Hepatitis C virus (HCV) depends on host lipid metabolism and lipid droplets (LDs) for genome replication, assembly, and particle production, yet how LD structure and lipid utilization change over the course of infection remains incompletely understood. Here, we investigated the temporal remodeling of LD-associated metabolic pathways during HCV JFH-1 infection of human hepatoma Huh7 cells. HCV infection transiently increased LD fluorescence intensity at 24 hours post-infection (hpi), followed by normalization or relative loss of LD signal at later time points. Concomitantly, LDs became progressively clustered and enlargement during late infection, despite reduced protein levels of the canonical LD fusion proteins CIDEA, CIDEB, and CIDEC, suggesting that HCV-induced LD enlargement occurs through CIDE-independent mechanisms. Transcriptomic, RT-qPCR, and immunoblot analyses revealed time-dependent regulation of genes and proteins involved in LD structure, triglyceride synthesis, lipolysis, lipid uptake, and mitochondrial fatty acid utilization. Subcellular fractionation demonstrated preferential accumulation of fatty acids in mitochondrial fractions at 24-72 hpi. This redistribution was accompanied by increased oxygen consumption rate, elevated extracellular acidification, and progressive reactive oxygen species accumulation, indicating infection-associated metabolic activation and oxidative stress. Pharmacological inhibition of DGAT1-dependent LD biogenesis, LIPA-dependent lysosomal lipid hydrolysis, LIPE/HSL-dependent lipolysis, or CPT1-dependent mitochondrial fatty acid transport markedly reduced mitochondrial fatty acid accumulation and suppressed HCV-induced respiratory activity. Inhibition of LIPA or LIPE/HSL reduced both HCV RNA and core protein levels, whereas inhibition of CPT1 or DGAT1 had more pronounced effects on core protein than on viral RNA. Together, these findings support a model in which HCV dynamically remodels LDs, mobilizes LD-associated fatty acids, and redirects them toward mitochondria to support infection-associated metabolism and downstream stages of the viral life cycle. Lipid hydrolysis and mitochondrial fatty acid trafficking therefore represent potential host-directed targets for limiting HCV infection. SIGNIFIGANCE:Hepatitis C virus depends on host lipid metabolism for replication, assembly, and production of infectious particles, but how it uses lipid droplets over time remains incompletely understood. This study shows that hepatitis C virus dynamically remodels lipid droplets, causing an early increase in lipid storage followed by droplet enlargement and mobilization of fatty acids during later infection. The released fatty acids preferentially accumulate in mitochondria, where they are associated with increased cellular respiration and oxidative stress. Blocking lipid droplet formation, lipid breakdown, or fatty acid transport to mitochondria reduced this metabolic response and decreased viral RNA or core protein accumulation. Inhibition of lysosomal acid lipase and hormone-sensitive lipase suppressed both viral RNA and protein levels. These findings identify lipid droplet breakdown and mitochondrial fatty acid trafficking as important host processes used by hepatitis C virus and as potential targets for host-directed antiviral intervention.

Background: Varicella-zoster virus (VZV) is a clinically important human neurotropic herpesvirus that causes varicella (chickenpox) as primary infection, typically in children. After primary infection, VZV establishes lifelong latency in sensory ganglia and can later reactivate and cause herpes zoster (shingles). Globally, varicella and shingles affect a substantial number of individuals each year. Although people with shingles can recover without specific antiviral treatment, the persistent, often severe pain associated with the disease can lead to depression and significantly decrease quality of life. Currently, acyclovir and its derivatives or similar compounds are the primary FDA-approved treatments for VZV infection. However, resistance to acyclovir, often because of mutations in the viral thymidine kinase, is increasingly observed in clinical settings. Therefore, alternative antiviral strategies against VZV are needed. Propolis (also known as "bee glue"), a resin-like substance produced by bees, has well-documented antibacterial and anti-inflammatory properties. Herein, we investigated the antiviral potential of propolis against VZV in in vitro systems including a cell culture model, human skin, and human dorsal root ganglia (DRG) tissue. Methods: ARPE-19 cells, human fetal skin, DRG tissues, and the VZV pOka-Luc-GFP strain (expressing luciferase and GFP for viral monitoring) were used. Propolis (Brazilian green propolis in DMSO) cytotoxicity was assessed with MTT assays. Viral replication was quantified with time-dependent luciferase assays. RNA sequencing (RNA-seq) was performed to analyze differential gene expression in propolis-treated (0.0625% and 0.125%) and control cells (with/ without VZV), and was followed by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses. Additionally, an acyclovir-resistant VZV mutant (VZV-delTK, lacking the thymidine kinase gene ORF36) was generated with BAC technology for a mechanistic comparison of propolis and acyclovir. Results: MTT assays indicated that propolis at <= 0.125% had low cytotoxicity to ARPE-19 cells. Luciferase assays demonstrated that propolis inhibited VZV replication in a concentration-dependent manner. This efficacy was consistent in skin and DRG tissues, in which 0.1% propolis approached acyclovir's antiviral activity. RNA-seq revealed that propolis altered host gene expression. Among the 207-1035 differentially expressed genes identified across concentrations, the enriched pathways included glycolysis/gluconeogenesis, calcium signaling, and ferroptosis. Propolis also inhibited VZV RNA transcription and splicing. Notably, propolis effectively inhibited the acyclovir-resistant strain VZV-delTK, whereas acyclovir did not, thus confirming the treatments' distinct mechanisms. Conclusions: Propolis exhibited significant antiviral activity against VZV in both cellular and tissue models, through a mechanism distinct from that of acyclovir. These results support further investigation of propolis as a potential anti-VZV agent with a novel mechanism of action.

The human angiotensin-converting enzyme 2 (hACE2) is the primary receptor for the entry of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Some human alleles of ACE2 exhibit an improved affinity for the SARS-CoV-2 Spike protein. However, the impact of ACE2 polymorphisms on SARS-CoV-2 infection remains unclear. Our previous study predicted that G431 and S514 in the receptor-binding domain (RBD) of SARS-CoV-2 S1 domain are important for S protein stability, and that S protein residues G496 and F497 and ACE2 residues D355 and Y41 are critical for the RBD-ACE2 interaction. In this study, we explored the potential of hACE2-derived neutralizing peptides as a therapeutic strategy against SARS-CoV-2 and investigated how ACE2 polymorphisms affect RBD-ACE2 binding affinity. We applied computational saturation mutagenesis to systematically screen the binding affinity changes among all possible ACE2 missense mutations within the ACE2-Wuhan-S1 complex. Mutations at ACE2 residues D355 and Y41 were predicted to weaken binding affinity, whereas those at N330 and D30 enhanced it. We identified six ACE2 regions (19-49, 65-102, 320-333, 348-359, 378-395, and 552-563) to be vital for ACE2-RBD interaction. We synthesized peptides corresponding to these six regions and tested them using a pseudotyped viral particle system and dot blot assay. Three peptides were confirmed to bind with the S protein, and four exhibited inhibitory effects. We aligned ACE2-Wuhan-S1 and ACE2-Omicron-S1 complexes, conducted correlation analysis, and observed similar binding patterns, suggesting that these peptides also have the potential to neutralize Omicron strains.IMPORTANCESARS-CoV-2 continues its global spread. In this research, we identified six regions within ACE2 that are vital for interaction with the viral S receptor-binding domain and have the potential to neutralize SARS-CoV-2 infection. Among the six peptides derived from ACE2, three were confirmed to bind with the S protein of the Wuhan strain, and four exhibited inhibitory effects on the Wuhan strain SARS-CoV-2. We also found ACE2 residues D355 and Y41 as weakening affinity, and N330 and D30 as enhancing it. We also aligned this complex with the ACE2-Omicron-S1 complex, performed correlation analyses, and compared their patterns of stability changes upon mutations and obtained similar results, indicating that these peptides may also be effective against Omicron variants. These results provide insight into the role of ACE2 polymorphism in viral entry and suggest that hACE2-derived peptides may offer a promising therapeutic strategy against SARS-CoV-2, demonstrating strong consistency between our computational predictions and experimental outcomes.

Endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) represent fundamental cellular adaptive mechanisms that maintain protein homeostasis and metabolic balance. Many RNA viruses, particularly flaviviruses such as dengue virus (DENV), Zika virus (ZIKV), West Nile virus (WNV), yellow fever virus (YFV), and Japanese encephalitis virus (JEV), extensively remodel the ER to establish replication compartments and assemble progeny virions. This massive reorganization disrupts ER homeostasis, leading to UPR activation. Emerging evidence reveals that flaviviruses not only trigger but also manipulate the three UPR branches—PERK, IRE1, and ATF6—to optimize viral translation, replication, and egress. In parallel, flavivirus infection profoundly alters host lipid metabolism and promotes dynamic changes in lipid droplets (LDs), key organelles that mediate lipid storage and serve as scaffolds for viral replication and assembly. The UPR intimately connects to LD biogenesis through transcriptional and translational programs mediated by XBP1, ATF4, and ATF6, thereby coupling ER stress responses to lipid remodeling and energy homeostasis. This intricate crosstalk between UPR and LDs creates a metabolic and structural niche favorable for viral replication but detrimental to host cell integrity. This review provides a comprehensive analysis of the molecular mechanisms by which flaviviruses exploit ER stress and the UPR to reprogram lipid metabolism and LD dynamics. We highlight the dual role of UPR signaling in promoting adaptive lipid synthesis and initiating cell death under prolonged stress, discuss recent insights into ER–LD interactions during flavivirus infection, and explore therapeutic opportunities targeting UPR–lipid metabolic pathways as broad-spectrum antiviral strategies. Understanding this interconnected network will advance our knowledge of viral pathogenesis and identify new avenues for host-directed antiviral intervention.

Pathogenic variants of MSH3 can increase mutational load within colorectal cells, which may drive initiation and progression of colorectal cancer (CRC). We identified several variants within MSH3 among CRCs from African Americans (AA). To predict and assess the functional significance of these MSH3 variants, we employed a combination of in silico analyses and in vitro functionality assays. Our objective was to elucidate the correlation between computational predictions and functional outcomes.MethodsA CRISPR-Cas9 knock-in approach was used to introduce and generate specific point mutations in Exons 21, 22, and 23 of MSH3 in the wild-type (WT) MSH3 CRC cell line SW620, which was confirmed with Sanger sequencing. We employed cell proliferation, microsatellite instability assays, and whole genome sequencing to assess biological and genetic consequences. We utilized immunofluorescence, Western blot, and coimmunoprecipitation methods to assess subcellular localization and differences in heterodimer MSH2 binding between WT and variant MSH3 proteins.ResultsWe previously identified six novel, potentially pathogenic nonsynonymous variants (c.G1237A, c.C2759T, c.G1397A, c.G2926A, c.C3028T, and c.G3241A) within six exons (Exons 8 (E413K), 9 (S466N), 20 (S920F), 21 (E976K), 22 (H1010Y), and 23 (E1081K), respectively) of MSH3 among AA CRCs as assessed by computational bioinformatic and molecular dynamic simulation analysis. We successfully knocked in three of the MSH3 variants (Exons 21, 22, and 23). Biological phenotypic assays revealed no observable changes in cell morphology or proliferation between WT and MSH3-variant knocked-in cells, and no differences were observed in microsatellite assays. Subcellular localization of variant MSH3 protein was unaffected compared with WT, whereas the interaction between MSH3 and MSH2 was not impacted. Short tandem repeats (STRs), or microsatellites, are short DNA motifs of two to six base pairs repeated consecutively, and they serve as powerful genetic markers for studying inheritance patterns and disease-associated repeat instability. In our analysis, STR profiling revealed both repeat expansions and contractions across multiple motifs, with tetranucleotide repeats showing the most pronounced alterations. Notably, loci flanking LINC00550 (ATTT), FBXL7 (AGAT), and DUSP28 (CTTT/GTTT) displayed consistent repeat expansions (+1.5 to +2), whereas intronic regions within GPC5, TMEM232, and ABCA13 exhibited contractions. Trinucleotide STRs revealed a mix of instability, with repeat gains at SLC25A12 and losses near GTF3C3 and AKAP12. Pentanucleotide and hexanucleotide motifs were more stable overall, but expansions were still noted at CNTN5, CPEB1, and CBSL, and contractions at SGO1 and FER. These findings highlight the bidirectional and motif-specific nature of STR instability driven by MSH3 deficiency. Our results support the utility of STR-based assays as sensitive tools to detect nonclassical MSI events and deepen our understanding of MSH3 ' s role in preserving microsatellite integrity across the genome.ConclusionThis study investigated the functional consequences of MSH3 variants identified among AA CRCs. While CRISPR-Cas9 knock-in of MSH3 variants (Exons 21, 22, and 23) in SW620 cells did not alter cell morphology, proliferation, protein localization, or MSH2 binding, we observed genetic changes that collectively underscore the bidirectional nature of STR instability in MSH3-deficient cells and reinforce this protein ' s pivotal role in suppressing slippage at longer repeat motifs. This study advances our understanding of how MSH3 deficiency contributes to genomic instability beyond canonically defined MSI loci, offering novel insights into the mutational landscapes of MMR-deficient tumors and how these MSH3 mutations can potentially contribute to the outcome of AA CRC patients.

ABSTRACTSARS‐CoV‐2 Envelope (E) protein is critical in viral assembly, release, and virulence. E gene was considered highly conserved and evolving slowly. Pan‐sarbecoviruses–conserved regions in the E gene have been used as targets for various RT‐PCR assays to detect SARS‐CoV‐2. It remains elusive whether SARS‐CoV‐2 variants of concern (VOCs) have accumulated significant E mutations that may affect protein stability and diagnostic RT‐PCR assays. Herein we aimed to perform a comprehensive genetic analysis on the conservation and diversity of the E gene of SARS‐CoV‐2 and its VOCs in comparison with other human coronaviruses (HCoVs). In silico analysis of 20 326 HCoV E gene sequences retrieved from GenBank and GISAID suggests that SARS‐CoV‐2 E gene has multiple pan‐HCoVs– and pan‐SARS‐CoV‐2–conserved positions but accumulates significant mutations in VOC B.1.351 and Omicron strains. Mutations were often found in the 5′ and 3′ variable regions, whereas the central region is conserved. Nucleotide changes C109U and A114G may lead to potential failure of first‐line SARS‐CoV‐2 diagnostic/screening assays. Nucleotide change C212U and its concomitant amino acid substitution Pro71Leu (i.e., C212U/Pro71Leu) is a hallmark mutation of B.1.351 variants, while C26U/Thr9Ile is characteristic of all Omicron variants. Later Omicron subvariants, such as XBB.1.5 and EG.5, additionally acquired the A31G/Thr11Ala mutation, as was confirmed by whole genome sequencing of SARS‐CoV‐2 in 118 pediatric cases. Wild‐type E protein exhibits cytotoxicity to cells, but the mutations Thr9Ile, Thr11Ala, Thr9Ile + Thr11Ala, or Pro71Leu reduces its cytotoxicity. The Thr9Ile + Thr11Ala mutation stabilizes the E proteins of Omicron variants, while Pro71Leu alters the cellular distribution of the E protein, reducing its colocalization with the Golgi body. Altogether, this study not only sheds light on the conservation and diversity of the E gene in SARS‐CoV‐2 and its VOCs but also informs the improvement and development of SARS‐CoV‐2 or pan‐HCoVs screening and diagnostic assays.

Zika virus (ZIKV) is primarily transmitted through mosquito bites and, occasionally, via breast milk, making postnatal ZIKV infections common among newborns and infants, particularly in tropical regions. Previous studies, including ours, have demonstrated that neonatal ZIKV infection can be fatal, highlighting a severe health issue of ZIKV in newborns. However, the pathogenesis and functional outcomes of postnatal ZIKV infection remain largely unexplored. The mechanisms underlying organ failure in infected neonates are still unknown. Here, we investigated postnatal ZIKV (PRVABC59) infection in neonatal mice and found significant cardiac abnormalities. Electrocardiogram (EKG) analysis revealed extended P-R intervals (indicative of the atrioventricular block), widened QRS complexes (suggesting intraventricular block), and elevated ST wave (a biomarker of myocardium impairment), implying defects in myocardial conduction. In addition, ZIKV infection caused increased levels of cTnT, cTnI, CK, CK-MB, CCL2, CXCL9, and CXCL10-biomarkers associated with cardiovascular diseases and infarction-like myocardial pathology. To further elucidate the underlying mechanisms, we analyzed cytokine and chemokine responses and observed a significant increase in multiple inflammatory mediators, including M-CSF, LIF, IL-6, IL-15, CCL2, CCL4, CCL5, CCL11, CXCL1, CXCL9, CXCL10, TNF-α, and VEGF. Notably, ZIKV infection also led to the degradation of connexin 43 (Cx43), a critical protein involved in heart development and intercellular communication among myocardial cells. In summary, our neonatal mouse model of ZIKV infection suggests that ZIKV-induced myocarditis and cardiac dysfunction may contribute to fatal outcomes in newborns. These findings provide new insights into ZIKV pathogenesis and underscore the need for further research into its impact on the cardiovascular system in early life.IMPORTANCEZika virus (ZIKV) is a known teratogen responsible for microcephaly in neonates born to mothers infected during pregnancy. Mouse models have been instrumental in elucidating ZIKV pathogenesis; however, most published studies utilize interferon (IFN)-compromised animals, either genetically modified or antibody-treated. In this study, we employed immunocompetent neonatal mice to investigate postnatal ZIKV infection and uncovered its impact on heart function. We detected high viral loads in heart tissue at early, middle, and late stages of infection using RT-qPCR. Electrocardiogram (EKG) analysis demonstrated cardiac dysfunction, including conduction abnormalities. At the same time, elevated levels of cTnT, cTnI, CK, CK-MB, LDH, α-HBDH, CCL2, and CXCL10-hallmarks of cardiovascular pathology-suggested inflammatory responses associated with heart failure. These findings indicate that neonatal mortality following postnatal ZIKV infection may be driven by virus-induced cardiac dysfunction. Our results provide new insights into ZIKV pathogenesis, suggesting that postnatal ZIKV infection poses a significant risk for severe cardiac disease in neonates.

The maintenance of DNA sequence integrity is critical to avoid accumulation of cancer-causing mutations. Inactivation of DNA Mismatch Repair (MMR) genes (e.g., MLH1 and MSH2) is common among many cancers, including colorectal cancer (CRC) and is the driver of classic microsatellite instability (MSI) in tumors. Somatic MSH3 alterations have been linked to a specific form of MSI called elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) that is associated with patient poor prognosis and elevated among African American (AA) rectal cancer patients. Genetic variants of MSH3 and their pathogenicity vary among different populations, such as among AA, which are not well-represented in publicly available databases. Targeted exome sequencing of MSH3 among AA CRC samples followed by computational bioinformatic pipeline and molecular dynamic simulation analysis approach confirmed six identified MSH3 variants (c.G1237A, c.C2759T, c.G1397A, c.G2926A, c.C3028T, c.G3241A) that corresponded to MSH3 amino-acid changes (p.E413K; p.S466N; p.S920F; p.E976K; p.H1010Y; p.E1081K). All identified MSH3 variants were non-synonymous, novel, pathogenic, and show loss or gain of hydrogen bonding, ionic bonding, hydrophobic bonding, and disulfide bonding and have a deleterious effect on the structure of MSH3 protein. Some variants were located within the ATPase site of MSH3, affecting ATP hydrolysis that is critical for MSH3′s function. Other variants were in the MSH3-MSH2 interacting domain, important for MSH3’s binding to MSH2. Overall, our data suggest that these variants among AA CRC patients affect the function of MSH3 making them pathogenic and likely contributing to the development or advancement of CRC among AA. Further clarifying functional studies will be necessary to fully understand the impact of these variants on MSH3 function and CRC development in AA patients.

The global impact of emerging viral infections emphasizes the urgent need for effective broad-spectrum antivirals. The cellular organelle, lipid droplet (LD), is utilized by many types of viruses for replication, but its reduction does not affect cell survival. Therefore, LD is a potential target for developing broad-spectrum antivirals. In this study, we found that 2-bromopalmitate (2 BP), a previously defined palmitoylation inhibitor, depletes LD across all studied cell lines and exerts remarkable antiviral effects on different coronaviruses. We comprehensively utilized 2 BP, alongside other palmitoylation inhibitors such as cerulenin and 2-fluoro palmitic acid (2-FPA), as well as the enhancer palmostatin B and evaluated their impact on LD and the replication of human coronaviruses (hCoV-229E, hCoV-Oc43) and murine hepatitis virus (MHV-A59) at non-cytotoxic concentrations. While cerulenin and 2-FPA exhibited moderate inhibition of viral replication, 2 BP exhibited a much stronger suppressive effect on MHV-A59 replication, although they share similar inhibitory effects on palmitoylation. As expected, palmostatin B significantly enhanced viral replication, it failed to rescue the inhibitory effects of 2 BP, whereas it effectively counteracted the effects of cerulenin and 2-FPA. This suggests that the mechanism that 2 BP used to inhibit viral replication is beyond palmitoylation inhibition. Further investigations unveil that 2 BP uniquely depletes LDs, a phenomenon not exhibited by 2-FPA and cerulenin. Importantly, the depletion of LDs was closely associated with the inhibition of viral replication because the addition of oleic acid to 2 BP significantly rescued LD depletion and its inhibitory effects on MHV-A59. Our findings indicate that the inhibitory effects of 2 BP on viral replication primarily stem from LD disruption rather than palmitoylation inhibition. Intriguingly, fatty acid (FA) assays demonstrated that 2 BP reduces the FA level in mitochondria while concurrently increasing FA levels in the cytoplasm. These results highlight the crucial role of LDs in viral replication and uncover a novel biological activity of 2 BP. These insights contribute to the development of broad-spectrum antiviral strategies. IMPORTANCE:In our study, we conducted a comparative investigation into the antiviral effects of palmitoylation inhibitors including 2-bromopalmitate (2-BP), 2-fluoro palmitic acid (2-FPA), and cerulenin. Surprisingly, we discovered that 2-BP has superior inhibitory effects on viral replication compared to 2-FPA and cerulenin. However, their inhibitory effects on palmitoylation were the same. Intrigued by this finding, we delved deeper into the underlying mechanism of 2-BP's potent antiviral activity, and we unveiled a novel biological activity of 2-BP: depletion of lipid droplets (LDs). Importantly, we also highlighted the crucial role of LDs in viral replication. Our insights shed new light on the antiviral mechanism of LD depletion paving the way for the development of broad-spectrum antiviral strategies by targeting LDs.

This study investigates the role of circular RNAs (circRNAs) in the context of Varicella-Zoster Virus (VZV) lytic infection. We employ two sequencing technologies, short-read sequencing and long-read sequencing, following RNase R treatment on VZV-infected neuroblastoma cells to identify and characterize both cellular and viral circRNAs. Our large scanning analysis identifies and subsequent experiments confirm 200 VZV circRNAs. Moreover, we discover numerous VZV latency-associated transcripts (VLTs)-like circRNAs (circVLTslytic), which contain multiple exons and different isoforms within the same back-splicing breakpoint. To understand the functional significance of these circVLTslytic, we utilize the Bacteria Artificial Chromosome system to disrupt the expression of viral circRNAs in genomic DNA location. We reveal that the sequence flanking circVLTs' 5' splice donor plays a pivotal role as a cis-acting element in the formation of circVLTslytic. The circVLTslytic is dispensable for VZV replication, but the mutation downstream of circVLTslytic exon 5 leads to increased acyclovir sensitivity in VZV infection models. This suggests that circVLTslytic may have a role in modulating the sensitivity to antiviral treatment. The findings shed new insight into the regulation of cellular and viral transcription during VZV lytic infection, emphasizing the intricate interplay between circRNAs and viral processes.

Human cytomegalovirus (HCMV) replication relies on a nucleocapsid coat of the 150kDa, subfamily-specific tegument phosphoprotein (pp150) to regulate cytoplasmic virion maturation. While recent structural studies revealed pp150-capsid interactions, the role of specific amino-acids involved in these interactions have not been established experimentally. In this study, pp150 and the small capsid protein (SCP), one of pp150’s binding partners found atop the major capsid protein (MCP), were subjected to mutational and structural analyses. Mutations to clusters of polar or hydrophobic residues along the pp150-SCP interface abolished viral replication, with no replication detected in mutant virus-infected cells. Notably, a single amino acid mutation (pp150 K255E) at the pp150-MCP interface significantly attenuated viral replication, unlike in pp150-deletion mutants where capsids degraded outside host nuclei. These functionally significant mutations targeting pp150-capsid interactions, particularly the pp150 K255E replication-attenuated mutant, can be explored to overcome the historical challenges of developing effective antivirals and vaccines against HCMV infection.

The primary challenge posed by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is COVID-19-related mortality, often exacerbated by additional medical complications, such as COVID-19-associated kidney injuries (CAKIs). Up to half of COVID-19 patients experience kidney complications, with those facing acute respiratory failure and kidney injury having the worst overall prognosis. Despite the significant impact of CAKI on COVID-19-related mortality and its enduring effects in long COVID, the underlying causes and molecular mechanisms of CAKI remain elusive. In this study, we identified a functional relationship between the expression of the SARS-CoV-2 ORF3a protein and inflammation-driven apoptotic death of renal tubular epithelial cells in patients with CAKI. We demonstrate in vitro that ORF3a independently induces renal cell-specific apoptotic cell death, as evidenced by the elevation of kidney injury molecule-1 (KIM-1) and the activation of NF-kB-mediated proinflammatory cytokine (TNFα and IL-6) production. By examining kidney tissues of SARS-CoV-2-infected K18-ACE2 transgenic mice, we observed a similar correlation between ORF3a-induced cytopathic changes and kidney injury. This correlation was further validated through reconstitution of the ORF3a effects via direct adenoviral injection into mouse kidneys. Through medicinal analysis, we identified a natural compound, glycyrrhizin (GL4419), which not only blocks viral replication in renal cells, but also mitigates ORF3a-induced renal cell death by inhibiting activation of a high mobility group box 1 (HMGB1) protein, leading to a reduction of KIM-1. Moreover, ORF3a interacts with HMGB1. Overproduction or downregulation of hmgb1 expression results in correlative changes in renal cellular KIM-1 response and respective cytokine production, implicating a crucial role of HMGB1 in ORF3a-inflicted kidney injuries. Our data suggest a direct functional link between ORF3a and kidney injury, highlighting ORF3a as a unique therapeutic target contributing to CAKI. IMPORTANCE:The major challenge of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection during the pandemic is COVID-19-related mortality, which has tragically claimed millions of lives. COVID-19-associated morbidity and mortality are often exacerbated by pre-existing medical conditions, such as chronic kidney diseases (CKDs), or the development of acute kidney injury (AKI) due to COVID-19, collectively known as COVID-19-associated kidney injuries (CAKIs). Patients who experience acute respiratory failure with CAKI have the poorest clinical outcomes, including increased mortality. Despite these alarming clinical findings, there is a critical gap in our understanding of the underlying causes of CAKI. Our study establishes a direct correlation between the expression of the SARS-CoV-2 viral ORF3a protein and kidney injury induced by ORF3a linking to CAKI. This functional relationship was initially observed in our clinical studies of COVID-19 patients with AKI and was further validated through animal and in vitro cellular studies, either by expressing ORF3a alone or in the context of viral infection. By elucidating this functional relationship and its underlying mechanistic pathways, our research deepens the understanding of COVID-19-associated kidney diseases and presents potential therapeutic avenues to address the healthcare challenges faced by individuals with underlying conditions.

Lipid droplets (LDs) are cellular organelles derived from the endoplasmic reticulum (ER), serving as lipid storage sites crucial for maintaining cellular lipid homeostasis. Recent attention has been drawn to their roles in viral replication and their interactions with viruses. However, the precise biological functions of LDs in viral replication and pathogenesis remain incompletely understood. To elucidate the interaction between LDs and viruses, it is imperative to comprehend the biogenesis of LDs and their dynamic interactions with other organelles. In this review, we explore the intricate pathways involved in LD biogenies within the cytoplasm, encompassing the uptake of fatty acid from nutrients facilitated by CD36-mediated membranous protein (FABP/FATP)-FA complexes, and FA synthesis via glycolysis in the cytoplasm and the TCL cycle in mitochondria. While LD biogenesis primarily occurs in the ER, matured LDs are intricately linked to multiple organelles. Viral infections can lead to diverse consequences in terms of LD status within cells post-infection, potentially involving the breakdown of LDs through the activation of lipophagy. However, the exact mechanisms underlying LD destruction or accumulation by viruses remain elusive. The significance of LDs in viral replication renders them effective targets for developing broad-spectrum antivirals. Moreover, considering that reducing neutral lipids in LDs is a strategy for anti-obesity treatment, LD depletion may not pose harm to cells. This presents LDs as promising antiviral targets for developing therapeutics that are minimally or non-toxic to the host.

Circular RNA (circRNA), a newly identified important component of the transcriptome, is formed by covalently bonded single-stranded RNA through back splicing (1) or other unknown mechanisms. It has been reported that circRNA plays important biological functions including microRNA (miRNA) sponges, parental gene expression regulators, and the translation template (2–5). Viral circRNAs have been recently identified from cells infected with different DNA viruses, such as Epstein-Barr virus (EBV) (6, 7), Kaposi’s sarcoma-associated herpesvirus (KSHV) (8), human papillomaviruses (HPVs) (9), and human cytomegalovirus (HCMV) (10). Coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARSCoV-2), has become a worldwide pandemic and poses a high threat to global health. We unprecedentedly identified viral circRNAs from cells that were infected by different coronaviruses, including SARS-CoV-2, SARS-CoV, and Middle East respiratory syndrome (MERS)-CoV (11). However, the biogenesis of circRNAs from coronavirus is still unknown. Murine hepatitis virus (MHV), a betacoronavirus, has been used in a mouse model to study human coronaviruses (12). Gribble et al. (13) reported that an RNA proofreading exoribonuclease, nsp14-ExoN, encoded by the MHV genome contributes to RNA recombination. In their study, deep transcriptome sequencing (RNA-seq) was performed on murine DBT cells infected either with wild-type MHV (MHV-WT) or with nsp14-ExoN inactive mutant MHV (MHV-ExoN2) (Table 1). We hypothesized that nsp14-ExoN may mediate the biogenesis of MHV circRNAs. To systematically test this hypothesis, we analyzed RNA from MHV-WTor MHV-ExoN2-infected cells and virioncontaining supernatants. Currently used methods of identifying circRNA are established on the examination of the back-splicing junction (BSJ) (14). ViReMa is one such tool that can quickly and sensitively identify viral RNA splicing junctions, including forward-splicing junctions (FSJs) and BSJs from next-generation sequencing data (15). Therefore, we applied ViReMa to assess the abundance of MHV-derived BSJs and FSJs and mapped the breakpoints to their respective genomic locations (Fig. 1A to D). In summary, there are two significant hot spots of FSJs and BSJs: (i) distant splicing between the 39 and the 59 ends of the genome corresponding to the N gene and the untranslated region (UTR) and (ii) local splicing in regions of MHV. The statistical analysis of genome coverage shows that about 23,043,062 to 56,476,922 nucleotides of each sample were mapped to the MHV genome (Table 1), suggesting that the numbers of MHV RNA molecules in the samples are comparable. To assess the effect of nsp14-ExoN loss of function, we calculated the junction Editor Haidong Gu, Wayne State University Copyright © 2023 Yang et al. This is an openaccess article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Qiyi Tang, qiyi.tang@howard.edu, or Hua Zhu, hua.zhu@rutgers.edu. The authors declare no conflict of interest.

Human cytomegalovirus (HCMV) is a widespread pathogen that poses significant risks to immunocompromised individuals. Its genome spans over 230 kbp and potentially encodes over 200 open-reading frames. The HCMV transcriptome consists of various types of RNAs, including messenger RNAs (mRNAs), long non-coding RNAs (lncRNAs), circular RNAs (circRNAs), and microRNAs (miRNAs), with emerging insights into their biological functions. HCMV mRNAs are involved in crucial viral processes, such as viral replication, transcription, and translation regulation, as well as immune modulation and other effects on host cells. Additionally, four lncRNAs (RNA1.2, RNA2.7, RNA4.9, and RNA5.0) have been identified in HCMV, which play important roles in lytic replication like bypassing acute antiviral responses, promoting cell movement and viral spread, and maintaining HCMV latency. CircRNAs have gained attention for their important and diverse biological functions, including association with different diseases, acting as microRNA sponges, regulating parental gene expression, and serving as translation templates. Remarkably, HCMV encodes miRNAs which play critical roles in silencing human genes and other functions. This review gives an overview of human cytomegalovirus and current research on the HCMV transcriptome during lytic and latent infection.

Objective Dengue viruses (DENV) and Zika viruses (ZIKV) are transmitted from human to human or from non-human primates to humans by mosquito biting, so the viral interaction with mosquito cells is one key step within the viral life cycle. Therefore, our objective is to know how DENV or ZIKV interacts with mosquito cells. Methods Immunofluorescence assay and a direct visualization system are combined to monitor the syncytial or congregative effects of DENVs and ZIKVs on C6/36 cells. we studied the cytopathic effects of DENVs and ZIKVs on the mosquito cells, C6/36 which are widely used in the laboratory for the infections of DENV and ZIKV. Results Our results show that all strains of DENV-1 and DENV-2, most DENV-4 and some DENV-3 strains caused syncytial effects on C6/36 cells, while some DENV-3 and DENV-4 strains, and all the tested ZIKV strains caused cell congregation after infection but no cell fusion. In addition, we detected a range of pH environments from 6.0 to 8.0 that support the virus-caused cell fusion and figured out that the optimal pH condition is 7.5 at which the viral production is also the best. Furthermore, viral replication may be required for DENV's syncytial effects on C6/36 cells because the UV-inactivated virus failed to cause cell fusion. Conclusion Syncytial and congregative effects of DENV and ZIKV on the Aedes albopictus cells differ among the viral strains. Syncytial effects of DENV on C6/36 are important for viral replication.

ABSTRACTSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ORF3a protein plays a vital role in viral pathogenesis and coronavirus disease 2019 (COVID-19). Like the spike protein, ORF3a mutates frequently, and certain variants are associated with the severity of COVID-19. Given the clinical significance and functional implications of ORF3a mutations, we conducted a comprehensive mutagenesis study targeting various known functional elements and revealed two distinctive types of ORF3a proteins based on their subcellular localizations: ORF3a proteins primarily localize on the lysosomal membrane (L-ORF3a) and those present in the endoplasmic reticulum (E-ORF3a). The objective of this study was to contrast the functional and mechanistic distinctions between these two types of ORF3a proteins. We examined six distinct ORF3a mutants and assessed their effects on cellular oxidative stress, nuclear factor kappa B-induced cytokine production, and cell death. Mechanistically, we explored ORF3a-induced ER stress, autophagy, and interactions with relevant cellular proteins. Our findings indicate that ORF3a proteins induce cytopathic effects through a similar mechanism, irrespective of their subcellular location. However, E-ORF3a proteins elicit more pronounced cytopathic effects despite their lower abundance and minimal impact on ER stress and autophagy when compared to L-ORF3a proteins. This discrepancy is attributed to ER-associated degradation since ORF3a proteins bind to a ubiquitin E3 ligase TRIM59. Inhibition of the 26S proteasome partially restores the protein levels of E-ORF3a and cellular ER stress response. This suggests that even a small quantity of ORF3a can lead to significant cytopathic effects due to the delicate nature of ER. Our study underscores the intricate interplay of dynamic cellular signaling within these two subcellular compartments in response to ORF3a.IMPORTANCEThe severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has tragically claimed millions of lives through coronavirus disease 2019 (COVID-19), and there remains a critical gap in our understanding of the precise molecular mechanisms responsible for the associated fatality. One key viral factor of interest is the SARS-CoV-2 ORF3a protein, which has been identified as a potent inducer of host cellular proinflammatory responses capable of triggering the catastrophic cytokine storm, a primary contributor to COVID-19-related deaths. Moreover, ORF3a, much like the spike protein, exhibits a propensity for frequent mutations, with certain variants linked to the severity of COVID-19. Our previous research unveiled two distinct types of ORF3a mutant proteins, categorized by their subcellular localizations, setting the stage for a comparative investigation into the functional and mechanistic disparities between these two types of ORF3a variants. Given the clinical significance and functional implications of the natural ORF3a mutations, the findings of this study promise to provide invaluable insights into the potential roles undertaken by these mutant ORF3a proteins in the pathogenesis of COVID-19.

ABSTRACT The emergence of viral infections with global impact highlights the urgent need for broad-spectrum antivirals. In this study, we evaluated the effect of palmitoylation inhibitors [2-bromopalmitate (2-BP), cerulenin, and 2-fluoro palmitic acid (2-FPA)] and the enhancer palmostatin B on the replication of human coronaviruses (hCoV-229E, hCoV-Oc43) and murine hepatitis virus (MHV-A59) at non-cytotoxic concentrations. The results demonstrated that 2-BP strongly suppressed MHV-A59 replication, while cerulenin and 2-FPA only moderately inhibited viral replication. Palmostatin B significantly enhanced viral replication. Notably, 2-BP exhibited superior efficacy. Interestingly, palmostatin B failed to rescue the inhibitory effects of 2-BP but effectively rescued cerulenin and 2-FPA, suggesting additional biological activities of 2-BP beyond palmitoylation inhibition. Furthermore, we discovered that 2-BP specifically disrupted lipid droplets (LDs), and this LD disruption was correlated with viral replication inhibition. Based on our findings, we conclude that the inhibitory effects of 2-BP on viral replication primarily stem from LD disruption rather than palmitoylation inhibition. Therefore, we revealed the crucial role of LDs in the viral replication. Our study provides insights into the development of wide-spectrum antiviral strategies.

Zika virus (ZIKV) is a teratogen causing devastating sequelae to the newborns who suffer a congenital ZIKV infection while it brings about only mild symptoms to the health-competent older children or adults. Mouse models have played an important role in mechanistic and pathogenic studies of ZIKV. ABSTRACT Zika virus (ZIKV) is transmitted mostly via mosquito bites and no vaccine is available, so it may reemerge. We and others previously demonstrated that neonatal infection of ZIKV results in heart failure and can be fatal. Animal models implicated ZIKV involvement in viral heart diseases. It is unknown whether and how ZIKV causes heart failure in adults. Herein, we studied the effects of ZIKV infection on the heart function of adult A129 mice. First, we found that ZIKV productively infects the rat-, mouse-, or human-originated heart cell lines and caused ubiquitination-mediated degradation of and distortive effects on connexin 43 (Cx43) protein that is important for communications between cardiomyocytes. Second, ZIKV infection caused 100% death of the A129 mice with decreasing body weight, worsening health score, shrugging fur, and paralysis. The viral replication was detected in multiple organs. In searching for the viral effects on heart of the A129 mice, we found that ZIKV infection resulted in the increase of cardiac muscle enzymes, implicating a viral acute myocardial injury. ZIKV-caused heart injury was also demonstrated by electrocardiogram (ECG) showing widened and fragmented QRS waves, prolonged PR interval, and slower heart rate. The intercalated disc (ICD) between two cardiomyocytes was destroyed, as shown by the electronic microscopy, and the Cx43 distribution in the ICDs was less organized in the ZIKV-infected mice compared to that in the phosphate-buffered saline (PBS)-treated mice. Consistently, ZIKV productively infected the heart of A129 mice and decreased Cx43 protein. Therefore, we demonstrated that ZIKV infection caused heart failure, which might lead to fatal sequelae in ZIKV-infected A129 mice. IMPORTANCE Zika virus (ZIKV) is a teratogen causing devastating sequelae to the newborns who suffer a congenital ZIKV infection while it brings about only mild symptoms to the health-competent older children or adults. Mouse models have played an important role in mechanistic and pathogenic studies of ZIKV. In this study, we employed 3 to 4 week-old A129 mice for ZIKV infection. RT-qPCR assays discovered that ZIKV replicated in multiple organs, including the heart. As a result of ZIKV infection, the A129 mice experienced weight loss, health score worsening, paralysis, and deaths. We revealed that the ZIKV infection caused abnormal electrocardiogram presentations, increased cardiac muscle enzymes, downregulated Cx43, and destroyed the gap junction and the intercalated disc between the cardiomyocytes, implicating that ZIKV may cause an acute myocardial injury in A129 mice. Therefore, our data imply that ZIKV infection may jeopardize the immunocompromised population with a severe clinical consequence, such as heart defect.