Viral Exit Research Articles

Enterovirus-A71 exploits RAB11 to recruit chaperones for virus morphogenesis

BackgroundEnterovirus 71 (EV-A71) causes Hand, Foot and Mouth Disease (HFMD) in children and has been associated with neurological complications. The molecular mechanisms involved in EV-A71 pathogenesis have remained elusive.MethodsA siRNA screen in EV-A71 infected-motor neurons was performed targeting 112 genes involved in intracellular membrane trafficking, followed by validation of the top four hits using deconvoluted siRNA. Downstream approaches including viral entry by-pass, intracellular viral genome quantification by qPCR, Western blot analyses, and Luciferase reporter assays allowed determine the stage of the infection cycle the top candidate, RAB11A was involved in. Proximity ligation assay, co-immunoprecipitation and multiplex confocal imaging were employed to study interactions between viral components and RAB11A. Dominant negative and constitutively active RAB11A constructs were used to determine the importance of the protein’s GTPase activity during EV-A71 infection. Mass spectrometry and protein interaction analyses were employed for the identification of RAB11A’s host interacting partners during infection.ResultsSmall GTPase RAB11A was identified as a novel pro-viral host factor during EV-A71 infection. RAB11A and RAB11B isoforms were interchangeably exploited by strains from major EV-A71 genogroups and by Coxsackievirus A16, another major causative agent of HFMD. We showed that RAB11A was not involved in viral entry, IRES-mediated protein translation, viral genome replication, and virus exit. RAB11A co-localized with replication organelles where it interacted with structural and non-structural viral components. Over-expression of dominant negative (S25N; GDP-bound) and constitutively active (Q70L; GTP-bound) RAB11A mutants had no effect on EV-A71 infection outcome, ruling out RAB11A’s involvement in intracellular trafficking of viral or host components. Instead, decreased ratio of intracellular mature viral particles to viral RNA copies and increased VP0:VP2 ratio in siRAB11-treated cells supported a role in provirion maturation hallmarked by VP0 cleavage into VP2 and VP4. Finally, chaperones, not trafficking and transporter proteins, were found to be RAB11A’s top interacting partners during EV-A71 infection. Among which, CCT8 subunit from the chaperone complex TRiC/CCT was further validated and shown to interact with viral structural proteins specifically, representing yet another novel pro-viral host factor during EV-A71 infection.ConclusionsThis study describes a novel, unconventional role for RAB11A during viral infection where it participates in the complex process of virus morphogenesis by recruiting essential chaperone proteins.

Natural variation in infection specificity of Caenorhabditis briggsae isolates by two RNA viruses.

Antagonistic relationships such as host-virus interactions potentially lead to rapid evolution and specificity in interactions. The Orsay virus is so far the only horizontal virus naturally infecting the nematode C. elegans. In contrast, several related RNA viruses infect its congener C. briggsae, including Santeuil (SANTV) and Le Blanc (LEBV) viruses. Here we focus on the host's intraspecific variation in sensitivity to these two intestinal viruses. Many temperate-origin C. briggsae strains, including JU1264 and JU1498, are sensitive to both, while many tropical strains, such as AF16, are resistant to both. Interestingly, some C. briggsae strains exhibit a specific resistance, such as the HK104 strain, specifically resistant to LEBV. The viral sensitivity pattern matches the strains' geographic and genomic relationships. The heavily infected strains mount a seemingly normal small RNA response that is insufficient to suppress viral infection, while the resistant strains show no small RNA response, suggesting an early block in viral entry or replication. We use a genetic approach from the host side to map genomic regions participating in viral resistance polymorphisms. Using Advanced Intercrossed Recombinant Inbred Lines (RILs) between virus-resistant AF16 and SANTV-sensitive HK104, we detect Quantitative Trait Loci (QTLs) on chromosomes IV and III. Building RILs between virus-sensitive JU1498 and LEBV-resistant HK104 followed by bulk segregant analysis, we identify a chromosome II QTL. In both cases, further introgressions of the regions confirmed the QTLs. This diversity provides an avenue for studying virus entry, replication, and exit mechanisms, as well as host-virus specificity and the host response to a specific virus infection.

Inhibition of nervous necrosis virus replication by Shewanella putrefaciens Pdp11 extract

One of the most relevant infectious diseases affecting farmed fish is the viral encephalopathy and retinopathy, caused by the nervous necrosis virus (NNV). The two commercial vaccines available against this virus have been designed to protect European seabass against the infection with a particular NNV species, RGNNV. Therefore, the development of strategies to protect different fish species against different viruses is a key issue for the aquaculture industry. In this regard, there are numerous studies focused on the use of probiotics, although their antiviral activity has been poorly investigated. Shewanella putrefaciens Pdp11, SpPdp11, is a probiotic with proven positive effects on gilthead seabream and Senegalese sole, protecting those species against several bacterial pathogens; however, its antiviral activity remains to be investigated. The current study is a step forward in the use of probiotics against viral infections, evaluating the anti-RGNNV activity of sonicated-SpPdp11 extracts both, in vitro and in vivo. According to our results, SpPdp11 extracts compromised RGNNV multiplication in E11 cells, affecting viral assembly and/or exit rather than genome replication. Furthermore, this antiviral activity may be produced by the capacity of the SpPdp11-extract to induce an immune response in treated cells. This anti-RGNNV activity has been confirmed in vivo, since 82% of fish fed with the SpPdp11-supplemented diet survived an experimental-RGNNV infection, whereas the survival rate of fish fed with the control diet was 64%. These results suggest that SpPdp11-supplemented feeding can be a promising prophylactic tool against RGNNV and encourage further research on other fish species and viral pathogens.

Get in and get out: Remodeling of the cellular actin cytoskeleton upon HIV-1 infection.

The human immunodeficiency virus type 1 (HIV-1) is an intracellular pathogen whose replication cycle strictly depends on the host cell molecular machinery. HIV-1 crosses twice the plasma membrane, to get in and to get out of the cell. Therefore, the first and the last line of intracellular component encountered by the virus is the cortical actin network. Here, we review the role of actin and actin-related proteins in HIV-1 entry, assembly, budding, and release. We first highlight the mechanisms controlling actin polymerization at the entry site that promote the clustering of HIV-1 receptors, a crucial step for the virus to fuse with the plasma membrane. Then, we describe how actin is transiently depolymerized locally to allow the capsid to cross the actin cortex, before migrating towards the nucleus. Finally, we review the role of several actin-binding proteins in actin remodeling events required for membrane deformation and curvature at the viral assembly site as well as for virus release. Strikingly, it appears that common actin-regulating pathways are involved in viral entry and exit. However, while the role of actin remodeling during entry is well understood, this is not the case during exit. We discuss remaining challenges regarding the actin-dependent mechanisms involved in HIV-1 entry and exit, and how they could be overcome.

The SARS-CoV-2 accessory protein Orf3a is not an ion channel, but does interact with trafficking proteins.

The severe acute respiratory syndrome associated coronavirus 2 (SARS-CoV-2) and SARS-CoV-1 accessory protein Orf3a colocalizes with markers of the plasma membrane, endocytic pathway, and Golgi apparatus. Some reports have led to annotation of both Orf3a proteins as viroporins. Here, we show that neither SARS-CoV-2 nor SARS-CoV-1 Orf3a form functional ion conducting pores and that the conductances measured are common contaminants in overexpression and with high levels of protein in reconstitution studies. Cryo-EM structures of both SARS-CoV-2 and SARS-CoV-1 Orf3a display a narrow constriction and the presence of a positively charged aqueous vestibule, which would not favor cation permeation. We observe enrichment of the late endosomal marker Rab7 upon SARS-CoV-2 Orf3a overexpression, and co-immunoprecipitation with VPS39. Interestingly, SARS-CoV-1 Orf3a does not cause the same cellular phenotype as SARS-CoV-2 Orf3a and does not interact with VPS39. To explain this difference, we find that a divergent, unstructured loop of SARS-CoV-2 Orf3a facilitates its binding with VPS39, a HOPS complex tethering protein involved in late endosome and autophagosome fusion with lysosomes. We suggest that the added loop enhances SARS-CoV-2 Orf3a's ability to co-opt host cellular trafficking mechanisms for viral exit or host immune evasion.

Transcriptome profiling of colorectal tumors from patients with sepsis reveals an ethnic basis for viral infection risk and sepsis progression

Mortality from cancer-associated sepsis varies by cancer site and host responses to sepsis are heterogenous. Native Hawaiians have the highest mortality risk from cancer-associated sepsis and colorectal cancer (CRC), even though they demonstrate lower CRC incidence compared to other ethnicities. We conducted a retrospective transcriptomic analysis of CRC tumors and adjacent non-tumor tissue from adult patients of Native Hawaiian and Japanese ethnicity who died from cancer-associated sepsis. We examined differential gene expression in relation to patient survival and sepsis disease etiology. Native Hawaiian CRC patients diagnosed with sepsis had a median survival of 5 (IQR 4–49) months, compared to 117 (IQR 30–146) months for Japanese patients. Transcriptomic analyses identified two distinct sepsis gene signatures classified as early response and late response sepsis genes that were significantly altered in the Native Hawaiian cohort. Analysis of canonical pathways revealed significant up and downregulation in mechanisms of viral exit from host cells (p = 4.52E−04) and epithelial junction remodeling (p = 4.01E−05). Key genes including elongation initiation factor pathway genes, GSK3B, and regulatory associated protein of mTOR (RPTOR) genes that protect cells from infection were significantly downregulated in Native Hawaiians. Genes promoting sepsis progression including CLOCK, PPBP and Rho family GTPASE 2 (RND2) were upregulated in Native Hawaiian patients. Our transcriptomic approach advances understanding of sepsis heterogeneity by revealing a role of genetic background and defining patient subgroups with altered early and late biological responses to sepsis. This study is the first to investigate differential gene expression in CRC-associated sepsis patients in relation to ethnicity. Our findings may lead to personalized approaches in stratifying patient mortality risk for sepsis and in the development of effective targeted therapies for sepsis.

The physical interactome between Peregrinus maidis proteins and the maize mosaic virus glycoprotein provides insights into the cellular biology of a rhabdovirus in the insect vector.

Rhabdovirus glycoproteins (G) serve multifunctional roles in virus entry, assembly, and exit from animal cells. We hypothesize that maize mosaic virus (MMV) G is required for invasion, infection, and spread in Peregrinus maidis, the planthopper vector. Using a membrane-based yeast two-hybrid assay, we identified 107 P. maidis proteins that physically interacted with MMV G, of which approximately 53% matched proteins with known functions including endocytosis, vesicle-mediated transport, protein synthesis and turnover, nuclear export, metabolism and host defense. Physical interaction networks among conserved proteins indicated a possible cellular coordination of processes associated with MMV G translation, protein folding and trafficking. Non-annotated proteins contained predicted functional sites, including a diverse array of ligand binding sites. Cyclophilin A and apolipophorin III co-immunoprecipitated with MMV G, and each showed different patterns of localization with G in insect cells. This study describes the first protein interactome for a rhabdovirus spike protein and insect vector.

Asgard archaea shed light on the evolutionary origins of the eukaryotic ubiquitin-ESCRT machinery

The ESCRT machinery, comprising of multiple proteins and subcomplexes, is crucial for membrane remodelling in eukaryotic cells, in processes that include ubiquitin-mediated multivesicular body formation, membrane repair, cytokinetic abscission, and virus exit from host cells. This ESCRT system appears to have simpler, ancient origins, since many archaeal species possess homologues of ESCRT-III and Vps4, the components that execute the final membrane scission reaction, where they have been shown to play roles in cytokinesis, extracellular vesicle formation and viral egress. Remarkably, metagenome assemblies of Asgard archaea, the closest known living relatives of eukaryotes, were recently shown to encode homologues of the entire cascade involved in ubiquitin-mediated membrane remodelling, including ubiquitin itself, components of the ESCRT-I and ESCRT-II subcomplexes, and ESCRT-III and Vps4. Here, we explore the phylogeny, structure, and biochemistry of Asgard homologues of the ESCRT machinery and the associated ubiquitylation system. We provide evidence for the ESCRT-I and ESCRT-II subcomplexes being involved in ubiquitin-directed recruitment of ESCRT-III, as it is in eukaryotes. Taken together, our analyses suggest a pre-eukaryotic origin for the ubiquitin-coupled ESCRT system and a likely path of ESCRT evolution via a series of gene duplication and diversification events.

Potential Antiviral Strategy Exploiting Dependence of SARS-CoV-2 Replication on Lysosome-Based Pathway.

The recent novel coronavirus (SARS-CoV-2) disease (COVID-19) outbreak created a severe public health burden worldwide. Unfortunately, the SARS-CoV-2 variant is still spreading at an unprecedented speed in many countries and regions. There is still a lack of effective treatment for moderate and severe COVID-19 patients, due to a lack of understanding of the SARS-CoV-2 life cycle. Lysosomes, which act as “garbage disposals” for nearly all types of eukaryotic cells, were shown in numerous studies to support SARS-CoV-2 replication. Lysosome-associated pathways are required for virus entry and exit during replication. In this review, we summarize experimental evidence demonstrating a correlation between lysosomal function and SARS-CoV-2 replication, and the development of lysosomal perturbation drugs as anti-SARS-CoV-2 agents.

The Effect of Glycosylation Modulators on the Trafficking and Interaction of Spike Protein S1 Subunit and Angiotensin‐Converting Enzyme 2

The betacoronavirus severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) is the cause of the ongoing worldwide pandemic. The spike (S) glycoprotein of SARS‐CoV‐2 is a heavily glycosylated trimer that binds to host cell receptor angiotensin‐converting enzyme 2 (ACE2), initiating viral entry. Evidence also shows that the S glycoprotein interacts with other receptors including dipeptidyl peptidase IV (DPPIV). Glycosylation is a post‐translational modification important for proper folding and trafficking of membrane and secretory proteins. It plays a critical role not only for host cell proteins, but also in viral virulence, immunity evasion and receptor binding capabilities. This study focused on the modulation of glycosylation and its effects on the trafficking of the S1 subunit, ACE2 and DPPIV, in addition to its effect on their interactions. For this purpose, the S1 subunit was expressed in COS‐1 cells in the presence or absence of N‐butyldeoxynojirimycin (NB‐DNJ), an inhibitor of the ER‐located a‐glucosidases I and II, 1‐deoxymannojirimycin (dMM), an inhibitor of Golgi‐mannosidase I, or benzyl‐N‐acetyl‐α‐galactosaminide (benzyl‐GalNac), an inhibitor of O‐glycosylation. The data demonstrate that NB‐DNJ affects substantially the trafficking and secretion of S1 protein, while its overall synthetic levels remained unchanged. A similar effect was observed upon treatment of the cells with the benzyl‐GalNac. On the contrary, the presence of dMM in the culture medium did not affect the secretion of S1. Together, the data suggest that folding events implicating calnexin in the ER, the site of action of NB‐DNJ, as well as O‐glycans that are modulated by benzyl‐GalNac are crucial for the secretion of the S1 protein. We further investigated the trafficking of ACE2 and DPPIV in lung Calu‐3 and intestinal Caco‐2 cells in the presence or absence of the glycosylation modulators and their interaction with S1. The data demonstrate a reduced binding capacity of S1 to ACE2 in the presence of NB‐DNJ and dMM, while this interaction was enhanced in Calu‐3 cells upon inhibition of O‐glycosylation by benzyl‐GalNac. We conclude that glycosylation modulators differentially act on the secretory pathway of S1 and the receptors. By virtue of its negative impact on both the secretion of S1 as well as interaction with its receptor ACE2, NB‐DNJ may be considered as a potential therapeutic drug that may act at the level of viral entry and exit.

Protein Disulfide Isomerase A3 Regulates Influenza Neuraminidase Activity and Influenza Burden in the Lung.

Influenza (IAV) neuraminidase (NA) is a glycoprotein required for the viral exit from the cell. NA requires disulfide bonds for proper function. We have recently demonstrated that protein disulfide isomerase (PDI)A3 is required for oxidative folding of IAV hemagglutinin (HA), and viral propagation. However, it not known whether PDIs are required for NA maturation or if these interactions represent a putative target for the treatment of influenza infection. We sought to determine whether PDIA3 is required for disulfide bonds of NA, its activity, and propagation of the virus. Requirement of disulfides for NA oligomerization and activity were determined using biotin switch and redox assays in WT and PDIA3−/− in A549 cells. A PDI specific inhibitor (LOC14) was utilized to determine the requirement of PDIs in NA activity, IAV burden, and inflammatory response in A549 and primary mouse tracheal epithelial cells. Mice were treated with the inhibitor LOC14 and subsequently examined for IAV burden, NA activity, cytokine, and immune response. IAV-NA interacts with PDIA3 and this interaction is required for NA activity. PDIA3 ablation or inhibition decreased NA activity, viral burden, and inflammatory response in lung epithelial cells. LOC14 treatment significantly attenuated the influenza-induced inflammatory response in mice including the overall viral burden. These results provide evidence for PDIA3 inhibition suppressing NA activity, potentially providing a novel platform for host-targeted antiviral therapies.

A pH-Induced Reversible Conformational Switch Able to Control the Photocurrent Efficiency in a Peptide Supramolecular System.

External stimuli are potent tools that Nature uses to control protein function and activity. For instance, during viral entry and exit, pH variations are known to trigger large protein conformational changes. In Nature, also the electron transfer (ET) properties of ET proteins are influenced by pH-induced conformational changes. In this work, a pH-controlled, reversible 310 -helix to α-helix conversion (from acidic to highly basic pH values and vice versa) of a peptide supramolecular system built on a gold surface is described. The effect of pH on the ability of the peptide SAM to generate a photocurrent was investigated, with particular focus on the effect of the pH-induced conformational change on photocurrent efficiency. The films were characterized by electrochemical and spectroscopic techniques, and were found to be very stable over time, also in contact with a solution. They were also able to generate current under illumination, with an efficiency that is the highest recorded so far with biomolecular systems.

A Targeted Computational Screen of the SWEETLEAD Database Reveals FDA-Approved Compounds with Anti-Dengue Viral Activity.

Affordable and effective antiviral therapies are needed worldwide, especially against agents such as dengue virus that are endemic in underserved regions. Many antiviral compounds have been studied in cultured cells but are unsuitable for clinical applications due to pharmacokinetic profiles, side effects, or inconsistent efficacy across dengue serotypes. Such tool compounds can, however, aid in identifying clinically useful treatments. Here, computational screening (Rapid Overlay of Chemical Structures) was used to identify entries in an in silico database of safe-in-human compounds (SWEETLEAD) that display high chemical similarities to known inhibitors of dengue virus. Inhibitors of the dengue proteinase NS2B/3, the dengue capsid, and the host autophagy pathway were used as query compounds. Three FDA-approved compounds that resemble the tool molecules structurally, cause little toxicity, and display strong antiviral activity in cultured cells were selected for further analysis. Pyrimethamine (50% inhibitory concentration [IC50] = 1.2 μM), like the dengue proteinase inhibitor ARDP0006 to which it shows structural similarity, inhibited intramolecular NS2B/3 cleavage. Lack of toxicity early in infection allowed testing in mice, in which pyrimethamine also reduced viral loads. Niclosamide (IC50 = 0.28 μM), like dengue core inhibitor ST-148, affected structural components of the virion and inhibited early processes during infection. Vandetanib (IC50 = 1.6 μM), like cellular autophagy inhibitor spautin-1, blocked viral exit from cells and could be shown to extend survival in vivo Thus, three FDA-approved compounds with promising utility for repurposing to treat dengue virus infections and their potential mechanisms were identified using computational tools and minimal phenotypic screening.IMPORTANCE No antiviral therapeutics are currently available for dengue virus infections. By computationally overlaying the three-dimensional (3D) chemical structures of compounds known to inhibit dengue virus over those of compounds known to be safe in humans, we identified three FDA-approved compounds that are attractive candidates for repurposing as antivirals. We identified targets for two previously identified antiviral compounds and revealed a previously unknown potential anti-dengue compound, vandetanib. This computational approach to analyze a highly curated library of structures has the benefits of speed and cost efficiency. It also leverages mechanistic work with query compounds used in biomedical research to provide strong hypotheses for the antiviral mechanisms of the safer hit compounds. This workflow to identify compounds with known safety profiles can be expanded to any biological activity for which a small-molecule query compound has been identified, potentially expediting the translation of basic research to clinical interventions.

Clinical and Proteomic Correlates of Plasma ACE2 (Angiotensin-Converting Enzyme 2) in Human Heart Failure.

ACE2 (angiotensin-converting enzyme 2) is a key component of the renin-angiotensin-aldosterone system. Yet, little is known about the clinical and biologic correlates of circulating ACE2 levels in humans. We assessed the clinical and proteomic correlates of plasma (soluble) ACE2 protein levels in human heart failure. We measured plasma ACE2 using a modified aptamer assay among PHFS (Penn Heart Failure Study) participants (n=2248). We performed an association study of ACE2 against ≈5000 other plasma proteins measured with the SomaScan platform. Plasma ACE2 was not associated with ACE inhibitor and angiotensin-receptor blocker use. Plasma ACE2 was associated with older age, male sex, diabetes mellitus, a lower estimated glomerular filtration rate, worse New York Heart Association class, a history of coronary artery bypass surgery, and higher pro-BNP (pro-B-type natriuretic peptide) levels. Plasma ACE2 exhibited associations with 1011 other plasma proteins. In pathway overrepresentation analyses, top canonical pathways associated with plasma ACE2 included clathrin-mediated endocytosis signaling, actin cytoskeleton signaling, mechanisms of viral exit from host cells, EIF2 (eukaryotic initiation factor 2) signaling, and the protein ubiquitination pathway. In conclusion, in humans with heart failure, plasma ACE2 is associated with various clinical factors known to be associated with severe coronavirus disease 2019 (COVID-19), including older age, male sex, and diabetes mellitus, but is not associated with ACE inhibitor and angiotensin-receptor blocker use. Plasma ACE2 protein levels are prominently associated with multiple cellular pathways involved in cellular endocytosis, exocytosis, and intracellular protein trafficking. Whether these have a causal relationship with ACE2 or are relevant to novel coronavirus-2 infection remains to be assessed in future studies.

Host cell p53 associates with the feline calicivirus major viral capsid protein VP1, the protease-polymerase NS6/7, and the double-stranded RNA playing a role in virus replication

p53 is implicated in several cellular pathways such as induction of cell-cycle arrest, differentiation, senescence, and apoptosis. p53 is activated by a broad range of stress signals, including viral infections. While some viruses activate p53, others induce its inactivation, and occasionally p53 is differentially modulated during the replicative cycle. During calicivirus infections, apoptosis is required for virus exit and spread into the host; yet, the role of p53 during infection is unknown. By confocal microscopy, we found that p53 associates with FCV VP1, the protease-polymerase NS6/7, and the dsRNA. This interaction was further confirmed by proximity ligation assays, suggesting that p53 participates in the FCV replication. Knocked-down of p53 expression in CrFK cells before infection, resulted in a strong reduction of the non-structural protein levels and a decrease of the viral progeny production. These results indicate that p53 is associated with the viral replication complex and is required for an efficient FCV replication.

Hepatitis B Virus Exploits ERGIC-53 in Conjunction with COPII to Exit Cells.

Several decades after its discovery, the hepatitis B virus (HBV) still displays one of the most successful pathogens in human populations worldwide. The identification and characterization of interactions between cellular and pathogenic components are essential for the development of antiviral treatments. Due to its small-sized genome, HBV highly depends on cellular functions to produce and export progeny particles. Deploying biochemical-silencing methods and molecular interaction studies in HBV-expressing liver cells, we herein identified the cellular ERGIC-53, a high-mannose-specific lectin, and distinct components of the endoplasmic reticulum (ER) export machinery COPII as crucial factors of viral trafficking and egress. Whereas the COPII subunits Sec24A, Sec23B and Sar1 are needed for both viral and subviral HBV particle exit, ERGIC-53 appears as an exclusive element of viral particle propagation, therefore interacting with the N146-glycan of the HBV envelope in a productive manner. Cell-imaging studies pointed to ER-derived, subcellular compartments where HBV assembly initiates. Moreover, our findings provide evidence that HBV exploits the functions of ERGIC-53 and Sec24A after the envelopment of nucleocapsids at these compartments in conjunction with endosomal sorting complexes required for transport (ESCRT) components. These data reveal novel insights into HBV assembly and trafficking, illustrating therapeutic prospects for intervening with the viral life cycle.

Characterisation of the transcriptome and proteome of SARS-CoV-2 reveals a cell passage induced in-frame deletion of the furin-like cleavage site from the spike glycoprotein

BackgroundSARS-CoV-2 is a recently emerged respiratory pathogen that has significantly impacted global human health. We wanted to rapidly characterise the transcriptomic, proteomic and phosphoproteomic landscape of this novel coronavirus to provide a fundamental description of the virus’s genomic and proteomic potential.MethodsWe used direct RNA sequencing to determine the transcriptome of SARS-CoV-2 grown in Vero E6 cells which is widely used to propagate the novel coronavirus. The viral transcriptome was analysed using a recently developed ORF-centric pipeline. Allied to this, we used tandem mass spectrometry to investigate the proteome and phosphoproteome of the same virally infected cells.ResultsOur integrated analysis revealed that the viral transcripts (i.e. subgenomic mRNAs) generally fitted the expected transcription model for coronaviruses. Importantly, a 24 nt in-frame deletion was detected in over half of the subgenomic mRNAs encoding the spike (S) glycoprotein and was predicted to remove a proposed furin cleavage site from the S glycoprotein. Tandem mass spectrometry identified over 500 viral peptides and 44 phosphopeptides in virus-infected cells, covering almost all proteins predicted to be encoded by the SARS-CoV-2 genome, including peptides unique to the deleted variant of the S glycoprotein.ConclusionsDetection of an apparently viable deletion in the furin cleavage site of the S glycoprotein, a leading vaccine target, shows that this and other regions of SARS-CoV-2 proteins may readily mutate. The furin site directs cleavage of the S glycoprotein into functional subunits during virus entry or exit and likely contributes strongly to the pathogenesis and zoonosis of this virus. Our data emphasises that the viral genome sequence should be carefully monitored during the growth of viral stocks for research, animal challenge models and, potentially, in clinical samples. Such variations may result in different levels of virulence, morbidity and mortality.

Creating Contacts Between Replication and Movement at Plasmodesmata - A Role for Membrane Contact Sites in Plant Virus Infections?

To infect their hosts and cause disease, plant viruses must replicate within cells and move throughout the plant both locally and systemically. RNA virus replication occurs on the surface of various cellular membranes, whose shape and composition become extensively modified in the process. Membrane contact sites (MCS) can mediate non-vesicular lipid-shuttling between different membranes and viruses co-opt components of these structures to make their membrane environment suitable for replication. Whereas animal viruses exit and enter cells when moving throughout their host, the rigid wall of plant cells obstructs this pathway and plant viruses therefore move between cells symplastically through plasmodesmata (PD). PD are membranous channels connecting nearly all plant cells and are now viewed to constitute a specialized type of endoplasmic reticulum (ER)-plasma membrane (PM) MCS themselves. Thus, both replication and movement of plant viruses rely on MCS. However, recent work also suggests that for some viruses, replication and movement are closely coupled at ER-PM MCS at the entrances of PD. Movement-coupled replication at PD may be distinct from the main bulk of replication and virus accumulation, which produces progeny virions for plant-to-plant transmission. Thus, MCS play a central role in plant virus infections, and may provide a link between two essential steps in the viral life cycle, replication and movement. Here, we provide an overview of plant virus-MCS interactions identified to date, and place these in the context of the connection between viral replication and cell-to-cell movement.

The Hepatitis B Virus Envelope Proteins: Molecular Gymnastics Throughout the Viral Life Cycle.

New hepatitis B virions released from infected hepatocytes are the result of an intricate maturation process that starts with the formation of the nucleocapsid providing a confined space where the viral DNA genome is synthesized via reverse transcription. Virion assembly is finalized by the enclosure of the icosahedral nucleocapsid within a heterogeneous envelope. The latter contains integral membrane proteins of three sizes, collectively known as hepatitis B surface antigen, and adopts multiple conformations in the course of the viral life cycle. The nucleocapsid conformation depends on the reverse transcription status of the genome, which in turn controls nucleocapsid interaction with the envelope proteins for virus exit. In addition, after secretion the virions undergo a distinct maturation step during which a topological switch of the large envelope protein confers infectivity. Here we review molecular determinants for envelopment and models that postulate molecular signals encoded in the capsid scaffold conducive or adverse to the recruitment of envelope proteins.

Unraveling the Genomic-Epigenomic Interaction Landscape in Triple Negative and Non-Triple Negative Breast Cancer.

Background: The recent surge of next generation sequencing of breast cancer genomes has enabled development of comprehensive catalogues of somatic mutations and expanded the molecular classification of subtypes of breast cancer. However, somatic mutations and gene expression data have not been leveraged and integrated with epigenomic data to unravel the genomic-epigenomic interaction landscape of triple negative breast cancer (TNBC) and non-triple negative breast cancer (non-TNBC). Methods: We performed integrative data analysis combining somatic mutation, epigenomic and gene expression data from The Cancer Genome Atlas (TCGA) to unravel the possible oncogenic interactions between genomic and epigenomic variation in TNBC and non-TNBC. We hypothesized that within breast cancers, there are differences in somatic mutation, DNA methylation and gene expression signatures between TNBC and non-TNBC. We further hypothesized that genomic and epigenomic alterations affect gene regulatory networks and signaling pathways driving the two types of breast cancer. Results: The investigation revealed somatic mutated, epigenomic and gene expression signatures unique to TNBC and non-TNBC and signatures distinguishing the two types of breast cancer. In addition, the investigation revealed molecular networks and signaling pathways enriched for somatic mutations and epigenomic changes unique to each type of breast cancer. The most significant pathways for TNBC were: retinal biosynthesis, BAG2, LXR/RXR, EIF2 and P2Y purigenic receptor signaling pathways. The most significant pathways for non-TNBC were: UVB-induced MAPK, PCP, Apelin endothelial, Endoplasmatic reticulum stress and mechanisms of viral exit from host signaling Pathways. Conclusion: The investigation revealed integrated genomic, epigenomic and gene expression signatures and signing pathways unique to TNBC and non-TNBC, and a gene signature distinguishing the two types of breast cancer. The study demonstrates that integrative analysis of multi-omics data is a powerful approach for unravelling the genomic-epigenomic interaction landscape in TNBC and non-TNBC.