Biology subjects

Samolej, J.

Publications and source records attributed to Samolej, J..

4 recordsLinked to original sources

bioRxiv2022

Herpes simplex virus 1 expressing GFP-tagged virion host shutoff (vhs) protein uncouples the activities of degradation and nuclear retention of the infected cell transcriptome

Virion host shutoff (vhs) protein is an endoribonuclease encoded by herpes simplex virus 1 (HSV1). Vhs causes a number of changes to the infected cell environment that favour translation of late (L) virus proteins: cellular mRNAs are degraded, immediate-early (IE) and early (E) viral transcripts are sequestered in the nucleus with polyA binding protein (PABPC1), and dsRNA is degraded to help dampen the PKR-dependent stress response. To further our understanding of the cell biology of vhs, we constructed a virus expressing vhs tagged at its C-terminus with GFP. When first expressed, vhs-GFP localised to juxtanuclear clusters, and later it colocalised and interacted with its binding partner VP16, and was packaged into virions. Despite vhs-GFP maintaining activity when expressed in isolation, it failed to degrade mRNA or relocalise PABPC1 during infection, while viral transcript levels were similar to those seen for a vhs knockout virus. PKR phosphorylation was also enhanced in vhs-GFP infected cells, in line with a failure to degrade dsRNA. Nonetheless, mRNA FISH revealed that as in Wt but not {Delta}vhs infection, IE and E, but not L transcripts were retained in the nucleus of vhs-GFP infected cells at late times. Moreover, a representative cellular transcript which is ordinarily highly susceptible to vhs degradation, was also retained in the nucleus. These results reveal that the vhs-induced nuclear retention of the infected cell transcriptome is dependent on vhs expression but not on its endoribonuclease activity, uncoupling these two functions of vhs. ImportanceLike many viruses, herpes simplex virus 1 (HSV1) expresses an endoribonuclease, the virion host shutoff (vhs) protein, which regulates the RNA environment of the infected cell and facilitates the classical cascade of virus protein translation. It does this by causing the degradation of some mRNA molecules and the nuclear retention of others. Here we describe a virus expressing vhs tagged at its C-terminus with green fluorescent protein (GFP) and show that the vhs-GFP fusion protein retains the physical properties of native vhs, but does not induce the degradation of mRNA. Nonetheless, vhs-GFP maintains the ability to trap the infected cell transcriptome in the nucleus, proving for the first time that mRNA degradation is not a prerequisite for vhs effects on the nuclear transcriptome. This virus has therefore uncoupled the nuclear retention and degradation activities of vhs, providing new understanding of vhs during infection.

microbiology↗

bioRxiv2021

Vaccinia Virus Arrests and Shifts the Cell Cycle

Modulation of the host cell cycle is a common strategy used by viruses to create a pro-replicative environment. To facilitate viral genome replication, vaccinia virus (VACV) has been reported to alter cell cycle regulation and trigger the host cell DNA damage response. However, the cellular factors and viral effectors that mediate these changes remain unknown. Here, we set out to investigate the effect of VACV infection on cell proliferation and host cell cycle progression. Using a subset of VACV mutants we characterize the stage of infection required for inhibition of cell proliferation and define the viral effectors required to dysregulate the host cell cycle. Consistent with previous studies, we show that VACV inhibits, and subsequently shifts the host cell cycle. We demonstrate that these two phenomena are independent of one another, with viral early genes being responsible for cell cycle inhibition, and post-replicative viral gene(s) responsible for the cell cycle shift. Extending previous findings, we show that the viral kinase F10 is required to activate the DNA damage checkpoint and that the viral B1/B12 (pseudo) kinases mediate degradation of checkpoint effectors p53 and p21 during infection. We conclude that VACV modulates host cell proliferation and host cell cycle progression through temporal expression of multiple VACV effector proteins.

microbiology↗

bioRxiv2020

Novel role for ESCRT-III component CHMP4C in the integrity of the endocytic network utilized for herpes simplex virus envelopment

Enveloped viruses exploit cellular trafficking pathways for their morphogenesis, providing potential scope for the development of new antiviral therapies. We have previously shown that herpes simplex virus 1 (HSV1) utilises recycling endocytic membranes as the source of its envelope, in a process involving four Rab GTPases. To identify novel factors involved in HSV1 envelopment, we have screened an siRNA library targeting over eighty human trafficking proteins including coat proteins, adaptor proteins, fusion factors, fission factors and Rab effectors. Depletion of eleven factors reduced virus yield by 20- to 100-fold, including three early secretory pathway proteins; four late secretory pathway proteins; and four endocytic pathway proteins, three of which are membrane fission factors. Five of the eleven targets were chosen for further analysis in virus infection where it was found that the absence of only one, the fission factor CHMP4C, known for its role in the cytokinesis checkpoint, specifically reduced virus production at the final stage of morphogenesis. Ultrastructural and confocal microscopy of CHMP4C-depleted, HSV1-infected cells, showed an accumulation of endocytic membranes; extensive tubulation of recycling, transferrin receptor-positive endosomes indicative of aberrant fission; and a failure in virus envelopment. No effect on the late endocytic pathway was detected, while exogenous CHMP4C was shown to localise to recycling endosomes. Taken together, these data reveal a novel role for the CHMP4C fission factor in the integrity of the recycling endosomal network, which has been unveiled through the dependence of HSV1 on these membranes for the acquisition of their envelopes. ImportanceCellular transport pathways play a fundamental role in secretion and membrane biogenesis. Enveloped viruses exploit these pathways to direct their membrane proteins to sites of envelopment, and as such, are powerful tools for unravelling subtle activities of trafficking factors, potentially pinpointing therapeutic targets. Using the sensitive biological readout of virus production, over eighty trafficking factors involved in diverse and poorly defined cellular processes have been screened for involvement in the complex process of HSV1 envelopment. Out of eleven potential targets, CHMP4C - a key component in the cell-cycle abscission checkpoint - stood out as being required for the physical process of virus wrapping in endocytic tubules, where it was shown to localise. In the absence of CHMP4C, recycling endocytic membranes failed to undergo scission, causing transient tubulation and accumulation of membranes and unwrapped virus. These data reveal a new role for this important cellular factor in the biogenesis of recycling endocytic membranes.

cell biology↗

bioRxiv2019

Mimicry embedding for advanced neuralnetwork training of 3D biomedical micrographs

The use of deep neural networks (DNNs) for analysis of complex biomedical images shows great promise but is hampered by a lack of large verified datasets for rapid network evolution. Here we present a novel \"mimicry embedding\" strategy for rapid application of neural network architecture-based analysis of biomedical imaging datasets. Embedding of a novel biological dataset, such that it mimics a verified dataset, enables efficient deep learning and seamless architecture switching. We apply this strategy across various microbiological phenotypes; from super-resolved viruses to in vivo parasitic infections. We demonstrate that mimicry embedding enables efficient and accurate analysis of three-dimensional microscopy datasets. The results suggest that transfer learning from pre-trained network data may be a powerful general strategy for analysis of heterogeneous biomedical imaging datasets.

bioinformatics↗