GTPase activity of porcine Mx1 plays a dominant role in inhibiting the N-Nsp9 interaction and thus inhibiting PRRSV replication.

Porcine Mx1 is a type of interferon-induced GTPase that inhibits the replication of certain RNA viruses. However, the antiviral effects and the underlying mechanism of porcine Mx1 for porcine reproductive and respiratory syndrome virus (PRRSV) remain unknown. In this study, we demonstrated that porcine Mx1 could significantly inhibit PRRSV replication in MARC-145 cells. By Mx1 segment analysis, it was indicated that the GTPase domain (68-341aa) was the functional area to inhibit PRRSV replication and that Mx1 interacted with the PRRSV-N protein through the GTPase domain (68-341aa) in the cytoplasm. Amino acid residues K295 and K299 in the G domain of Mx1 were the key sites for Mx1-N interaction while mutant proteins Mx1(K295A) and Mx1(K299A) still partially inhibited PRRSV replication. Furthermore, we found that the GTPase activity of Mx1 was dominant for Mx1 to inhibit PRRSV replication but was not essential for Mx1-N interaction. Finally, mechanistic studies demonstrated that the GTPase activity of Mx1 played a dominant role in inhibiting the N-Nsp9 interaction and that the interaction between Mx1 and N partially inhibited the N-Nsp9 interaction. We propose that the complete anti-PRRSV mechanism of porcine Mx1 contains a two-step process: Mx1 binds to the PRRSV-N protein and subsequently disrupts the N-Nsp9 interaction by a process requiring the GTPase activity of Mx1. Taken together, the results of our experiments describe for the first time a novel mechanism by which porcine Mx1 evolves to inhibit PRRSV replication. IMPORTANCE: Mx1 protein is a key mediator of the interferon-induced antiviral response against a wide range of viruses. How porcine Mx1 affects the replication of porcine reproductive and respiratory syndrome virus (PRRSV) and its biological function has not been studied. Here, we show that Mx1 protein inhibits PRRSV replication by interfering with N-Nsp9 interaction. Furthermore, the GTPase activity of porcine Mx1 plays a dominant role and the Mx1-N interaction plays an assistant role in this interference process. This study uncovers a novel mechanism evolved by porcine Mx1 to exert anti-PRRSV activities.

Oligomeric assembly of the C-terminal and transmembrane region of SARS-CoV-2 nsp3.

As for all single-stranded, positive-sense RNA (+RNA) viruses, intracellular RNA synthesis relies on extensive remodeling of host cell membranes that leads to the formation of specialized structures. In the case of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) coronavirus causing COVID-19, endoplasmic reticulum membranes are modified, resulting in the formation of double-membrane vesicles (DMVs), which contain the viral dsRNA intermediate and constitute membrane-bound replication organelles. The non-structural and transmembrane protein nsp3 is a key player in the biogenesis of DMVs and, therefore, represents an interesting antiviral target. However, as an integral transmembrane protein, it is challenging to express for structural biology. The C-terminus of nsp3 encompasses all the membrane-spanning, -interacting, and -remodeling elements. By using a cell-free expression system, we successfully produced the C-terminal region of nsp3 (nsp3C) and reconstituted purified nsp3C into phospholipid nanodiscs, opening the way for structural studies. Negative-stain transmission electron microscopy revealed the presence of nsp3C oligomers very similar to the region abutting and spanning the membrane on the cytosolic side of DMVs in a recent subtomogram average of the SARS-CoV-2 nsp3-4 pore (1). AlphaFold-predicted structural models fit particularly well with our experimental data and support a pore-forming hexameric assembly. Altogether, our data give unprecedented clues to understand the structural organization of nsp3, the principal component that shapes the molecular pore that spans the DMVs and is required for the export of RNA in vivo. IMPORTANCE: Membrane remodeling is at the heart of intracellular replication for single-stranded, positive-sense RNA viruses. In the case of coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), this leads to the formation of a network of double-membrane vesicles (DMVs). Targeting DMV biogenesis offers promising prospects for antiviral therapies. This requires a better understanding of the molecular mechanisms and proteins involved. Three non-structural proteins (nsp3, nsp4, and nsp6) direct the intracellular membrane rearrangements upon SARS-CoV-2 infection. All of them contain transmembrane helices. The nsp3 component, the largest and multi-functional protein of the virus, plays an essential role in this process. Aiming to understand its structural organization, we used a cell-free protein synthesis assay to produce and reconstitute the C-terminal part of nsp3 (nsp3C) including transmembrane domains into phospholipid nanodiscs. Our work reveals the oligomeric organization of one key player in the biogenesis of SARS-CoV-2 DMVs, providing basis for the design of future antiviral strategies.

Chikungunya virus infection disrupts MHC-I antigen presentation via nonstructural protein 2.

Infection by chikungunya virus (CHIKV), a mosquito-borne alphavirus, causes severe polyarthralgia and polymyalgia, which can last in some people for months to years. Chronic CHIKV disease signs and symptoms are associated with the persistence of viral nucleic acid and antigen in tissues. Like humans and nonhuman primates, CHIKV infection in mice results in the development of robust adaptive antiviral immune responses. Despite this, joint tissue fibroblasts survive CHIKV infection and can support persistent viral replication, suggesting that they escape immune surveillance. Here, using a recombinant CHIKV strain encoding the fluorescent protein VENUS with an embedded CD8+ T cell epitope, SIINFEKL, we observed a marked loss of both MHC class I (MHC-I) surface expression and antigen presentation by CHIKV-infected joint tissue fibroblasts. Both in vivo and ex vivo infected joint tissue fibroblasts displayed reduced cell surface levels of H2-Kb and H2-Db MHC-I proteins while maintaining similar levels of other cell surface proteins. Mutations within the methyl transferase-like domain of the CHIKV nonstructural protein 2 (nsP2) increased MHC-I cell surface expression and antigen presentation efficiency by CHIKV-infected cells. Moreover, expression of WT nsP2 alone, but not nsP2 with mutations in the methyltransferase-like domain, resulted in decreased MHC-I antigen presentation efficiency. MHC-I surface expression and antigen presentation was rescued by replacing VENUS-SIINFEKL with SIINFEKL tethered to ß2-microglobulin in the CHIKV genome, which bypasses the requirement for peptide processing and TAP-mediated peptide transport into the endoplasmic reticulum. Collectively, this work suggests that CHIKV escapes the surveillance of antiviral CD8+ T cells, in part, by nsP2-mediated disruption of MHC-I antigen presentation.

NS1-mediated enhancement of MVC transcription and replication promoted by KAT5/H4K12ac.

Histone modifications function in both cellular and viral gene expression. However, the roles of acetyltransferases and histone acetylation in parvoviral infection remain poorly understood. In the current study, we found the histone deacetylase (HDAC) inhibitor, trichostatin A (TSA), promoted the replication and transcription of parvovirus minute virus of canines (MVC). Notably, the expression of host acetyltransferases KAT5, GTF3C4, and KAT2A was increased in MVC infection, as well as H4 acetylation (H4K12ac). KAT5 is not only responsible for H4K12ac but also crucial for viral replication and transcription. The viral nonstructural protein NS1 interacted with KAT5 and enhanced its expression. Further study showed that Y44 in KAT5, which may be tyrosine-phosphorylated, is indispensable for NS1-mediated enhancement of KAT5 and efficient MVC replication. The data demonstrated that NS1 interacted with KAT5, which resulted in an enhanced H4K12ac level to promote viral replication and transcription, implying the epigenetic addition of H4K12ac in viral chromatin-like structure by KAT5 is vital for MVC replication.IMPORTANCEParvoviral genomes are chromatinized with host histones. Therefore, histone acetylation and related acetyltransferases are required for the virus to modify histones and open densely packed chromatin structures. This study illustrated that histone acetylation status is important for MVC replication and transcription and revealed a novel mechanism that the viral nonstructural protein NS1 hijacks the host acetyltransferase KAT5 to enhance histone acetylation of H4K12ac, which relies on a potential tyrosine phosphorylation site, Y44 in KAT5. Other parvoviruses share a similar genome organization and coding potential and may adapt a similar strategy for efficient viral replication and transcription.

Ubiquitin-specific proteinase 1 stabilizes PRRSV nonstructural protein Nsp1ß to promote viral replication by regulating K48 ubiquitination.

The porcine reproductive and respiratory syndrome virus (PRRSV) can lead to severe reproductive problems in sows, pneumonia in weaned piglets, and increased mortality, significantly negatively impacting the economy. Post-translational changes are essential for the host-dependent replication and long-term infection of PRRSV. Uncertainty surrounds the function of the ubiquitin network in PRRSV infection. Here, we screened 10 deubiquitinating enzyme inhibitors and found that the ubiquitin-specific proteinase 1 (USP1) inhibitor ML323 significantly inhibited PRRSV replication in vitro. Importantly, we found that USP1 interacts with nonstructural protein 1ß (Nsp1ß) and deubiquitinates its K48 to increase protein stability, thereby improving PRRSV replication and viral titer. Among them, lysine at position 45 is essential for Nsp1ß protein stability. In addition, deficiency of USP1 significantly reduced viral replication. Moreover, ML323 loses antagonism to PRRSV rSD16-K45R. This study reveals the mechanism by which PRRSV recruits the host factor USP1 to promote viral replication, providing a new target for PRRSV defense.IMPORTANCEDeubiquitinating enzymes are critical factors in regulating host innate immunity. The porcine reproductive and respiratory syndrome virus (PRRSV) nonstructural protein 1ß (Nsp1ß) is essential for producing viral subgenomic mRNA and controlling the host immune system. The host inhibits PRRSV proliferation by ubiquitinating Nsp1ß, and conversely, PRRSV recruits the host protein ubiquitin-specific proteinase 1 (USP1) to remove this restriction. Our results demonstrate the binding of USP1 to Nsp1ß, revealing a balance of antagonism between PRRSV and the host. Our research identifies a brand-new PRRSV escape mechanism from the immune response.

Kinetic characterization of human mRNA guanine-N7 methyltransferase.

The 5'-mRNA-cap formation is a conserved process in protection of mRNA in eukaryotic cells, resulting in mRNA stability and efficient translation. In humans, two methyltransferases, RNA cap guanine-N7 methyltransferase (hRNMT) and cap-specific nucleoside-2'-O-methyltransferase 1 (hCMTr1) methylate the mRNA resulting in cap0 (N7mGpppN-RNA) and cap1 (N7mGpppN2'-Om-RNA) formation, respectively. Coronaviruses mimic this process by capping their RNA to evade human immune systems. The coronaviral nonstructural proteins, nsp14 and nsp10-nsp16, catalyze the same reactions as hRNMT and hCMTr1, respectively. These two viral enzymes are important targets for development of inhibitor-based antiviral therapeutics. However, assessing the selectivity of such inhibitors against human corresponding proteins is crucial. Human RNMTs have been implicated in proliferation of cancer cells and are also potential targets for development of anticancer therapeutics. Here, we report the development and optimization of a radiometric assay for hRNMT, full kinetic characterization of its activity, and optimization of the assay for high-throughput screening with a Z-factor of 0.79. This enables selectivity determination for a large number of hits from various screening of coronaviral methyltransferases, and also screening hRNMT for discovery of inhibitors and chemical probes that potentially could be used to further investigate the roles RNMTs play in cancers.

[Advances in the coronavirus nonstructural protein 13].

Coronaviruses pose significant threats to animal and human health, leading to the development of various infectious diseases. It is critical to develop effective vaccines and antiviral medicines to prevent and treat these diseases. The coronavirus genome encodes several types of proteins, including structural, nonstructural, and accessory proteins. Among them, nonstructural protein 13 (NSP13) helicase plays a crucial role in regulating viral replication and the innate immune response of the host. Therefore, it serves as a vital target for the development of anti-coronavirus drugs. This paper presents a comprehensive review of NSP13 research, covering its source, structure, sequence conservation, unwinding mechanism, enzyme inhibitors, protein interaction, and immune regulation. Additionally, the paper analyzes the current challenges in NSP13 research and aims to provide a theoretical foundation for the development of broad-spectrum antiviral drugs targeting NSP13.

Landscape of protein-protein interactions during hepatitis C virus assembly and release.

Assembly of infectious hepatitis C virus (HCV) particles requires multiple cellular proteins including for instance apolipoprotein E (ApoE). To describe these protein-protein interactions, we performed an affinity purification mass spectrometry screen of HCV-infected cells. We used functional viral constructs with epitope-tagged envelope protein 2 (E2), protein (p) 7, or nonstructural protein 4B (NS4B) as well as cells expressing a tagged variant of ApoE. We also evaluated assembly stage-dependent remodeling of protein complexes by using viral mutants carrying point mutations abrogating particle production at distinct steps of the HCV particle production cascade. Five ApoE binding proteins, 12 p7 binders, 7 primary E2 interactors, and 24 proteins interacting with NS4B were detected. Cell-derived PREB, STT3B, and SPCS2 as well as viral NS2 interacted with both p7 and E2. Only GTF3C3 interacted with E2 and NS4B, highlighting that HCV assembly and replication complexes exhibit largely distinct interactomes. An HCV core protein mutation, preventing core protein decoration of lipid droplets, profoundly altered the E2 interactome. In cells replicating this mutant, E2 interactions with HSPA5, STT3A/B, RAD23A/B, and ZNF860 were significantly enhanced, suggesting that E2 protein interactions partly depend on core protein functions. Bioinformatic and functional studies including STRING network analyses, RNA interference, and ectopic expression support a role of Rad23A and Rad23B in facilitating HCV infectious virus production. Both Rad23A and Rad23B are involved in the endoplasmic reticulum (ER)-associated protein degradation (ERAD). Collectively, our results provide a map of host proteins interacting with HCV assembly proteins, and they give evidence for the involvement of ER protein folding machineries and the ERAD pathway in the late stages of the HCV replication cycle.IMPORTANCEHepatitis C virus (HCV) establishes chronic infections in the majority of exposed individuals. This capacity likely depends on viral immune evasion strategies. One feature likely contributing to persistence is the formation of so-called lipo-viro particles. These peculiar virions consist of viral structural proteins and cellular lipids and lipoproteins, the latter of which aid in viral attachment and cell entry and likely antibody escape. To learn about how lipo-viro particles are coined, here, we provide a comprehensive overview of protein-protein interactions in virus-producing cells. We identify numerous novel and specific HCV E2, p7, and cellular apolipoprotein E-interacting proteins. Pathway analyses of these interactors show that proteins participating in processes such as endoplasmic reticulum (ER) protein folding, ER-associated protein degradation, and glycosylation are heavily engaged in virus production. Moreover, we find that the proteome of HCV replication sites is distinct from the assembly proteome, suggesting that transport process likely shuttles viral RNA to assembly sites.

ZNF283, a Krüppel-associated box zinc finger protein, inhibits RNA synthesis of porcine reproductive and respiratory syndrome virus by interacting with Nsp9 and Nsp10.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a viral pathogen with substantial economic implications for the global swine industry. The existing vaccination strategies and antiviral drugs offer limited protection. Replication of the viral RNA genome encompasses a complex series of steps, wherein a replication complex is assembled from various components derived from both viral and cellular sources, as well as from the viral genomic RNA template. In this study, we found that ZNF283, a Krüppel-associated box (KRAB) containing zinc finger protein, was upregulated in PRRSV-infected Marc-145 cells and porcine alveolar macrophages and that ZNF283 inhibited PRRSV replication and RNA synthesis. We also found that ZNF283 interacts with the viral proteins Nsp9, an RNA-dependent RNA polymerase, and Nsp10, a helicase. The main regions involved in the interaction between ZNF283 and Nsp9 were determined to be the KRAB domain of ZNF283 and amino acids 178-449 of Nsp9. The KRAB domain of ZNF283 plays a role in facilitating Nsp10 binding. In addition, ZNF283 may have an affinity for the 3' untranslated region of PRRSV. These findings suggest that ZNF283 is an antiviral factor that inhibits PRRSV infection and extend our understanding of the interactions between KRAB-containing zinc finger proteins and viruses.

Zika virus restriction of host antioxidant response is mediated by intracellular NS1 and reveals its ability to upregulate Bach1 expression.

Zika virus (ZIKV), has gained global attention due to its association with severe disorders, including microcephaly and congenital Zika syndrome. We investigated the role of ZIKV nonstructural protein 1 (NS1) in altering the host's antioxidant response. Using a stable cell line expressing NS1, we found that NS1 significantly reduced the expression of antioxidant-related genes, including heme oxygenase 1 (HO-1), NAD(P)H quinone dehydrogenase 1 (NQO1), and sequestosome-1 (SQSTM1), which are regulated NRF2. Interestingly, this effect was attributed to increased expression of BACH1, a factor that competes with NRF2 for binding to certain antioxidant responsive elements (ARE). Thus, ZIKV NS1-mediated disruption of the antioxidant system is linked to BACH1 overexpression. These findings offer insights into ZIKV pathogenesis and suggest potential therapeutic strategies targeting the NRF2-BACH1 axis.

A structural analysis of the nsp9 protein from the coronavirus MERS CoV reveals a conserved RNA binding interface.

Middle East respiratory syndrome coronavirus (MERS CoV) and severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) are RNA viruses from the Betacoronavirus family that cause serious respiratory illness in humans. One of the conserved non-structural proteins encoded for by the coronavirus family is non-structural protein 9 (nsp9). Nsp9 plays an important role in the RNA capping process of the viral genome, where it is covalently linked to viral RNA (known as RNAylation) by the conserved viral polymerase, nsp12. Nsp9 also directly binds to RNA; we have recently revealed a distinct RNA recognition interface in the SARS CoV-2 nsp9 by using a combination of nuclear magnetic resonance spectroscopy and biolayer interferometry. In this study, we have utilized a similar methodology to determine a structural model of RNA binding of the related MERS CoV. Based on these data, we uncover important similarities and differences to SARS CoV-2 nsp9 and other coronavirus nsp9 proteins. Our findings that replacing key RNA binding residues in MERS CoV nsp9 affects RNAylation efficiency indicate that recognition of RNA may play a role in the capping process of the virus.

Differential specificity of SARS-CoV-2 main protease variants on peptide versus protein-based substrates.

The SARS-CoV-2 main protease (Mpro ) holds significant importance as a biological target in combating coronaviruses due to its importance in virus replication. Considering the emergence of novel SARS-CoV-2 variants and the mutations observed in the Mpro sequence, we hypothesized that these mutations may have a potential impact on the protease's specificity. To test this, we expressed Mpro corresponding to the original strain and variants Beta1, Beta2, and Omicron and analyzed their activity on protein-based and peptide substrates. Although we observed differential activity on the protein-based substrate, there was very little difference when analyzed on the peptide substrate. We conclude that mutations on the Mpro sequence, despite having a minor effect on a peptide substrate cleavage, did not change the catalytic site environment enough to build resistance to inhibition. Therefore, we propose that inhibitors initially designed for the Mpro of the original strain will be effective in all the variants. Thus, Mpro is likely to continue to be a target of therapeutic interest as mutations in its sequence are rare and, as we show here, have a minor effect on the protease's recognition of peptide-based molecules.

Ser235 phosphorylation of hepatitis C virus NS5A is required for NS5A dimerization and drug resistance.

Hepatitis C virus (HCV) infection is recognized as a major causative agent of chronic hepatitis, cirrhosis, and hepatocellular carcinoma. HCV non-structural protein 5A (NS5A) is a dimeric phosphoprotein with a hyperphosphorylated form to act as a switch that regulates HCV replication and assembly. NS5A inhibitors have been utilized as the scaffold for combination therapy of direct-acting antiviral agents (DAA). However, the mode of action of NS5A inhibitors is still unclear due to the lack of mechanistic detail regarding NS5A phosphorylation and dimerization in the HCV life cycle. It has been demonstrated that phosphorylation of NS5A at Ser235 is essential for RNA replication of the JFH1 strain. In this report, we found that NS5A phosphomimetic Ser235 substitution (Ser-to-Asp mutation) formed a dimer that was resistant to disruption by NS5A inhibitors as was the NS5A resistance-associated substitution Y93H. Phosphorylation of NS5A at Ser235 residue was required for the interaction of two NS5A-WT molecules in JFH1-based cell culture system but not absolutely required for dimerization of the NS5A-Y93H mutant. Interestingly, HCV nonstructural proteins from the subgenomic replicon NS3-5A was required for NS5A-WT dimerization but not required for NS5A-Y93H dimerization. Our data suggest that spontaneous Ser235 phosphorylation of NS5A and ensuing dimerization account for resistance of the JFH1/NS5A-Y93H mutant to NS5A inhibitors.

Host IP3R channels are dispensable for rotavirus Ca2+ signaling but critical for intercellular Ca2+ waves that prime uninfected cells for rapid virus spread.

IMPORTANCE: Many viruses exploit host Ca2+ signaling to facilitate their replication; however, little is known about how Ca2+ signals from different host and viral channels contribute to the overall dysregulation of Ca2+ signaling or promote virus replication. Using cells lacking IP3R, a host ER Ca2+ channel, we delineated intracellular Ca2+ signals within virus-infected cells and intercellular Ca2+ waves (ICWs), which increased Ca2+ signaling in neighboring, uninfected cells. In infected cells, IP3R was dispensable for rotavirus-induced Ca2+ signaling and replication, suggesting the rotavirus NSP4 viroporin supplies these signals. However, IP3R-mediated ICWs increase rotavirus replication kinetics and spread, indicating that the Ca2+ signals from the ICWs may prime nearby uninfected cells to better support virus replication upon eventual infection. This "pre-emptive priming" of uninfected cells by exploiting host intercellular pathways in the vicinity of virus-infected cells represents a novel mechanism for viral reprogramming of the host to gain a replication advantage.

Atypical activation of the RNA sensor MDA5 by hepatitis C virus.

Hepatitis C virus (HCV) is a significant human pathogen that can cause a number of serious diseases including chronic inflammation of the liver, cirrhosis, and hepatocellular carcinoma. A key enzyme in the HCV life cycle is the nonstructural protein 5B (NS5B), which functions as an RNA-dependent RNA polymerase (RdRp) responsible for replicating the viral RNA genome. In their recent study, Dansako and colleagues showed that HCV NS5B induces type I interferon via activation of the RNA receptor MDA5, an activity that was dependent on the RdRp enzymatic activity but independent of viral RNA replication. Their data further indicated that the NS5B enzymes of HCV and the related GB virus-B produce cellular double-stranded RNA (dsRNA) species with potential immunostimulatory activity. These findings unveil an unconventional mechanism of activation of MDA5-mediated host immunity by viral RdRp enzymes, which is expected to spur new research directions in viral immunology.

Ser38-His93-Asn91 triad confers resistance of JFH1 HCV NS5A-Y93H variant to NS5A inhibitors.

HCV NS5A is a dimeric phosphoprotein involved in HCV replication. NS5A inhibitors are among direct-acting antivirals (DAA) for HCV therapy. The Y93H mutant of NS5A is resistant to NS5A inhibitors, but the precise mechanism remains unclear. In this report, we proposed a Ser38-His93-Asn91 triad to dissect the mechanism. Using pymol 1.3 software, the homology structure of JFH1 NS5A was determined based on the dimer structure of genotype 1b extracted from the database Protein DataBank (www.ebi.ac.uk/pdbsum) with codes 1ZH1 and 3FQM/3FQQ. FLAG-NS5A-WT failed to form dimer in the absence of nonstructural proteins from subgenomic replicon (NS3-5A); however, FLAG-NS5A-Y93H was able to form dimer without the aid of NS3-5A. The Ser38-His93-Asn91 triad in the dimer of the Y93H variant predicts a structural crash of the cleft receiving the NS5A inhibitor daclatasvir. The dimerization assay revealed that the existence of JFH1-NS5A-1ZH1 and -3FQM homology dimers depended on each other for existence and that both NS5A-WT 1ZH1 and 3FQM dimers cooperated to facilitate RNA replication. However, NS5A-Y93H 1ZH1 alone could form dimer and conduct RNA replication in the absence of the 3FQM structure. In conclusion, this study provides novel insight into the functional significance of the Ser38-His93-Asn91 triad in resistance of the Y93H variant to NS5A inhibitors.

Amino acid substitution L232F in non-structural protein 6 identified as a possible human-adaptive mutation in clade B MERS coronaviruses.

IMPORTANCE: Viral host adaptation plays an important role in inter-species transmission of coronaviruses and influenza viruses. Multiple human-adaptive mutations have been identified in influenza viruses but not so far in MERS-CoV that circulates widely in dromedary camels in the Arabian Peninsula leading to zoonotic transmission. Here, we analyzed clade B MERS-CoV sequences and identified an amino acid substitution L232F in nsp6 that repeatedly occurs in human MERS-CoV. Using a loss-of-function reverse genetics approach, we found the nsp6 L232F conferred increased viral replication competence in vitro, in cultures of the upper human respiratory tract ex vivo, and in lungs of mice infected in vivo. Our results showed that nsp6 L232F may be an adaptive mutation associated with zoonotic transmission of MERS-CoV. This study highlighted the capacity of MERS-CoV to adapt to transmission to humans and also the need for continued surveillance of MERS-CoV in camels.

Emerging variants of SARS-CoV-2 NSP10 highlight strong functional conservation of its binding to two non-structural proteins, NSP14 and NSP16.

The coronavirus SARS-CoV-2 protects its RNA from being recognized by host immune responses by methylation of its 5' end, also known as capping. This process is carried out by two enzymes, non-structural protein 16 (NSP16) containing 2'-O-methyltransferase and NSP14 through its N7 methyltransferase activity, which are essential for the replication of the viral genome as well as evading the host's innate immunity. NSP10 acts as a crucial cofactor and stimulator of NSP14 and NSP16. To further understand the role of NSP10, we carried out a comprehensive analysis of >13 million globally collected whole-genome sequences (WGS) of SARS-CoV-2 obtained from the Global Initiative Sharing All Influenza Data (GISAID) and compared it with the reference genome Wuhan/WIV04/2019 to identify all currently known variants in NSP10. T12I, T102I, and A104V in NSP10 have been identified as the three most frequent variants and characterized using X-ray crystallography, biophysical assays, and enhanced sampling simulations. In contrast to other proteins such as spike and NSP6, NSP10 is significantly less prone to mutation due to its crucial role in replication. The functional effects of the variants were examined for their impact on the binding affinity and stability of both NSP14-NSP10 and NSP16-NSP10 complexes. These results highlight the limited changes induced by variant evolution in NSP10 and reflect on the critical roles NSP10 plays during the SARS-CoV-2 life cycle. These results also indicate that there is limited capacity for the virus to overcome inhibitors targeting NSP10 via the generation of variants in inhibitor binding pockets.

Identification and characterization of Sofosbuvir-resistant mutations of hepatitis C virus genotype 3a replicon.

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease worldwide. Among its 8 genotypes (GT), GT3 has a relatively lower sustained virological response to highly effective direct-acting antiviral agents (DAA). Sofosbuvir (SOF), an anti-NS5B polymerase inhibitor, is a core component of many anti-HCV DAA cocktail regimens, and its resistant mutations are rare in clinics because these mutations usually severely impair the NS5B polymerase activity, including a mutation S282T in NS5B, the most frequently reported SOF-resistant mutation. In this study, we selected SOF-resistant variants of a previously developed GT3 subgenomic replicon (PR87A7). Two mutations were identified in the viral genome of SOF-resistant PR87A7 variants, Q606R in nontargeted NS3 and S282T in targeted N5SB. We demonstrated that Q606R could rescue the replication defect of S282T in PR87A7, and the resulting double mutant confers the SOF resistance. Finally, we showed that NS3-606R could not compensate for the replication defect of S282T in other GTs. In conclusion, we identified a novel GT3-specific combination of two mutations that confers SOF resistance. Our result calls for attention to potential mutations that may arise in nontargeted viral proteins during the SOF-based DAA treatment of chronic HCV.

Direct Dengue Virus Genome Sequencing from Antigen NS1 Rapid Diagnostic Tests: A Proof-of-Concept with the Standard Q Dengue Duo Assay.

With nearly half of the world's population being at risk of infection, dengue virus represents a major global health issue. The use of dengue antigen rapid diagnostic tests (Ag-RDTs) represents an alternative to PCR methods for the diagnosis of acute infections since they display excellent sensitivities and specificities and can be performed outside the laboratory. The high genetic diversity of the dengue virus genome represents a challenge for vaccine development, and the progressive expansion of this virus into previously nonendemic regions justifies the implementation of a genomic surveillance program. In this proof-of-concept study, we show the feasibility of sequencing dengue virus genomes directly from positive Ag-RDT (Standard Q Dengue Duo Test assay, n = 7) cassettes stored up to 31 days at room temperature after testing. For 5 of the 7 samples, a high number of reads were obtained allowing phylogenetic analyses to be carried out to determine not only the serotypes (dengue 1, 2, 3 and 4 were detected) but also the genotypes. Furthermore, in one sample, our unbiased metagenomic next-generation sequencing approach made it possible to detect epizootic hemorrhagic disease virus sequences, an arthropod-transmitted virus in ruminants. To conclude, as such an approach requires no cold storage or freezing of samples, dengue Ag-RDTs represent a very pragmatic and robust alternative for the genomic surveillance of dengue virus.