Viral Proteins Research Articles

The roles of rabies virus structural proteins in immune evasion and implications for vaccine development

Rabies is a zoonotic infectious disease that targets the nervous system of human and animals and has about 100% fatality rate without treatment. Rabies virus is a bullet-like viral particle composed of five structural proteins, including nucleoprotein (N), phosphorylated protein (P), matrix protein (M), glycoprotein (G), and large subunit (L) of RNA-dependent RNA polymerase. These multifunctional viral proteins also play critical roles in the immune escape by inhibiting specific immune responses in the host, resulting in massive replication of the virus in the nervous system and abnormal behaviors of patients such as brain dysfunction and hydrophobia, which ultimately lead to the death of patients. Herein, the role of five structural proteins of rabies virus in the viral replication and immune escape and its implication for the development of vaccines were systemically reviewed, so as to shed light on the understanding of pathogenic mechanism of rabies virus.

The Pentamer glycoprotein complex inhibits viral Immediate Early transcription during Human Cytomegalovirus infections

The Pentamer complex of Human Cytomegalovirus (HCMV) consists of the viral glycoproteins gH, gL, UL128, UL130, and UL131 and is incorporated into infectious virions. HCMV strains propagated extensively in vitro in fibroblasts carry UL128, UL130, or UL131 alleles that do not make a functional complex and thus lack Pentamer function. Adding functional Pentamer to such strains decreases virus growth in fibroblasts. Here, we show that the Pentamer inhibits productive HCMV replication in fibroblasts by repressing viral Immediate Early (IE) transcription. We show that ectopic expression of the viral IE1 protein, a target of Pentamer-mediated transcriptional repression, complements the growth defect of a Pentamer-positive virus. Furthermore, we show that the Pentamer also represses viral IE transcription in cell types where HCMV in vitro latency is studied. Finally, we identify UL130 as a functional subunit of the Pentamer for IE transcriptional repression and demonstrate that cyclic AMP Response Element (CRE) and NFkB sites within the Major Immediate Early Promoter that drives IE1 transcription contribute to this repression. We conclude that the HCMV Pentamer represses viral IE transcription.

SNX27:Retromer:ESCPE-1-mediated early endosomal tubulation impacts cytomegalovirus replication

IntroductionCytomegaloviruses (CMVs) extensively reorganize the membrane system of the cell and establish a new structure as large as the cell nucleus called the assembly compartment (AC). Our previous studies on murine CMV (MCMV)-infected fibroblasts indicated that the inner part of the AC contains rearranged early endosomes, recycling endosomes, endosomal recycling compartments and trans-Golgi membrane structures that are extensively tubulated, including the expansion and retention of tubular Rab10 elements. An essential process that initiates Rab10-associated tubulation is cargo sorting and retrieval mediated by SNX27, Retromer, and ESCPE-1 (endosomal SNX-BAR sorting complex for promoting exit 1) complexes.ObjectiveThe aim of this study was to investigate the role of SNX27:Retromer:ESCPE-1 complexes in the biogenesis of pre-AC in MCMV-infected cells and subsequently their role in secondary envelopment and release of infectious virions.ResultsHere we show that SNX27:Retromer:ESCPE1-mediated tubulation is essential for the establishment of a Rab10-decorated subset of membranes within the pre-AC, a function that requires an intact F3 subdomain of the SNX27 FERM domain. Suppression of SNX27-mediated functions resulted in an almost tenfold decrease in the release of infectious virions. However, these effects cannot be directly linked to the contribution of SNX27:Retromer:ESCPE-1-dependent tubulation to the secondary envelopment, as suppression of these components, including the F3-FERM domain, led to a decrease in MCMV protein expression and inhibited the progression of the replication cycle.ConclusionThis study demonstrates a novel and important function of membrane tubulation within the pre-AC associated with the control of viral protein expression.

Palmitoylation of SARS-CoV-2 Envelope protein is central to virus particle formation.

The Envelope (E) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an integral structural protein in the virus particles. However, its role in the assembly of virions and the underlying molecular mechanisms are yet to be elucidated, including whether the function of E protein is regulated by post-translational modifications. In the present study, we report that SARS-CoV-2 E protein is palmitoylated at C40, C43, and C44 by palmitoyltransferases zDHHC3, 6, 12, 15, and 20. Mutating these three cysteines to serines (C40/43/44S) reduced the stability of E protein, decreased the interaction of E with structural proteins Spike, Membrane, and Nucleocapsid, and thereby inhibited the production of virus-like particles (VLPs) and VLP-mediated luciferase transcriptional delivery. Specifically, the C40/43/44S mutation of E protein reduced the density of VLPs. Collectively, these results demonstrate that palmitoylation of E protein is vital for its function in the assembly of SARS-CoV-2 particles.IMPORTANCEIn this study, we systematically examined the biochemistry of palmitoylation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) E protein and demonstrated that palmitoylation of SARS-CoV-2 E protein is required for virus-like particle (VLP) production and maintaining normal particle density. These results suggest that palmitoylated E protein is central for proper morphogenesis of SARS-CoV-2 VLPs in densities required for viral infectivity. This study presents a significant advancement in the understanding of how palmitoylation of viral proteins is vital for assembling SARS-CoV-2 particles and supports that palmitoyl acyltransferases can be potential therapeutic targets for the development of SARS-CoV-2 inhibitors.

Conserved linear motif within the immediate early protein ORF45 promotes its engagement with KSHV lytic cycle-promoting forkhead transcription factors, FOXK1 and FOXK2.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a gammaherpesvirus that can cause several cancers, such as Kaposi sarcoma and primary effusion lymphoma (PEL). We and others have recently demonstrated that Forkhead box (FOX) transcription factors can be dysregulated by KSHV, and they can affect KSHV infection. Herein, we focus on dissecting the role of two FOXK subfamily members, FOXK1 and FOXK2, in the KSHV life cycle. FOXK proteins are key host regulators of cellular functions, yet their role in KSHV infection remains unknown. Here, we demonstrated that both FOXK proteins are essential for efficient KSHV lytic reactivation in PEL cells. FOXK1 and FOXK2 are unique as they are the only FOX proteins that contain a Forkhead-associated (FHA) domain. The FHA domain is a specialized protein binding domain that recognizes a short linear serine/threonine-rich (S/T) motif. Through an unbiased motif survey, we found that KSHV viral protein ORF45 and its gammaherpesvirus homologs contain a putative FHA-binding motif. ORF45 is an immediate early tegument protein, vital for lytic reactivation and virus production. We demonstrated that ORF45 uses its novel conserved motif to interact with the FHA domain containing FOXK factors in the nucleus of infected cells. A single-point mutation of the conserved threonine residue in the motif within ORF45 abolished the ORF45-FOXK1/2 interaction. Our data indicates that FOXK proteins interact with ORF45 homologs encoded by murine gammaherpesvirus 68 (MHV68) and Rhesus macaque rhadinovirus (RRV), and that the FHA domains of FOXK proteins are sufficient for their interactions, highlighting a conserved mechanism.IMPORTANCEThe dysregulation of Forkhead transcription factors contributes to many different human diseases, including cancers, but their impact on herpesvirus lifecycle and pathogenesis is less understood. Our study uncovers a critical pro-lytic function of the FOXK subfamily and its requirement for KSHV lytic reactivation in PEL. We found that FOXK proteins bind to a key immediate early KSHV protein ORF45 using its novel short linear S/T motif. Notably, even though ORF45 homologs in gammaherpesviruses are highly diverse, we identified a similar S/T short linear motif in ORF45 homologs and also showed an evolutionary conserved interaction between FOXK proteins and ORF45 homologs of MHV68 and RRV. Our study provides a basis for future studies in animal models to evaluate the role of FOXK proteins and the impact of their interactions with ORF45 in gammaherpesvirus infection and pathogenesis. Targeting these interactions could allow a novel way to limit gammaherpesvirus infections.

PhageScanner: a reconfigurable machine learning framework for bacteriophage genomic and metagenomic feature annotation

Bacteriophages are the most prolific organisms on Earth, yet many of their genomes and assemblies from metagenomic sources lack protein sequences with identified functions. While most bacteriophage proteins are structural proteins, categorized as Phage Virion Proteins (PVPs), a considerable number remain unclassified. Complicating matters further, traditional lab-based methods for PVP identification can be tedious. To expedite the process of identifying PVPs, machine-learning models are increasingly being employed. Existing tools have developed models for predicting PVPs from protein sequences as input. However, none of these efforts have built software allowing for both genomic and metagenomic data as input. In addition, there is currently no framework available for easily curating data and creating new types of machine learning models. In response, we introduce PhageScanner, an open-source platform that streamlines data collection for genomic and metagenomic datasets, model training and testing, and includes a prediction pipeline for annotating genomic and metagenomic data. PhageScanner also features a graphical user interface (GUI) for visualizing annotations on genomic and metagenomic data. We further introduce a BLAST-based classifier that outperforms ML-based models and an efficient Long Short-Term Memory (LSTM) classifier. We then showcase the capabilities of PhageScanner by predicting PVPs in six previously uncharacterized bacteriophage genomes. In addition, we create a new model that predicts phage-encoded toxins within bacteriophage genomes, thus displaying the utility of the framework.

Exogenous interactome analysis of bovine viral diarrhea virus-host using network based-approach and identification of hub genes and important pathways involved in virus pathogenesis

Bovine viral diarrhea (BVD) is one of the most important diseases in livestock, caused by BVD virus (BVDV). During the pathogenesis of the virus, many interactions occur between host and viral proteins. Studying these interactions can help better understand the pathogenesis of the virus, identify putative functional proteins, and find new treatment and prevention strategies. To this aim, a BVDV-host protein-protein interaction (PPI) network map was constructed using Cytoscape and analyzed with cytoHubba, Kyoto Encyclopedia of Genes and Genomics (KEGG), Gene Ontology (GO), and Protein Analysis Through Evolutionary Relationships (PANTHER). Npro with 125 connections had the greatest number of interactions with host proteins. CD46, EEF-2, and TXN genes were detected as hub genes using different ranking algorithms in cytoHubba. BVDV interactions with its host mainly focus on targeting translation, protein synthesis, and cellular metabolism pathways. Different classes of proteins including translational proteins, nucleic acid metabolism proteins, metabolite interconversion enzymes, and protein-modifying enzymes are affected by BVDV. These findings improve our understanding of the effects of the virus on the cell. Hub genes and key pathways identified in the present study can serve as targets for novel BVDV prevention or treatment strategies.

Two phylogenetic cohorts of the nucleocapsid protein NP and their correlation with the host range of influenza A viruses

Influenza A virus has a wide natural areal among birds, mammals and humans. One of the main regulatory adaptors of the virus host range is the major NP protein of the viral nucleocapsid. Phylogenetic analysis of the NP protein of different viruses has revealed the existence of two phylogenetic cohorts in human influenza virus population. Cohort I includes classical human viruses that caused epidemics in 1957, 1968, 1977. Cohort II includes the H1N1/2009pdm virus, which had a mixed avian-swine origin, but caused global human pandemic. Also, the highly virulent H5N1 avian influenza virus emerged in 2021 and caused outbreaks of lethal infections in mammals, including humans, appeared to have the NP gene of the second phylogenetic cohort and, therefore, by the type of adaptation to human is similar to the H1N1/2009pdm virus and seems to possess a high epidemic potential for humans. The data obtained shed light on pathways and dynamics of avian influenza viruses adaptation to humans and propose phylogenetic algorithm for systemic monitoring of dangerous virus strains to predict epidemic harbingers and take immediate preventive measures.

Bispecific Antibodies Provide Broad Neutralization of Emerging Beta-Coronaviruses by Targeting ACE2 and Viral Spikes

Human coronaviruses such as SARS-CoV, MERS-CoV, and SARS-CoV-2 have recurrently emerged as significant pathogens, causing severe respiratory illnesses and presenting challenges to monoclonal antibody therapeutics due to their rapid evolution, particularly the diverse variants of SARS-CoV-2. In this study, we utilized ‘Knob-into-Hole’ and ‘IgG-scFv’ technologies to engineer bispecific antibodies (bsAbs) that target both the viral receptor and spike protein, enhancing their neutralization breadth and potency. Our bsAbs, combining anti-SARS-CoV-2 or anti-MERS-CoV antibodies with an anti-ACE2 antibody, demonstrated effective neutralization across a range of SARS-CoV-2 variants, SARS-CoV and MERS-CoV in both pseudovirus and authentic virus assays. Notably, the ‘IgG-scFv’ bsAbs format exhibited superior binding and neutralization capabilities compared to the ‘Knob-into-Hole’ configurations. The most effective of these, ‘IgG-scFv’ H11B11_m336, displayed exceptional neutralization potency against a panel of 24 pseudotyped Beta-Coronaviruses, with IC50 values ranging from 0.001 to 0.183 μg/mL. Overall, our findings underscore the potential of bsAbs as an effective strategy to meet the immediate challenges posed by existing and emerging pathogens, thereby enhancing global pandemic preparedness.

Strategic Design of Tryptophan-Aspartic Acid-Containing Peptide Inhibitors Using Coronin 1 as a Template: Inhibition of Fusion by Enhancing Acyl Chain Order.

Enveloped viruses enter the host cell by fusing at the cell membrane or entering the cell via endocytosis and fusing at the endosome. Conventional inhibitors target the viral fusion protein to inactivate it for inducing fusion. These target-specific vis-à-vis virus-specific inhibitors fail to display their inhibitory efficacy against emerging and remerging viral infections. This necessitates the need to develop broad-spectrum entry inhibitors that are effective irrespective of the virus. Using a broad range of targeting techniques, the fusion inhibitors can modify the physical characteristics of the viral membrane, making it less prone to fusion. We have previously shown that two tryptophan-aspartic acid (WD)-containing hydrophobic peptides, TG-23 and GG-21, from coronin 1, a phagosomal protein, inhibit membrane fusion by modulating membrane organization and dynamics. In the present work, we designed two WD-containing hydrophilic peptides, QG-22 and AG-22, using coronin 1 as a template and evaluated their fusion inhibitory efficacies in the absence and presence of membrane cholesterol. Our results demonstrate that QG-22 and AG-22 inhibit membrane fusion irrespective of the concentration of membrane cholesterol. Our measurements of depth-dependent membrane organization and dynamics reveal that they impede fusion by enhancing the acyl chain order. Overall, our results validate the hypothesis of designing fusion inhibitors by modulating the membrane's physical properties. In addition, it demonstrates that chain hydrophobicity might not be a critical determinant for the development of peptide-based fusion inhibitors.

In-Depth Host Cell Protein Analysis and Viral Protein Impurity Monitoring in Adeno-Associated Virus-Based Gene Therapy Products Using Optimized Wide Window Data-Dependent Acquisition Method.

Compared to other protein therapeutics, there is currently limited knowledge about the residual host cell proteins (HCPs) in adeno-associated virus (AAV)-based gene therapy products. This is primarily due to the lack of a robust and sensitive mass spectrometry-based method for HCP analysis in AAV samples. Existing liquid chromatography-mass spectrometry methods used for analyzing HCPs in therapeutic monoclonal antibodies (mAbs) often cannot be directly applied to AAVs, due to some unique characteristics of AAV samples encountered during their development such as limited sample availability/protein concentration and the presence of surfactants. In this study, we have developed a novel workflow for robust and in-depth HCP analysis of AAV samples by combining wide-window data-dependent acquisition for improved low-abundance HCP detection with single-pot, solid-phase-enhanced sample preparation (SP3) for low-input sample preparation. Using this newly developed method, we were able to detect more than 650 HCPs in a commercial AAV1 sample with a high quantitative reproducibility. This represents a greater than 5-fold increase in HCP protein identification compared to an in-solution digestion method followed by traditional data-dependent acquisition. Similar benefits can also be achieved for other AAV serotypes that were produced internally and purified through different processes. The detection limit of this method is as low as 0.06 ng/mL, enabling more comprehensive HCP coverage in AAV samples. Moreover, for the first time, we have identified several process-related viral proteins, such as Rep 78 and E4. These proteins need to be closely monitored during AAV process development as they may present a greater risk for immunogenicity compared to HCPs that are derived from human HEK293 cells.

Targeting SARS-CoV-2 proteins: In Silico Investigation with Polypyridyl-based Zn(II)-Curcumin Complexes.

Herein, we have selected eight Zn(II)-based complexes viz., [Zn(bpy)(acac)Cl] (1), [Zn(phen)(acac)Cl] (2), [Zn(dppz)(acac)Cl] (3), [Zn(dppn)(acac)Cl] (4), [Zn(bpy)(cur)Cl] (5), [Zn(phen)(cur)Cl] (6), [Zn(dppz)(cur)Cl] (7), [Zn(dppn)(cur)Cl] (8), where bpy=2,2'-bipyridine, phen=1,10-phenanthroline, dppz=benzo[i]dipyrido[3,2-a:2',3'-c]phenazine, dppn=naphtho[2,3-i]dipyrido[3,2-a:2',3'-c]phenazine, acac=acetylacetonate, cur=curcumin and performed in silico molecular docking studies with the viral proteins, i.e., spike protein (S), Angiotensin-converting enzyme II Receptor protein (ACE2), nucleocapsid protein (N), main protease protein (Mpro), and RNA-dependent RNA polymerase protein (RdRp) of SARS-CoV-2. The binding energy calculations, visualization of the docking orientation, and analysis of the interactions revealed that these complexes could be potential inhibitors of the viral proteins. Among complexes 1-8, complex 6 showed the strongest binding affinity with S and ACE2 proteins. 4 exerted better binding affinity in the case of the N protein, whereas 8 presented the highest binding affinities with Mpro and RdRp among all the complexes. Overall, the study indicated that Zn(II) complexes have the potential as alternative and viable therapeutic solutions for COVID-19.

Characterization of bi-segmented and tri-segmented recombinant Pichinde virus particles.

Mammarenaviruses include several highly virulent pathogens (e.g., Lassa virus) capable of causing severe hemorrhagic fever diseases for which there are no approved vaccines and limited treatment options. Mammarenaviruses are enveloped, bi-segmented ambisense RNA viruses. There is limited knowledge about cellular proteins incorporated into progeny virion particles and their potential biological roles in viral infection. Pichinde virus (PICV) is a prototypic arenavirus used to characterize mammarenavirus replication and pathogenesis. We have developed a recombinant PICV with a tri-segmented RNA genome as a viral vector platform. Whether the tri-segmented virion differs from the wild-type bi-segmented one in viral particle morphology and protein composition has not been addressed. In this study, recombinant PICV (rPICV) virions with a bi-segmented (rP18bi) and a tri-segmented (rP18tri) genome were purified by density-gradient ultracentrifugation and analyzed by cryo-electron microscopy and mass spectrometry. Both virion types are pleomorphic with spherical morphology and have no significant difference in size despite rP18tri having denser particles. Both virion types also contain similar sets of cellular proteins. Among the highly enriched virion-associated cellular proteins are components of the endosomal sorting complex required for transport pathway and vesicle trafficking, such as ALIX, Tsg101, VPS, CHMP, and Ras-associated binding proteins, which have known functions in virus assembly and budding. Other enriched cellular proteins include peripheral and transmembrane proteins, chaperone proteins, and ribosomal proteins; their biological roles in viral infection warrant further analysis. Our study provides important insights into mammarenavirus particle formation and aids in the future development of viral vectors and antiviral discovery.IMPORTANCEMammarenaviruses, such as Lassa virus, are enveloped RNA viruses that can cause severe hemorrhagic fever diseases (Lassa fever) with no approved vaccine and limited therapeutic options. Cellular proteins incorporated into progeny virion particles and their biological roles in mammarenavirus infection have not been well characterized. Pichinde virus (PICV) is a prototypic mammarenavirus used as a surrogate model for Lassa fever. We used cryo-electron microscopy and proteomic analysis to characterize the morphology and protein contents of the purified PICV particles that package either two (bi-segmented) or three (tri-segmented) genomic RNA segments. Our results demonstrate a similar virion morphology but different particle density for the bi- and tri-segmented viral particles and reveal major virion-associated cellular proteins. This study provides important insights into the virus-host interactions that can be used for antiviral development and optimizing arenavirus-based vaccine vectors.

Lactoferrin: A Promising Therapeutic Molecule against Human Papillomavirus

Lactoferrin is a multifunctional glycoprotein naturally found in mammalian secretions, predominantly in colostrum and milk. As a key component of dairy foods, lactoferrin enhances viral protection and boosts human health, owing to its fundamental properties including antiviral, anti-inflammatory, and immune-modulatory effects. Importantly, the antiviral effect of lactoferrin has been shown against a range of viruses causing serious infections and threatening human health. One of the viruses that lactoferrin exerts significant antiviral effects on is the human papillomavirus (HPV), which is the most prevalent transmitted infection affecting a myriad of people around the world. Lactoferrin has a high potential to inhibit HPV via different mechanisms, including direct binding to viral envelope proteins or their cell receptors, thereby hindering viral entry and immune stimulation by triggering the release of some immune-related molecules through the body, such as lymphocytes. Along with HPV, lactoferrin also can inhibit a range of viruses including coronaviruses and hepatitis viruses in the same manner. Here, we overview the current knowledge of lactoferrin and its effects on HPV and other viral infections.

The Epstein-Barr virus nuclear antigen 1 variant associated with nasopharyngeal carcinoma defines the sequence criteria for serologic risk prediction.

Antibodies to select Epstein-Barr virus (EBV) proteins can diagnose early-stage nasopharyngeal carcinoma (NPC). We have previously shown that IgA against EBV nuclear antigen 1 (EBNA1) can predict incident NPC in high- and intermediate-risk cohorts 4 years pre-diagnosis. Here, we tested EBNA1 variants, with mutants, to define the sequence requirements for an NPC risk assay. Mammalian-expressed constructs were developed to represent EBNA1 variants 487V and 487A which can differ by ≥15 amino acids in the N- and C-termini. Denatured lysates were evaluated by a refined IgA and IgG immunoblot assay in a case-control study using pre-diagnostic NPC sera from two independent cohorts in Singapore and Shanghai, P.R. China. At 95% sensitivity, 487V yielded a 94.9% specificity compared to 86.1% for 487A. EBNA1 deleted for the conserved glycine-alanine repeats (GAr) reduced false positives by 22.8%. NPC sera reacted more strongly to the C-terminus than healthy controls, but the C-terminal construct (a.a. 390-641) showed lower specificity (84.8%) than the EBNA1 GAr deleted construct (92.4%) at 95% sensitivity. Although EBNA1 IgA was present in healthy sera, most epitopes localized to the immunodominant GAr. We conclude that a refined EBNA1 antigen deleted for the GAr but with residues consistently detected in Southeast Asian NPC tumors is optimal for risk prediction with an extended sojourn time of 7.5 years. Furthermore, distinct EBNA1 serologic profiles enhanced the utility of the EBNA1 IgA assay for risk stratification. This illustrates the importance of serologically relevant EBNA1 sequences for NPC risk prediction and early detection.

Phytochemicals of Different Medicinal Herbs as Potential Inhibitors Against Dengue Serotype 2 Virus: A Computational Approach.

Dengue is one of the major mosquito-borne infectious diseases of the present century, reported to affect about 100-400 million people globally. The lack of effective therapeutic options has inspired several in vitro and in silico studies for the search of antivirals. Our previous study revealed the anti-dengue activity of different plant extracts from Plumeria alba, Bacopa monnieri, Vitex negundo, and Ancistrocladus heyneanus. Therefore, the current in silico study was designed to identify the phytochemicals present in the aforementioned plants, which are possibly responsible for the anti-dengue activity. Different plant databases as well as relevant literature were explored to find out the major compounds present in the above-stated plants followed by screening of the retrieved phytochemicals for the assessment of their binding affinity against different dengue viral proteins via molecular docking. The best poses of protein-ligand complexes obtained after molecular docking were selected for the calculation of binding free energy via MM-GBSA method. Based on the highest docking score and binding energy, six complexes were considered for further analysis. To analyze the stability of the complex, 100ns molecular dynamics (MD) simulations were carried out using Desmond module in the Schrodinger suite. The MD simulation analysis showed that four compounds viz. liriodendrin, bacopaside VII, isoorientin, and cynaroside exhibited stability with viral targets including the RdRp, NS3 helicase, and E protein indicating their potential as novel anti-dengue antivirals.

Highly-Efficient Selection of Aptamers for Quantitative Fluorescence Detecting Multiple IAV Subtypes.

Influenza A virus (IAV) can cause infectious respiratory diseases in humans and animals. IAVs mutate rapidly through antigenic drift and shift, resulting in the emergence of numerous IAV subtypes and significant challenges for IAV detection. Therefore, achieving the simultaneous detection of multiple IAVs is crucial. In this work, three specific aptamers targeting the hemagglutination (HA) protein of the influenza A H5N1, H7N9, and H9N2 viruses were screened using a multichannel magnetic microfluidic chip. The aptamers exhibit nanomolar affinity and excellent specificity for the HA protein of H5N1, H7N9, and H9N2 viruses. Furthermore, three specific aptamers were truncated and labeled with different fluorescence markers to realize fluorescence quantitative detection of influenza A H5N1, H7N9, and H9N2 viruses through an aptamer sandwich assay in 1 h. The limit of detection (LOD) of the developed method is 0.38 TCID50/mL for the H5N1 virus, 0.75 TCID50/mL for the H7N9 virus, and 1.14 TCID50/mL for the H9N2 virus. The detection method has excellent specificity, strong anti-interference ability, and good reproducibility. This work provides a sensitive quantitative detection method for the H5N1, H7N9, and H9N2 viruses, enabling quantitative fluorescence detection for multiple IAV subtypes.

Release of P-TEFb from the Super Elongation Complex promotes HIV-1 latency reversal.

The persistence of HIV-1 in long-lived latent reservoirs during suppressive antiretroviral therapy (ART) remains one of the principal barriers to a functional cure. Blocks to transcriptional elongation play a central role in maintaining the latent state, and several latency reversal strategies focus on the release of positive transcription elongation factor b (P-TEFb) from sequestration by negative regulatory complexes, such as the 7SK complex and BRD4. Another major cellular reservoir of P-TEFb is in Super Elongation Complexes (SECs), which play broad regulatory roles in host gene expression. Still, it is unknown if the release of P-TEFb from SECs is a viable latency reversal strategy. Here, we demonstrate that the SEC is not required for HIV-1 replication in primary CD4+ T cells and that a small molecular inhibitor of the P-TEFb/SEC interaction (termed KL-2) increases viral transcription. KL-2 acts synergistically with other latency reversing agents (LRAs) to reactivate viral transcription in several cell line models of latency in a manner that is, at least in part, dependent on the viral Tat protein. Finally, we demonstrate that KL-2 enhances viral reactivation in peripheral blood mononuclear cells (PBMCs) from people living with HIV (PLWH) on suppressive ART, most notably in combination with inhibitor of apoptosis protein antagonists (IAPi). Taken together, these results suggest that the release of P-TEFb from cellular SECs may be a novel route for HIV-1 latency reactivation.

Blockade of endothelin receptors mitigates SARS-CoV-2-induced osteoarthritis.

Joint pain and osteoarthritis can occur as coronavirus disease 2019 (COVID-19) sequelae after infection. However, little is known about the damage to articular cartilage. Here we illustrate knee joint damage after wild-type, Delta and Omicron variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in vivo. Rapid joint injury with cystic lesions at the osteochondral junction was observed in two patients with post-COVID osteoarthritis and recapitulated in a golden Syrian hamster model. SARS-CoV-2-activated endothelin-1 signalling increased vascular permeability and caused viral spike proteins leakage into the subchondral bone. Osteoclast activation, chondrocyte dropout and cyst formation were confirmed histologically. The US Food and Drug Administration-approved endothelin receptor antagonist, macitentan, mitigated cystic lesions and preserved chondrocyte number in the acute phase of viral infection in hamsters. Delayed macitentan treatment at post-acute infection phase alleviated chondrocyte senescence and restored subchondral bone loss. It is worth noting that it could also attenuate viral spike-induced joint pain. Our work suggests endothelin receptor blockade as a novel therapeutic strategy for post-COVID arthritis.

Defense Responses Induced by Viral Movement Protein and Its Nuclear Localization Modulate Virus Cell-to-Cell Transport

Defense Responses Induced by Viral Movement Protein and Its Nuclear Localization Modulate Virus Cell-to-Cell Transport