Effect of Strain Variations on Lassa Virus Z Protein-Mediated Human RIG-I Inhibition.

Mammarenaviruses include several known human pathogens, such as the prototypic lymphocytic choriomeningitis virus (LCMV) that can cause neurological diseases and Lassa virus (LASV) that causes endemic hemorrhagic fever infection. LASV-infected patients show diverse clinical manifestations ranging from asymptomatic infection to hemorrhage, multi-organ failures and death, the mechanisms of which have not been well characterized. We have previously shown that the matrix protein Z of pathogenic arenaviruses, including LASV and LCMV, can strongly inhibit the ability of the innate immune protein RIG-I to suppress type I interferon (IFN-I) expression, which serves as a mechanism of viral immune evasion and virulence. Here, we show that Z proteins of diverse LASV isolates derived from rodents and humans have a high degree of sequence variations at their N- and C-terminal regions and produce variable degrees of inhibition of human RIG-I (hRIG-I) function in an established IFN-ß promoter-driven luciferase (LUC) reporter assay. Additionally, we show that Z proteins of four known LCMV strains can also inhibit hRIG-I at variable degrees of efficiency. Collectively, our results confirm that Z proteins of pathogenic LASV and LCMV can inhibit hRIG-I and suggest that strain variations of the Z proteins can influence their efficiency to suppress host innate immunity that might contribute to viral virulence and disease heterogeneity.

DNA-Barcoded pMHC Tetramers for Detection of Single Antigen-Specific T Cells by Digital PCR.

Antigen-specific T cells are found at low frequencies in circulation but carry important diagnostic information as liquid biomarkers in numerous biomedical settings, such as monitoring the efficacy of vaccines and cancer immunotherapies. To enable detection of antigen-specific T cells with high sensitivity, we develop peptide-MHC (pMHC) tetramers labeled with DNA barcodes to detect single T cells by droplet digital PCR (ddPCR). We show that site-specific conjugation of DNA via photocleavable linkers allows barcoded tetramers to stain T cells with similar avidity compared to conventional fluorescent tetramers and efficient recovery of barcodes by light with no loss in cell viability. We design an orthogonal panel of DNA-barcoded tetramers to simultaneously detect multiple antigen-specific T cell populations, including from a mouse model of viral infection, and discriminate single cancer-specific T cells with high diagnostic sensitivity and specificity. This approach of DNA-barcoding can be broadened to encompass additional rare cells for monitoring immunological health at the single cell level.

[First evidence of lymphocytic choriomeningitis virus (Arenavirus) infection in Mus musculus rodents captured in the urban area of the municipality of Sincelejo, Sucre, Colombia].

INTRODUCTION: The lymphocytic choriomeningitis virus is an Old World arenavirus that infects Mus musculus, and can cause congenital hydrocephalus, chorioretinitis and multisystemic failure in transplant human recipients. Although the disease has not been clinically diagnosed in Colombia yet, there have been reports of infection with the Pichindé virus in rodents from Cauca and Valle del Cauca departments, and with the Guanarito virus in rodents from Córdoba department. OBJECTIVE: To identify the lymphocytic choriomeningitis virus from Mus musculus captured in the municipality of Sincelejo. MATERIALS AND METHODS: We evaluated 80 samples of plasma by ELISA using antigen from lymphocytic choriomeningitis virus. Additionally, a nested RT-PCR was performed to seropositive and seronegative samples for the S-segment. RESULTS: We found a 10% seroprevalence (8/80) and the viral genome was detected in 16 brain samples; the alignment (BLAST) and the phylogenetic analysis (MrBayes, version 3.2.2) confirmed the presence of the lymphocytic choriomeningitis virus. CONCLUSION: The results indicated that human infection with the lymphocytic choriomeningitis virus in humans could occur in the urban area of Sincelejo, although no cases have been reported so far.

Primera evidencia de infección por el virus de la coriomeningitis linfocítica (arenavirus) en roedores Mus musculus capturados en la zona urbana del municipio de Sincelejo, Sucre, Colombia / First evidence of lymphocytic choriomeningitis virus (Arenavirus) infection in Mus musculus rodents captured in the urban area of the municipality of Sincelejo, Sucre, Colombia

Resumen Introducción. El virus de la coriomeningitis linfocítica es un arenavirus del Viejo Mundo que se hospeda en el ratón casero (Mus musculus), y puede causar infecciones congénitas, hidrocefalia, coriorretinitis y falla orgánica múltiple en pacientes receptores de trasplantes. En Colombia aún no se ha reportado la enfermedad mediante diagnóstico clínico, pero en estudios serológicos se ha detectado la infección por el virus Pichindé en roedores en los departamentos del Cauca y Valle del Cauca, y por el virus Guanarito, en roedores en Córdoba. Objetivo. Detectar el virus de la coriomeningitis linfocítica en M. musculus en el municipio de Sincelejo. Materiales y métodos. Se evaluaron 80 muestras de plasma mediante la prueba ELISA usando antígeno del virus de la coriomeningitis linfocítica. Además, se empleó la reacción en cadena de la polimerasa con transcriptasa inversa (RT-PCR) anidada en muestras de animales seropositivos y seronegativos para la detección del segmento S. Resultados. Se encontró una seroprevalencia de 10% (8/80) y se detectó el genoma viral en 16 muestras de cerebro; el alineamiento (en la Basic Local Alignment Search Tool, BLAST) y el análisis filogenético (mediante el programa MrBayes, versión 3.2.2) confirmaron que correspondía al virus de la coriomeningitis linfocítica. Conclusión. Los resultados indicaron que la infección por el virus de la coriomeningitis linfocítica en humanos podría ocurrir en el área urbana de Sincelejo, aunque hasta la fecha no se hayan reportado casos.

Abstract Introduction: The lymphocytic choriomeningitis virus is an Old World arenavirus that infects Mus musculus, and can cause congenital hydrocephalus, chorioretinitis and multisystemic failure in transplant human recipients. Although the disease has not been clinically diagnosed in Colombia yet, there have been reports of infection with the Pichindé virus in rodents from Cauca and Valle del Cauca departments, and with the Guanarito virus in rodents from Córdoba department. Objective: To identify the lymphocytic choriomeningitis virus from Mus musculus captured in the municipality of Sincelejo. Materials and methods: We evaluated 80 samples of plasma by ELISA using antigen from lymphocytic choriomeningitis virus. Additionally, a nested RT-PCR was performed to seropositive and seronegative samples for the S-segment. Results: We found a 10% seroprevalence (8/80) and the viral genome was detected in 16 brain samples; the alignment (BLAST) and the phylogenetic analysis (MrBayes, version 3.2.2) confirmed the presence of the lymphocytic choriomeningitis virus. Conclusion: The results indicated that human infection with the lymphocytic choriomeningitis virus in humans could occur in the urban area of Sincelejo, although no cases have been reported so far.

Crystal structure of the prefusion surface glycoprotein of the prototypic arenavirus LCMV.

Arenaviruses exist worldwide and can cause hemorrhagic fever and neurologic disease. A single glycoprotein expressed on the viral surface mediates entry into target cells. This glycoprotein, termed GPC, contains a membrane-associated signal peptide, a receptor-binding subunit termed GP1 and a fusion-mediating subunit termed GP2. Although GPC is a critical target of antibodies and vaccines, the structure of the metastable GP1-GP2 prefusion complex has remained elusive for all arenaviruses. Here we describe the crystal structure of the fully glycosylated prefusion GP1-GP2 complex of the prototypic arenavirus LCMV at 3.5 Å. This structure reveals the conformational changes that the arenavirus glycoprotein must undergo to cause fusion and illustrates the fusion regions and potential oligomeric states.

Pseudotyping lentiviral vectors with lymphocytic choriomeningitis virus glycoproteins for transduction of dendritic cells and in vivo immunization.

Lentiviral vectors (LVs) are promising delivery systems for gene therapy, and they can be further engineered to increase their potential for effectively delivering transgenes to desired cell populations. Here, we have engineered LVs pseudotyped with envelope glycoproteins derived from lymphocytic choriomeningitis virus (LCMV) for antigen delivery to elicit vaccine-directed immune responses. Two variants, LCMV-WE and LCMV-Arm53b, were evaluated for their ability to mediate LV-based cellular transduction in vitro. LCMV-WE with a leucine residue at position 260 (260L) is known for its high-affinity binding with a cellular receptor, α-dystroglycan (α-DG), whereas LCMV-Arm53b has low-affinity binding resulting from a phenylalanine residue at the same position. In contrast to LCMV-Arm53b, we found that LVs pseudotyped with LCMV-WE could transduce 293T cells and murine dendritic cells much more efficiently based, at least in part, on their favorable interaction with α-DG. In mice, LCMV-WE-bearing LVs encoding a model antigen, invariant chain ovalbumin, could elicit substantial antigen-specific CD8(+) T cell immune response. The response could be further enhanced by a homologous boosting immunization with the same vector. These findings offer evidence to support the potential utilization of LCMV-WE-bearing LVs for vectored vaccines against cancer and infectious diseases.

Tunicamycin inhibits diabetes.

BACKGROUND: Autoimmune diseases are characterized by a breakdown of immunologic tolerance, and this breakdown can lead to life-threatening or lifelong disorders. Moreover; drugs that are used to treat these diseases are few in number and are associated with many serious adverse effects. METHODS: We used the rat insulin promoter-glycoprotein mouse model to analyze the role of tunicamycin in the process of autoimmune diabetes; the P14 mouse model to analyze the effect of tunicamycin on CD8(+) T cells; chop knockout mice to analyze the role of tunicamycin on an endoplasmic reticulum stress model; and fluorescence-activated cell sorting, quantitative real-time polymerase chain reaction, and histologic methods. RESULTS: We found that a single dose of tunicamycin reduced the activation and pancreatic infiltration of CD8(+) T cells. This activity delayed the incidence of virus-induced diabetes and improved survival rates. CONCLUSION: Tunicamycin may offer therapeutic opportunities for T cell-mediated autoimmune diseases such as diabetes.

Self-association of lymphocytic choriomeningitis virus nucleoprotein is mediated by its N-terminal region and is not required for its anti-interferon function.

Arenaviruses have a bisegmented, negative-strand RNA genome. Both the large (L) and small (S) genome segments use an ambisense coding strategy to direct the synthesis of two viral proteins. The L segment encodes the virus polymerase (L protein) and the matrix Z protein, whereas the S segment encodes the nucleoprotein (NP) and the glycoprotein precursor (GPC). NPs are the most abundant viral protein in infected cells and virions and encapsidate genomic RNA species to form an NP-RNA complex that, together with the virus L polymerase, forms the virus ribonucleoprotein (RNP) core capable of directing both replication and transcription of the viral genome. RNP formation predicts a self-association property of NPs. Here we document self-association (homotypic interaction) of the NP of the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV), as well as those of the hemorrhagic fever (HF) arenaviruses Lassa virus (LASV) and Machupo virus (MACV). We also show heterotypic interaction between NPs from both closely (LCMV and LASV) and distantly (LCMV and MACV) genetically related arenaviruses. LCMV NP self-association was dependent on the presence of single-stranded RNA and mediated by an N-terminal region of the NP that did not overlap with the previously described C-terminal NP domain involved in either counteracting the host type I interferon response or interacting with LCMV Z.

X-ray structure of the arenavirus glycoprotein GP2 in its postfusion hairpin conformation.

Arenaviruses are important agents of zoonotic disease worldwide. The virions expose a tripartite envelope glycoprotein complex at their surface, formed by the glycoprotein subunits GP1, GP2 and the stable signal peptide. This complex is responsible for binding to target cells and for the subsequent fusion of viral and host-cell membranes for entry. During this process, the acidic environment of the endosome triggers a fusogenic conformational change in the transmembrane GP2 subunit of the complex. We report here the crystal structure of the recombinant GP2 ectodomain of the lymphocytic choriomeningitis virus, the arenavirus type species, at 1.8-Å resolution. The structure shows the characteristic trimeric coiled coil present in class I viral fusion proteins, with a central stutter that allows a close structural alignment with most of the available structures of class I and III viral fusion proteins. The structure further shows a number of intrachain salt bridges stabilizing the postfusion hairpin conformation, one of which involves an aspartic acid that appears released from a critical interaction with the stable signal peptide upon low pH activation.

Molecular characterization of the genes coding for glycoprotein and L protein of lymphocytic choriomeningitis virus strain MX.

Lymphocytic choriomeningitis virus (LCMV) is the prototype Arenavirus with ambisense coding strategy. We have previously described a new MX strain LCMV and determined the primary structure of the genes coding for the nucleoprotein and RING finger Z protein. In this report, we describe amplification and sequencing of the entire coding sequences of additional MX genes, the glycoprotein precursor (GPC) and L protein. The obtained MX GPC cDNA sequence was 1,615 nucleotides long and contained an ORF, which encodes the GPC precursor of 498 amino acids. MX L polymerase cDNA sequence was 6,668 nucleotides long and predicted ORF encodes the L polymerase of 2,209 amino acids. Nucleotide and deduced amino acid sequences were compared with the known GPC and L sequences and the comparison revealed that both genes shared the highest amino acid identity with Armstrong strain. Phylogenetic analysis confirmed that MX represents a separate LCMV strain. The GPC and L genes products contained several characteristic conserved regions. On the other hand, we have observed numerous differences in predicted protein sequences, which distinguish MX LCMV from other LCMV strains and might be of potential biological significance.

A retroviral packaging cell line for pseudotype vectors based on glioma-infiltrating progenitor cells.

BACKGROUND: Early clinical trials for gene therapy of human gliomas with retroviral packaging cells (PC) have been hampered by low transduction efficacy and lack of dissemination of PC within the tumor. In the current approach, these issues have been addressed by creating a stable packaging cell line for retroviral vectors pseudotyped with glycoproteins of lymphocytic choriomeningitis virus (LCMV) based on tumor-infiltrating progenitor cells. METHODS: Tumor-infiltrating progenitor cells, which had been isolated from adult rat bone marrow (BM-TIC), were modified to stably express Gag-Pol proteins of moloney murine leukemia virus (Mo-MLV) and glycoproteins of LCMV. Packaging of a retroviral vector was measured by titration experiments on human fibroblast cells as well as on mouse and human glioma cell lines. Additionally, gene transfer was tested in a rat glioma model in vivo. RESULTS: The BM-TIC-derived packaging cell line (BM-TIPC) produced retroviral vectors with titers between 2-8 x 10(3) transducing units (TU)/ml. Extended culturing of BM-TIPC over several weeks and freezing/thawing of cells did not affect vector titers. No replication-competent retrovirus was released from BM-TIPC. In a rat glioma model, BM-TIPC infiltrated the tumors extensively and with high specificity. Moreover, BM-TIPC mediated transduction of glioma cells in vivo. CONCLUSION: This proof-of-principle study shows that primary adult progenitor cells with tumor-infiltrating capacity can be genetically modified to stably produce retroviral LCMV pseudotype vectors. These BM-TIPC may be a useful tool to enhance specificity and efficacy of gene transfer to gliomas in patients.

Old World and clade C New World arenaviruses mimic the molecular mechanism of receptor recognition used by alpha-dystroglycan's host-derived ligands.

alpha-Dystroglycan (DG) is an important cellular receptor for extracellular matrix (ECM) proteins and also serves as the receptor for Old World arenaviruses Lassa fever virus (LFV) and lymphocytic choriomeningitis virus (LCMV) and clade C New World arenaviruses. In the host cell, alpha-DG is subject to a remarkably complex pattern of O glycosylation that is crucial for its interactions with ECM proteins. Two of these unusual sugar modifications, protein O mannosylation and glycan modifications involving the putative glycosyltransferase LARGE, have recently been implicated in arenavirus binding. Considering the complexity of alpha-DG O glycosylation, our present study was aimed at the identification of the specific O-linked glycans on alpha-DG that are recognized by arenaviruses. As previously shown for LCMV, we found that protein O mannosylation of alpha-DG is crucial for the binding of arenaviruses of distinct phylogenetic origins, including LFV, Mobala virus, and clade C New World arenaviruses. In contrast to the highly conserved requirement for O mannosylation, more generic O glycans present on alpha-DG are dispensable for arenavirus binding. Despite the critical role of O-mannosyl glycans for arenavirus binding under normal conditions, the overexpression of LARGE in cells deficient in O mannosylation resulted in highly glycosylated alpha-DG that was functional as a receptor for arenaviruses. Thus, modifications by LARGE but not O-mannosyl glycans themselves are most likely the crucial structures recognized by arenaviruses. Together, the data demonstrate that arenaviruses recognize the same highly conserved O-glycan structures on alpha-DG involved in ECM protein binding, indicating a strikingly similar mechanism of receptor recognition by pathogen- and host-derived ligands.

Mapping the landscape of the lymphocytic choriomeningitis virus stable signal peptide reveals novel functional domains.

The stable signal peptide (SSP) of the lymphocytic choriomeningitis virus surface glycoprotein precursor has several unique characteristics. The SSP is unusually long, at 58 amino acids, and contains two hydrophobic domains, and its sequence is highly conserved among both Old and New World arenaviruses. To better understand the functions of the SSP, a panel of point and deletion mutants was created by in vitro mutagenesis to target the highly conserved elements within the SSP. We were also able to confirm critical residues required for separate SSP functions by trans-complementation. Using these approaches, it was possible to resolve functional domains of the SSP. In characterizing our SSP mutants, we discovered that the SSP is involved in several distinct functions within the viral life cycle, beyond translocation of the viral surface glycoprotein precursor into the endoplasmic reticulum lumen. The SSP is required for efficient glycoprotein expression, posttranslational maturation cleavage of GP1 and GP2 by SKI-1/S1P protease, glycoprotein transport to the cell surface plasma membrane, formation of infectious virus particles, and acid pH-dependent glycoprotein-mediated cell fusion.

Nonneutralizing antibodies binding to the surface glycoprotein of lymphocytic choriomeningitis virus reduce early virus spread.

The biological relevance of nonneutralizing antibodies elicited early after infection with noncytopathic persistence-prone viruses is unclear. We demonstrate that cytotoxic T lymphocyte-deficient TgH(KL25) mice, which are transgenic for the heavy chain of the lymphocytic choriomeningitis virus (LCMV)-neutralizing monoclonal antibody KL25, mount a focused neutralizing antibody response following LCMV infection, and that this results in the emergence of neutralization escape virus variants. Further investigation revealed that some of the escape variants that arose early after infection could still bind to the selecting antibody. In contrast, no antibody binding could be detected for late isolates, indicating that binding, but nonneutralizing, antibodies exerted a selective pressure on the virus. Infection of naive TgH(KL25) mice with distinct escape viruses differing in their antibody-binding properties revealed that nonneutralizing antibodies accelerated clearance of antibody-binding virus variants in a partly complement-dependent manner. Virus variants that did not bind antibodies were not affected. We therefore conclude that nonneutralizing antibodies binding to the same antigenic site as neutralizing antibodies are biologically relevant by limiting early viral spread.

Direct stimulation of T lymphocytes by immunosomes: virus-like particles decorated with T cell receptor/CD3 ligands plus costimulatory molecules.

Many infectious viruses coevolved with the vertebrate immune system. During the assembly of enveloped viruses, lipid ordered domains of the host cell plasma membrane, called lipid rafts, frequently function as a natural meeting point for viral proteins. The role of lipid rafts in the organization of complex combinations of immune receptors during antigen presentation and T cell signaling is widely recognized. In our studies, we determined whether lipid rafts, virus budding, and molecular interactions during T cell activation could be brought into a novel context to create artificial antigen-presenting particles. We show here that cell-free virus-like particles (VLP) expressing a surrogate TCR/CD3 ligand (OKT3scFv) and the costimulator CD80 polyclonally activate human T cells independently of accessory cells. VLP expressing the glycoprotein epitope 33-41 of the lymphocytic choriomeningitis virus in the context of H-2D(b) activate and expand naïve, antigen-specific CD8(+) T lymphocytes and differentiate them into cytotoxic effector cells. Efficient targeting of T cell ligands to lipid rafts and ultimately to VLP is achieved by C-terminal introduction of glycosyl phosphatidyl inositol acceptor sequences, replacing transmembrane and intracellular domains. In this work, basic functions of immunostimulatory molecules meet virus biology and translate into a reductionist antigen-specific T lymphocyte-stimulating vehicle, which we refer to as immunosomes. A large variety of agonistic and antagonistic accessory molecules on genuine antigen-presenting cells may complicate the predictable manipulation of T cells as well as the analysis of selected receptor combinations, making immunosomes potentially useful reagents for such purposes in the future.

Identification of an N-terminal trimeric coiled-coil core within arenavirus glycoprotein 2 permits assignment to class I viral fusion proteins.

The lymphocytic choriomeningitis virus (LCMV) glycoprotein (GP) consists of the transmembrane subunit GP-2 and the receptor binding subunit GP-1. Both are synthesized as one precursor protein and stay noncovalently attached after cleavage. In this study, we determined the oligomeric state of the LCMV GP and expressed it in two different conformations suitable for structural analysis. Sequence analysis of GP-2 identified a trimeric heptad repeat pattern containing an N-terminal alpha-helix. An alpha-helical peptide matching this region formed a stable oligomer as revealed by gel filtration chromatography and dynamic light scattering. In contrast, a second alpha-helical peptide corresponding to a predicted C-terminal alpha-helix within GP-2 did not oligomerize. Refolding of the complete GP-2 ectodomain revealed trimeric all-alpha complexes probably representing the six-helix bundle state that is considered a hallmark of class I viral fusion proteins. Based on these results, we generated a construct consisting of the complete uncleavable LCMV GP ectodomain fused C-terminally to the trimeric motif of fibritin. Gel filtration analysis of the secreted fusion protein identified two complexes of approximately 230 and approximately 440 kDa. Both complexes bound to a set of conformational and linear antibodies. Cross-linking confirmed the 230-kDa complex to be a trimer. The 440-kDa complexes were found to represent disulfide-linked pairs of trimers, since partial reduction converted them to a complex species migrating at 250 kDa. By electron microscopy, the 230-kDa complexes appeared as single spherical particles and showed no signs of rosette formation. Our results clearly demonstrate that the arenavirus GP is a trimer and must be considered a member of the class I viral fusion protein family.

Recovery of an arenavirus entirely from RNA polymerase I/II-driven cDNA.

The prototypic arenavirus lymphocytic choriomeningitis virus has been a primary workhorse of viral immunologists for almost a century, and it has served as an important model for studying basic principles of arenavirus molecular biology. Its negative-stranded bisegmented RNA genome has, however, posed a major obstacle to attempts at manipulating the infectious virus by reverse genetic techniques. Here, we report the recovery of infectious lymphocytic choriomeningitis virus (the immunosuppressive strain clone 13) entirely from cDNA. Intracellular transcription of the short and the long viral genome segment from polymerase (pol) I-driven vectors and coexpression of the minimal viral-transacting factors NP and L from pol II-driven plasmids resulted in the efficient formation of infectious virus with genetic tags in both genome segments. The cDNA-derived viruses behaved identically to wild-type virus in both cell culture and infected mice. Importantly, they caused a chronic infection and suppressed the adaptive immune response to an unrelated third-party virus. This technology provides an important basis for investigating viral determinants of persistent infection and immunosuppression. In addition, our findings demonstrate that pol I/II-based vector systems may represent an efficient alternative strategy for the recovery of cytoplasmic negative-strand RNA viruses from cDNA.

Long-lived signal peptide of lymphocytic choriomeningitis virus glycoprotein pGP-C.

Signal peptides (SPs) direct nascent secretory and membrane proteins to the membrane of the endoplasmic reticulum. They are usually cleaved from the nascent polypeptide by signal peptidase and then further proteolytically processed. The SP of the pre-glycoprotein (pGP-C) of the lymphocytic choriomeningitis virus SPGP-C (signal peptide of pGP-C) shows different properties: 1) The SPGP-C is unusually long (58 amino acid residues) and contains two hydrophobic segments interrupted by a lysine residue. 2) The SPGP-C is cleaved only from a subset of pGP-C proteins. A substantial portion of pGP-C accumulates that still contains the SPGP-C.3)The cleaved SPGP-C is rather long-lived (t(1/2) of more than 6 h). 4) The cleaved SPGP-C resides in the membrane and is resistant to digestion with proteinase K even in the presence of detergents, suggesting a very compact structure. 5) SPGP-C accumulates in virus particles. These unusual features of the cleaved SPGP-C suggest that SPGP-C not only targets the nascent pGP-C to the endoplasmic reticulum membrane but also has additional functions in lymphocytic choriomeningitis virus life cycle.

Self-assembly properties of a model RING domain.

RING domains act in a variety of essential cellular processes but have no general function ascribed to them. Here, we observe that purified arenaviral protein Z, constituted almost entirely by its RING domain, self-assembles in vitro into spherical structures that resemble functional bodies formed by Z in infected cells. By using a variety of biophysical methods we provide a thermodynamic and kinetic framework for the RING-dependent self-assembly of Z. Assembly appears coupled to substantial conformational reorganization and changes in zinc coordination of site II of the RING. Thus, the rate-limiting nature of conformational reorganization observed in the folding of monomeric proteins can also apply to the assembly of macromolecular scaffolds. These studies describe a unique mechanism of nonfibrillar homogeneous self-assembly and suggest a general function of RINGs in the formation of macromolecular scaffolds that are positioned to integrate biochemical processes in cells.

Zooming in on the hydrophobic ridge of H-2D(b): implications for the conformational variability of bound peptides.

Class I major histocompatibility complex (MHC) molecules, which display intracellularly processed peptides on the cell surface for scanning by T-cell receptors (TCRs), are extraordinarily polymorphic. MHC polymorphism is believed to result from natural selection, since individuals heterozygous at the corresponding loci can cope with a larger number of pathogens. Here, we present the crystal structures of the murine MHC molecule H-2D(b) in complex with the peptides gp276 and np396 from the lymphocytic choriomeningitis virus (LCMV), solved at 2.18 A and 2.20 A resolution, respectively. The most prominent feature of H-2D(b) is a hydrophobic ridge that cuts across its antigen-binding site, which is conserved in the L(d)-like family of class I MHC molecules. The comparison with previously solved crystal structures of peptide/H-2D(b) complexes shows that the hydrophobic ridge focuses the conformational variability of the bound peptides in a "hot-spot", which could allow optimal TCR interaction and discrimination. This finding suggests a functional reason for the conservation of this structural element.