Virus Receptor Research Articles

An ACE2 decamer viral trap as a durable intervention solution for current and future SARS-CoV

ABSTRACT The capacity of SARS-CoV-2 to evolve poses challenges to conventional prevention and treatment options such as vaccination and monoclonal antibodies, as they rely on viral receptor binding domain (RBD) sequences from previous strains. Additionally, animal CoVs, especially those of the SARS family, are now appreciated as a constant pandemic threat. We present here a new antiviral approach featuring inhalation delivery of a recombinant viral trap composed of ten copies of angiotensin-converting enzyme 2 (ACE2) fused to the IgM Fc. This ACE2 decamer viral trap is designed to inhibit SARS-CoV-2 entry function, regardless of viral RBD sequence variations as shown by its high neutralization potency against all known SARS-CoV-2 variants, including Omicron BQ.1, BQ.1.1, XBB.1 and XBB.1.5. In addition, it demonstrates potency against SARS-CoV-1, human NL63, as well as bat and pangolin CoVs. The multivalent trap is effective in both prophylactic and therapeutic settings since a single intranasal dosing confers protection in human ACE2 transgenic mice against viral challenges. Lastly, this molecule is stable at ambient temperature for more than twelve weeks and can sustain physical stress from aerosolization. These results demonstrate the potential of a decameric ACE2 viral trap as an inhalation solution for ACE2-dependent coronaviruses of current and future pandemic concerns.

Neutralization of Different Variants of SARS-CoV-2 by a F(ab')2 Preparation from Sera of Horses Immunized with the Viral Receptor Binding Domain.

The Receptor Binding Domain (RBD) of SARS-CoV-2, the virus responsible for the COVID-19 pandemic, is the functional region of the viral Spike protein (S), which is involved in cell attachment to target cells. The virus has accumulated progressively mutations in its genome, particularly in the RBD region, many of them associated with immune evasion of the host neutralizing antibodies. Some of the viral lineages derived from this evolution have been classified as Variant of Interest (VOI) or Concern (VOC). The neutralizing capacity of a F(ab')2 preparation from sera of horses immunized with viral RBD was evaluated by lytic plaque reduction assay against different SARS-CoV-2 variants. A F(ab')2 preparation of a hyperimmune serum after nine immunizations with RBD exhibited a high titer of neutralizing antibodies against the ancestral-like strain (1/18,528). A reduction in the titer of the F(ab')2 preparation was observed against the different variants tested compared to the neutralizing activity against the ancestral-like strain. The highest reduction in the neutralization titer was observed for the Omicron VOC (4.7-fold), followed by the Mu VOI (2.6), Delta VOC (1.8-fold), and Gamma VOC (1.5). Even if a progressive reduction in the neutralizing antibodies titer against the different variants evaluated was observed, the serum still exhibited a neutralizing titer against the Mu VOI and the Omicron VOC (1/7113 and 1/3918, respectively), the evaluated strains most resistant to neutralization. Therefore, the preparation retained neutralizing activity against all the strains tested.

Research progress of common respiratory virus receptor binding sites

Respiratory viral infections are an important public health problem worldwide, with complex mechanisms of infection, and the key to infection lies in the specific binding between respiratory viruses and receptors. This article provides an overview of the progress in the study of receptors for respiratory viruses, such as coronavirus and influenza virus (IV), with a focus on the binding sites of receptors such as angiotensin-converting enzyme 2 (ACE2) and sialic acid (SA) to respiratory viruses and the role of receptor diversity in respiratory viral infections. An in-depth study of the binding sites between viruses and receptors will help to understand the molecular mechanism of respiratory viral infections and provide a theoretical basis for disease prevention and control and the development of new therapeutic targets.

Emerging insights into the structure-activity relationship of water-soluble polysaccharides in antiviral therapy

This review provides an overview of recent advancements in understanding the antiviral activities and mechanism of action of water-soluble polysaccharides (WSPs). Water-soluble polysaccharides are complex carbohydrate molecules derived from various natural sources, including plants, microorganisms, and fungi. Their unique structural characteristics make them intriguing candidates for antiviral therapeutics. However, in this review, we highlight the emerging insights into the multifaceted antiviral properties of water-soluble polysaccharides. These substances have exhibited the ability to hinder the activity of various viruses, encompassing both enveloped and non-enveloped types. Their mechanisms of action encompass interference with viral attachment and entry, inhibition of viral replication, modulation of the host immune response, and even direct virucidal effects. Furthermore, we delve into the structural features of water-soluble polysaccharides that underpin their antiviral potential. Factors such as molecular weight, branching patterns, sulfation, and acetylation play pivotal roles in their antiviral activity. Additionally, we explore the importance of water-soluble polysaccharide conformation, size, shape, solubility, charge density, and stereochemistry in influencing their interactions with viral particles and host cell receptors. Moreover, this review provides the growing significance of water-soluble polysaccharides as promising antiviral agents and emphasizes the need for further research to harness their full therapeutic potential in the fight against viral diseases.

Actin cytoskeleton remodeling disrupts physical barriers to infection and presents entry receptors to respiratory syncytial virus.

RSV is the leading cause of infant hospitalizations and a significant cause of paediatric and geriatric morbidity worldwide. Recently, we reported that insulin-like growth factor 1 receptor (IGF1R) was a receptor for respiratory syncytial virus (RSV) in airway epithelial cells and that activation of IGF1R recruited the coreceptor, nucleolin (NCL), to the cell surface. Cilia and mucus that line the airways pose a significant barrier to viral and bacterial infection. The cortical actin cytoskeleton has been shown by others to mediate RSV entry, so we studied the roles of the RSV receptors and actin remodelling during virus entry. We found that IGF1R expression and phosphorylation were associated with the ability of RSV to infect cells. Confocal immunofluorescence imaging showed that actin projections, a hallmark of macropinocytosis, formed around viral particles 30 min after infection. Consistent with prior reports we also found that virus particles were internalized into early endosome antigen-1 positive endosomes within 90 min. Inhibiting actin polymerization significantly reduced viral titre by approximately ten-fold. Inhibiting PI3 kinase and PKCζ in stratified air-liquid interface (ALI) models of the airway epithelium had similar effects on reducing the actin remodelling observed during infection and attenuating viral entry. Actin projections were associated with NCL interacting with RSV particles resting on apical cilia of the ALIs. We conclude that macropinocytosis-like actin projections protrude through normally protective cilia and mucus layers of stratified airway epithelium that helps present the IGF1R receptor and the NCL coreceptor to RSV particles waiting at the surface.

Assessing risk of Bombali virus spillover to humans by mutagenesis analysis of viral glycoprotein

Ebolaviruses, such as the highly pathogenic Ebola virus (EBOV), belong to the family Filoviridae and have caused outbreaks of severe and fatal hemorrhagic fever over the past years. Bombali virus (BOMV) is a newly discovered ebolavirus from bats with unknown potential to infect humans. For most ebolaviruses, the interaction between viral glycoprotein (GP) and host receptor Niemann-Pick C1 (NPC1) is crucial for viral entry and determines host tropism. Here, we analyzed the BOMV GP-mediated virus entry into human cells using our recently developed EBOV transcription- and replication-competent virus-like particle (trVLP) system. We demonstrated that while BOMV GP can be efficiently incorporated into trVLPs, it is inefficient in mediating trVLP entry into human cells. However, BOMV GP-mediated virus entry into human cells can be significantly enhanced by a few mutations in the NPC1-binding domain of GP. Furthermore, we showed that these mutations increase the binding of BOMV-GP to human NPC1. In summary, our results suggested that although wild-type BOMV does not efficiently infect human cells, the emergence of mutations in viral GP may boost its ability to spill over to humans, highlighting the importance of monitoring BOMV GP evolution in preventing potential BOMV spillover events.

CD4 is an important host factor for Japanese encephalitis virus entry and replication in PK-15 cells

Japanese encephalitis virus (JEV) is a flavivirus that is spread through mosquito bites and is the leading cause of viral encephalitis in Asia. JEV can infect a variety of cell types; however, crucial receptor molecules remain unclear. The purpose of this study was to determine whether porcine CD4 protein is a receptor protein that impacts JEV entry into PK15 cells and subsequent viral replication. We confirmed the interaction between the JEV E protein and the CD4 protein through Co-IP, virus binding and internalization, antibody blocking, and overexpression and created a PK-15 cell line with CD4 gene knockdown by CRISPR/Cas9. The results show that CD4 interacts with JEV E and that CD4 knockdown cells altered virus adsorption and internalization, drastically reducing virus attachment. The level of viral transcription in CD4 antibody-blocked cells, vs. control cells, was decreased by 49.1%. Based on these results, we believe that CD4 is a receptor protein for JEVs. Furthermore, most viral receptors appear to be associated with lipid rafts, and colocalization studies demonstrate the presence of CD4 protein on lipid rafts. RT‒qPCR and WB results show that virus replication was suppressed in PK-15-CD4KD cells. The difference in viral titer between KD and WT PK-15 cells peaked at 24 h, and the viral titer in WT PK-15 cells was 5.6 × 106, whereas in PK-15-CD4KD cells, it was only 1.8 × 106, a 64% drop, demonstrating that CD4 deficiency has an effect on the process of viral replication. These findings suggest that JEV enters porcine kidney cells via lipid raft-colocalized CD4, and the proliferation process is positively correlated with CD4.

TurboID-Based Proximity Labeling: A Method to Decipher Protein-Protein Interactions in Plants.

Proteins form complex networks through interaction to drive biological processes. Thus, dissecting protein-protein interactions (PPIs) is essential for interpreting cellular processes. To overcome the drawbacks of traditional approaches for analyzing PPIs, enzyme-catalyzed proximity labeling (PL) techniques based on peroxidases or biotin ligases have been developed and successfully utilized in mammalian systems. However, the use of toxic H2O2 in peroxidase-based PL, the requirement of long incubation time (16-24h), and higher incubation temperature (37°C) with biotin in BioID-based PL significantly restricted their applications in plants. TurboID-based PL, a recently developed approach, circumvents the limitations of these methods by providing rapid PL of proteins under room temperature. We recently optimized the use of TurboID-based PL in plants and demonstrated that it performs better than BioID in labeling endogenous proteins. Here, we describe a step-by-step protocol for TurboID-based PL in studying PPIs in planta, including Agrobacterium-based transient expression of proteins, biotin treatment, protein extraction, removal of free biotin, quantification, and enrichment of the biotinylated proteins by affinity purification. We describe the PL using plant viral immune receptor N, which belongs to the nucleotide-binding leucine-rich repeat (NLR) class of immune receptors, as a model. The method described could be easily adapted to study PPI networks of other proteins in Nicotiana benthamiana and provides valuable information for future application of TurboID-based PL in other plant species.

Brain metastasis-associated fibroblasts secrete fucosylated PVR/CD155 that induces breast cancer invasion

Brain metastasis cancer-associated fibroblasts (bmCAFs) are emerging as crucial players in the development of breast cancer brain metastasis (BCBM), but our understanding of the underlying molecular mechanisms is limited. In this study, we aim to elucidate the pathological contributions of fucosylation (the post-translational modification of proteins by the dietary sugar L-fucose) to tumor-stromal interactions that drive the development of BCBM. Here, we report that patient-derived bmCAFs secrete high levels of polio virus receptor (PVR), which enhance the invasive capacity of BC cells. Mechanistically, we find that HIF1α transcriptionally upregulates fucosyltransferase 11, which fucosylates PVR, triggering its secretion from bmCAFs. Global phosphoproteomic analysis of BC cells followed by functional verification identifies cell-cell junction and actin cytoskeletal signaling as modulated by bmCAF-secreted, -fucosylated PVR. Our findings delineate a hypoxia- and fucosylation-regulated mechanism by which bmCAFs contribute to the invasiveness of BCBM in the brain.

Efficient SARS-CoV-2 infection antagonization by rhACE2 ectodomain multimerized onto the Avidin-Nucleic-Acid-NanoASsembly

Nanodecoy systems based on analogues of viral cellular receptors assembled onto fluid lipid-based membranes of nano/extravescicles are potential new tools to complement classic therapeutic or preventive antiviral approaches. The need for lipid-based membranes for transmembrane receptor anchorage may pose technical challenges along industrial translation, calling for alternative geometries for receptor multimerization. Here we developed a semisynthetic self-assembling SARS-CoV-2 nanodecoy by multimerizing the biotin labelled virus cell receptor -ACE2- ectodomain onto a poly-avidin nanoparticle (NP) based on the Avidin-Nucleic-Acid-NanoASsembly-ANANAS. The ability of the assembly to prevent SARS-CoV-2 infection in human lung cells and the affinity of the ACE2:viral receptor-binding domain (RBD) interaction were measured at different ACE2:NP ratios. At ACE2:NP = 30, 90 % SARS-CoV-2 infection inhibition at ACE2 nanomolar concentration was registered on both Wuhan and Omicron variants, with ten-fold higher potency than the monomeric protein. Lower and higher ACE2 densities were less efficient suggesting that functional recognition between multi-ligand NPs and multi-receptor virus surfaces requires optimal geometrical relationships. In vivo studies in mice showed that the biodistribution and safety profiles of the nanodecoy are potentially suitable for preventing viral infection upon nasal instillation. Viral receptor multimerization using ANANAS is a convenient process which, in principle, could be rapidly adapted to counteract also other viral infections.

Engineering a highly durable adeno-associated virus receptor for analytical applications

Adeno-associated virus (AAV) is a major viral vector used in gene therapy. There are multiple AAV serotypes, and many engineered AAV serotypes are developed to alter their tissue tropisms with capsid modification. The universal AAV receptor (AAVR) is an essential receptor for multiple AAV serotypes. Since most AAV serotypes used in gene therapy infect cells via interaction with AAVR, the quantification of the vector-binding ability of AAV to AAVR could be an important quality check for therapeutic AAV vectors. To enable a steady evaluation of the AAV-AAVR interaction, we created an engineered AAVR through mutagenesis. Engineered AAVR showed high durability against acid while retaining its AAV-binding activity. An affinity chromatography column with the engineered AAVR was also developed. This column enabled repeated binding and acid dissociation measurements of AAVR with various AAV serotypes. Our data showed that the binding affinities of AAV2 to AAVR were diverse among serotypes, providing insight into the relationship with the infection efficiency of AAV vectors. Thus, this affinity column can be used in process development for quality checks, quantitating capsid titers, and affinity purification of AAV vectors. Furthermore, this column may serve as a useful tool in novel AAV vector capsid engineering.

HDL mimetics protect Alzheimer’s patients carrying APOE ε4 from SARS-CoV-2 invasion

Patients with Alzheimer’s disease (AD) inheriting Apolipoprotein (APOE) ε4 are susceptible to COVID-19, but the underlying mechanism of how such a neurodegenerative disease promotes respiratory vulnerability to viral infections remains insufficiently understood. The uncovering of genetic basis of COVID-19 outcome holds the potential to establish disease models for therapeutic development. Here, using human iPSC-derived type II alveolar epithelial cells (hAECII) from AD patients with APOE ε4 and healthy individuals, we showed that AECII of AD had a greater level of SARS-CoV-2 invasion but not replication, along with increased expressions of viral receptor and co-receptors, while reduced pulmonary surfactant proteins. Since low serum HDL-C levels have been implicated in the onset of both AD and COVID-19, we further revealed that HDL mimetics, including 4 F dimeric peptide and its phospholipid conjugate pHDL, were effective in protecting AECII bearing APOE ε4 against SARS-CoV-2 invasion. In AD, concomitant with improved AD pathological phenotypes, HDL mimetics specifically restored the defective basal SP-D, a pulmonary innate immune lipoprotein that targets glycans of the spike protein to neutralize invading viruses. Moreover, HDL mimetics demonstrated strong SP-D-independent virucidal effects, which additionally restricted the entry of coronaviral particles into non-AD AECII models and lungs of human ACE2-transgenic mice in response to Alpha and Omicron variants of SARS-CoV-2. Our work offers critical insights into the respiratory sensitivity of AD patients carrying APOE ε4 to viral infection and repurpose HDL-based therapeutics as potential preventive interventions in respiratory viral pandemics.

Virtual Screening Based on Docking and Molecular Dynamics Simulations of Potential Ebola Receptor Inhibitors

AbstractGalidesivir (BCX4430) is known to treat different viruses under Filoviridae family and it has proved as a potential antiviral drug against Ebola virus. Therefore, for treating Ebola virus in particular there is a need for the identification a drug compound. In this work, derivatives of Pyrrolopyrimidine are designed to investigate their antiviral activity against Ebola virus receptors (VP24, VP30, VP35 and VP40). FBDD‐based virtual screening resulted in eight compounds, based on pharmacokinetics and Density Functional Theory (DFT) analysis; four compounds were shortlisted. Further, docking simulation resulted that, compound 4 exhibited −9.8, −9.6, −9.7, −9.5 kcal/mol binding energy against each Ebola receptor, respectively. The predicted docking results of the above compound against each receptor were better than BCX4430. Docking results were validated and compared to BCX4430 by performing Molecular Dynamic simulations at 100 ns, in order to study the Root Mean Square Deviation, Root Mean Square Fluctuation, Radius of Gyration and MM/PBSA of the docked complexes. Therefore, the study lays the foundation for the designed and screened compound to act as a probable drug compound against Ebola.

'SAXS-osmometer' method provides measurement of DNA pressure in viral capsids and delivers an empirical equation of state.

We present a novel method that provides a measurement of DNA pressure in viral capsids using small angle X-ray scattering (SAXS). This method, unlike our previous assay, does not require triggering genome release with a viral receptor. Thus, it can be used to determine the existence of a pressurized genome state in a wide range of virus systems, even if the receptor is not known, leading to a better understanding of the processes of viral genome uncoating and encapsidation in the course of infection. Furthermore, by measuring DNA pressure for a collection of bacteriophages with varying DNA packing densities, we derived an empirical equation of state (EOS) that accurately predicts the relation between the capsid pressure and the packaged DNA density and includes the contribution of both DNA-DNA interaction energy and DNA bending stress to the total DNA pressure. We believe that our SAXS-osmometer method and the EOS, combined, provide the necessary tools to investigate physico-chemical properties of confined DNA condensates and mechanisms of infection, and may also provide essential data for the design of viral vectors in gene therapy applications and development of antivirals that target the pressurized genome state.

Bacterial outer membrane vesicles bound to bacteriophages modulate neutrophil responses to bacterial infection.

Pseudomonas aeruginosa is a major human pathogen, particularly effective at colonizing the airways of patients with cystic fibrosis. Bacteriophages are highly abundant at infection sites, but their impact on mammalian immunity remains unclear. We previously showed that Pf4, a temperate filamentous bacteriophage produced by P. aeruginosa, modifies the innate immune response to P. aeruginosa infections via TLR3 signaling, but the underlying mechanisms remained unclear. Notably, Pf4 is a single-stranded DNA and lysogenic phage, and its production does not typically result in lysis of its bacterial host. We identified previously that internalization of Pf4 by human or murine immune cells triggers maladaptive viral pattern recognition receptors and resulted in bacterial persistence based on the presence of phage RNA. We report now that Pf4 phage dampens inflammatory responses to bacterial endotoxin and that this is mediated in part via bacterial vesicles attached to phage particles. Outer membrane vesicles (OMVs) are produced by Gram-negative bacteria and play a key role in host pathogen interaction. Recently, evidence has emerged that OMVs differentially package small RNAs. In this study, we show that Pf4 are decorated with OMVs that remain affixed to Pf4 despite of purification steps. These phages are endocytosed by human cells and delivered to endosomal vesicles. We demonstrate that short RNAs within the OMVs form hairpin structures that trigger TLR3-dependent type I interferon production and antagonize production of antibacterial cytokines and chemokines. In particular, Pf4 phages inhibit CXCL5, preventing efficient neutrophil chemotaxis in response to endotoxin. Moreover, blocking IFNAR or TLR3 signaling abrogates the effect of Pf4 bound to OMVs on macrophage activation. In a murine acute pneumonia model, mice treated with Pf4 associated with OMVs show significantly less neutrophil infiltration in BAL fluid than mice treated with purified Pf4. These changes in macrophage phenotype are functionally relevant: conditioned media from cells exposed to Pf4 decorated with OMVs are significantly less effective at inducing neutrophil migration in vitro and in vivo. These results suggest that Pf4 phages alter innate immunity to bacterial endotoxin and OMVs, potentially dampening inflammation at sites of bacterial colonization or infection.

Structure-function analyses reveal key molecular determinants of HIV-1 CRF01_AE resistance to the entry inhibitor temsavir

The HIV-1 entry inhibitor temsavir prevents the viral receptor CD4 (cluster of differentiation 4) from interacting with the envelope glycoprotein (Env) and blocks its conformational changes. To do this, temsavir relies on the presence of a residue with small side chain at position 375 in Env and is unable to neutralize viral strains like CRF01_AE carrying His375. Here we investigate the mechanism of temsavir resistance and show that residue 375 is not the sole determinant of resistance. At least six additional residues within the gp120 inner domain layers, including five distant from the drug-binding pocket, contribute to resistance. A detailed structure-function analysis using engineered viruses and soluble trimer variants reveals that the molecular basis of resistance is mediated by crosstalk between His375 and the inner domain layers. Furthermore, our data confirm that temsavir can adjust its binding mode to accommodate changes in Env conformation, a property that likely contributes to its broad antiviral activity.

Evolving antibody evasion and receptor affinity of the Omicron BA.2.75 sublineage of SARS-CoV-2

SARS-CoV-2 Omicron BA.2.75 has diversified into multiple subvariants with additional spike mutations and several are expanding in prevalence, particularly CH.1.1 and BN.1. Here, we investigated the viral receptor affinities and neutralization evasion properties of major BA.2.75 subvariants actively circulating in different regions worldwide. We found two distinct evolutionary pathways and three newly identified mutations that shaped the virological features of these subvariants. One phenotypic group exhibited a discernible decrease in viral receptor affinities, but a noteworthy increase in resistance to antibody neutralization, as exemplified by CH.1.1, which is apparently as resistant as XBB.1.5. In contrast, a second group demonstrated a substantial increase in viral receptor affinity but only a moderate increase in antibody evasion, as exemplified by BN.1. We also observed that all prevalent SARS-CoV-2 variants in the circulation presently, except for BN.1, exhibit profound levels of antibody evasion, suggesting this is the dominant determinant of virus transmissibility today.

Nanobodies against porcine CD163 as PRRSV broad inhibitor

PRRSV (Porcine Reproductive and Respiratory Syndrome Virus) is a major swine pathogen causing economic losses. To the date, effective broad PRRSV inhibitory strategies have not been available in practice yet. Targeting the key viral receptor CD163 to block PRRSV entry has emerged as an alternative approach beside vaccines for PRRSV inhibition. As an effective therapeutic tool, nanoantibodies (Nbs) have been widely used in antiviral research. In this study, a phage display VHH library was constructed for the selection of Nbs against porcine CD163 scavenger receptor cysteine-rich 5–9 domain (SRCR5–9). After five rounds of bio-panning and indirect ELISA, seven CD163-specific Nbs (Nb1-Nb7) were identified. All obtained Nbs displayed strong affinity to CD163 receptor and excellent antiviral activity. In particular, Nb2 exhibited significant broad inhibitory effects on variable PRRSV lineages and downregulated virus-related NF-κB signaling. Further studies suggested that Nbs mainly exerted antiviral functions by interfering with virus attachment stage, and also decreased the transcription of CD163. The conformational epitopes recognized by Nbs were localized in the SRCR5 domain of CD163, a crucial region in PRRSV infection. Overall, our findings provide a novel insight into the biofunction of CD163 in antiviral infection and the development of broad-spectrum strategies against PRRSV.

Proximity-dependent mapping of the HCMV US28 interactome identifies RhoGEF signaling as a requirement for efficient viral reactivation.

Human cytomegalovirus (HCMV) encodes multiple putative G protein-coupled receptors (GPCRs). US28 functions as a viral chemokine receptor and is expressed during both latent and lytic phases of virus infection. US28 actively promotes cellular migration, transformation, and plays a major role in mediating viral latency and reactivation; however, knowledge about the interaction partners involved in these processes is still incomplete. Herein, we utilized a proximity-dependent biotinylating enzyme (TurboID) to characterize the US28 interactome when expressed in isolation, and during both latent (CD34+ hematopoietic progenitor cells) and lytic (fibroblasts) HCMV infection. Our analyses indicate that the US28 signalosome converges with RhoA and EGFR signal transduction pathways, sharing multiple mediators that are major actors in processes such as cellular proliferation and differentiation. Integral members of the US28 signaling complex were validated in functional assays by immunoblot and small-molecule inhibitors. Importantly, we identified RhoGEFs as key US28 signaling intermediaries. In vitro latency and reactivation assays utilizing primary CD34+ hematopoietic progenitor cells (HPCs) treated with the small-molecule inhibitors Rhosin or Y16 indicated that US28 -RhoGEF interactions are required for efficient viral reactivation. These findings were recapitulated in vivo using a humanized mouse model where inhibition of RhoGEFs resulted in a failure of the virus to reactivate. Together, our data identifies multiple new proteins in the US28 interactome that play major roles in viral latency and reactivation, highlights the utility of proximity-sensor labeling to characterize protein interactomes, and provides insight into targets for the development of novel anti-HCMV therapeutics.

Retroviral Replicating Vectors Mediated Prodrug Activator Gene Therapy in a Gastric Cancer Model.

Retroviral replicating vectors (RRVs) selectively replicate and can specifically introduce prodrug-activating genes into tumor cells, whereby subsequent prodrug administration induces the death of the infected tumor cells. We assessed the ability of two distinct RRVs generated from amphotropic murine leukemia virus (AMLV) and gibbon ape leukemia virus (GALV), which infect cells via type-III sodium-dependent phosphate transporters, PiT-2 and PiT-1, respectively, to infect human gastric cancer (GC) cells. A quantitative RT-PCR showed that all tested GC cell lines had higher expression levels of PiT-2 than PiT-1. Accordingly, AMLV, encoding a green fluorescent protein gene, infected and replicated more efficiently than GALV in most GC cell lines, whereas both RRVs had a low infection rate in human fibroblasts. RRV encoding a cytosine deaminase prodrug activator gene, which converts the prodrug 5-flucytosine (5-FC) to the active drug 5-fluorouracil, showed that AMLV promoted superior 5-FC-induced cytotoxicity compared with GALV, which correlated with the viral receptor expression level and viral spread. In MKN-74 subcutaneous xenograft models, AMLV had significant antitumor effects compared with GALV. Furthermore, in the MKN-74 recurrent tumor model in which 5-FC was discontinued, the resumption of 5-FC administration reduced the tumor volume. Thus, RRV-mediated prodrug activator gene therapy might be beneficial for treating human GC.