Biology subjects

Bosse, J.

Publications and source records attributed to Bosse, J..

5 recordsLinked to original sources

bioRxiv2025

The nuclear egress complex of MHV-68 is not essential for nuclear egress but mediates C-capsid specificity

Herpesvirus capsids must exit the nucleus to undergo additional maturation steps in the cytoplasm, such as secondary envelopment. This process is orchestrated by the nuclear egress complex (NEC), a conserved heterodimer that deforms the inner nuclear membrane and facilitates capsid egress. While NEC proteins are critical for the release of capsids into the cytoplasm and, therefore, efficient replication in [a]-, {beta}-, and {gamma}-herpesviruses, residual infectivity has been observed in NEC-deficient mutants of certain herpesviruses, including the [a]-herpesvirus Pseudorabies virus (PrV). To investigate the role of the NEC in murine gammaherpesvirus 68 (MHV-68), a mutant virus lacking most of the nucleoplasmic NEC component ORF69 ({Delta}ORF69) was generated. Unlike previous studies relying on transfection-based systems, this study demonstrates that NEC-deficient MHV-68 remains replication-competent, albeit with a significant replication defect. Despite the absence of ORF69, the mutant virus produced infectious progeny in non-complementing cells. Quantitative electron microscopy revealed regular nuclear capsid assembly and DNA packaging. However, while the parental virus primarily exported mature, genome-filled C-capsids, the {Delta}ORF69 mutant exhibited cytoplasmic and extracellular emergence of all capsid forms. These findings indicate that the MHV-68 NEC functions as a quality control checkpoint, selectively facilitating the nuclear egress of mature capsids. In the absence of ORF69, some capsids still egress, likely through alternative, less efficient pathways. Inhibition of cyclin-dependent kinases reduced parental and mutant virus spread, suggesting that {gamma}-herpesviruses exploit host-dependent mechanisms for an alternative nuclear egress route. ImportanceGammaherpesviruses, such as Epstein-Barr virus and Kaposis sarcoma-associated herpesvirus, establish lifelong infections and can contribute to cancer. A crucial step in the viral life cycle depends on the nuclear egress of capsids, a process mediated by the viral nuclear egress complex (NEC). Surprisingly, murine gammaherpesvirus 68 (MHV-68) can translocate capsids from the nucleus to the cytoplasm even when a key NEC component, nucleoplasmic ORF69, is disrupted. Deleting most of ORF69 reduced viral replication but did not entirely block nuclear egress. Instead, the mutant virus allowed both mature and immature capsids to leave the nucleus, suggesting that the NEC functions as a quality control checkpoint for mature capsids. Furthermore, host cell kinases seem to enable alternative routes for egress that do not rely on the NEC. These findings further support the notion that NEC is not strictly necessary and suggest the potential exploitation of host-virus interactions for antiviral approaches.

microbiology↗

bioRxiv2025

Dissecting the role of RNA-binding proteins in early herpes simplex virus 1 transcription using acute protein depletion

Herpes simplex virus 1 (HSV-1) infects about 50-80% of the entire human population and persists in the neurons of affected individuals. A fraction of affected individuals suffer from recurrent cold sores caused by reactivating virus, in rare but severe cases the virus can cause encephalitis. During lytic infection, the virus relies on host factors such as RNA polymerase II and accessory proteins involved in transcription to express its genes and ensure successful replication. In general, RNA molecules in cells are bound by RNA-binding proteins (RBPs) during their entire lifecycle. Importantly, RBPs are increasingly described to also regulate transcription, an aspect long time outside the scope of investigations, especially during viral infections. Here, we examined the impact of five nuclear proteins (FUBP1, SLBP, SFPQ, SPT5 and SAF-B) with known RNA-binding activities on HSV-1 transcription. Additionally, we evaluated their importance for human adenovirus C5 (HAdV) growth to assess whether these host factors are specific to HSV-1 infections or might have broader relevance for the general transcription of dsDNA viruses. We show that the transcriptional elongation factor SPT5 coded by SUPT5H accumulates on HSV-1 genomes early during the infection and is required for the transcription of the immediate-early gene UL54. Its depletion affects also HAdV replication, indicating a general role in transcription of viruses that depend on the host transcriptional machinery. In contrast, depletion of the transcriptional repressor and paraspeckle protein SFPQ reduces UL54 RNA levels in HSV-1 infection, but does not cause significant changes in HAdV growth. Since SFPQ does not co-localize with HSV-1 genomes, this suggests a function not directly associated to viral DNA.

molecular biology↗

bioRxiv2022

Actinobacillus pleuropneumoniae encodes multiple phase-variable DNA methyltransferases that comprise distinct phasevarions

ABSTRACTActinobacillus pleuropneumoniae is the cause of porcine pleuropneumonia, a severe respiratory tract infection that is responsible for major economic losses to the swine industry. Many host-adapted bacterial pathogens encode systems known as phasevarions (phase- variable regulons). Phasevarions result from variable expression of cytoplasmic DNA methyltransferases. Variable expression results in genome-wide methylation differences within a bacterial population, leading to altered expression of multiple genes via epigenetic mechanisms. Our examination of a diverse population of A. pleuropneumoniae strains determined that Type I and Type III DNA methyltransferases with the hallmarks of phase variation were present in this species. We demonstrate that phase variation is occurring in these methyltransferase, and show associations between particular Type III methyltransferase alleles and serovar. Using Pacific BioSciences Single-Molecule, Real-Time (SMRT) sequencing and Oxford Nanopore sequencing, we demonstrate the presence of the first ever characterised phase-variable, cytosine-specific Type III DNA methyltransferase. Phase variation of distinct Type III DNA methyltransferase variants results in the regulation of distinct phasevarions, and in multiple phenotypic differences relevant to pathobiology. Our characterisation of these newly described phasevarions in A. pleuropneumoniae will aid in the selection of stably expressed antigens, and direct and inform development of a rationally designed subunit vaccine against this major veterinary pathogen.

microbiology↗

bioRxiv2021

Development of a novel glycoengineering platform for the rapid production of conjugate vaccines.

Antimicrobial resistance (AMR) is threatening the lives of millions worldwide. Antibiotics which once saved countless lives, are now failing, ushering in vaccines development as a current global imperative. Conjugate vaccines produced either by chemical synthesis or biologically in Escherichia coli cells, have been demonstrated to be safe and efficacious in protection against several deadly bacterial diseases. However, conjugate vaccines assembly and production have several shortcomings which hinders their wider availability. Here, we developed a tool, Mobile-element Assisted Glycoconjugation by Insertion on Chromosome, MAGIC, a novel method that overcomes the limitations of the current conjugate vaccine design method(s). We demonstrate at least 2-fold increase in glycoconjugate yield via MAGIC when compared to conventional bioconjugate method(s). Furthermore, the modularity of the MAGIC platform also allowed us to perform glycoengineering in genetically intractable bacterial species other than E. coli. The MAGIC system promises a rapid, robust and versatile method to develop vaccines against bacteria, especially AMR pathogens, and could be applied for biopreparedness.

microbiology↗

bioRxiv2019

Human cytomegalovirus nuclear capsid motility is non-directed and independent of nuclear actin bundles

Herpesvirus genome replication, capsid assembly and packaging take place in the host cell nucleus. Matured capsids leave the nucleus through a unique envelopment-de-envelopment process at the nuclear membranes called nuclear egress. How assembled and DNA-containing herpesvirus capsids reach the sites of nuclear egress is however still controversially discussed, as host chromatin that marginalizes during infection might constitute a major barrier. For alphaherpesviruses, previous work has suggested that nuclear capsids use active transport mediated by nuclear filamentous actin (F-actin). However, direct evidence for nuclear capsid motility on nuclear F-actin was missing. Our subsequent work did not detect nuclear F-actin associated with motile capsids, but instead found evidence for chromatin remodeling to facilitate passive capsid diffusion. A recent report described that human cyto-megalovirus, a betaherpesvirus, induces nuclear F-actin and that the motor protein myosin V localizes to these structures. Direct evidence of capsid recruitment to these structures and motility on them was however missing. In this study, we tested the functional role of HCMV-induced, nuclear actin assemblies for capsid transport. We did not observe transport events along nuclear F-actin. Instead, reproduction of nuclear F-actin was only possible using strong overexpression of the fluorescent marker LifeAct-mCherry-NLS. Also, two alternative fluo-rescent F-actin markers did not detect F-actin in HCMV-infected cells. Furthermore, single particle tracking of nuclear HCMV capsids showed no indication for active transport, which is in line with previous work on alphaherpesviruses.

microbiology↗