Nonstructural Research Articles

Mechanical mechanism for the translocation of hexameric and nonstructural helicases: Dependence on physical parameters.

Three recently observed facts of the translocation of actual hexameric and nonstructural (NS) helicases are related to the various physical quantities and are in accordance with the recently proposed mechanical mechanism: a) the translocation of hexameric helicases might be led by either the N-terminal domain (NTD) or C-terminal domain (CTD) depending on which domain has a smaller central pore, b) the translocation speed (vt) of the ring-shaped helicases and NS helicases decreased with decreasing applied tension, and c) a large difference in the vt of the NS helicase was observed for the helicase translocating on DNA and RNA. These findings are the effects of the physical quantities of the helicase/nuclei acid strands on the translocation of helicases and are difficult to explain with biochemical models. We predict that a similar behavior as described in b) and c) is also shown by hexameric helicases. The validity of the mechanical mechanism is demonstrated in simulation experiments.

Impact of Respiratory Syncytial Virus Infection on Host Functions: Implications for Antiviral Strategies.

Respiratory syncytial virus (RSV) is one of the leading causes of viral respiratory tract infection in infants, the elderly, and the immunocompromised worldwide, causing more deaths each year than influenza. Years of research into RSV since its discovery over 60 yr ago have elucidated detailed mechanisms of the host-pathogen interface. RSV infection elicits widespread transcriptomic and proteomic changes, which both mediate the host innate and adaptive immune responses to infection, and reflect RSV's ability to circumvent the host stress responses, including stress granule formation, endoplasmic reticulum stress, oxidative stress, and programmed cell death. The combination of these events can severely impact on human lungs, resulting in airway remodeling and pathophysiology. The RSV membrane envelope glycoproteins (fusion F and attachment G), matrix (M) and nonstructural (NS) 1 and 2 proteins play key roles in modulating host cell functions to promote the infectious cycle. This review presents a comprehensive overview of how RSV impacts the host response to infection and how detailed knowledge of the mechanisms thereof can inform the development of new approaches to develop RSV vaccines and therapeutics.

1H-Imidazole-2,5-Dicarboxamides as NS4A Peptidomimetics: Identification of a New Approach to Inhibit HCV-NS3 Protease.

The nonstructural (NS) protein NS3/4A protease is a critical factor for hepatitis C virus (HCV) maturation that requires activation by NS4A. Synthetic peptide mutants of NS4A were found to inhibit NS3 function. The bridging from peptide inhibitors to heterocyclic peptidomimetics of NS4A has not been considered in the literature and, therefore, we decided to explore this strategy for developing a new class of NS3 inhibitors. In this report, a structure-based design approach was used to convert the bound form of NS4A into 1H-imidazole-2,5-dicarboxamide derivatives as first generation peptidomimetics. This scaffold mimics the buried amino acid sequence Ile-25` to Arg-28` at the core of NS4A21`–33` needed to activate the NS3 protease. Some of the synthesized compounds (Coded MOC) were able to compete with and displace NS4A21`–33` for binding to NS3. For instance, N5-(4-guanidinobutyl)-N2-(n-hexyl)-1H-imidazole-2,5-dicarboxamide (MOC-24) inhibited the binding of NS4A21`–33` with a competition half maximal inhibitory concentration (IC50) of 1.9 ± 0.12 µM in a fluorescence anisotropy assay and stabilized the denaturation of NS3 by increasing the aggregation temperature (40% compared to NS4A21`–33`). MOC-24 also inhibited NS3 protease activity in a fluorometric assay. Molecular dynamics simulations were conducted to rationalize the differences in structure–activity relationship (SAR) between the active MOC-24 and the inactive MOC-26. Our data show that MOC compounds are possibly the first examples of NS4A peptidomimetics that have demonstrated promising activities against NS3 proteins.

Dengue virus non-structural protein 1 activates the p38 MAPK pathway to decrease barrier integrity in primary human endothelial cells.

Dengue virus (DENV) causes an estimated 390 million infections worldwide annually, with severe forms of disease marked by vascular leakage. Endothelial cells (EC) are directly responsible for vascular homeostasis and are highly responsive to circulating mediators but are not commonly infected. DENV encodes seven non-structural (NS) proteins; with only one of those, NS1, secreted from infected cells and accumulating in the blood of patients. NS1 has been implicated in the pathogenesis of vascular permeability, but the mechanism is not completely understood. Here we used primary endothelial cells and an array of in vitro approaches to study the effect of NS1 in disease-relevant human ECs. Confocal microscopy demonstrated rapid NS1 internalization by ECs into endosomes with accumulation over time. Transcriptomic and pathway analysis showed significant changes in functions associated with EC homeostasis and vascular permeability. Functional significance of this activation was assessed by trans-endothelial electrical resistance and showed that NS1 induced rapid and transient loss in EC barrier function within 3 h post-treatment. To understand the molecular mechanism by which NS1 induced EC activation, we evaluated the stress-sensing p38 MAPK pathway known to be directly involved in EC permeability and inflammation. WB analysis of NS1-stimulated ECs showed clear activation of p38 MAPK and downstream effectors MAPKAPK-2 and HSP27 with chemical inhibition of the p38 MAP kinase pathway restoring barrier function. Our results suggest that DENV NS1 may be involved in the pathogenesis of severe dengue by activating the p38 MAPK in ECs, promoting increased permeability that characterizes severe disease.

Genetic characterization of canine parvovirus type 2c from domestic dogs in Korea.

Canine parvovirus type 2 (CPV‐2) is an aetiological agent that causes acute haemorrhagic enteritis and fatal myocarditis in dogs. Since CPV‐2 first emerged in the late 1970s, its rapid evolution has resulted in three antigenic variants: CPV‐2a, CPV‐2b and CPV‐2c. Here, we report, for the first time in Korea, two cases of CPV‐2c infection in two dogs with severe diarrhoea. The complete open reading frame (4,269nt) of CPV‐2, encoding both non‐structural (NS) and structural (VP) proteins, was sequenced. Based on the amino acid Gln present at residue 426 of the VP2 gene, these strains were typed as CPV‐2c, and were named Korea CPV‐2c_1 and Korea CPV‐2c_2. These strains shared 99.48% reciprocal nucleotide sequence identity and had the highest nucleotide identity (99.77%–99.34%) with Asian CPV strains isolated in China, Italy (found in a dog imported from Thailand), and Vietnam from 2013 to 2017. Phylogenetic analysis based on the non‐structural (NS1) and capsid (VP2) genes revealed that Korean CPV‐2c strains clustered closely to Asian CPV strains, and separately from strains isolated in Europe, South America and North America. Amino acid changes never reported before were observed in NS1 (Thr70Pro, Cys287Tyr), VP1 (Lys17Arg, Phe33Leu) and VP2 (Gln365His, Ala516Val). Additional observed mutations, including Phe267Tyr, Tyr324Ile and Gln370Arg, have been previously reported in the recent CPV‐2c strains with Asian origins. These results suggest that the Korean CPV‐2c strains were potentially introduced via neighbouring Asian countries.

Identification of Zika Virus NS5 Novel Inhibitors through Virtual Screening and Docking Studies

Objective: Screening of ZINC inhibitors library for Zika virus (ZIKV) Non-Structural 5 (NS5) protein as potential drug target. Study Design: Cross sectional.Place and Duration of Study: The study was carried out at Department of Biological Sciences of National University of Medical Sciences, Rawalpindi, Pakistan from December 2018 to March 2019.Materials and Methods: NS5 protein was obtained from Protein databank (PDB ID: 5TMH) and screened against ZINC library of 11,193 drug-like molecules for NS5 and 3 ligands were identified based on optimum binding energy. MOE, PyMOL and CLUSTALW were used for docking studies and structural analysis.Results: Out of 11, 193 compounds, three ligands were observed to interact with residues of the Methyl Transferase (MT) domain of NS5. These ligands fit in the MT domain by making hydrogen and hydrophobic interactions in the active site and S-adenosyl-methionine (SAM) binding pocket.Conclusion: Hence, upon experimental validation, these ligands can be utilized as potential inhibitors against NS5 MT activity to control ZIKV viral replication and ultimately control the disease. How to cite this: Faheem M, Jamal SB. Identification of Zika Virus NS5 Novel Inhibitors through Virtual Screening and Docking Studies. Life and Science. 2020; 1(1): 3-7. doi:https://doi.org/10.37185/L&S.1.1.42

Ferulic Acid Protects against Porcine Parvovirus Infection-Induced Apoptosis by Suppressing the Nuclear Factor-κB Inflammasome Axis and Toll-Like Receptor 4 via Nonstructural Protein 1.

Background Porcine parvovirus (PPV) infection-induced apoptosis was recently identified as an important pathological factor in PPV-induced placental tissue damage, resulting in reproduction failure. In the present study, we demonstrate the possible involvement of toll-like receptor (TLR) 4 and nuclear factor (NF)-κB inflammasome activation in PPV infection-induced apoptosis and the protective potential of ferulic acid (FA). PPV infection significantly activated the expression levels of TLR4, NF-κB, MyD88, and interleukin (IL)-6. However, FA ameliorated the pathological process, prevented histological alterations, and inhibited the apoptosis rate in porcine kidney (PK-15) cells infected with PPV. Results FA inhibited PPV infection-induced inflammasome activation as shown by decreases in the expression of NF-κB, MyD88, and IL-6. FA also downregulated nonstructural (NS) 1 protein expression in infected PK-15 cells. Conclusions FA downregulated NS1 and TLR4 signaling, prevented the overproduction of reactive oxygen species, and suppressed the NF-κB inflammasome axis to inhibit PPV-induced apoptosis in PK-15 cells.

Seismic safety of valuable non-structural elements in RC buildings: Floor Response Spectrum approaches

The seismic performances of non-structural (NS) components belonging to the category of “valuable elements”, i.e. elements characterized by high value in terms of economic, cultural or strategic purposes, represent nowadays a crucial aspect in seismic safety design and assessment of new and existing buildings. Actually, most valuable NS elements are simply-supported objects, which can be classified as acceleration-sensitive NS elements. Frequently, these elements can be considered dynamically uncoupled from the primary structure to which they are connected, thereby justifying the Floor Response Spectrum methods usually adopted in literature and by most of building codes. This paper presents an extensive parametric study of floor response spectra obtained by linear and nonlinear numerical modeling of RC structures. Two sets of 30 horizontal ground motion scaled acceleration records are generated according to the Ultimate Limit State (ULS) and the Damage Limitation State (DLS) spectra adopted for building design. The numerical floor response spectra and those proposed by codes and international standards are critically compared, and the Peak Floor Acceleration (PFA) and the Peak Floor Velocity (PFV) profiles along the building height are discussed. Finally, a simplified method based on the “stability charts” is developed to assess the seismic safety of free standing NS elements located at the upper floors of the host buildings.

Diaphorina citri densovirus is a persistently infecting virus with a hybrid genome organization and unique transcription strategy.

Diaphorina citri densovirus (DcDV) is an ambisense densovirus with a 5071 nt genome. Phylogenetic analysis places DcDV in an intermediate position between those in the Ambidensovirus and Iteradensovirus genera, a finding that is consistent with the observation that DcDV possesses an Iteradensoviris-like non-structural (NS) protein-gene cassette, but a capsid-protein (VP) gene cassette resembling those of other ambisense densoviruses. DcDV is maternally transmitted to 100 % of the progeny of infected female Diaphorina citri, and the progeny of infected females carry DcDV as a persistent infection without outward phenotypic effects. We were unable to infect naïve individuals by oral inoculation, however low levels of transient viral replication are detected following intrathoracic injection of DcDV virions into uninfected D. citri insects. Transcript mapping indicates that DcDV produces one transcript each from the NS and VP gene cassettes and that these transcripts are polyadenylated at internal sites to produce a ~2.2 kb transcript encoding the NS proteins and a ~2.4 kb transcript encoding the VP proteins. Additionally, we found that transcriptional readthrough leads to the production of longer non-canonical transcripts from both genomic strands.

Characterising the morphological characters and carbohydrate metabolism of oat culms and their association with lodging resistance.

Lodging resistance can be improved by enhancing the mechanical strength of culms, and culm carbohydrates could improve this mechanical strength. Culm carbohydrates can regulate development of the culm and affect its toughness. The present study determines the relationship between lodging and carbohydrate content in oat culms. Field experiments were conducted in alpine regions in 2017 and 2018 using three oat varieties with different lodging resistance. Lodging-related morphological characteristics were directly determined and culm carbohydrate content and enzyme activity related to cellulose synthesis and sucrose metabolism were evaluated with ultraviolet spectrophotometry. Results showed that the lower the gravity height or the lower ratio of gravity height to plant height, the stronger the lodging resistance of the varieties. Higher culm nonstructural (NSC) and structural (SC) carbohydrate content contributed to the ability of culms to resist lodging, especially the content of cellulose and sucrose. PCA showed that sucrose metabolism and SC content were closely related to lodging resistance. Correlation analysis showed that the lodging index (LI) was significantly negatively correlated with NSC. Sucrose content was highly and significantly positively correlated with NSC. Additionally, the activities of sucrose phosphate synthase (SPS) and sucrose synthase (SS) were highly and significantly positively correlated with sucrose and cellulose content. The relationship between field characters and oat lodging, as well as the regulatory mechanism of carbohydrate content on lodging resistance of the culm are discussed.

Characterization and Phylodynamics of Reassortant H12Nx Viruses in Northern Eurasia.

Wild waterfowl birds are known to be the main reservoir for a variety of avian influenza viruses of different subtypes. Some subtypes, such as H2Nx, H8Nx, H12Nx, and H14Nx, occur relatively rarely in nature. During 10-year long-term surveillance, we isolated five rare H12N5 and one H12N2 viruses in three different distinct geographic regions of Northern Eurasia and studied their characteristics. H12N2 from the Far East region was a double reassortant containing hemagglutinin (HA), non-structural (NS) and nucleoprotein (NP) segments of the American lineage and others from the classical Eurasian avian-like lineage. H12N5 viruses contain Eurasian lineage segments. We suggest a phylogeographical scheme for reassortment events associated with geographical groups of aquatic birds and their migration flyways. The H12N2 virus is of particular interest as this subtype has been found in common teal in the Russian Far East region, and it has a strong relation to North American avian influenza virus lineages, clearly showing that viral exchange of segments between the two continents does occur. Our results emphasize the importance of Avian Influenza Virus (AIV) surveillance in Northern Eurasia for the annual screening of virus characteristics, including the genetic constellation of rare virus subtypes, to understand the evolutionary ecology of AIV.

Zika virus utilises the ubiquitin-proteasome pathway during infection of mosquito and human cells

Arthropod-borne viruses are able to infect vertebrate and invertebrate hosts. One such arbovirus is Zika virus (family Flaviviridae) that is mainly transmitted to humans by Aedes mosquitoes causing febrile illness and congenital Zika syndrome in infants. An interplay between host and virus proteins enables ZIKV to manipulate its host's cellular machineries in order to facilitate infection and evade antiviral responses. A possible mechanism it utilises is the ubiquitin-proteasome pathway (UPP) where target proteins are ubiquitinated and subsequently degraded by the proteasome. Results of proteomics analysis of Ae. aegypti cell lines (AF5) stably expressing V5-tagged ZIKV capsid (C), anchored capsid (AC) or non-structural 3 (NS3) proteins revealed that these viral proteins interact with effector proteins of the UPP. One of these proteins is TER94 an AAA-ATPase that acts as a chaperone segregating ubiquitinated proteins to the proteasome complex. Knockdown experiments of TER94 or its human ortholog VCP using dsRNA or siRNAs showed reduced virus replication in AF5 or A549 cells. Using small molecule inhibitors of UPP proteins also diminished ZIKV replication. Inhibiting different stages of the pathway have identified critical steps during early stages of infection. The ubiquitination of lysine-rich ZIKV C and its interaction with TER94/VCP could be one of the many universal strategies that the virus employs when it switches between mosquito and human hosts. Understanding how ZIKV is able to infect both human and insect species could provide novel strategies for prevention and therapeutics. Keywords: Zika virus, Aedes aegypti, ubiquitin-proteasome pathway, mosquito, virus-host interaction

An attenuated Zika virus NS4B protein mutant is a potent inducer of antiviral immune responses

Live attenuated vaccines (LAVs) are one of the most important strategies to control flavivirus diseases. The flavivirus nonstructural (NS) 4B proteins are a critical component of both the virus replication complex and evasion of host innate immunity. Here we have used site-directed mutagenesis of residues in the highly conserved N-terminal and central hydrophobic regions of Zika virus (ZIKV) NS4B protein to identify candidate attenuating mutations. Three single-site mutants were generated, of which the NS4B-C100S mutant was more attenuated than the other two mutants (NS4B-C100A and NS4B-P36A) in two immunocompromised mouse models of fatal ZIKV disease. The ZIKV NS4B-C100S mutant triggered stronger type 1 interferons and interleukin-6 production, and higher ZIKV-specific CD4+ and CD8+ T-cell responses, but induced similar titers of neutralization antibodies compared with the parent wild-type ZIKV strain and a previously reported candidate ZIKV LAV with a 10-nucleotide deletion in 3′-UTR (ZIKV-3′UTR-Δ10). Vaccination with ZIKV NS4B-C100S protected mice from subsequent WT ZIKV challenge. Furthermore, either passive immunization with ZIKV NS4B-C100S immune sera or active immunization with ZIKV NS4B-C100S followed by the depletion of T cells affords full protection from lethal WT ZIKV challenge. In summary, our results suggest that the ZIKV NS4B-C100S mutant may serve as a candidate ZIKV LAV due to its attenuated phenotype and high immunogenicity.

Respiratory syncytial virus nonstructural proteins 1 and 2: Exceptional disrupters of innate immune responses.

Human respiratory syncytial virus (RSV) is the most important cause of acute lower respiratory tract disease in infants worldwide. As a first line of defense against respiratory infections, innate immune responses, including the production of type I and III interferons (IFNs), play an important role. Upon infection with RSV, multiple pattern recognition receptors (PRRs) can recognize RSV-derived pathogen-associated molecular patterns (PAMPs) and mount innate immune responses. Retinoic-acid-inducible gene-I (RIG-I) and nucleotide-binding oligomerization domain-containing protein 2 (NOD2) have been identified as important innate receptors to mount type I IFNs during RSV infection. However, type I IFN levels remain surprisingly low during RSV infection despite strong viral replication. The poor induction of type I IFNs can be attributed to the cooperative activity of 2 unique, nonstructural (NS) proteins of RSV, i.e., NS1 and NS2. These viral proteins have been shown to suppress both the production and signaling of type I and III IFNs by counteracting a plethora of key host innate signaling proteins. Moreover, increasing numbers of IFN-stimulated genes (ISGs) are being identified as targets of the NS proteins in recent years, highlighting an underexplored protein family in the identification of NS target proteins. To understand the diverse effector functions of NS1 and NS2, Goswami and colleagues proposed the hypothesis of the NS degradasome (NSD) complex, a multiprotein complex made up of, at least, NS1 and NS2. Furthermore, the crystal structure of NS1 was resolved recently and, remarkably, identified NS1 as a structural paralogue of the RSV matrix protein. Unfortunately, no structural data on NS2 have been published so far. In this review, we briefly describe the PRRs that mount innate immune responses upon RSV infection and provide an overview of the various effector functions of NS1 and NS2. Furthermore, we discuss the ubiquitination effector functions of NS1 and NS2, which are in line with the hypothesis that the NSD shares features with the canonical 26S proteasome.

Ribavirin induces hepatitis C virus genome mutations in chronic hepatitis patients who failed to respond to prior daclatasvir plus asunaprevir therapy.

Ribavirin (RBV) induces nucleotide (nt) substitutions in hepatitis C virus (HCV) genome nonstructural (NS) regions. Although emergence of drug resistance-associated variants is associated with direct-acting antiviral treatment failure, the effect of RBV on genome substitutions in such patients is unknown. Genotype 1b HCV subgenomic replicon cells were treated with RBV for 120 hours. Six patients with chronic genotype 1b with HCV-infected patients who failed to respond to prior daclatasvir plus asunaprevir (DCV/ASV) therapy were treated with 12 weeks of sofosbuvir and ledipasvir plus RBV after 4 weeks of RBV monotherapy. RBV-induced genome mutations in the HCV NS region (nt3493-9301) in replicon cells and in patients during 4 weeks of RBV monotherapy were analyzed by deep sequencing. RBV-associated G-to-A and C-to-U transitions increased in a dose-dependent manner in HCV replicon cells after the RBV treatment. In patients with prior DCV/ASV treatment failures, the median serum HCV RNA level was 6.25 ± 0.31 log IU/mL at the start of RBV therapy and decreased significantly to 5.95 ± 0.4 log IU/mL (P = .03) after 4 weeks of RBV monotherapy. Although predominant HCV genome substitutions rates were similar between nontreatment and RBV-treatment periods (0.042 and 0.031 per base pair, respectively; P = .248), the frequencies of G-to-A and C-to-U transitions significantly increased after RBV monotherapy. These transitions were enriched, particularly within the HCV NS3 region in all patients. RBV treatment induces G-to-A and C-to-U transitions in the HCV genome even in chronic patients with hepatitis C with prior DCV/ASV treatment failures.

Use of an Outbred Rat Hepacivirus Challenge Model for Design and Evaluation of Efficacy of Different Immunization Strategies for Hepatitis C Virus.

Background and AimsThe lack of immunocompetent small animal models for hepatitis C virus (HCV) has greatly hindered the development of effective vaccines. Using rodent hepacivirus (RHV), a homolog of HCV that shares many characteristics of HCV infection, we report the development and application of an RHV outbred rat model for HCV vaccine development.Approach and ResultsSimian adenovirus (ChAdOx1) encoding a genetic immune enhancer (truncated shark class II invariant chain) fused to the nonstructural (NS) proteins NS3‐NS5B from RHV (ChAd‐NS) was used to vaccinate Sprague‐Dawley rats, resulting in high levels of cluster of differentiation 8–positive (CD8+) T‐cell responses. Following RHV challenge (using 10 or 100 times the minimum infectious dose), 42% of vaccinated rats cleared infection within 6‐8 weeks, while all mock vaccinated controls became infected with high‐level viremia postchallenge. A single, 7‐fold higher dose of ChAd‐NS increased efficacy to 67%. Boosting with ChAd‐NS or with a plasmid encoding the same NS3‐NS5B antigens increased efficacy to 100% and 83%, respectively. A ChAdOx1 vector encoding structural antigens (ChAd‐S) was also constructed. ChAd‐S alone showed no efficacy. Strikingly, when combined with ChAd‐NS, ChAD‐S produced 83% efficacy. Protection was associated with a strong CD8+ interferon gamma–positive recall response against NS4. Next‐generation sequencing of a putative RHV escape mutant in a vaccinated rat identified mutations in both identified immunodominant CD8+ T‐cell epitopes.ConclusionsA simian adenovirus vector vaccine strategy is effective at inducing complete protective immunity in the rat RHV model. The RHV Sprague‐Dawley rat challenge model enables comparative testing of vaccine platforms and antigens and identification of correlates of protection and thereby provides a small animal experimental framework to guide the development of an effective vaccine for HCV in humans.

Suppression of Zika Virus Infection in the Brain by the Antiretroviral Drug Rilpivirine.

Zika virus (ZIKV) infection is associated with microcephaly in neonates and Guillain-Barré syndrome in adults. ZIKV produces a class of nonstructural (NS) regulatory proteins that play a critical role in viral transcription and replication, including NS5, which possesses RNA-dependent RNA polymerase (RdRp) activity. Here we demonstrate that rilpivirine (RPV), a non-nucleoside reverse transcriptase inhibitor (NNRTI) used in the treatment of HIV-1 infection, inhibits the enzymatic activity of NS5 and suppresses ZIKV infection and replication in primary human astrocytes. Similarly, other members of the NNRTI family, including etravirine and efavirenz, showed inhibitory effects on viral infection of brain cells. Site-directed mutagenesis identified 14 amino acid residues within the NS5 RdRp domain (AA265-903), which are important for the RPV interaction and the inhibition of NS5 polymerase activity. Administration of RPV to ZIKV-infected interferon-alpha/beta receptor (IFN-A/R) knockout mice improved the clinical outcome and prevented ZIKV-induced mortality. Histopathological examination of the brains from infected animals revealed that RPV reduced ZIKV RNA levels in the hippocampus, frontal cortex, thalamus, and cerebellum. Repurposing of NNRTIs, such as RPV, for the inhibition of ZIKV replication offers a possible therapeutic strategy for the prevention and treatment of ZIKV-associated disease.

HCV NS5A hyperphosphorylation is involved in viral translation modulation

Hepatitis C virus (HCV) non-structural (NS) 5A protein is a multifunctional phosphoprotein. NS5A exists as hypo- and hyper-phosphorylated forms and the dynamic transitions between these two states are involved in the functions of NS5A. Hyperphosphorylation occurs primarily at six serine residues within the low complexity sequence I of NS5A. We previously showed that NS5A downregulates viral translation. In this study, we investigated the role of NS5A hyperphosphorylation in translation modulation. By analyzing the effects of phospho-ablative and phospho-mimetic mutants of the six serine residues on translation, we showed that NS5A hyperphospho-ablative mutation at all six serine residues can no longer downregulate viral translation. We then studied the effects of phospho-mutations at each of the six serine residues on translation. We found that phosphorylation of S222, S225, S235 is not involved in translation downregulation by NS5A. In contrast, NS5A with alanine mutations at S229 or S238 can no longer downregulate translation, whereas S229D or S238D mutations have no effect. Interestingly, S232D NS5A, but not S232A, abrogates translation downregulation by NS5A. Since dimerization of NS5A plays an important role in its functions, we also studied the effects of phospho-mutants of S229, S232, and S238 on dimerization in a protein-protein interaction assay. We showed that phopho-mimetic S229D or S238D mutations enhances NS5A dimerization, whereas the phospho-ablative mutations of these two residues have no effect. Neither phospho-ablative nor phopho-mimetic mutations of S232 affect dimerization. These results indicate that phosphorylation of NS5A at S229, S232, and S238 is involved in viral translation regulation and NS5A dimerization.

Complete genome sequence of a novel reassortant H3N3 avian influenza virus.

Aquatic birds are known to be a reservoir for the most common influenza A viruses (IAVs). In the annual surveillance program, we collected the feces of migratory birds for the detection of IAVs in South Korea in November 2016. A novel reassorted H3N3 avian influenza virus (AIV) containing genes from viruses of wild and domestic birds was identified and named A/aquatic bird/South Korea/sw006/2016(H3N3). The polymerase basic 2 (PB2) and non-structural (NS) genes of this isolate are most closely related to those of wild-bird-origin AIV, while the polymerase basic 1 (PB1), polymerase acidic (PA), hemagglutinin (HA), nucleoprotein (NP), neuraminidase (NA), and matrix (M) genes are most closely related to those of domestic-bird-origin AIV. A/aquatic bird/South Korea/sw006/2016 contains PA, NP, M, and NS genes were most closely related to those of AIV subtype H4 and PB2, PB1, and HA genes that are most closely related to those of AIV subtype H3N8, while the NA gene was most closely related to those of subtype H10, which was recently detected in humans in China. These results suggest that novel reassortment of AIV strains occurred due to interaction between wild and domestic birds. Hence, we emphasize the need for continued surveillance of avian influenza virus in bird populations.

A Luciferase-fluorescent Reporter Influenza Virus for Live Imaging and Quantification of Viral Infection.

Influenza A viruses (IAVs) cause human respiratory disease that is associated with significant health and economic consequences. As with other viruses, studying IAV requires the use of laborious secondary approaches to detect the presence of the virus in infected cells and/or in animal models of infection. This limitation has been recently circumvented with the generation of recombinant IAVs expressing easily traceable fluorescent or bioluminescent (luciferase) reporter proteins. However, researchers have been forced to select fluorescent or luciferase reporter genes due to the restricted capacity of the IAV genome for including foreign sequences. To overcome this limitation, we have generated a recombinant replication-competent bi-reporter IAV (BIRFLU) stably expressing both a fluorescent and a luciferase reporter gene to easily track IAV infections in vitro and in vivo. To this end, the viral non-structural (NS) and hemagglutinin (HA) viral segments of influenza A/Puerto Rico/8/34 H1N1 (PR8) were modified to encode the fluorescent Venus and the bioluminescent Nanoluc luciferase proteins, respectively. Here, we describe the use of BIRFLU in a mouse model of IAV infection and the detection of both reporter genes using an in vivo imaging system. Notably, we have observed a good correlation between the expressions of both reporters and viral replication. The combination of cutting-edge techniques in molecular biology, animal research and imaging technologies, provides researchers the unique opportunity to use this tool for influenza research, including the study of virus-host interactions and dynamics of viral infections. Importantly, the feasibility to genetically alter the viral genome to express two foreign genes from different viral segments opens up opportunities to use this approach for: (i) the development of novel IAV vaccines, (ii) the generation of recombinant IAVs that can be used as vaccine vectors for the treatment of other human pathogen infections.