Structural basis for the inhibition of HIV-1 Nef by a high-affinity binding single-domain antibody.

BACKGROUND: The HIV-1 Nef protein is essential for AIDS pathogenesis by its interaction with host cell surface receptors and signaling factors. Despite its critical role as a virulence factor Nef is not targeted by current antiviral strategies. RESULTS: We have determined the crystal structure of the complex formed by a camelid single-domain antibody fragment, termed sdAb19, bound to HIV-1 Nef together with a stabilizing SH3 domain. sdAb19 forms a stoichiometric 1:1 complex with Nef and binds to a conformationally conserved surface at the C-terminus of Nef that overlaps with functionally important interaction sites involved in Nef-induced perturbations of signaling and trafficking pathways. The antibody fragment binds Nef with low nanomolar affinity, which could be attenuated to micromolar affinity range by site-directed mutagenesis of key interaction residues in sdAb19. Fusion of the SH3 domain to sdAb19, termed Neffin, leads to a significantly increased affinity for Nef and formation of a stoichiometric 2:2 Nef-Neffin complex. The 19 kDa Neffin protein inhibits all functions of Nef as CD4 and MHC-I downregulation, association with Pak2, and the increase in virus infectivity and replication. CONCLUSIONS: Together, sdAb19 and Neffin thus represent efficient tools for the rational development of antiviral strategies against HIV-1 Nef.

Single-domain antibody-SH3 fusions for efficient neutralization of HIV-1 Nef functions.

HIV-1 Nef is essential for AIDS pathogenesis, but this viral protein is not targeted by antiviral strategies. The functions of Nef are largely related to perturbations of intracellular trafficking and signaling pathways through leucine-based and polyproline motifs that are required for interactions with clathrin-associated adaptor protein complexes and SH3 domain-containing proteins, such as the phagocyte-specific kinase Hck. We previously described a single-domain antibody (sdAb) targeting Nef and inhibiting many, but not all, of its biological activities. We now report a further development of this anti-Nef strategy through the demonstration of the remarkable inhibitory activity of artificial Nef ligands, called Neffins, comprised of the anti-Nef sdAb fused to modified SH3 domains. The Neffins inhibited all key activities of Nef, including Nef-mediated CD4 and major histocompatibility complex class I (MHC-I) cell surface downregulation and enhancement of virus infectivity. When expressed in T lymphocytes, Neffins specifically inhibited the Nef-induced mislocalization of the Lck kinase, which contributes to the alteration of the formation of the immunological synapse. In macrophages, Neffins inhibited the Nef-induced formation of multinucleated giant cells and podosome rosettes, and it counteracted the inhibitory activity of Nef on phagocytosis. Since we show here that these effects of Nef on macrophage and T cell functions were both dependent on the leucine-based and polyproline motifs, we confirmed that Neffins disrupted interactions of Nef with both AP complexes and Hck. These results demonstrate that it is possible to inhibit all functions of Nef, both in T lymphocytes and macrophages, with a single ligand that represents an efficient tool to develop new antiviral strategies targeting Nef.

Nucleophosmin phosphorylation by v-cyclin-CDK6 controls KSHV latency.

Nucleophosmin (NPM) is a multifunctional nuclear phosphoprotein and a histone chaperone implicated in chromatin organization and transcription control. Oncogenic Kaposi's sarcoma herpesvirus (KSHV) is the etiological agent of Kaposi's sarcoma, primary effusion lymphoma (PEL) and multicentric Castleman disease (MCD). In the infected host cell KSHV displays two modes of infection, the latency and productive viral replication phases, involving extensive viral DNA replication and gene expression. A sustained balance between latency and reactivation to the productive infection state is essential for viral persistence and KSHV pathogenesis. Our study demonstrates that the KSHV v-cyclin and cellular CDK6 kinase phosphorylate NPM on threonine 199 (Thr199) in de novo and naturally KSHV-infected cells and that NPM is phosphorylated to the same site in primary KS tumors. Furthermore, v-cyclin-mediated phosphorylation of NPM engages the interaction between NPM and the latency-associated nuclear antigen LANA, a KSHV-encoded repressor of viral lytic replication. Strikingly, depletion of NPM in PEL cells leads to viral reactivation, and production of new infectious virus particles. Moreover, the phosphorylation of NPM negatively correlates with the level of spontaneous viral reactivation in PEL cells. This work demonstrates that NPM is a critical regulator of KSHV latency via functional interactions with v-cyclin and LANA.

Viral oncogene-induced DNA damage response is activated in Kaposi sarcoma tumorigenesis.

Kaposi sarcoma is a tumor consisting of Kaposi sarcoma herpesvirus (KSHV)-infected tumor cells that express endothelial cell (EC) markers and viral genes like v-cyclin, vFLIP, and LANA. Despite a strong link between KSHV infection and certain neoplasms, de novo virus infection of human primary cells does not readily lead to cellular transformation. We have studied the consequences of expression of v-cyclin in primary and immortalized human dermal microvascular ECs. We show that v-cyclin, which is a homolog of cellular D-type cyclins, induces replicative stress in ECs, which leads to senescence and activation of the DNA damage response. We find that antiproliferative checkpoints are activated upon KSHV infection of ECs, and in early-stage but not late-stage lesions of clinical Kaposi sarcoma specimens. These are some of the first results suggesting that DNA damage checkpoint response also functions as an anticancer barrier in virally induced cancers.

Phosphorylation of the cyclin-dependent kinase inhibitor p21Cip1 on serine 130 is essential for viral cyclin-mediated bypass of a p21Cip1-imposed G1 arrest.

K cyclin encoded by Kaposi's sarcoma-associated herpesvirus confers resistance to the cyclin-dependent kinase (cdk) inhibitors p16Ink4A, p21Cip1, and p27Kip1 on the associated cdk6. We have previously shown that K cyclin expression enforces S-phase entry on cells overexpressing p27Kip1 by promoting phosphorylation of p27Kip1 on threonine 187, triggering p27Kip1 down-regulation. Since p21Cip1 acts in a manner similar to that of p27Kip1, we have investigated the subversion of a p21Cip1-induced G1 arrest by K cyclin. Here, we show that p21Cip1 is associated with K cyclin both in overexpression models and in primary effusion lymphoma cells and is a substrate of the K cyclin/cdk6 complex, resulting in phosphorylation of p21Cip1 on serine 130. This phosphoform of p21Cip1 appeared unable to associate with cdk2 in vivo. We further demonstrate that phosphorylation on serine 130 is essential for K cyclin-mediated release of a p21Cip1-imposed G1 arrest. Moreover, we show that under physiological conditions of cell cycle arrest due to elevated levels of p21Cip1 resulting from oxidative stress, K cyclin expression enabled S-phase entry and was associated with p21Cip1 phosphorylation and partial restoration of cdk2 kinase activity. Thus, expression of the viral cyclin enables cells to subvert the cell cycle inhibitory function of p21Cip1 by promoting cdk6-dependent phosphorylation of this antiproliferative protein.

Cell signaling pathways engaged by KSHV.

Kaposi's sarcoma herpesvirus (KSHV) is the eighth human herpesvirus discovered in 1994 from Kaposi's sarcoma lesion of an AIDS patient. The strong molecular and epidemiological links associating KSHV with Kaposi's sarcoma and certain lymphoproliferative disorders indicate that KSHV is required for the development of these malignancies. Although KSHV is equipped to manipulate and deregulate several cellular signaling pathways, it is not yet understood how this leads to cell transformation. Profound understanding of the interplay of viral and cellular factors in KSHV-infected cells will provide valuable information on the mechanisms of viral tumorigenesis and enable development of efficient targeted therapies for virus-induced cancers. This review focuses on the cellular signaling pathways that KSHV gene products impinge on and discusses their putative contribution to tumorigenesis.

Preferred SH3 domain partners of ADAM metalloproteases include shared and ADAM-specific SH3 interactions.

A disintegrin and metalloproteinases (ADAMs) constitute a protein family essential for extracellular signaling and regulation of cell adhesion. Catalytic activity of ADAMs and their predicted potential for Src-homology 3 (SH3) domain binding show a strong correlation. Here we present a comprehensive characterization of SH3 binding capacity and preferences of the catalytically active ADAMs 8, 9, 10, 12, 15, 17, and 19. Our results revealed several novel interactions, and also confirmed many previously reported ones. Many of the identified SH3 interaction partners were shared by several ADAMs, whereas some were ADAM-specific. Most of the ADAM-interacting SH3 proteins were adapter proteins or kinases, typically associated with sorting and endocytosis. Novel SH3 interactions revealed in this study include TOCA1 and CIP4 as preferred partners of ADAM8, and RIMBP1 as a partner of ADAM19. Our results suggest that common as well as distinct mechanisms are involved in regulation and execution of ADAM signaling, and provide a useful framework for addressing the pathways that connect ADAMs to normal and aberrant cell behavior.

Immunogenicity of an inactivated mycobacterial vaccine for the prevention of HIV-associated tuberculosis: a randomized, controlled trial.

OBJECTIVE: Prior to the widespread use of Mycobacterium bovis, Bacille Calmette-Guerin (BCG), inactivated whole cell mycobacterial vaccines had been shown effective in the prevention of tuberculosis. The present study was conducted to determine the safety and immunogenicity of an inactivated whole cell mycobacterial vaccine in persons with HIV infection.DESIGN Randomized, controlled trial. METHODS: A total of 39 HIV-positive patients with prior BCG immunization and CD4 cell counts >/= 200 x 10(6) cells/l were randomized to five doses of inactivated Mycobacterium vaccae (MV) vaccine or control vaccine (CV). Lymphocyte proliferation (LPA) and interferon gamma (IFN-gamma) responses to mycobacterial antigens were assayed at baseline, after three and five doses of vaccine and > 1 year later. Parallel studies were conducted in 10 HIV-negative subjects with prior BCG immunization. RESULTS: Among HIV-positive patients, 19 MV recipients had higher LPA and IFN-gamma responses to MV sonicate than 20 CV recipients after three and five doses of vaccine and > 1 year later. LPA responses to Mycobacterium tuberculosis whole cell lysate increased over time in both groups consistent with prior BCG immunization and current antiretroviral therapy; after three doses, responses were boosted to higher levels in MV subjects than CV subjects. LPA responses to WCL were also boosted in HIV-negative MV recipients. Immunization was safe and had no adverse effects on HIV viral load or CD4 cell count. CONCLUSIONS: In BCG-primed, HIV-positive and HIV-negative subjects, MV induces durable cellular immune responses to a new mycobacterial antigen and boosts pre-existing responses to WCL. MV is a candidate for clinical trials for the prevention of HIV-associated tuberculosis.

KSHV viral cyclin interferes with T-cell development and induces lymphoma through Cdk6 and Notch activation in vivo.

Kaposi's sarcoma herpesvirus (KSHV)-encoded v-cyclin, a homolog of cellular cyclin D2, activates cellular CDK6, promotes G1-S transition of the cell cycle, induces DNA damage, apoptosis, autophagy and is reported to have oncogenic potential. Here we show that in vivo expression of v-cyclin in the B- and T-cell lymphocyte compartments results in a markedly low survival due to high penetrance of early-onset T-cell lymphoma and pancarditis. The v-cyclin transgenic mice have smaller pre-tumorigenic lymphoid organs, showing decreased cellularity, and increased proliferation and apoptosis. Furthermore, v-cyclin expression resulted in decreased amounts of CD3-expressing mature T-cells in the secondary lymphoid organs concurrent with alterations in the T-cell subpopulations of the thymus. This suggests that v-cyclin interferes with normal T-cell development. As the Notch pathway is recognized for its role in both T-cell development and lymphoma initiation, we addressed the role of Notch in the v-cyclin-induced alterations. Fittingly, we demonstrate induction of Notch3 and Hes1 in the pre-tumorigenic thymi and lymphomas of v-cyclin expressing mice, and show that lymphoma growth and viability are dependent on activated Notch signaling. Notch3 transcription and growth of the lymphomas was dependent on CDK6, as determined by silencing of CDK6 expression or chemical inhibition, respectively. Our work here reveals a viral cyclin-CDK6 complex as an upstream regulator of Notch receptor, suggesting that cyclins can play a role in the initiation of Notch-dependent lymphomagenesis.

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.

KSHV viral cyclin inactivates p27KIP1 through Ser10 and Thr187 phosphorylation in proliferating primary effusion lymphomas.

Kaposi sarcoma herpesvirus (KSHV) infection is consistently associated with primary effusion lymphomas (PELs) that are non-Hodgkin lymphomas of B-cell origin. All PEL cells are latently infected with KSHV and express latent viral proteins such as the viral cyclin (v-cyclin), which has previously been implicated in down-regulation of cell-cycle inhibitor p27(KIP1) levels via phosphorylation on Thr187. PEL cells retain high levels of p27(KIP1) but yet proliferate actively, which has left the biologic significance of this p27(KIP1) destabilization somewhat elusive. We have recently demonstrated that v-cyclin and p27(KIP1) stably associate in PEL cells. Here we demonstrate that v-cyclin together with its kinase partner CDK6 phosphorylates the associated p27(KIP1) in PEL cells, which represent a biologically relevant model system for KSHV pathobiology. During latent viral replication p27(KIP1) was phosphorylated by v-cyclin-CDK6 predominantly on Ser10, which enhances its cytoplasmic localization. Interestingly, upon reactivation of KSHV lytic cycle, v-cyclin-CDK6 phosphorylated p27(KIP1) on Thr187, which resulted in down-regulation of p27(KIP1) protein levels. These findings indicate that v-cyclin modulates the cell-cycle inhibitory function of p27(KIP1) by phosphorylation in PELs, and also suggest a novel role for v-cyclin in the lytic reactivation of KSHV.

KSHV viral cyclin binds to p27KIP1 in primary effusion lymphomas.

Primary effusion lymphomas (PELs) represent a unique non-Hodgkin lymphoma that is consistently infected by Kaposi sarcoma herpesvirus (KSHV). PEL cells express high levels of the cell cycle inhibitor p27(KIP1) and yet proliferate actively. KSHV genome encodes a viral cyclin homolog, v-cyclin, which has previously been implicated in down-regulation of p27(KIP1) levels. To address how PEL cells can tolerate high p27(KIP1) levels, we investigated functional interactions between v-cyclin and p27(KIP1) using PEL-derived cell lines as a model system. Here we demonstrate that v-cyclin and p27(KIP1) stably associate in PEL cells in vivo suggesting an attractive model by which p27(KIP1) is inactivated in the actively proliferating PEL cells. Moreover, we show that v-cyclin and cyclin-dependent kinase 6 (CDK6) form an active kinase without p27(KIP1) and that CDK6 is the in vivo catalytic subunit of v-cyclin in PEL cells. These findings suggest that KSHV may promote oncogenesis in PEL by expressing v-cyclin, which both overrides negative cell cycle controls present in the PEL precursor cells and induces a strong proliferative signal via CDK6 kinase activity.