Host mRNA Research Articles

P921 FMT in UC is associated with a decrease in Bacteroides-2 enterotype and response with baseline RNA and microbiota signatures

Abstract Background The efficacy of faecal microbiota transplantation (FMT) in UC has been reported to be donor-, patient- and procedure-dependent (Rees et al., 2022). The RESTORE-UC trial [NCT03110289] aimed to improve the outcome of FMT in patients with active UC by donor preselection on microbiota level, a strict anaerobic preparation and repeated FMT administration. The trial was prematurely stopped for futility (Caenepeel et al., 2022). We investigated changes in resp. biopsy and faecal samples obtained host transcriptomics and microbiota profiles from baseline to primary endpoint (PE) at week 8 to understand reasons for the observed lack of efficacy. Methods Active UC patients (total Mayo score 4-10 with endoscopic sub-score ³2, n=72) were randomly allocated to receive 4 anaerobic-prepared superdonor (S) FMT or autologous (A) FMT. Primary endpoint was defined as steroid-free clinical remission (Total Mayo ≤ 2, with no sub-score >1). Host RNA extractions were performed from mucosal biopsies collected at week 0 (n=63) and 8 (n=54) using the Qiagen AllPrep DNA/RNA Mini kit, and sequenced using Illumina HiSeq4000. Sequencing data was further processed (read-count filtering, normalization) and further analyzed using DESeq2 package. Corresponding faecal samples (resp. n=62 and n=50) were submitted to DNA extractions using MagAttract PowerMicrobiome DNA/RNA kit on an automated extraction platform, followed by library prep and 16S rDNA-sequencing using Illumina MiSeq. The obtained sequences were subjected to the DADA2 pipeline in R and the Quantitative Microbial Composition (QMP) was quantified by flow cytometry. Results Eight responders were observed: 3 after S-FMT and 5 after A-FMT, as well as 58 non-responders considering the intention-to-treat population. Responders to FMT tended to cluster at baseline (adonis p=0.34) and PCA analysis on the top 500 mucosal genes with the highest variance (Fig.1), showed a significant host effect at week 8 between responders and non-responders (adonis p<0.05). Likewise, PCA analysis of the currently available QMP (Fig.2) showed a trend towards clustering by response at week 0 and 8 resp. adonis p=0.18 and p=0.11). An overall decrease in Bacteroides-2 enterotype prevalence was observed (Fig.3) over both treatment groups and independently from reaching the PE (All McNemar's p=0.077). Conclusion A trend towards clustering of responders on host mRNA level and QMP was observed at baseline as well as at the primary endpoint, showing a potential role for pre-FMT patient selection by phenotype. Moreover, the dysbiotic enterotype Bacteroides-2 seems to be decreased after FMT. However, further analyses are mandatory to identify specific predictors of response and the origin of the microbiota shift.

Anti-microbial host factor HDAC6 is antagonised by the influenza A virus through host caspases and viral PA.

Histone deacetylase 6 (HDAC6), through the repertoire of its substrate proteins, plays a critical role in human physiology, and an aberrant function of HDAC6 contributes to various pathophysiological conditions. HDAC6 is also known to be an anti-microbial host factor and has been implicated in restricting or clearing the infection of various human viral and bacterial pathogens. However, the state and the mechanisms of its antagonism in infected cells are not understood. Here, we demonstrate that influenza A virus (IAV) antagonises HDAC6 by recruiting both viral and host components. We found that HDAC6 mRNA expression, and consequently, the HDAC6 polypeptide expression is downregulated in human lung epithelial cells during early stage of IAV infection but can be rescued by depleting the expression of viral polymerase acidic (PA) protein, a subunit of IAV RNA polymerase. In addition, during later stage of the infection, the HDAC6 polypeptide undergoes caspase-mediated cleavage at two sites, generating two cleaved fragments. Both these fragments disappeared when the expression of caspase 3 was depleted in infected cells, whereas only second fragment disappeared when the expression of caspase 6 was depleted. But both fragments disappeared and the level of full-length HDAC6 polypeptide was rescued when the expression of PA was depleted in infected cells. Collectively, these data indicated that IAV antagonises the HDAC6 by decreasing its expression level in infected cells, both at mRNA and polypeptide level via PA gene, which has been implicated in auxiliary functions like degradation of host mRNA and induction of apoptosis.

Genome-Wide Identification, Quantification, and Validation of Differentially Expressed miRNAs in Eggplant (Solanum melongena L.) Based on Their Response to Ralstonia solanacearum Infection.

MicroRNAs (miRNAs), a type of short noncoding RNA molecule (21-23 nucleotides), mediate repressive gene regulation through RNA silencing at the posttranscriptional level and play an important role in the defense response to abiotic and biotic stresses. miRNAs of the plant system have been studied in model crops for their diverse regulatory role while less is known about their significance in other plants whose genome and transcriptome data are scarce in the database, including eggplant (Solanum melongena L.). In the present study, a next-generation sequencing platform was used for the sequencing of miRNA, and real-time quantitative PCR for miRNAs was used to validate the gene expression patterns of miRNAs in Solanum melongena plantlets infected with the bacterial wilt-causing pathogen Ralstonia solanacearum (R. solanacearum). Sequence analyses showed the presence of 375 miRNAs belonging to 29 conserved families. The miR414 is highly conserved miRNA across the plant system while miR5658 and miR5021 were found exclusively in Arabidopsis thaliana surprisingly, these miRNAs were found in eggplants too. The most abundant families were miR5658 and miR414. Ppt-miR414, hvu-miR444b, stu-miR8020, and sly miR5303 were upregulated in Pusa purple long (PPL) (susceptible) at 48 h postinfection, followed by a decline after 96 h postinfection. A similar trend was obtained in ath-miR414, stu-mir5303h, alymiR847-5p, far-miR1134, ath-miR5021, ath-miR5658, osa-miR2873c, lja-miR7530, stu-miR7997c, and gra-miR8741 but at very low levels after infection in the susceptible variety, indicating their negative role in the suppression of host immunity. On the other hand, osa-miR2873c was found to be slightly increased after 96 hpi from 48 hpi. Most of the miRNAs under study showed relatively lower expression in the resistant variety Arka Nidhi after infection than in the susceptible variety. These results shed light on a deeper regulatory role of miRNAs and their targets in regulation of the plant response to bacterial infection. The present experiment and their results suggested that the higher expression of miRNA leads to a decline in host mRNA and thus shows susceptibility.

A potential role for SARS-CoV-2 small viral RNAs in targeting host microRNAs and modulating gene expression

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease (COVID-19) in humans, with symptoms ranging from mild to severe, including fatality. The molecular mechanisms surrounding the effects of viral infection on the host RNA machinery remain poorly characterized. We used a comparative transcriptomics approach to investigate the effects of SARS-CoV-2 infection on the host mRNA and sRNA expression machinery in a human lung epithelial cell line (Calu-3) and an African green monkey kidney cell line (Vero-E6). Upon infection, we observed global changes in host gene expression and differential expression of dozens of host miRNAs, many with known links to viral infection and immune response. Additionally, we discovered an expanded landscape of more than a hundred SARS-CoV-2-derived small viral RNAs (svRNAs) predicted to interact with differentially expressed host mRNAs and miRNAs. svRNAs are derived from distinct regions of the viral genome and sequence signatures suggest they are produced by a non-canonical biogenesis pathway. 52 of the 67 svRNAs identified in Calu-3 cells are predicted to interact with differentially expressed miRNAs, with many svRNAs having multiple targets. Accordingly, we speculate that these svRNAs may play a role in SARS-CoV-2 propagation by modulating post-transcriptional gene regulation, and that methods for antagonizing them may have therapeutic value.

SARS‐CoV‐2 Non-structural protein 1(NSP1) mutation virulence and natural selection: Evolutionary trends in the six continents

ObjectiveRapid transmission and reproduction of RNA viruses prepare conducive conditions to have a high rate of mutations in their genetic sequence. The viral mutations make adapt the severe acute respiratory syndrome coronavirus 2 in the host environment and help the evolution of the virus then also caused a high mortality rate by the virus that threatens worldwide health. Mutations and adaptation help the virus to escape confrontations that are done against it. MethodsIn the present study, we analyzed 6,510,947 sequences of non-structural protein 1 as one of the conserved regions of the virus to find out frequent mutations and substitute amino acids in comparison with the wild type. NSP1 mutations rate divided into continents were different. ResultsBased on this continental categorization, E87D in global vision and also in Europe notably increased. The E87D mutation has signed up to January 2022 as the first frequent mutation observed. The remarkable mutations, H110Y and R24C have the second and third frequencies, respectively. ConclusionAccording to the important role of non-structural protein 1 on the host mRNA translation, developing drug design against the protein could be so hopeful to find more effective ways the control and then treatment of the global pandemic coronavirus disease 2019.

Revealing druggable cryptic pockets in the Nsp1 of SARS-CoV-2 and other β-coronaviruses by simulations and crystallography.

Non-structural protein 1 (Nsp1) is a main pathogenicity factor of α- and β-coronaviruses. Nsp1 of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) suppresses the host gene expression by sterically blocking 40S host ribosomal subunits and promoting host mRNA degradation. This mechanism leads to the downregulation of the translation-mediated innate immune response in host cells, ultimately mediating the observed immune evasion capabilities of SARS-CoV-2. Here, by combining extensive molecular dynamics simulations, fragment screening and crystallography, we reveal druggable pockets in Nsp1. Structural and computational solvent mapping analyses indicate the partial crypticity of these newly discovered and druggable binding sites. The results of fragment-based screening via X-ray crystallography confirm the druggability of the major pocket of Nsp1. Finally, we show how the targeting of this pocket could disrupt the Nsp1-mRNA complex and open a novel avenue to design new inhibitors for other Nsp1s present in homologous β-coronaviruses.

Buffy Coat Transcriptomic Analysis Reveals Alterations in Host Cell Protein Synthesis and Cell Cycle in Severe COVID-19 Patients.

Transcriptome studies have reported the dysregulation of cell cycle-related genes and the global inhibition of host mRNA translation in COVID-19 cases. However, the key genes and cellular mechanisms that are most affected by the severe outcome of this disease remain unclear. For this work, the RNA-seq approach was used to study the differential expression in buffy coat cells of two groups of people infected with SARS-CoV-2: (a) Mild, with mild symptoms; and (b) SARS (Severe Acute Respiratory Syndrome), who were admitted to the intensive care unit with the severe COVID-19 outcome. Transcriptomic analysis revealed 1009 up-regulated and 501 down-regulated genes in the SARS group, with 10% of both being composed of long non-coding RNA. Ribosome and cell cycle pathways were enriched among down-regulated genes. The most connected proteins among the differentially expressed genes involved transport dysregulation, proteasome degradation, interferon response, cytokinesis failure, and host translation inhibition. Furthermore, interactome analysis showed Fibrillarin to be one of the key genes affected by SARS-CoV-2. This protein interacts directly with the N protein and long non-coding RNAs affecting transcription, translation, and ribosomal processes. This work reveals a group of dysregulated processes, including translation and cell cycle, as key pathways altered in severe COVID-19 outcomes.

RNase L activation in the cytoplasm induces aberrant processing of mRNAs in the nucleus.

The antiviral endoribonuclease, RNase L, is activated by the mammalian innate immune response to destroy host and viral RNA to ultimately reduce viral gene expression. Herein, we show that RNase L and RNase L-mediated mRNA decay are primarily localized to the cytoplasm. Consequently, RNA-binding proteins (RBPs) translocate from the cytoplasm to the nucleus upon RNase L activation due to the presence of intact nuclear RNA. The re-localization of RBPs to the nucleus coincides with global alterations to RNA processing in the nucleus. While affecting many host mRNAs, these alterations are pronounced in mRNAs encoding type I and type III interferons and correlate with their retention in the nucleus and reduction in interferon protein production. Similar RNA processing defects also occur during infection with either dengue virus or SARS-CoV-2 when RNase L is activated. These findings reveal that the distribution of RBPs between the nucleus and cytosol is dictated by the availability of RNA in each compartment. Thus, viral infections that trigger RNase L-mediated cytoplasmic RNA in the cytoplasm also alter RNA processing in the nucleus, resulting in an ingenious multi-step immune block to protein biogenesis.

HSV-1 and influenza infection induce linear and circular splicing of the long NEAT1 isoform.

The herpes simplex virus 1 (HSV-1) virion host shut-off (vhs) protein cleaves both cellular and viral mRNAs by a translation-initiation-dependent mechanism, which should spare circular RNAs (circRNAs). Here, we show that vhs-mediated degradation of linear mRNAs leads to an enrichment of circRNAs relative to linear mRNAs during HSV-1 infection. This was also observed in influenza A virus (IAV) infection, likely due to degradation of linear host mRNAs mediated by the IAV PA-X protein and cap-snatching RNA-dependent RNA polymerase. For most circRNAs, enrichment was not due to increased circRNA synthesis but due to a general loss of linear RNAs. In contrast, biogenesis of a circRNA originating from the long isoform (NEAT1_2) of the nuclear paraspeckle assembly transcript 1 (NEAT1) was induced both in HSV-1 infection-in a vhs-independent manner-and in IAV infection. This was associated with induction of novel linear splicing of NEAT1_2 both within and downstream of the circRNA. NEAT1_2 forms a scaffold for paraspeckles, nuclear bodies located in the interchromatin space, must likely remain unspliced for paraspeckle assembly and is up-regulated in HSV-1 and IAV infection. We show that NEAT1_2 splicing and up-regulation can be induced by ectopic co-expression of the HSV-1 immediate-early proteins ICP22 and ICP27, potentially linking increased expression and splicing of NEAT1_2. To identify other conditions with NEAT1_2 splicing, we performed a large-scale screen of published RNA-seq data. This uncovered both induction of NEAT1_2 splicing and poly(A) read-through similar to HSV-1 and IAV infection in cancer cells upon inhibition or knockdown of CDK7 or the MED1 subunit of the Mediator complex phosphorylated by CDK7. In summary, our study reveals induction of novel circular and linear NEAT1_2 splicing isoforms as a common characteristic of HSV-1 and IAV infection and highlights a potential role of CDK7 in HSV-1 or IAV infection.

Different RNA Elements Control Viral Protein Synthesis in Polerovirus Isolates Evolved in Separate Geographical Regions.

Most plant viruses lack the 5′-cap and 3′-poly(A) structures, which are common in their host mRNAs, and are crucial for translation initiation. Thus, alternative translation initiation mechanisms were identified for viral mRNAs, one of these being controlled by an RNA element in their 3′-ends that is able to enhance mRNA cap-independent translation (3′-CITE). The 3′-CITEs are modular and transferable RNA elements. In the case of poleroviruses, the mechanism of translation initiation of their RNAs in the host cell is still unclear; thus, it was studied for one of its members, cucurbit aphid-borne yellows virus (CABYV). We determined that efficient CABYV RNA translation requires the presence of a 3′-CITE in its 3′-UTR. We showed that this 3′-CITE requires the presence of the 5′-UTR in cis for its eIF4E-independent activity. Efficient virus multiplication depended on 3′-CITE activity. In CABYV isolates belonging to the three phylogenetic groups identified so far, the 3′-CITEs differ, and recombination prediction analyses suggest that these 3′-CITEs have been acquired through recombination with an unknown donor. Since these isolates have evolved in different geographical regions, this may suggest that their respective 3′-CITEs are possibly better adapted to each region. We propose that translation of other polerovirus genomes may also be 3′-CITE-dependent.

Two Ligand-Binding Sites on SARS-CoV-2 Non-Structural Protein 1 Revealed by Fragment-Based X-ray Screening.

The regular reappearance of coronavirus (CoV) outbreaks over the past 20 years has caused significant health consequences and financial burdens worldwide. The most recent and still ongoing novel CoV pandemic, caused by Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) has brought a range of devastating consequences. Due to the exceptionally fast development of vaccines, the mortality rate of the virus has been curbed to a significant extent. However, the limitations of vaccination efficiency and applicability, coupled with the still high infection rate, emphasise the urgent need for discovering safe and effective antivirals against SARS-CoV-2 by suppressing its replication or attenuating its virulence. Non-structural protein 1 (nsp1), a unique viral and conserved leader protein, is a crucial virulence factor for causing host mRNA degradation, suppressing interferon (IFN) expression and host antiviral signalling pathways. In view of the essential role of nsp1 in the CoV life cycle, it is regarded as an exploitable target for antiviral drug discovery. Here, we report a variety of fragment hits against the N-terminal domain of SARS-CoV-2 nsp1 identified by fragment-based screening via X-ray crystallography. We also determined the structure of nsp1 at atomic resolution (0.99 Å). Binding affinities of hits against nsp1 and potential stabilisation were determined by orthogonal biophysical assays such as microscale thermophoresis and thermal shift assays. We identified two ligand-binding sites on nsp1, one deep and one shallow pocket, which are not conserved between the three medically relevant SARS, SARS-CoV-2 and MERS coronaviruses. Our study provides an excellent starting point for the development of more potent nsp1-targeting inhibitors and functional studies on SARS-CoV-2 nsp1.

Using a 29-mRNA Host Response Classifier To Detect Bacterial Coinfections and Predict Outcomes in COVID-19 Patients Presenting to the Emergency Department.

Clinicians in the emergency department (ED) face challenges in concurrently assessing patients with suspected COVID-19 infection, detecting bacterial coinfection, and determining illness severity since current practices require separate workflows. Here, we explore the accuracy of the IMX-BVN-3/IMX-SEV-3 29 mRNA host response classifiers in simultaneously detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and bacterial coinfections and predicting clinical severity of COVID-19. A total of 161 patients with PCR-confirmed COVID-19 (52.2% female; median age, 50.0 years; 51% hospitalized; 5.6% deaths) were enrolled at the Stanford Hospital ED. RNA was extracted (2.5 mL whole blood in PAXgene blood RNA), and 29 host mRNAs in response to the infection were quantified using Nanostring nCounter. The IMX-BVN-3 classifier identified SARS-CoV-2 infection in 151 patients with a sensitivity of 93.8%. Six of 10 patients undetected by the classifier had positive COVID tests more than 9 days prior to enrollment, and the remaining patients oscillated between positive and negative results in subsequent tests. The classifier also predicted that 6 (3.7%) patients had a bacterial coinfection. Clinical adjudication confirmed that 5/6 (83.3%) of the patients had bacterial infections, i.e., Clostridioides difficile colitis (n = 1), urinary tract infection (n = 1), and clinically diagnosed bacterial infections (n = 3), for a specificity of 99.4%. Two of 101 (2.8%) patients in the IMX-SEV-3 "Low" severity classification and 7/60 (11.7%) in the "Moderate" severity classification died within 30 days of enrollment. IMX-BVN-3/IMX-SEV-3 classifiers accurately identified patients with COVID-19 and bacterial coinfections and predicted patients' risk of death. A point-of-care version of these classifiers, under development, could improve ED patient management, including more accurate treatment decisions and optimized resource utilization. IMPORTANCE We assay the utility of the single-test IMX-BVN-3/IMX-SEV-3 classifiers that require just 2.5 mL of patient blood in concurrently detecting viral and bacterial infections as well as predicting the severity and 30-day outcome from the infection. A point-of-care device, in development, will circumvent the need for blood culturing and drastically reduce the time needed to detect an infection. This will negate the need for empirical use of broad-spectrum antibiotics and allow for antibiotic use stewardship. Additionally, accurate classification of the severity of infection and the prediction of 30-day severe outcomes will allow for appropriate allocation of hospital resources.

Analysis of nested alternate open reading frames and their encoded proteins.

Transcriptional and post-transcriptional mechanisms diversify the proteome beyond gene number, while maintaining a sequence relationship between original and altered proteins. A new mechanism breaks this paradigm, generating novel proteins by translating alternative open reading frames (Alt-ORFs) within canonical host mRNAs. Uniquely, ‘alt-proteins’ lack sequence homology with host ORF-derived proteins. We show global amino acid frequencies, and consequent biochemical characteristics of Alt-ORFs nested within host ORFs (nAlt-ORFs), are genetically-driven, and predicted by summation of frequencies of hundreds of encompassing host codon-pairs. Analysis of 101 human nAlt-ORFs of length ≥150 codons confirms the theoretical predictions, revealing an extraordinarily high median isoelectric point (pI) of 11.68, due to anomalous charged amino acid levels. Also, nAlt-ORF proteins exhibit a >2-fold preference for reading frame 2 versus 3, predicted mitochondrial and nuclear localization, and elevated codon adaptation index indicative of natural selection. Our results provide a theoretical and conceptual framework for exploration of these largely unannotated, but potentially significant, alternative ORFs and their encoded proteins.

Sodium Selenite As Potential Adjuvant Therapy for COVID-19.

The review considers the role that selenium plays in RNA virus infections and, in particular, COVID-19. Many RNA viruses are selenium dependent because antisense interactions arise between viral RNAs and host mRNA regions containing the selencysteine insertion sequence to cause selenium deficiency, oxidative stress, immune response impairment, etc. Sodium selenite is a licensed selenium-containing product and is widely used in medicine, veterinary, and agriculture. Its advantages include the following. Sodium selenite rapidly penetrates through cell membranes in all tissues of the body; is intensely involved in metabolic processes accompanied by oxidation of sulfur-containing cell proteins; exerts an antiaggregation effect by reducing thromboxane activity; interrupts the contact of a virion (SARS-CoV-1 and SARS-CoV-2) with the membrane of a healthy cell; and suppresses NF-κB activity, which significantly increases in coronavirus infections. Arguments supporting the use of sodium selenite as adjuvant therapy in COVID-19 are discussed.

Epitranscriptome profiling of spleen mRNA m6A methylation reveals pathways of host responses to malaria parasite infection.

N6 -Methyladenosine (m6A), the most abundant mammalian mRNA modification, has been reported to modulate various viral infections. Although it has been confirmed that RNA modifications can also modulate the replication and development of different parasites, the role of the RNA epitranscriptome in the regulation of host response post parasite infection remains to be elucidated. Here we report host spleen m6A epitranscriptome landscapes induced by different strains of the malaria parasite Plasmodium yoelii. We found that malaria parasite infection dramatically changes host spleen m6A mRNA modification and gene expression. Additionally, malaria parasite infection reprograms host immune response pathways by regulating the m6A modification enzymes. Collectively, our study is the first characterization of host spleen m6A methylome triggered by malaria parasite infections, and our data identify m6A modifications as significant transcriptome-wide marks during host-parasite interactions. We demonstrate that host mRNA methylation machinery can sense and respond to malaria parasite infections, and provide new insights into epitranscriptomic mechanisms underlying parasite-induced pathogenesis.

A tRNA-derived fragment present in E. coli OMVs regulates host cell gene expression and proliferation.

RNA-sequencing has led to a spectacular increase in the repertoire of bacterial sRNAs and improved our understanding of their biological functions. Bacterial sRNAs have also been found in outer membrane vesicles (OMVs), raising questions about their potential involvement in bacteria-host relationship, but few studies have documented this issue. Recent RNA-Sequencing analyses of bacterial RNA unveiled the existence of abundant very small RNAs (vsRNAs) shorter than 16 nt. These especially include tRNA fragments (tRFs) that are selectively loaded in OMVs and are predicted to target host mRNAs. Here, in Escherichia coli (E. coli), we report the existence of an abundant vsRNA, Ile-tRF-5X, which is selectively modulated by environmental stress, while remaining unaffected by inhibition of transcription or translation. Ile-tRF-5X is released through OMVs and can be transferred to human HCT116 cells, where it promoted MAP3K4 expression. Our findings provide a novel perspective and paradigm on the existing symbiosis between bacteria and human cells.

Detection of Viral Infection and Bacterial Coinfection and Superinfection in Coronavirus Disease 2019 Patients Presenting to the Emergency Department Using the 29-mRNA Host Response Classifier IMX-BVN-3: A Multicenter Study.

BackgroundIdentification of bacterial coinfection in patients with coronavirus disease 2019 (COVID-19) facilitates appropriate initiation or withholding of antibiotics. The Inflammatix Bacterial Viral Noninfected (IMX-BVN) classifier determines the likelihood of bacterial and viral infections. In a multicenter study, we investigated whether IMX-BVN version 3 (IMX-BVN-3) identifies patients with COVID-19 and bacterial coinfections or superinfections.MethodsPatients with polymerase chain reaction–confirmed COVID-19 were enrolled in Berlin, Germany; Basel, Switzerland; and Cleveland, Ohio upon emergency department or hospital admission. PAXgene Blood RNA was extracted and 29 host mRNAs were quantified. IMX-BVN-3 categorized patients into very unlikely, unlikely, possible, and very likely bacterial and viral interpretation bands. IMX-BVN-3 results were compared with clinically adjudicated infection status.ResultsIMX-BVN-3 categorized 102 of 111 (91.9%) COVID-19 patients into very likely or possible, 7 (6.3%) into unlikely, and 2 (1.8%) into very unlikely viral bands. Approximately 94% of patients had IMX-BVN-3 unlikely or very unlikely bacterial results. Among 7 (6.3%) patients with possible (n = 4) or very likely (n = 3) bacterial results, 6 (85.7%) had clinically adjudicated bacterial coinfection or superinfection. Overall, 19 of 111 subjects for whom adjudication was performed had a bacterial infection; 7 of these showed a very likely or likely bacterial result in IMX-BVN-3.ConclusionsIMX-BVN-3 identified COVID-19 patients as virally infected and identified bacterial coinfections and superinfections. Future studies will determine whether a point-of-care version of the classifier may improve the management of COVID-19 patients, including appropriate antibiotic use.

Bidirectional mRNA transfer between Cuscuta australis and its hosts.

The holoparasitic dodder (Cuscuta spp.) is able to transfer mRNA and certain plant pathogens (e.g., viruses and bacteria) from the host plant. “Candidatus Liberibacter asiaticus,” the phloem-limited causative agent of citrus Huanglongbing, can be transferred from citrus to periwinkle (Catharanthus roseus) mediated by dodder. However, characterization of mRNA transport between dodder and citrus/periwinkle remains unclear. In this study, we sequenced transcriptomes of dodder and its parasitizing host, sweet orange (Citrus sinensis “Newhall”) and periwinkle (Catharanthus roseus), to identify and characterize mRNA transfer between dodder and the host plant during parasitism. The mRNA transfer between dodder and citrus/periwinkle was bidirectional and most of the transfer events occurred in the interface tissue. Compared with the citrus–dodder system, mRNA transfer in the periwinkle–dodder system was more frequent. Function classification revealed that a large number of mRNAs transferred between dodder and citrus/periwinkle were involved in secondary metabolism and stress response. Dodder transcripts encoding proteins associated with microtubule-based processes and cell wall biogenesis were transferred to host tissues. In addition, transcripts involved in translational elongation, plasmodesmata, and the auxin-activated signaling pathway were transmitted between dodder and citrus/periwinkle. In particular, transcripts involved in shoot system development and flower development were transferred between the host and dodder in both directions. The high abundance of dodder-origin transcripts, encoding MIP aquaporin protein, and S-adenosylmethionine synthetase 1 protein, in citrus and periwinkle tissues indicated they could play an important biological role in dodder–host interaction. In addition, the uptake of host mRNAs by dodder, especially those involved in seed germination and flower development, could be beneficial for the reproduction of dodder. The results of this study provide new insights into the RNA-based interaction between dodder and host plants.

CHIKV infection reprograms codon optimality to favor viral RNA translation by altering the tRNA epitranscriptome

Ample evidence indicates that codon usage bias regulates gene expression. How viruses, such as the emerging mosquito-borne Chikungunya virus (CHIKV), express their genomes at high levels despite an enrichment in rare codons remains a puzzling question. Using ribosome footprinting, we analyze translational changes that occur upon CHIKV infection. We show that CHIKV infection induces codon-specific reprogramming of the host translation machinery to favor the translation of viral RNA genomes over host mRNAs with an otherwise optimal codon usage. This reprogramming was mostly apparent at the endoplasmic reticulum, where CHIKV RNAs show high ribosome occupancy. Mechanistically, it involves CHIKV-induced overexpression of KIAA1456, an enzyme that modifies the wobble U34 position in the anticodon of tRNAs, which is required for proper decoding of codons that are highly enriched in CHIKV RNAs. Our findings demonstrate an unprecedented interplay of viruses with the host tRNA epitranscriptome to adapt the host translation machinery to viral production.

Bunyaviral N Proteins Localize at RNA Processing Bodies and Stress Granules: The Enigma of Cytoplasmic Sources of Capped RNA for Cap Snatching

Most cytoplasmic-replicating negative-strand RNA viruses (NSVs) initiate genome transcription by cap snatching. The source of host mRNAs from which the cytoplasmic NSVs snatch capped-RNA leader sequences has remained elusive. Earlier reports have pointed towards cytoplasmic-RNA processing bodies (P body, PB), although several questions have remained unsolved. Here, the nucleocapsid (N) protein of plant- and animal-infecting members of the order Bunyavirales, in casu Tomato spotted wilt virus (TSWV), Rice stripe virus (RSV), Sin nombre virus (SNV), Crimean-Congo hemorrhagic fever virus (CCHFV) and Schmallenberg virus (SBV) have been expressed and localized in cells of their respective plant and animal hosts. All N proteins localized to PBs as well as stress granules (SGs), but extensively to docking stages of PB and SG. TSWV and RSV N proteins also co-localized with Ran GTPase-activating protein 2 (RanGAP2), a nucleo-cytoplasmic shuttling factor, in the perinuclear region, and partly in the nucleus when co-expressed with its WPP domain containing a nuclear-localization signal. Upon silencing of PB and SG components individually or concomitantly, replication levels of a TSWV minireplicon, as measured by the expression of a GFP reporter gene, ranged from a 30% reduction to a four-fold increase. Upon the silencing of RanGAP homologs in planta, replication of the TSWV minireplicon was reduced by 75%. During in vivo cap-donor competition experiments, TSWV used transcripts destined to PB and SG, but also functional transcripts engaged in translation. Altogether, the results implicate a more complex situation in which, besides PB, additional cytoplasmic sources are used during transcription/cap snatching of cytoplasmic-replicating and segmented NSVs.