Deep mutational scanning reveals sequence to function constraints for SWEET family transporters.

Protein science is entering a transformative phase enabled by deep mutational scans that provide an unbiased view of the residue level interactions that mediate function. However, it has yet to be extensively used to characterize the mutational and evolutionary landscapes of plant proteins. Here, we apply the method to explore sequence-function relationships within the sugar transporter AtSWEET13. DMS results describe how mutational interrogation throughout different regions of the protein affects AtSWEET13 abundance and transport function. Our results identify novel transport-enhancing mutations that are validated using the FRET sensor assays. Extending DMS results to phylogenetic analyses reveal the role of transmembrane helix 4 (TM4) which makes the SWEET family transporters distinct from prokaryotic SemiSWEETs. We show that transmembrane helix 4 is intolerant to motif swapping with other clade-specific SWEET TM4 compositions, despite accommodating single point-mutations towards aromatic and charged polar amino acids. We further show that the transfer learning approaches based on physics and ML based In silico variant prediction tools have limited utility for engineering plant proteins as they were unable to reproduce our experimental results. We conclude that DMS can produce datasets which, when combined with the right predictive computational frameworks, can direct plant engineering efforts through derivative phenotype selection and evolutionary insights.

Sequence basis for selectivity of ephrin-B2 ligand for Eph receptors and pathogenic henipavirus G glycoproteins.

IMPORTANCE: Ephrin-B2 (EFNB2) is a ligand for six Eph receptors in humans and regulates multiple cell developmental and signaling processes. It also functions as the cell entry receptor for Nipah virus and Hendra virus, zoonotic viruses that can cause respiratory and/or neurological symptoms in humans with high mortality. Here, we investigate the sequence basis of EFNB2 specificity for binding the Nipah virus attachment G glycoprotein over Eph receptors. We then use this information to engineer EFNB2 as a soluble decoy receptor that specifically binds the attachment glycoproteins of the Nipah virus and other related henipaviruses to neutralize infection. These findings further mechanistic understanding of protein selectivity and may facilitate the development of diagnostics or therapeutics against henipavirus infection.

Identification of a 1.4-kb-Long Sequence Located in the nsp12 and nsp13 Coding Regions of SARS-CoV-2 Genomic RNA That Mediates Efficient Viral RNA Packaging.

The specific packaging of the viral RNA genome into virus particles is an essential step in the replication cycle of coronaviruses (CoVs). Using a single-cycle, replicable severe acute respiratory syndrome CoV-2 (SARS-CoV-2) mutant, we demonstrated the preferential packaging of the SARS-CoV-2 genomic RNA into purified virus particles. Furthermore, based on the sequence of an efficiently packaged defective interfering RNA of SARS-CoV, a closely related CoV, that was generated after serial passages of SARS-CoV in cell culture, we designed a series of replication-competent SARS-CoV-2 minigenome RNAs to identify the specific viral RNA region that is important for SARS-CoV-2 RNA packaging into virus particles. We showed that a 1.4-kb-long sequence, derived from the nsp12 and nsp13 coding regions of the SARS-CoV-2 genomic RNA, is required for the efficient packaging of SARS-CoV-2 minigenome RNA into SARS-CoV-2 particles. In addition, we also showed that the presence of possibly the entire 1.4-kb-long sequence is important for the efficient packaging of SARS-CoV-2 RNA. Our findings highlight the differences between the RNA packaging sequence identified in SARS-CoV-2, a Sarbecovirus, and the packaging signal of mouse hepatitis virus (MHV), an Embecovirus, which is a 95-nt-long sequence located at the nsp15 coding region of MHV genomic RNA. Collectively, our data imply that both the location and the sequence/structural features of the RNA element(s) that drives the selective and efficient packaging of viral genomic RNA are not conserved among the subgenera Embecovirus and Sarbecovirus within the Betacoronavirus genus. IMPORTANCE Elucidating the mechanism of SARS-CoV-2 RNA packaging into virus particles is important for the rational design of antiviral drugs that inhibit this vital step in the replication cycle of CoVs. However, our knowledge about the RNA packaging mechanism in SARS-CoV-2, including the identification of the viral RNA region important for SARS-CoV-2 RNA packaging, is limited, primarily due to the logistical challenges of handing SARS-CoV-2 in biosafety level 3 (BSL3) facilities. Our study, using a single-cycle, replicable SARS-CoV-2 mutant, which can be handled in a BSL2 lab, demonstrated the preferential packaging of full-length SARS-CoV-2 genomic RNA into virus particles and identified a specific 1.4-kb-long RNA region in SARS-CoV-2 genomic RNA that is required for the efficient packaging of SARS-CoV-2 RNA into virus particles. The information generated in our study could be valuable for clarifying the mechanisms of SARS-CoV-2 RNA packaging and for the development of targeted therapeutics against SARS-CoV-2 and other related CoVs.

The Sequence Basis for Selectivity of Ephrin-B2 Ligand for Eph Receptors and Pathogenic Henipavirus G Glycoproteins: Selective Ephrin-B2 Decoys for Nipah and Hendra Virus.

Ephrin-B2 (EFNB2) is a ligand for six Eph receptors in humans and functions as a cell entry receptor for several henipaviruses including Nipah virus (NiV), a pathogenic zoonotic virus with pandemic potential. To understand the sequence basis of promiscuity for EFNB2 binding to the attachment glycoprotein of NiV (NiV-G) and Eph receptors, we performed deep mutagenesis on EFNB2 to identify mutations that enhance binding to NiV-G over EphB2, one of the highest affinity Eph receptors. The mutations highlight how different EFNB2 conformations are selected by NiV-G versus EphB2. Specificity mutations are enriched at the base of the G-H binding loop of EFNB2, especially surrounding a phenylalanine hinge upon which the G-H loop pivots, and at a phenylalanine hook that rotates away from the EFNB2 core to engage Eph receptors. One EFNB2 mutant, D62Q, possesses pan-specificity to the attachment glycoproteins of closely related henipaviruses and has markedly diminished binding to the six Eph receptors. However, EFNB2-D62Q has high residual binding to EphB3 and EphB4. A second deep mutational scan of EFNB2 identified combinatorial mutations to further enhance specificity to NiV-G. A triple mutant of soluble EFNB2, D62Q-Q130L-V167L, has minimal binding to Eph receptors but maintains binding, albeit reduced, to NiV-G. Soluble EFNB2 decoy receptors carrying the specificity mutations were potent neutralizers of chimeric henipaviruses. These findings demonstrate how specific residue changes at the shared binding interface of a promiscuous ligand (EFNB2) can influence selectivity for multiple receptors, and may also offer insight towards the development of henipavirus therapeutics and diagnostics.

Mechanistic insight into the efficient packaging of antigenomic S RNA into Rift Valley fever virus particles.

Rift Valley fever virus (RVFV), a bunyavirus, has a single-stranded, negative-sense tri-segmented RNA genome, consisting of L, M and S RNAs. An infectious virion carries two envelope glycoproteins, Gn and Gc, along with ribonucleoprotein complexes composed of encapsidated viral RNA segments. The antigenomic S RNA, which serves as the template of the mRNA encoding a nonstructural protein, NSs, an interferon antagonist, is also efficiently packaged into RVFV particles. An interaction between Gn and viral ribonucleoprotein complexes, including the direct binding of Gn to viral RNAs, drives viral RNA packaging into RVFV particles. To understand the mechanism of efficient antigenomic S RNA packaging in RVFV, we identified the regions in viral RNAs that directly interact with Gn by performing UV-crosslinking and immunoprecipitation of RVFV-infected cell lysates with anti-Gn antibody followed by high-throughput sequencing analysis (CLIP-seq analysis). Our data suggested the presence of multiple Gn-binding sites in RVFV RNAs, including a prominent Gn-binding site within the 3' noncoding region of the antigenomic S RNA. We found that the efficient packaging of antigenomic S RNA was abrogated in a RVFV mutant lacking a part of this prominent Gn-binding site within the 3' noncoding region. Also, the mutant RVFV, but not the parental RVFV, triggered the early induction of interferon-ß mRNA expression after infection. These data suggest that the direct binding of Gn to the RNA element within the 3' noncoding region of the antigenomic S RNA promoted the efficient packaging of antigenomic S RNA into virions. Furthermore, the efficient packaging of antigenomic S RNA into RVFV particles, driven by the RNA element, facilitated the synthesis of viral mRNA encoding NSs immediately after infection, resulting in the suppression of interferon-ß mRNA expression.

An ACE2 decoy can be administered by inhalation and potently targets omicron variants of SARS-CoV-2.

Monoclonal antibodies targeting the SARS-CoV-2 spike (S) neutralize infection and are efficacious for the treatment of COVID-19. However, SARS-CoV-2 variants, notably sublineages of B.1.1.529/omicron, have emerged that escape antibodies in clinical use. As an alternative, soluble decoy receptors based on the host entry receptor ACE2 broadly bind and block S from SARS-CoV-2 variants and related betacoronaviruses. The high-affinity and catalytically active decoy sACE22 .v2.4-IgG1 was previously shown to be effective against SARS-CoV-2 variants when administered intravenously. Here, inhalation of aerosolized sACE22 .v2.4-IgG1 increased survival and ameliorated lung injury in K18-hACE2 mice inoculated with P.1/gamma virus. Loss of catalytic activity reduced the decoy's therapeutic efficacy, which was further confirmed by intravenous administration, supporting dual mechanisms of action: direct blocking of S and turnover of ACE2 substrates associated with lung injury and inflammation. Furthermore, sACE22 .v2.4-IgG1 tightly binds and neutralizes BA.1, BA.2, and BA.4/BA.5 omicron and protects K18-hACE2 mice inoculated with a high dose of BA.1 omicron virus. Overall, the therapeutic potential of sACE22 .v2.4-IgG1 is demonstrated by the inhalation route and broad neutralization potency persists against highly divergent SARS-CoV-2 variants.

Parsing the role of NSP1 in SARS-CoV-2 infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) leads to shutoff of protein synthesis, and nsp1, a central shutoff factor in coronaviruses, inhibits cellular mRNA translation. However, the diverse molecular mechanisms employed by nsp1 as well as its functional importance are unresolved. By overexpressing various nsp1 mutants and generating a SARS-CoV-2 mutant, we show that nsp1, through inhibition of translation and induction of mRNA degradation, targets translated cellular mRNA and is the main driver of host shutoff during infection. The propagation of nsp1 mutant virus is inhibited exclusively in cells with intact interferon (IFN) pathway as well as in vivo, in hamsters, and this attenuation is associated with stronger induction of type I IFN response. Therefore, although nsp1's shutoff activity is broad, it plays an essential role, specifically in counteracting the IFN response. Overall, our results reveal the multifaceted approach nsp1 uses to shut off cellular protein synthesis and uncover nsp1's explicit role in blocking the IFN response.

An engineered ACE2 decoy receptor can be administered by inhalation and potently targets the BA.1 and BA.2 omicron variants of SARS-CoV-2.

Monoclonal antibodies targeting the SARS-CoV-2 spike (S) glycoprotein neutralize infection and are efficacious for the treatment of mild-to-moderate COVID-19. However, SARS-CoV-2 variants have emerged that partially or fully escape monoclonal antibodies in clinical use. Notably, the BA.2 sublineage of B.1.1.529/omicron escapes nearly all monoclonal antibodies currently authorized for therapeutic treatment of COVID-19. Decoy receptors, which are based on soluble forms of the host entry receptor ACE2, are an alternative strategy that broadly bind and block S from SARS-CoV-2 variants and related betacoronaviruses. The high-affinity and catalytically active decoy sACE2 2 .v2.4-IgG1 was previously shown to be effective in vivo against SARS-CoV-2 variants when administered intravenously. Here, the inhalation of sACE2 2 .v2.4-IgG1 is found to increase survival and ameliorate lung injury in K18-hACE2 transgenic mice inoculated with a lethal dose of the virulent P.1/gamma virus. Loss of catalytic activity reduced the decoy’s therapeutic efficacy supporting dual mechanisms of action: direct blocking of viral S and turnover of ACE2 substrates associated with lung injury and inflammation. Binding of sACE2 2 .v2.4-IgG1 remained tight to S of BA.1 omicron, despite BA.1 omicron having extensive mutations, and binding exceeded that of four monoclonal antibodies approved for clinical use. BA.1 pseudovirus and authentic virus were neutralized at picomolar concentrations. Finally, tight binding was maintained against S from the BA.2 omicron sublineage, which differs from S of BA.1 by 26 mutations. Overall, the therapeutic potential of sACE2 2 .v2.4-IgG1 is further confirmed by inhalation route and broad neutralization potency persists against increasingly divergent SARS-CoV-2 variants.

Parsing the role of NSP1 in SARS-CoV-2 infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the ongoing coronavirus disease 19 (COVID-19) pandemic. Despite its urgency, we still do not fully understand the molecular basis of SARS-CoV-2 pathogenesis and its ability to antagonize innate immune responses. SARS-CoV-2 leads to shutoff of cellular protein synthesis and over-expression of nsp1, a central shutoff factor in coronaviruses, inhibits cellular gene translation. However, the diverse molecular mechanisms nsp1 employs as well as its functional importance in infection are still unresolved. By overexpressing various nsp1 mutants and generating a SARS-CoV-2 mutant in which nsp1 does not bind ribosomes, we untangle the effects of nsp1. We uncover that nsp1, through inhibition of translation and induction of mRNA degradation, is the main driver of host shutoff during SARS-CoV-2 infection. Furthermore, we find the propagation of nsp1 mutant virus is inhibited specifically in cells with intact interferon (IFN) response as well as in-vivo , in infected hamsters, and this attenuation is associated with stronger induction of type I IFN response. This illustrates that nsp1 shutoff activity has an essential role mainly in counteracting the IFN response. Overall, our results reveal the multifaceted approach nsp1 uses to shut off cellular protein synthesis and uncover the central role it plays in SARS-CoV-2 pathogenesis, explicitly through blockage of the IFN response.

An Enhanced Decoding Algorithm for Coded Compressed Sensing with Applications to Unsourced Random Access.

Unsourced random access (URA) has emerged as a pragmatic framework for next-generation distributed sensor networks. Within URA, concatenated coding structures are often employed to ensure that the central base station can accurately recover the set of sent codewords during a given transmission period. Many URA algorithms employ independent inner and outer decoders, which can help reduce computational complexity at the expense of a decay in performance. In this article, an enhanced decoding algorithm is presented for a concatenated coding structure consisting of a wide range of inner codes and an outer tree-based code. It is shown that this algorithmic enhancement has the potential to simultaneously improve error performance and decrease the computational complexity of the decoder. This enhanced decoding algorithm is applied to two existing URA algorithms, and the performance benefits of the algorithm are characterized. Findings are supported by numerical simulations.

Characterization of the Molecular Interactions That Govern the Packaging of Viral RNA Segments into Rift Valley Fever Phlebovirus Particles.

Rift Valley fever phlebovirus (RVFV) has a single-stranded, negative-sense RNA genome, consisting of L, M, and S segments. The virion carries two envelope glycoproteins, Gn and Gc, along with ribonucleoprotein complexes (RNPs), composed of encapsidated genomes carrying N protein and the viral polymerase, L protein. A quantitative analysis of the profile of viral RNA segments packaged into RVFV particles showed that all three genomic RNA segments had similar packaging abilities, whereas among antigenomic RNA segments, the antigenomic S RNA, which serves as the template for the transcription of mRNA expressing the RVFV virulence factor, NSs, displayed a significantly higher packaging ability. To delineate the factor(s) governing the packaging of RVFV RNA segments, we characterized the interactions between Gn and viral RNPs in RVFV-infected cells. Coimmunoprecipitation analysis demonstrated the interaction of Gn with N protein, L protein, and viral RNAs in RVFV-infected cells. Furthermore, UV-cross-linking and immunoprecipitation analysis revealed, for the first time in bunyaviruses, the presence of a direct interaction between Gn and all the viral RNA segments in RVFV-infected cells. Notably, analysis of the ability of Gn to bind to RVFV RNA segments indicated a positive correlation with their respective packaging abilities and highlighted a binding preference of Gn for antigenomic S RNA, among the antigenomic RNA segments, suggesting the presence of a selection mechanism for antigenomic S RNA incorporation into infectious RVFV particles. Collectively, the results of our study illuminate the importance of a direct interaction between Gn and viral RNA segments in determining their efficiency of incorporation into RVFV particles. IMPORTANCE Rift Valley fever phlebovirus, a bunyavirus, is a mosquito-borne, segmented RNA virus that can cause severe disease in humans and ruminants. An essential step in RVFV life cycle is the packaging of viral RNA segments to produce infectious virus particles for dissemination to new hosts. However, there are key gaps in knowledge regarding the mechanisms that regulate viral RNA packaging efficiency in bunyaviruses. Our studies investigating the mechanism of RNA packaging in RVFV revealed the presence of a direct interaction between the viral envelope glycoprotein, Gn, and the viral RNA segments in infected cells, for the first time in bunyaviruses. Furthermore, our data strongly indicate a critical role for the direct interaction between Gn and viral RNAs in determining the efficiency of incorporation of viral RNA segments into RVFV particles. Clarifying the fundamental mechanisms of RNA packaging in RVFV would be valuable for the development of antivirals and live-attenuated vaccines.

Deep Mutational Scanning of Viral Glycoproteins and Their Host Receptors.

Deep mutational scanning or deep mutagenesis is a powerful tool for understanding the sequence diversity available to viruses for adaptation in a laboratory setting. It generally involves tracking an in vitro selection of protein sequence variants with deep sequencing to map mutational effects based on changes in sequence abundance. Coupled with any of a number of selection strategies, deep mutagenesis can explore the mutational diversity available to viral glycoproteins, which mediate critical roles in cell entry and are exposed to the humoral arm of the host immune response. Mutational landscapes of viral glycoproteins for host cell attachment and membrane fusion reveal extensive epistasis and potential escape mutations to neutralizing antibodies or other therapeutics, as well as aiding in the design of optimized immunogens for eliciting broadly protective immunity. While less explored, deep mutational scans of host receptors further assist in understanding virus-host protein interactions. Critical residues on the host receptors for engaging with viral spikes are readily identified and may help with structural modeling. Furthermore, mutations may be found for engineering soluble decoy receptors as neutralizing agents that specifically bind viral targets with tight affinity and limited potential for viral escape. By untangling the complexities of how sequence contributes to viral glycoprotein and host receptor interactions, deep mutational scanning is impacting ideas and strategies at multiple levels for combatting circulating and emergent virus strains.

An engineered decoy receptor for SARS-CoV-2 broadly binds protein S sequence variants.

The spike S of SARS-CoV-2 recognizes ACE2 on the host cell membrane to initiate entry. Soluble decoy receptors, in which the ACE2 ectodomain is engineered to block S with high affinity, potently neutralize infection and, because of close similarity with the natural receptor, hold out the promise of being broadly active against virus variants without opportunity for escape. Here, we directly test this hypothesis. We find that an engineered decoy receptor, sACE22v2.4, tightly binds S of SARS-associated viruses from humans and bats, despite the ACE2-binding surface being a region of high diversity. Saturation mutagenesis of the receptor-binding domain followed by in vitro selection, with wild-type ACE2 and the engineered decoy competing for binding sites, failed to find S mutants that discriminate in favor of the wild-type receptor. We conclude that resistance to engineered decoys will be rare and that decoys may be active against future outbreaks of SARS-associated betacoronaviruses.

An Affordable Neurosurgical Training System for Neurosurgical Residents; The Indian Perspective.

CONTEXT: Neurosurgical training in India. AIMS: To establish a sustainable, functional, and relatively inexpensive neurosurgical training system. METHODS AND MATERIALS: The training system involved using a relatively inexpensive stereoscopic microscope and ophthalmological microinstruments , including two jewellers' forceps and a microscissors. Training modules were designed using commonly available materials such as gauze, latex gloves, and chicken wings. STATISTICAL ANALYSIS USED: None. RESULTS: The system proved to be easy to construct, easy-to-use, and highly compact. This system allowed for residents to practice microsuturing and vessel anastomosis and allows the resident to be familiarized with operating under the microscope. Thereby , developing the hand-eye coordination and precise motor control required for neurosurgery as well as the art of appropriate tissue handling. CONCLUSIONS: The inexpensive nature of this training system and its inherent simplicity allows for a larger portion of neurosurgical residents in India, without access to sophisticated skills labs, to practice their skills in a controlled environment, without compromising on their ward duties.

Isolation and characterization of SARS-CoV-2 from the first US COVID-19 patient.

The etiologic agent of the outbreak of pneumonia in Wuhan China was identified as severe acute respiratory syndrome associated coronavirus 2 (SARS-CoV-2) in January, 2020. The first US patient was diagnosed by the State of Washington and the US Centers for Disease Control and Prevention on January 20, 2020. We isolated virus from nasopharyngeal and oropharyngeal specimens, and characterized the viral sequence, replication properties, and cell culture tropism. We found that the virus replicates to high titer in Vero-CCL81 cells and Vero E6 cells in the absence of trypsin. We also deposited the virus into two virus repositories, making it broadly available to the public health and research communities. We hope that open access to this important reagent will expedite development of medical countermeasures.

An Infectious cDNA Clone of SARS-CoV-2.

The ongoing pandemic of COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), underscores the urgency to develop experimental systems for studying this virus and identifying countermeasures. We report a reverse genetic system for SARS-CoV-2. Seven complimentary DNA (cDNA) fragments spanning the SARS-CoV-2 genome were assembled into a full-genome cDNA. RNA transcribed from the full-genome cDNA was highly infectious after electroporation into cells, producing 2.9 × 106 plaque-forming unit (PFU)/mL of virus. Compared with a clinical isolate, the infectious-clone-derived SARS-CoV-2 (icSARS-CoV-2) exhibited similar plaque morphology, viral RNA profile, and replication kinetics. Additionally, icSARS-CoV-2 retained engineered molecular markers and did not acquire other mutations. We generated a stable mNeonGreen SARS-CoV-2 (icSARS-CoV-2-mNG) by introducing this reporter gene into ORF7 of the viral genome. icSARS-CoV-2-mNG was successfully used to evaluate the antiviral activities of interferon (IFN). Collectively, the reverse genetic system and reporter virus provide key reagents to study SARS-CoV-2 and develop countermeasures.

Severe Acute Respiratory Syndrome Coronavirus 2 from Patient with Coronavirus Disease, United States.

The etiologic agent of an outbreak of pneumonia in Wuhan, China, was identified as severe acute respiratory syndrome coronavirus 2 in January 2020. A patient in the United States was given a diagnosis of infection with this virus by the state of Washington and the US Centers for Disease Control and Prevention on January 20, 2020. We isolated virus from nasopharyngeal and oropharyngeal specimens from this patient and characterized the viral sequence, replication properties, and cell culture tropism. We found that the virus replicates to high titer in Vero-CCL81 cells and Vero E6 cells in the absence of trypsin. We also deposited the virus into 2 virus repositories, making it broadly available to the public health and research communities. We hope that open access to this reagent will expedite development of medical countermeasures.

An engineered decoy receptor for SARS-CoV-2 broadly binds protein S sequence variants.

The spike S of SARS-CoV-2 recognizes ACE2 on the host cell membrane to initiate entry. Soluble decoy receptors, in which the ACE2 ectodomain is engineered to block S with high affinity, potently neutralize infection and, due to close similarity with the natural receptor, hold out the promise of being broadly active against virus variants without opportunity for escape. Here, we directly test this hypothesis. We find an engineered decoy receptor, sACE2 2 .v2.4, tightly binds S of SARS-associated viruses from humans and bats, despite the ACE2-binding surface being a region of high diversity. Saturation mutagenesis of the receptor-binding domain (RBD) followed by in vitro selection, with wild type ACE2 and the engineered decoy competing for binding sites, failed to find S mutants that discriminate in favor of the wild type receptor. Variant N501Y in the RBD, which has emerged in a rapidly spreading lineage (B.1.1.7) in England, enhances affinity for wild type ACE2 20-fold but remains tightly bound to engineered sACE22.v2.4. We conclude that resistance to engineered decoys will be rare and that decoys may be active against future outbreaks of SARS-associated betacoronaviruses.

A strand-specific real-time quantitative RT-PCR assay for distinguishing the genomic and antigenomic RNAs of Rift Valley fever phlebovirus.

Rift Valley Fever phlebovirus (RVFV), genus Phlebovirus, family Phenuiviridae, order Bunyavirales, has a single-stranded, negative-sense RNA genome, consisting of L, M and S segments. Here, we report the establishment of a strand-specific, quantitative reverse transcription (RT)-PCR assay system that can selectively distinguish between the genomic and antigenomic RNAs of each of the three viral RNA segments produced in RVFV-infected cells. To circumvent the obstacle of primer-independent cDNA synthesis during RT, we used a tagged, strand-specific RT primer, carrying a non-viral 'tag' sequence at the 5' end, which ensured the strand-specificity through the selective amplification of only the tagged cDNA in the real-time PCR assay. We used this assay system to examine the kinetics of intracellular accumulation of genomic and antigenomic viral RNAs in mammalian cells infected with the MP-12 strain of RVFV. The genomic RNA copy numbers, for all three viral RNA segments, were higher than that of their corresponding antigenomic RNAs throughout the time-course of infection, with a notable exception, wherein the M segment genomic and antigenomic RNAs exhibited similar copy numbers at specific times post-infection. Overall, this assay system could be a useful tool to gain an insight into the mechanisms of RNA replication and packaging in RVFV.

Interplay between coronavirus, a cytoplasmic RNA virus, and nonsense-mediated mRNA decay pathway.

Coronaviruses (CoVs), including severe acute respiratory syndrome CoV and Middle East respiratory syndrome CoV, are enveloped RNA viruses that carry a large positive-sense single-stranded RNA genome and cause a variety of diseases in humans and domestic animals. Very little is known about the host pathways that regulate the stability of CoV mRNAs, which carry some unusual features. Nonsense-mediated decay (NMD) is a eukaryotic RNA surveillance pathway that detects mRNAs harboring aberrant features and targets them for degradation. Although CoV mRNAs are of cytoplasmic origin, the presence of several NMD-inducing features (including multiple ORFs with internal termination codons that create a long 3' untranslated region) in CoV mRNAs led us to explore the interplay between the NMD pathway and CoVs. Our study using murine hepatitis virus as a model CoV showed that CoV mRNAs are recognized by the NMD pathway as a substrate, resulting in their degradation. Furthermore, CoV replication induced the inhibition of the NMD pathway, and N protein (a viral structural protein) had an NMD inhibitory function that protected viral mRNAs from rapid decay. Our data further suggest that the NMD pathway interferes with optimal viral replication by degrading viral mRNAs early in infection, before sufficient accumulation of N protein. Our study presents clear evidence for the biological importance of the NMD pathway in controlling the stability of mRNAs and the efficiency of replication of a cytoplasmic RNA virus.