Viral Replication Research Articles

Activity and cryo-EM structure of the polymerase domain of the human norovirus ProPol precursor.

Human norovirus (HuNV) is a leading cause of acute gastroenteritis worldwide with most infections caused by genogroup I and genogroup II (GII) viruses. Replication of HuNV generates both precursor and mature proteins during processing of the viral polyprotein that are essential to the viral lifecycle. One such precursor is protease-polymerase (ProPol), a multi-functional enzyme comprised of the norovirus protease and polymerase proteins. This work investigated HuNV ProPol by determining the de novo polymerase activity, protein structure, and antiviral inhibition profile. The GII ProPol de novo enzymatic efficiencies (kcat/Km) for RNA templates and ribonucleotides were equal or superior to those of mature GII Pol on all templates measured. Furthermore, GII ProPol was the only enzyme form active on a poly(A) template. The first structure of the polymerase domain of HuNV ProPol in the unliganded state was determined by cryo-electron microscopy at a resolution of 2.6 Å. The active site and overall architecture of ProPol are similar to those of mature Pol. In addition, both galidesivir triphosphate and PPNDS inhibited polymerase activity of GII ProPol, with respective half-maximal inhibitory concentration (IC50) values of 247.5 µM and 3.8 µM. In both instances, the IC50 obtained with ProPol was greater than that of mature Pol, indicating that ProPol can exhibit different responses to antivirals. This study provides evidence that HuNV ProPol possesses overlapping and unique enzyme properties compared with mature Pol and will aid our understanding of the replication cycle of the virus.IMPORTANCEDespite human norovirus (HuNV) being a leading cause of acute gastroenteritis, the molecular mechanisms surrounding replication are not well understood. Reports have shown that HuNV replication generates precursor proteins from the viral polyprotein, one of which is the protease-polymerase (ProPol). This precursor is important for viral replication; however, the polymerase activity and structural differences between the precursor and mature forms of the polymerase remain to be determined. We show that substrate specificity and polymerase activity of ProPol overlap with, but is distinct from, the mature polymerase. We employ cryo-electron microscopy to resolve the first structure of the polymerase domain of ProPol. This shows a polymerase architecture similar to mature Pol, indicating that the interaction of the precursor with substrates likely defines its activity. We also show that ProPol responds differently to antivirals than mature polymerase. Altogether, these findings enhance our understanding of the function of the important norovirus ProPol precursor.

Apoptotic Caspases Suppress Expression of Endogenous Retroviruses in HPV31+ Cells That Are Associated with Activation of an Innate Immune Response

Avoidance of an immune response is critical to completion of the human papillomavirus (HPV) life cycle, which occurs in the stratified epithelium and is linked to epithelial differentiation. We previously demonstrated that high-risk HPVs use apoptotic caspases to suppress an antiviral innate immune response during the productive phase of the life cycle. We found that caspase-8 and caspase-3 suppress a type I IFN-β and type III IFN-λ response by disabling the MDA5/MAVS double-stranded RNA (dsRNA) sensing pathway, indicating that immunogenic RNAs increase upon differentiation in HPV+ cells. In this study, we demonstrate that caspase inhibition results in aberrant transcription of a subset of endogenous retroviruses (ERVs) that have been shown to activate an IFN response through dsRNA-sensing pathways. We show that the increase in ERV transcription is accompanied by an enrichment in dsRNA formation. Additionally, we demonstrate that the robust increase in ERV expression requires activation of the JAK/STAT-signaling pathway, indicating that this subset of ERVs is IFN-inducible. Overall, these results suggest a model by which caspase activity blocks the reactivation of ERVs through the JAK/STAT pathway, protecting HPV+ cells from an increase in immunogenic dsRNAs that otherwise would trigger an IFN response that inhibits productive viral replication.

Viral RNA Interactome: The Ultimate Researcher’s Guide to RNA–Protein Interactions

RNA molecules in the cell are bound by a multitude of RNA-binding proteins (RBPs) with a variety of regulatory consequences. Often, interactions with these RNA-binding proteins are facilitated by the complex secondary and tertiary structures of RNA molecules. Viral RNAs especially are known to be heavily structured and interact with many RBPs, with roles including genome packaging, immune evasion, enhancing replication and transcription, and increasing translation efficiency. As such, the RNA–protein interactome represents a critical facet of the viral replication cycle. Characterization of these interactions is necessary for the development of novel therapeutics targeted at the disruption of essential replication cycle events. In this review, we aim to summarize the various roles of RNA structures in shaping the RNA–protein interactome, the regulatory roles of these interactions, as well as up-to-date methods developed for the characterization of the interactome and directions for novel, RNA-directed therapeutics.

SARS-CoV-2 persistence: A potential catalyst for age-associated neurodegenerative diseases

The persistence of RNA viruses in the brain is increasingly recognized as a significant factor in the progression of age-related neurodegenerative diseases. This phenomenon is particularly evident in infections caused by various neurotropic and non-neurotropic viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The COVID-19 pandemic has underscored the urgent need to explore the complex relationship between viral persistence and neurological decline. Growing evidence indicates that SARS-CoV-2 affects brain structure and function, although the precise molecular mechanisms remain poorly understood. Chronic neuroinflammation induced by viral infections is thought to accelerate age-related neurodegeneration. While the immune system typically clears many viral infections in the brain, some viruses establish chronic infections, leading to restricted viral replication. These persistent infections can exacerbate neuroinflammation and contribute to ongoing neuronal damage, key drivers of age-related neurodegeneration. This review explores current knowledge on how SARS-CoV-2 infiltrates the brain, evades immune defenses, and persists within brain cells, potentially using them as viral reservoirs. As individuals age, the cumulative effects of such viral infections may accelerate cognitive decline and increase vulnerability to neurodegenerative diseases such as Alzheimer’s and Parkinson’s. Understanding the molecular mechanisms of viral persistence and its long-term impact on brain health is crucial for developing targeted therapies to combat these age-related diseases.

Single-cell transcriptional analysis of murine norovirus infection in a human intestinal cell line.

Noroviruses are a major agent of acute gastroenteritis in humans, but host cell requirements for efficient replication in vitro have not been established. We engineered a human intestinal cell line (designated mCD300lf-hCaco2) expressing the murine norovirus (MNV) receptor, mouse CD300lf to become fully permissive for MNV replication. To explore the replicative machinery and host response of these cells, we performed a single-cell RNA sequencing (scRNA-seq) transcriptomics analysis of an MNV infection over time. Marked similarities were observed between certain global features of MNV infection in human cells compared to those previously reported in mouse cells by whole population transcriptomics such as downregulation of ribosome biogenesis, mitochondrial dysfunction, and cell cycle preference for G1. Our scRNA-seq analysis allowed further resolution of an infected cell population into distinct clusters with varying levels of viral RNA and interferon-stimulated gene ISG15 transcripts. Cells with high viral replication displayed downregulated ribosomal protein small (RPS) and large (RPL) genes and mitochondrial complexes I, III, IV, and V genes during exponential viral propagation. Ferritin subunit genes FTL and FTH1 were also downregulated during active MNV replication, suggesting that inhibition of iron metabolism may increase replication efficiency. Consistent with this, transcriptional activation of these genes with ferric ammonium citrate and overexpression of FTL lowered virus yields. Comparative studies of cells that support varying levels of norovirus replication efficiency, as determined by scRNA-seq may lead to improved human cell-based culture systems and effective viral interventions.IMPORTANCEHuman noroviruses cause acute gastroenteritis in all age groups. Vaccines and antiviral drugs are not yet available, in part, because it is difficult to propagate the viruses causing human disease in standard laboratory cell culture systems. In contrast, a norovirus found in mice [murine norovirus (MNV)] replicates efficiently in murine-based cell culture and has served as a model system. In this study, we established a new human intestinal cell line that was genetically modified to express the murine norovirus receptor so that the human cells became permissive to murine norovirus infection. We then defined the host response to MNV infection in the engineered human cell line at a single-cell resolution and identified cellular genes associated with the highest levels of MNV replication. This study may lead to the improvement of the current human norovirus cell culture systems and help to identify norovirus-host interactions that could be targeted for antiviral drugs.

A CRISPR-Cas9 knockout screening identifies IRF2 as a key driver of OAS3/RNase L-mediated RNA decay during viral infection

OAS-RNase L is a double-stranded RNA-induced antiviral pathway triggered in response to diverse viral infections. Upon activation, OAS-RNase L suppresses virus replication by promoting the decay of host and viral RNAs and inducing translational shutdown. However, whether OASs and RNase L are the only factors involved in this pathway remains unclear. Here, we develop CRISPR-Translate, a FACS-based genome-wide CRISPR-Cas9 knockout screening method that uses translation levels as a readout and identifies IRF2 as a key regulator of OAS3. Mechanistically, we demonstrate that IRF2 promotes basal expression of OAS3 in unstressed cells, allowing a rapid activation of RNase L following viral infection. Furthermore, IRF2 works in concert with the interferon response through STAT2 to further enhance OAS3 expression. We propose that IRF2-induced RNase L is critical in enabling cells to mount a rapid antiviral response immediately after viral infection, serving as the initial line of defense. This rapid response provides host cells the necessary time to activate additional antiviral signaling pathways, forming secondary defense waves.

Protein phosphatase 1 suppresses PKR/EIF2α signaling during human cytomegalovirus infection.

Human cytomegalovirus (HCMV) is a ubiquitous pathogen that infects the majority of the world's population. Lytic HCMV replication in immunocompromised individuals or neonates can lead to severe disease in multiple organ systems and even death. The establishment of lytic replication is driven by the first viral proteins expressed upon infection, the immediate early proteins, which play a key role in creating an intracellular environment conducive to virus replication. Two immediate early proteins, the functional orthologs pTRS1 and pIRS1, stimulate immediate early gene expression by suppressing antiviral PKR/eIF2α signaling and enhance the translation of viral mRNAs independent of PKR antagonism. To better understand the molecular functions of pTRS1, we used proximity labeling proteomics to identify proteins that interact with pTRS1 in infected cells. Multiple novel host and viral interactors were identified, including the catalytic subunits of the protein phosphatase 1 (PP1) holoenzyme. Mutations to a PP1 catalytic subunit known to disrupt binding to PP1 regulatory subunits decreased binding to pTRS1. pTRS1 immune complexes contained phosphatase activity, and inhibition of phosphatase activity in transfected or infected cells reversed the ability of pTRS1 to inhibit the antiviral kinase PKR. Depletion of individual PP1 catalytic subunits decreased virus replication and increased the phosphorylation of the PKR substrate eIF2α. Taken together, our data suggest potential novel functions for pTRS1 and define a novel role for PP1 as an antagonist of the antiviral PKR/eIF2α signaling axis during HCMV infection.IMPORTANCEThe human cytomegalovirus (HCMV) pTRS1 and pIRS1 proteins are critical regulators of HCMV replication, both during primary infection and during reactivation from viral latency. Thus, defining the molecular functions of pTRS1/pIRS1 is important for understanding the molecular events controlling HCMV replication and viral disease. These data provide new insights into potential pTRS1 functional roles, providing a starting point for others to understand new features of infected cell biology. Another important result of this study is the finding that specific protein phosphatase 1 (PP1) regulatory subunits are required to suppress PKR/eIF2α signaling, a critical cellular innate immune defense to viral infection. These data lay the groundwork for future efforts to discover therapeutics that disrupt pTRS1 interaction with PP1 allowing cellular defenses to limit HCMV replication and disease.

Cyclooxygenase-2/prostaglandin E2 pathway orchestrates the replication of infectious bronchitis virus in chicken tracheal explants

ABSTRACT In this study, we investigated the localized pathogenesis of infectious bronchitis virus (IBV) in chicken tracheal organ cultures (TOCs), focusing on the role of inducible cyclooxygenase (COX-2). Two divergent IBV strains, respiratory Connecticut (Conn) A5968 and nephropathogenic Delmarva (DMV)/1639, were studied at 6, 12, 24, and 48 hours post-infection (hpi). Various treatments including exogenous prostaglandin (PGE)2, a selective COX-2 antagonist (SC-236), and inhibitors of PGE2 receptors and Janus kinase (JAK) were administered. IBV genome load and antigen expression were quantified using real-time quantitative PCR and immunohistochemistry. COX-2, interferon (IFN)-α, IFN-β, interleukin (IL)−1β, IL-6, and inducible nitric oxide synthase (iNOS) expressions were measured, along with PGE2 and COX-2 concentrations. IBV genome load and protein expression peaked at 12 and 24 hpi, respectively. Conn A5968-infected TOCs exhibited continuous COX-2 expression for up to 24 hpi, extended PGE2 production up to 48 hpi, and reduced inflammatory cytokine expression. In contrast, DMV/1639-infected TOCs displayed heightened inflammatory cytokine expression, brief COX-2 expression, and PGE2 production. Treatment with IFN-γ, SC-236, PGE2 receptor inhibitors, or JAK inhibitors reduced IBV infection and lesion scores, whereas exogenous PGE2 or IFN-γ pretreatment with a JAK-2 inhibitor augmented infection. These findings shed light on the innate immune regulation of IBV infection in the trachea, highlighting the involvement of the COX-2/PGE2 pathway. IMPORTANCE Understanding the localized pathogenesis of infectious bronchitis virus (IBV) within the trachea of chickens is crucial for developing effective control strategies against this prevalent poultry pathogen. This study sheds light on the role of inducible cyclooxygenase (COX-2) and prostaglandin (PGE)2 in IBV pathogenesis using chicken tracheal organ culture (TOC) models. The findings reveal distinct patterns of COX-2 expression, PGE2 production, and immune responses associated with different IBV strains, highlighting the complexity of host-virus interactions. Furthermore, the identification of specific inhibitors targeting the COX-2/PGE2 pathway and Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway provides potential therapeutic avenues for mitigating IBV infection in poultry. Overall, this study contributes to our understanding of the innate immune regulation of IBV infection within the trachea, laying the groundwork for the development of targeted interventions to control IBV outbreaks in poultry populations.

ATPase Valosin-Containing Protein (VCP) Is Involved During the Replication and Egress of Sialodacryoadenitis Virus (SDAV) in Neurons

Sialodacryoadenitis virus (SDAV) has been identified as the etiological agent responsible for the respiratory system and salivary gland infections in rats. The existing literature on SDAV infections is insufficient to address the topic adequately, particularly in relation to the central nervous system. In order to ascertain how SDAV gains access to neuronal cells and subsequently exits, our attention was focused on the small molecule valosin-containing protein (VCP), which is an ATPase. VCP is acknowledged for its function in the ubiquitin-mediated proteasomal degradation of proteins, including those of viral origin. To ascertain the potential influence of VCP on SDAV replication and egress, high-content screening was employed to determine the viral titer and protein content. Western blot analysis was employed to ascertain the relative expression of VCP. Real-time imaging of SDAV-infected cells and confocal imaging for qualitative morphological analysis were conducted. The Eeyarestatin I (EerI) inhibitor was employed to disrupt VCP involvement in the endoplasmic reticulum-associated protein degradation pathway (ERAD) in both pre- and post-incubation systems, with concentrations of 5 μM/mL and 25 μM/mL, respectively. We demonstrated for the first time that SDAV productively replicates in cultured primary neurons. VCP expression is markedly elevated during SDAV infection. The application of 5 μM/mL EerI in the post-treatment system yielded a statistically significant inhibition of the SDAV yield. It is likely that this modulates the efficacy of virion assembly by arresting viral proteins in the submembrane area.

Molecular depiction and functional delineation of E3 ubiquitin ligase MARCH5 in yellowtail clownfish (Amphiprion clarkii)

Membrane-associated Ring-CH 5 (MARCH5) is a mitochondrial E3 ubiquitin ligase playing a key role in the regulation of mitochondrial dynamics. In mammals, MARCH5 negatively regulates mitochondrial antiviral signaling (MAVS) protein aggregation during viral infection and hampers downstream type I interferon signaling to prevent excessive immune activation. However, its precise functional role in the teleost immune system remains unclear. This study investigated the molecular characteristics and immune response of the MARCH5 ortholog in Amphiprion clarkii (A. clarkii; AcMARCH5). The predicted AcMARCH5 protein sequence consists of 287 amino acids with a molecular weight of 32.02 kDa and a theoretical isoelectric point of 9.11. It contains four C-terminal transmembrane (TM) domains and an N-terminal RING cysteine-histidine (CH) domain, which directly regulates ubiquitin transfer. Multiple sequence alignment revealed a high level of conservation between AcMARCH5 and its orthologs in other vertebrate species. Under normal physiological conditions, AcMARCH5 showed the highest mRNA expression in the muscle, brain, and kidney tissues of A. clarkii. Upon stimulation with polyinosinic:polycytidylic acid (Poly I:C), lipopolysaccharide (LPS), and Vibrio harveyi, AcMARCH5 expression was drastically modulated. Functional assays showed that overexpression of AcMARCH5 in fathead minnow (FHM) cells downregulated antiviral gene expression, accompanied by enhanced viral hemorrhagic septicemia virus (VHSV) replication. In murine macrophages, AcMARCH5 overexpression markedly reduced the production of pro-inflammatory cytokines in response to poly I:C treatment. Additionally, AcMARCH5 exhibited an anti-apoptotic effect in H2O2-treated FHM cells. Collectively, these results suggest that AcMARCH5 may play a role in maintaining cellular homeostasis under disease and stress conditions in A. clarkii.

BiP/GRP78 is a pro-viral factor for diverse dsDNA viruses that promotes the survival and proliferation of cells upon KSHV infection.

The Endoplasmic Reticulum (ER)-resident HSP70 chaperone BiP (HSPA5) plays a crucial role in maintaining and restoring protein folding homeostasis in the ER. BiP's function is often dysregulated in cancer and virus-infected cells, conferring pro-oncogenic and pro-viral advantages. We explored BiP's functions during infection by the Kaposi's sarcoma-associated herpesvirus (KSHV), an oncogenic gamma-herpesvirus associated with cancers of immunocompromised patients. Our findings reveal that BiP protein levels are upregulated in infected epithelial cells during the lytic phase of KSHV infection. This upregulation occurs independently of the unfolded protein response (UPR), a major signaling pathway that regulates BiP availability. Genetic and pharmacological inhibition of BiP halts KSHV viral replication and reduces the proliferation and survival of KSHV-infected cells. Notably, inhibition of BiP limits the spread of other alpha- and beta-herpesviruses and poxviruses with minimal toxicity for normal cells. Our work suggests that BiP is a potential target for developing broad-spectrum antiviral therapies against double-stranded DNA viruses and a promising candidate for therapeutic intervention in KSHV-related malignancies.

Elucidation of the Pharmacological Development and Action Mechanism of Remdesivir and Paxlovid in Combatting COVID-19

COVID-19, the disease identified in 2019 as coronavirus disease, stems from an infection by severe acute respiratory syndrome coronavirus 2, or SARS-CoV-2, which possesses a single-stranded RNA genome. Since the onset of the pandemic attributed to COVID-19, a variety of vaccines and antiviral medications have received approval from international regulatory bodies. Two notable antiviral agents, remdesivir and paxlovid, serve as treatments for viral infections. Initially developed to combat Ebola, remdesivir has also demonstrated efficacy against a broad spectrum of coronaviruses (CoV). It is a nucleoside analogue and functions by being incorporated into the RNA chain of the virus and thus induce the early termination of RNA synthesis. Paxlovid comprises two pharmacological agents, nirmatrelvir and ritonavir. The mechanism of nirmatrelvir involves the specific targeting and inhibition of the viral protease’s active site, thereby impeding the viral replication process. By inhibiting the cytochrome CYP3A4 enzyme which metabolites nirmatrelvir, ritonavir acts as a pharmacokinetic enhancer in this combination. The initial section of this review provides a comprehensive analysis of SARS-CoV-2, detailing the mechanisms of its pathogenesis and the subsequent immune responses elicited by the body. Then this article generalizes the history of development, the structure, and the mode of action of remdesivir and paxlovid.

Mitochondrial Dysfunction and Metabolic Disturbances Induced by Viral Infections

Viruses are intracellular parasites that utilize organelles, signaling pathways, and the bioenergetics machinery of the cell to replicate the genome and synthesize proteins to build up new viral particles. Mitochondria are key to supporting the virus life cycle by sustaining energy production, metabolism, and synthesis of macromolecules. Mitochondria also contribute to the antiviral innate immune response. Here, we describe the different mechanisms involved in virus–mitochondria interactions. We analyze the effects of viral infections on the metabolism of glucose in the Warburg phenotype, glutamine, and fatty acids. We also describe how viruses directly regulate mitochondrial function through modulation of the activity of the electron transport chain, the generation of reactive oxygen species, the balance between fission and fusion, and the regulation of voltage-dependent anion channels. In addition, we discuss the evasion strategies used to avoid mitochondrial-associated mechanisms that inhibit viral replication. Overall, this review aims to provide a comprehensive view of how viruses modulate mitochondrial function to maintain their replicative capabilities.

Stimulator of interferon genes (STING) inhibits coronavirus infection by disrupting viral replication organelles.

Stimulator of interferon genes (STING) is an endoplasmic reticulum (ER) protein that plays a crucial role in cytosolic DNA-mediated innate immunity. Both STING agonists and antagonists have demonstrated their ability to enhance mouse survival against coronavirus, however, the physiological role of endogenous STING in coronavirus infection remains unclear. Our research unveils that STING inhibits coronavirus replication by impeding the formation of the ER-derived double-membrane vesicles (DMVs), the organelles in which coronavirus replicates. We found that STING was still capable of inhibiting coronavirus OC43 infection in cells, regardless of the knockout of cGAS or MAVS, or blocking type I interferon receptor. Moreover, STING disrupted the interaction between two crucial proteins, NSP4 and NSP6, involved in DMV formation, leading to the disruption of DMV formation. Taken together, our study sheds light on a novel antiviral role of STING in coronavirus infection, elucidating how it disrupts the formation of viral replication organelles, thereby impeding the replication process.

Chronic Hepatitis D Virus Infection and Its Treatment: A Narrative Review

More than 12 million individuals worldwide are chronically infected with the hepatitis D virus (HDV). HDV infection is the most severe form of viral hepatitis since it requires hepatitis B virus co-infection and accelerates progression to cirrhosis and hepatocellular carcinoma. Therefore, treatment modalities to slow the progression of the disease are essential but not yet available. In addition, no antiviral treatment to date has been shown to reliably eradicate HDV. Pegylated interferon (PEG-IFN) is the only universally used treatment to suppress HDV RNA replication and improve liver inflammation and fibrosis. This treatment can be completed in 12–18 months, but cure rates remain low, and success does not reliably increase with the addition of a nucleos(t)ide analog. PEG-IFN therapy is also limited by poor tolerability and multiple adverse effects, including neutropenia, thrombocytopenia, and neuropsychiatric symptoms. Newer antiviral therapies in development target unique aspects of HDV viral replication and show promising results in combination with PEG-IFN for long-term HDV RNA suppression. These newer antiviral therapies include buleviritide (which blocks HDV entry), lonafarnib (which prevents HDV assembly), and REP-2139 (which prevents HDV export). In this manuscript, we discuss the characteristics of HDV infection and review the new antiviral therapies approved for treatment and those under investigation.

Computational Assessment of Clinical Drugs against SARS-CoV-2: Foreseeing Molecular Mechanisms and Potent Mpro Inhibitors.

The emergence of new SARS-CoV-2 variants of concern (VOC) is a propulsion for accelerated potential therapeutic discovery. SARS-CoV-2's main protease (Mpro), essential for host cell viral replication, is a pre-eminent druggable protein target. Here, we perform extensive drug re-profiling of the comprehensive Excelra database, which compiles various under-trial drug candidates for COVID-19 treatment. For mechanistic understanding, the most promising screened-out molecules with targets are subjected to molecular dynamics (MD) simulations. Post-MD analyses demonstrate Darunavir, Ponatinib, and Tomivosertib forming a stable complex with Mpro, characterized by less fluctuation of Cα atoms, smooth and stable root-mean-square deviation (RMSD), and robust contact with the active site residues. Likewise, they all have lower binding free energy with Mpro, demonstrating strong affinity. In free energy landscape profiles, the distances from His41 and Cys145 exhibit a single energy minima basin, implying their preponderance in proximity to Mpro's catalytic dyad. Overall, the computational assessment earmarks promising candidates from the Excelra database, emphasizing on carrying out exhaustive biochemical experiments along with clinical trials. The work lays the foundation for potential therapeutic interventions in treating COVID-19.

Porcine reproductive and respiratory syndrome virus nonstructural protein 2 promotes the autophagic degradation of adaptor protein SH3KBP1 to antagonize host innate immune responses by enhancing K63-linked polyubiquitination of RIG-I.

Non-structural protein 2 (NSP2) of PRRSV is highly variable and plays crucial roles in the virus's life cycle. To elucidate the function of NSP2 during PRRSV infection, we identified SH3KBP1 as an NSP2-interacting host protein using mass spectrometry. Exogenous SH3KBP1 expression significantly inhibited PRRSV replication by enhancing IFN-I and related ISGs production. Conversely, SH3KBP1 knockdown promoted viral replication by downregulating IFN-I and ISGs levels. In vivo experiments revealed that Sh3kbp1-/- mice were more susceptible to VSV infection, exhibiting reduced serum IFN-β levels. Further investigation showed that SH3KBP1 enhances RIG-I signal transduction by increasing K63-linked polyubiquitination through interaction with the E3 ubiquitin ligase TRIM25. We also found that PRRSV infection and NSP2 overexpression induce the autophagic degradation of SH3KBP1, counteracting the host's innate immune response. A critical interaction site was identified within the third proline-rich motif in NSP2 (453PVPAPR458). Recombinant PRRSV lacking this motif displayed reduced virulence and decreased SH3KBP1 degradation. This study advances our understanding of how PRRSV interferes with the host immune response and offers valuable insights for development novel attenuated vaccines against PRRSV.

Phaleria macrocarpa Fruit Protein Aqueous Extract Affects Viral Entry, Virucidal Activity, and Progeny Release

Presence of acyclovir (ACV) resistant virus posed a major problem in treating virus infection. Alternative treatment with the ability to encounter infection of acyclovir-resistant virus is thus needed and possibly with a different mode of action from ACV. Hence, this study evaluates the antiviral effect of Phaleria macrocarpa (Scheff.) Boerl fruit protein aqueous extract (PMFPAE) against three different strains of human herpesvirus type-1 (HHV-1) including a clinical strain, a less pathogenic strain (KOS-1), and acyclovir (ACV) resistant mutant (UKM-1). PMFPAE displayed antiviral activity towards all the HHV-1 strains when post-treated with high selective indices (SIs) of 80.6, 50, and 35, respectively. Plaque reduction percentages were reduced in attachment and penetration assays following treatment with PMFPAE indicating the ability to deactivate the early phases of the HHV-1 replication cycle. The virucidal activity was also noted following treatment of the virus with PMFPAE and this is supported by damages to the virus envelope as observed by transmission electron microscopy (TEM). Incubation of virus-treated cells with PMFPAE for 24 hr, reduced progeny release in a dose-dependent manner. The study confirms the antiviral mode of action of P. macrocarpa fruit against HHV-1 strains and the ACV-mutant strain includes inhibition during virus entry represented as the early stages of viral replication, virucidal activity, and interfering with progeny release. PMFPAE mode of action is hence different from ACV and worthy for the development of future antiviral drugs.

Stress Can Induce Bovine Alpha-Herpesvirus 1 (BoHV-1) Reactivation from Latency

Bovine alpha-herpesvirus 1 (BoHV-1) is a significant problem for the cattle industry, in part because the virus establishes latency, and stressful stimuli increase the incidence of reactivation from latency. Sensory neurons in trigeminal ganglia and unknown cells in pharyngeal tonsils are importantsites for latency. Reactivation from latency can lead to reproductive problems in pregnant cows, virus transmission to young calves, suppression of immune responses, and bacterial pneumonia. BoHV-1 is also a significant cofactor in bovine respiratory disease (BRD). Stress, as mimicked by the synthetic corticosteroid dexamethasone, reproducibly initiates reactivation from latency. Stress-mediated activation of the glucocorticoid receptor (GR) stimulates viral replication and transactivation of viral promoters that drive the expression of infected cell protein 0 (bICP0) and bICP4. Notably, GR and Krüppel-like factor 15 (KLF15) form a feed-forward transcription loop that cooperatively transactivates immediate early transcription unit 1 (IEtu1 promoter). Two pioneer transcription factors, GR and KLF4, cooperatively transactivate the bICP0 early promoter. Pioneer transcription factors bind silent viral heterochromatin, remodel chromatin, and activate gene expression. Thus, wepredict that these novel transcription factors mediate early stages of BoHV-1 reactivation from latency.

MDA5 Is a Major Determinant of Developing Symptoms in Critically Ill COVID-19 Patients.

Apart from the skin and mucosal immune barrier, the first line of defense of the human immune system includes MDA5 (ifih1 gene) which acts as a cellular sensor protein for certain viruses including SARS-CoV-2. Upon binding with viral RNA, MDA5 activates cell-intrinsic innate immunity, humoral responses, and MAVS (mitochondrial antiviral signaling). MAVS signaling induces type I and III interferon (IFN) expressions that further induce ISGs (interferon stimulatory genes) expressions to initiate human cell-mediated immune responses and attenuate viral replication. SARS-CoV-2 counteracts by producing NSP1, NSP2, NSP3, NSP5, NSP7, NSP12, ORF3A, ORF9, N, and M protein and directs anti-MDA5 antibody production presumably to antagonize IFN signaling. Furthermore, COVID-19 resembles several diseases that carry anti-MDA5 antibodies and the current COVID-19 vaccines induced anti-MDA5 phenotypes in healthy individuals. GWAS (genome-wide association studies) identified several polymorphisms (SNPs) in the ifih1-ifn pathway genes including rs1990760 in ifih1 that are strongly associated with COVID-19, and the associated risk allele is correlated with reduced IFN production. The genetic association of SNPs in ifih1 and ifih1-ifn pathway genes reinforces the molecular findings of the critical roles of MDA5 in sensing SARS-CoV-2 and subsequently the IFN responses to inhibit viral replication and host immune evasion. Thus, MDA5 or its pathway genes could be targeted for therapeutic development of COVID-19.