Viral Proteins Research Articles

SERINC5 counters retroviruses and non-retroviruses.

SERINC5 (serine incorporator 5), a member of the serine incorporator family, has been identified as a retrovirus restriction factor that inhibits the fusion of virions with the plasma membrane, thus blocking the release of the viral core into target cells and subsequently attenuating viral infectivity. Several viruses, such as human immunodeficiency virus (HIV), murine leukemia virus (MLV), and equine infectious anemia virus (EIAV), have evolved mechanisms to antagonize the host protein SERINC5 through HIV Nef, MLV glycosylated Gag, and the EIAV S2 protein. These viral proteins degrade SERINC5 on the cell surface through the endolysosomal system. In addition to its direct antiviral ability, SERINC5 also modulates immunity to inhibit the replication of retroviruses and nonretroviruses. This review summarizes the interaction between SERINC5 and viral replication, providing a promising avenue for fighting viral diseases.

Computational Insights on the Assembly of the Dengue Virus Membrane-Capsid-RNA Complex.

Dengue virus, an arbovirus from the genus Flavivirus in the family Flaviviridae, forms a nucleocapsid structure through interactions between its genome and multiple copies of the capsid protein. Experimental studies have confirmed the interaction between the viral capsid protein and lipid droplets, indicating a protein-lipid interaction. Cryo-EM studies show that in immature viruses, the nucleocapsid is located close to the viral membrane. This study uses multiple MD simulations to explore the orientation of the capsid protein relative to the lipid membrane, focusing on how the protein's hydrophobic pocket interacts with the membrane. We also investigated the interaction between the capsid protein and RNA, considering the effects of sequence length and identity. Finally, we construct a model of the lipid-protein-RNA complex, demonstrating that the capsid protein's hydrophobic pocket interacts with the membrane, while the positively charged H4 helix interacts with the negatively charged RNA. This research may identify crucial interactions for immature virus particle formation and provide insights for future therapeutic interventions.

Accessory viral protein, V, of Newcastle Disease Virus binds dsRNA to facilitate immune evasion

Accessory viral protein, V, of Newcastle Disease Virus binds dsRNA to facilitate immune evasion

Supervised learning approaches for predicting Ebola-Human Protein-Protein interactions.

The goal of this research work is to predict protein-protein interactions (PPIs) between the Ebola virus and the host who is at risk of infection. Since there are very limited databases available on the Ebola virus; we have prepared a comprehensive database of all the PPIs between the Ebola virus and human proteins (EbolaInt). Our work focuses on the finding of some new protein-protein interactions between humans and the Ebola virus using some state- of-the-arts machine learning techniques. However, it is basically a two-class problem with a positive interacting dataset and a negative non-interacting dataset. These datasets contain various sequence-based human protein features such as structure of amino acid and conjoint triad and domain-related features. In this research, we have briefly discussed and used some well-known supervised learning approaches to predict PPIs between human proteins and Ebola virus proteins, including K-nearest neighbours (KNN), random forest (RF), support vector machine (SVM), and deep feed-forward multi-layer perceptron (DMLP) etc. We have validated our prediction results using gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Our goal with this prediction is to compare all other models' accuracy, precision, recall, and f1-score for predicting these PPIs. In the result section, DMLP is giving the highest accuracy along with the prediction of 2655 potential human target proteins.

Exploring Marine Natural Products as Antiviral Agents, Advances and Emerging Opportunities

Marine natural products have emerged as a promising source of antiviral agents, offering unique chemical diversity and bioactivity that are valuable for combatting viral infections. The recent advances in the discovery, characterization, and antiviral efficacy of marine-derived compounds, including peptides, polysaccharides, alkaloids, and terpenes, against viruses such as HIV, hepatitis, influenza, and coronaviruses. The mechanisms by which these marine natural products inhibit viral replication vary, targeting key viral proteins, entry pathways, and replication machinery, providing diverse modes of action that can help circumvent viral resistance. Advances in extraction, synthesis, and bioassay screening technologies have accelerated the identification of potent antiviral candidates from marine organisms, such as sponges, algae, fungi, and marine bacteria, the potential for novel marine-derived antiviral therapies is expanding, supported by new insights into molecular mechanisms and promising preclinical results. The advances, challenges remain, including sustainable harvesting, compound stability, and regulatory considerations. The perspectives in optimizing the drug-like properties of marine antivirals, scaling up production, and integrating these compounds into broader antiviral therapy pipelines, exploration of marine biodiversity holds substantial promise for addressing current and emerging viral threats.

The Development of a Novel Broad-Spectrum Influenza Polypeptide Vaccine Based on Multi-Epitope Tandem Sequences.

Polypeptide vaccines have the potential to improve immune responses by targeting conserved and weakly immunogenic regions in antigens. This study aimed to identify and evaluate the efficacy of a novel influenza universal vaccine candidate consisting of multiple polypeptides derived from highly conserved regions of influenza virus proteins hemagglutinin (HA), neuraminidase (NA), and matrix protein 2 (M2). Immunoinformatics tools were used to screen conserved epitopes from different influenza virus subtypes (H1N1, H3N2, H5N1, H7N9, H9N2, and IBV). A polypeptide vaccine, P125-H, was constructed by linking multiple epitopes using Ii-Key technology. The immunogenicity of P125-H was assessed in mice using MF59-adjuvanted P125-H via intraperitoneal injection. Hemagglutination inhibition (HI) and neutralizing antibody responses were measured, along with IFN-γ levels in spleen lymphocytes. Protective efficacy was evaluated using viral challenge with lethal doses of H1N1 and H7N9. Mice immunized with P125-H generated high levels of HI and neutralizing antibodies against multiple influenza strains. IFN-γ production was significantly elevated in spleen lymphocytes upon stimulation with the vaccine. P125-H protected mice from influenza infection, reducing weight loss and the viral load in the lungs, mitigating lung pathology, and decreasing mortality. The P125-H vaccine induced broad cross-protection against multiple influenza strains and elicited robust immune responses. It demonstrates strong potential as a candidate for a universal influenza vaccine.

Vaccinia growth factor-dependent modulation of the mTORC1-CAD axis upon nutrient restriction.

The molecular mechanisms by which vaccinia virus (VACV), the prototypical member of the poxviridae family, reprograms host cell metabolism remain largely unexplored. Additionally, cells sense and respond to fluctuating nutrient availability, thereby modulating metabolic pathways to ensure cellular homeostasis. Understanding how VACV modulates metabolic pathways in response to nutrient signals is crucial for understanding viral replication mechanisms, with the potential for developing antiviral therapies. In this study, we establish the importance of de novo pyrimidine synthesis during VACV infection. We report the significance of vaccinia growth factor (VGF), a viral early protein and a homolog of cellular epidermal growth factor (EGF), in enabling VACV to phosphorylate the key enzyme CAD of the de novo pyrimidine pathway at serine 1859, a site known to positively regulate CAD activity. Although nutrient-poor conditions typically inhibit mTORC1 activation, VACV activates CAD via the mTORC1-S6K1 signaling axis in a VGF-dependent manner, especially upon glutamine and asparagine limitation. However, unlike its cellular homolog EGF, the VGF peptide alone, in the absence of VACV infection, has minimal ability to activate CAD. This suggests the involvement of other viral factors yet to be identified. Our research provides a foundation for understanding the regulation of a significant metabolic pathway, de novo pyrimidine synthesis during VACV infection, shedding new light on viral regulation under distinct nutritional environments. This study not only has the potential to contribute to the advancement of antiviral treatments but also improve the development of VACV as an oncolytic agent and vaccine vector.IMPORTANCEViruses often reprogram host cell metabolism to facilitate replication. How poxviruses, such as the prototype member, vaccinia virus (VACV), modulate host cell metabolism is not well understood. Understanding how VACV affects these metabolic pathways is key to learning about viral replication and developing antiviral treatments. This study highlights the importance of de novo pyrimidine synthesis during VACV infection. We found that the vaccinia growth factor (VGF), a viral protein similar to the cellular epidermal growth factor (EGF), helps VACV activate the enzyme CAD of the de novo pyrimidine pathway. Upon nutrient limitation, VGF is needed for the activation of CAD through mTORC1-S6K signaling. VGF peptide alone is unable to activate this pathway independent of infection, suggesting the involvement of other viral factor(s). Our research not only sheds light on how VACV regulates metabolism but also holds promise for improving VACV as a cancer treatment and vaccine.

Molecular epidemiology of human papillomavirus variants in cervical cancer in India

Background & objectives Cervical cancer (CC) has been documented as the fourth most common cancer worldwide. Persistent infections with high-risk human papillomavirus (hr-HPV) have been suggested in the development of CC. Although prophylactic vaccines are available for the prevention of prevalent hr-HPV types, intra-type variations exist within a particular HPV type that has varying oncogenic potential as well as the mechanism of pathogenicity and varying neutralization by antibodies. Therefore, we carried out a systematic review to determine the distribution of HPV intra-typic variations in different geographical locations of India and their reported implications. Methods Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines were followed to retrieve relevant articles from the standard databases using appropriate keywords. Consequently, 17 articles were included in the current review after screening based on inclusion and exclusion criteria. Results The majority of articles included in this review reported variations within the HPV16 E6 gene, followed by the L1 and E7 genes. Analysis of available data indicated the differential regional distribution of some variations. These variations have also been reported to impact the biological functions of various viral proteins. Interpretation & conclusions The distribution of lineages varied with the different genomic regions sequenced. Additionally, there were certain unique and common variations in the HPV genome with respect to geographical regions. Hence, we suggest the identification of region-specific variations for the development of diagnostic and prognostic interventions.

RNA viruses, TLRs, and cytokines: the perfect storm?

The interactions between virus and the host immune response are nuanced and intricate. The cytokine response arguably plays a central role in dictating the outcome of virus infection, balancing inflammation and healing, which is crucial to resolving infection without destructive immunopathologies. Early innate immune responses are key to the generation of a beneficial or detrimental immune response. These initial responses are regulated by a plethora of surface bound, endosomal, and cytoplasmic innate immune receptors known as pattern recognition receptors. Of these, the Toll like receptors (TLRs) play an important role in the induction of cytokines during virus infection. Recognizing pathogen-associated molecular patterns (PAMPs) such as viral proteins and/or nucleotide sequences, the TLRs act as sentinels for the initiation and propagation of immune responses. TLRs are important receptors for initiating the innate response to single-stranded RNA (ssRNA) viruses like influenza A virus (IAV), severe acute respiratory syndrome coronavirus-1 (SARS-CoV-1), SARS-CoV-2, Middle East respiratory syndrome coronavirus (MERS-CoV), dengue virus and Ebola virus. Infection with these viruses is also associated with aberrant expression of proinflammatory cytokines that contribute to a harmful cytokine storm response. Herein we discuss the connections between these ssRNA viruses, cytokine storm, and the roles for TLRs.

Ultra-Sensitive Aptamer-Based Diagnostic Systems for Rapid Detection of All SARS-CoV-2 Variants.

The emergence of numerous SARS-CoV-2 variants, characterized by mutations in the viral RNA genome and target proteins, has presented challenges for accurate COVID-19 diagnosis. To address this, we developed universal aptamer probes capable of binding to the spike proteins of SARS-CoV-2 variants, including highly mutated strains like Omicron. These aptamers were identified through protein-based SELEX using spike proteins from three key variants (D614G-substituted Wuhan-Hu-1, Delta, and Omicron) and virus-based SELEX, known as viro-SELEX. Leveraging these universal aptamers, we created a highly sensitive lateral flow assay (LFA) and an ultra-sensitive molecular diagnostic platform that integrates a novel rapid PCR technique, enabling fast and reliable detection across all SARS-CoV-2 variants.

PROTAR Vaccine 2.0 generates influenza vaccines by degrading multiple viral proteins.

Manipulating viral protein stability using the cellular ubiquitin-proteasome system (UPS) represents a promising approach for developing live-attenuated vaccines. The first-generation proteolysis-targeting (PROTAR) vaccine had limitations, as it incorporates proteasome-targeting degrons (PTDs) at only the terminal ends of viral proteins, potentially restricting its broad application. Here we developed the next-generation PROTAR vaccine approach, referred to as PROTAR 2.0, which enabled flexible incorporation of PTDs at various genomic loci of influenza viruses, including internal regions and terminal ends. The PROTAR 2.0 influenza viruses maintained efficient replication in UPS-deficient cells for large-scale production but were attenuated by PTD-mediated proteasomal degradation of viral proteins in conventional cells. Incorporation of multiple PTDs into one virus generated optimized PROTAR 2.0 vaccine candidates. In animal models, PROTAR 2.0 vaccine candidates were highly attenuated and a single-dose intranasal immunization induced robust and broad immune responses that provided complete cross-reactive protection against both homologous and heterologous viral challenges.

Investigating the reassortment potential and pathogenicity of the S segment in Akabane virus using a reverse genetics system

BackgroundAkabane virus (AKAV) is an arthropod-borne virus that causes congenital malformations and neuropathology in cattle and sheep. In South Korea, AKAVs are classified into two main genogroups: K0505 and AKAV-7 strains. The K0505 strain infects pregnant cattle, leading to fetal abnormalities, while the AKAV-7 strain induces encephalomyelitis in post-natal cattle. The pathogenicities of K0505 and AKAV-7 strains differ significantly; however, the specific gene in the AKAV-7 strain that drives its pathogenicity remains unidentified. In this study, changes in viral replication and pathogenicity were investigated, particularly when the S segment of AKAV-7 was mutated using a T7 RNA polymerase-based reverse genetics (RG) system.ResultsThe rAKAV-7ΔNSs virus, with a deletion in the NSs protein of the wild-type AKAV-7 virus (wtAKAV-7), and the rAKAV-7(S-K0505) virus, where the S segment of wtAKAV-7 was reassorted with that from the wild type K0505 strain (wtK0505), were successfully rescued. The rAKAV-7ΔNSs virus demonstrated impaired replication in Vero cells and exhibited reduced mortality and RNA viral load in the organs of suckling mice compared to the wtAKAV-7. The rAKAV-7(S-K0505) virus displayed similar growth kinetics in Vero cells and showed no significant reduction in mortality rate in suckling mice compared to wtAKAV-7.ConclusionsThese observations suggest that the S segment, especially the NS protein, is associated with the pathogenicity of AKAV-7. Also, the results imply that the L and M segments might explain the differences in pathogenicity between the AKAV-7 and K0505 strains. Moreover, our findings indicate the potential for reassortment between distinct genogroups of AKAVs.

Repositioning of Furin inhibitors as potential drugs against SARS-CoV-2 through computational approaches

The recent spread of SARS-CoV-2 has led to serious concerns about newly emerging infectious coronaviruses. Drug repurposing is a practical method for rapid development of antiviral agents. The viral spike protein of SARS-CoV-2 binds to its major receptor ACE2 to promote membrane fusion. Following the entry process, the spike protein is further activated by cellular proteases such as TMPRSS2 and Furin to promote viral entry into human cells. A crucial factor in preventing SARS-CoV-2 from entering target cells using HIV-1 fusion inhibitors is the similarity between the fusion mechanisms of SARS-CoV-2 and HIV-1. In this investigation, the HIV-1 fusion inhibitors CMK, Luteolin, and Naphthofluorescein were selected to understand the molecular mode of interactions and binding energy of Furin with these experimental inhibitors. The binding affinity of the three inhibitors with Furin was verified by molecular docking studies. The docking scores of CMK, Luteolin and Naphthofluorescein are −7.4 kcal/mol, −9.3 kcal/mol, and −10.7 kcal/mol, respectively. Therefore, these compounds were subjected to MD, drug-likeness, ADMET, and MM-PBSA analysis. According to the results of a 200 ns MD simulation, all tested compounds show stability with the complex and can be employed as promising inhibitors targeting SARS-CoV-2 Furin protease. In addition, pharmacokinetic analysis revealed that these compounds possess favorable drug-likeness properties. Thus, this study of Furin inhibitors helps in the evaluation of these compounds for use as novel drugs against SARS-CoV-2.

Pseudotyped Viruses: A Useful Platform for Pre-Clinical Studies Conducted in a BSL-2 Laboratory Setting.

The study of pathogenic viruses has always posed significant biosafety challenges. In particular, the study of highly pathogenic viruses requires methods with low biological risk but relatively high sensitivity and convenience in detection. In recent years, pseudoviruses, which consist of a backbone of one virus and envelope proteins of another virus, have become one of the most widely used tools for exploring the mechanisms of viruses binding to cells, membrane fusion and viral entry, as well as for screening the libraries of antiviral substances, evaluating the potential of neutralizing monoclonal antibodies, developing neutralization tests, and therapeutic platforms. During the outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), pseudotyped virus-based assays played a pivotal role in advancing our understanding of virus-cell interactions and the role of its proteins in disease pathogenesis. Such tools facilitated the search for potential therapeutic agents and accelerated epidemiological studies on post-infection and post-vaccination humoral immunity. This review focuses on the use of pseudoviruses as a model for large-scale applications to study enveloped viruses.

Proteolysis-targeting influenza vaccine strains induce broad-spectrum immunity and in vivo protection.

Generating effective live vaccines from intact viruses remains challenging owing to considerations of safety and immunogenicity. Approaches that can be applied in a systematic manner are needed. Here we created a library of live attenuated influenza vaccines by using diverse cellular E3 ubiquitin ligases to generate proteolysis-targeting (PROTAR) influenza A viruses. PROTAR viruses were engineered to be attenuated by the ubiquitin-proteasome system, which mediates viral protein degradation in conventional host cells, but allows efficient replication in engineered cell lines for large-scale manufacturing. Depending on the degron-E3 ligase pairs, viruses showed varying degrees of attenuation. In animal models, PROTAR viruses were highly attenuated and elicited robust, broad, strain-dependent humoral, mucosal and cellular immunity. In addition, they provided cross-reactive protection against homologous and heterologous viral challenges. This study provides a systematic approach for developing safe and effective vaccines, with potential applications in designing live attenuated vaccines against other pathogens.

An Immunocytochemistry Method to Investigate the Translationally Active HIV Reservoir.

Despite the success of combination antiretroviral therapy (cART) to suppress HIV replication, HIV persists in a long-lived reservoir that can give rise to rebounding viremia upon cART cessation. The translationally active reservoir consists of HIV-infected cells that continue to produce viral proteins even in the presence of cART. These active reservoir cells are implicated in the resultant viremia upon cART cessation and likely contribute to chronic immune activation in people living with HIV (PLWH) on cART. Methodologies to quantify the active reservoir are needed. Here, an automated immunocytochemistry (ICC) assay coupled with computational image analysis to detect and quantify intracellular Gag capsid protein (CA) is described (CA-ICC). For this purpose, fixed cells were deposited on microscopy slides by the cytospin technique and stained with antibodies against CA by an automated stainer, followed by slide digitization. Nuclear staining was used to count the number of cells in the specimen, and the chromogenic signal was quantified to determine the percentage of CA-positive cells. In comparative analyses, digital ELISA, qPCR, and flow cytometry were used to validate CA-ICC. The specificity and sensitivity of CA-ICC were assessed by staining a cell line that expresses CA (MOLT IIIB) alongside a control cell line (Jurkat) devoid of this marker, as well as peripheral blood mononuclear cells (PBMCs) from HIV seronegative donors before or after ex vivo infection with an HIV laboratory strain. The sensitivity of CA-ICC was further assayed by spiking MOLT IIIB cells into uninfected Jurkat cells in limiting dilutions. In those analyses, CA-ICC could detect down to 10 CA-positive cells per million with a sensitivity superior to flow cytometry. To demonstrate the application of CA-ICC in pre-clinical research, bulk PBMCs obtained from mouse and non-human primate animal models were stained to detect HIV CA and SIV p27, respectively. The level of intracellular CA quantified by CA-ICC in PBMCs obtained from animal models was associated with plasma viral loads and cell-associated CA measured by qPCR and ELISA, respectively. The application of CA-ICC to evaluate the activity of small-molecule targeted activator of cell-kill (TACK) in clinical specimens is presented. Overall, CA-ICC offers a simple imaging method for specific and sensitive detection of CA-positive cells in bulk cell preparations.

Dual Inhibitors of SARS-CoV-2 3CL Protease and Human Cathepsin L Containing Glutamine Isosteres Are Anti-CoV-2 Agents.

SARS-CoV-2 3CL protease (Main protease) and human cathepsin L are proteases that play unique roles in the infection of human cells by SARS-CoV-2, the causative agent of COVID-19. Both proteases recognize leucine and other hydrophobic amino acids at the P2 position of a peptidomimetic inhibitor. At the P1 position, cathepsin L accepts many amino acid side chains, with a partial preference for phenylalanine, while 3CL-PR protease has a stringent specificity for glutamine or glutamine analogues. We have designed, synthesized, and evaluated peptidomimetic aldehyde dual-target (dual-acting) inhibitors using two peptide scaffolds based on those of two Pfizer 3CL-PR inhibitors, Nirmatrelvir, and PF-835321. Our inhibitors contain glutamine isosteres at the P1 position, including 2-pyridon-3-yl-alanine, 3-pyridinyl-alanine, and 1,3-oxazo-4-yl-alanine groups. Inhibition constants for these new inhibitors ranged from Ki = 0.6-18 nM (cathepsin L) and Ki = 2.6-124 nM (3CL-PR), for which inhibitors with the 2-pyridon-3-yl-alanal substituent were the most potent for 3CL-PR. The anti-CoV-2 activity of these inhibitors ranged from EC50 = 0.47-15 μM. X-ray structures of the peptidomimetic aldehyde inhibitors of 3CL-PR with similar scaffolds all demonstrated the formation of thiohemiacetals with Cys145, and hydrogen-bonding interactions with the heteroatoms of the pyridon-3-yl-alanyl group, as well as the nitrogen of the N-terminal indole and its appended carbonyl group at the P3 position. The absence of these hydrogen bonds for the inhibitors containing the 3-pyridinyl-alanyl and 1,3-oxazo-4-yl-alanyl groups was reflected in the less potent inhibition of the inhibitors with 3CL-PR. In summary, our studies demonstrate the value of a second generation of cysteine protease inhibitors that comprise a single agent that acts on both human cathepsin L and SARS-CoV-2 3CL protease. Such dual-target inhibitors will provide anti-COVID-19 drugs that remain active despite the development of resistance due to mutation of the viral protease. Such dual-target inhibitors are more likely to remain useful therapeutics despite the emergence of inactivating mutations in the viral protease because the human cathepsin L will not develop resistance. This particular dual-target approach is innovative since one of the targets is viral (3CL-PR) required for viral protein maturation and the other is human (hCatL) which enables viral infection.

Re-Examination Characterization and Screening of Stripe Rust Resistance Gene of Wheat TaPR1 Gene Family Based on the Transcriptome in Xinchun 32.

Pathogenesis-related protein-1 (PR1) encodes a water-soluble protein produced in plants after pathogen infection or abiotic stimulation. It plays a crucial role in plant-induced resistance by attacking pathogens, degrading cell wall macromolecules and pathogen toxins, and inhibiting the binding of viral coat proteins to plant receptor molecules. Compared to model plants, the mechanism of action of PR1 in wheat remains underexplored. In this study, the recently published wheat genome database (IWGSC RefSeq V2.1) was used to identify 83 genes in the TaPR1 gene family. Compared to previous work, the duplicate genes were removed and we corrected misannotated genes. Fourteen TaPR1 genes involved in the wheat-Pst interaction were identified based on RNA sequencing from Xinchun 32. The expression patterns of eight genes were validated using qRT-PCR, and the results showed that PR1 was highly expressed following Puccinia striiformis f. sp. tritici (Pst) infection. This study enhances previous research on wheat PR1, contributing to a more comprehensive understanding of the TaPR1 gene family and providing a reference for the screening of more broad-spectrum and high-resistance wheat populations.

Proteomic Analysis of Differentially Expressed Proteins in A549 Cells Infected with H9N2 Avian Influenza Virus.

Influenza A viruses (IAVs) are highly contagious pathogens that cause zoonotic disease with limited availability of antiviral therapies, presenting ongoing challenges to both public health and the livestock industry. Unveiling host proteins that are crucial to the IAV life cycle can help clarify mechanisms of viral replication and identify potential targets for developing alternative host-directed therapies. Using a four-dimensional (4D), label-free methodology coupled with bioinformatics analysis, we analyzed the expression patterns of cellular proteins that changed following H9N2 virus infection. Compared to the control group, the H9N2 infected group displayed 732 differentially expressed proteins (DEPs), with 298 proteins showing upregulation and 434 proteins showing downregulation. Gene Ontology (GO) functional analysis showed that DEPs were catalog in 11 biological processes, three cellular components, and eight molecular functions. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that DEPs were involved in processes including cytokine signaling pathways induced by virus infection and protein digestion and absorption. Proteins including TP53, DDX58, and STAT3 were among the top hub proteins in the protein-protein interaction (PPI) analysis, suggesting that these signaling cascades could be essential for the propagation of IAVs. Furthermore, the host protein SNAPIN was chosen to ascertain the accuracy of expression changes identified through a proteomic analysis. The results indicated that SNAPIN was downregulated following infection with IAVs both in vitro and in vivo, which is consistent with the proteomics results, suggesting that SNAPIN may serve as a key regulatory factor in the viral life cycle of IAVs. Our research delineates an extensive interaction map of IAV infection within the A549 cells, facilitating the discovery of pivotal proteins that contribute to the virus's propagation, potentially offering target candidates to screen for antiviral therapeutics.

Role of the Psi Packaging Signal and Dimerization Initiation Sequence in the Organization of Rous Sarcoma Virus Gag-gRNA Co-Condensates.

Retroviral genome selection and virion assembly remain promising targets for novel therapeutic intervention. Recent studies have demonstrated that the Gag proteins of Rous sarcoma virus (RSV) and human immunodeficiency virus type-1 (HIV-1) undergo nuclear trafficking, colocalize with nascent genomic viral RNA (gRNA) at transcription sites, may interact with host transcription factors, and display biophysical properties characteristic of biomolecular condensates. In the present work, we utilized a controlled in vitro condensate assay and advanced imaging approaches to investigate the effects of interactions between RSV Gag condensates and viral and nonviral RNAs on condensate abundance and organization. We observed that the psi (Ψ) packaging signal and the dimerization initiation sequence (DIS) had stabilizing effects on RSV Gag condensates, while RNAs lacking these features promoted or antagonized condensation, depending on local protein concentration and condensate architecture. An RNA containing Ψ, DIS, and the dimerization linkage structure (DLS) that is capable of stable dimer formation was observed to act as a bridge between RSV Gag condensates. These observations suggest additional, condensate-related roles for Gag-Ψ binding, gRNA dimerization, and Gag dimerization/multimerization in gRNA selection and packaging, representing a significant step forward in our understanding of how these interactions collectively facilitate efficient genome packaging.