ORF2 Protein Research Articles

The Acid Ceramidase Is a SARS-CoV-2 Host Factor

SARS-CoV-2 variants such as the delta or omicron variants, with higher transmission rates, accelerated the global COVID-19 pandemic. Thus, novel therapeutic strategies need to be deployed. The inhibition of acid sphingomyelinase (ASM), interfering with viral entry by fluoxetine was reported. Here, we described the acid ceramidase as an additional target of fluoxetine. To discover these effects, we synthesized an ASM-independent fluoxetine derivative, AKS466. High-resolution SARS-CoV-2–RNA FISH and RTqPCR analyses demonstrate that AKS466 down-regulates viral gene expression. It is shown that SARS-CoV-2 deacidifies the lysosomal pH using the ORF3 protein. However, treatment with AKS488 or fluoxetine lowers the lysosomal pH. Our biochemical results show that AKS466 localizes to the endo-lysosomal replication compartments of infected cells, and demonstrate the enrichment of the viral genomic, minus-stranded RNA and mRNAs there. Both fluoxetine and AKS466 inhibit the acid ceramidase activity, cause endo-lysosomal ceramide elevation, and interfere with viral replication. Furthermore, Ceranib-2, a specific acid ceramidase inhibitor, reduces SARS-CoV-2 replication and, most importantly, the exogenous supplementation of C6-ceramide interferes with viral replication. These results support the hypotheses that the acid ceramidase is a SARS-CoV-2 host factor.

Roles and functions of SARS-CoV-2 proteins in host immune evasion.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) evades the host immune system through a variety of regulatory mechanisms. The genome of SARS-CoV-2 encodes 16 non-structural proteins (NSPs), four structural proteins, and nine accessory proteins that play indispensable roles to suppress the production and signaling of type I and III interferons (IFNs). In this review, we discussed the functions and the underlying mechanisms of different proteins of SARS-CoV-2 that evade the host immune system by suppressing the IFN-β production and TANK-binding kinase 1 (TBK1)/interferon regulatory factor 3 (IRF3)/signal transducer and activator of transcription (STAT)1 and STAT2 phosphorylation. We also described different viral proteins inhibiting the nuclear translocation of IRF3, nuclear factor-κB (NF-κB), and STATs. To date, the following proteins of SARS-CoV-2 including NSP1, NSP6, NSP8, NSP12, NSP13, NSP14, NSP15, open reading frame (ORF)3a, ORF6, ORF8, ORF9b, ORF10, and Membrane (M) protein have been well studied. However, the detailed mechanisms of immune evasion by NSP5, ORF3b, ORF9c, and Nucleocapsid (N) proteins are not well elucidated. Additionally, we also elaborated the perspectives of SARS-CoV-2 proteins.

Long-term voluntary wheel running effects on markers of long interspersed nuclear element-1 in skeletal muscle, liver, and brain tissue of female rats.

We sought to determine the effects of long-term voluntary wheel running on markers of long interspersed nuclear element-1 (L1) in skeletal muscle, liver, and the hippocampus of female rats. In addition, markers of the cGAS-STING DNA-sensing pathway that results in inflammation were interrogated. Female Lewis rats (n = 34) were separated into one of three groups including a 6-mo-old group to serve as a young comparator group (CTL, n = 10), a group that had access to a running wheel for voluntary wheel running (EX, n = 12), and an age-matched group that did not (SED, n = 12). Both SED and EX groups were carried out from 6 mo to 15 mo of age. There were no significant differences in L1 mRNA expression for any of the tissues between groups. Methylation of the L1 promoter in the soleus and hippocampus was significantly higher in SED and EX than in CTL group (P < 0.05). ORF1p expression was higher in older SED and EX rats than in CTL rats for every tissue (P < 0.05). There were no differences between groups for L1 mRNA or cGAS-STING pathway markers. Our results suggest there is an increased ORF1 protein expression across tissues with aging that is not mitigated by voluntary wheel running. In addition, although previous data imply that L1 methylation changes may play a role in acute exercise for L1 RNA expression, this does not seem to occur during extended periods of voluntary wheel running.

Genomic Divergence Characterization and Quantitative Proteomics Exploration of Type 4 Porcine Astrovirus.

Porcine astrovirus (PAstV) has been identified as an important diarrheic pathogen with a broad global distribution. The PAstV is a potential pathogen to human beings and plays a role in public health. Until now, the divergence characteristics and pathogenesis of the PAstV are still not well known. In this study, the PAstV-4 strain PAstV/CH/2022/CM1 was isolated from the diarrheal feces of a piglet in Shanghai, which was identified to be a recombination of PAstV4/JPN (LC201612) and PAstV4/CHN (JX060808). A time tree based on the ORF2 protein of the astrovirus demonstrated that type 2–5 PAstV (PAstV-2 to 5) diverged from type 1 PAstV (PAstV-1) at a point from 1992 to 2000. To better understand the molecular basis of the virus, we sought to explore the host cell response to the PAstV/CH/2022/CM1 infection using proteomics. The results demonstrate that viral infection elicits global protein changes, and that the mitochondria seems to be a primary and an important target in viral infection. Importantly, there was crosstalk between autophagy and apoptosis, in which ATG7 might be the key mediator. In addition, the NOD-like receptor X1 (NLRX1) in the mitochondria was activated and participated in several important antiviral signaling pathways after the PAstV/CH/2022/CM1 infection, which was closely related to mitophagy. The NLRX1 may be a crucial protein for antagonizing a viral infection through autophagy, but this has yet to be validated. In conclusion, the data in this study provides more information for understanding the virus genomic characterization and the potential antiviral targets in a PAstV infection.

Nucleopore Traffic Is Hindered by SARS-CoV-2 ORF6 Protein to Efficiently Suppress IFN-β and IL-6 Secretion.

A weak production of INF-β along with an exacerbated release of pro-inflammatory cytokines have been reported during infection by the novel SARS-CoV-2 virus. SARS-CoV-2 encodes several proteins that are able to counteract the host immune system, which is believed to be one of the most important features contributing to the viral pathogenesis and development of a severe clinical outcomes. Previous reports demonstrated that the SARS-CoV-2 ORF6 protein strongly suppresses INF-β production by hindering the RIG-I, MDA-5, and MAVS signaling cascade. In the present study, we better characterized the mechanism by which the SARS-CoV-2 ORF6 counteracts IFN-β and interleukin-6 (IL-6), which plays a crucial role in the inflammation process associated with the viral infection. In the present study, we demonstrated that the SARS-CoV-2 ORF6 protein has evolved an alternative mechanism to guarantee host IFN-β and IL-6 suppression, in addition to the transcriptional control exerted on the genes. Indeed, a block in movement through the nucleopore of newly synthetized messenger RNA encoding the immune-modulatory cytokines IFN-β and IL-6 are reported here. The ORF6 accessory protein of SARS-CoV-2 displays a multifunctional activity and may represent one of the most important virulence factors. Where conventional antagonistic strategies of immune evasion—such as the suppression of specific transcription factors (e.g., IRF-3, STAT-1/2)—would not be sufficient, the SARS-CoV-2 ORF6 protein is the trump card for the virus, also blocking the movement of IFN-β and IL-6 mRNAs from nucleus to cytoplasm. Conversely, we showed that nuclear translocation of the NF-κB transcription factor is not affected by the ORF6 protein, although inhibition of its cytoplasmic activation occurred. Therefore, the ORF6 protein exerts a 360-degree inhibition of the antiviral response by blocking as many critical points as possible.

Whole-genome sequencing of SARS-CoV-2 reveals diverse mutations in circulating Alpha and Delta variants during the first, second, and third waves of COVID-19 in South Kivu, east of the Democratic Republic of the Congo

ObjectivesWe used whole-genome sequencing of SARS-CoV-2 to identify variants circulating in the Democratic Republic of the Congo and obtain molecular information useful for diagnosis, improving treatment, and general pandemic control strategies.MethodsA total of 74 SARS-CoV-2 isolates were sequenced using Oxford Nanopore platforms. Generated reads were processed to obtain consensus genome sequences. Sequences with more than 80% genome coverage were used for variant calling, phylogenetic analysis, and classification using Pangolin lineage annotation nomenclature.ResultsPhylogenetic analysis based on Pangolin classification clustered South Kivu sequences into seven lineages (A.23.1, B.1.1.6, B.1.214, B.1.617.2, B.1.351, C.16, and P.1). The Delta (B.1.617.2) variant was the most dominant and responsible for outbreaks during the third wave. Based on the Wuhan reference genome, 289 distinct mutations were detected, including 141 missenses, 123 synonymous, and 25 insertions/deletions when our isolates were mapped to the Wuhan reference strain. Most of these point mutations were located within the coding sequences of the SARS-CoV-2 genome that includes spike, ORF1ab, ORF3, and nucleocapsid protein genes. The most common mutation was D614G (1841A>G) observed in 61 sequences, followed by L4715L (14143 C>T) found in 60 sequences.ConclusionOur findings highlight multiple introductions of SARS-CoV-2 into South Kivu through different sources and subsequent circulation of variants in the province. These results emphasize the importance of timely monitoring of genetic variation and its effect on disease severity. This work set a foundation for the use of genomic surveillance as a tool for future global pandemic management and control.

Analysis of codon usage patterns in open reading frame 4 of hepatitis E viruses

BackgroundHepatitis E virus (HEV) is a member of the family Hepeviridae and causes acute HEV infections resulting in thousands of deaths worldwide. The zoonotic nature of HEV in addition to its tendency from human to human transmission has led scientists across the globe to work on its different aspects. HEV also accounts for about 30% mortality rates in case of pregnant women. The genome of HEV is organized into three open reading frames (ORFs): ORF1 ORF2 and ORF3. A reading frame encoded protein ORF4 has recently been discovered which is exclusive to GT 1 isolates of HEV. The ORF4 is suggested to play crucial role in pregnancy-associated pathology and enhanced replication. Though studies have documented the ORF4’s importance, the genetic features of ORF4 protein genes in terms of compositional patterns have not been elucidated. As codon usage performs critical role in establishment of the host–pathogen relationship, therefore, the present study reports the codon usage analysis (based on nucleotide sequences of HEV ORF4 available in the public database) in three hosts along with the factors influencing the codon usage patterns of the protein genes of ORF4 of HEV.ResultsThe nucleotide composition analysis indicated that ORF4 protein genes showed overrepresentation of C nucleotide and while A nucleotide was the least-represented, with random distribution of G and T(U) nucleotides. The relative synonymous codon usage (RSCU) analysis revealed biasness toward C/G-ended codons (over U/A) in all three natural HEV-hosts (human, rat and ferret). It was observed that all the ORF4 genes were richly endowed with GC content. Further, our results showed the occurrence of both coincidence and antagonistic codon usage patterns among HEV-hosts. The findings further emphasized that both mutational and selection forces influenced the codon usage patterns of ORF4 protein genes.ConclusionsTo the best of our knowledge, this is first bioinformatics study evaluating codon usage patterns in HEV ORF4 protein genes. The findings from this study are expected to increase our understanding toward significant factors involved in evolutionary changes of ORF4.

Phylogenetic Marker Selection and Protein Sequence Analysis of the ORF5 Gene Product of Grapevine Virus A.

Grapevine virus A (GVA), the type species of the Vitivirus genus, is one of the causal agents of the Kober stem grooving disease of the rugose wood complex and one of the most frequently detected viruses in grapevine. There is little information on GVA gene(s) marker useful for phylogenetic analysis. To this aim, a total of 403 leaf samples were collected from vineyards of East and West Azarbaijan provinces in the Northwestern provinces of Iran during 2014–2016 and tested by DAS-ELISA and RT-PCR using ORF5-specific primers. GVA was detected in 56 symptomatic samples, corresponding to 14% of infection, while it was not detected in asymptomatic samples. The ORF5 (p10) protein sequence of eight Iranian isolates was compared to other vitiviruses, showing that the most conserved region resides in the N-terminus, carrying an arginine-rich motif followed by a zinc-finger motif. Next, to define a robust phylogenetic marker representative of the whole genome sequence suitable for phylogenetic and evolutionary studies, phylogenetic trees based on the full genome sequences of all the available GVA isolates and on individual genomic regions were constructed and compared. ORF1, which encodes the RNA-dependent RNA polymerase, was found to be the best phylogenetic marker for GVA classification and evolution studies. These results can be used for further research on phylogenetic analyses, evolution history, epidemiology, and etiology of rugose wood complex, and to identify control measures against GVA and other vitiviruses.

The use of ORF2 and ORF3 proteins for creating an enzyme-linked immunosorbent assay for viral hepatitis E

Due to the lack of national analogies a prototype of enzyme-linked immunosorbent assay (ELISA) test system was developed for the qualitative determination of antibodies class G to hepatitis E virus (HEV) based on the using of antigenic polypeptides ORF2 and ORF3 of the 3rd genotype (FSBSI «I.I. Mechnikov Research Institute of Vaccines and Serums», Russia). The results of experimental ELISA conducted in parallel with the reference test system «DS-ELISA-ANTI-HEV-G» (NPO Diagnostic Systems, Russia) are presented. The working concentrations of ORF2 and ORF3 antigens were 2.5 µg/mL and 0.25 µg/mL, respectively. They were tested with 200 donors and patients’ serum samples. The article reflects the determination of sensitivity, specificity and reproducibility results. The developed test system demonstrated high efficiency in the detection of specific anti-HEV-IgG.

Mutational cascade of SARS-CoV-2 leading to evolution and emergence of omicron variant

BackgroundEmergence of new variant of SARS-CoV-2, namely omicron, has posed a global concern because of its high rate of transmissibility and mutations in its genome. Researchers worldwide are trying to understand the evolution and emergence of such variants to understand the mutational cascade events. MethodsWe have considered all omicron genomes (n = 302 genomes) available till 2nd December 2021 in the public repository of GISAID along with representatives of variants of concern (VOC), i.e., alpha, beta, gamma, delta, and omicron; variant of interest (VOI) mu and lambda; and variant under monitoring (VUM). Whole genome-based phylogeny and mutational analysis were performed to understand the evolution of SARS CoV-2 leading to emergence of omicron variant. ResultsWhole genome-based phylogeny depicted two phylogroups (PG-I and PG-II) forming variant specific clades except for gamma and VUM GH. Mutational analysis detected 18,261 mutations in the omicron variant, majority of which were non-synonymous mutations in spike (A67, T547K, D614G, H655Y, N679K, P681H, D796Y, N856K, Q954H), followed by RNA dependent RNA polymerase (rdrp) (A1892T, I189V, P314L, K38R, T492I, V57V), ORF6 (M19M) and nucleocapsid protein (RG203KR). ConclusionDelta and omicron have evolutionary diverged into distinct phylogroups and do not share a common ancestry. While, omicron shares common ancestry with VOI lambda and its evolution is mainly derived by the non-synonymous mutations.

Locus specific reduction of L1 expression in the cortices of individuals with amyotrophic lateral sclerosis

The activation and dysregulation of retrotransposons has been identified in the CNS of individuals with the fatal neurodegenerative disorder Amyotrophic lateral sclerosis (ALS). This includes elements from multiple different families and subfamilies of retrotransposons, however there is limited knowledge of the specific loci from which this expression occurs in ALS. The long interspersed element-1 (L1) is the only autonomous retrotransposon in the human genome and members of this family of elements maintain the ability to mobilise. Despite L1s contributing to 17% of the human genome only 80–100 L1s encode the required proteins for mobilisation and are retrotransposition competent. Identifying the specific loci from which L1 expression occurs will inform on the potential functional consequences of their expression, such as the potential for somatic retrotransposition or DNA damage caused by the endonuclease activity of the ORF2 protein of the L1. Here we characterised L1 loci expression using the L1EM tool (https://github.com/FenyoLab/L1EM) in RNA sequencing data from 518 samples across four tissues (motor cortex, frontal cortex, cerebellum and cervical spinal cord) in the Target ALS cohort obtained from the New York Genome Center. There was a significant reduction in total intact L1 expression (those that encode functional proteins) in two brain regions of individuals with ALS compared to controls and clustering of the ALS brain regions occurred based on their intact L1 expression profile. Although overall the levels of L1 expression were reduced in ALS/ALS with other neurological disorder (ND) there were individuals in which L1s were expressed at much higher levels than the rest of the ALS/ALSND cohort. Expressed L1 loci were more frequently located in introns compared to those not expressed and the level of L1 expression positively correlated with the expression of the gene in which it was located. Significant differences were observed in the expression profiles of L1s in ALS and specific features of these elements, such as location in the genome and whether or not they are intact, were significantly associated with those that were expressed in the cohort.

Characterization of the mitogenome of Gongronella sp. w5 reveals substantial variation in Mucoromycota.

Gongronella is a genus of fungi in Mucorales (Mucoromycota). Some of its members have important biotechnological applications, but until now, not a single mitogenome has been characterized in Gongronella. Here, we present the complete mitogenome assembly of Gongronella sp. w5, a soil isolate known to interact with plants and several fungi. Its 36,593-bp circular mitogenome encodes the large and small subunit rRNAs, 14 standard mitochondrial proteins, 24 tRNAs, three free-standing ORF proteins, and the RNA subunit of RNase P (rnpB). These genes arrange in an order novel to known fungal mitogenomes. Three group I introns are present in the cob, cox1, and nad5 genes, respectively, and they are probably acquired by horizontal gene transfer. Phylogenetic analysis based on mitochondrion-encoded proteins supports the grouping of Gongronella sp. w5 with Absidia glauca, forming the Cunninghamellaceae clade within Mucoromycota. Gongronella and most other Mucoromycota species are predicted to use the standard genetic code in mitochondrial translation, rather than code 4 assigned by GenBank. A comparison among seven publicly available mitogenomes in Mucoromycota reveals the presence of the same 14 typical protein-coding genes plus rnpB, yet substantial variation in mitogenome size, intron number, gene order, and orientation. In this comparison, the uniqueness of Gongronella is evident from similarly large differences to its closest phylogenetic neighbor, A. glauca. This study promotes our understanding of fungal evolution in Mucoromycota. KEY POINTS: • This study reports the first mitogenome in Gongronella, which presents a novel gene order. • Different Mucoromycota mitogenomes show substantial variation of gene organizations. • Most Mucoromycota species use the standard genetic code to translate mitochondrial genes.

Decoupling SARS-CoV-2 ORF6 localization and interferon antagonism.

Like many pathogenic viruses, SARS-CoV-2 must overcome interferon (IFN)-mediated host defenses for infection establishment. To achieve this, SARS-CoV-2 deploys overlapping mechanisms to antagonize IFN production and signaling. The strongest IFN antagonist is the accessory protein ORF6, which localizes to multiple membranous compartments, including the nuclear envelope, where it directly binds nuclear pore component Nup98-Rae1 to inhibit nuclear translocation of activated STAT1 and IRF3 transcription factors. However, this direct cause-and-effect relationship between ORF6 localization and IFN antagonism has yet to be explored experimentally. Here, we use extensive mutagenesis studies to define the structural determinants required for steady-state localization and demonstrate that mis-localized ORF6 variants still potently inhibit nuclear trafficking and IFN signaling. Additionally, expression of a peptide that mimics the ORF6-Nup98 interaction domain robustly blocked nuclear trafficking. Furthermore, pharmacologic and mutational approaches combined to suggest that ORF6 is likely a peripheral membrane protein, as opposed to being a transmembrane protein as previously speculated. Thus, ORF6 localization and IFN antagonism are independent activities, which raises the possibility that ORF6 may have additional functions within membrane networks to enhance virus replication. This article has an associated First Person interview with the first author of the paper.

Intrinsically disordered regions in the rodent hepevirus proteome.

Hepatitis E virus (HEV) is the causative agent of Hepatitis E infections across the world. Intrinsically disordered protein regions (IDPRs) or intrinsically disordered proteins (IDPs) are regions or proteins that are characterized by lack of definite structure. These IDPRs or IDPs play significant roles in a wide range of biological processes, such as cell cycle regulation, control of signaling pathways, etc. IDPR/IDP in proteins is associated with the virus's pathogenicity and infectivity. The prevalence of IDPR/IDP in rat HEV proteome remains undetermined. Hence, we examined the unstructured/disordered regions of the open reading frame (ORF) encoded proteins of rat HEV by analyzing the prevalence of intrinsic disorder. The intrinsic disorder propensity analysis showed that the different ORF proteins consisted of varying fraction of intrinsic disorder. The protein ORF3 was identified with maximum propensity for intrinsic disorder while the ORF6 protein had the least fraction of intrinsic disorder. The analysis revealed ORF6 as a structured protein (ORDP); ORF1 and ORF4 as moderately disordered proteins (IDPRs); and ORF3 and ORF5 as highly disordered proteins (IDPs). The protein ORF2 was found to be moderately as well as highly disordered using different predictors, thus, was categorized into both IDPR and IDP. Such disordered regions have important roles in pathogenesis and replication of viruses.

Processing and Subcellular Localization of the Hepatitis E Virus Replicase: Identification of Candidate Viral Factories.

Hepatitis E virus (HEV) is the major cause of acute hepatitis worldwide. HEV is a positive-sense RNA virus expressing three open reading frames (ORFs). ORF1 encodes the ORF1 non–structural polyprotein, the viral replicase which transcribes the full-length genome and a subgenomic RNA that encodes the structural ORF2 and ORF3 proteins. The present study is focused on the replication step with the aim to determine whether the ORF1 polyprotein is processed during the HEV lifecycle and to identify where the replication takes place inside the host cell. As no commercial antibody recognizes ORF1 in HEV-replicating cells, we aimed at inserting epitope tags within the ORF1 protein without impacting the virus replication efficacy. Two insertion sites located in the hypervariable region were thus selected to tolerate the V5 epitope while preserving HEV replication efficacy. Once integrated into the infectious full-length Kernow C-1 p6 strain, the V5 epitopes did neither impact the replication of genomic nor the production of subgenomic RNA. Also, the V5-tagged viral particles remained as infectious as the wildtype particles to Huh-7.5 cells. Next, the expression pattern of the V5-tagged ORF1 was compared in heterologous expression and replicative HEV systems. A high molecular weight protein (180 kDa) that was expressed in all three systems and that likely corresponds to the unprocessed form of ORF1 was detected up to 25 days after electroporation in the p6 cell culture system. Additionally, less abundant products of lower molecular weights were detected in both in cytoplasmic and nuclear compartments. Concurrently, the V5-tagged ORF1 was localized by confocal microscopy inside the cell nucleus but also as compact perinuclear substructures in which ORF2 and ORF3 proteins were detected. Importantly, using in situ hybridization (RNAScope ®), positive and negative-strand HEV RNAs were localized in the perinuclear substructures of HEV-producing cells. Finally, by simultaneous detection of HEV genomic RNAs and viral proteins in these substructures, we identified candidate HEV factories.

The dark proteome of rodent hepatitis E virus: Analysis of intrinsically disordered regions

Hepatitis E virus (HEV) is the causative agent of Hepatitis E infections across the world. Intrinsically disordered protein regions (IDPRs) or Intrinsically Disordered Protein (IDPs) are regions or proteins that are characterized by a lack of definite structure. These regions or proteins play significant roles in a wide range of biological processes, such as cell cycle regulation, control of signaling pathways, etc. IDPRs or IDPs in proteins are associated with the virus’s pathogenicity and infectivity. The occurrence of intrinsic disorder in the proteome of rat HEV remains to be elucidated, which prompted us to explore its dark proteome. In this study, the unstructured/disordered regions of ORF proteins of rat HEV have been examined. We have analyzed the prevalence of intrinsic disorder by using a set of computational predictors. The intrinsic disorder propensity analysis showed that the ORF proteins consisted of a varying fraction of intrinsic disorder. The ORF3 protein was identified with a maximum propensity for intrinsic disorder while the protein ORF6 showed the least propensity for the intrinsic disorder. Further, the analysis revealed ORF6 as highly structured protein (ORDP); ORF1 and ORF4 as moderately disordered proteins (IDPRs); and ORF3 and ORF5 as highly disordered proteins, categorizing them as ordered protein (ORDP), a protein having Intrinsically Disordered Region (IDPR) and Intrinsically Disordered Proteins (IDP) respectively. Such disordered regions may play several important roles in the pathogenesis and replication of viruses. Collectively, this comprehensive study data from our investigation suggested ORF protein’s role in the regulation and pathogenesis of rat herpesvirus.

Optimizing environmental safety and cell-killing potential of oncolytic Newcastle Disease virus with modifications of the V, F and HN genes.

Newcastle Disease Virus (NDV) is an avian RNA virus, which was shown to be effective and safe for use in oncolytic viral therapy for several tumour malignancies. The presence of a multi basic cleavage site (MBCS) in the fusion protein improved its oncolytic efficacy in vitro and in vivo. However, NDV with a MBCS can be virulent in poultry. We aimed to develop an NDV with a MBCS but with reduced virulence for poultry while remaining effective in killing human tumour cells. To this end, the open reading frame of the V protein, an avian specific type I interferon antagonist, was disrupted by introducing multiple mutations. NDV with a mutated V gene was attenuated in avian cells and chicken and duck eggs. Although this virus still killed tumour cells, the efficacy was reduced compared to the virulent NDV. Introduction of various mutations in the fusion (F) and hemagglutinin-neuraminidase (HN) genes slightly improved this efficacy. Taken together, these data demonstrated that NDV with a MBCS but with abrogation of the V protein ORF and mutations in the F and HN genes can be safe for evaluation in oncolytic viral therapy.

Development of Recombinant Infectious Hepatitis E Virus Harboring the nanoKAZ Gene and Its Application in Drug Screening.

Hepatitis E virus (HEV) is a quasi-enveloped virus with a single-stranded positive-sense RNA genome belonging to the family Hepeviridae. Studies of the molecular aspects of HEV and drug screening have benefited from the discovery of bioluminescent reporter genes. However, the stability of large foreign genes is difficult to maintain after insertion into the viral genome. Currently, ribavirin is used to treat HEV-infected patients who require antiviral therapy. This has several major drawbacks. Thus, the development of novel anti-HEV drugs is of great importance. We developed a system consisting of recombinant infectious HEV harboring a small luciferase gene (nanoKAZ) in the hypervariable region (HVR) of the open reading frame 1 (ORF1) (HEV-nanoKAZ). It replicated efficiently in cultured cells, was genetically stable, and had morphological characteristics similar to those of the parental virus. Both membrane-associated (eHEV-nanoKAZ) and membrane-unassociated (neHEV-nanoKAZ) particles were infectious. HEV particles circulating in the bloodstream and attaching to hepatocytes in HEV-infected patients are membrane-associated; thus, eHEV-nanoKAZ was applied in drug screening. The eHEV-nanoKAZ system covers at least the inhibitor of HEV entry and inhibitor of HEV RNA replication. Four drugs with anti-HEV activity were identified. Their effectiveness in cultured cells was confirmed in naive and HEV-producing PLC/PRF/5 cells. Two hit drugs (azithromycin and ritonavir) strongly inhibited HEV production in culture supernatants, as well as intracellular expression of ORF2 protein, and may therefore be candidate novel anti-HEV drugs. The HEV-nanoKAZ system was developed and applied in drug screening and is expected to be useful for investigating the HEV life cycle. IMPORTANCE Bioluminescent reporter viruses are essential tools in molecular virological research. They have been widely used to investigate viral life cycles and in the development of antiviral drugs. For drug screening, the use of a bioluminescent reporter virus helps shorten the time required to perform the assay. A system, consisting of recombinant infectious HEV harboring the nanoKAZ gene in the HVR of ORF1 (HEV-nanoKAZ), was developed in this study and was successfully applied to drug screening in which four hit drugs with anti-HEV activity were identified. The results of this study provide evidence supporting the use of this system in more variable HEV studies. In addition, both forms of viral particles (eHEV-nanoKAZ and neHEV-nanoKAZ) are infectious, which will enable their application in HEV studies requiring both forms of viral particles, such as in the investigation of unknown HEV receptors and the elucidation of host factors important for HEV entry.

Sequence to structural analysis of ORF5 protein in Norway rat Hepatitis E Virus.

Hepatitis E virus (HEV) is a major causative agent of acute hepatitis in developing countries. The Norway rat HEV genome consists of six open reading frames (ORFs), i.e., ORF1, ORF2, ORF3, ORF4, ORF5 and ORF6. The additional reading frame encoded protein ORF5 is attributed to life cycle of rat HEV. The ORFF5 protein’s function remains undetermined. Therefore, it is of interest to analyze the ORF5 protein for its physiochemical properties, primary structure, secondary structure, tertiary structure and functional characteristics using bioinformatics tools. Analysis of the ORF5 protein revealed it as highly unstable, hydrophilic with basic pI. The ORF5 protein consisted mostly of Arg, Pro, Ser, Leu and Gly. The 3D structural homology model of the ORF5 protein generated showed mixed α/β structural fold with predominance of coils. Structural analysis revealed the presence of clefts, pores and a tunnel. This data will help in the sequence, structure and functional annotation of ORF5.

Host E3 ligase HUWE1 attenuates the proapoptotic activity of the MERS-CoV accessory protein ORF3 by promoting its ubiquitin-dependent degradation

With the outbreak of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), coronaviruses have begun to attract great attention across the world. Of the known human coronaviruses, however, Middle East respiratory syndrome coronavirus (MERS-CoV) is the most lethal. Coronavirus proteins can be divided into three groups: nonstructural proteins, structural proteins, and accessory proteins. While the number of each of these proteins varies greatly among different coronaviruses, accessory proteins are most closely related to the pathogenicity of the virus. We found for the first time that the ORF3 accessory protein of MERS-CoV, which closely resembles the ORF3a proteins of severe acute respiratory syndrome coronavirus and SARS-CoV-2, has the ability to induce apoptosis in cells in a dose-dependent manner. Through bioinformatics analysis and validation, we revealed that ORF3 is an unstable protein and has a shorter half-life in cells compared to that of severe acute respiratory syndrome coronavirus and SARS-CoV-2 ORF3a proteins. After screening, we identified a host E3 ligase, HUWE1, that specifically induces MERS-CoV ORF3 protein ubiquitination and degradation through the ubiquitin–proteasome system. This results in the diminished ability of ORF3 to induce apoptosis, which might partially explain the lower spread of MERS-CoV compared to other coronaviruses. In summary, this study reveals a pathological function of MERS-CoV ORF3 protein and identifies a potential host antiviral protein, HUWE1, with an ability to antagonize MERS-CoV pathogenesis by inducing ORF3 degradation, thus enriching our knowledge of the pathogenesis of MERS-CoV and suggesting new targets and strategies for clinical development of drugs for MERS-CoV treatment.