ICTV Virus Taxonomy Profile: Hantaviridae 2024.

Hantaviridae is a family for negative-sense RNA viruses with genomes of about 10.5-14.6 kb. These viruses are maintained in and/or transmitted by fish, reptiles, and mammals. Several orthohantaviruses can infect humans, causing mild, severe, and sometimes-fatal diseases. Hantavirids produce enveloped virions containing three single-stranded RNA segments with open reading frames that encode a nucleoprotein (N), a glycoprotein precursor (GPC), and a large (L) protein containing an RNA-directed RNA polymerase (RdRP) domain. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Hantaviridae, which is available at ictv.global/report/hantaviridae.

Complete genome analysis of a novel narnavirus in sweet viburnum (Viburnum odoratissimum).

Trees and shrubs provide important ecological services. However, few studies have surveyed the virome in trees and shrubs. In this study, we discovered a new positive-sense RNA virus originating from Viburnum odoratissimum, which we named "Vo narna-like virus". The complete genome of Vo narna-like virus is 3,451 nt in length with an open reading frame (ORF) encoding the RNA-dependent RNA polymerase (RdRP) protein. Phylogenetic analysis placed this virus within the betanarnavirus clade, sharing 53.63% amino acid sequence identity with its closest relative, Qingdao RNA virus 2. The complete sequence of the virus was confirmed by rapid amplification of cDNA ends (RACE) and Sanger sequencing. Small interfering RNA (siRNA) analysis indicated that this virus interacts with the RNA interference (RNAi) pathway of V. odoratissimum. This is the first report of a narnavirus in V. odoratissimum.

Evolution of a Human-Specific De Novo Open Reading Frame and Its Linked Transcriptional Silencer.

In the human genome, two short open reading frames (ORFs) separated by a transcriptional silencer and a small intervening sequence stem from the gene SMIM45. The two ORFs show different translational characteristics, and they also show divergent patterns of evolutionary development. The studies presented here describe the evolution of the components of SMIM45. One ORF consists of an ultra-conserved 68 amino acid (aa) sequence, whose origins can be traced beyond the evolutionary age of divergence of the elephant shark, ~462 MYA. The silencer also has ancient origins, but it has a complex and divergent pattern of evolutionary formation, as it overlaps both at the 68 aa ORF and the intervening sequence. The other ORF consists of 107 aa. It develops during primate evolution but is found to originate de novo from an ancestral non-coding genomic region with root origins within the Afrothere clade of placental mammals, whose evolutionary age of divergence is ~99 MYA. The formation of the complete 107 aa ORF during primate evolution is outlined, whereby sequence development is found to occur through biased mutations, with disruptive random mutations that also occur but lead to a dead-end. The 107 aa ORF is of particular significance, as there is evidence to suggest it is a protein that may function in human brain development. Its evolutionary formation presents a view of a human-specific ORF and its linked silencer that were predetermined in non-primate ancestral species. The genomic position of the silencer offers interesting possibilities for the regulation of transcription of the 107 aa ORF. A hypothesis is presented with respect to possible spatiotemporal expression of the 107 aa ORF in embryonic tissues.

ICTV Virus Taxonomy Profile: Fusariviridae 2024.

Fusariviridae is a family of mono-segmented, positive-sense RNA viruses with genome sizes of 5.9-10.7 kb. Most genomic RNAs are bicistronic, but exceptions have up to four predicted ORFs. In bicistronic genomes, the 5'-proximal ORF codes for a single protein with both RNA-directed RNA polymerase (RdRP) and RNA helicase (Hel) domains; little is known about the protein encoded by the second ORF. Fusarivirids do not appear to form virions. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Fusariviridae, which is available at ictv.global/report/fusariviridae.

sOCP: a framework predicting smORF coding potential based on TIS and in-frame features and effectively applied in the human genome.

Small open reading frames (smORFs) have been acknowledged to play various roles on essential biological pathways and affect human beings from diabetes to tumorigenesis. Predicting smORFs in silico is quite a prerequisite for processing the omics data. Here, we proposed the smORF-coding-potential-predicting framework, sOCP, which provides functions to construct a model for predicting novel smORFs in some species. The sOCP model constructed in human was based on in-frame features and the nucleotide bias around the start codon, and the small feature subset was proved to be competent enough and avoid overfitting problems for complicated models. It showed more advanced prediction metrics than previous methods and could correlate closely with experimental evidence in a heterogeneous dataset. The model was applied to Rattus norvegicus and exhibited satisfactory performance. We then scanned smORFs with ATG and non-ATG start codons from the human genome and generated a database containing about a million novel smORFs with coding potential. Around 72 000 smORFs are located on the lncRNA regions of the genome. The smORF-encoded peptides may be involved in biological pathways rare for canonical proteins, including glucocorticoid catabolic process and the prokaryotic defense system. Our work provides a model and database for human smORF investigation and a convenient tool for further smORF prediction in other species.

Proteins à la carte: riboproteogenomic exploration of bacterial N-terminal proteoform expression.

In recent years, it has become evident that the true complexity of bacterial proteomes remains underestimated. Gene annotation tools are known to propagate biases and overlook certain classes of truly expressed proteins, particularly proteoforms-protein isoforms arising from a single gene. Recent (re-)annotation efforts heavily rely on ribosome profiling by providing a direct readout of translation to fully describe bacterial proteomes. In this study, we employ a robust riboproteogenomic pipeline to conduct a systematic census of expressed N-terminal proteoform pairs, representing two isoforms encoded by a single gene raised by annotated and alternative translation initiation, in Salmonella. Intriguingly, conditional-dependent changes in relative utilization of annotated and alternative translation initiation sites (TIS) were observed in several cases. This suggests that TIS selection is subject to regulatory control, adding yet another layer of complexity to our understanding of bacterial proteomes. IMPORTANCE: With the emerging theme of genes within genes comprising the existence of alternative open reading frames (ORFs) generated by translation initiation at in-frame start codons, mechanisms that control the relative utilization of annotated and alternative TIS need to be unraveled and our molecular understanding of resulting proteoforms broadened. Utilizing complementary ribosome profiling strategies to map ORF boundaries, we uncovered dual-encoding ORFs generated by in-frame TIS usage in Salmonella. Besides demonstrating that alternative TIS usage may generate proteoforms with different characteristics, such as differential localization and specialized function, quantitative aspects of conditional retapamulin-assisted ribosome profiling (Ribo-RET) translation initiation maps offer unprecedented insights into the relative utilization of annotated and alternative TIS, enabling the exploration of gene regulatory mechanisms that control TIS usage and, consequently, the translation of N-terminal proteoform pairs.

A novel sORF gene mutant strain of Yersinia pestis vaccine EV76 offers enhanced safety and improved protection against plague.

We recently identified two virulence-associated small open reading frames (sORF) of Yersinia pestis, named yp1 and yp2, and null mutants of each individual genes were highly attenuated in virulence. Plague vaccine strain EV76 is known for strong reactogenicity, making it not suitable for use in humans. To improve the immune safety of EV76, three mutant strains of EV76, Δyp1, Δyp2, and Δyp1&yp2 were constructed and their virulence attenuation, immunogenicity, and protective efficacy in mice were evaluated. All mutant strains were attenuated by the subcutaneous (s.c.) route and exhibited more rapid clearance in tissues than the parental strain EV76. Under iron overload conditions, only the mice infected with EV76Δyp1 survived, accompanied by less draining lymph nodes damage than those infected by EV76. Analysis of cytokines secreted by splenocytes of immunized mice found that EV76Δyp2 induced higher secretion of multiple cytokines including TNF-α, IL-2, and IL-12p70 than EV76. On day 42, EV76Δyp2 or EV76Δyp1&yp2 immunized mice exhibited similar protective efficacy as EV76 when exposed to Y. pestis 201, both via s.c. or intranasal (i.n.) routes of administration. Moreover, when exposed to 200-400 LD50 Y. pestis strain 201Δcaf1 (non-encapsulated Y. pestis), EV76Δyp2 or EV76Δyp1&yp2 are able to afford about 50% protection to i.n. challenges, significantly better than the protection afforded by EV76. On 120 day, mice immunized with EV76Δyp2 or EV76Δyp1&yp2 cleared the i.n. challenge of Y. pestis 201-lux as quickly as those immunized with EV76, demonstrating 90-100% protection. Our results demonstrated that deletion of the yp2 gene is an effective strategy to attenuate virulence of Y. pestis EV76 while improving immunogenicity. Furthermore, EV76Δyp2 is a promising candidate for conferring protection against the pneumonic and bubonic forms of plague.

PRPF19 mRNA Encodes a Small Open Reading Frame That Is Important for Viability of Human Cells.

High-throughput ribosome profiling demonstrates the translation of thousands of small open reading frames located in the 5' untranslated regions of messenger RNAs (upstream ORFs). Upstream ORF can both perform a regulatory function by influencing the translation of the downstream main ORF and encode a small functional protein or microprotein. In this work, we showed that the 5' untranslated region of the PRPF19 mRNA encodes an upstream ORF that is translated in human cells. Inactivation of this upstream ORF reduces the viability of human cells.

Complete genome sequence of a novel dsRNA virus from the phytopathogenic fungus Fusarium oxysporum.

Fusarium oxysporum is a widespread plant pathogen that causes fusarium wilt and fusarium root rot in many economically significant crops. Here, a novel dsRNA virus tentatively named "Fusarium oxysporum virus 1" (FoV1) was identified in F. oxysporum strain 3S-18. The genome of FoV1 is 2,944 nucleotides (nt) in length and contains two non-overlapping open reading frames (ORF1 and 2). The larger of these, ORF2, encodes an RNA-dependent RNA polymerase (RdRp) of 590 amino acids with a molecular mass of 67.52 kDa. ORF1 encodes a putative nucleocapsid protein consisting of 134 amino acids with a molecular mass of 34.25 kDa. The RdRp domain of FoV1 shares 60.00% to 84.24% sequence identity with non-segmented dsRNA viruses. Phylogenetic analysis further suggested that FoV1 is a new member of the proposed genus "Unirnavirus" accommodating unclassified monopartite dsRNA viruses.

Genome characterization of a novel narnavirus infecting the plant-pathogenic fungus Ustilaginoidea virens.

Mycoviruses are viruses that infect fungi and oomycetes. They are widespread in all major groups of plant-pathogenic fungi and oomycetes. To date, only the full genome of dsRNA mycoviruses and the contigs of positive-sense single-stranded RNA (+ssRNA) mycoviruses have been reported in Ustilaginoidea virens, which is the notorious causal agent of rice false smut (RFS). Here, we report the molecular characterization of a novel +ssRNA mycovirus, Ustilaginoidea virens narnavirus 4 (UvNV4), isolated from U. virens strain Uv418. UvNV4 has a genome of 3,131 nucleotides (nt) and possesses an open reading frame (ORF) predicted to encode an RNA-dependent RNA polymerase (RdRp) of 1,017 amino acids (aa) sequence with a molecular mass of 116.6 kDa. BLASTp analysis revealed that the RdRp showed 50.34% aa sequence identity to that of the previously described Zhangzhou Narna tick virus 1. Phylogenetic analysis indicated that UvNV4 is closely related to members of the family Narnaviridae. Taken together, these results clearly demonstrate that UvNV4 is a novel +ssRNA virus infecting U. virens.

Complete genome sequence of a novel alternavirus isolated from the phytopathogenic fungus Colletotrichum fioriniae.

A novel double-strand RNA (dsRNA) mycovirus, named "Colletotrichum fioriniae alternavirus1" (CfAV1), was isolated from the strain CX7 of Colletotrichum fioriniae, the causal agent of walnut anthracnose. The complete genome of CfAV1 is composed of three dsRNA segments: dsRNA1 (3528 bp), dsRNA2 (2485 bp), and dsRNA3 (2481 bp). The RNA-dependent RNA polymerase (RdRp) is encoded by dsRNA1, while both dsRNA2 and dsRNA3 encode hypothetical proteins. Based on multiple sequence alignments and phylogenetic analysis, CfAV1 is identified as a new member of the family Alternaviridae. This is the first report of an alternavirus that infects the phytopathogenic fungus C. fioriniae.

Complete genome characterization of cacao leafroll virus, a newly described cacao-infecting polerovirus.

The complete genome sequence of cacao leafroll virus (CaLRV; family Solemoviridae, genus Polerovirus) was determined by high-throughput sequencing of total RNA isolated from symptomatic cacao Theobroma cacao L. plants (n = 4). The CaLRV genome sequences ranged from 5,976 to 5,997 nucleotides (nt) in length and contained seven open reading frames (ORFs). Nucleotide and amino acid (aa) sequence comparisons showed that, among selected well-characterized poleroviruses, the CaLRV genome shared the highest nt sequence identity of 62% with that of potato leafroll virus (PLRV, NC_076505). A comparison of the predicted aa sequence of the CaLRV coat protein indicated that cotton leafroll dwarf virus (CLRDV, NC_014545) and melon aphid-borne yellows virus (MABYV, NC_010809) were the closest relatives, sharing 57% aa sequence identity. Bayesian phylogenetic analysis based on complete genome sequences showed that CaLRV grouped with well-characterized poleroviruses that cause diseases of cereal and vegetable crops. During the course of publishing this work, the nearly complete genome sequence of a member of the same polerovirus species, referred to as "cacao polerovirus" (OR605721), with which CaLRV shares 99% nt sequence identity, was reported.

Molecular and biological characterization of a novel partitivirus from Talaromyces pinophilus.

Talaromyces spp. have a worldwide distribution, are ecologically diverse and have been isolated from numerous different substrates. Talaromyces spp. are considered biotechnologically important due to their ability to produce a range of enzymes and pigments. Talaromyces pinophilus, belonging to genus Talaromyces and family Trichocomaceae, is known for producing several important bioactive metabolites. Here we report the isolation and characterisation of a partitivirus from T. pinophilus which we have nominated Talaromyces pinophilus partitivirus-1 (TpPV-1). TpPV-1 possesses a genome consisting of three double stranded (ds) RNA segments i.e., dsRNAs1-3, 1824 bp, 1638 bp and 1451 bp respectively, which are encapsidated in icosahedral particles 35 nm in diameter. Both dsRNA1 and dsRNA2 contain a single open reading frame (ORF) encoding respectively a 572 amino acid (aa) protein of 65 kDa and a 504 aa protein of 50 kDa. The third segment (dsRNA3) is potentially a satellite RNA. Phylogenetic analysis revealed that the TpPV-1 belongs to the family Partitiviridae in the proposed genus Zetapartitivirus. TpPV-1 infection decreases the mycelial growth rate of the host fungus and alters pigmentation as indicated by time course experiments performed on a range of different solid media comparing virus-infected and virus-free isogenic lines. This is the first report of mycovirus infection in T. pinophilus and may provide insights into understanding the effect of the mycovirus on the production of enzymes and pigments by the host fungus.

Setup and Applications of Modular Protein Expression Toolboxes (MoPET) for Mammalian Systems.

The design and generation of an optimal protein expression construct is the first and essential step in the characterization of any protein of interest. However, the exchange and modification of the coding and/or noncoding elements to analyze their effect on protein function or generating the optimal result can be a tedious and time-consuming process using standard molecular biology cloning methods. To streamline the process to generate defined expression constructs or libraries of otherwise difficult to express proteins, the Modular Protein Expression Toolbox (MoPET) has been developed (Weber E, PloS One 12(5):e0176314, 2017). The system applies Golden Gate cloning as an assembly method and follows the standardized modular cloning (MoClo) principle (Weber E, PloS One 6(2):e16765, 2011). This cloning platform allows highly efficient DNA assembly of pre-defined, standardized functional DNA modules effecting protein expression with a focus on minimizing the cloning burden in coding regions. The original MoPET system consists of 53 defined DNA modules divided into eight functional main classes and can be flexibly expanded dependent on the need of the experimenter and expression host. However, already with a limited set of only 53 modules, 792,000 different constructs can be rationally designed or used to generate combinatorial expression optimization libraries. We provide here a detailed protocol for the (1) design and generation of level 0 basic parts, (2) generation of defined expressions constructs, and (3) generation of combinatorial expression libraries.

ProTInSeq: transposon insertion tracking by ultra-deep DNA sequencing to identify translated large and small ORFs.

Identifying open reading frames (ORFs) being translated is not a trivial task. ProTInSeq is a technique designed to characterize proteomes by sequencing transposon insertions engineered to express a selection marker when they occur in-frame within a protein-coding gene. In the bacterium Mycoplasma pneumoniae, ProTInSeq identifies 83% of its annotated proteins, along with 5 proteins and 153 small ORF-encoded proteins (SEPs; ≤100 aa) that were not previously annotated. Moreover, ProTInSeq can be utilized for detecting translational noise, as well as for relative quantification and transmembrane topology estimation of fitness and non-essential proteins. By integrating various identification approaches, the number of initially annotated SEPs in this bacterium increases from 27 to 329, with a quarter of them predicted to possess antimicrobial potential. Herein, we describe a methodology complementary to Ribo-Seq and mass spectroscopy that can identify SEPs while providing other insights in a proteome with a flexible and cost-effective DNA ultra-deep sequencing approach.

A Precision Therapy Approach for Retinitis Pigmentosa 11 Using Splice-Switching Antisense Oligonucleotides to Restore the Open Reading Frame of PRPF31.

Retinitis pigmentosa 11 is an untreatable, dominantly inherited retinal disease caused by heterozygous mutations in pre-mRNA processing factor 31 PRPF31. The expression level of PRPF31 is linked to incomplete penetrance in affected families; mutation carriers with higher PRPF31 expression can remain asymptomatic. The current study explores an antisense oligonucleotide exon skipping strategy to treat RP11 caused by truncating mutations within PRPF31 exon 12 since it does not appear to encode any domains essential for PRPF31 protein function. Cells derived from a patient carrying a PRPF31 1205C>A nonsense mutation were investigated; PRPF31 transcripts encoded by the 1205C>A allele were undetectable due to nonsense-mediated mRNA decay, resulting in a 46% reduction in PRPF31 mRNA, relative to healthy donor cells. Antisense oligonucleotide-induced skipping of exon 12 rescued the open reading frame with consequent 1.7-fold PRPF31 mRNA upregulation in the RP11 patient fibroblasts. The level of PRPF31 upregulation met the predicted therapeutic threshold of expression inferred in a non-penetrant carrier family member harbouring the same mutation. This study demonstrated increased PRPF31 expression and retention of the nuclear translocation capability for the induced PRPF31 isoform. Future studies should evaluate the function of the induced PRPF31 protein on pre-mRNA splicing in retinal cells to validate the therapeutic approach for amenable RP11-causing mutations.

cirCodAn: A GHMM-based tool for accurate prediction of coding regions in circRNA.

Studies focusing on characterizing circRNAs with the potential to translate into peptides are quickly advancing. It is helping to elucidate the roles played by circRNAs in several biological processes, especially in the emergence and development of diseases. While various tools are accessible for predicting coding regions within linear sequences, none have demonstrated accurate open reading frame detection in circular sequences, such as circRNAs. Here, we present cirCodAn, a novel tool designed to predict coding regions in circRNAs. We evaluated the performance of cirCodAn using datasets of circRNAs with strong translation evidence and showed that cirCodAn outperformed the other tools available to perform a similar task. Our findings demonstrate the applicability of cirCodAn to identify coding regions in circRNAs, which reveals the potential of use of cirCodAn in future research focusing on elucidating the biological roles of circRNAs and their encoded proteins. cirCodAn is freely available at https://github.com/denilsonfbar/cirCodAn.

Widespread stable noncanonical peptides identified by integrated analyses of ribosome profiling and ORF features.

Studies have revealed dozens of functional peptides in putative 'noncoding' regions and raised the question of how many proteins are encoded by noncanonical open reading frames (ORFs). Here, we comprehensively annotate genome-wide translated ORFs across five eukaryotes (human, mouse, zebrafish, worm, and yeast) by analyzing ribosome profiling data. We develop a logistic regression model named PepScore based on ORF features (expected length, encoded domain, and conservation) to calculate the probability that the encoded peptide is stable in humans. Systematic ectopic expression validates PepScore and shows that stable complex-associating microproteins can be encoded in 5'/3' untranslated regions and overlapping coding regions of mRNAs besides annotated noncoding RNAs. Stable noncanonical proteins follow conventional rules and localize to different subcellular compartments. Inhibition of proteasomal/lysosomal degradation pathways can stabilize some peptides especially those with moderate PepScores, but cannot rescue the expression of short ones with low PepScores suggesting they are directly degraded by cellular proteases. The majority of human noncanonical peptides with high PepScores show longer lengths but low conservation across species/mammals, and hundreds contain trait-associated genetic variants. Our study presents a statistical framework to identify stable noncanonical peptides in the genome and provides a valuable resource for functional characterization of noncanonical translation during development and disease.

Single nucleotide polymorphisms (SNPs) in the open reading frame (ORF) of prion protein gene (PRNP) in Nigerian livestock species.

BACKGROUND: Prion diseases, also known as transmissible spongiform encephalopathies (TSEs) remain one of the deleterious disorders, which have affected several animal species. Polymorphism of the prion protein (PRNP) gene majorly determines the susceptibility of animals to TSEs. However, only limited studies have examined the variation in PRNP gene in different Nigerian livestock species. Thus, this study aimed to identify the polymorphism of PRNP gene in Nigerian livestock species (including camel, dog, horse, goat, and sheep). We sequenced the open reading frame (ORF) of 65 camels, 31 village dogs and 12 horses from Nigeria and compared with PRNP sequences of 886 individuals retrieved from public databases. RESULTS: All the 994 individuals were assigned into 162 haplotypes. The sheep had the highest number of haplotypes (n = 54), and the camel had the lowest (n = 7). Phylogenetic tree further confirmed clustering of Nigerian individuals into their various species. We detected five non-synonymous SNPs of PRNP comprising of G9A, G10A, C11G, G12C, and T669C shared by all Nigerian livestock species and were in Hardy-Weinberg Equilibrium (HWE). The amino acid changes in these five non-synonymous SNP were all "benign" via Polyphen-2 program. Three SNPs G34C, T699C, and C738G occurred only in Nigerian dogs while C16G, G502A, G503A, and C681A in Nigerian horse. In addition, C50T was detected only in goats and sheep. CONCLUSION: Our study serves as the first to simultaneously investigate the polymorphism of PRNP gene in Nigerian livestock species and provides relevant information that could be adopted in programs targeted at breeding for prion diseases resistance.

The complete genome sequences of a negative single-stranded RNA virus and a double-stranded RNA virus coinfecting the entomopathogenic fungus Beauveria bassiana Vuillemin.

Beauveria bassiana Vuillemin is an entomopathogenic fungus that has been developed as a biological insecticide. B. bassiana can be infected by single or multiple mycoviruses, most of which are double-stranded RNA (dsRNA) viruses, while infections with single-stranded RNA (ssRNA) viruses, especially negative single-stranded RNA (-ssRNA) viruses, have been observed less frequently. In the present study, we sequenced and analyzed the complete genomes of two new different mycoviruses coinfecting a single B. bassiana strain: a -ssRNA virus which we have named "Beauveria bassiana negative-strand RNA virus 1" (BbNSRV1), and a dsRNA virus, which we have named "Beauveria bassiana orthocurvulavirus 1" (BbOCuV1). The genome of BbNSRV1 consists of a single segment of negative-sense, single-stranded RNA with a length of 6169 nt, containing a single open reading frame (ORF) encoding a putative RNA-dependent RNA polymerase (RdRp) with 1949 aa (220.1 kDa). BLASTx analysis showed that the RdRp had the highest sequence similarity (59.79%) to that of Plasmopara viticola lesion associated mononegaambi virus 2, a member of the family Mymonaviridae. This is the first report of a -ssRNA mycovirus infecting B. bassiana. The genome of BbOCuV1 consists of two dsRNA segments, 2164 bp and 1765 bp in length, respectively, with dsRNA1 encoding a protein with conserved RdRp motifs and 70.75% sequence identity to the putative RdRp of the taxonomically unassigned mycovirus Fusarium graminearum virus 5 (FgV5), and the dsRNA2 encoding a putative coat protein with sequence identity 64.26% to the corresponding protein of the FgV5. Phylogenetic analysis indicated that BbOCuV1 belongs to a taxonomically unassigned group of dsRNA mycoviruses related to members of the families Curvulaviridae and Partitiviridae. Hence, it might be the member of a new family that remains to be named and formally recognized.