Journal of Virology最新文献

The global challenge of multidrug-resistant Pseudomonas aeruginosa demands innovative anti-virulence approaches. We characterize PemR, a novel transcriptional regulator encoded by P. aeruginosa phage PAYQ66, which orchestrates multimodal virulence attenuation in P. aeruginosa. Biochemical analyses demonstrated that PemR directly binds to the mvfR promoter, resulting in significant repression of this key quorum-sensing regulator. This repression, in turn, induces profound metabolic reprogramming by redirecting metabolic flux away from PQS biosynthesis toward catechol accumulation. PemR globally attenuates virulence phenotypes, including pyocyanin production, rhamnolipid synthesis, motility, and biofilm formation. Transcriptomic profiling further reveals that PemR upregulates rsmA to suppress the type VI secretion system, thereby potentially modulating the host's interaction with competing bacteria in polymicrobial environments. The motility analysis shows that PemR suppresses bacterial swimming, swarming, and twitching at gene expression levels. Overall, infection models demonstrate that PemR significantly inhibits bacterial virulence. As the first reported phage-encoded transcriptional hijacker of the mvfR regulon, PemR concurrently disrupts quorum sensing and bacterial pathogenicity, offering a strategic blueprint for novel anti-virulence therapeutics targeting multidrug-resistant pathogens.IMPORTANCEThis study identifies a novel bacteriophage-encoded regulator PemR that simultaneously disrupts multiple virulence pathways in the opportunistic pathogen P. aeruginosa. By hijacking a key bacterial quorum-sensing (QS) system and reprogramming host metabolism, PemR significantly reduces pathogenicity without killing the bacteria. This work reveals a sophisticated strategy phages use to manipulate their hosts and provides a promising blueprint for developing next-generation anti-virulence therapeutics. Such approaches aim to disarm dangerous bacteria rather than eliminate them, potentially slowing the emergence of antibiotic resistance and offering new strategies against multidrug-resistant infections.

Viruses remodel metabolic processes and utilize host lipids for different stages of their life cycle. Our earlier studies have shown that the flavivirus Japanese encephalitis virus (JEV) downmodulates several key proteins involved in sterol and lipid biosynthesis. Through a lipidome analysis, we report that virus infection dysregulates nearly 41% of the cellular lipids in mouse embryonic fibroblasts (MEFs). This manifests as down-modulation of cholesterol, cholesterol esters, and glycerolipids and upregulation of ceramides and several phospholipids. Significant transcriptional downregulation of cholesterol biosynthetic pathway genes was observed in JEV-infected MEFs and mouse bone marrow-derived macrophages (BMDMs). This effect was dependent upon an active interferon (IFN) signaling node. Both knockdown and pharmacological inhibition of 7-dehydrocholesterol reductase (Dhcr7), a key regulator of the cholesterol biosynthesis pathway, exerted potent antiviral effects by blocking viral replication and enhancing IFN signaling. Direct supplementation with the Dhcr7 substrate, 7-dehydrocholesterol (7DHC), showed similar antiviral effects. A partial inhibition of virus replication was also observed in cells deficient for IFN signaling, highlighting an IFN-independent antiviral role of Dhcr7 inhibition. Overall, these findings underscore the potential of cholesterol biosynthetic pathway inhibition as an antiviral strategy for JEV.IMPORTANCEJEV, a mosquito-borne virus, is a leading global cause of virus-induced encephalitis with a significant disease burden in Southeast Asia. In this study, we observe that the cellular lipid composition changes in virus-infected cells, with lower levels of cholesterol and cholesterol esters. We also observe that specific genes of the cholesterol biosynthesis pathway are decreased, and this depends on the activation of the antiviral interferon (IFN) response. We have characterized one specific downregulated gene Dhcr7, which catalyzes the conversion of 7-dehydrocholesterol (7DHC) to cholesterol. Depletion of the Dhcr7-specific mRNA, inhibition through drugs, or adding the substrate 7DHC further enhanced the IFN response and blocked virus replication. Our study highlights that downregulation of the cholesterol biosynthetic pathway has the potential to be developed into an antiviral strategy.  .

African swine fever virus (ASFV) employs sophisticated regulatory strategies to manipulate host cell apoptosis, a process critical for its pathogenesis and immune evasion; however, the mechanisms underlying this process remain incompletely understood. Here, we report a novel mechanism by which the ASFV-encoded envelope protein CD2v suppresses apoptosis by activating the TPL2 (tumor progression locus 2)-MEK (mitogen-activated protein kinase kinase)-ERK (extracellular signal-regulated kinase) signaling axis, leading to proteasomal degradation of the pro-apoptotic protein Bim_EL in primary porcine alveolar macrophages and wild boar lung (WSL) cells. We further demonstrated that ASFV infection triggers ERK1/2-dependent phosphorylation and degradation of Bim_EL, a process independent of viral replication and mediated by viral structural components. A targeted screen identified CD2v as the key viral protein driving this pathway. Both the purified extracellular domain of CD2v (Asp17-Tyr206) and virion-associated CD2v activated TPL2-MEK-ERK signaling without requiring internalization into cells, resulting in Bim_EL downregulation and subsequent suppression of apoptosis. Crucially, CRISPR-Cas9-mediated knockout of CD2v abolished ASFV-induced ERK1/2 activation and consequential Bim_EL degradation. Furthermore, we discovered that soluble CD2v released from ASFV-infected cells can activate this signaling axis in uninfected bystander cells, thereby inhibiting apoptosis distantly. This paracrine function, alongside its intrinsic role in directly infected cells, enables CD2v to establish a pro-survival microenvironment conducive to viral propagation. Our findings uncover a multifaceted anti-apoptotic mechanism employed by ASFV, expanding the functional repertoire of CD2v and providing new insights into ASFV pathogenesis with potential therapeutic implications.IMPORTANCEThis study elucidates a distinct mechanism of apoptosis inhibition by African swine fever virus (ASFV), a pathogen that causes a devastating disease in swine. We identify the ASFV CD2v protein as a key suppressor of cell death that operates by hijacking the host TPL2-MEK-ERK signaling pathway to degrade the pro-apoptotic protein Bim_EL. Importantly, CD2v mediates this effect not only within infected cells but also, in a soluble form, on surrounding uninfected bystander cells. This dual action helps create a protective, pro-survival cellular environment that facilitates viral spread and persistence. Understanding this novel apoptotic suppression mechanism advances our knowledge of ASFV-host interactions and highlights potential new avenues for therapeutic intervention.

Establishing a rapid, broad-spectrum antiviral state in human cells offers a promising strategy to combat viral infections, especially when vaccines or pathogen-specific treatments are unavailable. Here, we evaluate immunostimulatory nucleic acid nanoparticles (iNANPs), identified as potent innate immune activators, for their ability to induce protective antiviral states. By mimicking pathogen-associated molecular patterns, iNANPs engage intracellular pattern recognition receptors to stimulate type I and III interferon responses. We tested iNANPs for antiviral efficacy against a replication-incompetent lentiviral vector pseudotyped with vesicular stomatitis virus (VSV) G protein, as well as replication-competent viruses, including VSV, Sendai virus (SeV), and respiratory syncytial virus (RSV). These viruses vary in their mechanisms of innate immune activation and evasion, providing a robust system to assess iNANP activity. Our results demonstrate that iNANPs dramatically restrict viral infection via induction of a robust IFN response, establishing an antiviral state that impairs replication of all tested viruses. This study highlights the potential of iNANPs as a broad-spectrum antiviral prophylactic platform.IMPORTANCEEstablishing a rapid, broad-spectrum antiviral state in human cells offers a promising strategy to combat viral infections, especially when vaccines or pathogen-specific treatments are unavailable. Here, we evaluate immunostimulatory nucleic acid nanoparticles (iNANPs) identified as potent innate immune activators for their ability to induce protective antiviral states. The highly modular design and tunable physicochemical properties of iNANPs with defined architectures and compositions can be tailored to engage specific innate immune sensors. This same modularity allows a seamless "plug-and-play" integration of diverse therapeutic nucleic acids, as well as other therapeutics and small molecules, directly into the iNANP structure. We tested iNANPs for antiviral efficacy against a replication-incompetent lentiviral vector pseudotyped with vesicular stomatitis virus (VSV) G protein, as well as replication-competent viruses, including VSV, Sendai virus (SeV), and respiratory syncytial virus (RSV). Our results demonstrate that iNANPs significantly restrict viral infection, highlighting their potential as a broad-spectrum antiviral prophylactic platform.

The internal ribosome entry site (IRES) is a cis-acting structural element found in many viral mRNAs, which mediates cap-independent translation by recruiting various RNA-binding proteins and IRES trans-acting factors (ITAFs). Foot-and-mouth disease virus (FMDV), a significant member of the Picornaviridae family, contains a functional IRES element that contributes to viral protein translation and RNA synthesis. Here, we uncover a previously unrecognized mechanism in which DEAD-box RNA helicase 5 (DDX5) functions as a novel ITAF, inhibiting FMDV translation and viral RNA synthesis through two distinct strategies. First, DDX5 binds to the D4 domain of the IRES, suppressing FMDV IRES-driven translation by blocking the assembly of 80S ribosome. Second, DDX5 interacts with the viral RNA-dependent RNA polymerase 3D^pol and 3'UTR of FMDV, disrupting viral RNA synthesis. Conversely, the inhibitory effect of DDX5 was counteracted by viral precursor protein 3ABCD-mediated proteolysis of 3C^pro. Furthermore, the functional importance of DDX5 in FMDV pathogenicity was further validated in vivo experiments. These findings enhance our understanding of how viruses exploit or antagonize cellular factors to regulate IRES-driven translation and provide new insights into translational control during viral infection.

Importance: Picornaviruses have evolved various strategies to compete and dominate host protein synthesis machinery, often bypassing cap-dependent mRNA translation. Foot-and-mouth disease virus (FMDV), a highly contagious member of the Picornaviridae family, is a globally significant pathogen responsible for severe epidemics in cloven-hoofed animals, posing substantial economic and agricultural threats. In this study, we identified DEAD-box RNA helicase 5 (DDX5) as a novel IRES trans-acting factor that plays a critical role in the translational regulation of FMDV. Specifically, DDX5 was found to negatively modulate FMDV IRES-driven translation and suppress viral RNA replication during infection. Furthermore, we elucidated a novel viral counteraction mechanism in which DDX5 is cleaved by the viral precursor molecule 3ABCD through proteolytic activity. These findings provide new insights into the complex interplay between viral and host factors, advancing our understanding of translational control during picornavirus infection and offering potential avenues for the development of antiviral strategies.

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.

Hepatitis E virus (HEV) is the most common cause of acute viral hepatitis worldwide. Filamin A (FLNa), a cytoskeletal protein, is involved in cytoskeleton remodeling to construct a barrier to infection and participates in virus entry and release. However, how HEV enters host cells and how it is sensed by pattern recognition receptors are largely unexplored. In this study, the role of FLNa during HEV infection was evaluated in patients with HEV infection, animal models, and cell cultures. Notably, HEV interacted with FLNa at the early stage of infection and remarkably inhibited the expression of FLNa in vivo and in vitro. Its knockdown inhibited the proteolytic degradation of IκB, thus blocking the nuclear translocation of NF-κB. As a result, robust viral replication occurred, and numerous virions were released. Furthermore, the inhibition of FLNa suppressed ubiquitination-mediated degradation to aggravate apoptosis and inflammatory responses. Results indicated that FLNa plays a critical role in the remodeling of the cytoskeletal network during HEV infection. This role may be responsible for the easy entry and escape of HEV from sensing by innate immunity.IMPORTANCEHepatitis E virus (HEV) is the most common pathogen of acute viral hepatitis. The mechanisms by which HEV enters host cells and is sensed by pattern recognition receptors are largely unexplored. In the present study, filamin A (FLNa), a cytoskeletal protein, was significantly inhibited in patients with HEV infection, animal models, and cell cultures. The knockdown of FLNa facilitates viral replication by blocking the nuclear translocation of NF-κB while inhibiting ubiquitination-mediated degradation to aggravate apoptosis and inflammatory responses. This work demonstrates that FLNa plays a critical role in the remodeling of the cytoskeletal network during HEV infection. Such remodeling may be responsible for the entry and escape of HEV from sensing by innate immunity.

Alphaviruses have positive-strand RNA genomes that mimic cellular mRNAs, and their translation results in the synthesis of nonstructural (ns) polyprotein, the precursor of viral replicase. The ns polyprotein is processed by its protease activity to form an early replicase complex, responsible for the synthesis of negative-strand RNA that forms a double-stranded RNA (dsRNA) replication intermediate with the RNA genome. The following processing results in the formation of a late replicase complex responsible for the synthesis of positive-strand RNAs. Replication complexes are anchored to membranes, and dsRNA is shielded from cellular pattern recognition receptors. Nevertheless, alphavirus infection triggers a type I interferon response; this is partly due to the ability of replicases to utilize cellular RNAs as templates for synthesis of specific dsRNAs (rPAMPs). Here, we demonstrate that replicases of 11 alphaviruses, representing most of the antigenic complexes of alphaviruses, are all capable of rPAMP synthesis in human cells and that some replicases also do the same in mosquito cells. The levels of rPAMPs generally correlate with the efficiency of viral RNA synthesis and are increased by mutations slowing down the processing of the ns polyprotein. For different strains of Semliki Forest virus, the elevated synthesis of rPAMPs correlates with a previously reported virulent phenotype, while for mutants of chikungunya virus, the situation is reversed. Thus, synthesis of rPAMPs is a universal property of alphavirus replicases; these molecules are used to regulate virus infection, and their functional impact depends on their amount as well as the virus species.IMPORTANCEAlphaviruses are important mosquito-borne emerging pathogens. Their ability to interact with cellular defenses, including type I IFN, is crucial for infection. Here, we found that alphavirus replicases have a universal ability to synthesize type I IFN-inducing RNAs using non-viral templates, and that their synthesis varies greatly among viruses and their strains. Production of these RNAs was increased by mutations slowing down the maturation of the viral replicase. The abundance of non-viral type I IFN-inducing RNAs correlated with neurovirulence of Semliki Forest virus, indicating their role in virus pathogenicity; however, for chikungunya virus, their excess correlated with virus attenuation. These data are important to promote the understanding of mechanisms of alphavirus pathogenesis and virus interactions with the host immune system. As alphaviruses represent promising platforms for development of advanced mRNA vaccines, the data can also be used for rational optimization of alphavirus-based vaccine candidates.

Vaccinia virus (VACV) confers cross-protective immunity against monkeypox virus (MPXV), the causative agent of mpox, and has therefore been extensively exploited as a preventive vaccine. VACV Tiantan strain (VTT) is a second-generation smallpox vaccine used in China in the last century, and there are consistent efforts to minimize its virulence and ensure its best safety for potential clinical applications. In this study, an attenuated VACV rVTT△C12K2△A45 was constructed by deletion of gene segments related to virulence genes, host range genes, immune regulatory genes, and other functional genes from the VTT genome by genetic engineering. Attenuation characteristics of rVTT△C12K2△A45 were confirmed by smaller plaque size, lower replication capacity in various mammalian cell lines along with tests for neurotoxicity in mice, and lesion formation on rabbit skin. Immunization in BALB/c mice with rVTT△C12K2△A45 induced both anti-MPXV and anti-VACV neutralizing antibodies. Animals vaccinated with rVTT△C12K2△A45 showed lower MPXV viral loads in the lungs and genital organs compared to the non-immunized mice.IMPORTANCEThe World Health Organization declared monkeypox a "Public Health Emergency of International Concern" twice, in 2022 and 2024, respectively. Smallpox vaccines have shown efficacy in protecting against monkeypox because of the cross-protective immunity among orthopoxviruses. The vaccinia virus Tiantan strain (VTT) played a critical role in China's smallpox eradication campaign. Here, we construct an attenuated vaccinia virus by deletion of different ranges of genes in the VTT genome. This attenuated vaccinia virus replicates like its parental VTT strain in production CEF cells but is severely impaired in human-derived cells like 2BS, MRC-5, and WI-38 cells. Meanwhile, this virus shows significantly reduced virulence in small animals. Animals vaccinated with this attenuated vaccinia virus showed lower monkeypox virus (MPXV) viral loads in the lungs and genital organs compared to the non-immunized mice after MPXV challenge. Our data suggest the potential of this genetically engineered VTT strain as a MPXV vaccine candidate.

The 2022 outbreak of monkeypox virus (MPXV), a double-stranded DNA virus, is remarkable for an unusually high number of single-nucleotide substitutions compared to earlier strains, with a strong bias toward C→T and G→A transitions consistent with the APOBEC3 cytidine deaminase activity. While APOBEC3-induced mutagenesis is well documented at the DNA level, its potential impact on MPXV RNA transcripts remains unclear. To assess whether APOBEC3 enzymes act on MPXV RNA, we analyzed RNA-seq data from infected samples. The enrichment of APOBEC signature substitutions among high-frequency mismatched positions led us to consider two possibilities: RNA editing at hotspots or fixed DNA mutations. Multiple lines of evidence support the conclusion that these substitutions arise from DNA-level mutagenesis rather than RNA editing. These include a substantial number of G→A substitutions remaining after normalization by gene strand direction, a largely neutral impact of substitutions on protein-coding sequences, the lack of positional correlation with transcriptional features or RNA secondary structure typically associated with APOBEC action hotspots, and an overlap with known genomic mutations in MPXV strains. Analysis of the nucleotide context of observed substitutions indicated that APOBEC3A or APOBEC3B was likely a driver of DNA-level mutagenesis.IMPORTANCEThe 2022 monkeypox virus (MPXV) outbreak showed an unusually high number of mutations thought to result from human antiviral enzymes of the APOBEC3 family. While such mutations have been clearly documented in the viral DNA, whether APOBEC3 also edits viral messenger RNA molecules remained unclear. In this study, we analyzed multiple publicly available MPXV RNA sequencing datasets to address this question. We found that the apparent APOBEC-like changes in RNA are best explained by fixed DNA mutations rather than active RNA editing. This finding helps clarify how MPXV evolves and adapts, suggesting that APOBEC3's role in shaping the virus likely operates at the DNA level. Understanding where and how these mutations occur provides insight into the virus's interaction with the human immune system and informs future studies on viral evolution and antiviral defenses.