Comparison of RNA synthesis initiation properties of non-segmented negative strand RNA virus polymerases

It is generally thought that the promoters of non-segmented, negative strand RNA viruses (nsNSVs) direct the polymerase to initiate RNA synthesis exclusively opposite the 3´ terminal nucleotide of the genome RNA by a de novo (primer independent) initiation mechanism. However, recent studies have revealed that there is diversity between different nsNSVs with pneumovirus promoters directing the polymerase to initiate at positions 1 and 3 of the genome, and ebolavirus polymerases being able to initiate at position 2 on the template. Studies with other RNA viruses have shown that polymerases that engage in de novo initiation opposite position 1 typically have structural features to stabilize the initiation complex and ensure efficient and accurate initiation. This raised the question of whether different nsNSV polymerases have evolved fundamentally different structural properties to facilitate initiation at different sites on their promoters. Here we examined the functional properties of polymerases of respiratory syncytial virus (RSV), a pneumovirus, human parainfluenza virus type 3 (PIV-3), a paramyxovirus, and Marburg virus (MARV), a filovirus, both on their cognate promoters and on promoters of other viruses. We found that in contrast to the RSV polymerase, which initiated at positions 1 and 3 of its promoter, the PIV-3 and MARV polymerases initiated exclusively at position 1 on their cognate promoters. However, all three polymerases could recognize and initiate from heterologous promoters, with the promoter sequence playing a key role in determining initiation site selection. In addition to examining de novo initiation, we also compared the ability of the RSV and PIV-3 polymerases to engage in back-priming, an activity in which the promoter template is folded into a secondary structure and nucleotides are added to the template 3´ end. This analysis showed that whereas the RSV polymerase was promiscuous in back-priming activity, the PIV-3 polymerase generated barely detectable levels of back-primed product, irrespective of promoter template sequence. Overall, this study shows that the polymerases from these three nsNSV families are fundamentally similar in their initiation properties, but have differences in their abilities to engage in back-priming.

RNA Modifications in Genomic RNA of Influenza A Virus and the Relationship between RNA Modifications and Viral Infection

Recent studies about the transcriptome-wide presence of RNA modifications have revealed their importance in many cellular functions. Nevertheless, information about RNA modifications in viral RNA is scarce, especially for negative-strand RNA viruses. Here we provide a catalog of RNA modifications including m1A, ac4C, m7G, inosine, and pseudouridine on RNA derived from an influenza A virus infected into A549 cells, as studied by RNA immunoprecipitation followed by deep-sequencing. Possible regions with RNA modifications were found in the negative-strand segments of viral genomic RNA. In addition, our analyses of previously published data revealed that the expression levels of the host factors for RNA modifications were affected by an infection with influenza A virus, and some of the host factors likely have a proviral effect. RNA modification is a novel aspect of host–virus interactions leading to the discovery of previously unrecognized viral pathogenicity mechanisms and has the potential to aid the development of novel antivirals.

Predicting Infectivity: Comparing Four PCR-based Assays to Detect Culturable SARS-CoV-2 in Clinical Samples

With the COVID-19 pandemic caused by SARS-CoV-2 now in its second year, there remains an urgent need for diagnostic testing that can identify infected individuals, particularly those who harbor infectious virus. Various RT-PCR strategies have been proposed to identify specific viral RNA species that may predict the presence of infectious virus, including detection of transcriptional intermediates (e.g. subgenomic RNA [sgRNA]) and replicative intermediates (e.g. negative-strand RNA species). Using a novel primer/probe set for detection of subgenomic (sg)E transcripts, we successfully identified 100% of specimens containing culturable SARS-CoV-2 from a set of 126 clinical samples (total sgE CT values ranging from 12.3-37.5). This assay showed superior performance compared to a previously published sgRNA assay and to a negative-strand RNA assay, both of which failed to detect target RNA in a subset of samples from which we isolated live virus. In addition, total levels of viral RNA (genome, negative-strand, and sgE) detected with the WHO/Charite primer-probe set correlated closely with levels of infectious virus. Specifically, infectious virus was not detected in samples with a CT above 31.0. Clinical samples with higher levels of viral RNA also displayed cytopathic effect (CPE) more quickly than those with lower levels of viral RNA. Finally, we found that the infectivity of SARS-CoV-2 samples is significantly dependent on the cell type used for viral isolation, as Vero E6 cells expressing TMRPSS2 extended the analytical sensitivity of isolation by more than 3 CT compared to parental Vero E6 cells and resulted in faster isolation. Our work shows that using a total viral RNA Ct cut-off of >31 or specifically testing for sgRNA can serve as an effective rule-out test for viral infectivity.

Type I interferon receptor-independent interferon-α induction upon infection with a variety of negative-strand RNA viruses

Type I interferons (IFNs) are a first line of defence against viral infections. Upon infection, a first small wave of early type I IFN, mainly IFN-β and particularly IFN-α4, are induced and bind to the type I IFN receptor (IFNAR) to amplify the IFN response. It was shown for several viruses that robust type I IFN responses require this positive feedback loop via the IFNAR. Recently, we showed that infection of IFNAR knockout mice with the orthomyxovirus Thogoto virus lacking the ML open reading frame (THOV(ML-)) results in the expression of unexpected high amounts of type I IFN. To investigate if IFNAR-independent IFN responses are unique for THOV(ML-), we performed infection experiments with several negative-strand RNA viruses using different routes and dosages for infection. A variety of these viruses induced type I IFN responses IFNAR-independently when using the intraperitoneal (i.p.) route for infection. In vitro studies demonstrated that myeloid dendritic cells (mDC) are capable of producing IFNAR-independent IFN-α responses that are dependent on the expression of the adaptor protein mitochondrial antiviral-signalling protein (MAVS) whereas pDC where entirely depending on the IFNAR feedback loop in vitro. Thus, depending on dose and route of infection, the IFNAR feedback loop is not strictly necessary for robust type I IFN expression and an IFNAR-independent type I IFN production might be the rule rather than the exception for infections with numerous negative-strand RNA viruses.

An increased ratio of SARS-CoV-2 positive to negative sense genomic and subgenomic RNAs within routine diagnostic upper respiratory tract swabs may be a marker of virion shedding

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread rapidly in the global population since its emergence in humans in late 2019. Replication of SARS-CoV-2 is characterised by transcription and replication of genomic length RNA and shorter subgenomic RNAs to produce virus proteins and ultimately progeny virions. Here we explore the pattern of both genome-length and subgenomic RNAs and positive and negative strand SARS-CoV-2 RNAs in diagnostic nasopharyngeal swabs using sensitive probe based PCR assays as well as Ampliseq panels designed to target subgenomic RNAs. Using these assays, we measured the ratios of genomic to subgenomic RNAs as well as the ratios of positive to negative strand RNAs in SARS-CoV-2 positive nasopharyngeal swab samples. We found that while subgenomic RNAs and negative strand RNA can be readily detected in swab samples taken up to 19 and 17 days post symptom onset respectively, and therefore their detection alone is not likely an indicator of active SARS-CoV-2 replication. However, the ratios of genomic-length to subgenomic RNA and also of positive to negative strand RNA were elevated in some swabs, particularly those collected around the onset of clinical symptoms or in an individual with decreasing PCR Cts in successive swab samples. We tentatively conclude that it may be possible to refine such molecular assays to help determine if active replication of virus is occurring and progeny virions likely present in a SARS-CoV-2 positive individual. Assays targeting subgenomic N or ORF7a RNAs as well as strand specific ORF7a total genome-length and subgenomic RNAs may be the most sensitive for this purpose as these targets were consistently the most abundant in the swab samples.

A synergistic effect between 3′ terminal noncoding and adjacent coding regions of influenza A virus HA segment on template preference

The influenza A virus genome is comprised of eight single-stranded negative-sense viral RNA (vRNA) segments. Each of the eight vRNA segments contains segment-specific nonconserved noncoding regions (NCRs) of similar sequence and length in different influenza A virus strains. However, in the subtype-determinant segments, encoding haemagglutinin (HA) and neuraminidase (NA), the segment-specific noncoding regions are subtype-specific, varying significantly in sequence and length at both the 3´ and 5´ termini among different subtypes. The significance of these subtype-specific noncoding regions (ssNCR) in the influenza virus replication cycle is not fully understood. In this study, we show that truncations of the 3´-end H1-subtype-specific noncoding region (H1-ssNCR) resulted in recombinant viruses with decreased HA vRNA replication and attenuated growth phenotype, although the vRNA replication was not affected in single-template RNP reconstitution assays. The attenuated viruses were unstable and point mutations at nucleotide position 76 or 56 in the adjacent coding region of HA vRNA were found after serial passage. The mutations restored the HA vRNA replication and reversed the attenuated virus growth phenotype. We propose that the terminal noncoding and adjacent coding regions act synergistically to ensure optimal levels of HA vRNA replication in a multi-segment environment. These results, provide novel insights into the role of the 3´-end nonconserved noncoding regions and adjacent coding regions on template preference in multiple-segmented negative-strand RNA viruses. IMPORTANCE While most influenza A virus vRNA segments contain segment-specific nonconserved noncoding regions of similar length and sequence, these regions vary considerably both in length and sequence in the segments encoding HA and NA, the two major antigenic determinants of influenza A viruses. In this study, we investigated the function of the 3´-end H1-ssNCR and observed a synergistic effect between the 3´-end H1-ssNCR nucleotides and adjacent coding nucleotide(s) of HA segment on template preference in a multi-segment environment. The results unravel an additional level of complexity in the regulation of RNA replication in multiple-segmented negative-strand RNA viruses.

The Bunyavirales: The Plant-Infecting Counterparts

Negative-strand (-) RNA viruses (NSVs) comprise a large and diverse group of viruses that are generally divided in those with non-segmented and those with segmented genomes. Whereas most NSVs infect animals and humans, the smaller group of the plant-infecting counterparts is expanding, with many causing devastating diseases worldwide, affecting a large number of major bulk and high-value food crops. In 2018, the taxonomy of segmented NSVs faced a major reorganization with the establishment of the order Bunyavirales. This article overviews the major plant viruses that are part of the order, i.e., orthospoviruses (Tospoviridae), tenuiviruses (Phenuiviridae), and emaraviruses (Fimoviridae), and provides updates on the more recent ongoing research. Features shared with the animal-infecting counterparts are mentioned, however, special attention is given to their adaptation to plant hosts and vector transmission, including intra/intercellular trafficking and viral counter defense to antiviral RNAi.

COVID-19: CRISPR/Cas-like System of nsp3 Promotes the Mutant Recombination and Drug Resistance

<p>Patients with novel coronavirus pneumonia usually suffer from bacterial and fungal infections, and the drug resistance problem caused by the pandemic is becoming more and more serious. Simultaneously, the SARS-COV-2 virus has a rapid mutation phenomenon, and somegene coding regions by mutation and recombination may be related to the drug resistance of the virus. Therefore, studying the relationship between the co-infection of bacteria and fungi and the evolution of SARS-COV-2 has important guiding significance for preventing a pandemic. We found that the SARS-COV-2 virus's nsp3 protein had a CRISPR/Cas 9 (II-B)-like function by searching for conserved domains. The system could target and edit the negative-strand RNA of SARS-COV-2. We speculated that the crRNA (CRISPR RNA) produced by the CRISPR/Cas system of Pseudomonas aeruginosa carried the genetic information of the conserved domains of bacteriophages and Pseudomonas, including drug resistance. After the phage lysed the Pseudomonas, the crRNA was released and attached to the fungal spores, and then invaded the patient's cells along with the spores or hyphae. nsp3 synthesized and assembled 4Fe-4S, iron-containing molecules bound to the cas4 domain, in the mitochondria of phagocytes. The iron came from hemoglobin attacked by the SARS-COV-2 virus protein. The nsp3 protein bound the crRNA in the phagocytic cytoplasm. It targeted the negative-strand RNA of SARS-COV-2, inserting conserved domain gene fragments into the negative-strand RNA through editing and splicing. Since the Cas protein had no codon checking function, the cutting and splicing would destroy the protein-coding information in the original RNA coding region, causing mutation and recombination of the SARS-COV-2 virus genome. If crRNA carried the drug resistance gene fragments of bacteria or phage, SARS-COV-2 would have similar drug resistance. Because of the growing problem of drug resistance in COVID-19 patients, we should pay attention to preventing fungi and bacteria co-infection. Avoid the CRISPR/Cas-like system of the novel coronavirus to cause rapid mutation and recombination and increased the drug resistance problem of SARS-COV-2.</p>

COVID-19: CRISPR/Cas-like System of nsp3 Promotes the Mutant Recombination and Drug Resistance

<p>Patients with novel coronavirus pneumonia usually suffer from bacterial and fungal infections, and the drug resistance problem caused by the pandemic is becoming more and more serious. Simultaneously, the SARS-COV-2 virus has a rapid mutation phenomenon, and somegene coding regions by mutation and recombination may be related to the drug resistance of the virus. Therefore, studying the relationship between the co-infection of bacteria and fungi and the evolution of SARS-COV-2 has important guiding significance for preventing a pandemic. We found that the SARS-COV-2 virus's nsp3 protein had a CRISPR/Cas 9 (II-B)-like function by searching for conserved domains. The system could target and edit the negative-strand RNA of SARS-COV-2. We speculated that the crRNA (CRISPR RNA) produced by the CRISPR/Cas system of Pseudomonas aeruginosa carried the genetic information of the conserved domains of bacteriophages and Pseudomonas, including drug resistance. After the phage lysed the Pseudomonas, the crRNA was released and attached to the fungal spores, and then invaded the patient's cells along with the spores or hyphae. nsp3 synthesized and assembled 4Fe-4S, iron-containing molecules bound to the cas4 domain, in the mitochondria of phagocytes. The iron came from hemoglobin attacked by the SARS-COV-2 virus protein. The nsp3 protein bound the crRNA in the phagocytic cytoplasm. It targeted the negative-strand RNA of SARS-COV-2, inserting conserved domain gene fragments into the negative-strand RNA through editing and splicing. Since the Cas protein had no codon checking function, the cutting and splicing would destroy the protein-coding information in the original RNA coding region, causing mutation and recombination of the SARS-COV-2 virus genome. If crRNA carried the drug resistance gene fragments of bacteria or phage, SARS-COV-2 would have similar drug resistance. Because of the growing problem of drug resistance in COVID-19 patients, we should pay attention to preventing fungi and bacteria co-infection. Avoid the CRISPR/Cas-like system of the novel coronavirus to cause rapid mutation and recombination and increased the drug resistance problem of SARS-COV-2.</p>