Intranasal administration of adenoviral vaccines expressing SARS-CoV-2 spike protein improves vaccine immunity in mouse models.

The ongoing SARS-CoV-2 pandemic is controlled but not halted by public health measures and mass vaccination strategies which have exclusively relied on intramuscular vaccines. Intranasal vaccines can prime or recruit to the respiratory epithelium mucosal immune cells capable of preventing infection. Here we report a comprehensive series of studies on this concept using various mouse models, including HLA class II-humanized transgenic strains. We found that a single intranasal (i.n.) dose of serotype-5 adenoviral vectors expressing either the receptor binding domain (Ad5-RBD) or the complete ectodomain (Ad5-S) of the SARS-CoV-2 spike protein was effective in inducing i) serum and bronchoalveolar lavage (BAL) anti-spike IgA and IgG, ii) robust SARS-CoV-2-neutralizing activity in the serum and BAL, iii) rigorous spike-directed T helper 1 cell/cytotoxic T cell immunity, and iv) protection of mice from a challenge with the SARS-CoV-2 beta variant. Intramuscular (i.m.) Ad5-RBD or Ad5-S administration did not induce serum or BAL IgA, and resulted in lower neutralizing titers in the serum. Moreover, prior immunity induced by an intramuscular mRNA vaccine could be potently enhanced and modulated towards a mucosal IgA response by an i.n. Ad5-S booster. Notably, Ad5 DNA was found in the liver or spleen after i.m. but not i.n. administration, indicating a lack of systemic spread of the vaccine vector, which has been associated with a risk of thrombotic thrombocytopenia. Unlike in otherwise genetically identical HLA-DQ6 mice, in HLA-DQ8 mice Ad5-RBD vaccine was inferior to Ad5-S, suggesting that the RBD fragment does not contain a sufficient collection of helper-T cell epitopes to constitute an optimal vaccine antigen. Our data add to previous promising preclinical results on intranasal SARS-CoV-2 vaccination and support the potential of this approach to elicit mucosal immunity for preventing transmission of SARS-CoV-2.

Structure of SNX9 SH3 in complex with a viral ligand reveals the molecular basis of its unique specificity for alanine-containing class I SH3 motifs.

Class I SH3 domain-binding motifs generally comply with the consensus sequence [R/K]xØPxxP, the hydrophobic residue Ø being proline or leucine. We have studied the unusual Ø = Ala-specificity of SNX9 SH3 by determining its complex structure with a peptide present in eastern equine encephalitis virus (EEEV) nsP3. The structure revealed the length and composition of the n-Src loop as important factors determining specificity. We also compared the affinities of EEEV nsP3 peptide, its mutants, and cellular ligands to SNX9 SH3. These data suggest that nsP3 has evolved to minimize reduction of conformational entropy upon binding, hence acquiring stronger affinity, enabling takeover of SNX9. The RxAPxxP motif was also found in human T cell leukemia virus-1 (HTLV-1) Gag polyprotein. We found that this motif was required for efficient HTLV-1 infection, and that the specificity of SNX9 SH3 for the RxAPxxP core binding motif was importantly involved in this process.

Evolutionary plasticity of SH3 domain binding by Nef proteins of the HIV-1/SIVcpz lentiviral lineage.

The accessory protein Nef of human and simian immunodeficiency viruses (HIV and SIV) is an important pathogenicity factor known to interact with cellular protein kinases and other signaling proteins. A canonical SH3 domain binding motif in Nef is required for most of these interactions. For example, HIV-1 Nef activates the tyrosine kinase Hck by tightly binding to its SH3 domain. An archetypal contact between a negatively charged SH3 residue and a highly conserved arginine in Nef (Arg77) plays a key role here. Combining structural analyses with functional assays, we here show that Nef proteins have also developed a distinct structural strategy-termed the "R-clamp"-that favors the formation of this salt bridge via buttressing Arg77. Comparison of evolutionarily diverse Nef proteins revealed that several distinct R-clamps have evolved that are functionally equivalent but differ in the side chain compositions of Nef residues 83 and 120. Whereas a similar R-clamp design is shared by Nef proteins of HIV-1 groups M, O, and P, as well as SIVgor, the Nef proteins of SIV from the Eastern chimpanzee subspecies (SIVcpzP.t.s.) exclusively utilize another type of R-clamp. By contrast, SIV of Central chimpanzees (SIVcpzP.t.t.) and HIV-1 group N strains show more heterogenous R-clamp design principles, including a non-functional evolutionary intermediate of the aforementioned two classes. These data add to our understanding of the structural basis of SH3 binding and kinase deregulation by Nef, and provide an interesting example of primate lentiviral protein evolution.

T cell circuits that sense antigen density with an ultrasensitive threshold.

Overexpressed tumor-associated antigens [for example, epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2)] are attractive targets for therapeutic T cells, but toxic "off-tumor" cross-reaction with normal tissues that express low levels of target antigen can occur with chimeric antigen receptor (CAR)-T cells. Inspired by natural ultrasensitive response circuits, we engineered a two-step positive-feedback circuit that allows human cytotoxic T cells to discriminate targets on the basis of a sigmoidal antigen-density threshold. In this circuit, a low-affinity synthetic Notch receptor for HER2 controls the expression of a high-affinity CAR for HER2. Increasing HER2 density thus has cooperative effects on T cells-it increases both CAR expression and activation-leading to a sigmoidal response. T cells with this circuit show sharp discrimination between target cells expressing normal amounts of HER2 and cancer cells expressing 100 times as much HER2, both in vitro and in vivo.

HIV-1 Nef-Induced Secretion of the Proinflammatory Protease TACE into Extracellular Vesicles Is Mediated by Raf-1 and Can Be Suppressed by Clinical Protein Kinase Inhibitors.

Chronic immune activation is an important driver of human immunodeficiency virus type 1 (HIV-1) pathogenesis and has been associated with the presence of tumor necrosis factor-α converting enzyme (TACE) in extracellular vesicles (EVs) circulating in infected individuals. We have recently shown that activation of the Src-family tyrosine kinase hematopoietic cell kinase (Hck) by HIV-1 Nef can trigger the packaging of TACE into EVs via an unconventional protein secretion pathway. Using a panel of HIV-1 Nef mutants and natural HIV-2 and simian immunodeficiency virus (SIV) Nef alleles, we now show that the capacity to promote TACE secretion depends on the superior ability of HIV-1-like Nef alleles to induce Hck kinase activity, whereas other Nef effector functions are dispensable. Strikingly, among the numerous Src-family downstream effectors, serine/threonine kinase Raf-1 was found to be necessary and alone sufficient to trigger the secretion of TACE into EVs. These data reveal the involvement of Raf-1 in regulation of unconventional protein secretion and highlight the importance of Raf-1 as a cellular effector of Nef, thereby suggesting a novel rationale for testing pharmacological inhibitors of the Raf-MAPK pathway to treat HIV-associated immune activation.IMPORTANCE Chronic immune activation contributes to the immunopathogenesis of human immunodeficiency virus type 1 (HIV-1) infection and is associated with poor recovery of the immune system despite potent antiretroviral therapy, which is observed in 10% to 40% drug-treated patients depending on the definition of immune reconstitution. We have previously shown that the HIV pathogenicity factor Nef can promote loading of the proinflammatory protease TACE into extracellular vesicles (EVs), and the levels of such TACE-containing EVs circulating in the blood correlate with low CD4 lymphocyte counts in HIV patients receiving antiretroviral therapy. Here, we show that Nef promotes uploading of TACE into EVs by triggering unconventional secretion via activation of the Hck/Raf/mitogen-activated protein kinase (MAPK) cascade. We find that several pharmaceutical inhibitors of these kinases that are currently in clinical use for other diseases can potently suppress this pathogenic deregulation and could thus provide a novel strategy for treating HIV-associated immune activation.

MC159 of Molluscum Contagiosum Virus Suppresses Autophagy by Recruiting Cellular SH3BP4 via an SH3 Domain-Mediated Interaction.

MC159 is a viral FLIP (FLICE inhibitory protein) encoded by the molluscum contagiosum virus (MCV) enabling MCV to evade antiviral immunity and to establish persistent infections in humans. Here, we show that MC159 contains a functional SH3 binding motif, which mediates avid and selective binding to SH3BP4, a signaling protein known to regulate endocytic trafficking and suppress cellular autophagy. The capacity to bind SH3BP4 was dispensable for regulation of NF-κB-mediated transcription and suppression of proapoptotic caspase activation but contributed to inhibition of amino acid starvation-induced autophagy by MC159. These results provide new insights into the cellular functions of MC159 and reveal SH3BP4 as a novel host cell factor targeted by a viral immune evasion protein.IMPORTANCE After the eradication of smallpox, molluscum contagiosum virus (MCV) is the only poxvirus restricted to infecting humans. MCV infection is common and causes benign skin lesions that usually resolve spontaneously but may persist for years and grow large, especially in immunocompromised individuals. While not life threatening, MCV infections pose a significant global health burden. No vaccine or specific anti-MCV therapy is available. MCV encodes several proteins that enable it to evade antiviral immunity, a notable example of which is the MC159 protein. In this study, we describe a novel mechanism of action for MC159 involving hijacking of a host cell protein called SH3BP4 to suppress autophagy, a cellular recycling mechanism important for antiviral immunity. This study contributes to our understanding of the host cell interactions of MCV and the molecular function of MC159.

Tyrosine phosphorylation directs TACE into extracellular vesicles via unconventional secretion.

When studying how HIV-1 Nef can promote packaging of the proinflammatory transmembrane protease TACE (tumor necrosis factor-α converting enzyme) into extracellular vesicles (EVs) we have revealed a novel tyrosine kinase-regulated unconventional protein secretion (UPS) pathway for TACE. When TACE was expressed without its trafficking cofactor iRhom allosteric Hck activation by Nef triggered translocation of TACE into EVs. This process was insensitive to blocking of classical secretion by inhibiting endoplasmic reticulum (ER) to Golgi transport, and involved a distinct form of TACE devoid of normal glycosylation and incompletely processed for prodomain removal. Like most other examples of UPS this process was Golgi reassembly stacking protein (GRASP)-dependent but was not associated with ER stress. These data indicate that Hck-activated UPS provides an alternative pathway for TACE secretion that can bypass iRhom-dependent ER to Golgi transfer, and suggest that tyrosine phosphorylation might have a more general role in regulating UPS.

ANKRD54 preferentially selects Bruton's Tyrosine Kinase (BTK) from a Human Src-Homology 3 (SH3) domain library.

Bruton's Tyrosine Kinase (BTK) is a cytoplasmic protein tyrosine kinase with a fundamental role in B-lymphocyte development and activation. The nucleocytoplasmic shuttling of BTK is specifically modulated by the Ankyrin Repeat Domain 54 (ANKRD54) protein and the interaction is known to be exclusively SH3-dependent. To identify the spectrum of the ANKRD54 SH3-interactome, we applied phage-display screening of a library containing all the 296 human SH3 domains. The BTK-SH3 domain was the prime interactor. Quantitative western blotting analysis demonstrated the accuracy of the screening procedure. Revealing the spectrum and specificity of ANKRD54-interactome is a critical step toward functional analysis in cells and tissues.

Structural Basis of the High Affinity Interaction between the Alphavirus Nonstructural Protein-3 (nsP3) and the SH3 Domain of Amphiphysin-2.

We show that a peptide from Chikungunya virus nsP3 protein spanning residues 1728-1744 binds the amphiphysin-2 (BIN1) Src homology-3 (SH3) domain with an unusually high affinity (Kd 24 nm). Our NMR solution complex structure together with isothermal titration calorimetry data on several related viral and cellular peptide ligands reveal that this exceptional affinity originates from interactions between multiple basic residues in the target peptide and the extensive negatively charged binding surface of amphiphysin-2 SH3. Remarkably, these arginines show no fixed conformation in the complex structure, indicating that a transient or fluctuating polyelectrostatic interaction accounts for this affinity. Thus, via optimization of such dynamic electrostatic forces, viral peptides have evolved a superior binding affinity for amphiphysin-2 SH3 compared with typical cellular ligands, such as dynamin, thereby enabling hijacking of amphiphysin-2 SH3-regulated host cell processes by these viruses. Moreover, our data show that the previously described consensus sequence PXRPXR for amphiphysin SH3 ligands is inaccurate and instead define it as an extended Class II binding motif PXXPXRpXR, where additional positive charges between the two constant arginine residues can give rise to extraordinary high SH3 binding affinity.

HIV-Nef and ADAM17-Containing Plasma Extracellular Vesicles Induce and Correlate with Immune Pathogenesis in Chronic HIV Infection.

Antiretroviral therapy (ART) efficiently suppresses HIV replication but immune activation and low CD4 T cell counts often persist. The underlying mechanism of this ART-resistant pathogenesis is not clear. We observed that levels of plasma extracellular vesicles (pEV) are strongly elevated in HIV infection and do not decline during ART. Surprisingly, these vesicles contained the viral accessory proteins Nef and Vpu, which are assumed to be not expressed under efficient ART, as well as pro-inflammatory effectors, including activated ADAM17. HIV pEV were characterized by the presence of activated αvß3 and absence of CD81 and Tsg101. Correlating with immune activation, peripheral monocytes ingested large amounts of pEV, giving rise to an increased population of CD1c(+) CD14(+) cells that secreted inflammatory cytokines. Importantly, the pro-inflammatory content, particularly ADAM17 activity, correlated with low T cell counts. Preliminary evidence suggested that HIV pEV derived from peripheral mononuclear cells and from an unknown myeloid cell population. In summary we propose an important role of pro-inflammatory pEV in chronic HIV infection due to ongoing viral Nef activity.

Nuclear Translocation of Crk Adaptor Proteins by the Influenza A Virus NS1 Protein.

The non-structural protein-1 (NS1) of many influenza A strains, especially those of avian origin, contains an SH3 ligand motif, which binds tightly to the cellular adaptor proteins Crk (Chicken tumor virus number 10 (CT10) regulator of kinase) and Crk-like adapter protein (CrkL). This interaction has been shown to potentiate NS1-induced activation of the phosphatidylinositol 3-kinase (PI3K), but additional effects on the host cell physiology may exist. Here we show that NS1 can induce an efficient translocation of Crk proteins from the cytoplasm into the nucleus, which results in an altered pattern of nuclear protein tyrosine phosphorylation. This was not observed using NS1 proteins deficient in SH3 binding or engineered to be exclusively cytoplasmic, indicating a physical role for NS1 as a carrier in the nuclear translocation of Crk. These data further emphasize the role of Crk proteins as host cell interaction partners of NS1, and highlight the potential for host cell manipulation gained by a viral protein simply via acquiring a short SH3 binding motif.

MicroRNA-Attenuated Clone of Virulent Semliki Forest Virus Overcomes Antiviral Type I Interferon in Resistant Mouse CT-2A Glioma.

UNLABELLED: Glioblastoma is a terminal disease with no effective treatment currently available. Among the new therapy candidates are oncolytic viruses capable of selectively replicating in cancer cells, causing tumor lysis and inducing adaptive immune responses against the tumor. However, tumor antiviral responses, primarily mediated by type I interferon (IFN-I), remain a key problem that severely restricts viral replication and oncolysis. We show here that the Semliki Forest virus (SFV) strain SFV4, which causes lethal encephalitis in mice, is able to infect and replicate independent of the IFN-I defense in mouse glioblastoma cells and cell lines originating from primary human glioblastoma patient samples. The ability to tolerate IFN-I was retained in SFV4-miRT124 cells, a derivative cell line of strain SFV4 with a restricted capacity to replicate in neurons due to insertion of target sites for neuronal microRNA 124. The IFN-I tolerance was associated with the viral nsp3-nsp4 gene region and distinct from the genetic loci responsible for SFV neurovirulence. In contrast to the naturally attenuated strain SFV A7(74) and its derivatives, SFV4-miRT124 displayed increased oncolytic potency in CT-2A murine astrocytoma cells and in the human glioblastoma cell lines pretreated with IFN-I. Following a single intraperitoneal injection of SFV4-miRT124 into C57BL/6 mice bearing CT-2A orthotopic gliomas, the virus homed to the brain and was amplified in the tumor, resulting in significant tumor growth inhibition and improved survival. IMPORTANCE: Although progress has been made in development of replicative oncolytic viruses, information regarding their overall therapeutic potency in a clinical setting is still lacking. This could be at least partially dependent on the IFN-I sensitivity of the viruses used. Here, we show that the conditionally replicating SFV4-miRT124 virus shares the IFN-I tolerance of the pathogenic wild-type SFV, thereby allowing efficient targeting of a glioma that is refractory to naturally attenuated therapy vector strains sensitive to IFN-I. This is the first evidence of orthotopic syngeneic mouse glioma eradication following peripheral alphavirus administration. Our findings indicate a clear benefit in harnessing the wild-type virus replicative potency in development of next-generation oncolytic alphaviruses.

The C terminus of NS1 protein of influenza A/WSN/1933(H1N1) virus modulates antiviral responses in infected human macrophages and mice.

Non-structural protein NS1 of influenza A viruses interacts with cellular factors through its N-terminal RNA-binding, middle effector and C-terminal non-structured domains. NS1 attenuates antiviral responses in infected cells and thereby secures efficient virus replication. Some influenza strains express C-terminally truncated NS1 proteins due to nonsense mutations in the NS1 gene. To understand the role of the NS1 C-terminal region in regulation of antiviral responses, we engineered influenza viruses expressing C-terminally truncated NS1 proteins using A/WSN/33(H1N1) reverse genetics and tested them in human macrophages and in mice. We showed that a WSN virus expressing NS1 with a 28 aa deletion from its C terminus is a more powerful inducer of antiviral responses than the virus expressing full-length NS1, or one with a 10 aa truncation of NS1 in vitro. Thus, our findings suggest that the C-terminal region of NS1 is essential for regulation of antiviral responses. Moreover, viruses expressing truncated NS1 proteins could be good vaccine candidates.

Hepatitis C virus NS5A protein blocks epidermal growth factor receptor degradation via a proline motif- dependent interaction.

Hepatitis C virus (HCV) establishes a persistent infection that in many cases leads to cirrhosis and hepatocellular carcinoma. The non-structural 5A protein (NS5A) has been implicated in this process as it contains a C-terminal polyproline motif (termed P2) that binds to Src homology 3 (SH3) domains to regulate cellular signalling and trafficking pathways. We have shown previously that NS5A impaired epidermal growth factor (EGF) receptor (EGFR) endocytosis, thereby inhibiting EGF-stimulated EGFR degradation by a mechanism that remained unclear. As EGFR has been implicated in HCV cell entry and trafficking of the receptor involves several SH3-domain containing proteins, we investigated in more detail the mechanisms by which NS5A perturbs EGFR trafficking. We demonstrated that the P2 motif was required for the NS5A-mediated disruption to EGFR trafficking. We further demonstrated that the P2 motif was required for an interaction between NS5A and CMS, a homologue of CIN85 that has previously been implicated in EGFR endocytosis. We provided evidence that CMS was involved in the NS5A-mediated perturbation of EGFR trafficking. We also showed that NS5A effected a loss of EGFR ubiquitination in a P2-motif-dependent fashion. These data provide clues to the mechanism by which NS5A regulates the trafficking of a key cellular receptor and demonstrate for the first time the ability of NS5A to regulate host cell ubiquitination pathways.

Hepatitis C virus NS5A inhibits mixed lineage kinase 3 to block apoptosis.

Hepatitis C virus (HCV) infection results in the activation of numerous stress responses including oxidative stress, with the potential to induce an apoptotic state. Previously we have shown that HCV attenuates the stress-induced, p38MAPK-mediated up-regulation of the K(+) channel Kv2.1, to maintain the survival of infected cells in the face of cellular stress. We demonstrated that this effect was mediated by HCV non-structural 5A (NS5A) protein, which impaired p38MAPK activity through a polyproline motif-dependent interaction, resulting in reduction of phosphorylation activation of Kv2.1. In this study, we investigated the host cell proteins targeted by NS5A to mediate Kv2.1 inhibition. We screened a phage-display library expressing the entire complement of human SH3 domains for novel NS5A-host cell interactions. This analysis identified mixed lineage kinase 3 (MLK3) as a putative NS5A interacting partner. MLK3 is a serine/threonine protein kinase that is a member of the MAPK kinase kinase (MAP3K) family and activates p38MAPK. An NS5A-MLK3 interaction was confirmed by co-immunoprecipitation and Western blot analysis. We further demonstrate a novel role of MLK3 in the modulation of Kv2.1 activity, whereby MLK3 overexpression leads to the up-regulation of channel activity. Accordingly, coexpression of NS5A suppressed this stimulation. Additionally we demonstrate that overexpression of MLK3 induced apoptosis, which was also counteracted by NS5A. We conclude that NS5A targets MLK3 with multiple downstream consequences for both apoptosis and K(+) homeostasis.

MicroRNA-mediated suppression of oncolytic adenovirus replication in human liver.

MicroRNAs (miRNAs) are important and ubiquitous regulators of gene expression that can suppress their target genes by translational inhibition as well as mRNA destruction. Cell type-specific miRNA expression patterns have been successfully exploited for targeting the expression of experimental and therapeutic gene constructs, for example to reduce pathogenic effects of cancer virotherapy in normal tissues. In order to avoid liver damage associated with systemic or intrahepatic delivery of oncolytic adenoviruses we have introduced the concept of suppressing adenovirus replication in hepatic cells by inserting target elements for the liver-specific miR122 into the viral genome. Here we show using ex vivo cultured tissue specimens that six perfectly complementary miR122 target sites in the 3' untranslated region of the viral E1A gene are sufficient in the absence of any other genetic modifications to prevent productive replication of serotype 5 adenovirus (Ad5) in normal human liver. This modification did not compromise the replicative capacity of the modified virus in cancer tissue derived from a colon carcinoma liver metastasis or its oncolytic potency in a human lung cancer xenograft mouse model. Unlike wild-type Ad5, the modified virus did not result in increased serum levels of liver enzymes in infected mice. These results provide a strong preclinical proof of concept for the use of miR122 target sites for reducing the risk of liver damage caused by oncolytic adenoviruses, and suggest that ectopic miR122 target elements should be considered as an additional safety measure included in any therapeutic virus or viral vector posing potential hazard to the liver.

HIV Nef, paxillin, and Pak1/2 regulate activation and secretion of TACE/ADAM10 proteases.

The HIV Nef protein recruits the polycomb protein Eed and mimics an integrin receptor signal for reasons that are not entirely clear. Here we demonstrate that Nef and Eed complex with the integrin effector paxillin to recruit and activate TNFα converting enzyme (TACE alias ADAM 17) and its close relative ADAM10. The activated proteases cleaved proTNFα and were shuttled into extracellular vesicles (EVs). Peripheral blood mononuclear cells that ingested these EVs released TNFα. Analyzing the mechanism, we found that Pak2, an established host cell effector of Nef, phosphorylated paxillin on Ser272/274 to induce TACE-paxillin association and shuttling into EVs via lipid rafts. Conversely, Pak1 phosphorylated paxillin on Ser258, which inhibited TACE association and lipid raft transfer. Interestingly, melanoma cells used an identical mechanism to shuttle predominantly ADAM10 into EVs. We conclude that HIV-1 and cancer cells exploit a paxillin/integrin-controlled mechanism to release TACE/ADAM10-containing vesicles, ensuring better proliferation/growth conditions in their microenvironment.

Attenuation of Semliki Forest virus neurovirulence by microRNA-mediated detargeting.

Artificial target sequences for tissue-specific miRNAs have recently been introduced as a new means for altering the tissue tropism of viral replication. This approach can be used to improve the safety of oncolytic viruses for cancer virotherapy by restricting their replication in unwanted tissues, such as the liver. Semliki Forest virus (SFV) is a positive-strand RNA virus and, similar to the related alphaviruses, like Sindbis virus, has potential as a gene therapy vector and an oncolytic virotherapy agent, but this potential is limited by the neurovirulence of these alphaviruses. Here, we have generated a replicative SFV4 carrying six tandem targets for the neuron-specific miR124 between the viral nonstructural protein 3 and 4 (nsp3 and nsp4) genes. When administered intraperitoneally into adult BALB/c mice, SFV4-miRT124 displayed an attenuated spread into the central nervous system (CNS) and greatly increased survival. Peripheral replication was not affected, indicating neuron-specific attenuation. Moreover, a strong protective SFV immunity was elicited in these animals. Intracranial infection of adult mice with SFV4-miRT124 showed greatly reduced infection of neurons in the brain but led to the infection of oligodendrocytes in the corpus callosum. Taken together, our data show that miR124-mediated attenuation of neurovirulence is a feasible and promising strategy for generating safer oncolytic alphavirus virotherapy agents.

High-affinity target binding engineered via fusion of a single-domain antibody fragment with a ligand-tailored SH3 domain.

Monoclonal and recombinant antibodies are ubiquitous tools in diagnostics, therapeutics, and biotechnology. However, their biochemical properties lack optimal robustness, their bacterial production is not easy, and possibilities to create multifunctional fusion proteins based on them are limited. Moreover, the binding affinities of antibodies towards their antigens are suboptimal for many applications where they are commonly used. To address these issues we have made use of the concept of creating high binding affinity based on multivalent target recognition via exploiting some of the best features of immunoglobulins (Ig) and non-Ig-derived ligand-binding domains. We have constructed a small protein, named Neffin, comprised of a 118 aa llama Ig heavy chain variable domain fragment (VHH) fused to a ligand-tailored 57 aa SH3 domain. Neffin could be readily produced in large amounts (>18 mg/L) in the cytoplasm of E. coli, and bound with a subpicomolar affinity (K(d) 0.54 pM) to its target, the HIV-1 Nef protein. When expressed in human cells Neffin could potently inhibit Nef function. Similar VHH-SH3 fusion proteins could be targeted against many other proteins of interest and could have widespread use in diverse medical and biotechnology applications where biochemical robustness and strong binding affinity are required.

Comprehensive analysis of interactions between the Src-associated protein in mitosis of 68 kDa and the human Src-homology 3 proteome.

The protein Sam68 is involved in many cellular processes such as cell-cycle regulation, RNA metabolism, or signal transduction. Sam68 comprises a central RNA-binding domain flanked by unstructured tails containing docking sites for signalling proteins including seven proline-rich sequences (denoted P0 to P6) as potential SH3-domain binding motifs. To comprehensively assess Sam68-SH3-interactions, we applied a phage-display screening of a library containing all approx. 300 human SH3 domains. Thereby we identified five new (from intersectin 2, the osteoclast stimulating factor OSF, nephrocystin, sorting nexin 9, and CIN85) and seven already known high-confidence Sam68-ligands (mainly from the Src-kinase family), as well as several lower-affinity binders. Interaction of the high-affinity Sam68-binders was confirmed in independent assays in vitro (phage-ELISA, GST-pull-down) and in vivo (FACS-based FRET-analysis with CFP- and YFP-tagged proteins). Fine-mapping analyses with peptides established P0, P3, P4, and P5 as exclusive docking-sites for SH3 domains, which showed varying preferences for these motifs. Mutational analyses identified individual residues within the proline-rich motifs being crucial for the interactions. Based on these data, we generated a Sam68-mutant incapable of interacting with SH3 domains any more, as subsequently demonstrated by FRET-analyses. In conclusion, we present a thorough characterization of Sam68's interplay with the SH3 proteome. The observed interaction between Sam68 and OSF complements the known Sam68-Src and OSF-Src interactions. Thus, we propose, that Sam68 functions as a classical scaffold protein in this context, assembling components of an osteoclast-specific signalling pathway.