Chemically Tagging Cargo for Specific Packaging inside and on the Surface of Virus-like Particles.

Virus-like particles (VLPs) have untapped potential for packaging and delivery of macromolecular cargo. To be a broadly useful platform, there needs to be a strategy for attaching macromolecules to the inside or the outside of the VLP with minimal modification of the platform or cargo. Here, we repurpose antiviral compounds that bind to hepatitis B virus (HBV) capsids to create a chemical tag to noncovalently attach cargo to the VLP. Our tag consists of a capsid assembly modulator, HAP13, connected to a linker terminating in maleimide. Our cargo is a green fluorescent protein (GFP) with a single addressable cysteine, a feature that can be engineered in many proteins. The HAP-GFP construct maintained HAP's intrinsic ability to bind HBV capsids and accelerate assembly. We investigated the capacity of HAP-GFP to coassemble with HBV capsid protein and bind to preassembled capsids. HAP-GFP binding was concentration-dependent, sensitive to capsid stability, and dependent on linker length. Long linkers had the greatest activity to bind capsids, while short linkers impeded assembly and damaged intact capsids. In coassembly reactions, >20 HAP-GFP molecules were presented on the outside and inside of the capsid, concentrating the cargo by more than 100-fold compared to bulk solution. We also tested an HAP-GFP with a cleavable linker so that external GFP molecules could be removed, resulting in exclusive internal packaging. These results demonstrate a generalizable strategy for attaching cargo to a VLP, supporting development of HBV as a modular VLP platform.

Extracellular vesicle isolation methods identify distinct HIV-1 particles released from chronically infected T-cells.

The current study analyzed the intersecting biophysical, biochemical, and functional properties of extracellular particles (EPs) with the human immunodeficiency virus type-1 (HIV-1) beyond the currently accepted size range for HIV-1. We isolated five fractions (Frac-A through Frac-E) from HIV-infected cells by sequential differential ultracentrifugation (DUC). All fractions showed a heterogeneous size distribution with median particle sizes greater than 100 nm for Frac-A through Frac-D but not for Frac-E, which contained small EPs with an average size well below 50 nm. Synchronized and released cultures contained large infectious EPs in Frac-A, with markers of amphisomes and viral components. Additionally, Frac-E uniquely contained EPs positive for CD63, HSP70, and HIV-1 proteins. Despite its small average size, Frac-E contained membrane-protected viral integrase, detectable only after SDS treatment, indicating that it is enclosed in vesicles. Single particle analysis with dSTORM further supported these findings as CD63, HIV-1 integrase, and the viral surface envelope (Env) glycoprotein (gp) colocalized on the same Frac-E particles. Surprisingly, Frac-E EPs were infectious, and infectivity was significantly reduced by immunodepleting Frac-E with anti-CD63, indicating the presence of this protein on the surface of infectious small EPs in Frac-E. To our knowledge, this is the first time that extracellular vesicle (EV) isolation methods have identified infectious small HIV-1 particles (smHIV-1) that are under 50 nm. Collectively, our data indicate that the crossroads between EPs and HIV-1 potentially extend beyond the currently accepted biophysical properties of HIV-1, which may have further implications for viral pathogenesis.

The Conserved YPX3L Motif in the BK Polyomavirus VP1 Protein Is Important for Viral Particle Assembly but Not for Its Secretion into Extracellular Vesicles.

The BK polyomavirus (BKPyV) is a small DNA non-enveloped virus whose infection is asymptomatic in most of the world's adult population. However, in cases of immunosuppression, the reactivation of the virus can cause various complications, and in particular, nephropathies in kidney transplant recipients or hemorrhagic cystitis in bone marrow transplant recipients. Recently, it was demonstrated that BKPyV virions can use extracellular vesicles to collectively traffic in and out of cells, thus exiting producing cells without cell lysis and entering target cells by diversified entry routes. By a comparison to other naked viruses, we investigated the possibility that BKPyV virions recruit the Endosomal-Sorting Complexes Required for Transport (ESCRT) machinery through late domains in order to hijack extracellular vesicles. We identified a single potential late domain in the BKPyV structural proteins, a YPX3L motif in the VP1 protein, and used pseudovirions to study the effect of point mutations found in a BKPyV clinical isolate or known to ablate the interaction of such a domain with the ESCRT machinery. Our results suggest that this domain is not involved in BKPyV association with extracellular vesicles but is crucial for capsomere interaction and thus viral particle assembly.

HIV Immunocapture Reveals Particles Expressed in Semen Under Integrase Strand Transfer Inhibitor-Based Therapy Are Largely Myeloid Cell-Derived and Disparate.

As use of human immunodeficiency virus (HIV) integrase strand transfer inhibitors (INSTI) increases and formulations are being developed for maintenance therapies and chemoprophylaxis, assessing virus suppression under INSTI-based regimens in prevention-relevant biologic compartments, such as the male genital tract, is timely. We used cell-source marker virion immunocapture to examine amplification of particle RNA then assessed the phylogenetic relatedness of seminal and blood viral sequences from men with HIV who were prescribed INSTI-based regimens. Seminal plasma immunocaptures yielded amplifiable virion RNA from 13 of 24 (54%) men, and the sequences were primarily associated with markers indicative of macrophage and resident dendritic cell sources. Genetic distances were greatest (>2%) between seminal virions and circulating proviruses, pointing to ongoing low-level expression from tissue-resident cells. While the low levels in semen predict an improbable likelihood of transmission, viruses with large genetic distances are expressed under potent INSTI therapy and have implications for determining epidemiologic linkages if adherence is suboptimal.

Comprehensive proteomic analysis of HCoV-OC43 virions and virus-modulated extracellular vesicles.

Viruses are obligate parasites that depend on the cellular machinery for their propagation. Several viruses also incorporate cellular proteins that facilitate viral spread. Defining these cellular proteins is critical to decipher viral life cycles and delineate novel therapeutic strategies. While numerous studies have explored the importance of host proteins in coronavirus spread, information about their presence in mature virions is limited. In this study, we developed a protocol to highly enrich mature HCoV-OC43 virions and characterize them by proteomics. Recognizing that cells release extracellular vesicles whose content is modulated by viruses, and given our ability to separate virions from these vesicles, we also analyzed their protein content in both uninfected and infected cells. We uncovered 69 unique cellular proteins associated with virions including 31 high-confidence hits. These proteins primarily regulate RNA metabolism, enzymatic activities, vesicular transport, cell adhesion, metabolite interconversion, and translation. We further discovered that the virus had a profound impact on exosome composition, incorporating 47 novel cellular proteins (11 high confidence) and excluding 92 others (61 high confidence) in virus-associated extracellular vesicles compared to uninfected cells. Moreover, a dsiRNA screen revealed that 11 of 18 select targets significantly impacted viral yields, including proteins found in virions or extracellular vesicles. Overall, this study provides new and important insights into the incorporation of numerous host proteins into HCoV-OC43 virions, their biological significance, and the ability of the virus to modulate extracellular vesicles. IMPORTANCE: In recent years, coronaviruses have dominated global attention, making it crucial to develop methods to control them and prevent future pandemics. Besides viral proteins, host proteins play a significant role in viral propagation and offer potential therapeutic targets. Targeting host proteins is advantageous because they are less likely to mutate and develop resistance compared to viral proteins, a common issue with many antiviral treatments. In this study, we examined the protein content of the less virulent biosafety level 2 HCoV-OC43 virus as a stand-in for the more virulent SARS-CoV-2. Our findings reveal that several cellular proteins incorporated into the virion regulate viral spread. In addition, we report that the virus extensively modulates the content of extracellular vesicles, enhancing viral dissemination. This underscores the critical interplay between the virus, host proteins, and extracellular vesicles.

HIV-1 Vpr combats the PU.1-driven antiviral response in primary human macrophages.

HIV-1 Vpr promotes efficient spread of HIV-1 from macrophages to T cells by transcriptionally downmodulating restriction factors that target HIV-1 Envelope protein (Env). Here we find that Vpr induces broad transcriptomic changes by targeting PU.1, a transcription factor necessary for expression of host innate immune response genes, including those that target Env. Consistent with this, we find silencing PU.1 in infected macrophages lacking Vpr rescues Env. Vpr downmodulates PU.1 through a proteasomal degradation pathway that depends on physical interactions with PU.1 and DCAF1, a component of the Cul4A E3 ubiquitin ligase. The capacity for Vpr to target PU.1 is highly conserved across primate lentiviruses. In addition to impacting infected cells, we find that Vpr suppresses expression of innate immune response genes in uninfected bystander cells, and that virion-associated Vpr can degrade PU.1. Together, we demonstrate Vpr counteracts PU.1 in macrophages to blunt antiviral immune responses and promote viral spread.

Assembly of respiratory syncytial virus matrix protein lattice and its coordination with fusion glycoprotein trimers.

Respiratory syncytial virus (RSV) is an enveloped, filamentous, negative-strand RNA virus that causes significant respiratory illness worldwide. RSV vaccines are available, however there is still significant need for research to support the development of vaccines and therapeutics against RSV and related Mononegavirales viruses. Individual virions vary in size, with an average diameter of ~130 nm and ranging from ~500 nm to over 10 µm in length. Though the general arrangement of structural proteins in virions is known, we use cryo-electron tomography and sub-tomogram averaging to determine the molecular organization of RSV structural proteins. We show that the peripheral membrane-associated RSV matrix (M) protein is arranged in a packed helical-like lattice of M-dimers. We report that RSV F glycoprotein is frequently observed as pairs of trimers oriented in an anti-parallel conformation to support potential interactions between trimers. Our sub-tomogram averages indicate the positioning of F-trimer pairs is correlated with the underlying M lattice. These results provide insight into RSV virion organization and may aid in the development of RSV vaccines and anti-viral targets.

Deciphering the Thermal Stability of Bacteriophage MS2-Derived Virus-like Particle and Its Engineered Variant.

RNA bacteriophage MS2-derived virus-like particles (VLPs) have been widely used in biomedical research as model systems to study virus assembly, structure-function relationships, vaccine development, and drug delivery. Considering the diverse utility of these VLPs, a systemic engineering approach has been utilized to generate smaller particles with optimal serum stability and tissue penetrance. Additionally, it is crucial to demonstrate the overall stability of these mini MS2 VLPs, ensuring cargo protection until they reach their target cell/organ. However, no detailed analysis of the thermal stability and heat-induced disassembly of MS2 VLPs has yet been attempted. In this work, we investigated the thermal stability of both wild-type (WT) MS2 VLP and its "mini" variant containing S37P mutation (mini MS2 VLP). The mini MS2 VLP exhibits a higher capsid melting temperature (Tm) when compared to its WT MS2 VLP counterpart, possibly attributed to its smaller interdimer angle. Our study presents that the thermal unfolding of MS2 VLPs follows a sequential process involving particle destabilization, nucleic acid exposure/melting, and disassembly of VLP. This observation underscores the disruption of cooperative intersubunit interactions and protein-nucleic acid interactions, shedding light on the mechanism of heat-induced VLP disassembly.

Construing the function of N-terminal domain of D29 mycobacteriophage LysA endolysin in phage lytic efficiency and proliferation.

Endolysins produced by bacteriophages hydrolyze host cell wall peptidoglycan to release newly assembled virions. D29 mycobacteriophage specifically infects mycobacteria including the pathogenic Mycobacterium tuberculosis. D29 encodes LysA endolysin, which hydrolyzes mycobacterial cell wall peptidoglycan. We previously showed that LysA harbors two catalytic domains (N-terminal domain [NTD] and lysozyme-like domain [LD]) and a C-terminal cell wall binding domain (CTD). While the importance of LD and CTD in mycobacteriophage biology has been examined in great detail, NTD has largely remained unexplored. Here, to address NTD's significance in D29 physiology, we generated NTD-deficient D29 (D29∆NTD) by deleting the NTD-coding region from D29 genome using CRISPY-BRED. We show that D29∆NTD is viable, but has a longer latent period, and a remarkably reduced burst size and plaque size. A large number of phages were found to be trapped in the host during the D29∆NTD-mediated cell lysis event. Such poor release of progeny phages during host cell lysis strongly suggests that NTD-deficient LysA produced by D29∆NTD, despite having catalytically-active LD, is unable to efficiently lyse host bacteria. We thus conclude that LysA NTD is essential for optimal release of progeny virions, thereby playing an extremely vital role in phage physiology and phage propagation in the environment.

Exploring the Effects of Intersubunit Interface Mutations on Virus-Like Particle Structure and Stability.

Virus-like particles (VLPs) from bacteriophage MS2 provide a platform to study protein self-assembly and create engineered systems for drug delivery. Here, we aim to understand the impact of intersubunit interface mutations on the local and global structure and function of MS2-based VLPs. In previous work, our lab identified locally supercharged double mutants [T71K/G73R] that concentrate positive charge at capsid pores, enhancing uptake into mammalian cells. To study the effects of particle size on cellular internalization, we combined these double mutants with a single point mutation [S37P] that was previously reported to switch particle geometry from T = 3 to T = 1 icosahedral symmetry. These new variants retained their enhanced cellular uptake activity and could deliver small-molecule drugs with efficacy levels similar to our first-generation capsids. Surprisingly, these engineered triple mutants exhibit increased thermostability and unexpected geometry, producing T = 3 particles instead of the anticipated T = 1 assemblies. Transmission electron microscopy revealed various capsid assembly states, including wild-type (T = 3), T = 1, and rod-like particles, that could be accessed using different combinations of these point mutations. Molecular dynamics experiments recapitulated the structural rationale in silico for the single point mutation [S37P] forming a T = 1 virus-like particle and showed that this assembly state was not favored when combined with mutations that favor rod-like architectures. Through this work, we investigated how interdimer interface dynamics influence VLP size and morphology and how these properties affect particle function in applications such as drug delivery.

One-Step Purification Strategy for Cowpea Chlorotic Mottle Virus-Like Particles Produced by the IC-BEVS.

Virus-like particles (VLP) of the cowpea chlorotic mottle virus (CCMV), a plant virus, have been shown to be safe and noncytotoxic vehicles for delivering various cargos, including nucleic acids and peptides, and as scaffolds for presenting epitopes. Thus, CCMV-VLP have acquired increasing attention to be used in fields such as gene therapy, drug delivery, and vaccine development. Regardless of their production method, most reports purify CCMV-VLP through a series of ultracentrifugation steps using sucrose density gradient ultracentrifugation, which is a complex and time-consuming process. Here, the use of anion exchange chromatography is described as a one-step protocol for purification of CCMV-VLP produced by the insect cell-baculovirus expression vector system (IC-BEVS).

Evaluating HIV-1 Infectivity and Virion Maturation across Varied Producer Cells with a Novel FRET-Based Detection and Quantification Assay.

The maturation of HIV-1 virions is a crucial process in viral replication. Although T-cells are a primary source of virus production, much of our understanding of virion maturation comes from studies using the HEK293T human embryonic kidney cell line. Notably, there is a lack of comparative analyses between T-cells and HEK293T cells in terms of virion maturation efficiency in existing literature. We previously developed an advanced virion visualization system based on the FRET principle, enabling the effective distinction between immature and mature virions via fluorescence microscopy. In this study, we utilized pseudotyped, single-round infectious viruses tagged with FRET labels (HIV-1 Gag-iFRET∆Env) derived from Jurkat (a human T-lymphocyte cell line) and HEK293T cells to evaluate their virion maturation rates. HEK293T-derived virions demonstrated a maturity rate of 81.79%, consistent with other studies and our previous findings. However, virions originating from Jurkat cells demonstrated a significantly reduced maturation rate of 68.67% (p < 0.0001). Correspondingly, viruses produced from Jurkat cells exhibited significantly reduced infectivity compared to those derived from HEK293T cells, with the relative infectivity measured at 65.3%. This finding is consistent with the observed relative maturation rate of viruses produced by Jurkat cells. These findings suggest that initiation of virion maturation directly correlates with viral infectivity. Our observation highlights the dynamic nature of virus-host interactions and their implications for virion production and infectivity.

HIV-1 Gag Compact form Stabilized by Intramolecular Interactions is Crucial for Infectious Particle Production.

HIV-1 Gag polyprotein plays a pivotal role in assembly and budding of new particles, by specifically packaging two copies of viral gRNA in the host cell cytoplasm and selecting the cell plasma membrane for budding. Both gRNA and membrane selections are thought to be mediated by the compact form of Gag. This compact form binds to gRNA through both its matrix (MA) and nucleocapsid (NC) domains in the cytoplasm. At the plasma membrane, the membrane competes with gRNA for Gag binding, resulting in a transition to the extended form of Gag found in immature particles with MA bound to membrane lipids and NC to gRNA. The Gag compact form was previously evidenced in vitro. Here, we demonstrated the compact form of Gag in cells by confocal microscopy, using a bimolecular fluorescence complementation approach with a split-GFP bipartite system. Using wild-type Gag and Gag mutants, we showed that the compact form is highly dependent on the binding of MA and NC domains to RNA, as well as on interactions between MA and CA domains. In contrast, Gag multimerization appears to be less critical for the accumulation of the compact form. Finally, mutations altering the formation of Gag compact form led to a strong reduction in viral particle production and infectivity, revealing its key role in the production of infectious viral particles.

Epitranscriptomic cytidine methylation of the hepatitis B viral RNA is essential for viral reverse transcription and particle production.

Epitranscriptomic RNA modifications have emerged as important regulators of the fate and function of viral RNAs. One prominent modification, the cytidine methylation 5-methylcytidine (m5C), is found on the RNA of HIV-1, where m5C enhances the translation of HIV-1 RNA. However, whether m5C functionally enhances the RNA of other pathogenic viruses remains elusive. Here, we surveyed a panel of commonly found RNA modifications on the RNA of hepatitis B virus (HBV) and found that HBV RNA is enriched with m5C as well as ten other modifications, at stoichiometries much higher than host messenger RNA (mRNA). Intriguingly, m5C is mostly found on the epsilon hairpin, an RNA element required for viral RNA encapsidation and reverse transcription, with these m5C mainly deposited by the cellular methyltransferase NSUN2. Loss of m5C from HBV RNA due to NSUN2 depletion resulted in a partial decrease in viral core protein (HBc) production, accompanied by a near-complete loss of the reverse transcribed viral DNA. Similarly, mutations introduced to remove the methylated cytidines resulted in a loss of HBc production and reverse transcription. Furthermore, pharmacological disruption of m5C deposition led to a significant decrease in HBV replication. Thus, our data indicate m5C methylations as a critical mediator of the epsilon elements' function in HBV virion production and reverse transcription, suggesting the therapeutic potential of targeting the m5C methyltransfer process on HBV epsilon as an antiviral strategy.

Applying Flow Virometry to Study the HIV Envelope Glycoprotein and Differences Across HIV Model Systems.

The HIV envelope glycoprotein (Env) is a trimeric protein that facilitates viral binding and fusion with target cells. As the sole viral protein on the HIV surface, Env is important both for immune responses to HIV and in vaccine designs. Targeting Env in clinical applications is challenging due to its heavy glycosylation, high genetic variability, conformational camouflage, and its low abundance on virions. Thus, there is a critical need to better understand this protein. Flow virometry (FV) is a useful methodology for phenotyping the virion surface in a high-throughput, single virion manner. To demonstrate the utility of FV to characterize Env, we stained HIV virions with a panel of 85 monoclonal antibodies targeting different regions of Env. A broad range of antibodies yielded robust staining of Env, with V3 antibodies showing the highest quantitative staining. A subset of antibodies tested in parallel on viruses produced in CD4+ T cell lines, HEK293T cells, and primary cells showed that the cellular model of virus production can impact Env detection. Finally, in addition to being able to highlight Env heterogeneity on virions, we show FV can sensitively detect differences in Env conformation when soluble CD4 is added to virions before staining.

Characterization of a lipid-based jumbo phage compartment as a hub for early phage infection.

Viral genomes are most vulnerable to cellular defenses at the start of the infection. A family of jumbo phages related to phage ΦKZ, which infects Pseudomonas aeruginosa, assembles a protein-based phage nucleus to protect replicating phage DNA, but how it is protected prior to phage nucleus assembly is unclear. We find that host proteins related to membrane and lipid biology interact with injected phage protein, clustering in an early phage infection (EPI) vesicle. The injected virion RNA polymerase (vRNAP) executes early gene expression until phage genome separation from the vRNAP and the EPI vesicle, moving into the nascent proteinaceous phage nucleus. Enzymes involved in DNA replication and CRISPR/restriction immune nucleases are excluded by the EPI vesicle. We propose that the EPI vesicle is rapidly constructed with injected phage proteins, phage DNA, host lipids, and host membrane proteins to enable genome protection, early transcription, localized translation, and to ensure faithful genome transfer to the proteinaceous nucleus.

LDL receptor in alphavirus entry: structural analysis and implications for antiviral therapy.

Various low-density lipoprotein receptors (LPRs) have been identified as entry factors for alphaviruses, and structures of the corresponding virion-receptor complexes have been determined. Here, we analyze the similarities and differences in the receptor binding modes of multiple alphaviruses to understand their ability to infect a wide range of hosts. We further discuss the challenges associated with the development of broad-spectrum treatment strategies against a diverse range of alphaviruses.

BmHSP19.9 targeting P6.9 and VLF-1 to mediate the formation of defective progeny viruses in the silkworm antiviral variety 871C.

The 871C silkworm strain exhibits a high level of resistance to Bombyx mori nucleopolyhedrovirus (BmNPV), making it a valuable variety for the sericulture industry. Understanding the underlying mechanism of its resistance holds great biological significance and economic value in addressing viral disease risks in sericulture. Initially, we infected the resistant strain 871C and its control strain 871 with BmNPV and conducted secondary infection experiments using the progeny occlusion bodies (OBs). As a result, a significant decrease in pathogenicity was observed. Electron microscopy analysis revealed that 871C produces progeny virions with defective DNA packaging, reducing virulence following BmNPV infection. Blood proteomic identification of the silkworm variety 871C and control 871 after BmNPV infection demonstrated the crucial role of the viral proteins P6.9 and VLF-1 in the production of defective viruses by impeding the proper encapsulation of viral DNA. Additionally, we discovered that BmHSP19.9 interacts with P6.9 and VLF-1 and that its expression is significantly upregulated after BmNPV infection. BmHSP19.9 exhibits strong antiviral activity, in part by preventing the entry of the proteins P6.9 and VLF-1 into the nucleus, thereby hindering viral nucleocapsid and viral DNA assembly. Our findings indicate that the antiviral silkworm strain 871C inhibits BmNPV proliferation by upregulating Bmhsp19.9 and impeding the nuclear localization of the viral proteins P6.9 and VLF-1, leading to the production of defective viral particles. This study offers a comprehensive analysis of the antiviral mechanism in silkworms from a viral perspective, providing a crucial theoretical foundation for future antiviral research and the breeding of resistant silkworm strains.

A virally encoded high-resolution screen of cytomegalovirus dependencies.

Genetic screens have transformed our ability to interrogate cellular factor requirements for viral infections1,2, but most current approaches are limited in their sensitivity, biased towards early stages of infection and provide only simplistic phenotypic information that is often based on survival of infected cells2-4. Here, by engineering human cytomegalovirus to express single guide RNA libraries directly from the viral genome, we developed virus-encoded CRISPR-based direct readout screening (VECOS), a sensitive, versatile, viral-centric approach that enables profiling of different stages of viral infection in a pooled format. Using this approach, we identified hundreds of host dependency and restriction factors and quantified their direct effects on viral genome replication, viral particle secretion and infectiousness of secreted particles, providing a multi-dimensional perspective on virus-host interactions. These high-resolution measurements reveal that perturbations altering late stages in the life cycle of human cytomegalovirus (HCMV) mostly regulate viral particle quality rather than quantity, establishing correct virion assembly as a critical stage that is heavily reliant on virus-host interactions. Overall, VECOS facilitates systematic high-resolution dissection of the role of human proteins during the infection cycle, providing a roadmap for in-depth study of host-herpesvirus interactions.

Intrinsic Disorder in the Host Proteins Entrapped in Rabies Virus Particles.

A proteomics analysis of purified rabies virus (RABV) revealed 47 entrapped host proteins within the viral particles. Out of these, 11 proteins were highly disordered. Our study was particularly focused on five of the RABV-entrapped mouse proteins with the highest levels of disorder: Neuromodulin, Chmp4b, DnaJB6, Vps37B, and Wasl. We extensively utilized bioinformatics tools, such as FuzDrop, D2P2, UniProt, RIDAO, STRING, AlphaFold, and ELM, for a comprehensive analysis of the intrinsic disorder propensity of these proteins. Our analysis suggested that these disordered host proteins might play a significant role in facilitating the rabies virus pathogenicity, immune system evasion, and the development of antiviral drug resistance. Our study highlighted the complex interaction of the virus with its host, with a focus on how the intrinsic disorder can play a crucial role in virus pathogenic processes, and suggested that these intrinsically disordered proteins (IDPs) and disorder-related host interactions can also be a potential target for therapeutic strategies.