Biology subjects

Jobe, F.

Publications and source records attributed to Jobe, F..

3 recordsLinked to original sources

bioRxiv2023

Viral pIC-pocketing: RSV sequestration of eIF4F Initiation Complexes into bi-phasic biomolecular condensates

Orthopneumoviruses characteristically form membrane-less cytoplasmic inclusion bodies (IBs) wherein RNA replication and transcription occur. Herein, we report a strategy whereby the orthopneumoviruses sequester various components of the eiF4F Initiation Complex machinery into viral IBs to facilitate translation of their own mRNAs; pIC-pocketing. Mass spectrometry analysis of sub-cellular fractions from RSV-infected cells identified significant modification of the cellular translation machinery; however; interestingly, ribopuromycylation assays showed no changes to global levels of translation. Electron micrographs of RSV-infected cells revealed bi-phasic organisation of IBs; specifically, spherical "droplets" nested within the larger inclusion. Using correlative light and electron microscopy (CLEM), combined with fluorescence in situ hybridisation (FISH), we showed that the observed bi-phasic morphology represents functional compartmentalisation of the IB and that these domains are synonymous with the previously reported inclusion body associated granules (IBAGs). Detailed analysis demonstrated that IBAGs concentrate nascent viral mRNA, the viral M2-1 protein as well as many components of the eIF4F complex, involved in translation initiation. Interestingly, although ribopuromycylation-based imaging indicates the majority of viral mRNA translation likely occurs in the cytoplasm, there was some evidence for intra-IBAG translation, consistent with the likely presence of ribosomes in a subset of IBAGs imaged by electron microscopy. The mechanistic basis for this pathway was subsequently determined; the viral M2-1 protein interacting with eukaryotic translation initiation factor 4G (eIF4G) to facilitate its transport between the cytoplasm and the separate phases of the viral IB. In summary, our data shows that IBs function to spatially regulate early steps in viral translation within a highly selective biphasic liquid organelle.

microbiology↗

bioRxiv2020

Respiratory syncytial virus sequesters NF-κB subunit p65 to cytoplasmic inclusion bodies to inhibit innate immune signalling

Viruses routinely employ strategies to prevent the activation of innate immune signalling in infected cells. RSV is no exception, encoding two accessory proteins (NS1 and NS2) which are well established to block Interferon signalling. However, RSV-encoded mechanisms for inhibiting NF-{kappa}B signalling are less well characterised. In this study we identified RSV-mediated antagonism of this pathway, independent of the NS1 and NS2 proteins, and indeed distinct from other known viral mechanisms of NF-{kappa}B inhibition. In both human and bovine RSV infected cells we demonstrated that the P65 subunit of NF-{kappa}B is rerouted to perinuclear puncta in the cytoplasm, puncta which are synonymous with viral inclusion bodies (IBs), the site for viral RNA replication. Captured P65 was unable to translocate to the nucleus or transactivate a NF-{kappa}B reporter following TNF- stimulation, confirming the immune-antagonistic nature of this sequestration. Subsequently, we used correlative light electron microscopy (CLEM) to colocalise RSV N protein and P65 within bRSV IBs; granular, membraneless regions of cytoplasm with liquid organelle-like properties. Additional characterisation of bRSV IBs indicated that although they are likely formed by liquid-liquid phase separation (LLPS), they have a differential sensitivity to hypotonic shock proportional to their size. Together, these data identify a novel mechanism for viral antagonism of innate immune signalling which relies on sequestration of the NF-{kappa}B subunit p65 to a biomolecular condensate - a mechanism conserved across the Orthopneumovirus genus and not host-cell specific. More generally they provide additional evidence that RNA virus IBs are important immunomodulatory complexes within infected cells. Impact summaryMany viruses replicate almost entirely in the cytoplasm of infected cells, without too many direct interactions with the nucleus. Examples include respiratory syncytial virus (RSV), measles, Ebola and Nipah; however, how these pathogens are able to compartmentalise their life cycle to provide favourable conditions for replication and to avoid the litany of antiviral detection mechanisms in the cytoplasm remains relatively uncharacterised. In this paper we show that bovine RSV (bRSV), which infects cattle, does this by generating inclusion bodies in the cytoplasm of infected cells. These organelles are unusually membrane-less; likely forming by a process called liquid-liquid phase separation which involves macro-molecular interactions between the viral proteins N and P. We also showed that these organelles, otherwise known as inclusion bodies (IBs), are able to capture important innate immune transcription factors (in this case NF-KB), blocking the normal signalling processes that tell the nucleus the cell is infected. Using fluorescent bioimaging and a combination of confocal and electron microscopy we then characterised this interaction in detail, also confirming that human RSV (hRSV) employs the same mechanism. Like hRSV, bRSV viral RNA replication also takes place in the IB, likely meaning these organelles are a functionally conserved feature of orthopneumoviruses.

microbiology↗

bioRxiv2019

A single dose of antibody-drug conjugate cures a stage 1 model of African trypanosomiasis.

Infections of humans and livestock with African trypanosomes are treated with drugs introduced decades ago that are not always fully effective and often have severe side effects. Here, the trypanosome haptoglobin-haemoglobin receptor (HpHbR) has been exploited as a route of uptake for an antibody-drug conjugate (ADC) that is completely effective against Trypanosoma brucei in the standard mouse model of infection. Recombinant human anti-HpHbR monoclonal antibodies were isolated and shown to be internalised in a receptor-dependent manner. Antibodies were conjugated to a pyrrolobenzodiazepine (PBD) toxin and killed T. brucei in vitro at picomolar concentrations. A single therapeutic dose (0.25 mg/kg) of a HpHbR antibody-PBD conjugate completely cured a T. brucei mouse infection within 2 days with no re-emergence of infection over a subsequent time course of 77 days. These experiments provide a demonstration of how ADCs can be exploited to treat protozoal diseases that desperately require new therapeutics. Author SummaryHere we show that antibody-drug conjugates (ADCs) can be re-purposed from cancer immunotherapeutics to anti-protozoals by changing the specificity of the immunoglobulin to target a trypanosome cell surface receptor. Trypanosomes were used as a model system due to the availability of receptor null cell lines that allowed the unambiguous demonstration that ADCs targeted to a parasite surface receptor could be specifically internalised via receptor-mediated endocytosis. A single low dose of the resulting ADC was able to cure a stage 1 mouse model of trypanosome infection. We have used toxins and conjugation chemistry that are identical to anti cancer ADCs demonstrating the ability to piggy-back onto the huge research efforts and resources that are being invested in the development of such ADCs. The potential for development of ADCs against a wide range of human pathogens is vast, where only epitope binding sites need vary in order to provide selectivity. This provides a far-reaching opportunity for the rapid development of novel anti-protozoals for the targeted killing of a wide range of pathogens that cause disease worldwide, especially in developing countries.

microbiology↗