machinery system to produce three types of RNA and two forms of HDAg, and hijack HBsAg for its secretion and de novo entry. Previously, interferon-α was the only recommended therapy for HDV infection. In recent years, some new therapies targeting these regions, such as Bulevirtide, Lonafarnib, Nucleic acid polymers have appeared, with better curative effects and fewer adverse reactions.

Recruitment of dynein and kinesin to viral particles.

Dynein and kinesin are cytoskeletal motor proteins involved in transporting cellular cargos and viruses. Throughout viral infection, they actively participate in the virus life cycle in the cell during entry, genome replication, and departure. Through their retrograde and anterograde transport, dynein and kinesin assist in promoting viral infection as well as the cellular defense response. This review highlights the crucial roles kinesin and dynein play in facilitating viral proliferation and aims to exhibit these proteins as vital targets for drug discovery in exploring strategies for regulating their dual functions concerning involvements in various essential phases of viral infections and host cells' immune response.

Glycosylation of viral proteins: Implication in virus-host interaction and virulence.

Glycans are among the most important cell molecular components. However, given their structural diversity, their functions have not been fully explored. Glycosylation is a vital post-translational modification for various proteins. Many bacteria and viruses rely on N-linked and O-linked glycosylation to perform critical biological functions. The diverse functions of glycosylation on viral proteins during viral infections, including Dengue, Zika, influenza, and human immunodeficiency viruses as well as coronaviruses have been reported. N-linked glycosylation is the most common form of protein modification, and it modulates folding, transportation and receptor binding. Compared to N-linked glycosylation, the functions of O-linked viral protein glycosylation have not been comprehensively evaluated. In this review, we summarize findings on viral protein glycosylation, with particular attention to studies on N-linked glycosylation in viral life cycles. This review informs the development of virus-specific vaccines or inhibitors.

Immune response variables and viral mutations impact on COVID-19 reinfection and relapse.

The possibility of human reinfection with SARS-CoV-2, the coronavirus responsible for COVID-19, has not previously been thoroughly investigated. Although it is generally believed that virus-specific antibodies protect against COVID-19 pathogenesis, their duration of function and temporal activity remain unknown. Contrary to media reports that people retain protective antibody responses for a few months, science does not exclude reinfection and disease relapse shortly after initiating all immune responses during the primary onset of COVID-19. Despite production of antiviral antibodies, activated CD4+/CD8+ lymphocytes, and long-lived memory B cells, susceptibility to reinfection in humans for extended periods cannot be precluded due to repeated exposures to coronavirus or potential reactivation of the virus due to incomplete virus clearance. However, the mechanism of reinfection remains unknown. The biological characteristics of SARS-CoV-2, such as emergence of multiple mutations in the virus RNA molecules, transmissibility, rates of infection, reactivation and reinfection, can all affect the trajectory of the virus spread. Innate and adaptive immune response variables, differences in underlying diseases, and comorbidities, particularly in high risk individuals, can influence the dynamics of the virus infection. In this article, immune parameters and viral mutations pertaining to reinfection and disease relapse are reviewed and scientific gaps are discussed.

Novel therapeutic approaches toward Hantaan virus and its clinical features' similarity with COVID-19.

Zoonotic virus spill over in human community has been an intensive area of viral pathogenesis and the outbreak of Hantaan virus and severe acute respiratory syndrome coronavirus 2 (SARS CoV2) after late December 2019 caused a global threat. Hantaan virus is second to the COVID-19 outbreak in China with seven cases positive and one death. Both RNA viruses have opposite sense as in (-) for Hantaan virus and (+) for SARS CoV2 but have similarity in the pathogenesis and relevant clinical features including dry cough, high fever, shortness of breath, and SARS associated with pneumonia and certain reported cases with multiple organ failure. Although COVID-19 has global impact with high death toll, Hantaan virus has varyingly high mortality rate between 1% and 40%. Hence, there is a need to explore novel therapeutic targets in Hantaan virus due to its rapid evolution rate in its genetic makeup which governs virulence and target host cells. This review emphasizes the importance of structural and nonstructural proteins of Hantaan virus with relevant insight from SARS CoV2. The envelope glycoproteins such as Gn, Gc, and nucleocapsid protein (N) direct the viral assembly and replication in host cells. Therapeutic treatment has similarity in using ribavirin and extracorporeal membrane oxygenation but lack of efficacious treatment in both cases of SARAS CoV2 and Hantaan virus. Therefore, potential features regarding therapeutic targets for drug discovery for Hantaan viruses are discussed herewith. The conclusive description highlights that N protein is substantially involved in evoking immune response and induces symptoms and could be precursive target for drug discovery studies.

Contribution of hypoxia inducible factor-1 during viral infections.

Hypoxia-inducible factor 1 (HIF-1) is a transcription factor that plays critical roles during the cellular response to hypoxia. Under normoxic conditions, its function is tightly regulated by the degradation of its alpha subunit (HIF-1α), which impairs the formation of an active heterodimer in the nucleus that otherwise regulates the expression of numerous genes. Importantly, HIF-1 participates in both cancer and infectious diseases unveiling new therapeutic targets for those ailments. Here, we discuss aspects related to the activation of HIF-1, the effects of this transcription factor over immune system components, as well as the involvement of HIF-1 activity in response to viral infections in humans. Although HIF-1 is currently being assessed in numerous clinical settings as a potential therapy for different diseases, up to date, there are no clinical studies evaluating the pharmacological modulation of this transcription factor as a possible new antiviral treatment. However, based on the available evidence, clinical trials targeting this molecule are likely to occur soon. In this review we discuss the role of HIF-1 in viral immunity, the modulation of HIF-1 by different types of viruses, as well as the effects of HIF-1 over their life cycle and the potential use of HIF-1 as a new target for the treatment of viral infections.

COVID-19 update: The race to therapeutic development.

The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), represents an unprecedented challenge to global public health. At the time of this review, COVID-19 has been diagnosed in over 40 million cases and associated with 1.1 million deaths worldwide. Current management strategies for COVID-19 are largely supportive, and while there are more than 2000 interventional clinical trials registered with the U.S. National Library of Medicine (clinicaltrials.gov), results that can clarify benefits and risks of candidate therapies are only gradually becoming available. We herein describe recent advances in understanding SARS-CoV-2 pathobiology and potential therapeutic targets that are involved in viral entry into host cells, viral spread in the body, and the subsequent COVID-19 progression. We highlight two major lines of therapeutic strategies for COVID-19 treatment: 1) repurposing the existing drugs for use in COVID-19 patients, such as antiviral medications (e.g., remdesivir) and immunomodulators (e.g., dexamethasone) which were previously approved for other disease conditions, and 2) novel biological products that are designed to target specific molecules that are involved in SARS-CoV-2 viral entry, including neutralizing antibodies against the spike protein of SARS-CoV-2, such as REGN-COV2 (an antibody cocktail), as well as recombinant human soluble ACE2 protein to counteract SARS-CoV-2 binding to the transmembrane ACE2 receptor in target cells. Finally, we discuss potential drug resistance mechanisms and provide thoughts regarding clinical trial design to address the diversity in COVID-19 clinical manifestation. Of note, preventive vaccines, cell and gene therapies are not within the scope of the current review.

N-glycosylation in the Pre-Membrane Protein Is Essential for the Zika Virus Life Cycle.

Asparagine (N)-linked protein glycosylation plays an important role in protein synthesis and modification. Two Zika virus (ZIKV) structural proteins, the pre-membrane (prM) and envelope (E) protein are N-glycosylated. The prM protein of all ZIKV strains contains a single N-linked glycosylation site, while not all strains contain an N-linked site in the E protein. Our aim was to examine the impact of prM and E N-linked glycosylation on ZIKV infectivity and cell trafficking. Using a ZIKV infectious clone, we found that when the N-glycan sites were removed, the prM- and the prM/E-double mutants did not produce an infectious virus in the supernatant. Further, by using ZIKV prME constructs, we found that N-glycosylation was necessary for effective secretion of ZIKV virions. The absence of the N-glycan on prM or E caused protein aggregation in the rough endoplasmatic reticulum (ER) compartment. The aggregation was more pronounced for the prM-mutation, and the mutant virus lost the ER-Golgi intermediate compartment (ERGIC) localization. In addition, lack of the N-glycan on prM induced nuclear translocation of CCAAT-enhancer-binding protein homologous protein (CHOP), an ER stress marker. To conclude, we show that the prM N-glycan is essential for the ZIKV infectious cycle, and plays an important role in viral protein trafficking, protein folding, and virion assembly.

Multifaceted Functions of Host Cell Caveolae/Caveolin-1 in Virus Infections.

Virus infection has drawn extensive attention since it causes serious or even deadly diseases, consequently inducing a series of social and public health problems. Caveolin-1 is the most important structural protein of caveolae, a membrane invagination widely known for its role in endocytosis and subsequent cytoplasmic transportation. Caveolae/caveolin-1 is tightly associated with a wide range of biological processes, including cholesterol homeostasis, cell mechano-sensing, tumorigenesis, and signal transduction. Intriguingly, the versatile roles of caveolae/caveolin-1 in virus infections have increasingly been appreciated. Over the past few decades, more and more viruses have been identified to invade host cells via caveolae-mediated endocytosis, although other known pathways have been explored. The subsequent post-entry events, including trafficking, replication, assembly, and egress of a large number of viruses, are caveolae/caveolin-1-dependent. Deprivation of caveolae/caveolin-1 by drug application or gene editing leads to abnormalities in viral uptake, viral protein expression, or virion release, whereas the underlying mechanisms remain elusive and must be explored holistically to provide potential novel antiviral targets and strategies. This review recapitulates our current knowledge on how caveolae/caveolin-1 functions in every step of the viral infection cycle and various relevant signaling pathways, hoping to provide a new perspective for future viral cell biology research.

Viral G-quadruplexes: New frontiers in virus pathogenesis and antiviral therapy.

Viruses are the most abundant organisms on our planet, affecting all living beings: some of them are responsible for massive epidemics that concern health, national economies and the overall welfare of societies. Although advances in antiviral research have led to successful therapies against several human viruses, still some of them cannot be eradicated from the host and most of them do not have any treatment available. Consequently, innovative antiviral therapies are urgently needed. In the past few years, research on G-quadruplexes (G4s) in viruses has boomed, providing powerful evidence for the regulatory role of G4s in key viral steps. Comprehensive bioinformatics analyses have traced putative G4-forming sequences in the genome of almost all human viruses, showing that their distribution is statistically significant and their presence highly conserved. Since the genomes of viruses are remarkably variable, high conservation rates strongly suggest a crucial role of G4s in the viral replication cycle and evolution, emphasizing the possibility of targeting viral G4s as a new pharmacological approach in antiviral therapy. Recent studies have demonstrated the formation and function of G4s in pathogens responsible for serious diseases, such as HIV-1, Hepatitis B and C, Ebola viruses, to cite a few. In this chapter, we present the state of the art on the structural and functional characterization of viral G4s in RNA viruses, DNA viruses and retroviruses. We also present the G4 ligands that provide further details on the viral G4 role and which, showing promising antiviral activity, which could be exploited for the development of innovative antiviral agents.

The Multifarious Role of 14-3-3 Family of Proteins in Viral Replication.

The 14-3-3 proteins are a family of ubiquitous and exclusively eukaryotic proteins with an astoundingly significant number of binding partners. Their binding alters the activity, stability, localization, and phosphorylation state of a target protein. The association of 14-3-3 proteins with the regulation of a wide range of general and specific signaling pathways suggests their crucial role in health and disease. Recent studies have linked 14-3-3 to several RNA and DNA viruses that may contribute to the pathogenesis and progression of infections. Therefore, comprehensive knowledge of host-virus interactions is vital for understanding the viral life cycle and developing effective therapeutic strategies. Moreover, pharmaceutical research is already moving towards targeting host proteins in the control of virus pathogenesis. As such, targeting the right host protein to interrupt host-virus interactions could be an effective therapeutic strategy. In this review, we generated a 14-3-3 protein interactions roadmap in viruses, using the freely available Virusmentha network, an online virus-virus or virus-host interaction tool. Furthermore, we summarize the role of the 14-3-3 family in RNA and DNA viruses. The participation of 14-3-3 in viral infections underlines its significance as a key regulator for the expression of host and viral proteins.