The team began by screening natural compounds and testing ailanthone in African green monkey kidney Vero cells infected with HSV-1. Concentrations of 100 to 3,000 nanomolar reduced transcription of the viral immediate-early genes ICP0 and ICP4 and the late gene gD in a dose-dependent manner. The compound also reduced plaque formation and the concentration of progeny virus released into culture. Ailanthone had a half-maximal cytotoxic concentration above 10,000 nanomolar in these cells, while its half-maximal effective concentration against viral replication was 197.4 nanomolar, giving a selectivity index greater than 50. It also suppressed HSV-1-induced increases in the inflammatory cytokine genes IL-1B and TNF-α.

Antiviral activity was subsequently observed in human corneal epithelial cells, a model more directly relevant to the tissue affected by keratitis. Ailanthone concentrations of 100 to 500 nanomolar markedly reduced the cytopathic effects of HSV-1F, lowered expression of ICP0, ICP4 and gD, and reduced the viral induction of IL-1B and TNF-α. Its effective concentration in these cells was 50.16 nanomolar, with a cytotoxicity concentration above 10,000 nanomolar and a selectivity index greater than 100. The researchers also tested the compound in human retinal pigment epithelial ARPE-19 cells, where it reduced viral gene expression, plaque formation and progeny-virus titers while showing low cytotoxicity.

Activity was not restricted to the laboratory HSV-1F strain. In ARPE-19 cells, ailanthone concentration-dependently inhibited the late gD gene in the clinical acyclovir-resistant strain HSV-1/153, whereas 1,000 nanomolar acyclovir had little effect. The compound’s effective concentration was 35.05 nanomolar against HSV-1F and 227.4 nanomolar against HSV-1/153, with selectivity indices above 100 for both strains. A time-of-addition experiment indicated that ailanthone did not prevent attachment of HSV-1 to cells. It reduced entry under one experimental schedule and had its strongest effect on post-entry replication, where it lowered gD expression.

To investigate how the compound might work, the researchers combined computational target prediction with protein-interaction network analysis. They identified 36 targets shared by ailanthone and herpes simplex keratitis, then selected ITGB2 as a highly connected hub in the resulting network and as a top candidate in molecular docking. The predicted docking score for ailanthone and ITGB2 was −8.6 kcal per mole. Computational predictions alone cannot establish direct binding, so the team also performed a drug affinity responsive target stability assay in THP-1 cell lysates. Ailanthone protected ITGB2 from digestion by the protease pronase E in a dose-dependent manner, while acyclovir did not, providing biochemical evidence consistent with a direct interaction.

Functional experiments then tested whether ITGB2 was necessary for the antiviral effect. Three independent small interfering RNAs reduced ITGB2 messenger RNA and protein in THP-1 cells. Following HSV-1 infection, this knockdown lowered gD messenger RNA and protein and reduced the number of plaques produced when culture supernatants were transferred to fresh Vero cells. The converse pattern appeared when ITGB2 was overexpressed: elevated ITGB2 increased gD expression in infected cells, and it partially counteracted the reduction in gD protein caused by ailanthone in 293T and ARPE-19 cells. Together, these loss- and gain-of-function results support the conclusion that ailanthone’s antiviral activity is mediated at least partly by lowering ITGB2.

The researchers next tested ailanthone in an acute mouse model of HSV-1 keratitis. Six-week-old male BALB/c mice received a corneal scratch followed by application of HSV-1F, then received intraperitoneal ailanthone at 1 or 5 milligrams per kilogram or acyclovir at 5 milligrams per kilogram on days 1, 3 and 5 after infection. The methods section states that six animals were assigned to each group, although the discussion says five mice per group were used in the animal experiment. The 5-milligram-per-kilogram ailanthone treatment significantly reduced clinical disease scores, improved the appearance of the cornea under normal and cobalt-blue illumination, and lowered fluorescein staining scores on days 3 and 5. Hematoxylin and eosin staining at day 6 also indicated reduced corneal inflammation, and viral titers in ocular swabs were significantly lower than in infected mice given vehicle.

Analysis of mouse corneal tissue was consistent with the proposed mechanism. ITGB2 was upregulated in the corneas of infected animals, and ailanthone reduced its fluorescence intensity in a dose-dependent manner. Acyclovir did not significantly alter corneal ITGB2 levels. The investigators interpreted this as evidence that ailanthone differs from a direct viral DNA-replication inhibitor: the data are consistent with modulation of a host dependency factor rather than action solely through a viral enzyme. However, the study did not determine precisely how binding to ITGB2 leads to lower protein abundance. The authors note that transcriptional regulation, changes in messenger-RNA stability, or enhanced protein degradation could all be involved, and dedicated experiments would be required to distinguish among them.

Several limitations constrain how far the findings can be generalized. The cell experiments used established lines, including corneal epithelial and retinal pigment epithelial cells, rather than primary human corneal cells or an intact human immune system. The animal experiment examined acute primary infection in young male mice, whereas recurrent reactivation and the inflammation associated with repeated episodes are important features of human herpes simplex keratitis. Ailanthone was administered by intraperitoneal injection, but no topical ophthalmic formulation, corneal penetration, intraocular distribution, or optimized dosing regimen was evaluated. Long-term ocular toxicity and safety under repeated exposure also remain unknown. The natural compound may additionally affect multiple targets, and compensation among integrins could limit or alter its effects. The evidence therefore supports further preclinical investigation of ailanthone and ITGB2 as a host-directed antiviral strategy, not a clinical recommendation or a claim of human therapeutic benefit.

Subject of Research: Preclinical evaluation of ailanthone as a host-directed inhibitor of HSV-1 replication and herpes simplex keratitis.

Article Title: Natural compound ailanthone alleviates herpes simplex keratitis in mice by inhibiting HSV-1 replication

Article References: Chen, L., Yang, E., Zhang, X., Zhang, L., Wu, L., Chen, S., Jin, W., Qin, Q., Chen, R., Wu, J., & Chen, D. (2026). Natural compound ailanthone alleviates herpes simplex keratitis in mice by inhibiting HSV-1 replication. Frontiers in Pharmacology, 17, Article 1937590. https://doi.org/10.3389/fphar.2026.1937590

Image Credits: AI Generated

DOI: 10.3389/fphar.2026.1937590

Keywords: HSV-1, herpes simplex keratitis, ailanthone, ITGB2, host-directed antivirals, viral replication, corneal infection, mouse model, integrin beta 2, drug resistance, Natural, compound

Researchers used transcriptomic and functional analyses to investigate how dengue virus and VEEV interact with Wolbachia in Aedes mosquito cells. In cells without the symbiosis, the viruses induced ATPSβ1. The evidence indicates that this host protein supported viral binding through the viral envelope protein and increased intracellular ATP production, which facilitated viral replication. ATPSβ1 therefore appears to perform two roles during infection: promoting initial viral attachment and supplying energy needed for replication.

In Wolbachia-carrying cells, the symbiont overrode the virus-associated promotion of viral binding and repurposed ATPSβ1-driven ATP synthesis to fuel bacterial proliferation. This metabolic diversion strengthened the antiviral activity of Wolbachia. The authors characterized the process as a tug-of-war over host ATP, with a shared host protein serving both the virus and the symbiont.

Pharmacological experiments supported the importance of ATP metabolism in this interaction. Adenosine increased the density of Wolbachia strain wAlbB and strongly suppressed viral infection in Aedes aegypti and Aedes albopictus cells. Oligomycin reduced intracellular ATP and inhibited viral infection in Wolbachia-free A. aegypti cells. These findings were obtained in cell systems and do not by themselves demonstrate reduced mosquito-borne virus transmission or benefits in humans. The authors identify targeting ATPSβ1-driven metabolism as a possible route for curbing transmission, but that application remains to be validated beyond the tested cells.

Subject of Research: Wolbachia-mediated antiviral defense through host ATP metabolism in mosquito cells

Article Title: Wolbachia outcompetes mosquito-borne viruses by reprogramming host ATPSβ1-driven ATP metabolism toward symbiotic proliferation

Article References: Yuan, H., Zhang, Y., Zhao, Y., Wang, C., Zhou, Y., Ni, Q., Li, X., Zhang, B., Wang, Y., Cai, L., Liu, H., Wang, J., Gong, J., Zhang, M., Xi, Z., & Pan, X. (2026). Wolbachia outcompetes mosquito-borne viruses by reprogramming host ATPSβ1-driven ATP metabolism toward symbiotic proliferation. PLOS Pathogens, 22(10), e1014692. https://doi.org/10.1371/journal.ppat.1014692

Image Credits: AI Generated

DOI: 10.1371/journal.ppat.1014692

Keywords: Wolbachia, ATPSβ1, ATP metabolism, dengue virus, Venezuelan equine encephalitis virus, Aedes aegypti, Aedes albopictus, mosquito-borne viruses, antiviral defense, viral replication, outcompetes, mosquito-borne

Vpr is one of four HIV-1-specific accessory proteins, alongside Vif, Nef and Vpu, that support viral replication and pathogenicity in vivo. First characterized in 1987, it is unusually conserved across HIV-1 strains and divergent primate lentiviruses. Loss-of-function mutants can regain full-length Vpr during prolonged transmission in primates or humans, indicating strong evolutionary selection for its function.

Structural studies, principally using nuclear magnetic resonance spectroscopy and later crystallography, have resolved three alpha-helical regions: residues 17–33, 40–48 and 55–83. The first two helices are largely polar, whereas helix 3 is amphipathic, allowing it to associate with lipid membranes and helping explain the perinuclear location of Vpr in host cells. The protein’s ability to manipulate the host ubiquitin-proteasome system is central to many of its effects.

Vpr acts as an adaptor for the CUL4-DDB1-DCAF1 ubiquitin ligase complex, commonly called CRLCul4. A crystal structure showed that helices 1 and 3 engage DCAF1, also known as Vpr-binding protein, while helix 2 contacts uracil-DNA glycosylase 2. This arrangement allows Vpr to recruit cellular proteins for proteasomal degradation.

Among the substrates are UNG2, exonuclease 1 and HLTF, all connected to DNA repair. Their depletion compromises genome maintenance, activates ATR- and Chk1/2-dependent checkpoint signaling and inhibits CDC25C, reducing cyclin B1-CDK1 activity and holding cells at G2/M. Vpr may also act directly on CDC25C and on regulators including WEE1, 14-3-3 proteins and PP2A through pathways that do not require the proteasome.

Cell-cycle arrest can benefit HIV-1 because the viral long terminal repeat, which contains the proviral promoter, is more active in G2-arrested cells. Vpr consequently boosts viral replication in proliferating cell lines and primary CD4+ T cells, whereas terminally differentiated macrophages already occupy G0 and do not show the same benefit. The same structural helix is central to another major function.

Vpr delivered by the incoming virion can insert partially into the outer mitochondrial membrane, dissipate membrane potential and release apoptogenic factors including apoptosis-inducing factor and cytochrome c. Cytochrome c then supports caspase-9 activation. Vpr also binds adenine nucleotide translocase, disturbing mitochondrial homeostasis and increasing reactive oxygen species, which can activate ATR-Chk1 DNA-damage signaling.

Experiments in Jurkat cells and peripheral blood mononuclear cells have linked this cytotoxicity to possible CD4+ T-cell depletion, but clinical validation remains limited. The coupling of G2 arrest and apoptosis creates a delicate evolutionary balance. Arrest can raise proviral transcription and favor production of progeny virions, whereas killing an infected producer cell may limit sustained viral output.

Studies cited in the review indicate that cell-cycle arrest generally precedes and peaks before strong apoptotic activity. Vpr may therefore calibrate the timing of transcriptional amplification and cellular death rather than simply promote one outcome. This distinction matters because the review concludes that no single established Vpr function has been validated as the driver of HIV-1 infection or AIDS.

Many findings derive from tumor-derived lines, primary cells, overexpression systems or laboratory infection models, and discrepancies often arise when findings from these systems are compared. The authors caution that physiological relevance cannot be assumed from an in vitro phenotype alone. Vpr can also enhance the expression and stability of the HIV-1 envelope protein, Env, particularly in macrophages, a major target cell and potential viral reservoir.

In primary macrophages, DCAF1 binding is required for Vpr-mediated Env upregulation. Vpr inhibits endosome-related degradation of Env by promoting CRLCul4-dependent proteasomal degradation of LAPTM5, a lysosomal transmembrane protein; loss of LAPTM5 reduces lysosomal clearance of Env and improves the infectivity and cell-to-cell transmission of newly formed virions among macrophages. Vpr also depletes the endonuclease Dicer and host factors including gelsolin and PU.1.

In macrophages, Vpr and Nef can reduce the surface mannose receptor, which otherwise destabilizes Env and impairs virion assembly and release. Together, these mechanisms may increase infectious virion production and facilitate transfer between macrophages and CD4+ T cells, although most detailed evidence remains from in vitro systems. These macrophage findings also connect Vpr to several routes of HIV-1 dissemination.

Phagocytosis of infected T cells can be more efficient than direct infection for delivering HIV-1 to macrophages, while infected macrophages can present Env-dependent virions on their surfaces and transmit virus to CD4+ T cells. Direct contact between infected T cells and uninfected macrophages can also promote membrane fusion and formation of multinucleated giant cells, which have been observed in infected patients and can be highly virus-productive. Vpr-mediated elevation of Env therefore has consequences beyond particle production: it may influence cell-to-cell spread, macrophage reservoirs and the efficiency of transfer between immune-cell compartments.

The review does not establish that these pathways are sufficient to sustain viral persistence in vivo, and it does not provide evidence for a clinical intervention targeting Vpr. Host-cell signaling offers another route by which Vpr may support viral replication. Virion-packaged Vpr activates nuclear factor of activated T cells without requiring the upstream calcium flux normally initiated by plasma-membrane receptors, allowing productive infection of Jurkat cells, primary T cells and macrophages.

Vpr also promotes proteasome-dependent degradation of interferon regulatory factor 3, a central component of type I interferon production. Its effects on nuclear factor kappa B are unresolved. Some studies report that Vpr and DCAF1 inhibit nuclear-kappa-B translocation, while others show activation through TAK1, IKKα/β, DNA-damage signaling or interactions with cell-cycle machinery.

Donor macrophages in one experiment differed markedly in their response, suggesting intrinsic biological variation. The review says the net effect remains to be validated even though the convergent cellular outcome may be enhanced proviral transcription and replication. Associations between particular Vpr variants and long-term nonprogressors, people who delay or avoid progression to AIDS, point to a potentially important role in pathogenesis.

Candidate variants include Q3R, N16H, T19A, Q65R, F72L/A, R77Q/A, R80A and R90N/K. Q65R and R80A disrupt the Vpr-DCAF1 interface and therefore impair recruitment of CRLCul4, potentially disabling numerous Vpr activities. Yet T19A and R90N retain wild-type activity across known functions, and R77Q, previously associated with weaker apoptosis, was later found in HUT78 cells to retain G2/M arrest and apoptosis.

Its apparent instability in a proviral expression system, together with stronger arrest and cytotoxicity at low expression, may indicate that some variants alter the timing of producer-cell death rather than simply removing a function. These mixed phenotypes reinforce the idea that Vpr’s pathogenic effects may arise from coordinated, context-dependent activities rather than one mechanism. Newer functions may help explain unresolved links between Vpr and disease progression.

Vpr can promote proteasomal depletion of the methylcytosine dioxygenase TET2, affecting epigenetic regulation of interleukin-6 expression; a clinical cohort has also associated high Vpr detection with increased interleukin-6 and aggravated AIDS pathogenesis. Vpr can deplete PU.1, which links Env expression to suppression of innate immune responses in macrophages, and target SLF2, a regulator of the SMC5-SMC6 complex, in CEM-CD4 and primary CD4+ T cells. Vpr also has context-dependent effects on LINE-1 retrotransposition.

HIV-1 infection increased LINE-1 activity in a Vif- and Vpr-dependent manner in Jurkat cells, and L1 DNA accumulated in infected primary CD4+ T cells. Extracellular Vpr enhanced L1 replication in mouse models and patient-derived samples, whereas intracellular Vpr directly bound LINE-1 ORF2p and inhibited its reverse-transcriptase activity. The review notes that evidence connecting LINE-1 modulation to AIDS progression is still lacking.

Overall, the synthesis presents Vpr as an evolutionarily conserved coordinator of replication, cytotoxicity, immune disruption and persistence, but not yet as a clinically proven causal target; questions remain about its activity in primary cells, donor-dependent signaling effects, the balance between viral amplification and infected-cell death, and whether any of its functions can be therapeutically separated from those needed to maintain viral fitness.

Subject of Research: Multifunctional roles of HIV-1 Vpr in viral replication, immune disruption and AIDS pathogenesis

Article Title: HIV-1 Vpr: a multifunctional accessory protein with potential contributions to AIDS pathogenesis and persistence

Article References: Du, J., Li, Q., Wang, S.-H., Zhao, Y., & Zhao, K. (2026). HIV-1 Vpr: a multifunctional accessory protein with potential contributions to AIDS pathogenesis and persistence. Frontiers in Microbiology, 17, Article 1971957. https://doi.org/10.3389/fmicb.2026.1971957

Image Credits: AI Generated

DOI: 10.3389/fmicb.2026.1971957

Keywords: HIV-1, Vpr protein, AIDS pathogenesis, viral replication, G2/M arrest, apoptosis, immune evasion, macrophages, LINE-1 retrotransposition, long-term nonprogressors, multifunctional, accessory

The protein at the center of the study is non-structural protein 1, universally abbreviated NS1. In mosquito-borne orthoflaviviruses, NS1 is something of a molecular multitasker. Inside infected cells, it functions as a dimer and participates in viral RNA replication, associating with the replication complexes that assemble on intracellular membranes. At the same time, a fraction of NS1 travels through the host secretory pathway and is released from the cell, either as a soluble hexameric lipoparticle or tethered to the outer leaflet of the plasma membrane. Secreted NS1 is a major player in disease: it helps the virus evade immune responses, modulates complement activation, and has been implicated in the vascular leakage and endothelial dysfunction associated with severe dengue and with the neurological complications of Zika infection.

Given this central role, the team led by Bodine Bezemer, Pascal Miesen, and Ronald van Rij set out to map the interaction partners of Zika virus NS1 in infected cells. Rather than studying the protein in isolation, they engineered a recombinant Zika virus in which NS1 carries a small molecular handle, a FLAG tag, that allows the protein and everything bound to it to be pulled out of infected cell lysates and identified. They infected two biologically distinct cell types with this tagged virus: Huh7 cells, a well-established liver-derived line commonly used for flavivirus work, and human neural progenitor cells, which are directly relevant to the neurodevelopmental defects seen in congenital Zika syndrome.

Quantitative mass spectrometry of the pulled-down material revealed a strikingly consistent picture across both cell types. Among the NS1 interactors were multiple prolyl hydroxylase enzymes, along with enzymes involved in glycosylation, components of the endoplasmic reticulum-associated degradation pathway, and proteins with roles in antiviral immunity. The presence of prolyl hydroxylases was particularly intriguing. These enzymes, best known for adding hydroxyl groups to proline residues in collagen and for their role in cellular oxygen sensing through modification of hypoxia-inducible factors, catalyze a post-translational modification that had not previously been appreciated as important for flavivirus NS1.

The mass spectrometry data went further than merely hinting at an interaction. The analyses detected hydroxyproline residues on NS1 itself, providing direct evidence that the viral protein is actually modified by these enzymes rather than simply passing near them. This observation reframed the interaction as a bona fide enzymatic substrate relationship and raised an immediate functional question: what happens to NS1, and to the virus, if prolyl hydroxylation is blocked?

To answer that question, the researchers turned to chemical inhibitors of prolyl hydroxylases. When cells infected with Zika virus were treated with these compounds, the consequences for NS1 were visible and specific. The protein’s intracellular trafficking was impaired, and its localization to the plasma membrane, a critical step on the route to secretion, was reduced. Consistent with a defect in the virus’s ability to produce and release functional NS1, treatment also lowered intracellular Zika virus RNA levels and reduced infectious virus titers. Together, the interaction data and the inhibitor experiments established prolyl hydroxylation as a modification that the virus depends on for the normal handling of NS1 within the secretory pathway.

To pin the effect down to individual amino acids, the team used site-directed mutagenesis to alter specific proline residues within the NS1 sequence and then rescued mutant viruses. Substituting the proline at position 281 produced a clean and interpretable phenotype: secretion of NS1 from infected cells was impaired, yet viral replication itself, measured by RNA levels and infectious output, was unaffected. This dissociation is mechanistically informative. It indicates that the hydroxylation at this position is not required for the intracellular replication functions of NS1 but is specifically needed for the protein to mature and exit the cell, most likely by stabilizing a conformation or trafficking state that the secretory machinery recognizes.

The second mutant told a different story. Replacing the proline at position 267 strongly dysregulated NS1 expression, and, notably, the mutation reverted to the wild-type sequence after only a single passage in culture. Such rapid reversion is a classic signature of a mutation that imposes a severe fitness cost on the virus. The interpretation offered by the authors is that proline 267 is so central to NS1’s structure or function that any virus carrying the substitution replicates poorly enough to be rapidly outcompeted by revertants in the population. While the precise biochemical role of this residue remains to be defined, its evolutionary inflexibility underscores how tightly constrained the NS1 sequence is around its modification sites.

Taken together, the study identifies prolyl hydroxylation as an essential post-translational modification of Zika virus NS1 and embeds it in a broader network of host factors that the virus co-opts during infection. The co-occurrence of prolyl hydroxylases, glycosylation enzymes, and ER-associated degradation proteins among the NS1 interactors suggests that NS1’s journey through the endoplasmic reticulum and Golgi apparatus is choreographed by a series of host-driven chemical edits, of which hydroxylation is now one of the best supported. Because secreted NS1 contributes substantially to the immune evasion and pathogenic features of orthoflavivirus infections, any process required for its secretion is, by extension, a potential point of therapeutic leverage.

The work also raises questions that extend beyond Zika virus. NS1 is highly conserved among mosquito-borne orthoflaviviruses, and the proline residues examined in this study are positioned within regions of the protein that are shared across dengue, West Nile, and yellow fever viruses. Whether prolyl hydroxylation similarly governs the maturation and secretion of NS1 in these related pathogens is an obvious next step, and one with practical implications: if a single host enzymatic activity supports the secretion of NS1 from multiple medically important viruses, inhibitors of that activity could offer a strategy with breadth across the orthoflavivirus family. For now, the study provides a concrete molecular foothold, showing that a small chemical decoration on a single proline residue can determine whether a viral protein reaches the cell surface, and demonstrating once again that the fine details of virus-host biochemistry often hide the most consequential vulnerabilities.

Subject of Research: Prolyl hydroxylation of Zika virus non-structural protein 1 and its role in viral maturation and secretion

Article Title: Prolyl hydroxylation is required for maturation and secretion of Zika virus non-structural protein 1

Article References: Bezemer, B., Overheul, G. J., Stevens, M., Spruijt, C. G., Vermeulen, M., Miesen, P., & van Rij, R. P. (2026). Prolyl hydroxylation is required for maturation and secretion of Zika virus non-structural protein 1. npj Viruses. https://doi.org/10.1038/s44298-026-00248-8

Image Credits: AI Generated

DOI: 10.1038/s44298-026-00248-8

Keywords: Zika virus, NS1 protein, prolyl hydroxylation, orthoflavivirus, post-translational modification, mass spectrometry, viral secretion, prolyl hydroxylases, mutagenesis, viral replication, immune evasion, npj Viruses

The review frames viral emergence as a cascade of linked molecular gates rather than a single event. Viral surface proteins determine attachment and entry, but compatibility between viral glycoproteins and host receptors does not guarantee productive infection. Intracellular host factors, polymerase activity, codon usage, replication organelles, interferon sensitivity and immune evasion proteins collectively determine whether progeny virus can be produced and transmitted. For SARS-CoV-2, the convergence of ACE2 engagement, spike priming by TMPRSS2, furin-site biology, replication-complex efficiency and immune evasion created a biologically favourable foundation for pandemic expansion. The same logic explains why a virus may be detected frequently at the animal-human interface yet fail to establish sustained human transmission, whereas another virus with modest ecological exposure may spread explosively once critical within-host barriers are crossed.

At the family level, the review identifies distinct molecular architectures that shape emergence risk. Coronaviruses combine large RNA genomes, recombination, broad mammalian reservoirs and spike-mediated receptor flexibility. Orthomyxoviruses such as influenza A possess segmented genomes that permit reassortment, enabling rapid antigenic and host-range shifts. Filoviruses and paramyxoviruses have non-segmented negative-sense RNA genomes, but their glycoproteins and immune-antagonist proteins facilitate severe disease in susceptible hosts. Flaviviruses and alphaviruses are shaped by vector ecology, climate-sensitive transmission and envelope-protein adaptation. Orthopoxviruses demonstrate that large DNA viruses can still undergo epidemiologically meaningful evolution under transmission pressure, as mpox has shown under new contact networks. This family-level view is not merely taxonomic; it provides a practical map for prototype-pathogen research, in which studying one coronavirus polymerase, protease or spike architecture can inform assay development and therapeutic design across related coronaviruses.

Entry is the first molecular gate through which an emerging virus must pass. Enveloped viruses use surface glycoproteins to bind receptors or attachment factors and then undergo fusion at the plasma membrane or within endosomal compartments. For henipaviruses, ephrin-B2 and ephrin-B3 receptor usage helps explain endothelial and neuronal tropism. For filoviruses, macropinocytosis, glycoprotein processing and NPC1-dependent endosomal entry are central determinants of permissiveness. However, tropism is also determined by host proteases, attachment factors, glycan presentation and innate-state context. A receptor may be expressed in a tissue yet fail to support efficient infection if the proteolytic environment or replication cofactors are absent. Conversely, viruses can exploit multiple entry routes or evolve altered receptor-binding surfaces that expand their host range. This explains why entry studies should be integrated with organoid models, primary cells, pseudovirus assays and animal-interface surveillance rather than being interpreted from receptor-binding data alone.

Replication machinery constitutes a second determinant of emergence. RNA-dependent RNA polymerases, viral proteases, nucleocapsid proteins and replication organelles determine not only viral yield but also mutation rate, recombination potential and sensitivity to nucleoside analogues. Coronaviruses are unusual among RNA viruses because their replication complex includes proofreading activity, enabling large genomes while still permitting adaptive diversity. This balance of fidelity and flexibility is one reason SARS-CoV-2 could accumulate meaningful mutations without losing genome integrity. Assembly mechanisms differ across viral families but often expose vulnerabilities: coronavirus assembly occurs at ER-Golgi intermediate compartments through coordinated interactions among M, E, S and N proteins; influenza assembly requires packaging of eight genome segments, creating opportunities for reassortment but also imposing strict compatibility constraints; filovirus budding depends heavily on matrix protein VP40 and host ESCRT machinery. Host-factor screens and proteomics have expanded the field from single viral proteins to systems-level replication networks. A SARS-CoV-2 protein-interaction map identified host pathways that could be repurposing targets, while CRISPR screens revealed TMEM41B and other host factors as pan-coronavirus dependencies.

Innate immunity imposes one of the strongest barriers to emergence, and successful emerging viruses frequently encode proteins that mask pathogen-associated molecular patterns, block adaptor proteins, degrade signalling molecules, suppress STAT activation, remodel stress responses or antagonise antigen presentation. These mechanisms are not incidental virulence accessories; they are often prerequisites for efficient replication in a new host. Convergent immune-evasion logic can be seen across unrelated viruses: influenza A NS1 inhibits antiviral RNA sensing and interferon responses; Ebola virus VP35 suppresses RIG-I-like receptor signalling while VP24 interferes with interferon-induced nuclear import; coronaviruses deploy several antagonists, including nsp1-mediated host translation suppression and accessory proteins that disrupt interferon signalling; henipavirus V, W and C proteins impair interferon production or response; and orthopoxviruses encode multiple soluble and intracellular immunomodulators. Pathogenesis results from both direct viral injury and dysregulated host response, with severe disease reflecting tropism for endothelial, respiratory, neurological or immune cells alongside excessive inflammation, vascular leakage, coagulopathy or immunopathology.

Evolutionary dynamics determine whether control measures remain effective over time. SARS-CoV-2 demonstrated that variants can accumulate spike mutations that alter transmissibility, antigenicity and neutralisation while maintaining receptor engagement. Omicron showed that immune escape can become a dominant population-level advantage in settings of widespread infection- or vaccine-derived immunity, yet escape must remain compatible with entry and replication fitness. Influenza viruses exemplify the risk of segmented genomes: reassortment between avian, swine and human viruses can generate new constellations of internal genes and surface antigens, while point mutations in HA, PB2 and other segments influence receptor preference and mammalian adaptation. Highly pathogenic H5N1 clade 2.3.4.4b has increased concern because infection in mammals creates additional opportunities for adaptation, even though sustained human-to-human transmission has not been established. The review argues that genomic surveillance should evaluate variants as phenotypic hypotheses rather than as lists of mutations, because a mutation in a receptor-binding domain, polymerase, protease, matrix protein or immune antagonist may be important only in a specific genomic background.

Rapid diagnostics are central to containment, but performance is conditional. RT-qPCR is sensitive and scalable, yet primer mismatch, sampling time, specimen quality and infrastructure can reduce effectiveness. Programmable CRISPR assays can shorten redesign cycles, whereas metagenomic next-generation sequencing offers agnostic detection when the causative agent is unknown, divergent or coinfecting; neither replaces prospective clinical validation, contamination control or external quality assurance. Outbreak genomics has transformed reconstruction for Ebola, Zika, yellow fever, SARS-CoV-2 and mpox by linking sequence data to transmission dynamics, revealing introductions, hidden transmission, superspreading and international dissemination. The value of genomic data depends on representative sampling, metadata quality, rapid deposition, analytic capacity and equitable participation by affected regions. On the countermeasure side, the COVID-19 pandemic demonstrated both the power and fragility of platform approaches: mRNA, adenoviral-vector and protein-based vaccines can be developed quickly when antigen design, manufacturing capacity, clinical trial networks and regulatory pathways are prepared in advance, but antigenic evolution can reduce neutralisation, requiring booster strategies, updated antigens or broader immunogen design. Direct-acting antivirals are most useful when they target conserved enzymatic functions, have acceptable safety profiles and can be delivered early in infection, as remdesivir and nirmatrelvir-ritonavir showed in selected clinical settings, while molnupiravir demonstrated more modest clinical utility and raises different resistance and mutagenesis considerations.

For filoviruses, the PALM trial established that monoclonal antibody therapies can improve outcomes for Ebola virus disease when delivered with supportive care, shifting the field from empiricism towards evidence-based outbreak therapeutics. For mpox, tecovirimat has been widely used because of orthopoxvirus rationale and animal-rule approval, but recent human trial evidence for clade I mpox did not demonstrate faster lesion resolution, highlighting the need for outbreak-embedded randomised trials rather than reliance on plausible mechanism alone. The evidence base remains structurally uneven: SARS-CoV-2 offers abundant structural, clinical and population data, while Lassa virus, Crimean-Congo haemorrhagic fever virus, hantaviruses and Rift Valley fever virus remain comparatively under-characterised. This asymmetry can make publication volume appear equivalent to biological importance. Lassa-virus diversity and immune evasion complicate vaccine generalisation; CCHFV combines tick ecology with incomplete correlates of protection; hantavirus pathogenesis centres on endothelial dysfunction despite limited specific therapy; and RVFV risk spans livestock, vectors and human disease. Conflicting evidence commonly arises from model choice, because receptor overexpression can exaggerate permissiveness, pseudoviruses isolate entry while omitting replication and immune antagonism, organoids improve tissue relevance but incompletely reproduce systemic immunity, and animal models may not capture human transmission.

The review proposes a deliberately modular sequence-to-decision standard in which a novel signal passes through five stages: detection and metadata review; family and structural triage; functional testing of entry, replication and innate responses; explicit evidence grading; and a proportionate decision about surveillance, assay updating, countermeasure screening or clinical research. Artificial intelligence can accelerate protein-structure prediction, variant prioritisation, epitope screening and surveillance anomaly detection, yet confidence scores do not establish biological activity, host range or clinical relevance, and training-data bias, poor representation of disordered complexes and limited prospective validation require that AI outputs remain auditable hypotheses tested against experimental and epidemiological data. Across virus families, three implementation priorities recur: assay standardisation so that variant phenotyping does not depend on isolated laboratories using incomparable methods; predefined connections between molecular evidence and public-health thresholds, such as repeated detection of a high-risk virus in a new host or convergent mutations at receptor-binding sites; and sustainable capacity in genomic sequencing, biobanking, bioinformatics, clinical trials and vaccine delivery that must exist before a crisis, especially in regions where spillover and neglected outbreaks are most likely to occur. Molecular systems analysis cannot predict emergence with certainty, but evidence-graded integration can shorten the interval between a credible signal and a proportionate response while reducing both false reassurance and unjustified alarm.

Subject of Research: Molecular mechanisms of viral emergence, immune evasion and cross-species adaptation across major virus families, with a sequence-to-decision framework for precision countermeasures

Article Title: Molecular systems of emerging viruses: replication, host immune evasion, cross-species adaptation and precision countermeasures

Article References: Hejran, A. B., Niazi, P., & Sangin, M. Y. (2026). Molecular systems of emerging viruses: replication, host immune evasion, cross-species adaptation and precision countermeasures. Frontiers in Virology, 6, Article 1926007. https://doi.org/10.3389/fviro.2026.1926007

Image Credits: AI Generated

DOI: 10.3389/fviro.2026.1926007

Keywords: emerging viruses, systems virology, SARS-CoV-2, immune evasion, cross-species transmission, genomic surveillance, CRISPR diagnostics, vaccine platforms, antivirals, One Health, zoonotic spillover, viral replication

Powassan virus exists in two distinct lineages. Lineage I corresponds to the classic Powassan virus, historically associated with Ixodes cookei ticks and small mammals in the northeastern and midwestern parts of the continent, while lineage II corresponds to deer tick virus, carried primarily by Ixodes scapularis, the same blacklegged tick that transmits Lyme disease bacteria. Despite high prevalence of infection in ticks and evidence of a broad geographic distribution, fewer than 50 human cases are detected annually, a figure that has long implied a substantial burden of undetected asymptomatic infections. What has been missing, the authors note, is any systematic experimental link between the virus’s genetic diversity and its disease potential.

To address that gap, the team assembled a geographically diverse panel of isolates and subjected each to a battery of in vitro assays. They measured plaque morphology, a classic virological readout in which the visible zones of cell death caused by viral spread reflect how efficiently a virus moves from cell to cell. They then quantified replication kinetics across multiple mammalian cell lines, including baby hamster kidney cells and deer mouse kidney cells, using standardized culture conditions and defined multiplicities of infection. These assays allowed the researchers to ask whether viruses drawn from different lineages and different places behave in measurably different ways when placed in identical laboratory environments.

The answer was emphatically yes. Plaque size varied significantly among the isolates, and the variation was not random. Viruses belonging to lineage II that were isolated in the Northeast generated significantly smaller plaques than both lineage I viruses and lineage II viruses from the Midwest. In flavivirology, plaque size can correlate with growth rate, cell-to-cell spread mechanisms, and sometimes virulence, although the relationship is context-dependent and should be interpreted cautiously. The fact that northeastern deer tick viruses form characteristically smaller plaques suggests that this geographically defined subset of the Powassan virus population carries functional differences that are detectable even under the uniform conditions of cell culture.

Replication assays reinforced the picture of a phenotypically diverse virus population. Across all cell lines tested, the isolates showed broad differences in how efficiently they multiplied, and lineage I viruses often reached significantly higher titers than their lineage II counterparts. This is a notable finding because it means that two viruses circulating in the same general region, infecting the same types of ticks and potentially the same host species, can differ markedly in their fundamental growth properties. If such differences carry over into infection of vertebrate hosts or tick vectors, they could influence transmission efficiency, tissue tropism, or the likelihood that an infection produces symptomatic disease.

To explore whether these viruses can adapt to a rodent host relevant to their natural ecology, the researchers serially passaged all of the isolates on deer mouse kidney cells. Deer mice, Peromyscus maniculatus, are plausible amplifying hosts in the sylvatic cycle of Powassan virus, so changes that emerge during adaptation to their cells may hint at host-specific selective pressures. After serial passage, sequencing revealed that two residues in the viral envelope protein, at positions 164 and 308, had changed in two of the viruses. The envelope protein is the major surface glycoprotein of flaviviruses, mediating receptor binding and membrane fusion, so substitutions in this protein can alter host range, entry efficiency, and antibody sensitivity. The authors are careful to state that the functional role of these particular mutations remains unknown, and further work will be needed to determine whether they represent genuine host adaptation or incidental changes arising during laboratory culture.

The study’s design has important strengths. By using 17 isolates from 14 locations rather than a handful of laboratory-adapted reference strains, the team captured a snapshot of natural variation that is often lost when virologists work with a single prototype virus. Serial passage experiments and standardized assays performed in parallel reduce the risk that observed differences are artifacts of inconsistent handling. The inclusion of a rodent cell line ties the work to the ecological context in which the virus actually circulates, between ticks and small mammals, with humans serving as incidental, dead-end hosts.

At the same time, the authors are appropriately measured about what the findings do and do not demonstrate. Differences in plaque size and replication titer are in vitro phenotypes, measured in cell culture conditions that simplify the enormously complex environment of a living host. The degree to which these laboratory traits reflect differences in transmission dynamics or human pathogenesis remains to be determined. Establishing that connection will require infection studies in animal models, comparisons with clinical outcomes in human cases, and possibly reverse genetics experiments in which the envelope substitutions at positions 164 and 308 are engineered into infectious clones to test their effects directly.

The public health stakes of this line of research are considerable. Powassan virus can cause severe encephalitis, and there is no approved antiviral treatment or widely deployed human vaccine. Roughly 10 percent of recognized severe cases are fatal, and many survivors suffer lasting neurological damage. As blacklegged ticks expand their range and infection prevalence in ticks rises in endemic areas, understanding whether certain viral lineages or geographic subpopulations pose greater risks becomes a pressing question. A virus population that is genetically and phenotypically diverse, as this study suggests, is also a population with more raw material for evolutionary change, including changes that could affect transmissibility or virulence.

The work also offers a foundation for surveillance. If in vitro phenotypes can eventually be mapped onto specific genetic signatures, public health laboratories could potentially gauge the biological characteristics of newly detected viruses from sequence data alone, without the need to culture each isolate. In the nearer term, the Colorado State team’s findings serve as a corrective to any assumption that Powassan virus is a monolithic threat. The virus circulating in North American ticks is a collection of distinct biological entities, and the ones infecting a hiker in Maine may behave quite differently, at least in the laboratory, from those in Wisconsin or Ontario. Sorting out which of those laboratory differences matter for human disease is now the field’s central task.

Subject of Research: Phenotypic diversity of North American Powassan virus isolates in cell culture

Article Title: North American Powassan virus encompasses diverse in vitro phenotypes

Article References: Gallichotte, E. N., McMinn, R. J., Kimball, K. X., E. Trammell, C., Marsico, B., Savitt, A., Piantadosi, A., Telford, S. R., & Ebel, G. D. (2026). North American Powassan virus encompasses diverse in vitro phenotypes. Virology Journal. https://doi.org/10.1186/s12985-026-03330-y

Image Credits: AI Generated

DOI: 10.1186/s12985-026-03330-y

Keywords: Powassan virus, tick-borne virus, orthoflavivirus, deer tick virus, plaque size, viral replication, envelope protein, lineage I, lineage II, virus evolution, tick-borne encephalitis, in vitro phenotypes

The discovery emerged from a systematic examination of how the GBP family behaves during HSV-1 infection. When the researchers depleted GBP7 from infected cells, viral replication dropped markedly, an outcome opposite to what would be expected from a conventional antiviral factor. The effect was not subtle. Knockdown of GBP7 produced significant antiviral consequences, and the team traced this activity to the enzyme’s catalytic core: when its GTPase function was disabled, the proviral effect vanished. In other words, it is not merely the presence of GBP7 but its enzymatic activity that matters, a detail that would later prove central to both the mechanism and the therapeutic strategy that grew out of the work.

To determine whether the phenomenon extended beyond the culture dish, the investigators turned to mice engineered to lack the GBP7 gene. When these GBP7-deficient animals were infected with HSV-1, they fared substantially better than their wild-type counterparts. Survival rates climbed, and the clinical symptoms of infection were milder across the course of the disease. The in vivo data confirmed that the proviral role observed in cells translates into a meaningful determinant of disease severity in a living host. For a gene induced by interferon, the canonical signal of antiviral mobilization, to instead enhance viral fitness in vivo represents an unexpected twist in the biology of this protein family.

The search for a mechanism led the team to the translational apparatus of the host cell itself. Using immunoprecipitation followed by mass spectrometry, a technique that captures a bait protein along with everything physically bound to it and identifies the passengers, the researchers found that GBP7 associates with eukaryotic translation elongation factor 1 alpha 1, or eEF1A1. This factor is one of the most abundant and essential components of the protein synthesis machinery, responsible for delivering aminoacylated transfer RNAs to the ribosome during each elongation cycle. Binding between GBP7 and eEF1A1 was subsequently validated with direct affinity assays, establishing that the interaction is physical and specific rather than an artifact of the screening method.

What makes the partnership consequential is its effect on the enzymology of translation. eEF1A1 is itself a GTPase: each cycle of tRNA delivery requires the factor to bind GTP, and the energy of GTP hydrolysis drives the conformational changes that allow the factor to release the tRNA into the ribosomal acceptor site and dissociate for another round. The study found that GBP7 uses its own GTPase activity to assist eEF1A1 in this hydrolysis step, effectively speeding up the elongation factor’s catalytic cycle. With eEF1A1 working faster, the overall rate of protein translation in the cell rises. For a virus like HSV-1, which commandeers the host translational machinery to mass-produce its own proteins, a turbocharged elongation factor is a direct growth advantage.

The consequences for the virus follow logically from the biochemistry. With GBP7 present and active, eEF1A1 accelerates protein synthesis, viral proteins accumulate more rapidly, and viral replication proceeds at a higher rate, deepening the infection. When GBP7 is removed, or when its GTPase activity is lost, the boost disappears and the virus replicates more poorly. This mechanistic chain, from GTPase-assisted GTP hydrolysis to enhanced elongation to increased viral output, provides a coherent explanation for every observation in the study, from the cell culture knockdown experiments to the improved survival of GBP7-deficient mice. It also reframes GBP7 as part of a growing appreciation that interferon-stimulated genes can cut both ways, sometimes serving pathogens that have learned to exploit the host’s defensive mobilization.

Identifying a host factor that a virus exploits naturally raises the question of whether that dependency can be turned against the pathogen. Because the proviral effect requires GBP7’s catalytic activity, the enzyme’s GTP-binding and hydrolysis site presents an obvious drug target. The team pursued this avenue through high-throughput virtual screening, computationally docking large libraries of candidate molecules against the protein in search of compounds predicted to occupy and disable its active site. Among the hits that emerged was S-adenosylhomocysteine, or SAH, a well-known metabolite that is produced naturally in cellular methylation reactions as the byproduct of S-adenosylmethionine-dependent methyltransferase activity.

Experimental testing confirmed the computational prediction. SAH bound and inhibited GBP7, and in cells infected with HSV-1 the compound displayed potent antiviral activity, suppressing replication in a manner consistent with the loss of GBP7’s proviral function. Notably, the authors report that SAH showed broad-spectrum potential, suggesting that the GBP7-eEF1A1 axis may be exploited by viruses beyond herpes simplex virus 1. If that holds up in further studies, a strategy aimed at dampening this host pathway could offer a template for antivirals that are harder for viruses to escape, since the target is a host protein rather than a viral one. Host-targeted antivirals generally impose a higher genetic barrier to resistance, because the pathogen cannot simply mutate the drugged protein.

The findings also carry implications for how the GBP family is understood. GBPs have been studied most intensively in the context of immunity to intracellular bacteria and parasites, where several members direct autophagy, inflammasome activation and direct antimicrobial attack. GBP7 had remained comparatively obscure, and the new work assigns it a defined and surprising role: an interferon-induced GTPase that potentiates translation elongation through a direct physical partnership with eEF1A1. That a family member deployed during antiviral signaling would instead facilitate a viral infection underscores how incomplete the functional annotation of interferon-stimulated genes remains, and it raises the possibility that other GBPs or related GTPases may harbor similarly counterintuitive activities in different infection contexts.

For the field of antiviral research, the study offers both a mechanism and a starting point. The GBP7-eEF1A1 axis is now a defined node at which viral replication can be throttled, and SAH provides a chemical lead whose activity against HSV-1 can be optimized and whose spectrum can be mapped. At the same time, many questions remain open, including precisely how GBP7’s GTPase cycle is coupled to eEF1A1’s hydrolysis step, whether the interaction is regulated during the interferon response, and how inhibiting the axis in patients might affect normal cellular protein synthesis, given eEF1A1’s essential housekeeping role. What the work establishes firmly is that the boundary between host defense and viral exploitation is thinner than the textbook account of the GBPs suggests, and that a protein summoned to the front lines of the immune response can end up serving the enemy’s supply chain.

Subject of Research: The role of the GTPase GBP7 and the translation factor eEF1A1 in herpes simplex virus 1 replication and host-targeted antiviral therapy

Article Title: GTPase GBP7 promotes HSV-1 infection by interacting with and activating eEF1A1 to accelerate the protein translation process

Article References: Wang, J., Man, L., Rong, B., Lu, K., Cheng, L., He, J., Li, H., Su, H., Lin, X., Li, R., Zou, M., Yang, C., & Liu, S. (2026). GTPase GBP7 promotes HSV-1 infection by interacting with and activating eEF1A1 to accelerate the protein translation process. PLOS Pathogens, 22(10), e1014652. https://doi.org/10.1371/journal.ppat.1014652

Image Credits: AI Generated

DOI: 10.1371/journal.ppat.1014652

Keywords: GBP7, guanylate-binding proteins, HSV-1, herpes simplex virus, eEF1A1, protein translation, GTPase, interferon, host-targeted antivirals, S-adenosylhomocysteine, viral replication, PLOS Pathogens

NS2A belongs to the nonstructural complement of proteins encoded by orthoflaviviruses, a genus that includes Zika, dengue, yellow fever, Japanese encephalitis, and West Nile viruses. These viruses carry their genetic material as a single positive-sense RNA strand, which upon entry into a host cell is translated into one long polyprotein. Viral and host proteases then slice this polyprotein into the individual structural and nonstructural proteins. NS2A is among the smallest and least understood of these products. It is markedly hydrophobic, meaning it embeds itself in cellular membranes, and it has resisted both structural determination and routine laboratory detection because, until recently, no protein-specific antibodies were available to track it.

Despite these technical obstacles, genetic studies had long hinted that NS2A was doing double duty. Mutations in the protein could impair the replication of the viral RNA genome, while other changes seemed to disrupt the assembly and release of new virions, the infectious particles that spread the infection to neighboring cells. Whether one form of the protein performed both tasks, or whether the protein existed in functionally distinct states, remained unknown. The new work addressed this question directly by taking advantage of endogenously tagged viral RNAs, in which a detectable molecular tag was engineered into the viral genome itself so that the NS2A protein could be visualized during authentic infection without relying on antibodies that did not exist.

Using this tagging strategy, the researchers made the central observation of the study: during Zika virus infection, two NS2A isoforms are present in infected cells. The first is the full-length protein, complete with its entire C-terminal region. The second is a shorter version, truncated at its C-terminus, which the team named NS2Aα. The existence of this truncated species had been suspected from earlier work on related viruses, but its origin and biological purpose had not been firmly established. Its detection in the context of genuine viral infection, rather than in simplified expression systems, lent considerable weight to the conclusion that both forms are genuine products of the viral life cycle.

The team then asked how the two isoforms arise. Because orthoflaviviruses do not encode a large arsenal of processing enzymes, the most likely candidate was the viral protease, the enzyme complex responsible for cleaving the polyprotein at defined junctions. Phylogenetic and conservation analyses across the Orthoflavivirus genus, combined with direct polyprotein cleavage assays, supported this interpretation. The cleavage site that separates the full-length protein from its truncated counterpart appears to be conserved across the genus, suggesting that the production of two NS2A isoforms is not a quirk of Zika virus alone but a shared feature of this entire group of medically important pathogens.

Conservation of this kind is usually a strong signal of functional importance. If a processing event is maintained across viruses as diverse as dengue, Zika, and yellow fever, it implies that natural selection has preserved it because the viruses that carry it reproduce more successfully. The corollary is that interfering with the generation of either isoform might cripple the virus, a possibility that immediately interests antiviral researchers. A protease cleavage site that must be cut precisely, and whose disruption halts particle production, represents a potential vulnerability that small-molecule drugs could in principle exploit.

The functional dissection of the two isoforms formed the core of the study. Using established assays for viral RNA replication and for the production of infectious particles, the researchers showed that the two forms do not overlap in their responsibilities. Full-length NS2A is required for viral RNA replication, the process by which the viral genome is copied many times over inside specialized membrane structures within the infected cell. The truncated isoform NS2Aα, by contrast, is important for virion assembly, the later stage in which newly synthesized RNA is packaged into particles that can exit the cell and infect new hosts. Neither isoform could substitute for the other in these experiments, establishing a mutually exclusive division of labor.

This division of labor makes considerable mechanistic sense when viewed against what is known about flavivirus replication architecture. RNA replication occurs in virus-induced membrane compartments where the replication complex concentrates the enzymes and templates needed for genome synthesis, and the membrane topology of full-length NS2A is thought to help organize these structures. Virion assembly, meanwhile, takes place at the endoplasmic reticulum, where the capsid protein condenses around viral RNA and the particle buds into the lumen before trafficking through the secretory pathway. A C-terminally truncated NS2A, lacking the segment present in the full-length protein, would present a different membrane topology and a different set of interaction surfaces, plausibly better suited to recruiting assembly factors than to supporting the replication complex.

The broader significance of the work lies in reframing how virologists think about small hydrophobic viral proteins. NS2A has often been treated as a single entity whose many phenotypes reflect one protein wearing many hats. The new data indicate instead that the virus deliberately generates two molecular species from the same genomic region, each optimized for a distinct stage of infection. This strategy is economical, extracting two functions from one stretch of sequence, and it also introduces a regulatory dimension: the ratio of the two isoforms, governed by the efficiency of the protease cleavage event, could influence the balance between genome amplification and particle production at different phases of infection.

For the field of antiviral development, the study opens several concrete avenues. Compounds that block the cleavage event generating NS2Aα would be expected to impair virion assembly without directly touching RNA replication, offering a mechanism of action distinct from existing nucleoside analogs and protease inhibitors aimed at other flaviviral targets. Conversely, molecules that destabilize full-length NS2A would strike at genome replication. Because both isoforms and their generating cleavage site appear conserved across the Orthoflavivirus genus, a single intervention might hold promise against multiple viruses, from dengue to West Nile. The work also provides the research community with tagged viral RNA tools and a clearer conceptual map of NS2A function, resources likely to accelerate the structural studies that this elusive protein has so long resisted. As Zika and its relatives continue to circulate in tropical and temperate regions alike, understanding the molecular choreography of proteins as small and slippery as NS2A is a necessary step toward therapies that can keep pace with them.

Subject of Research: The dual roles of Zika virus NS2A protein isoforms in viral RNA replication and virion assembly

Article Title: Zika virus produces two isoforms of the NS2A protein that play mutually exclusive roles in viral replication and virion assembly

Article References: Landry, B. N., Klann, O. J., Hauch, H. C., Bemis, C. L., Owen, J. E., Harris, J., Barnard, T. R., Neufeldt, C. J., & Sagan, S. M. (2026). Zika virus produces two isoforms of the NS2A protein that play mutually exclusive roles in viral replication and virion assembly. PLOS Pathogens, 22(10), e1014609. https://doi.org/10.1371/journal.ppat.1014609

Image Credits: AI Generated

DOI: 10.1371/journal.ppat.1014609

Keywords: Zika virus, NS2A, orthoflavivirus, viral replication, virion assembly, viral protease, polyprotein processing, PLOS Pathogens, flavivirus, NS2Aα, antiviral targets, virology

The research team set out to determine whether dampening this kinase pathway could influence the course of infection with A/Puerto Rico/8/1934, the laboratory H1N1 strain universally abbreviated as PR8. Their experimental design was deliberately multi-layered. In cell culture, they infected susceptible cells with PR8 and treated them with GSK583 at defined concentrations, then quantified viral RNA and viral protein accumulation inside the cells. To measure the production of infectious progeny, they performed plaque-forming assays on culture supernatants, a classical virological technique in which diluted samples are overlaid onto fresh monolayers of cells and each infectious particle gives rise to a visible zone of cytopathic effect. In parallel, they moved to a mouse model of PR8 infection, treating animals early after inoculation and tracking body weight, disease severity, pulmonary viral load, and inflammatory markers over the course of the experiment.

The cellular results were consistent and pointed in a single direction. GSK583 reduced both the amount of viral RNA and the abundance of viral protein within infected cells, and supernatants collected from treated cultures contained significantly fewer plaque-forming units, indicating that the compound suppressed the release of infectious virions rather than merely distorting intracellular readouts. Importantly, the investigators used time-of-addition experiments to pinpoint where in the viral life cycle the drug exerted its effect. In this approach, the compound is added at different times relative to infection — before the virus is introduced, during the attachment phase, or only after adsorption has been completed. If a drug directly inactivates virions or blocks their binding to cell surface receptors, it must be present early to show activity. GSK583 failed to directly inactivate PR8 and did not measurably inhibit viral attachment under the conditions tested; instead, its antiviral effect became detectable only after the virus had adsorbed to cells. This pattern is characteristic of a compound that interferes with post-entry stages of replication or with host signaling pathways that the virus co-opts.

Having established antiviral activity in vitro, the team turned to the mouse model, where the stakes for host-directed therapy are higher because the immune system contributes both to viral clearance and to tissue damage. When GSK583 was administered early after infection, treated animals experienced alleviated disease severity compared with untreated controls. Quantitative assessment of lung tissue revealed reduced signals for the viral nucleoprotein — both its RNA transcripts and the antigen itself — indicating that viral replication in the respiratory tract was genuinely blunted, not simply redistributed. Alongside the reduction in viral load, the treated mice showed lower inflammatory readouts in the lung. This dual effect is notable because excessive inflammation is a hallmark of severe influenza, contributing to acute lung injury and, in the worst cases, acute respiratory distress syndrome, a syndrome that the authors explicitly reference in framing the clinical significance of their work.

The mechanistic core of the study concerns the RIPK2–MKK7–JNK signaling module. RIPK2 is a kinase with well-documented roles in innate immunity, particularly as an adaptor that transduces signals from NOD-like pattern recognition receptors, and the mitogen-activated protein kinase cascade it engages includes MKK7, a dual-specificity kinase that phosphorylates and activates JNK. JNK, in turn, is a stress-activated protein kinase whose phosphorylation status is a sensitive indicator of inflammatory and stress signaling intensity. Using protein interaction assays, the investigators demonstrated that the physical association between RIPK2 and MKK7 was linked to PR8 replication-related readouts under their experimental conditions, and that JNK phosphorylation tracked with viral replication. Genetic knockdown experiments and pharmacological inhibition corroborated the involvement of this axis, providing convergent lines of evidence rather than relying on a single technique.

Against this mechanistic backdrop, the effect of GSK583 became interpretable. Treatment with the compound was accompanied by a reduction in the RIPK2–MKK7 interaction and a corresponding decrease in JNK phosphorylation, findings consistent with the hypothesis that the drug works by loosening a signaling complex that influenza virus either requires or exploits. The authors are careful in their language here: they describe the data as consistent with a potential link between the signaling module and the observed antiviral effect, rather than claiming definitive causation. This restraint is scientifically appropriate, because RIPK2 has pleiotropic functions and inhibitor specificity is never absolute, a caveat the team acknowledges directly when they call for further studies to define the mechanistic specificity of the pathway in influenza infection.

Beyond the JNK arm, the study documented effects on a second major inflammatory pathway. GSK583 treatment reduced the phosphorylation of NF-κB p65, the canonical subunit of the transcription factor that drives expression of a large suite of pro-inflammatory genes, and it lowered the transcript levels of selected inflammatory cytokines in infected cells. The intersection of these two pathways is significant for influenza biology. The JNK and NF-κB cascades are both activated during influenza infection, both contribute to cytokine storm pathology, and both have been implicated in supporting various stages of the viral life cycle in prior literature. A single compound that dampens phosphorylation events in both cascades while simultaneously reducing viral replication therefore offers a pharmacologically attractive profile, at least in the models examined.

The translational implications deserve careful framing. GSK583 was developed in an industrial setting as a RIPK2 inhibitor, and its repurposing for influenza represents the kind of cross-indication thinking that host-directed antiviral research increasingly encourages. Because the target is a host protein, the theoretical barrier to viral escape is much higher than for virus-targeted drugs: the virus cannot simply mutate its own polymerase to render a host-directed compound obsolete. Moreover, a host-directed agent could in principle retain activity against multiple influenza subtypes and even against unrelated respiratory viruses that co-opt overlapping signaling networks. However, host-directed approaches carry their own risks, since interfering with innate immune signaling can impair genuine host defense, and the therapeutic window must be established empirically. The mouse data showing alleviated disease severity with early treatment are encouraging on this point, but early intervention windows are a known limitation of antiviral therapy in practice, because patients frequently present after peak viral replication has occurred.

The study’s methodological rigor is worth noting for readers assessing the strength of the conclusions. Cytotoxicity considerations were built into the design, with the abstract referencing the half-maximal cytotoxic concentration, the half-maximal inhibitory concentration, and the selectivity index — standard pharmacological metrics that distinguish genuine antiviral activity from nonspecific toxicity. Statistical analyses employed Student’s t-tests, analysis of variance, and log-rank tests as appropriate to the data structure, and all animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee of Huazhong University of Science and Technology. The authors also flag that the paper was released as an accepted manuscript under accelerated sharing, subject to final editorial production, and they explicitly position their findings as proof-of-concept rather than as a validated therapeutic strategy.

What, then, does this work contribute to the field? It extends the map of host factors that influenza virus interacts with, placing RIPK2-associated signaling squarely within the network of pathways whose manipulation affects viral replication. It provides a concrete pharmacological tool — a compound with an existing development history — that the community can use to probe this pathway further. And it demonstrates, in both cellular and animal systems, that attenuating this signaling module can simultaneously reduce viral load and inflammatory output, addressing the two faces of influenza pathology in a single intervention. The open questions are equally clear: whether the RIPK2–MKK7 interaction is a direct requirement of the virus or an indirect consequence of altered cellular physiology, whether the effect generalizes beyond PR8 to circulating human strains and other subtypes, and whether the therapeutic window in mammals is wide enough to support clinical development. The authors’ call for further studies on mechanistic specificity and broader applicability is the appropriate next step, and the present findings supply a well-documented starting point for that work.

Subject of Research: Host-directed antiviral activity of the RIPK2 inhibitor GSK583 against A/PR/8 H1N1 influenza virus infection and associated inflammatory signaling

Article Title: GSK583 attenuates viral and inflammatory readouts in an A/PR/8 H1N1 infection model

Article References: Kuang, J., Zhou, J., Xiang, J., Ren, F., Fu, J.-J., Zhu, G., Li, Z., Ruan, H., Tan, X., Zhang, W., Zhou, M., Xu, J., Yang, Q., & Shang, Y. (2026). GSK583 attenuates viral and inflammatory readouts in an A/PR/8 H1N1 infection model. Virology Journal. https://doi.org/10.1186/s12985-026-03283-2

Image Credits: AI Generated

DOI: 10.1186/s12985-026-03283-2

Keywords: GSK583, RIPK2, influenza A virus, H1N1, JNK signaling, MKK7, NF-κB, host-directed antivirals, PR8 mouse model, inflammation, viral replication, antiviral therapy

Cowpea mild mottle virus, formally named Carlavirus vignae, is a betaflexivirus with a flexible filamentous particle and a single-stranded, positive-sense RNA genome. It infects soybean and other legume crops across tropical and subtropical regions, causing mosaic symptoms, leaf mottling and yield losses that make it a growing concern for soybean production. Like all positive-sense RNA viruses, it must co-opt host cell machinery to copy its genome, yet for betaflexiviruses the identity of the plant proteins involved in that process has remained largely a mystery. The new study, led by Larissa G. Zanardo, F. Murilo Zerbini and Claudine M. Carvalho of the Universidade Federal de Viçosa and collaborators at several Brazilian institutions, set out to close that gap by searching systematically for soybean proteins that contact the viral replication machinery.

The team’s starting point was the viral RNA-dependent RNA polymerase, the enzyme at the heart of genome replication. For many RNA viruses, the polymerase sits at the center of a multiprotein replication complex that also includes host factors, membranes and viral accessory proteins. Identifying which host proteins are pulled into this complex is therefore a critical step toward understanding, and potentially disrupting, the infection cycle. To find such partners, the researchers constructed a soybean complementary DNA library and screened it against the CPMMV polymerase domain using a yeast two-hybrid assay, a technique in which two candidate proteins are fused to separate halves of a transcription factor; if the proteins interact, the reassembled factor switches on reporter genes that make the yeast colonies change color.

Controls were carefully built into the screen. The viral polymerase domain fused to the yeast DNA-binding domain proved neither toxic to the yeast cells nor capable of switching on reporters on its own, ruling out false positives from self-activation. When the full soybean library was interrogated, one clone that repeatedly came through the screen encoded GmASMT8, a member of the N-acetylserotonin O-methyltransferase family. These enzymes are best known in plants for catalyzing steps in the melatonin biosynthesis pathway, converting N-acetylserotonin into melatonin, a molecule implicated in stress responses and development. A role for such an enzyme in viral replication had not previously been described for this virus family.

To confirm that the interaction was genuine rather than an artifact of the yeast system, the researchers turned to bimolecular fluorescence complementation. In this approach, the two candidate proteins are each fused to one nonfluorescent half of a fluorescent protein and expressed together in plant cells; fluorescence is restored only if the proteins come into close proximity. The assay again supported a physical association between the CPMMV polymerase domain and GmASMT8, providing independent evidence in a plant cellular environment that the two proteins meet during infection.

Subcellular localization studies then revealed where these encounters take place. GmASMT8 was found distributed in the cytoplasm, the expected location for a soluble metabolic enzyme. The viral polymerase domain, by contrast, formed punctate structures associated with the endoplasmic reticulum, a pattern consistent with the membrane-associated replication sites that many positive-sense RNA viruses build on ER membranes. The cytoplasmic distribution of GmASMT8 overlaps with these ER-associated viral structures, making the cytoplasm the plausible arena in which the host enzyme and the viral polymerase interact to support genome replication.

Correlation alone does not establish function, so the team next asked whether GmASMT8 actually matters for viral multiplication. Gene expression analysis showed that GmASMT8 transcript levels rise during CPMMV infection, indicating that the plant’s own program responds to the virus by producing more of the enzyme. The researchers then manipulated the gene in both directions. When GmASMT8 was overexpressed in soybean protoplasts, single plant cells stripped of their walls that are a standard platform for testing viral replication, CPMMV accumulated to higher levels than in control cells. Conversely, when GmASMT8 expression was knocked down in whole soybean plants using virus-induced gene silencing, a technique that harnesses an unrelated viral vector, in this case bean pod mottle virus, to trigger the plant’s RNA interference machinery against a target gene, viral accumulation dropped markedly.

The silencing experiments were validated with appropriate controls. A construct targeting the phytoene desaturase gene produced the characteristic photobleached phenotype, confirming that the vector system efficiently silenced soybean genes, and quantitative measurements confirmed that GmASMT8 transcripts were reduced in plants receiving the GmASMT8 silencing construct over multiple time points. Statistical comparisons across treatments supported the conclusion that reduced GmASMT8 levels translate into reduced viral loads. Taken together, the gain-of-function and loss-of-function results position GmASMT8 as a positive regulator of CPMMV accumulation, meaning the virus benefits from the presence of this host enzyme throughout its infection cycle.

The broader significance of the work lies in what it reveals about a poorly charted corner of plant virology. Betaflexiviridae includes economically important genera such as Carlavirus, Potexvirus, Trichovirus and Foveavirus, whose members infect crops ranging from potatoes and grapes to fruit trees and ornamentals. Despite this agricultural importance, the host factors that support betaflexivirus replication have remained largely unknown, in contrast to the extensive host-factor catalogs available for viruses such as potyviruses or tombusviruses. By identifying GmASMT8 as a partner of the CPMMV polymerase domain, the Brazilian team has delivered the first report of a host protein interacting with the replication enzyme of any Betaflexiviridae member, establishing a foundation for comparative studies across the family.

The study also raises intriguing questions about why a melatonin-biosynthetic enzyme would serve a virus. One possibility is that GmASMT8 contributes a biochemical activity that directly assists RNA synthesis or the assembly of replication complexes. Another is that the virus manipulates the enzyme’s normal role in stress hormone metabolism, perhaps dampening antiviral responses that depend on melatonin or related signaling molecules. Distinguishing between these mechanisms will require further experiments, but the practical implications are already apparent. Host factors that viruses depend on are attractive targets for resistance breeding and genome editing, because disrupting a host protein that a virus needs can confer broad and durable resistance while imposing minimal cost on the plant. If GmASMT8 proves dispensable for normal soybean growth under field conditions, reducing its availability to the virus, or blocking the protein-protein interaction with the viral polymerase, could become a strategy for protecting soybean crops against cowpea mild mottle virus. For now, the study stands as a reminder that even well-studied cellular enzymes can harbor hidden roles in the arms race between plants and their pathogens.

Subject of Research: Host factor GmASMT8 in the replication of the betaflexivirus Carlavirus vignae in soybean

Article Title: The betaflexivirus Carlavirus vignae recruits N-acetylserotonin O-methyltransferase 8 (GmASMT8) during its replication in soybean

Article References: Zanardo, L. G., Barbosa, T. M. C., Alves, M. S., Queiroz, S. S., Bruckner, F. P., Silva, F. N., Zerbini, F. M., & Carvalho, C. M. (2026). The betaflexivirus Carlavirus vignae recruits N-acetylserotonin O-methyltransferase 8 (GmASMT8) during its replication in soybean. Virology Journal. https://doi.org/10.1186/s12985-026-03293-0

Image Credits: AI Generated

DOI: 10.1186/s12985-026-03293-0

Keywords: CPMMV, GmASMT8, Betaflexiviridae, Carlavirus, soybean, RNA-dependent RNA polymerase, viral replication, host-virus interaction, yeast two-hybrid, virus-induced gene silencing, plant virology, melatonin biosynthesis