Antiviral Properties of the NSAID Drug Naproxen Targeting the Nucleoprotein of SARS-CoV-2 Coronavirus.

There is an urgent need for specific antiviral treatments directed against SARS-CoV-2 to prevent the most severe forms of COVID-19. By drug repurposing, affordable therapeutics could be supplied worldwide in the present pandemic context. Targeting the nucleoprotein N of the SARS-CoV-2 coronavirus could be a strategy to impede viral replication and possibly other essential functions associated with viral N. The antiviral properties of naproxen, a non-steroidal anti-inflammatory drug (NSAID) that was previously demonstrated to be active against Influenza A virus, were evaluated against SARS-CoV-2. Intrinsic fluorescence spectroscopy, fluorescence anisotropy, and dynamic light scattering assays demonstrated naproxen binding to the nucleoprotein of SARS-Cov-2 as predicted by molecular modeling. Naproxen impeded recombinant N oligomerization and inhibited viral replication in infected cells. In VeroE6 cells and reconstituted human primary respiratory epithelium models of SARS-CoV-2 infection, naproxen specifically inhibited viral replication and protected the bronchial epithelia against SARS-CoV-2-induced damage. No inhibition of viral replication was observed with paracetamol or the COX-2 inhibitor celecoxib. Thus, among the NSAID tested, only naproxen combined antiviral and anti-inflammatory properties. Naproxen addition to the standard of care could be beneficial in a clinical setting, as tested in an ongoing clinical study.

Targeting ebola virus VP40 protein through novel inhibitors: exploring the structural and dynamic perspectives on molecular landscapes.

Ebola filovirus (EBOV) is one of the deadliest known infectious agents, and a cause of Western African epidemics from 2013 to 2016. The virus has infected nearly 3000 humans and almost 1900 have died. In the past few years, various small molecules have been discovered to display efficiency against EBOV and some of them have progressed towards clinical trials. Even though continuous attempts have been made to find antiEBOV therapeutics, no potential drugs are yet approved against this viral infection. The development of small antiviral inhibitors has gained tremendous attention in the attempt to overcome EVD. With this background, we seek to offer molecular insights into EBOV VP40 protein inhibition, using all atom molecular mechanics methodology and binding free energy calculations. We have selected five novel reported inhibitors against VP40 protein, namely Comp1, Comp2, Comp3, Comp4, and Comp5, and explored their binding against the same target. It was evident from the analysis that all the inhibitors displayed stability in complex with VP40 protein; however, Comp1 exhibited enhanced stability and compactness. Comp1 unveiled favorable binding, which accounted for positive correlation motions in the active site residues. Likewise, Comp1 revealed the most promising binding (ΔGbind - 40.3504 kcal/mol) as compared to the other four inhibitors, which disclosed relatively less favorable ΔGbind. The highest binding energy of Comp1 to VP40 protein can be primarily endorsed to the upsurge in van der Waals energy by ΔEvdW - 37.1609 kcal/mol and Coulomb energy by ΔEele - 52.7332 kcal/mol. Also, the hydrogen bond network is robust in Comp1-VP40 complex, with four hydrogen bonds, whilst it is less in other inhibitors. The outcomes from this report may assist in the advancement of novel VP40 inhibitors with high selectivity and potency for EVD therapeutics.

A Novel Ebola Virus VP40 Matrix Protein-Based Screening for Identification of Novel Candidate Medical Countermeasures.

Filoviruses, such as Ebola virus and Marburg virus, are of significant human health concern. From 2013 to 2016, Ebola virus caused 11,323 fatalities in Western Africa. Since 2018, two Ebola virus disease outbreaks in the Democratic Republic of the Congo resulted in 2354 fatalities. Although there is progress in medical countermeasure (MCM) development (in particular, vaccines and antibody-based therapeutics), the need for efficacious small-molecule therapeutics remains unmet. Here we describe a novel high-throughput screening assay to identify inhibitors of Ebola virus VP40 matrix protein association with viral particle assembly sites on the interior of the host cell plasma membrane. Using this assay, we screened nearly 3000 small molecules and identified several molecules with the desired inhibitory properties. In secondary assays, one identified compound, sangivamycin, inhibited not only Ebola viral infectivity but also that of other viruses. This finding indicates that it is possible for this new VP40-based screening method to identify highly potent MCMs against Ebola virus and its relatives.

Paratope Duality and Gullying are Among the Atypical Recognition Mechanisms Used by a Trio of Nanobodies to Differentiate Ebolavirus Nucleoproteins.

We had previously shown that three anti-Marburg virus nanobodies (VHH or single-domain antibody [sdAb]) targeted a cryptotope within an alpha-helical assembly at the nucleoprotein (NP) C-terminus that was conserved through half a century of viral evolution. Here, we wished to determine whether an anti-Ebola virus sdAb, that was cross-reactive within the Ebolavirus genus, recognized a similar structural feature upstream of the ebolavirus NP C-terminus. In addition, we sought to determine whether the specificities of a less cross-reactive anti-Zaire ebolavirus sdAb and a totally specific anti-Sudan ebolavirus sdAb were the result of exclusion from this region. Binding and X-ray crystallographic studies revealed that the primary determinant of cross-reactivity did indeed appear to be a preference for the helical feature. Specificity, in the case of the Zaire ebolavirus-specific sdAb, arose from the footprint shifting away from the helices to engage more variable residues. While both sdAbs used CDRs, they also had atypical side-on approaches, with framework 2 helping to accommodate parts of the epitope in sizeable paratope gullies. The Sudan ebolavirus-specific sdAb was more remarkable and appeared to bind two C-terminal domains simultaneously via nonoverlapping epitopes-"paratope duality." One mode involved paratope gullying, whereas the other involved only CDRs, with CDR3 restructuring to wedge in between opposing walls of an interdomain crevice. The varied routes used by sdAbs to engage antigens discovered here deepen our appreciation of the small scaffold's architectural versatility and also reveal lucrative opportunities within the ebolavirus NP C-termini that might be leveraged for diagnostics and novel therapeutic targeting.

Regulation of Hazara virus growth through apoptosis inhibition by viral nucleoprotein.

Hazara virus (HAZV) is closely related to Crimean-Congo hemorrhagic fever virus (CCHFV), but differs in that it is non-pathogenic to humans. Since HAZV was isolated for the first time in 1954, the biological characteristics of this virus, particularly its behavior within culture cells, have not been well-studied, despite its importance as a surrogate model for CCHFV. Nucleoprotein (N) is the main component of viral nucleocapsid and is the most abundant virion protein, it is believed to play a pivotal role in the viral lifecycle. Generation of a series of anti-HAZV N monoclonal antibodies has enabled us to directly examine the involvement of this protein on viral growth. Observation of HAZV-infected cells revealed that this infection caused apoptosis, which was further characterized by DNA ladder and elevated caspase-3/7 activity. HAZV titers initially increased in cell culture, but after reaching the peak titer began to rapidly decline. HAZV particles were found to be very unstable in culture medium at 37 °C, and virus particles tend to lose infectivity at that point. HAZV N appears to inhibit apoptosis, thus can potentially support efficient viral propagation.

Total Synthesis, Biological Evaluation, and Target Identification of Rare Abies Sesquiterpenoids.

Abiespiroside A (1), beshanzuenone C (2), and beshanzuenone D (3) belong to the Abies sesquiterpenoid family. Beshanzuenones C (2) and D (3) are isolated from the critically endangered Chinese fir tree species Abies beshanzuensis and demonstrated weak inhibiting activity against protein tyrosine phosphatase 1B (PTP1B). We describe herein the first total syntheses of these Abies sesquiterpenoids relying on the sustainable and inexpensive chiral pool molecule (+)-carvone. The syntheses feature a palladium-catalyzed hydrocarbonylative lactonization to install the 6,6-fused bicyclic ring system and a Dreiding-Schmidt reaction to build the oxaspirolactone moiety of these target molecules. Our chemical total syntheses of these Abies sesquiterpenoids have enabled (i) the validation of beshanzuenone C's weak PTP1B inhibiting potency, (ii) identification of new synthetic analogs with promising and selective protein tyrosine phosphatase SHP2 inhibiting potency, and (iii) preparation of azide-tagged probe molecules for target identification via a chemoproteomic approach. The latter has resulted in the identification and evaluation of DNA polymerase epsilon subunit 3 (POLE3) as one of the novel cellular targets of these Abies sesquiterpenoids and their analogs. More importantly, via POLE3 inactivation by probe molecule 29 and knockdown experiment, we further demonstrated that targeting POLE3 with small molecules may be a novel strategy for chemosensitization to DNA damaging drugs such as etoposide in cancer.

Development of Locked Nucleic Acid Antisense Oligonucleotides Targeting Ebola Viral Proteins and Host Factor Niemann-Pick C1.

The Ebola virus is a zoonotic pathogen that can cause severe hemorrhagic fever in humans, with up to 90% lethality. The deadly 2014 Ebola outbreak quickly made an unprecedented impact on human lives. While several vaccines and therapeutics are under development, current approaches contain several limitations, such as virus mutational escape, need for formulation or refrigeration, poor scalability, long lead-time, and high cost. To address these challenges, we developed locked nucleic acid (LNA)-modified antisense oligonucleotides (ASOs) to target critical Ebola viral proteins and the human intracellular host protein Niemann-Pick C1 (NPC1), required for viral entry into infected cells. We generated noninfectious viral luciferase reporter assays to identify LNA ASOs that inhibit translation of Ebola viral proteins in vitro and in human cells. We demonstrated specific inhibition of key Ebola genes VP24 and nucleoprotein, which inhibit a proper immune response and promote Ebola virus replication, respectively. We also identified LNA ASOs targeting human host factor NPC1 and demonstrated reduced infection by chimeric vesicular stomatitis virus harboring the Ebola glycoprotein, which directly binds to NPC1 for viral infection. These results support further in vivo testing of LNA ASOs in infectious Ebola virus disease animal models as potential therapeutic modalities for treatment of Ebola.

Tratamiento antigripal: fármacos actualmente utilizados y nuevos agentes en desarrollo / Anti-influenza treatment: drugs currently used and under development

La gripe es una enfermedad contagiosa altamente prevalente y con significativa morbimortalidad. El tratamiento disponible con fármacos antivirales, de ser administrado de forma precoz, puede reducir el riesgo de complicaciones severas; sin embargo, muchos tipos de virus desarrollan resistencia a estos fármacos, reduciendo notablemente su efectividad. Ha habido un gran interés en el desarrollo de nuevas opciones terapéuticas para combatir la enfermedad. Una gran variedad de fármacos han demostrado tener actividad antiinfluenza, pero aún no están disponibles para su uso en la clínica. Muchos de ellos tienen como objetivo componentes del virus, mientras que otros son dirigidos a elementos de la célula huésped que participan en el ciclo viral. Modular los componentes del huésped es una estrategia que minimiza el desarrollo de cepas resistentes, dado que estos no están sujetos a la variabilidad genética que tiene el virus. Por otro lado, la principal desventaja es que existe un mayor riesgo de efectos secundarios asociados al tratamiento. El objetivo de la presente revisión es describir los principales agentes farmacológicos disponibles en la actualidad, así como los nuevos fármacos en estudio con potencial beneficio en el tratamiento de la gripe

Influenza is a very common contagious disease that carries significant morbidity and mortality. Treatment with antiviral drugs is available, which if administered early, can reduce the risk of severe complications. However, many virus types develop resistance to those drugs, leading to a notable loss of efficacy. There has been great interest in the development of new drugs to combat this disease. A wide range of drugs has shown anti-influenza activity, but they are not yet available for use in the clinic. Many of these target viral components, which others are aimed at elements in the host cell which participate in the viral cycle. Modulating host components is a strategy which minimizes the development of resistance, since host components are not subject to the genetic variability of the virus. The main disadvantage is the risk of treatment-related side effects. The aim of this review is to describe the main pharmacological agents currently available and new drugs in the pipeline with potential benefit in the treatment of influenza

In silico and in vitro methods to identify ebola virus VP35-dsRNA inhibitors.

Ebola virus continues to be problematic as sporadic outbreaks in Africa continue to arise, and as terrorist organizations have considered the virus for bioterrorism use. Several proteins within the virus have been targeted for antiviral chemotherapy, including VP35, a dsRNA binding protein that promotes viral replication, protects dsRNA from degradation, and prevents detection of the viral genome by immune complexes. To augment the scope of our antiviral research, we have now employed molecular modeling techniques to enrich the population of compounds for further testing in vitro. In the initial docking of a static VP35 structure with an 80,000 compound library, 40 compounds were selected, of which four compounds inhibited VP35 with IC50 <200µM, with the best compounds having an IC50 of 20µM. By superimposing 26 VP35 structures, we determined four aspartic acid residues were highly flexible and the docking was repeated under flexible parameters. Of 14 compounds chosen for testing, five compounds inhibited VP35 with IC50 <200µM and one compound with an IC50 of 4µM. These studies demonstrate the value of docking in silico for enriching compounds for testing in vitro, and specifically using multiple structures as a guide for detecting flexibility and provide a foundation for further development of small molecule inhibitors directed towards VP35.

Knock-Down of Endogenous Bornavirus-Like Nucleoprotein 1 Inhibits Cell Growth and Induces Apoptosis in Human Oligodendroglia Cells.

Endogenous bornavirus-like nucleoprotein elements (EBLNs) have been discovered in the genomes of various animals including humans, whose functions have been seldom studied. To explore the biological functions of human EBLNs, we constructed a lentiviral vector expressing a short-hairpin RNA against human EBLN1, which successfully inhibited EBLN1 expression by above 80% in infected human oligodendroglia cells (OL cells). We found that EBLN1 silencing suppressed cell proliferation, induced G2/M phase arrest, and promoted apoptosis in OL cells. Gene expression profiling demonstrated that 1067 genes were up-regulated, and 2004 were down-regulated after EBLN1 silencing. The top 10 most upregulated genes were PI3, RND3, BLZF1, SOD2, EPGN, SBSN, INSIG1, OSMR, CREB3L2, and MSMO1, and the top 10 most-downregulated genes were KRTAP2-4, FLRT2, DIDO1, FAT4, ESCO2, ZNF804A, SUV420H1, ZC3H4, YAE1D1, and NCOA5. Pathway analysis revealed that these differentially expressed genes were mainly involved in pathways related to the cell cycle, the mitogen-activated protein kinase pathway, p53 signaling, and apoptosis. The gene expression profiles were validated by using quantitative reverse transcription polymerase chain reaction (RT-PCR) for detecting these 20 most-changed genes. Three genes closely related to glioma, RND3, OSMR, and CREB3L2, were significantly upregulated and might be the key factors in EBLN1 regulating the proliferation and apoptosis of OL cells. This study provides evidence that EBLN1 plays a key role in regulating cell life and death, thereby opening several avenues of investigation regarding EBLN1 in the future.

Implication of NPM1 phosphorylation and preclinical evaluation of the nucleoprotein antagonist N6L in prostate cancer.

Despite the advent of several new treatment options over the past years, advanced/metastatic prostate carcinoma (PCa) still remains incurable, which justifies the search for novel targets and therapeutic molecules. Nucleophosmin (NPM1) is a shuttling nucleoprotein involved in tumor growth and its targeting could be a potential approach for cancer therapy. We previously demonstrated that the multivalent pseudopeptide N6L binds to NPM1 potently affecting in vitro and in vivo tumor cell growth of various tumor types as well as angiogenesis. Furthermore, NPM1 binds to androgen receptor (AR) and modulate its activity. In this study, we first investigated the implication of the NPM1 and its Thr199 and Thr234/237 phosphorylated forms in PCa. We showed that phosphorylated forms of NPM1 interact with androgen receptor (AR) in nucleoplasm. N6L treatment of prostate tumor cells led to inhibition of NPM1 phosphorylation in conjunction with inhibition of AR activity. We also found that total and phosphorylated NPM1 were overexpressed in castration-resistant PCa. Assessment of the potential therapeutic role of N6L in PCa indicated that N6L inhibited tumor growth both in vitro and in vivo when used either alone or in combination with the standard-of-care first- (hormonotherapy) and second-line (docetaxel) treatments for advanced PCa. Our findings reveal the role of Thr199 and Thr234/237 phosphorylated NPM1 in PCa progression and define N6L as a new drug candidate for PCa therapy.

Small Molecular Inhibitors Targeting Chromatin Regulating Proteins for Cancer.

Chromatin-regulating proteins modulate nucleosome structure by either modifying histones covalently or disrupting DNA-protein interaction directly with ATP hydrolysis. Evidence has shown that chromatin-regulating proteins play critical roles in regulation of molecular processes using DNA as template, including gene expression, DNA replication, DNA damage repair, and chromosome integrity. In most of human malignancies, chromatin-regulating proteins have been shown as functional oncogenes. In some scenarios, chromatin-regulating proteins also could have tumor suppressive functions. Thereby, small molecular inhibitors targeting chromatin-regulating proteins could be used for cancer therapies. Numerous small molecular inhibitors against chromatin-regulating proteins are recently developed by academic and industrial groups. These compounds are evaluated for antitumor effects in vitro and in vivo. Some of them have shown great potential to become a therapeutic drug for cancer, and is currently evaluated in clinical trials. A few compounds have been approved for clinical use in cancer treatment. In this review, we will focus on the recent progress on the development of small inhibitors of chromatin-regulating proteins for cancer therapy.

Influenza virus nucleoprotein: structure, RNA binding, oligomerization and antiviral drug target.

The nucleoprotein (NP) of influenza virus covers the viral RNA entirely and it is this NP-RNA complex that is the template for transcription and replication by the viral polymerase. Purified NP forms a dynamic equilibrium between monomers and small oligomers, but only the monomers can oligomerize onto RNA. Therefore, drugs that stabilize the monomers or that induce abnormal oligomerization may have an antiviral effect, as would drugs that interfere with RNA binding. Crystal structures have been produced for monomeric and dimeric mutants, and for trimers and tetramers; high-resolution electron microscopy structures have also been calculated for the viral NP-RNA complex. We explain how these structures and the dynamic oligomerization equilibrium of NP can be and have been used for anti-influenza drug development.

Adeno-associated viruses serotype 2-mediated RNA interference efficiently inhibits rabies virus replication in vitro and in vivo.

To investigate the potential of adeno-associated viruses serotype 2 (AAV2)-mediated RNA interference (RNAi) as an antiviral agent against rabies, recombinant AAV2 vectors expressing siRNA targeting the nucleoprotein (N) gene of rabies virus (RABV) (rAAV-N796) were constructed and evaluated. When NA cells pretreated with rAAV-N796 were challenged with RABV, there was a 37.8 ± 3.4% to 55.1 ± 5.3% reduction in RABV virus titer. When cells pre-challenged with RABV were treated with rAAV-N796, there was a 4.4 ± 1.4 to 28.8 ± 3.2% reduction in RABV virus titer. Relative quantification of RABV transcripts using real-time PCR and Western blot revealed that the knockdown of RABV-N gene transcripts was based on the rAAV-N796 inoculation titer. When any NA cells were treated with rAAV-N796 before or after challenged with RABV, significant reduction in virus titer was observed in both administrations. Mice treated intracerebrally with rAAV-N796 exhibited 50 ± 5.3 and 62.5 ± 4.7% protection when challenged intracerebrally or intramuscally, respectively, with lethal RABV. When mice treated intramuscularly with rAAV-N796 were challenged intramuscularly with lethal RABV, they exhibited 37.5 ± 3.7% protection. When mice were intracerebrally and intramuscularly with rAAV-N796 24 hr after exposure to RABV infection, they exhibited 25 ± 4.1% protection The N gene mRNA levels in the brains of challenged mice with three different administrations were reduced (55, 68, 32 and 25%, respectively). These results indicated that AAV2 vector-mediated siRNA delivery in vitro in NA cells inhibited RABV multiplication, inhibited RABV multiplication in vivo in the mice brain and imparted partial protection against lethal rabies. So, it may have a potential to be used as an alternative antiviral approach against rabies.

Structure-based discovery of the novel antiviral properties of naproxen against the nucleoprotein of influenza A virus.

The nucleoprotein (NP) binds the viral RNA genome and associates with the polymerase in a ribonucleoprotein complex (RNP) required for transcription and replication of influenza A virus. NP has no cellular counterpart, and the NP sequence is highly conserved, which led to considering NP a hot target in the search for antivirals. We report here that monomeric nucleoprotein can be inhibited by a small molecule binding in its RNA binding groove, resulting in a novel antiviral against influenza A virus. We identified naproxen, an anti-inflammatory drug that targeted the nucleoprotein to inhibit NP-RNA association required for NP function, by virtual screening. Further docking and molecular dynamics (MD) simulations identified in the RNA groove two NP-naproxen complexes of similar levels of interaction energy. The predicted naproxen binding sites were tested using the Y148A, R152A, R355A, and R361A proteins carrying single-point mutations. Surface plasmon resonance, fluorescence, and other in vitro experiments supported the notion that naproxen binds at a site identified by MD simulations and showed that naproxen competed with RNA binding to wild-type (WT) NP and protected active monomers of the nucleoprotein against proteolytic cleavage. Naproxen protected Madin-Darby canine kidney (MDCK) cells against viral challenges with the H1N1 and H3N2 viral strains and was much more effective than other cyclooxygenase inhibitors in decreasing viral titers of MDCK cells. In a mouse model of intranasal infection, naproxen treatment decreased the viral titers in mice lungs. In conclusion, naproxen is a promising lead compound for novel antivirals against influenza A virus that targets the nucleoprotein in its RNA binding groove.

Small interfering RNAs targeting the rabies virus nucleoprotein gene.

Rabies virus (RABV) infection continues to be a global threat to human and animal health, yet no curative therapy has been developed. RNA interference (RNAi) therapy, which silences expression of specific target genes, represents a promising approach for treating viral infections in mammalian hosts. We designed six small interfering (si)RNAs (N473, N580, N783, N796, N799 and N1227) that target the conserved region of the RABV challenge virus standard (CVS)-11 strain nucleoprotein (N) gene. Using a plasmid-based transient expression model, we demonstrated that N796, N580 and N799 were capable of significantly inhibiting viral replication in vitro and in vivo. These three siRNAs effectively suppressed RABV expression in infected baby hamster kidney-21 (BHK-21) cells, as evidenced by direct immunofluorescence assay, viral titer measurements, real-time PCR, and Western blotting. In addition, liposome-mediated siRNA expression plasmid delivery to RABV-infected mice significantly increased survival, compared to a non-liposome-mediated delivery method. Collectively, our results showed that the three siRNAs, N796, N580 and N799, targeting the N gene could potently inhibit RABV CVS-11 reproduction. These siRNAs have the potential to be developed into new and effective prophylactic anti-RABV drugs.

Development of an anti-influenza drug screening assay targeting nucleoproteins with tryptophan fluorescence quenching.

Recent studies have shown that NP (nucleoprotein), which possesses multiple functions in the viral life cycle, is a new potential anti-influenza drug target. NP inhibitors reliably induce conformational changes in NPs, and these changes may confer inhibition of the influenza virus. The six conserved tryptophan residues in NP can be used as an intrinsic probe to monitor the change in fluorescence of the tryptophan residues in the protein upon binding to an NP inhibitor. In the present study, we found that the fluorescence of recombinant NP proteins was quenched following the binding of available NP inhibitors (such as nucleozin) in a concentration- and time-dependent manner, which suggests that the inhibitor induced conformational changes in the NPs. The minimal fluorescence-quenching effect and weak binding constant of nucleozin to the swine-origin influenza virus H1N1pdm09 (SOIV) NP revealed that the SOIV is resistant to nucleozin. We have used the fluorescence-quenching property of tryptophans in NPs that were bound to ligands in a 96-well-plate-based drug screen to assess the ability of promising small molecules to interact with NPs and have identified one new anti-influenza drug, CSV0C001018, with a high SI value. This convenient method for drug screening may facilitate the development of antiviral drugs that target viruses other than the influenza virus, such as HIV and HBV.

Identification of influenza A nucleoprotein as an antiviral target.

Influenza A remains a significant public health challenge because of the emergence of antigenically shifted or highly virulent strains. Antiviral resistance to available drugs such as adamantanes or neuraminidase inhibitors has appeared rapidly, creating a need for new antiviral targets and new drugs for influenza virus infections. Using forward chemical genetics, we have identified influenza A nucleoprotein (NP) as a druggable target and found a small-molecule compound, nucleozin, that triggers the aggregation of NP and inhibits its nuclear accumulation. Nucleozin impeded influenza A virus replication in vitro with a nanomolar median effective concentration (EC(50)) and protected mice challenged with lethal doses of avian influenza A H5N1. Our results demonstrate that viral NP is a valid target for the development of small-molecule therapies.

Short hairpin RNA targeting NP mRNA inhibiting Newcastle disease virus production and other viral structural mRNA transcription.

Newcastle disease virus (NDV), formally recognized as avian paramyxovirus 1 (APMV-1), is the etiological agent of Newcastle disease (ND), an affliction which can cause severe losses in the poultry industry. Better understanding of the molecular basis of viral structural genes involved with production should contribute significantly toward the development of improved prophylactic and therapeutic reagents to control the infection. Here we show that a short hairpin RNA (shRNA) eukaryotic expression vector targeting nucleocapsid (NP) gene of NDV can potently inhibit NDV production in both primary cells and embryonated chicken eggs. Moreover, shRNA specific for NP abolished the accumulation of not only the corresponding mRNA but also P, HN, F, M gene mRNA. The findings reveal that newly synthesized NP mRNA is essential for NDV transcription and replication, and provide a basis for the development of shRNAs as a prophylaxis and therapy for NDV infection in poultry.

No essential role for tripeptidyl peptidase II for the processing of LCMV-derived T cell epitopes.

The proteasome is critically involved in the production of MHC class I-restricted T cell epitopes. Approximately 20% of all peptides generated by the proteasome are too large for direct presentation by MHC class I molecules. Reits et al. (Immunity 2004. 20: 495-506) suggested that a major portion of proteasomal products are larger than 15 amino acids and require further degradation by the tripeptidyl peptidase II (TPPII) before becoming ligands of MHC class I molecules. Using the well-characterized lymphocytic choriomeningitis virus (LCMV) model, the role of TPPII in the processing of several LCMV-derived T cell epitopes was investigated. In contrast to Reits' proposal, TPPII inhibition and TPPII overexpression experiments revealed that five out of six LCMV-derived CD8(+) T cell epitopes were not affected by inhibition of TPPII, while one epitope (GP276) was slightly reduced upon TPPII overexpression. Additionally, we demonstrated that the processing of two epitopes derived from ovalbumin and murine cytomegalovirus were not altered by TPPII inhibition. We propose that TPPII is not generally required for the production of MHC class I peptides, but the presentation of some peptides can be negatively affected by TPPII.