Biology subjects

Verhoeven, D.

Publications and source records attributed to Verhoeven, D..

7 recordsLinked to original sources

bioRxiv2025

Charge reversal at the Lhcb2 N-terminus impairs phosphorylation and PSI-LHCII complex formation

State transitions balance excitation-energy distribution between Photosystem I and Photosystem II in higher plants. Stn7-mediated phosphorylation of the N-terminus of the light-harvesting complex II protein Lhcb2 plays a central role in photosynthetic state transitions. However, it remains unclear how the intrinsic charge of this region, independent of its phosphorylation status, influences state transitions and thylakoid membrane organization. Here, we introduced specific charge-altering mutations in the Lhcb2 N-terminus of Arabidopsis thaliana in the lhcb2 knock-out background and analyzed their effects on LHCII phosphorylation, state transition dynamics, PSI-LHCII complex formation, and thylakoid ultrastructure. Substitution of a conserved positively charged arginine with a negatively charged glutamate (R2E) markedly reduced Lhcb1 and Lhcb2 phosphorylation and state transition efficiency, and abolished PSI-LHCII complex formation. In contrast, introducing a negative charge at a downstream position (Q9E) had no detectable effects. Electron microscopy revealed no significant changes in thylakoid organization in either mutant compared to WT Lhcb2 plants. Despite strongly reduced Lhcb1 and Lhcb2 phosphorylation in the R2E mutant, residual state transitions persisted, potentially mediated by Stn7-dependent phosphorylation of other target proteins. Together, these results provide insight into the role of N-terminal LHCII electrostatics in state transitions and thylakoid membrane organization in plants.

plant biology↗

bioRxiv2024

SARS-CoV-2 Mediated Inhibition Of Respiratory Syncytial Virus

With circulation of SARS-CoV-2, fears about coinfection with other respiratory viruses such as influenza and RSV were significant, but the opposite was observed. Distancing/barriers played a major role in reducing other viral co-infections, however, some infrequent co-infections still occurred. We investigated the relationship between SARS-CoV-2 and RSV during coinfection to understand how they might compete or synergize. We found only RSVs replication was significantly reduced when coinfected with SARS-CoV-2. Investigation of the mechanism revealed that the SARS-CoV-2 protein Nsp1 disrupts the RSV M2-2 protein but not the upstream M2-1 protein on the same biscistronic mRNA transcript. The impact of Nsp1 on M2-2 was not dependent on M2-2 being the second ORF in a bicistronic mRNA transcript, but likely from prevention of ribosomal termination-reinitiation necessary for M2-2 production. Additional viral ORFs from influenza A, influenza B, or Sendai virus dependent on the same or other ribosomal initiation mechanisms were tested and we found only influenza B M/M2 which likely uses a similar method as M2-2 was disrupted. Various M2-2 constructs, with/without the proposed site of ribosomal termination-reinitiating, co-transfected with Nsp1 and were in agreement that disruption to M2-2 expression occurs if the site of re-initiation was present upstream. These data not only suggest Sars-CoV-2 can outcompete RSV through suppression of M2, but may also point to potential ways to interfere with RSV by targeted therapies.

microbiology↗

bioRxiv2024

Use of equine H3N8 hemagglutinin as a broadly protective influenza vaccine immunogen

An efficacious universal influenza vaccine remains a long-sought goal for human health as current vaccines suffer from shortfalls such as mid to low efficacy and the need for yearly revisions in strains use to account for viral drift/shift. Horses undergo bi-annual vaccines for H3N8 equine influenza virus, and surveillance of sera from vaccinees demonstrated very broad reactivity and neutralization to many human seasonal influenza strains. Subsequently, vaccination of mice using the equine H3N8 Kentucky/1/91 strain of equine H3N8 vaccine induced similar broadly reactive and neutralizing serum antibodies to human seasonal strains and to high pathogenicity avian influenza strains. Challenge of mice vaccinated with equine H3N8 or recombinant hemagglutinin (HA) based on the same strain protected vaccinees from high-dose lethal virus challenges. Protection was associated with the presence of neutralizing antibodies to the HA head, esterase, and stem regions that was similar to that of antibodies generated in horses after vaccination. Vaccinated ferrets exhibited similar broadly reactive serum antibody responses that protected vaccinees from clinical signs of infection and viral-induced histopathology after challenge with influenza A/07/2009 (H1N1) pandemic virus. Taken together, these data suggest that vaccination with equine H3N8 vaccine induces broad protection against influenza without the need for non-influenza viral vectors, multiple HAs, or foreign protein scaffolds common to other universal influenza vaccine candidates.

immunology↗

bioRxiv2024

RESPIRATORY SYNCYTIAL VIRUS NON-STRUCTURAL PROTEIN EXPRESSION ARE LIMITED IN NEUTROPHILS

Respiratory Syncytial Virus (RSV) is a negative stranded RNA virus with a high incidence of secondary bacterial infections. RSV contains two broad immune inhibitory proteins Ns1 and Ns2 which are not present in any other viruses of the Mononegavirales Order. Here we report that expression of Ns2 is attenuated during RSV infection of neutrophils and that RSV is indeed infecting neutrophils rather than simply being phagocytosed by them. Infection was determined by intracellular staining and coinfection studies of uninfected Hep2 cells. The significant attenuation of Ns2 in vivo along with the low abundance of coinfected cells indicates that RSV infection is likely functionally non permissive in in vivo infection. The implications of RSV infection of neutrophils may explain the previously observed phenomenon of decreased phagocytosis in neutrophils exposed to RSV and the lack of Ns2 expression within neutrophils may provide avenues of study to attenuate viral infection through therapy development.

immunology↗

bioRxiv2024

ANALYSIS OF RESPIRATORY SYNCYTIAL VIRUS REVEALS CONSERVED RNA SECONDARY STRUCTURAL MOTIFS AND IMPACT VIRAL LIFECYCLE

An analysis that combined bioinformatics, comparative sequence/structural analysis, and experimental assays has been completed on respiratory syncytial virus (RSV). Both the genomic RNA and its reverse complement were studied using the novel bioinformatics pipeline ScanFold, which predicted 49 regions on RSV RNAs that appear to encode functional secondary structures (based on their unusually stable sequence order). Multiple motifs appear to be conserved between RSV and related virus strains, including one region within the F gene, which had a highly favorable overall prediction metric of a paired secondary structure. This motif was subjected to additional experimental analyses using SHAPE analysis to confirm ScanFold predicted secondary structure. In subsequent analysis, RSV F mRNA transcripts were made by in vitro transcription using T7 polymerase and transcripts which relaxed the predicted secondary structure yielded slightly higher mRNA transcripts and protein expression levels as wildtype F. However, using reverse genetics for comparison of viruses containing wildtype or relaxed F suggested that the predicted secondary structures may be critical for RSV replication in cells. To our knowledge, this is the first study to examine conserved RNA structures across multiple RSV strains and may help identify potential therapeutic targets to inhibit.

microbiology↗

bioRxiv2024

Divergent macrophage induced lung pathology occurs between infants and adults during respiratory viral infection

Young children, especially those aged 4 months to 2 years of age, frequently exhibit severe morbidity during respiratory viral infections. For influenza infections, macrophages/monocytes serve as front line defenses against early viral replication in the lungs until the adaptive immune system arrives to clear virus. However, infiltrating inflammatory monocytes are a significant cause of influenza induced lung pathology/morbidity. We utilized a young murine model of respiratory viral infections using 21-day-old mice to investigate the mechanisms driving the heightened influenza induced morbidity observed in human young children. We hypothesized that macrophages/monocytes responses to influenza would diverge between young and older mice despite our evidence that macrophages from both groups appear to control viral replication at similar rates. While inflammatory monocyte infiltration contributed to influenza induced morbidity/lung inflammation in adults, they did not appear to contribute to morbidity in young mice. Instead, young mice appeared to develop lung inflammation through a lack of interferon gamma (IFN{psi}) and infection of macrophage populations. In contrast, adult mice controlled early viral replication through macrophage populations (alveolar, interstitial, and inflammatory) and inflammatory monocytes. While intrinsic limitations in anti-viral cytokine responses, especially IFN{psi}, characterized the macrophage response to viral infection in young mice, the innate immune response to infection appears diminished compared to adults. This study highlights the intrinsic limitations in macrophage effector functions that may arise in young children but that also contribute to disease pathology.

immunology↗

bioRxiv2020

STREPTOCOCCUS PNEUMONIAE SEROTYPE 22F INFECTION IN RESPIRATORY SYNCYTIAL VIRUS INFECTED NEONATAL LAMBS ENHANCES MORBIDITY

Respiratory syncytial virus (RSV) is the primary cause of viral bronchiolitis resulting in hospitalization and a frequent cause of secondary respiratory bacterial infection, especially by Streptococcus pneumoniae (Spn) in infants. While murine studies have demonstrated enhanced morbidity during a viral/bacterial co-infection, human meta-studies have conflicting results. Moreover, little knowledge about the pathogenesis of emerging Spn serotype 22F, and especially the co-pathologies between RSV and Spn is known. Here, colostrum-deprived neonate lambs were divided into four groups. Two of the groups were nebulized with RSV M37, and the other two groups mock nebulized. At day 3 post-infection, one RSV group (RSV/Spn) and one mock-nebulized group (Spn only) were inoculated with Spn intratracheally. At day 6 post-infection, bacterial/viral loads were assessed along with histopathology and correlated with clinical symptoms. Lambs dually infected with RSV/Spn had higher RSV titers, but lower Spn. Additionally, lung lesions were observed to be more intense in the RSV/Spn group characterized by increased interalveolar wall thickness accompanied by neutrophil and lymphocyte infiltration. Despite lower Spn in lungs, co-infected lambs had more significant morbidity and histopathology, which correlated with a different cytokine response. Thus, enhanced disease severity during dual infection may be due to lesion development and altered immune responses rather than bacterial counts.

microbiology↗