Protein Open Reading Frame Research Articles

New Resources for the Specific and Sensitive Detection of the Emerging Tomato Brown Rugose Fruit Virus.

Plant viruses can evolve towards new pathogenic entities that may eventually cause outbreaks and become epidemics or even pandemics. Seven years ago, tomato brown rugose fruit virus (ToBRFV) emerged, overcoming the genetic resistance that had been employed for more than sixty years against tobamoviruses in tomato. Since then, ToBRFV has spread worldwide, producing significant losses in tomato crops. While new resistances are deployed, the only means of control is the implementation of effective prevention and eradication strategies. For this purpose, in this work, we have designed, assessed, and compared an array of tests for the specific and sensitive detection of the ToBRFV in leaf samples. First, two monoclonal antibodies were generated against a singular peptide of the ToBRFV coat protein; antibodies were utilized to devise a double-antibody-sandwich enzyme-linked immunosorbent assay (DAS-ELISA) test that sensitively detects this virus and has no cross-reactivity with other related tobamoviruses. Second, a real-time quantitative PCR (RT-qPCR) test targeting the RNA-dependent replicase open reading frame (ORF) was designed, and its performance and specificity validated in comparison with the CaTa28 and CSP1325 tests recommended by plant protection authorities in Europe. Third, in line with the tendency to use field-deployable diagnostic techniques, we developed and tested two sets of loop-mediated isothermal amplification (LAMP) primers to double-check the detection of the movement protein ORF of ToBRFV, and one set that works as an internal control. Finally, we compared all of these methods by employing a collection of samples with different ToBRFV loads to evaluate the overall performance of each test.

Intramolecular recombination enables the formation of hepatitis B virus (HBV) cccDNA in mice after HBV genome transfer using recombinant AAV vectors

The mouse is not a natural host of hepatitis B virus (HBV) infection and - despite engraftment of hepatocytes with the HBV receptor - does not support formation of HBV covalently closed circular (ccc) DNA serving as a template for viral transcription and permitting persistent infection. In a recent study, cccDNA formation in mouse hepatocytes has been described following an HBV genome delivery by a recombinant, adeno-associated virus vector (rAAV) (Lucifora et al., 2017). The integrity of HBV cccDNA, its origin and functionality, however, remained open. In this study, we investigated the identity, origin, and functionality of cccDNA established in mice infected with rAAV carrying 1.3-fold overlength HBV genomes. We show that replication of HBV genotypes A, B, C and D can be initiated in mouse livers, and that cccDNA derived from all genotypes is detected. Restriction enzyme and exonuclease digestion as well as sequencing analysis of cccDNA amplicons revealed authentic HBV cccDNA without any detectable alteration compared to cccDNA established after HBV infection of human liver cells. Mouse livers transduced with a core protein-deficient HBV using rAAV still supported cccDNA formation demonstrating that the genesis of cccDNA was independent of HBV replication. When mice were infected with an rAAV-HBV1.3 carrying premature stop codons in the 5′ but not in the 3′ core protein open reading frame, the stop codon was partially replaced by the wild-type sequence. This strongly indicated that intramolecular recombination, based on >900 identical base pairs residing at the both ends of the HBV1.3 transgene was the origin of cccDNA formation. Accordingly, we observed a constant loss of cccDNA molecules from mouse livers over time, while HBeAg levels increased over the first two weeks after rAAV-HBV1.3 infection and remained constant thereafter, suggesting a minor contribution of the cccDNA molecules formed to viral transcription and protein expression. In summary, our results provide strong evidence that intramolecular recombination of an overlength, linear HBV genome, but not HBV genome recycling, enables cccDNA formation in rAAV-HBV mouse models.

First Evidence of Function for Schistosoma japonicumriok-1 and RIOK-1.

Protein kinases are known as key molecules that regulate many biological processes in animals. The right open reading frame protein kinase (riok) genes are known to be essential regulators in model organisms such as the free-living nematode Caenorhabditis elegans. However, very little is known about their function in parasitic trematodes (flukes). In the present study, we characterized the riok-1 gene (Sj-riok-1) and the inferred protein (Sj-RIOK-1) in the parasitic blood fluke, Schistosoma japonicum. We gained a first insight into function of this gene/protein through double-stranded RNA interference (RNAi) and chemical inhibition. RNAi significantly reduced Sj-riok-1 transcription in both female and male worms compared with untreated control worms, and subtle morphological alterations were detected in the ovaries of female worms. Chemical knockdown of Sj-RIOK-1 with toyocamycin (a specific RIOK-1 inhibitor/probe) caused a substantial reduction in worm viability and a major accumulation of mature oocytes in the seminal receptacle (female worms), and of spermatozoa in the sperm vesicle (male worms). These phenotypic alterations indicate that the function of Sj-riok-1 is linked to developmental and/or reproductive processes in S. japonicum.

Small-Scale Plasma Membrane Preparation for the Analysis of Candida albicans Cdr1-mGFPHis.

The successful biochemical and biophysical characterization of ABC transporters depends heavily on the choice of the heterologous expression system. Over the past two decades, we have developed a yeast membrane protein expression platform that has been used to study many important fungal membrane proteins. The expression host Saccharomyces cerevisiae ADΔΔ is deleted in seven major endogenous ABC transporters and it contains the transcription factor Pdr1-3 with a gain-of-function mutation that enables the constitutive overexpression of heterologous membrane protein genes stably integrated as single copies at the genomic PDR5 locus. The creation of versatile plasmid vectors and the optimization of one-step cloning strategies enables the rapid and accurate cloning, mutagenesis, and expression of heterologous ABC transporters. Here, we describe the development and use of a novel protease-cleavable mGFPHis double tag (i.e., the monomeric yeast enhanced green fluorescent protein yEGFP3 fused to a six-histidine affinity purification tag) that was designed to avoid possible interference of the tag with the protein of interest and to increase the binding efficiency of the His tag to nickel-affinity resins. The fusion of mGFPHis to the membrane protein ORF (open reading frame) enables easy quantification of the protein by inspection of polyacrylamide gels and detection of degradation products retaining the mGFPHis tag. We demonstrate how this feature facilitates detergent screening for membrane protein solubilization. A protocol for the efficient, fast, and reliable isolation of the small-scale plasma membrane preparations of the C-terminally tagged Candida albicans multidrug efflux transporter Cdr1 overexpressed in S. cerevisiae ADΔΔ, is presented. This small-scale plasma membrane isolation protocol generates high-quality plasma membranes within a single working day. The plasma membrane preparations can be used to determine the enzyme activities of Cdr1 and Cdr1 mutant variants.

Characterization of ALTO-encoding circular RNAs expressed by Merkel cell polyomavirus and trichodysplasia spinulosa polyomavirus.

Circular RNAs (circRNAs) are a conserved class of RNAs with diverse functions, including serving as messenger RNAs that are translated into peptides. Here we describe circular RNAs generated by human polyomaviruses (HPyVs), some of which encode variants of the previously described alternative large T antigen open reading frame (ALTO) protein. Circular ALTO RNAs (circALTOs) can be detected in virus positive Merkel cell carcinoma (VP-MCC) cell lines and tumor samples. CircALTOs are stable, predominantly located in the cytoplasm, and N6-methyladenosine (m6A) modified. The translation of MCPyV circALTOs into ALTO protein is negatively regulated by MCPyV-generated miRNAs in cultured cells. MCPyV ALTO expression increases transcription from some recombinant promoters in vitro and upregulates the expression of multiple genes previously implicated in MCPyV pathogenesis. MCPyV circALTOs are enriched in exosomes derived from VP-MCC lines and circALTO-transfected 293T cells, and purified exosomes can mediate ALTO expression and transcriptional activation in MCPyV-negative cells. The related trichodysplasia spinulosa polyomavirus (TSPyV) also expresses a circALTO that can be detected in infected tissues and produces ALTO protein in cultured cells. Thus, human polyomavirus circRNAs are expressed in human tumors and infected tissues and express proteins that have the potential to modulate the infectious and tumorigenic properties of these viruses.

SARS‐CoV‐2 ORF8 Accessory Protein Dimerization Domains and Protein‐Protein Host Interaction

As the world continues to fight the coronavirus pandemic caused by SARS‐CoV‐2, we are gaining valuable insights by comparing this virus to other human coronaviruses such as SARS‐CoV and MERS‐CoV that have also caused outbreaks. Coronaviruses infect many animal species, but disease severity varies between strains. Viral accessory proteins are generally implicated in increased infectivity, pathogenicity, and virulence. The accessory protein open reading frame 8 (ORF8), although not essential for viral replication, appears to play a critical role in disease severity by inhibiting multiple pathways of the immune response. Furthermore, the ORF8 gene is found within a highly variable portion of the genome, increasing the possibility of dangerous mutant forms arising. Structure‐based design of therapeutics holds great promise for effective targeting of such rapidly evolving threats. Thus, we designed physical models and computer representations of ORF8, based on the published structure 7JTL, to better evaluate the structural features responsible for viral pathogenicity. ORF8 exists as a homodimer held together by amino acid residues that interact through hydrophobic interactions, hydrogen bonding, a salt bridge, and a disulfide bond. These interactions occur in a region referred to as the covalent interface. Another interface, referred to as the noncovalent interface, exists between separate homodimers and results in oligomerization. The homodimers in the oligomer are held together by an intermolecular beta sheet that is stabilized by an extensive hydrophobic surface. These two dimerization interfaces are unique to SARS‐CoV‐2 and have been proposed to be involved in the protein's ability to evade and suppress the host immune response. The computer and physical representations allow for better visualization of the ORF8 structure to evaluate the role of the multimeric structure. ORF8 has been found to interact with various host proteins, including Interleukin‐17 Receptor A (IL17RA), a pro‐inflammatory cytokine. To further corroborate the existence of this interaction and to better understand the role oligomerization might play in pathogenicity, we assessed the possible interactions of ORF8 (7JTL) and IL17RA (5N9B) through theoretical modeling using available tools such as HDOCK. A better understanding of these inter‐subunit interactions may improve our understanding of the unique mechanism the virus uses to evade the immune system and may be instrumental in the development of effective therapeutics.

Bioinformatic Prediction of an tRNASec Gene Nested inside an Elongation Factor SelB Gene in Alphaproteobacteria

In bacteria, selenocysteine (Sec) is incorporated into proteins via the recoding of a particular codon, the UGA stop codon in most cases. Sec-tRNASec is delivered to the ribosome by the Sec-dedicated elongation factor SelB that also recognizes a Sec-insertion sequence element following the codon on the mRNA. Since the excess of SelB may lead to sequestration of Sec-tRNASec under selenium deficiency or oxidative stress, the expression levels of SelB and tRNASec should be regulated. In this bioinformatic study, I analyzed the Rhizobiales SelB species because they were annotated to have a non-canonical C-terminal extension. I found that the open reading frame (ORF) of diverse Alphaproteobacteria selB genes includes an entire tRNASec sequence (selC) and overlaps with the start codon of the downstream ORF. A remnant tRNASec sequence was found in the Sinorhizobium meliloti selB genes whose products have a shorter C-terminal extension. Similar overlapping traits were found in Gammaproteobacteria and Nitrospirae. I hypothesized that once the tRNASec moiety is folded and processed, the expression of the full-length SelB may be repressed. This is the first report on a nested tRNA gene inside a protein ORF in bacteria.

Mucosal Influenza Vector Vaccine Carrying TB10.4 and HspX Antigens Provides Protection against Mycobacterium tuberculosis in Mice and Guinea Pigs.

New strategies providing protection against tuberculosis (TB) are still pending. The airborne nature of Mycobacterium tuberculosis (M.tb) infection assumes that the mucosal delivery of the TB vaccine could be a more promising strategy than the systemic route of immunization. We developed a mucosal TB vaccine candidate based on recombinant attenuated influenza vector (Flu/THSP) co-expressing truncated NS1 protein NS1(1–124) and a full-length TB10.4 and HspX proteins of M.tb within an NS1 protein open reading frame. The Flu/THSP vector was safe and stimulated a systemic TB-specific CD4+ and CD8+ T-cell immune response after intranasal immunization in mice. Double intranasal immunization with the Flu/THSP vector induced protection against two virulent M.tb strains equal to the effect of BCG subcutaneous injection in mice. In a guinea pig TB model, one intranasal immunization with Flu/THSP improved protection against M.tb when tested as a vaccine candidate for boosting BCG-primed immunity. Importantly, enhanced protection provided by a heterologous BCG-prime → Flu/THSP vector boost immunization scheme was associated with a significantly reduced lung and spleen bacterial burden (mean decrease of 0.77 lg CFU and 0.72 lg CFU, respectively) and improved lung pathology 8.5 weeks post-infection with virulent M.tb strain H37Rv.

ORF8 contributes to cytokine storm during SARS-CoV-2 infection by activating IL-17 pathway

SummaryRecently, COVID-19 caused by the novel coronavirus SARS-CoV-2 has brought great challenges to the world. More and more studies have shown that patients with severe COVID-19 may suffer from cytokine storm syndrome; however, there are few studies on its pathogenesis. Here we demonstrated that SARS-CoV-2 coding protein open reading frame 8 (ORF8) acted as a contributing factor to cytokine storm during COVID-19 infection. ORF8 could activate IL-17 signaling pathway and promote the expression of pro-inflammatory factors. Moreover, we demonstrated that treatment of IL17RA antibody protected mice from ORF8-induced inflammation. Our findings are helpful to understand the pathogenesis of cytokine storm caused by SARS-CoV-2 and provide a potential target for the development of COVID-19 therapeutic drugs.

Apoptosis Triggered by ORF3 Proteins of the Circoviridae Family.

Apoptosis, a form of the programmed cell death, is an indispensable defense mechanism regulating cellular homeostasis and is triggered by multiple stimuli. Because of the regulation of apoptosis in cellular homeostasis, viral proteins with apoptotic activity are particular foci of on antitumor therapy. One representative viral protein is the open reading frame 3 (ORF3) protein, also named as apoptin in the Circoviridae chicken anemia virus (CAV), and has the ability to induce tumor-specific apoptosis. Proteins encoded by ORF3 in other circovirus species, such as porcine circovirus (PCV) and duck circovirus (DuCV), have also been reported to induce apoptosis, with subtle differences in apoptotic activity based on cell types. This article is aimed at reviewing the latest research advancements in understanding ORF3 protein-mediated apoptosis mechanisms of Circoviridae from three perspectives: subcellular localization, interactions with host proteins, and participation in multiple apoptotic signaling pathways, providing a scientific basis for circovirus pathogenesis and a reference on its potential anticancer function.

Identifying Circular RNAs in HepG2 Expressing Genotype IV Swine Hepatitis E Virus ORF3 Via Whole Genome Sequencing

Swine hepatitis E (SHE) is a new type of zoonotic infectious disease caused by swine hepatitis E virus (SHEV). Open reading frame 3 (ORF3) is a key regulatory and virulent protein of SHEV. Circular RNAs (circRNAs) are a special kind of non-coding RNA molecule, which has a closed ring structure. In this study, to identify the circRNA profile in host cells affected by SHEV ORF3, adenovirus ADV4-ORF3 mediated the overexpression of ORF3 in HepG2 cells, whole genome sequencing was used to investigate the differentially expressed circRNAs, GO and KEGG were performed to enrichment analyze of differentially expressed circRNA-hosting gene, and Targetscan and miRanda softwares were used to analyze the interaction between circRNA and miRNA. The results showed adenovirus successfully mediated the overexpression of ORF3 in HepG2 cells, 1,105 up-regulation circRNAs and 1,556 down-regulation circRNAs were identified in ADV4-ORF3 infection group compared with the control. GO function enrichment analysis of differentially expressed circRNAs-hosting genes classified three main categories (cellular component, biological process and molecular function). KEGG pathway enrichment analysis scatter plot showed the pathway term of top20. The circRNAs with top10 number of BS sites for qRT-PCR validation were selected to confirmed, the results indicated that the up-regulated hsa_circ_0001423 and hsa_circ_0006404, and down-regulated of hsa_circ_0004833 and hsa_circ_0007444 were consistent with the sequencing data. Our findings first preliminarily found that ORF3 protein may affect triglyceride activation (GO:0006642) and riboflavin metabolism (ko00740) in HepG2 cells, which provides a scientific basis for further elucidating the effect of ORF3 on host lipid metabolism and the mechanism of SHEV infection.

Immunity, virus evolution, and effectiveness of SARS-CoV-2 vaccines.

Phylogenetic and pathogenesis studies of the severe acute respiratory syndrome-related coronaviruses (SARS-CoVs) strains have highlighted some specific mutations that could confer the RNA genome fitness advantages and immunological resistance for their rapid spread in the human population. The analyses of 30 kb RNA SARS-CoVs genome sequences, protein structures, and functions have provided us a perspective of how host-virus protein-protein complexes act to mediate virus infection. The open reading frame (ORF)1a and ORF1b translation yields 16 non-structural (nsp1-16) and 6 accessory proteins (p6, p7a, p8ab, p9b) with multiple functional domains. Viral proteins recruit over 300 host partners forming hetero-oligomeric complexes enabling the viral RNA synthesis, packing, and virion release. Many cellular host factors and the innate immune cells through pattern-recognition receptors and intracellular RNA sensor molecules act to inhibit virus entry and intracellular replication. However, non-structural ORF proteins hijack them and suppress interferon synthesis and its antiviral effects. Pro-inflammatory chemokines and cytokines storm leads to dysfunctional inflammation, lung injury, and several clinical symptoms in patients. During the global pandemic, COVID-19 patients were identified with non-synonymous substitution of G614D in the spike protein, indicating virus co-evolution in host cells. We review findings that suggest that host RNA editing and DNA repair systems, while carrying on recombination, mutation, and repair of viral RNA intermediates, may facilitate virus evolution. Understanding how the host cell RNA replication process may be driven by SARS-CoV-2 RNA genome fitness will help the testing of vaccines effectiveness to multiple independent mutated coronavirus strains that will emerge.

Genomic and Transcriptomic Survey Provides New Insight into the Organization and Transposition Activity of Highly Expressed LTR Retrotransposons of Sunflower (Helianthus annuus L.).

LTR retrotransposons (RTEs) play a crucial role in plant genome evolution and adaptation. Although RTEs are generally silenced in somatic plant tissues under non-stressed conditions, some expressed RTEs (exRTEs) escape genome defense mechanisms. As our understanding of exRTE organization in plants is rudimentary, we systematically surveyed the genomic and transcriptomic organization and mobilome (transposition) activity of sunflower (Helianthus annuus L.) exRTEs. We identified 44 transcribed RTEs in the sunflower genome and demonstrated their distinct genomic features: more recent insertion time, longer open reading frame (ORF) length, and smaller distance to neighboring genes. We showed that GAG-encoding ORFs are present at significantly higher frequencies in exRTEs, compared with non-expressed RTEs. Most exRTEs exhibit variation in copy number among sunflower cultivars and one exRTE Gagarin produces extrachromosomal circular DNA in seedling, demonstrating recent and ongoing transposition activity. Nanopore direct RNA sequencing of full-length RTE RNA revealed complex patterns of alternative splicing in RTE RNAs, resulting in isoforms that carry ORFs for distinct RTE proteins. Together, our study demonstrates that tens of expressed sunflower RTEs with specific genomic organization shape the hidden layer of the transcriptome, pointing to the evolution of specific strategies that circumvent existing genome defense mechanisms.

Genetic Adaptation of Coxsackievirus B1 during Persistent Infection in Pancreatic Cells.

Coxsackie B (CVB) viruses have been associated with type 1 diabetes. We have recently observed that CVB1 was linked to the initiation of the autoimmune process leading to type 1 diabetes in Finnish children. Viral persistency in the pancreas is currently considered as one possible mechanism. In the current study persistent infection was established in pancreatic ductal and beta cell lines (PANC-1 and 1.1B4) using four different CVB1 strains, including the prototype strain and three clinical isolates. We sequenced 5′ untranslated region (UTR) and regions coding for structural and non-structural proteins and the second single open reading frame (ORF) protein of all persisting CVB1 strains using next generation sequencing to identify mutations that are common for all of these strains. One mutation, K257R in VP1, was found from all persisting CVB1 strains. The mutations were mainly accumulated in viral structural proteins, especially at BC, DE, EF loops and C-terminus of viral capsid protein 1 (VP1), the puff region of VP2, the knob region of VP3 and infection-enhancing epitope of VP4. This showed that the capsid region of the viruses sustains various changes during persistency some of which could be hallmark(s) of persistency.

The Monoclonal Antibody Recognized the Open Reading Frame Protein in Porcine Circovirus Type 2-Infected Peripheral Blood Mononuclear Cells

The purpose of this study in the context of the open reading frame 3 (ORF3) protein of porcine circovirus type 2 (PCV2) was especially its location and its relation to the capsid protein and the apoptosis protein in PCV2-infected porcine peripheral blood mononuclear cells (PBMCs). To detect the ORF3 protein, monoclonal antibodies (mAbs) were generated in this study. The mAb 7D3 binds to the ORF3 peptide (residues 35–66) and the native ORF3 protein in PCV2-infected PBMCs, as shown by immunofluorescence assay (IFA). The data show that 3–5% of PBMCs were positive for ORF3 protein or p53 protein. Further, 78–82% of PBMCs were positive for the capsid. This study confirmed the ORF3 protein not only colocalized with the capsid protein but also colocalized with the p53 protein in PBMCs. Immunoassays were conducted in this study to detect the capsid protein, the ORF3 protein, anti-capsid IgG, and anti-ORF3 IgG. The data show the correlation (r = 0.758) of the ORF3 protein and the capsid protein in the blood samples from the PCV2-infected herd. However, each anti-viral protein IgG had a different curve of the profile in the same herd after vaccination. Overall, this study provides a blueprint to explore the ORF3 protein in PCV2-infected PBMCs.

Haemoglobin oxygen affinity in patients with severe COVID‐19 infection

Severely ill COVID-19 patients have an atypical form of respiratory distress. Acute respiratory distress syndrome (ARDS), while heterogenous, classically presents with severe hypoxia and decreased lung compliance.1 In SARS-CoV-2 coronavirus infection (COVID-19) lung mechanics and compliance are relatively well conserved until late in the disease course. Despite early preservation of lung compliance, the hypoxaemia in COVID-19 is severe and is ultimately the primary mechanism of multiple organ failure and death.2 The underlying pathology is due to COVID-19 entering cells via the ACE2 receptor. This receptor is expressed on many cells including alveolar epithelial cells and vascular endothelium resulting in a profound immune response and widespread endothelial dysfunction.3 We were both interested and concerned to read the report by Liu and Li.4 This in silico study used homology modelling and molecular docking algorithms to predict theoretical interactions between COVID-19 and haemoglobin (Hb). COVID-19 expresses a variety of open reading frame proteins including orf1ab, ORF3a, ORF6, ORF7a, ORF10 and ORF8. Liu and Li4 predict that these ORF proteins can interact with haemoglobin to reduce both oxygen (O2) affinity and total haemoglobin content. They report specifically that their modelling predicts a theoretical interaction between orf1ab, ORF10 and ORF3a and the haem moiety of the beta chain while ORF8 and viral surface glycoproteins can directly target the porphyrin. They assert that their data support the use of chloroquine and hydroxychloroquine as therapeutic agents. However, the methods used by Lui and Li have very recently been criticised — not least of all because they are unsupported by experimental evidence.5 It is unclear what testable physiological consequences might arise from an interaction between viral proteins and Hb but these are presumed to include a generalised loss of Hb and/or a change in O2 affinity. While mild anaemia and decreased Hb content have been reported,6,7 to our knowledge the effect of COVID-19 on O2 affinity has not been investigated. Multiple studies and national guidelines strongly support restrictive transfusion practice in the critically ill as a basic standard of care.8 It is possible that if the haemoglobin molecule was adversely affected by COVID-19, and the O2 affinity curve right-shifted, restrictive transfusion could be potentially harmful. Given the very high mortality of these ventilated COVID-19 patients, we considered evaluation of any effect on the O2 affinity curve as urgent to inform our practice. Physicians caring for patients infected with COVID-19 ordered an Hb O2 affinity assessment in 14 patients (four newly admitted from the emergency department and 10 patients receiving mechanical ventilation in critical care) which we compared to 11 age- and sex-matched controls as part of an audit to assess the utility of this investigation in the management of patients with severe disease (Guys and St Thomas’ Hospital Audit No. 10855; Table I). No extra samples or blood tests were taken from patients — rather relevant samples were identified after a routine full blood count had been reported. O2 saturation curves from control and COVID-19-positive patients are shown in Fig 1. It is clear that there are no differences in O2 affinity or co-operativity between samples from COVID-19 and matched controls. The P50 and Hill Slope values are given in Table I. However, as is common in critically ill patients, there is a substantial anaemia and loss of total haemoglobin. In summary, while the total O2-carrying capacity will certainly be reduced by the anaemia and loss of total haemoglobin, we see no evidence, using standard clinical measures in a small cohort of patients, to suggest that COVID-19 alters haemoglobin O2 affinity and there is no requirement to change our transfusion practice. We are grateful for the help of the Special Haematology and Haematology laboratory staff at Viapath, Guy's & St Thomas's NHS Foundation Trust. MJS is supported by the British Heart Foundation (RG/12/4/29426). The help of Felix Torrance with the curve-fitting equations is gratefully acknowledged. CCS and MJS conceived and designed the study; BJH provided access to Viapath analytical labs and haematology expertise; RA and HK were responsible for patient care; YD and SW analysed blood samples and patient data; MJS analysed the data and wrote the first draft; all authors revised and approved the final submission.

Structural Basis for Designing Multiepitope Vaccines Against COVID-19 Infection: In Silico Vaccine Design and Validation.

BackgroundThe novel coronavirus disease (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to the ongoing 2019-2020 pandemic. SARS-CoV-2 is a positive-sense single-stranded RNA coronavirus. Effective countermeasures against SARS-CoV-2 infection require the design and development of specific and effective vaccine candidates.ObjectiveTo address the urgent need for a SARS-CoV-2 vaccine, in the present study, we designed and validated one cytotoxic T lymphocyte (CTL) and one helper T lymphocyte (HTL) multi-epitope vaccine (MEV) against SARS-CoV-2 using various in silico methods.MethodsBoth designed MEVs are composed of CTL and HTL epitopes screened from 11 Open Reading Frame (ORF), structural and nonstructural proteins of the SARS-CoV-2 proteome. Both MEVs also carry potential B-cell linear and discontinuous epitopes as well as interferon gamma–inducing epitopes. To enhance the immune response of our vaccine design, truncated (residues 10-153) Onchocerca volvulus activation-associated secreted protein-1 was used as an adjuvant at the N termini of both MEVs. The tertiary models for both the designed MEVs were generated, refined, and further analyzed for stable molecular interaction with toll-like receptor 3. Codon-biased complementary DNA (cDNA) was generated for both MEVs and analyzed in silico for high level expression in a mammalian (human) host cell line.ResultsIn the present study, we screened and shortlisted 38 CTL, 33 HTL, and 12 B cell epitopes from the 11 ORF protein sequences of the SARS-CoV-2 proteome. Moreover, the molecular interactions of the screened epitopes with their respective human leukocyte antigen allele binders and the transporter associated with antigen processing (TAP) complex were positively validated. The shortlisted screened epitopes were utilized to design two novel MEVs against SARS-CoV-2. Further molecular models of both MEVs were prepared, and their stable molecular interactions with toll-like receptor 3 were positively validated. The codon-optimized cDNAs of both MEVs were also positively analyzed for high levels of overexpression in a human cell line.ConclusionsThe present study is highly significant in terms of the molecular design of prospective CTL and HTL vaccines against SARS-CoV-2 infection with potential to elicit cellular and humoral immune responses. The epitopes of the designed MEVs are predicted to cover the large human population worldwide (96.10%). Hence, both designed MEVs could be tried in vivo as potential vaccine candidates against SARS-CoV-2.

ELISA-based detection of Open Reading Frame protein 1 in patients at risk of developing lung cancer

BackgroundEarly detection of lung cancer significantly improves survival outcomes. Thus, lung cancer screening for high-risk individuals using low-dose CT scan (LDCT) is recommended. LDCT has several limitations, and often requires invasive follow up. Previously, we have developed an ELISA for measurement of Open Reading Frame 1 protein (ORF1p) in serum. We assessed whether ORF1p can be used as a risk assessment biomarker for patients at high risk for developing lung cancer. PatientsPatients with risk factors for lung cancer were enrolled in our study with consent under IRB approval. A total of 122 patients were included. The lung cancer cohort consisted of 38 patients with varying stages of cancer undergoing treatment. MethodsORF1p quantification was performed using our ELISA assay on serum samples. ResultsORF1p was significantly increased in the serum of patients with identified lung nodules compared to those without nodules (P = 0.0007). ORF1p was also significantly increased in patients who were recommended for follow up (P = 0.0004). When comparing the at-risk cohort to patients with lung cancer, there was not a significant difference in ORF1p levels. ConclusionORF1p can be used to identify patients at high risk of developing lung cancer and may provide an effective, non-invasive risk assessment marker to complement LDCT screening.

Multivesicular body sorting and the exosomal pathway are required for the release of rat hepatitis E virus from infected cells

Recent reports have shown that rat hepatitis E virus (HEV) is capable of infecting humans. We also successfully propagated rat HEV into human PLC/PRF/5 cells, raising the possibility of a similar mechanism shared by human HEV and rat HEV. Rat HEV has the proline-rich sequence, PxYPMP, in the open reading frame 3 (ORF3) protein that is indispensable for its release. However, the release mechanism remains unclear. The overexpression of dominant-negative (DN) mutant of vacuolar protein sorting (Vps)4A or Vps4B decreased rat HEV release to 23.9 % and 18.0 %, respectively. The release of rat HEV was decreased to 8.3 % in tumor susceptibility gene 101 (Tsg101)-depleted cells and to 31.5 % in apoptosis-linked gene 2-interacting protein X (Alix)-depleted cells. Although rat HEV ORF3 protein did not bind to Tsg101, we found a 90-kDa protein capable of binding to wild-type rat HEV ORF3 protein but not to ORF3 mutant with proline to leucine mutations in the PxYPMP motif. Rat HEV release was also decreased in Ras-associated binding 27A (Rab27A)- or hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs)-depleted cells (to 20.1 % and 18.5 %, respectively). In addition, the extracellular rat HEV levels in the infected PLC/PRF/5 cells were increased after treatment with Bafilomycin A1 and decreased after treatment with GW4869. These results indicate that rat HEV utilizes multivesicular body (MVB) sorting for its release and that the exosomal pathway is required for rat HEV egress. A host protein alternative to Tsg101 that can bind to rat HEV ORF3 should be explored in further study.

Use of the CRISPR-Cas9 System in Drosophila Cultured Cells to Introduce Fluorescent Tags into Endogenous Genes.

The CRISPR-Cas9 system makes it possible to cause double-strand breaks in specific regions, inducing repair. In the presence of a donor construct, repair can involve insertion or 'knock-in' of an exogenous cassette. One common application of knock-in technology is to generate cell lines expressing fluorescently tagged endogenous proteins. The standard approach relies on production of a donor plasmid with ∼500 to 1000 bp of homology on either side of an insertion cassette that contains the fluorescent protein open reading frame (ORF). We present two alternative methods for knock-in of fluorescent protein ORFs into Cas9-expressing Drosophila S2R+ cultured cells, the single-stranded DNA (ssDNA) Drop-In method and the CRISPaint universal donor method. Both methods eliminate the need to clone a large plasmid donor for each target. We discuss the advantages and limitations of the standard, ssDNA Drop-In, and CRISPaint methods for fluorescent protein tagging in Drosophila cultured cells. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Knock-in into Cas9-positive S2R+ cells using the ssDNA Drop-In approach Basic Protocol 2: Knock-in into Cas9-positive S2R+ cells by homology-independent insertion of universal donor plasmids that provide mNeonGreen (CRISPaint method) Support Protocol 1: sgRNA design and cloning Support Protocol 2: ssDNA donor synthesis Support Protocol 3: Transfection using Effectene Support Protocol 4: Electroporation of S2R+-MT::Cas9 Drosophila cells Support Protocol 5: Single-cell isolation of fluorescent cells using FACS.