Engineered DNA viruses are increasingly used as delivery vectors for gene therapy. Among these, the most promising viral vectors are those that do not naturally integrate into the cellular genome but instead persist as episomes, such as adeno-associated viral vectors (rAAV) and integration-deficient lentiviral vectors (IDLV). In this study, we characterize the role of replication protein A1 (RPA1), a subunit of the RPA heterotrimer, in regulating gene expression from mCherry-expressing rAAV and IDLV vectors. RPA1 is a single-stranded DNA (ssDNA)-binding protein involved in DNA repair and metabolism. Silencing RPA1 in cells led to increased reporter gene expression from both vector systems, suggesting that this protein negatively regulates viral gene expression. Ectopic expression of wild-type RPA1 reduced reporter expression, confirming that the phenotype was not due to off-target effects. Deletion and point mutant versions of RPA1 were employed to further investigate the genetic and functional requirements for the restriction phenotype observed in rAAV-transduced cells. Our results indicate that the restriction occurs in the nucleus, and depends on the ability of RPA1 to bind ssDNA. Similar results were obtained with self-complementary AAV and IDLV vectors. Furthermore, RPA1 depletion increased cellular DNA damage, while RPA1 overexpression restored basal damage levels in a ssDNA binding activity dependent manner. Importantly, the increase in reporter expression correlates with elevated DNA damage, implying that episomal vector DNA may integrate at double-strand breaks generated upon RPA1 loss. Ongoing studies aim to directly confirm this mechanism. Overall, this work identifies RPA1 as a key negative regulator of episomal vector expression, linking DNA repair with the control of transgene integration and expression.

The flavivirus nonstructural protein 5 (NS5) performs multiple functions that are essential to viral infection, including replicating and capping the viral RNA and antagonizing the host type I interferon (IFN) response. While flavivirus NS5 proteins inhibit IFN signaling through distinct mechanisms—implying evolutionary flexibility—how these activities coexist and evolve within the same protein remains poorly understood. We mapped the genetic determinants of Zika virus (ZIKV) NS5-mediated STAT2 antagonism and compared them to replication constraints defined by deep mutational scanning (DMS). Antagonism and replication determinants overlapped, and no single amino acid substitution disrupted antagonism without also impairing replication. By resolving both fitness landscapes in parallel, we identified three partially functional substitutions that, when combined, produced replication-competent viruses with markedly reduced IFN antagonism and severe attenuation in a humanized STAT2 mouse model. These findings reveal a fundamental constraint on viral evolution, show how multifunctional viral proteins can limit adaptability—even under immune pressure, and provide the clearest evidence to date that IFN antagonism is essential for in vivo pathogenesis.

The hepatitis E virus (HEV) poses a major global health challenge; however, no specific antiviral treatments are available, and the understanding of viral life cycle steps including cellular entry is very limited. While it is known that HEV infection depends on endolysosomal trafficking of virions to lysosomal compartments to initiate host cell entry, the underlying molecular determinants governing this process remain poorly understood.

Using a mini-arrayed CRISPR/Cas9 screen, we identified the lysosomal cholesterol transporter Niemann–Pick disease type C1 (NPC1) as a novel host dependency factor required for HEV infection. The importance of NPC1 for enveloped and non-enveloped HEV infection was confirmed through siRNA-mediated knockdown and CRIPSR/Cas9 mediated knockout. Moreover, pharmacological inhibition of NPC1 with itraconazole or U18666A efficiently lowered HEV infection of different strains and genotypes in vitro. Furthermore, NPC1 inhibition in primary human hepatocytes significantly decreased HEV infectivity and led to reduced viral RNA levels in feces in HEV-infected gerbils. Time-of-addition and subgenomic replicon assays revealed a role for NPC1 during viral entry without impacting replication. Ultimately, mechanistic studies using fluorescence microscopy indicate that NPC1 disruption leads to cholesterol accumulation and lysosomal enlargement, trapping viral particles and RNA within endo-/lysosomal compartments.

Collectively, our findings identify NPC1 as a critical host factor required for HEV entry and suggest that repurposing NPC1-targeting drugs could offer a promising new antiviral strategy for HEV treatment.

Mayaro virus (MAYV; genus Alphavirus, family Togaviridae) is a neglected tropical virus with distribution currently limited to Central and South America. Although MAYV infection can lead to a prolonged and debilitating polyarthralgia, there is limited surveillance and no approved antivirals or vaccines. Here, we compare the replication and transcription efficiencies of two MAYV strains with different geographic distributions, TRVL and BeAr, using a highly sensitive trans-replication system. In the trans-replication system, virus replicase proteins are uncoupled from the viral RNA template (i.e., the genomic strand). Initial experiments revealed that the replicase of the TRVL strain is more active in human cells, while the BeAr replicase is more active in mosquito cells. As mosquito and human cells are maintained at different temperatures, we investigated whether replicase activity is temperature-dependent. In human cells incubated at 28°C, TRVL replicase activity was more reduced than that of BeAr, indicating that TRVL replicase is more adapted to higher temperature conditions typical of the human host environment. Chimeric replicases with swapped nsP4, an RNA-dependent RNA polymerase, were constructed. The results with chimeric replicases indicated that if non-structural polyprotein P123 is derived from the BeAr strain, there is less efficient RNA replication in human cells, suggesting that determinants for different replicase activities do not reside within nsP4. Further, chimeric templates with exchanged 5’ UTRs between TRVL and BeAr were used to identify genomic regions responsible for different replicase activities. Replicases belonging to both strains generally replicated and transcribed their own, heterologous, and 5’ UTR-swapped template RNAs with similar efficiencies in human and mosquito cells. Furthermore, template cross-utilisation experiments demonstrated that both MAYV replicases can utilise template RNAs (i.e., genomic strand) of other alphaviruses, albeit with varying efficiencies. Additionally, replicases of other alphaviruses could utilise MAYV templates; however, efficiencies were substantially reliant on the cell type.

Hepatitis E virus (HEV) is a leading cause of acute viral hepatitis, with an estimated 20 million infections annually. HEV genotype 1 (HEV-1) infection, in particular, has been associated with severe disease progression and fulminant hepatitis in pregnant women. Currently, treatment options are limited, and despite notable research progress in recent years, key aspects of the molecular mechanisms and host–virus interactions remain unresolved.

A limiting factor for HEV-1 research has been the lack of an efficient in vitro cell culture system. Here, we established a recently described HEV-1 cell culture model using colorectal adenocarcinoma-derived Caco-2 cells. We were able to produce infectious viral particles, observe and validate infections in Caco-2 cells using immunofluorescence microscopy, and investigate replication efficiencies using a subgenomic HEV-1 replicon. Based on this cell culture model, we deepened our studies, focusing on the initial steps of the viral life cycle. Within the research of NPC1 as a host factor for HEV-3 infection (see the abstract by Emely Richter), we were able to confirm the effectiveness of the NPC1 inhibitor itraconazole during HEV-1 infection, postulating the role of NPC1 as a host factor for HEV-1. Additionally, we verified the effect of the pan-cathepsin inhibitor K11777, confirming the role of cathepsins in HEV-1 entry.

Furthermore, we investigated the effect of different interferon-alpha subtypes on HEV-1 replication. In the future, we want to deepen our research by looking into the mechanisms and pathways modulated during HEV-1 infection by integrating RNA-sequencing of HEV‑1 infected cells.

Overall, this project aims to elucidate the underlying viral and host mechanisms of HEV-1 infection and its pathogenesis.

Several members of the Schlafen (SLFN) protein family exhibit antiviral activity. The antiviral effects of SLFN11, SLFN13, and SLFN14 correlate with their capacity to inhibit translation of viral mRNAs enriched in rare codons, a process that requires their intrinsic endoribonuclease activity. SLFN11 and SLFN13 restrict viral replication by degrading tRNAs, thereby reducing the availability of these tRNAs below a critical threshold necessary for efficient translation of rare-codon–biased viral transcripts. In contrast, the molecular mechanism underlying the antiviral function of SLFN14 remains poorly defined. Previous studies have shown that SLFN14 does not selectively degrade rare-codon–rich mRNAs, suggesting that it may target ribosomal or transfer RNAs within ribosomes engaged in viral mRNA translation. To further elucidate the mechanism of SLFN14-mediated antiviral activity, we mapped the protein regions required for its function. Analysis of a panel of SLFN14 deletion mutants demonstrated that a C-terminally truncated variant (residues 1–373) retained both ribosome-binding and antiviral activity. Furthermore, a mutant lacking both the C-terminal region and an additional N-terminal segment (residues 121–328) remained capable of ribosome association and inhibition of HIV-1 replication. These findings indicate that the region encompassing the catalytic residues (clustered between residues 206 and 249), together with the ribosome-binding domain, is sufficient to mediate the antiviral activity of SLFN14.

Small DNA viruses such as hepatitis B virus (HBV) and adeno-associated virus (AAV) rely extensively on host cellular proteins and pathways to establish productive infections. The formation and maintenance of episomal transcriptional templates are key determinants of HBV persistence and the success of AAV-based gene therapy approaches. However, the production and expression of these episomes are rate-limiting steps in the replication cycles and are primarily regulated by host factors, many of which remain poorly characterized. Previously, our laboratory identified several cellular factors that negatively regulate HBV infection through a loss-of-function genetic screen. In the present study, we characterize the role of the Ku70/Ku80 heterodimer, two of the hits from that screen. Silencing of Ku70 or Ku80 led to increased viral gene expression at both mRNA and protein levels upon HBV infection, confirming their function as negative regulators. Similar effects were observed in AAV vector-transduced cells, indicating a broad-spectrum restrictive effect. Co-expression of wild-type Ku70 and Ku80 rescued the silencing phenotype, while mutants mislocalized to the cytoplasm or defective in DNA binding did not, suggesting that restriction occurs in the nucleus and is independent of Ku's DNA binding capacity. Mechanistic analyses by means o ChIP-qPCR revealed that Ku70 silencing does not alter cellular RNA polymerase II recruitment to the AAV genome, excluding transcriptional regulation as the targeted step. In contrast, Southern blot and qPCR assays demonstrated increased accumulation of viral episomal DNA in Ku70-silenced cells, suggesting that Ku70 negatively regulates viral episomal DNA content. In summary, our findings indicate that the Ku70/Ku80 heterodimer restricts the accumulation of viral episomal transcriptional templates, likely by influencing their formation and/or stability.

Background: We report a vaccine study on memory B/T cell responses after receipt of one of two different 2023 COVID-19 mRNA vaccine formulations.

Memory B/T cell responses over time to the SARS-CoV-2 strain Wuhan-Hu-1, Omicron subvariants BA.4/BA.5, XBB.1.5, and the mutated SARS-CoV-2 BA.2.86/Pirola variant (a predecessor of JN.1 and the KP.2 lineage/FLiRT in the 2024-2025 vaccine) were assessed.

Methods: The previously primed/immunized study subjects (n=50) received the 2023 bivalent ancestral strain/BA.4/BA.5 mRNA vaccine (Group A, n=15) or the monovalent XBB.1.5 mRNA vaccine (Group B, n=35). Blood was collected at baseline, 1 Month, 6 Months, and 12 Months post-vaccination for assessment of memory B cell (MBC) frequencies to different RBD and S antigens (memory B cell Elispot) or CD4⁺ T cell responses (IL-21/Tfh, IFNγ, and IL-2 by Fluorospot). Analyses were performed using the Wilcoxon Rank Sum or Signed Rank Tests.

Results: We found no significant differences in the antigen-specific MBC frequencies between Group A subjects and Group B subjects at any timepoints, except for the results at 6 months (higher MBC responses to all antigens in Group A subjects, p˂0.02).

We observed higher Wuhan-Hu-1 RBD- or S-specific MBC responses compared to all variant-specific MBC responses at all timepoints (p˂0.01), consistent with the “original antigenic sin” experience.

Importantly, we observed a largely reduced MBC response to the SARS-CoV-2 BA.2.86 (RBD, whole S protein) compared to all other MBC responses at all study timepoints (p≤0.01). At Month 1, the BA.2.86 RBD-specific MBC response was 3 to 4.8-fold lower compared to other RBD-specific MBC responses (p˂2E^-06). Limited difference between variant-specific CD4⁺ T cell responses was found.

Conclusion: The RBD and Spike-specific MBC responses to two different vaccine formulations demonstrated laboratory evidence of original antigenic sin. The largely restricted BA.2.86-specific MBC response suggested immune evasion and increased susceptibility to its arising subvariants.

While human astroviruses (HAstVs) are well-recognized as a significant cause of gastroenteritis in children, the elderly, and immunocompromised individuals, they have also been linked to severe neurological complications, such as encephalitis and meningitis, particularly in immunocompromised hosts. Disease onset begins with viral replication in the intestinal epithelium, but in some cases the infection may disseminate to the central nervous system. For other enteric viruses, association with extracellular vesicles (EVs) upon cell release has been identified as a factor promoting dissemination beyond the intestine and immune evasion. In addition to classic HAstVs, two novel HAstV clades containing neurotropic strains (VA and MLB) were described, with a less clear association with gastroenteritis.

Our work aims to explore the effects of novel MLB HAstV on the intestinal epithelium using human intestinal enteroids (HIEs) and determine the process of virus egress and the phenotype of released virions. MLB2 HAstV may infect differentiated HIEs, and released virions are strongly associated with EVs, without causing cell lysis. Infection of polarized HIEs on transwells shows that virus release predominantly occurs through the apical membrane as EV-associated virions, with a density of 1.10-1.13 g/cm³. Additionally, accumulation of virions with higher densities (1.17-1.19 g/cm³) is also observed in the basolateral compartment, even without a significant disruption of barrier permeability at the used MOI.

Overall, our results indicate that MLB2 HAstVs have the ability to infect and cross the epithelial barrier without causing significant cell damage. The HIE model may help to better elucidate MLB pathogenesis and understand which factors promote extraintestinal dissemination.

Funding: Spanish Ministry of Science and Innovation (PID2021-124023OB-I00; MICIU/AEI/10.13039/501100011033 and FEDER, UE) and Generalitat Valenciana (CIAPOS/2023/356, co-funded by European Social Fund ESF+). INSA-UB is María de Maeztu Unit of Excellence (CEX2021-001234-M; MICIN/AEI/FEDER, UE).

Viruses reprogram host gene expression through multiple mechanisms to establish infection. Among these, RNA modifications, which are chemical marks that shape RNA fate, play key roles in viral replication and immune responses. Coronaviruses remodel host RNA modifications, such as m⁶A and m⁵C, to favor replication. However, the role of N⁷-methylguanosine (m⁷G), one of the most abundant RNA marks, in coronavirus infection remains largely unclear. Although best known as the mRNA cap modification, m⁷G is also found internally in various RNA species, including rRNA, tRNA and mRNA. Internal m⁷G has emerging roles in cancer and neurological disease, but its functions in viral infection remain unknown. Here, we report a previously unrecognized function of the coronavirus nonstructural protein 14 (NSP14) in inducing internal m⁷G on host mRNA. NSP14 converts guanosine triphosphate (GTP) to m⁷G TP via its N7-methyltransferase domain, and the resulting m⁷GTP is incorporated co-transcriptionally by RNA polymerase II, producing widespread internal m⁷G across the host transcriptome. This activity is enhanced by the viral cofactor NSP10, which occurs predominantly in the nucleus and is conserved among alpha-, beta- and gamma-coronaviruses. Functionally, NSP14-driven internal m⁷G disrupts canonical splicing programs by promoting intron retention and creating novel splice junctions, with a bias toward genes governing genome stability, RNA metabolism and nuclear processes. Pharmacological interference with internal m⁷G deposition, through the inhibition of NSP14 or RNA polymerase II, impairs SARS-CoV-2 replication, indicating that the virus hijacks the host transcriptional machinery to support infection. Together, we reveal an epitranscriptomic strategy conserved among coronaviruses that reprograms host RNA processing and identify NSP14-induced internal m⁷G as a potential antiviral target.