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At least 19 recordsLinked to original sources

PubMed2024

Large language models improve annotation of prokaryotic viral proteins.

Viral genomes are poorly annotated in metagenomic samples, representing an obstacle to understanding viral diversity and function. Current annotation approaches rely on alignment-based sequence homology methods, which are limited by the paucity of characterized viral proteins and divergence among viral sequences. Here we show that protein language models can capture prokaryotic viral protein function, enabling new portions of viral sequence space to be assigned biologically meaningful labels. When applied to global ocean virome data, our classifier expanded the annotated fraction of viral protein families by 29%. Among previously unannotated sequences, we highlight the identification of an integrase defining a mobile element in marine picocyanobacteria and a capsid protein that anchors globally widespread viral elements. Furthermore, improved high-level functional annotation provides a means to characterize similarities in genomic organization among diverse viral sequences. Protein language models thus enhance remote homology detection of viral proteins, serving as a useful complement to existing approaches.

Viral Proteins↗

PubMed2026

Metaproteomic profiling reveals viral proteins and associated host proteomic alterations in glioblastoma.

Glioblastoma (GB) is a WHO grade 4 brain cancer with dismal prognosis, yet its aetiology remains poorly defined. Although viral involvement has been proposed, findings across studies remain inconsistent, reflecting inherent limitations of individual technologies and cohort size. Here we applied metaproteomic profiling to a publicly available GB proteome dataset (12 control, 21 adjacent, 159 tumour) and an independent cohort of 81 samples (37 control, 44 tumour) to detect viral proteins in tumour and controls tissues. Across cohorts, we detected viral proteins from diverse species, with human herpesviruses (HHV-1, 2, and 8) more frequently detected in GB tumours compared with control tissues. Analysis of the host tumour proteome revealed differential abundance of proteins related to transcriptional regulation, RNA processing, protein translation, immune responses, and mitochondrial-associated metabolism. Correlation analysis identified associations between viral and human proteins, with several linked to biological processes previously implicated in DNA virus-host interactions. Further stratification of tumour by HHV-1 status showed consistent alterations in proteins associated with mitochondrial-associated metabolism, protein turnover, and cell adhesion/signalling.In summary, this study demonstrates the feasibility of metaproteomics for detecting viral components in archival GB tissues. Using this approach, we observed differences in viral protein landscape across cohorts and identified associations between viral presence and host proteomic features, providing a protein-level framework for future studies of virus-host interactions in GB.

Humans↗

PubMed2027

Epitope Tagging and Coimmunoprecipitation to Identify Viral Protein Interactors.

Affinity purification-mass spectrometry (AP-MS) is a powerful proteomic approach for dissecting the interaction network between virus and host. Traditional AP-MS employs overexpression of viral proteins as baits to enrich host interactors. However, overexpressed viral proteins may mislocalize to inappropriate cellular compartments and trigger endoplasmic reticulum stress by overwhelming the protein-folding machinery, which leads to false identification of host factors. To overcome these limitations, we introduce an AP-MS strategy based on direct infection with an epitope-tagged chikungunya virus (CHIKV/myc-E2), which we used to successfully uncover two new antiviral factors in CHIKV cellular reservoirs-macrophages. In this protocol, we will describe this technique step by step: (1) design and construction of myc-tagged virus by advanced multi-fragment assembly, (2) in vitro transcription and preparation of infectious myc-tagged virus stocks, and (3) immunoprecipitation of myc-tagged viral protein and its interactome for mass spectrometry analysis. This strategy enables accurate identification of viral interactors in a physiologically relevant context, providing a framework for future proteomic studies using tagged viruses.

Chikungunya virus↗

PubMed2025

The glycoprotein quality control factor Malectin promotes coronavirus replication and viral protein biogenesis.

Coronaviruses (CoV) rewire host protein homeostasis (proteostasis) networks through interactions between viral nonstructural proteins (nsps) and host factors to promote infection. With the emergence of SARS-CoV-2, it is imperative to characterize host interactors shared across nsp homologs. Using quantitative proteomics and functional genetic screening, we identify conserved proteostasis interactors of nsp2 and nsp4 that serve pro-viral roles during infection of murine hepatitis virus - a model betacoronavirus. We uncover a glycoprotein quality control factor, Malectin (MLEC), which significantly reduces infectious titers when knocked down. During infection, nsp2 interacts with MLEC-associated proteins and the MLEC-interactome is drastically altered but retains association with the Oligosaccheryltransferase (OST) complex, a crucial component of viral glycoprotein production. MLEC promotes viral protein levels and genome replication through its quality control activity. Lastly, we show MLEC promotes SARS-CoV-2 replication. Our results reveal a role for MLEC in mediating CoV infection and identify a potential target for pan-CoV antivirals.

Biochemistry and Chemical Biology↗

PubMed2026

Uncovering viral protein acquisition events and human-specific folds with pairwise comparisons of predicted protein structures.

Pairwise sequence comparisons are at the center of molecular evolutionary analyses. However, viral pairwise comparisons are challenging because extreme mutation rates and evolutionary pressure cause genomes to diverge rapidly, limiting detectable sequence similarity to fewer than 3% of virus pairs. To overcome these limitations, we compared viruses based on structural similarity, using predicted protein structures from ColabFold and Foldseek to define protein fold clusters. We represented each virus genome by its protein structural content. Pairwise similarities between viruses were then quantified using the Jaccard index based on the presence or absence of protein fold clusters. Using a recently established viral protein fold database, we compared all pairs of eukaryotic viruses in RefSeq. This approach increased the proportion of comparable viral genome pairs from 2.4% to 16.5%. Using this protein-fold representation of viruses, we were able to accurately predict viral families with an average sensitivity of 85.9%. Investigation of viral families showing limited sensitivity with this approach uncovered a laterally transferred structural cluster (Rep/NS1) broadly shared across diverse viral families and found in the avian lineage of adenoviruses. Sequence homology suggests that this Rep was acquired from Parvoviridae, but the protein is mutant in the ATPase active site, indicating possible exaptation toward a purely DNA-binding function. In Gammapapillomaviruses, several E4 clusters were associated with human tropism. In summary, by representing viruses with structural protein clusters, we can classify highly divergent viruses, trace lateral gene transfer, and uncover features associated with viral host range.

Humans↗

PubMed2026

Performance comparison of rapid and native barcoding methods for Oxford Nanopore sequencing of Poliovirus Viral Protein 1 (VP1) amplicons.

Accurate and timely sequencing of poliovirus is critical for global eradication efforts, particularly for molecular epidemiology based on the typing region of the genome, viral protein 1 (VP1). While Oxford Nanopore Technologies (ONT) sequencing has expanded capabilities for poliovirus surveillance, the relative performance of different ONT library preparation methods, including ligation-based (Native Barcoding) and transposase-based (Rapid Barcoding) approaches, has not been systematically evaluated. In this study, we compared rapid barcoding and native barcoding workflows for sequencing VP1 amplicons from 17 type 2 poliovirus-positive samples, each processed in triplicate. Native barcoding generated significantly more sequencing output, producing approximately 2.3-fold greater total read yield than rapid barcoding, and demonstrated higher run-to-run reproducibility (R2 = 0.979-0.998 vs. 0.847-0.929, respectively; p < 0.001). In addition, native barcoding generated 80% of the total yield achieved by rapid barcoding within approximately 7 h, whereas rapid barcoding required approximately 40 h to reach the same output. Despite these differences, both methods produced identical VP1 consensus sequences across all samples, with comparable read quality (median per-base Q-scores of approximately Q17-Q18). Rapid barcoding provided substantial practical advantages, reducing hands-on library preparation time (55 vs. 200 min) and per-sample cost ($12.82 vs. $16.54), while simplifying workflow and reducing technical complexity. These findings indicate that sequencing yield may not be a determinant of downstream analytical outcomes for poliovirus VP1 ONT sequencing. Rapid barcoding therefore represents a cost-effective and efficient approach for routine poliovirus surveillance, whereas native barcoding remains advantageous in applications requiring rapid data generation or maximal sequencing depth.

Poliovirus↗

PubMed2026

A small viral protein suppresses immune amplification by two distinct mechanisms.

Diverse viral suppressors of RNA interference (RNAi) and RNA silencing (VSRs) interact directly with core protein and/or RNA components of the host RNAi pathway. However, the specific counter-defense function of any VSR biochemical activity is fully validated only when it is shown as essential for viral infection in the wild-type but not mutant hosts defective in antiviral RNAi. Here, we investigated the role of VSR activities for direct binding to small-interfering RNA duplexes (siRNA), long double-stranded RNA (dsRNA), or RNA-dependent RNA polymerase 1 (RDR1) during plant infection by wild-type and mutant cucumber mosaic virus (CMV), a positive-strand RNA virus expressing the 110-residue 2b protein as its VSR. We demonstrate that a C-terminally truncated 2b mutant (2b1-93) active in direct binding to siRNA and dsRNA, but not RDR1, was able to suppress the amplification of virus-derived siRNAs (vsiRNA) and antiviral RNAi mediated by RDR6, but not RDR1. By contrast, an N-terminally truncated 2b mutant (2b18-110) inactive in direct binding to siRNA or dsRNA was able to suppress vsiRNA amplification and antiviral RNAi mediated by RDR1, but not RDR6, and was less effective to promote systemic CMV infection and disease development than 2b1-93. Together, our results show that whereas RDR1 suppression requires direct binding of VSR-2b to RDR1, but not siRNA or dsRNA, RDR6 suppression depends on direct binding to siRNA and dsRNA, but not RDR1. Therefore, CMV, through its VSR-2b, suppresses two parallel vsiRNA amplification pathways by distinct molecular mechanisms, and this unique property may account for the unusually wide host range of CMV.IMPORTANCEHost amplification of antiviral immunity is essential for robust control of viral infections. However, little is known about the mechanisms that viruses have evolved to suppress immune amplification in plants. Here, we characterized whole plant infection by cucumber mosaic virus (CMV) with its viral suppressor of RNA interference (RNAi) mutated to become inactive in direct binding to small-interfering RNA duplexes (siRNA), long double-stranded RNA (dsRNA), or RNA-dependent RNA polymerase 1 (RDR1). We demonstrate maximal suppression of both RDR1- and RDR6-mediated antiviral RNAi amplification by the CMV 2b protein, a viral suppressor of RNAi (VSR). Notably, whereas RDR1 suppression requires direct binding of 2b to RDR1 but not siRNA or dsRNA, RDR6 suppression depends on direct binding to siRNA and dsRNA, but not RDR1. Our findings reveal a novel counter-defense strategy evolved by a wide host range positive-strand RNA virus to suppress two pathways of immune amplification by distinct mechanisms.

Cucumovirus↗

PubMed2025

The proxiome of a plant viral protein with dual targeting to mitochondria and chloroplasts revealed MAPK cascade and splicing components as proviral factors.

The coat protein (CP) of the melon necrotic spot virus (MNSV) is a multifunctional factor localized in the chloroplast, mitochondria, and cytoplasm, playing a critical role in overcoming plant defenses such as RNA silencing (RNAi) and the necrotic hypersensitive response. However, the molecular mechanisms through which CP interferes with plant defenses remain unclear. Identifying viral-host interactors can reveal how viruses exploit fundamental cellular processes and help elucidate viral survival strategies. Here, we employed a TurboID-based proximity labeling approach to identify interactors of both the wild-type MNSV CP and a cytoplasmic CP mutant lacking the dual transit peptide (ΔNtCP). Of the interactors, eight were selected for silencing. Notably, silencing MAP4K SIK1 and NbMAP3Kε1 kinases, and a splicing factor homolog NbSMU2 significantly reduced MNSV accumulation, suggesting a proviral role for these proteins in plants. Yeast two-hybrid and bimolecular fluorescence complementation assays confirmed the CP and ΔNtCP interaction with NbSMU2 and NbMAP3Kε1 but not with NbSIK1, which interacted with NbMAP3Kε1. These findings open up new possibilities for exploring how MNSV CP might modulate gene expression and MAPK, thereby facilitating MNSV infection.

Chloroplasts↗

PubMed2025

COG6 is an essential host factor for influenza A virus infection.

Influenza A virus (IAV) relies on the host cellular machinery to support its replication. Understanding these host dependencies can inform the development of novel antiviral strategies. In this study, we identified conserved oligomeric Golgi complex subunit 6 (COG6) as a novel host factor critical for IAV replication through a genome-wide clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) knockout screen. Disruption of COG6 significantly impaired viral replication. Mechanistically, COG6 supports IAV replication via two distinct means. First, consistent with the role of the COG complex in Golgi homeostasis, COG6 is required for the proper presentation of surface sialic acids, the primary receptor for IAV entry. Second, COG6 deficiency unexpectedly led to lysosome-dependent degradation of viral proteins. Notably, lysosomal activity was also upregulated in IAV-infected wild-type cells, albeit to a lesser extent than in COG6-deficient cells. Treatment with lysosomal inhibitors rescued viral protein stability in COG6 knockout cells. Protein interaction analysis further demonstrated that COG6-mediated stabilization of viral proteins did not rely on viral protein-COG6 interaction, refuting the hypothesis that COG6 acts as a shield factor to protect viral protein from lysosomal degradation. Moreover, knockout of other COG subunits produced similar antiviral effects, suggesting that an intact COG complex is required for IAV replication. Together, these findings uncover a critical role of the COG complex in regulating IAV replication and highlight a previously unappreciated functional link between the Golgi and lysosomes that could be exploited for treating IAV infections.IMPORTANCEDespite advances in virology, numerous host determinants facilitating influenza A virus (IAV) pathogenesis remain uncharacterized. Our study establishes conserved oligomeric Golgi complex subunit 6 (COG6) as a critical host factor promoting IAV infection through complementary mechanisms: receptor modulation and viral protein stabilization. This represents the first demonstration that the COG complex regulates viral pathogenesis through proteostasis mechanisms, fundamentally expanding our understanding of host-virus interactions at the organelle interface. These findings not only provide new perspectives on viral exploitation of Golgi trafficking networks but also identify potential therapeutic targets against evolving influenza strains.

Influenza A virus↗

PubMed2025

Development and Characterization of an Inducible Bacterial Artificial Chromosome System for Studying Lytic Replication and Pathogenesis of Kaposi's Sarcoma-Associated Herpesvirus.

Bacterial artificial chromosome (BAC) is widely used to manipulate herpesvirus genome and generate recombinant virus. Here, we developed a new KSHV BACmid, namely as iBAC, by replacing the EGFP with TET3G transactivator under EF1α promoter and inserted Tet response elements in the promoter of RTA in the original KSHV BAC16 clone and characterized KSHV lytic replication in SLK-iBAC cells. SLK-iBAC cells developed more efficient lytic replication and generated more progeny virus than iSLK-BAC16 cells upon the same conditions of doxycycline treatment. Since SLK-iBAC cells only occupied hygromycin selection marker, it is convenient to generate cellular gene knockout via lentivirus-mediated CRISPR-Cas9 or stably express viral or cellular gene via lentivirus followed by antibiotic selection, making iBAC system a better tool to identify cellular targets of viral proteins in the context of virus infection or study the role of viral or cellular genes for KSHV lytic replication and pathogenesis. In addition, iBAC is color-free and can be utilized to track subcellular localization of viral proteins or colocalization between different viral proteins by introducing fusing fluorescent proteins into the BAC backbone. Therefore, the new KSHV iBAC is a powerful inducible tool to study KSHV lytic replication and pathogenesis in cell model.

Chromosomes, Artificial, Bacterial↗

PubMed2026

PRMT3 restricts porcine epidemic diarrhea virus replication by disrupting the interaction between VAPA and the viral nucleocapsid protein.

Porcine epidemic diarrhea virus (PEDV) represents a severe threat to the global swine industry. Its infection process involves intricate virus-host interactions and immune evasion mechanisms, but effective therapeutic targets remain elusive. In this study, we identified protein arginine methyltransferase 3 (PRMT3) as a novel regulatory factor that significantly modulates PEDV infection via genome-wide CRISPR/Cas9 knockout library screening. Knockout or inhibition of PRMT3 markedly enhanced PEDV infection in multiple cell lines, including LLC-PK1, IPEC-J2, and primary porcine intestinal epithelial cells. Mechanistic investigations revealed that PRMT3 can restrict PEDV infection by interacting with vesicle-associated membrane protein-associated protein A (VAPA). Further analysis revealed that VAPA facilitates cholesterol transport through binding to oxysterol-binding protein (OSBP) and inhibits the autophagic degradation of the viral nucleocapsid (N) protein, with both processes being critical for promoting PEDV infection in host cells. A detailed analysis revealed that K52 within its major sperm protein (MSP) domain interacts with D404 and D405 in the two phenylalanines in an acidic tract (FFAT)-like motifs of the N protein, and these interactions proved essential for PEDV infection. In summary, this is the first study to identify and validate the PRMT3-VAPA-N protein autophagic degradation axis as a key pathway through which PRMT3 suppresses PEDV infection, with VAPA acting as an essential host factor for PEDV pathogenesis. These findings uncover novel signaling pathways and molecular targets for the development of anti-PEDV therapeutics.

Animals↗

PubMed2004

A bunyamwera virus minireplicon system in mosquito cells.

Artificial minigenomes are powerful tools for studying the replication and transcription of negative-strand RNA viruses. Bunyamwera virus (BUN; genus Orthobunyavirus, family Bunyaviridae) is an arbovirus that shows fundamental biological differences when replicating in mammalian versus mosquito cells. To study BUN RNA synthesis in mosquito cells, we developed a bacteriophage T7 RNA polymerase-based minireplicon system similar to that described previously for mammalian cells. An Aedes albopictus C6/36-derived mosquito cell line stably expressing T7 RNA polymerase was established. Viral proteins and artificial minigenomes (containing Renilla luciferase as a reporter) were transcribed and expressed in these cells from transfected T7 promoter-containing plasmids. Transcription of the minigenome required two viral proteins, the nucleocapsid protein N and the RNA-dependent RNA polymerase L, a situation similar to that in mammalian cells. However, unlike the situation in mammalian cells, the viral polymerase was not inhibited by the viral nonstructural protein NSs. We also report that promoter strength is different for vertebrate versus invertebrate cells. The development of this system opens the way for a detailed comparison of bunyavirus replication in cells of disparate phylogeny.

Aedes↗

PubMed2027

Production of Viral Particles from a Chikungunya Virus Infectious Clone.

Chikungunya virus (CHIKV) is a positive-sense single-stranded RNA virus, which poses challenges for its study and genetic manipulation. Because direct mutagenesis of viral RNA genomes is technically impractical, reverse genetics systems are essential tools for investigating viral biology. To enable such approaches, infectious clones containing a full-length cDNA copy of the viral genome are constructed. The cDNA is positioned under the control of a bacteriophage RNA polymerase promoter, allowing commercial RNA polymerases to use the linearized plasmid as a template for the in vitro transcription of full-length viral genomic RNA (gRNA). Importantly, positive-sense viral genomes serve as mRNAs for the translation of viral proteins in a cellular environment, meaning that these transcripts contain all the information required to initiate viral replication. Following transfection into permissive cultured cells, viral proteins are expressed, enabling genome replication and, ultimately, the recovery of infectious particles from the cell supernatant. Here, we describe a detailed procedure for generating CHIKV particles through plasmid linearization, in vitro transcription, and subsequent RNA transfection.

Chikungunya virus↗

PubMed2026

SARS-CoV-2 Orf3a protein interaction mapping using unnatural amino acid incorporation.

Mapping transient protein-protein interactions remain a major challenge in studying viral host-pathogen interfaces. While some virus-host interactions are stable and readily captured, the majority are highly dynamic, reflecting the need for viral proteins to engage distinct host factors at different stages of the life cycle. Here, we employ a protein engineering strategy based on the site-specific incorporation of the unnatural acid p-azido-L-phenylalanine (AzF) to enable photo-crosslinking proteomic analysis of the SARS-CoV-2 accessory protein Orf3a in live cells. Genetic installation of AzF at residue K198 of Orf3a permitted UV-induced covalent capture of proximal host interacting proteins, overcoming challenges associated with membrane localization and limited protein abundance. A total of 248 high-confidence Orf3a-interacting proteins were reproducibly identified and subjected to gene ontology analysis, revealing enrichment in innate immune signaling, antiviral defense, RNA processing, and viral replication-associated pathways. Orf3a is an accessory protein that functions as a viroporin and traffics across multiple cellular compartments, and was found to interact with host RNA helicases, RNA-binding proteins, immune regulators, and metabolic enzymes implicated in SARS-CoV-2 infection. Together, these results demonstrate that genetically encoded, site-specific photo-crosslinking enables selective capture of transient interactions that are often missed by nonspecific 254 nm UV crosslinking approaches and highlights Orf3a as a multifunctional protein that engages diverse host pathways. More broadly, this study establishes a generalizable framework for leveraging unnatural amino acid-based protein engineering approaches to interrogate dynamic host-pathogen interactions.

Humans↗

PubMed2026

Elevation-structured viral ecological strategies along glacier-fed rivers on the Qinghai-Tibet Plateau.

The Qinghai-Tibet Plateau, a climate-vulnerable source of Asia's major rivers, harbors underexplored viral communities critical to ecosystem functions. By integrating 597 metagenomes from the Yangtze, Yellow, Lancang, and Yarlung Tsangpo rivers with 85 public available glacial metagenomes (Tibetan Glacier Genome and Gene catalog), we built the Glacier-to-River Virome Catalogue, encompassing 36,358 vOTUs and 897,250 viral protein clusters, to decode viral adaptation and ecological influence across elevation gradients. Our results reveal that high-altitude conditions favor viruses with elevated Guanine-Cytosine content, larger genomes and more cold-adaptation genes. A central finding is a systematic viral lifestyle shift from temperate in glaciated regions to lytic viruses downstream, accompanied with decline of pathogens carrying antibiotic resistance genes along the glacier-to-river gradients. Further, viral auxiliary metabolic genes transition from glacier nutrient scavenging (e.g., nitrogen and sulfur transporters) to downstream mineralization processes (e.g., denitrification) in plains highlights their role in biogeochemical cycling. These findings position viruses as pivotal regulators of microbial community structural and functional dynamics to glacier-to-river gradient change and biogeochemistry in the Qinghai-Tibet Plateau, providing critical insights into climate response in vulnerable Asian water towers.

Ice Cover↗

PubMed2026

The human cytomegalovirus vGPCR UL33 is essential for efficient lytic replication in epithelial cells.

UNLABELLED: Human cytomegalovirus (HCMV) is a β-herpesvirus that is ubiquitous in the human population. HCMV has the largest genome of the human herpesviruses and encodes an array of genes that affect pathogenesis in different cell types. Given the ability of HCMV to replicate in a range of cell types, investigators have begun to identify viral proteins required for cell type-specific replication. There are four proteins encoded by HCMV that are homologous to G protein-coupled receptors (GPCRs); these viral GPCRs (vGPCRs) are UL33, UL78, US27, and US28. In this study, we find that deletion of all four vGPCR genes severely attenuates HCMV replication in primary human salivary gland epithelial cells and ARPE-19 retinal epithelial cells, as evidenced by decreases in viral gene expression and virus production. Deletion of UL33 from the HCMV genome also results in a failure to efficiently replicate in epithelial cells, and this defect is manifested by decreased levels of viral gene expression and virus production. We find that, similar to US28, UL33 constitutively activates Gαq signaling to high levels in epithelial cells. We also find that UL33 transcription is more complicated than originally believed, and there is the potential for the virus to utilize various 5' UTRs to create novel UL33 proteins that are all capable of constitutive Gαq signaling. Taken together, these studies provide novel molecular and biochemical data regarding UL33 expression, subcellular localization, and signaling, and indicate that UL33 activity is essential for efficient HCMV replication in cells of epithelial origin. IMPORTANCE: Human cytomegalovirus (HCMV) replicates in a number of cell types and tissues in vivo, and the viral genes involved in cell type-specific replication are just beginning to be elucidated. The HCMV-encoded viral G protein-coupled receptors (vGPCRs) UL33, UL78, US27, and US28 are proving to play important roles in multiple aspects of HCMV replication, including the establishment and maintenance of latency. Here, we demonstrate that the HCMV vGPCRs and UL33, in particular, play an important role in driving lytic replication in cells of epithelial origin, including those derived from the salivary gland. This work expands on potential functions of the vGPCRs, will drive future studies to understand mechanistically how they affect tropism, and provides a new target for future therapeutics.

Gαq↗

PubMed2026

SARS-CoV-2 3CLpro inhibits the replication of influenza viruses through the cleavage of NP and PA.

The co-circulation of multiple viruses can lead to distinct pathological outcomes, yet how severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection influences other viral infections remains poorly understood, despite its documented high frequency during the pandemic. In this study, we investigated how the proteolytic activity of SARS-CoV-2 3C-like protease (3CLpro) influences the replication of influenza A virus. In silico analysis identified candidate 3CLpro cleavage sites across numerous viral proteins, and biochemical assays confirmed that 3CLpro catalyzes the degradation of influenza virus nucleoprotein (NP) and polymerase acidic protein (PA) in a manner requiring its protease activity. This degradation of NP and PA, which are essential for viral genome packaging and transcription, disrupted the influenza replicative cycle and suppressed viral replication, both upon ectopic 3CLpro expression and during SARS-CoV-2 infection. Our data uncover a direct, enzyme-based mechanism by which SARS-CoV-2 can suppress influenza virus replication during coinfection. We provide a molecular explanation for the sharp, global decline in influenza activity observed during the COVID-19 pandemic and illustrate how enzymatic weapons of one virus can be repurposed to restrain a competing pathogen.

Virus Replication↗

PubMed2026

Rab10 coordinates SADS-CoV non-lytic egress through the ERGIC-TGN-lysosome trafficking pathway.

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is a bat-originated alphacoronavirus that causes devastating enteric disease in neonatal piglets and possesses significant potential for cross-species transmission. While the early stages of the coronavirus life cycle have been extensively characterized, the host factors indispensable for virion assembly and subsequent export remain largely enigmatic. Here, by performing a genome-wide CRISPR-Cas9 knockout screen using a recombinant icSADS-CoV-GFP reporter virus, we identified the small GTPase Rab10 as a critical host dependency factor for SADS-CoV infection. Viral life cycle analysis revealed that Rab10 is not required for viral attachment, entry, or initial genome replication, but is essential for the virion transport and non-lytic egress. Rab10 deficiency markedly reduced the extracellular release of viral RNA, viral proteins, and infectious progeny, as well as the secretion of SADS-CoV virus-like particles. Confocal imaging showed that Rab10 and viral protein-positive intracellular structures were associated with LMAN1, TGN46, and LAMP1 positive compartments. These findings support a model in which Rab10 coordinates a virus-containing vesicles trafficking pathway associated with ERGIC-TGN-lysosome compartments. Mechanistically, Rab10 facilitates the loading of the viral envelope (E) protein into transport vesicles derived from the ERGIC. Rab10 associates with the SADS-CoV E protein, and mapping analyses implicated the C-terminal PDZ-binding motif, particularly residue V75, in efficient Rab10 association and viral release. Collectively, our findings identify Rab10 as a host regulator of SADS-CoV non-lytic egress and highlight the E-Rab10 interaction and the vesicular trafficking machinery as a potential target for developing antiviral strategies.

Animals↗