Intranasal delivery of a self-adjuvanted nanovaccine composed of the curli filaments and the highly conserved M2e epitope confers protection against influenza a virus in mice.

Intranasal administration of vaccines is an attractive delivery route to fight viral respiratory infections. However, there are only a few intranasal vaccines used in human, emphasizing the critical need to identify novel safe mucosal adjuvants and antigen delivery systems to expand their usage. We recently revealed an immunostimulating nanoparticle based on a fragment (R4R5) of the Curli-specific gene A (CsgA) protein that confers protection against influenza A virus (IAV) when conjugated to three repeats of the highly conserved M2e epitope and administrated intramuscularly. Herein, the efficacy of this 3M2e-R4R5 nanovaccine was investigated upon administration by intranasal instillation. By triggering robust M2e-specific humoral and cellular responses, both systemic and locally in the respiratory tract, and by priming alveolar macrophages, the intranasal vaccine protected mice against a lethal IAV challenge without the use of additional adjuvant. Thus, CsgA-based nanostructures could serve as a safe and self-adjuvanted antigen delivery system for mucosal immunization.

Bionanocomposites with Enhanced Physical Properties from Curli Amyloid Assemblies and Cellulose Nanofibrils.

Proteinaceous amyloid fibrils are one of the stiffest biopolymers due to their extensive cross-ß-sheet quaternary structure, whereas cellulose nanofibrils (CNFs) exhibit interesting properties associated with their nanoscale size, morphology, large surface area, and biodegradability. Herein, CNFs were supplemented with amyloid fibrils assembled from the Curli-specific gene A (CsgA) protein, the main component of bacterial biofilms. The resulting composites showed superior mechanical properties, up to a 7-fold increase compared to unmodified CNF films. Wettability and thermogravimetric analyses demonstrated high surface hydrophobicity and robust thermal tolerance. Bulk spectroscopic characterization of CNF-CsgA films revealed key insights into the molecular organization within the bionanocomposites. Atomic force microscopy and photoinduced force microscopy revealed the high-resolution location of curli assemblies into the CNF films. This novel sustainable and cost-effective CNF-based bionanocomposites supplemented with intertwined bacterial amyloid fibrils opens novel directions for environmentally friendly applications demanding high mechanical, water-repelling properties, and thermal resistance.

Synthetic Multicomponent Nanovaccines Based on the Molecular Co-assembly of ß-Peptides Protect against Influenza A Virus.

Peptides with the ability to self-assemble into nanoparticles have emerged as an attractive strategy to design antigen delivery platforms for subunit vaccines. While toll-like receptor (TLR) agonists are promising immunostimulants, their use as soluble agents is limited by their rapid clearance and off-target inflammation. Herein, we harnessed molecular co-assembly to prepare multicomponent cross-ß-sheet peptide nanofilaments exposing an antigenic epitope derived from the influenza A virus and a TLR agonist. The TLR7 agonist imiquimod and the TLR9 agonist CpG were respectively functionalized on the assemblies by means of an orthogonal pre- or post-assembly conjugation strategy. The nanofilaments were readily uptaken by dendritic cells, and the TLR agonists retained their activity. Multicomponent nanovaccines induced a robust epitope-specific immune response and completely protected immunized mice from a lethal influenza A virus inoculation. This versatile bottom-up approach is promising for the preparation of synthetic vaccines with customized magnitude and polarization of the immune responses.

Engineered Curli Nanofilaments as a Self-Adjuvanted Antigen Delivery Platform.

Proteinaceous nanoparticles constitute efficient antigen delivery systems in vaccine formulations due to their size and repetitive nature that mimic most invading pathogens and promote immune activation. Nonetheless, the coadministration of an adjuvant with subunit nanovaccines is usually required to induce a robust, long-lasting, and protective immune response. Herein, the protein Curli-specific gene A (CsgA), which is known to self-assemble into nanofilaments contributing to bacterial biofilm, is exploited to engineer an intrinsically immunostimulatory antigen delivery platform. Three repeats of the M2e antigenic sequence from the influenza A virus matrix 2 protein are merged to the N-terminal domain of engineered CsgA proteins. These chimeric 3M2e-CsgA spontaneously self-assemble into antigen-displaying cross-ß-sheet nanofilaments that activate the heterodimeric toll-like receptors 2 and 1. The resulting nanofilaments are avidly internalized by antigen-presenting cells and stimulate the maturation of dendritic cells. Without the need of any additional adjuvants, both assemblies show robust humoral and cellular immune responses, which translate into complete protection against a lethal experimental infection with the H1N1 influenza virus. Notably, these CsgA-based nanovaccines induce neither overt systemic inflammation, nor reactogenicity, upon mice inoculation. These results highlight the potential of engineered CsgA nanostructures as self-adjuvanted, safe, and versatile antigen delivery systems to fight infectious diseases.

Vaccination Strategies Based on Bacterial Self-Assembling Proteins as Antigen Delivery Nanoscaffolds.

Vaccination has saved billions of human lives and has considerably reduced the economic burden associated with pandemic and endemic infectious diseases. Notwithstanding major advancements in recent decades, multitude diseases remain with no available effective vaccine. While subunit-based vaccines have shown great potential to address the safety concerns of live-attenuated vaccines, their limited immunogenicity remains a major drawback that still needs to be addressed for their use fighting infectious illnesses, autoimmune disorders, and/or cancer. Among the adjuvants and delivery systems for antigens, bacterial proteinaceous supramolecular structures have recently received considerable attention. The use of bacterial proteins with self-assembling properties to deliver antigens offers several advantages, including biocompatibility, stability, molecular specificity, symmetrical organization, and multivalency. Bacterial protein nanoassemblies closely simulate most invading pathogens, acting as an alarm signal for the immune system to mount an effective adaptive immune response. Their nanoscale architecture can be precisely controlled at the atomic level to produce a variety of nanostructures, allowing for infinite possibilities of organized antigen display. For the bottom-up design of the proteinaceous antigen delivery scaffolds, it is essential to understand how the structural and physicochemical properties of the nanoassemblies modulate the strength and polarization of the immune responses. The present review first describes the relationships between structure and the generated immune responses, before discussing potential and current clinical applications.

Self-Assembly of Flagellin into Immunostimulatory Ring-like Nanostructures as an Antigen Delivery System.

Proteinaceous nanoparticles represent attractive antigen carriers for vaccination as their size and repetitive antigen displays that mimic most viral particles enable efficient immune processing. However, these nanocarriers are often unable to stimulate efficiently the innate immune system, requiring coadministration with adjuvants to promote long-lasting protective immunity. The protein flagellin, which constitutes the primary constituent of the bacterial flagellum, has been widely evaluated as an antigen carrier due to its intrinsic adjuvant properties involving activation of the innate immune receptor Toll-like receptor 5 (TLR5). Although flagellin is known for its ability to self-assemble into micron-scale length nanotubes, few studies have evaluated the potential usage of flagellin-based nanostructures as immunostimulatory antigen carriers. In this study, we reported for the first time a strategy to guide the self-assembly of a flagellin protein from Bacillus subtilis, Hag, into lower aspect ratio nanoparticles by hindering non-covalent interactions responsible for its elongation into nanotubes. We observed that addition of an antigenic sequence derived from the influenza A virus (3M2e) at the C-terminus of this flagellin, as opposed to positioning the epitope into mid-sequence, precluded filament elongation and resulted in low aspect ratio ring-like nanostructures upon salting-out-induced self-assembly. These nanostructures displayed the antigen at their surface and shared morphological and structural characteristics with flagellin nanotubes, with a diameter of approximately 12 nm, and an α-helix-rich secondary structure. Flagellin ring-like nanostructures were efficiently internalized by antigen-presenting cells, and avidly activated the TLR5 in vitro as well as the innate and adaptive immune responses. Intranasal immunization of mice with these nanostructures resulted in the potentiation of the antigen-specific antibody response and protection against a lethal infection with the influenza A virus, illustrating the potential of these intrinsically immunostimulatory nanostructures as antigen carriers.

Recombinant Bacillus subtilis flagellin Hag is a potent immunostimulant with reduced proinflammatory properties compared to Salmonella enterica serovar Typhimurium FljB.

Flagellin constitutes a potential adjuvant for vaccines owing to its robust immunostimulatory properties. However, clinical trials have revealed that flagellin derived from Salmonella enterica serovar Typhimurium induces high levels of proinflammatory markers and substantial adverse effects. The flagellin from Bacillus subtilis, Hag, shares high sequence homology with Salmonella FljB within the D0 and D1 domains responsible for TLR5 engagement, while the D2 and D3 domains associated with an off-target immune response are absent. Accordingly, we compared the immunostimulatory and proinflammatory properties of Hag with FljB by harnessing an epitope from the matrix 2 protein (M2e) of the influenza virus. Both flagellins engaged TLR5, with FljB showing a 2.5-fold higher potency than Hag. Mice inoculation showed a robust FljB- or Hag-induced M2e-specific antibody response, with Hag demonstrating a decreased secretion of proinflammatory markers and reduced weight loss. This study revealed that flagellin Hag is a potent immunoadjuvant with reduced proinflammatory properties.

Immunogenicity and Protective Potential of Mucosal Vaccine Formulations Based on Conserved Epitopes of Influenza A Viruses Fused to an Innovative Ring Nanoplatform in Mice and Chickens.

Current inactivated vaccines against influenza A viruses (IAV) mainly induce immune responses against highly variable epitopes across strains and are mostly delivered parenterally, limiting the development of an effective mucosal immunity. In this study, we evaluated the potential of intranasal formulations incorporating conserved IAV epitopes, namely the long alpha helix (LAH) of the stalk domain of hemagglutinin and three tandem repeats of the ectodomain of the matrix protein 2 (3M2e), as universal mucosal anti-IAV vaccines in mice and chickens. The IAV epitopes were grafted to nanorings, a novel platform technology for mucosal vaccination formed by the nucleoprotein (N) of the respiratory syncytial virus, in fusion or not with the C-terminal end of the P97 protein (P97c), a recently identified Toll-like receptor 5 agonist. Fusion of LAH to nanorings boosted the generation of LAH-specific systemic and local antibody responses as well as cellular immunity in mice, whereas the carrier effect of nanorings was less pronounced towards 3M2e. Mice vaccinated with chimeric nanorings bearing IAV epitopes in fusion with P97c presented modest LAH- or M2e-specific IgG titers in serum and were unable to generate a mucosal humoral response. In contrast, N-3M2e or N-LAH nanorings admixed with Montanide™ gel (MG) triggered strong specific humoral responses, composed of serum type 1/type 2 IgG and mucosal IgG and IgA, as well as cellular responses dominated by type 1/type 17 cytokine profiles. All mice vaccinated with the [N-3M2e + N-LAH + MG] formulation survived an H1N1 challenge and the combination of both N-3M2e and N-LAH nanorings with MG enhanced the clinical and/or virological protective potential of the preparation in comparison to individual nanorings. Chickens vaccinated parenterally or mucosally with N-LAH and N-3M2e nanorings admixed with Montanide™ adjuvants developed a specific systemic humoral response, which nonetheless failed to confer protection against heterosubtypic challenge with a highly pathogenic H5N8 strain. Thus, while the combination of N-LAH and N-3M2e nanorings with Montanide™ adjuvants shows promise as a universal mucosal anti-IAV vaccine in the mouse model, further experiments have to be conducted to extend its efficacy to poultry.

Self-assembled peptide nanorod vaccine confers protection against influenza A virus.

Proteinaceous nanostructures have emerged as a promising strategy to develop safe and efficient subunit vaccines. The ability of synthetic ß-sheet self-assembling peptides to stabilize antigenic determinants and to potentiate the epitope-specific immune responses have highlighted their potential as an immunostimulating platform for antigen delivery. Nonetheless, the intrinsic polymorphism of the resulting cross-ß fibrils, their length in the microscale and their close structural similarity with pathological amyloids could limit their usage in vaccinology. In this study, we harnessed electrostatic capping motifs to control the self-assembly of a chimeric peptide comprising a 10-mer ß-sheet sequence and a highly conserved epitope derived from the influenza A virus (M2e). Self-assembly led to the formation of 100-200 nm long uniform nanorods (NRs) displaying the M2e epitope on their surface. These cross-ß assemblies differed from prototypical amyloid fibrils owing to low polydispersity, short length, non-binding to thioflavin T and Congo Red dyes, and incapacity to seed homologous amyloid assembly. M2e-NRs were efficiently uptaken by antigen presenting cells and the cross-ß quaternary architecture activated the Toll-like receptor 2 and stimulated dendritic cells. Mice subcutaneous immunization revealed a robust M2e-specific IgG response, which was dependent on self-assembly into NRs. Upon intranasal immunization in combination with the polymeric adjuvant montanide gel, M2e-NRs conferred complete protection with absence of clinical signs against a lethal experimental infection with the H1N1 influenza A virus. These findings indicate that by acting as an immunostimulator and delivery system, synthetic peptide-based NRs constitute a versatile self-adjuvanted nanoplatform for the delivery of subunit vaccines.

Identification of the nuclear and nucleolar localization signals of the Feline immunodeficiency virus Rev protein.

Lentivirus genomes code for a regulatory protein essential for virus replication termed Rev. The Rev protein binds to partially spliced and unspliced viral RNAs and mediates their nuclear export. Therefore, Rev possesses functional domains that enable its shuttling between the cytoplasm and the nucleus. The Feline immunodeficiency virus (FIV), a lentivirus, can lead to an immunodeficiency syndrome after a long incubation period, similar to that associated with the human immunodeficiency virus type 1 (HIV-1). The FIV Rev functional domains have been predicted only by homology with those of HIV-1 Rev. In the present study, the nuclear and nucleolar localization signals (NLS and NoLS, respectively) of the FIV Rev were examined. A series of FIV Rev deletion mutants fused to the enhanced green fluorescent protein (EGFP) were used to localize the NLS in a region spanning amino acids (aa) 81-100. By using alanine substitution mutants, basic residues present between the amino acids (aa) 84-99 of the FIV Rev protein sequence were identified to form the NLS, whereas those between aa 82-95 were associated with the NoLS function. These results further enhance our understanding of how Rev exerts its role in the replication cycle of lentiviruses.

Harnessing the Activation of Toll-Like Receptor 2/6 by Self-Assembled Cross-ß Fibrils to Design Adjuvanted Nanovaccines.

Protein fibrils characterized with a cross-ß-sheet quaternary structure have gained interest as nanomaterials in biomedicine, including in the design of subunit vaccines. Recent studies have shown that by conjugating an antigenic determinant to a self-assembling ß-peptide, the resulting supramolecular assemblies act as an antigen delivery system that potentiates the epitope-specific immune response. In this study, we used a ten-mer self-assembling sequence (I10) derived from an amyloidogenic peptide to biophysically and immunologically characterize a nanofibril-based vaccine against the influenza virus. The highly conserved epitope from the ectodomain of the matrix protein 2 (M2e) was elongated at the N-terminus of I10 by solid phase peptide synthesis. The chimeric M2e-I10 peptide readily self-assembled into unbranched, long, and twisted fibrils with a diameter between five and eight nm. These cross-ß nanoassemblies were cytocompatible and activated the heterodimeric Toll-like receptor (TLR) 2/6. Upon mice subcutaneous immunization, M2e-fibrils triggered a robust anti-M2e specific immune response, which was dependent on self-assembly and did not require the use of an adjuvant. Overall, this study describes the efficacy of cross-ß fibrils to activate the TLR 2/6 and to stimulate the epitope-specific immune response, supporting usage of these proteinaceous assemblies as a self-adjuvanted delivery system for antigens.

Characterization of Signal Sequences Determining the Nuclear/Nucleolar Import and Nuclear Export of the Caprine Arthritis-Encephalitis Virus Rev Protein.

Caprine arthritis-encephalitis virus (CAEV), a lentivirus, relies on the action of the Rev protein for its replication. The CAEV Rev fulfills its function by allowing the nuclear exportation of partially spliced or unspliced viral mRNAs. In this study, we characterized the nuclear and nucleolar localization signals (NLS and NoLS, respectively) and the nuclear export signal (NES) of the CAEV Rev protein. These signals are key actors in the nucleocytoplasmic shuttling of a lentiviral Rev protein. Several deletion and alanine substitution mutants were generated from a plasmid encoding the CAEV Rev wild-type protein that was fused to the enhanced green fluorescent protein (EGFP). Following cell transfection, images were captured by confocal microscopy and the fluorescence was quantified in the different cell compartments. The results showed that the NLS region is localized between amino acids (aa) 59 to 75, has a monopartite-like structure and is exclusively composed of arginine residues. The NoLS was found to be partially associated with the NLS. Finally, the CAEV Rev protein's NES mapped between aa 89 to 101, with an aa spacing between the hydrophobic residues that was found to be unconventional as compared to that of other retroviral Rev/Rev-like proteins.

Identification of a novel TLR5 agonist derived from the P97 protein of Mycoplasma hyopneumoniae.

By modulating specific immune responses against antigens, adjuvants are used in many vaccine preparations to enhance protective immunity. The C-terminal domain of the protein P97 (P97c) of Mycoplasma hyopneumoniae, which is the etiologic agent of porcine enzootic pneumonia, has been shown to increase the specific humoral response against an antigen when this antigen is merged with P97c and delivered by adenovectors. However, the immunostimulating mechanism of this protein remains unknown. In the present study, recombinantly expressed P97c triggered a concentration-dependent TLR5 activation and stimulates the production of interleukin-8 from HEK-Blue mTLR5 cells. Circular dichroism spectroscopy and prediction of 3-dimensional conformation exposed a relevant secondary and tertiary structural homology between P97c and flagellin, the known potent TLR5 agonist. P97c adjuvanticity was evaluated by fusing the conserved epitope of the ectodomain matrix 2 protein (M2e) of the influenza A virus to the protein. Mice immunized with P97c-3M2e revealed a high antibody titer against the M2e epitope associated with a mixed Th1/Th2 immune response. Overall, this study identifies a novel agonist of the pattern recognition receptor TLR5 and reveals that P97c is a potential adjuvant through the activation of the innate immune system.

Protein Supramolecular Structures: From Self-Assembly to Nanovaccine Design.

Life-inspired protein supramolecular assemblies have recently attracted considerable attention for the development of next-generation vaccines to fight against infectious diseases, as well as autoimmune diseases and cancer. Protein self-assembly enables atomic scale precision over the final architecture, with a remarkable diversity of structures and functionalities. Self-assembling protein nanovaccines are associated with numerous advantages, including biocompatibility, stability, molecular specificity and multivalency. Owing to their nanoscale size, proteinaceous nature, symmetrical organization and repetitive antigen display, protein assemblies closely mimic most invading pathogens, serving as danger signals for the immune system. Elucidating how the structural and physicochemical properties of the assemblies modulate the potency and the polarization of the immune responses is critical for bottom-up design of vaccines. In this context, this review briefly covers the fundamentals of supramolecular interactions involved in protein self-assembly and presents the strategies to design and functionalize these assemblies. Examples of advanced nanovaccines are presented, and properties of protein supramolecular structures enabling modulation of the immune responses are discussed. Combining the understanding of the self-assembly process at the molecular level with knowledge regarding the activation of the innate and adaptive immune responses will support the design of safe and effective nanovaccines.

The Jembrana disease virus Rev protein: Identification of nuclear and novel lentiviral nucleolar localization and nuclear export signals.

The lentiviral Rev protein, which is a regulatory protein essential for virus replication, has been first studied in the human immunodeficiency virus type 1 (HIV-1). The main function of Rev is to mediate the nuclear exportation of viral RNAs. To fulfill its function, Rev shuttles between the cytoplasm and the nucleus. The Jembrana disease virus (JDV), a lentivirus, is the etiologic agent of the Jembrana disease which was first described in Bali cattle in Indonesia in 1964. Despite the high mortality rate associated with JDV, this virus remains poorly studied. Herein the subcellular distribution of JDV Rev, the nuclear and nucleolar localization signals (NLS and NoLS, respectively) and the nuclear export signal (NES) of the protein were examined. JDV Rev fused to the enhanced green fluorescent protein (EGFP) predominantly localized to the cytoplasm and nucleolus of transfected cells, as determined by fluorescence microscopy analyses. Through transfection of a series of deletion mutants of JDV Rev, it was possible to localize the NLS/NoLS region between amino acids (aa) 74 to 105. By substituting basic residues with alanine within this sequence, we demonstrated that the JDV Rev NLS encompasses aa 76 to 86, and is exclusively composed of arginine residues, whereas a bipartite NoLS was observed for the first time in any retroviral Rev/Rev-like proteins. Finally, a NES was identified downstream of the NLS/NoLS and encompasses aa 116 to 128 of the JDV Rev protein. The JDV Rev NES was found to be of the protein kinase A inhibitor (PKI) class instead of the HIV-1 Rev class. It also corresponds to the most optimal consensus sequence of PKI NES and, as such, is novel among lentiviral Rev NES.

Guiding the Morphology of Amyloid Assemblies by Electrostatic Capping: from Polymorphic Twisted Fibrils to Uniform Nanorods.

Peptides that self-assemble into cross-ß-sheet amyloid structures constitute promising building blocks to construct highly ordered proteinaceous materials and nanoparticles. Nevertheless, the intrinsic polymorphism of amyloids and the difficulty of controlling self-assembly currently limit their usage. In this study, the effect of electrostatic interactions on the supramolecular organization of peptide assemblies is investigated to gain insights into the structural basis of the morphological diversities of amyloids. Different charged capping units are introduced at the N-terminus of a potent ß-sheet-forming sequence derived from the 20-29 segment of islet amyloid polypeptide, known to self-assemble into polymorphic fibrils. By tuning the charge and the electrostatic strength, different mesoscopic morphologies are obtained, including nanorods, rope-like fibrils, and twisted ribbons. Particularly, the addition of positive capping units leads to the formation of uniform rod-like assemblies, with lengths that can be modulated by the charge number. It is proposed that electrostatic repulsions between N-terminal positive charges hinder ß-sheet tape twisting, leading to a unique control over the size of these cytocompatible nanorods by protofilament growth frustration. This study reveals the high susceptibility of amyloid formation to subtle chemical modifications and opens to promising strategies to control the final architecture of proteinaceous assemblies from the peptide sequence.

Nanoparticle-Based Vaccines Against Respiratory Viruses.

The respiratory mucosa is the primary portal of entry for numerous viruses such as the respiratory syncytial virus, the influenza virus and the parainfluenza virus. These pathogens initially infect the upper respiratory tract and then reach the lower respiratory tract, leading to diseases. Vaccination is an affordable way to control the pathogenicity of viruses and constitutes the strategy of choice to fight against infections, including those leading to pulmonary diseases. Conventional vaccines based on live-attenuated pathogens present a risk of reversion to pathogenic virulence while inactivated pathogen vaccines often lead to a weak immune response. Subunit vaccines were developed to overcome these issues. However, these vaccines may suffer from a limited immunogenicity and, in most cases, the protection induced is only partial. A new generation of vaccines based on nanoparticles has shown great potential to address most of the limitations of conventional and subunit vaccines. This is due to recent advances in chemical and biological engineering, which allow the design of nanoparticles with a precise control over the size, shape, functionality and surface properties, leading to enhanced antigen presentation and strong immunogenicity. This short review provides an overview of the advantages associated with the use of nanoparticles as vaccine delivery platforms to immunize against respiratory viruses and highlights relevant examples demonstrating their potential as safe, effective and affordable vaccines.

Engineering and evaluation of amyloid assemblies as a nanovaccine against the Chikungunya virus.

The design of nanoparticles exposing a high density of antigens constitutes a promising strategy to address safety concerns of conventional life-attenuated vaccines as well as to increase the immunogenicity of subunit vaccines. In this study, we developed a fully synthetic nanovaccine based on an amyloid peptide sequence with high self-assembling properties. The immunogenic epitope E2EP3 from the E2 glycoprotein of the Chikungunya virus was used to evaluate the potential of a 10-mer peptide derived from an endogenous amyloidogenic polypeptide as a novel vaccine platform. Chimeric peptides, comprising the peptide antigen attached to the amyloid core by a short flexible linker, were prepared by solid phase synthesis. As observed using atomic force microscopy, these polypeptides self-assembled into linear and unbranched fibrils with a diameter ranging from 6 to 8 nm. A quaternary conformation rich in cross-ß-sheets characterized these assemblies, as demonstrated by circular dichroism spectroscopy and thioflavin T fluorescence. ELISA assays and transmission electronic microscopy of immunogold labeled-fibrils revealed a high density of the Chikungunya virus E2 glycoprotein derived epitope exposed on the fibril surface. These amyloid fibrils were cytocompatible and were efficiently uptaken by macrophages. Mice immunization revealed a robust IgG response against the E2EP3 epitope, which was dependent on self-assembly and did not require co-injection of the Alhydrogel adjuvant. These results indicate that cross-ß-sheet amyloid assemblies constitute suitable synthetic self-adjuvanted assemblies to anchor antigenic determinants and to increase the immunogenicity of peptide epitopes.

B23/nucleophosmin interacts with bovine immunodeficiency virus Rev protein and facilitates viral replication.

The bovine immunodeficiency virus (BIV) Rev shuttling protein contains nuclear/nucleolar localization signals and nuclear import/export mechanisms that are novel among lentivirus Rev proteins. Several viral proteins localize to the nucleolus, which may play a role in processes that are essential to the outcome of viral replication. Although BIV Rev localizes to the nucleoli of transfected/infected cells and colocalizes with one of its major proteins, nucleophosmin (NPM1, also known as B23), the role of the nucleolus and B23 in BIV replication remains to be determined. Here, we demonstrate for the first time that BIV Rev interacts with nucleolar phosphoprotein B23 in cells. Using small interfering RNA (siRNA) technology, we show that depletion of B23 expression inhibits virus production by BIV-infected cells, indicating that B23 plays an important role in BIV replication. The interaction between Rev and B23 may represent a potential new target for the development of antiviral drugs against lentiviruses.