Neutralization of rhesus cytomegalovirus IL-10 reduces horizontal transmission and alters long-term immunity.

Human cytomegalovirus (HCMV) causes severe disease in infants and immunocompromised people. There is no approved HCMV vaccine, and vaccine development strategies are complicated by evidence of both persistent infection and reinfection of people with prior immunity. The greatest emphasis has been placed on reducing transmission to seronegative pregnant women to prevent vertical transmission and its potentially severe sequelae. Increasing evidence suggests that the earliest host-HCMV interactions establish conditions for viral persistence, including evasion of host immune responses to the virus. Using a nonhuman primate model of HCMV infection, we show that rhesus macaques immunized against viral interleukin-10 (IL-10) manifest delayed rhesus cytomegalovirus (RhCMV) acquisition and altered immune responses to the infection when it does occur. Among animals with the greatest antiviral IL-10-neutralizing activity, the timing of RhCMV seroconversion was delayed by an average of 12 weeks. After acquisition, such animals displayed an antibody response to the new infection, which peaked as expected after 2 weeks but then declined rapidly. In contrast, surprisingly, vaccination with glycoprotein B (gB) protein had no discernible impact on these outcomes. Our results demonstrate that viral IL-10 is a key regulator of successful host immune responses to RhCMV. Viral IL-10 is, therefore, an important target for vaccine strategies against cytomegalovirus (CMV). Furthermore, given the immunoregulatory function of viral IL-10, targeting this protein may prove synergistic with other vaccine therapies and targets. Our study also provides additional evidence that the earliest host-CMV interactions can have a significant impact on the nature of persistent infection.

Cutting Edge: Endogenous IFN-ß Regulates Survival and Development of Transitional B Cells.

The transitional stage of B cell development is a formative stage in the spleen where autoreactive specificities are censored as B cells gain immune competence, but the intrinsic and extrinsic factors regulating survival of transitional stage 1 (T1) B cells are unknown. We report that B cell expression of IFN-ß is required for optimal survival and TLR7 responses of transitional B cells in the spleen and was overexpressed in T1 B cells from BXD2 lupus-prone mice. Single-cell gene expression analysis of B6 Ifnb+/+ versus B6 Ifnb-/- T1 B cells revealed heterogeneous expression of Ifnb in wild-type B cells and distinct gene expression patterns associated with endogenous IFN-ß. Single-cell analysis of BXD2 T1 B cells revealed that Ifnb is expressed in early T1 B cell development with subsequent upregulation of Tlr7 and Ifna1 Together, these data suggest that T1 B cell expression of IFN-ß plays a key role in regulating responsiveness to external factors.

Missense splice variant (g.20746A>G, p.Ile183Val) of interferon gamma receptor 1 (IFNGR1) coincidental with mycobacterial osteomyelitis - a screen of osteoarticular lesions.

Previously, dominant partial interferon-gamma receptor 1 (IFN-g-R1) susceptibility to environmental mycobacteria was found with IFNGR1 deletions or premature stop. Our aim was to search for IFNGR1 variants in patients with mycobacterial osteoarticular lesions. Biopsies from the patients were examined for acid-fast bacilli, inflammatory cell infiltration, and mycobacterial niacin. Mycobacterial rRNA was analyzed using a target-amplified rRNA probe test. Peripheral-blood-leukocyte genomic DNA was isolated from 19 patients using the QIAamp DNA Mini Kit, and all IFNGR1 exons were sequenced using an ABIPRISM 3130 device. After the discovery of an exon 5 variant, a Polish newborn population sample (n = 100) was assayed for the discovered variant. Splice sites and putative amino acid interactions were analyzed. All patients tested were positive for mycobacteria; one was heterozygous for the IFNGR1 exon 5 single-nucleotide-missense substitution (g.20746A>G, p.Ile183Val). No other variant was found. The splice analysis indicated the creation of an exonic splicing silencer, and alternatively, molecular graphics indicated that the p.Ile183Val might alter beta-strand packing (loss of van der Waals contacts; Val183/Pro205), possibly altering the IFN-g-R1/IFN-g-R2 interaction. The probability of non-deleterious variant was estimated as <10%. Heterozygous IFNGR1:p.Ile183Val (frequency 0.003%) was found to be coincidental with mycobacterial osteomyelitis. The small amount of variation detected in the patients with osteoarticular lesions indicates that screens should not yet be restricted: Intronic variants should be analyzed as well as the other genes affecting Type 1 T-helper-cell-mediated immunity.

The susceptibility of primary cultured rhesus macaque kidney epithelial cells to rhesus cytomegalovirus strains.

Kidney epithelial cells are common targets for human and rhesus cytomegalovirus (HCMV and RhCMV) in vivo, and represent an important reservoir for long-term CMV shedding in urine. To better understand the role of kidney epithelial cells in primate CMV natural history, primary cultures of rhesus macaque kidney epithelial cells (MKE) were established and tested for infectivity by five RhCMV strains, including two wild-type strains (UCD52 and UCD59) and three strains containing different coding contents in UL/b'. The latter strains included 180.92 [containing an intact RhUL128-RhUL130-R hUL131 (RhUL128L) locus but deleted for the UL/b' RhUL148-rh167-loci], 68-1 (RhUL128L-defective and fibroblast-tropic) and BRh68-1.2 (the RhUL128L-repaired version of 68-1). As demonstrated by RhCMV cytopathic effect, plaque formation, growth kinetics and early virus entry, we showed that MKE were differentially susceptible to RhCMV infection, related to UL/b' coding contents of the different strains. UCD52 and UCD59 replicated vigorously in MKE, 68-1 replicated poorly, and 180.92 grew with intermediate kinetics. Reconstitution of RhUL128L in 68-1 (BRh68-1.2) restored its replication efficiency in MKE as compared to UCD52 and UCD59, consistent with the essential role of UL128L for HCMV epithelial tropism. Further analysis revealed that the UL/b' UL148-rh167-loci deletion in 180.92 impaired RhUL132 (rh160) expression. Given that 180.92 retains an intact RhUL128L, but genetically or functionally lacks genes from RhUL132 (rh160) to rh167 in UL/b', its attenuated infection efficiency indicated that, along with RhUL128L, an additional protein(s) encoded within the UL/b' RhUL132 (rh160)-rh167 region (potentially, RhUL132 and/or RhUL148) is indispensable for efficient replication in MKE.

A Heterozygous RAB27A Mutation Associated with Delayed Cytolytic Granule Polarization and Hemophagocytic Lymphohistiocytosis.

Frequently fatal, primary hemophagocytic lymphohistiocytosis (HLH) occurs in infancy resulting from homozygous mutations in NK and CD8 T cell cytolytic pathway genes. Secondary HLH presents after infancy and may be associated with heterozygous mutations in HLH genes. We report two unrelated teenagers with HLH and an identical heterozygous RAB27A mutation (c.259G→C). We explore the contribution of this Rab27A missense (p.A87P) mutation on NK cell cytolytic function by cloning it into a lentiviral expression vector prior to introduction into the human NK-92 cell line. NK cell degranulation (CD107a expression), target cell conjugation, and K562 target cell lysis was compared between mutant- and wild-type-transduced NK-92 cells. Polarization of granzyme B to the immunologic synapse and interaction of mutant Rab27A (p.A87P) with Munc13-4 were explored by confocal microscopy and proximity ligation assay, respectively. Overexpression of the RAB27A mutation had no effect on cell conjugate formation between the NK and target cells but decreased NK cell cytolytic activity and degranulation. Moreover, the mutant Rab27A protein decreased binding to Munc13-4 and delayed granzyme B polarization toward the immunologic synapse. This heterozygous RAB27A mutation blurs the genetic distinction between primary and secondary HLH by contributing to HLH via a partial dominant-negative effect.

Cytokine Activation by Antibody Fragments Targeted to Cytokine-Receptor Signaling Complexes.

Exogenous cytokine therapy can induce systemic toxicity, which might be prevented by activating endogenously produced cytokines in local cell niches. Here we developed antibody-based activators of cytokine signaling (AcCS), which recognize cytokines only when they are bound to their cell surface receptors. AcCS were developed for type I interferons (IFNs), which induce cellular activities by binding to cell surface receptors IFNAR1 and IFNAR2. As a potential alternative to exogenous IFN therapy, AcCS were shown to potentiate the biological activities of natural IFNs by ∼100-fold. Biochemical and structural characterization demonstrates that the AcCS stabilize the IFN-IFNAR2 binary complex by recognizing an IFN-induced conformational change in IFNAR2. Using IFN mutants that disrupt IFNAR1 binding, AcCS were able to enhance IFN antiviral potency without activating antiproliferative responses. This suggests AcCS can be used to manipulate cytokine signaling for basic science and possibly for therapeutic applications.

VpreB serves as an invariant surrogate antigen for selecting immunoglobulin antigen-binding sites.

Developmental checkpoints eliminate B cells synthesizing defective immunoglobulin heavy (HC) and light (LC) chains. The first checkpoint tests for formation of a VpreB/λ5/µHC-containing preB-cell receptor (preBCR) and predicts whether µHCs will bind conventional LCs to form membrane IgM. VpreB and λ5 also create a sensing site that interacts with µHC antigen-binding region CDR-H3, but whether it plays a role in immunoglobulin repertoire selection and function is unknown. On a position-by-position basis, we analyzed the amino acid content of CDR-H3s from H chains cloned from living and apoptotic preB cells and from IgG:Antigen structures. Using a panel of DH gene-targeted mice, we show that progressively reducing CDR-H3 tyrosine content increasingly impairs preBCR checkpoint passage. Counting from cysteine at Framework 3 position 96, we found that VpreB particularly selects for tyrosine at CDR-H3 position 101, and that Y101 also binds antigen in IgG:Antigen structures. VpreB thus acts as an early invariant antigen. It selects for particular CDR-H3 amino acids and shapes the specificity of the IgG humoral response. This helps explain why some neutralizing antibodies against pathogens are readily produced while others are rare.

The molecular basis of IL-10 function: from receptor structure to the onset of signaling.

Assembly of the cell surface IL-10 receptor complex is the first step in initiating IL-10 signaling pathways that regulate intestinal inflammation, viral persistence and even tumor surveillance. The discovery of IL-10 homologs in the genomes of herpes viruses suggests IL-10 signaling pathways can be manipulated at the level of the receptor complex. This chapter will describe our current molecular understanding of IL-10 receptor assembly based on crystal structures and biochemical analyses of cellular and viral IL-10 receptor complexes.

Very early onset inflammatory bowel disease associated with aberrant trafficking of IL-10R1 and cure by T cell replete haploidentical bone marrow transplantation.

PURPOSE: Loss-of-function mutations in IL10 and IL10R cause very early onset inflammatory bowel disease (VEO-IBD). Here, we investigated the molecular pathomechanism of a novel intronic IL10RA mutation and describe a new therapeutic approach of T cell replete haploidentical hematopoietic stem cell transplantation (HSCT). METHODS: Clinical data were collected by chart review. Genotypes of IL10 and IL10R genes were determined by Sanger sequencing. Expression and function of mutated IL-10R1 were assessed by quantitative PCR, Western blot analysis, enzyme-linked immunosorbent assays, confocal microscopy, and flow cytometry. RESULTS: We identified a novel homozygous point mutation in intron 3 of the IL10RA (c.368-10C > G) in three related children with VEO-IBD. Bioinformatical analysis predicted an additional 3' splice site created by the mutation. Quantitative PCR analysis showed normal mRNA expression of mutated IL10RA. Sequencing of the patient's cDNA revealed an insertion of the last nine nucleotides of intron 3 as a result of aberrant splicing. Structure-based modeling suggested misfolding of mutated IL-10R1. Western blot analysis demonstrated a different N-linked glycosylation pattern of mutated protein. Immunofluorescence and FACS analysis revealed impaired expression of mutated IL-10R1 at the plasma membrane. In the absence of HLA-identical donors, T cell replete haploidentical HSCT was successfully performed in two patients. CONCLUSIONS: Our findings expand the spectrum of IL10R mutations in VEO-IBD and emphasize the need for genetic diagnosis of mutations in conserved non-coding sequences of candidate genes. Transplantation of haploidentical stem cells represents a curative therapy in IL-10R-deficient patients, but may be complicated by non-engraftment.

Epstein-Barr virus IL-10 engages IL-10R1 by a two-step mechanism leading to altered signaling properties.

Human interleukin-10 (hIL-10) is a pleiotropic cytokine that is able to suppress or activate cellular immune responses to protect the host from invading pathogens. Epstein-Barr virus (EBV) encodes a viral IL-10 (ebvIL-10) in its genome that has retained the immunosuppressive activities of hIL-10 but lost the ability to induce immunostimulatory activities on some cells. These functional differences are at least partially due to the ∼1000-fold difference in hIL-10 and ebvIL-10 binding affinity for the IL-10R1·IL-10R2 cell surface receptors. Despite weaker binding to IL-10R1, ebvIL-10 is more active than hIL-10 in inducing B-cell proliferation. To explore this counterintuitive observation further, a series of monomeric and dimeric ebvIL-10·hIL-10 chimeric proteins were produced and characterized for receptor binding and cellular proliferation on TF-1/hIL-10R1 cells that express high levels of the IL-10R1 chain. On this cell line, monomeric chimeras elicited cell proliferation in accordance with how tightly they bound to the IL-10R1 chain. In contrast, dimeric chimeras exhibiting the highest affinity for IL-10R1 exhibited reduced proliferative activity. These distinct activity profiles are correlated with kinetic analyses that reveal that the ebvIL-10 dimer is impaired in its ability to form a 1:2 ebvIL-10·IL-10R1 complex. As a result, the ebvIL-10 dimer functions like a monomer at low IL-10R1 levels, which prevents efficient signaling. At high IL-10R1 levels, the ebvIL-10 dimer is able to induce signaling responses greater than hIL-10. Thus, the ebvIL-10 dimer scaffold is essential to prevent activation of cells with low IL-10R1 levels but to maintain or enhance activity on cells with high IL-10R1 levels.

Interleukin-26: an IL-10-related cytokine produced by Th17 cells.

IL-26 is classified as a member of the IL-10 cytokine family because it has limited sequence homology to IL-10 and the IL-10-related cytokines. The human IL-26 gene, IL26, is located on chromosome 12q15 between the genes for two other important class-2 cytokines, IFNG (IFN-γ) and IL22 (IL-22). IL-26 is often co-expressed with IL-22 by activated T cells, especially Th17 cells. It signals through a heterodimeric receptor complex composed of the IL-20R1 and IL-10R2 chains. IL-26 receptors are primarily expressed on non-hematopoietic cell types, particularly epithelial cells. Signaling through IL-26 receptor complexes results in the activation of STAT1 and STAT3 with subsequent induction of IL-26-responsive genes. The biological functions of IL-26 have only begun to be defined.

Substitution of adenovirus serotype 3 hexon onto a serotype 5 oncolytic adenovirus reduces factor X binding, decreases liver tropism, and improves antitumor efficacy.

Following intravascular delivery, an important route of administration for many clinical applications, the liver is the predominant site of adenovirus serotype 5 (Ad5) sequestration, thereby posing a risk of toxicity. In this regard, it has recently been shown that the Ad5 capsid binds to the blood coagulation factor X (FX) via the Ad5 hexon protein. This interaction mediates the majority of Ad5 liver transduction. Patient FX levels can be diminished by the administration of warfarin, a vitamin K inhibitor in the liver that decreases FX production; however, warfarin is a potent anticoagulant and can have a number of undesired side effects. Therefore, genetic modification of the virus to ablate FX binding is the preferred approach. Modifications of the hexon protein, specifically within the hypervariable 5 (HVR5) and 7 (HVR7) regions, have produced Ad5 vectors that show minimal liver sequestration. Our laboratory has pioneered adenovirus hexon modifications, including insertion of peptide ligands into the hypervariable regions and substitution of the adenovirus hexon with hexon proteins from alternate serotypes. Substitution of the adenovirus serotype 3 (Ad3) hexon protein onto the Ad5 capsid has been further characterized with regard to its interaction with FX and incorporated into an infectivity-enhanced conditionally replicative adenovirus (CRAd). In vitro evaluation of these hexon-modified vectors showed decreased binding to FX and decreased cell transduction via FX-mediated pathways. Furthermore, in vivo biodistribution studies in mice exhibited a decrease in liver sequestration. With the use of xenograft tumor models, the antitumor efficacy of the hexon-modified CRAds was enhanced over nonmodified controls.

A docking model based on mass spectrometric and biochemical data describes phage packaging motor incorporation.

The molecular mechanism of scaffolding protein-mediated incorporation of one and only one DNA packaging motor/connector dodecamer at a unique vertex during lambdoid phage assembly has remained elusive because of the lack of structural information on how the connector and scaffolding proteins interact. We assembled and characterized a phi29 connector-scaffolding complex, which can be incorporated into procapsids during in vitro assembly. Native mass spectrometry revealed that the connector binds at most 12 scaffolding molecules, likely organized as six dimers. A data-driven docking model, using input from chemical cross-linking and mutagenesis data, suggested an interaction between the scaffolding protein and the exterior of the wide domain of the connector dodecamer. The connector binding region of the scaffolding protein lies upstream of the capsid binding region located at the C terminus. This arrangement allows the C terminus of scaffolding protein within the complex to both recruit capsid subunits and mediate the incorporation of the single connector vertex.

Isolation of flagellated bacteria implicated in Crohn's disease.

BACKGROUND: Serologic expression cloning has identified flagellins of the intestinal microbiota as immunodominant antigens in experimental colitis in mice and in individuals with Crohn's disease (CD). The present study was done to identify the microbial source of such flagellins. METHODS: Using a variety of isolation and culture approaches, a number of previously unknown flagellated bacteria were isolated. Based on 16S ribosomal DNA sequences, these bacteria fall into the family Lachnospiraceae of the phylum Firmicutes. RESULTS: Serum IgG from patients with CD and from mice with colitis reacted to the flagellins of these bacteria, and only their flagellins, whereas serum IgG from controls did not. The sequence of these flagellins demonstrate conserved amino- and carboxy-terminal domains that cluster phylogenetically and have a predicted 3D structure similar to Salmonella fliC, including an intact TLR5 binding site. The flagellin of 1 of these bacteria was likely O-glycosylated. CONCLUSIONS: The conserved immune response in both mouse and human to these previously unknown flagellins of the microbiota indicate that they play an important role in host-microbe interactions in the intestine.

Identification and characterization of a +1 frameshift observed during the expression of Epstein-Barr virus IL-10 in Escherichia coli.

Epstein-Barr virus IL-10 (ebvIL-10) mimics the biological functions of cellular IL-10 including a number of immunoinhibitory activities on diverse immune cells. Characterization of ebvIL-10 and several mutants, expressed in Escherichia coli, by gel filtration chromatography and mass spectrometry revealed a +1 frameshift upon ebvIL-10 expression. The frameshift is caused by the rare AGG codon at ebvIL-10 Arg159, which is followed by the most inefficient stop signal, UGAC. The frameshift was corrected by substituting the rare AGG codon with an abundant arginine codon, CGU, or by enhancing the level of tRNA that decodes the AGG codon. As a result, ebvIL-10 expression levels increased by approximately 3-fold and the purity of the protein improved from 85-95% to 98-99%. The correction of the frameshift has been essential for continuing structural and biophysical studies of ebvIL-10.

BiP/GRP78 is an intracellular target for MDA-7/IL-24 induction of cancer-specific apoptosis.

Melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL-24) is a unique member of the IL-10 gene family that induces cancer-selective growth suppression and apoptosis in a wide spectrum of human cancers in cell culture and animal models. Additionally, recent clinical trials confirm safety and document significant clinical activity of mda-7/IL-24 in patients with diverse solid cancers and melanomas. Despite intensive study the molecular basis of tumor-cell selectivity of mda-7/IL-24 is not well characterized. Using deletion analysis, a specific mutant of MDA-7/IL-24, M4, consisting of amino acids 104 to 206, is described that retains the cancer-specific growth-suppressive and apoptosis-inducing properties of the full-length protein. Employing rationally designed mutational analysis, we show that MDA-7/IL-24 and M4 physically interact with BiP/GRP78 through their C and F helices, localize in the endoplasmic reticulum, and activate p38 MAPK and GADD gene expression, culminating in cancer-selective apoptosis. These studies provide novel mechanistic insights into the discriminating antitumor activity of MDA-7/IL-24 by elucidating BiP/GRP78 as a defined intracellular target of action and present an unparalleled opportunity to develop improved therapeutic versions of this cancer-specific apoptosis-inducing cytokine.

The unique C termini of orthopoxvirus gamma interferon binding proteins are essential for ligand binding.

The orthopoxviruses ectromelia virus (ECTV) and vaccinia virus (VACV) express secreted gamma interferon binding proteins (IFN-gammaBPs) with homology to the ligand binding domains of the host's IFN-gamma receptor (IFN-gammaR1). Homology between these proteins is limited to the extracellular portions of the IFN-gammaR1 and the first approximately 200 amino acids of the IFN-gammaBPs. The remaining 60 amino acids at the C termini of the IFN-gammaBPs contain a single cysteine residue shown to be important in covalent dimerization of the secreted proteins. The function of the remaining C-terminal domain (CTD) has remained elusive, yet this region is conserved within all orthopoxvirus IFN-gammaBPs. Using a series of C-terminal deletion constructs, we have determined that the CTD is essential for IFN-gamma binding despite having no predicted homology to the IFN-gammaR1. Truncation of the ECTV IFN-gammaBP by more than two amino acid residues results in a complete loss of binding activity for both murine IFN-gamma and human IFN-gamma (hIFN-gamma), as measured by surface plasmon resonance (SPR) and bioassay. Equivalent truncation of the VACV IFN-gammaBP resulted in comparable loss of hIFN-gamma binding activity by SPR. Full-length IFN-gammaBPs were observed to form higher-ordered structures larger than the previously reported dimers. Mutants that were unable to bind IFN-gamma with high affinity in SPR experiments failed to assemble into these higher-ordered structures and migrated as dimers. We conclude that the unique CTD of orthopoxvirus IFN-gammaBPs is important for the assembly of covalent homodimers as well as the assembly of higher-ordered structures essential for IFN-gamma binding.

Unique aspects of mda-7/IL-24 antitumor bystander activity: establishing a role for secretion of MDA-7/IL-24 protein by normal cells.

Melanoma differentiation associated gene-7 (mda-7) was cloned using subtraction hybridization from terminally differentiated human melanoma cells. Based on structural and functional properties, mda-7 is now recognized as interleukin-24 (IL-24), a new member of the expanding IL-10 gene family. Unique properties of mda-7/IL-24 include its ability to selectively induce growth suppression, apoptosis and radiosensitization in diverse human cancer cells, without causing similar effects in normal cells. The utility of mda-7/IL-24, administered by means of a replication-incompetent adenovirus, as a gene therapy for cancer has recently received validation in patients, highlighting an important phenomenon initially observed in pancreatic tumor cells, namely a 'potent bystander apoptosis-inducing effect' in adjacent tumor cells not initially receiving this gene product. We presently investigated the contribution of mda-7/IL-24 secreted by normal cells in mediating this 'bystander effect', and document that normal cells induced to produce mda-7/IL-24 following infection with recombinant adenoviruses expressing this cytokine secrete mda-7/IL-24, which modifies the anchorage-independent growth, invasiveness, survival and sensitivity to radiation of cancer cells that contain functional IL-20/IL-22 receptors, but not in cancer cells that lack a complete set of receptors. Moreover, the combination of secreted mda-7/IL-24 and radiation engenders a 'bystander antitumor effect' not only in inherently mda-7/IL-24 or radiation-sensitive cancer cells, but also in tumor cells overexpressing the antiapoptotic proteins bcl-2 or bcl-x(L) and displaying resistance to either treatment alone. The present studies provide definitive evidence that secreted mda-7/IL-24 from normal cells can induce direct antitumor and radiation-enhancing effects that are dependent on the presence of canonical receptors for this cytokine on tumor cells. Moreover, we now describe a novel means of enhancing mda-7/IL-24's therapeutic potential by targeting normal cells to produce and release this cancer-specific apoptosis-inducing cytokine, a strategy that could be employed as an innovative way of using this unique gene product for treating metastatic disease.

Same structure, different function crystal structure of the Epstein-Barr virus IL-10 bound to the soluble IL-10R1 chain.

Human IL-10 (hIL-10) is a cytokine that modulates diverse immune responses. The Epstein-Barr virus (EBV) genome contains an IL-10 homolog (vIL-10) that shares high sequence and structural similarity with hIL-10. Although vIL-10 suppresses inflammatory responses like hIL-10, it cannot activate many other immunostimulatory functions performed by the cellular cytokine. These functional differences have been correlated with the approximately 1000-fold lower affinity of vIL-10, compared to hIL-10, for the IL-10R1 receptor chain. To define the structural basis for these observations, crystal structures of vIL-10 and a vIL-10 point mutant were determined bound to the soluble IL-10R1 receptor fragment (sIL-10R1) at 2.8 and 2.7 A resolution, respectively. The structures reveal that subtle changes in the conformation and dynamics of the vIL-10 AB and CD loops and an orientation change of vIL-10 on sIL-10R1 are the main factors responsible for vIL-10's reduced affinity for sIL-10R1 and its distinct biological profile.

An early stage of Mason-Pfizer monkey virus budding is regulated by the hydrophobicity of the Gag matrix domain core.

Intracellular capsid transport and release of Mason-Pfizer monkey virus are dependent on myristylation of the Gag matrix domain (MA). A myristylated MA mutant, in which Thr41 and Thr78 are replaced with isoleucines, assembles capsids that are transported to the plasma membrane but are blocked in an early budding step. Since the nuclear magnetic resonance structure of MA showed that these Thr residues point into the hydrophobic core of the protein, it was hypothesized that the T41I/T78I mutant was defective in release of myristic acid from the more hydrophobic core. In order to further investigate whether an increase in the hydrophobicity of the MA core modulates capsid-membrane interactions and viral budding, three tyrosine residues (11, 28, and 67), oriented toward the MA core, were replaced individually or in a pair-wise combination with the more hydrophobic phenylalanine residue(s). As a control, Tyr82, oriented toward the outer surface of MA, was also replaced with phenylalanine. These Tyr-to-Phe substitutions did not alter capsid assembly compared to wild type in a capsid assembly assay. Pulse-chase, immunofluorescence, and electron microscopy studies demonstrated that single substitutions of Tyr11, Tyr28, and Tyr67 recapitulated the T41I/T78I mutant phenotype of decreased budding kinetics and accumulation of capsids at the plasma membrane. MA double mutants with a combination of these Tyr substitutions exhibited a phenotype that was even more defective in budding. In contrast, MA mutants with Tyr82 replaced by Phe resulted in a transport-defective phenotype. These results strongly support the hypothesis that myristic acid is sequestered inside MA prior to capsid-membrane interactions.