Many viruses encode proteins whose major function is to evade or disable the host T cell response. Nevertheless, most viruses are readily detected by host T cells, and induce relatively strong T cell responses. Herein, we employ transgenic CD4(+) and CD8(+) T cells as sensors to evaluate in vitro and in vivo antigen presentation by coxsackievirus B3 (CVB3), and we show that this virus almost completely inhibits antigen presentation via the MHC class I pathway, thereby evading CD8(+) T cell immunity. In contrast, the presentation of CVB3-encoded MHC class II epitopes is relatively unencumbered, and CVB3 induces in vivo CD4(+) T cell responses that are, by several criteria, phenotypically normal. The cells display an effector phenotype and mature into multi-functional CVB3-specific memory CD4(+) T cells that expand dramatically following challenge infection and rapidly differentiate into secondary effector cells capable of secreting multiple cytokines. Our findings have implications for the efficiency of antigen cross-presentation during coxsackievirus infection.

A novel compound, TTP-8307, was identified as a potent inhibitor of the replication of several rhino- and enteroviruses. TTP-8307 inhibits viral RNA synthesis in a dose-dependent manner, without affecting polyprotein synthesis and/or processing. Drug-resistant variants of coxsackievirus B3 were all shown to carry at least one amino acid mutation in the nonstructural protein 3A. In particular, three mutations located in a nonstructured region preceding the hydrophobic domain (V45A, I54F, and H57Y) appeared to contribute to the drug-resistant phenotype. This region has previously been identified as a hot sport for mutations that resulted in resistance to enviroxime, the sole 3A-targeting enterovirus inhibitor reported thus far. This was corroborated by the fact that TTP-8307 and enviroxime proved cross-resistant. It is hypothesized that TTP-8307 and enviroxime disrupt proper interactions of 3A(B) with other viral or cellular proteins that are required for efficient replication.

The replication of coxsackievirus RNA occurs with rapid onset, starting approximately 2.5 h after infection. The mechanisms entailing the RNA replication of enteroviruses, like coxsackievirus and poliovirus, are highly conserved. These processes require two steps of RNA amplification:(i) complete synthesis of the negative-strand RNA using input RNA as the template and (ii) synthesis of the positive-strand RNA using the intermediate negative-strand RNA as the template. Successful enterovirus RNA replication requires all of the viral nonstructural proteins in their mature and precursor forms, as well as RNA secondary structures in the template. The encoded nonstructural proteins are responsible for RNA replication through multiple protein-protein interactions between viral and/or host proteins to mediate RNA synthesis, induce membranous vesicles, and deliver the replication complex to the template. The RNA secondary structures at the 5' and 3' termini of the template position the RNA replication complex at the initiation site(s) for both negative- and positive-strand RNA synthesis, thus providing binding sites for viral and host proteins that may functionally circularize the genome during RNA synthesis. Although considerable knowledge has been gained regarding the mechanism of enterovirus RNA synthesis, the complete steps in RNA replication have not been fully determined. The aim of this review is to summarize the current state of our knowledge and to present a model that encompasses the identified steps of enterovirus RNA replication.

Previous studies have suggested that coxsackievirus B (CVB) activates CD8(+) T cells in vivo, but the extent of this activation, and the antigen specificity of the CD8(+) T cells, remain uncertain. Furthermore, CVB-induced CD4(+) T cell responses have not been carefully investigated. Herein, we evaluate CD8(+) and CD4(+) T cell responses both in a secondary lymphoid organ (spleen) and in peripheral tissues (heart, pancreas), using a recombinant CVB3 (rCVB3.6) that encodes well-characterized CD8(+) and CD4(+) T cell epitopes. Despite reaching high levels in vivo, rCVB3.6 failed to trigger a marked expansion of CD8(+) or CD4(+) T cells, and T cell activation was surprisingly limited. Furthermore, epitope-specific effector functions could not be detected using highly sensitive in vivo and ex vivo assays. Moreover, MHC class I tetramer analysis indicated that our inability to detect CVB3-specific CD8(+) T cell responses could not be explained by the cells' being dysfunctional. In contrast to naïve T cells, epitope-specific memory CD8(+) and CD4(+) T cells proliferated markedly, indicating that both of the rCVB3.6-encoded epitopes were presented by their respective MHC molecules in vivo. These data are consistent with the observation that several CVB3 proteins can limit the presentation of viral epitopes on the surface of infected cells, and suggest that the level of MHC/peptide complex is sufficient to trigger memory, but not naïve, T cells. Finally, our findings have implications for the biological significance of cross-priming, a process thought by some to be important for the induction of antiviral CD8(+) T cell responses.

Both untranslated regions (UTRs) of plus-strand RNA virus genomes jointly control translation and replication of viral genomes. In the case of the Enterovirus genus of the Picornaviridae family, the 5'UTR consists of a cloverleaf-like terminus preceding the internal ribosomal entry site (IRES) and the 3' terminus is composed of a structured 3'UTR and poly(A). The IRES and poly(A) have been implicated in translation control, and all UTR structures, in addition to cis-acting genetic elements mapping to the open reading frame, have been assigned roles in RNA replication. Viral UTRs are recognized by viral and host cell RNA-binding proteins that may co-determine genome stability, translation, plus- and minus-strand RNA replication, and scaffolding of viral replication complexes within host cell substructures. In this report, we describe experiments with coxsackie B viruses with a cell type-specific propagation deficit in Sk-N-Mc neuroblastoma cells conferred by the combination of a heterologous IRES and altered 3'UTR. Serial passage of these constructs in Sk-N-Mc cells yielded genetic adaptation by mutations within the viral nonstructural proteins 3A and 3C. Our data implicate 3A and/or 3C or their precursors 3AB and/or 3CD in a functional complex with the IRES and 3'UTR that drives viral propagation. Adaptation to neuroblastoma cells suggests an involvement of cell type-specific host factors or the host cell cytoplasmic milieu in this phenomenon.

Two critical interactions within the poliovirus RNA replication complex are those of the RNA-dependent RNA polymerase 3D with the viral proteins 3AB and VPg. 3AB is a membrane-binding protein responsible for the localization of the polymerase to the membranous vesicles at which replication occurs. VPg (a peptide comprising the 3B region of 3AB) is the 22-residue soluble product of 3AB cleavage and serves as the protein primer for RNA replication. The detailed interactions of these proteins with the RNA-dependent RNA polymerase 3D were analyzed to elucidate the precise roles of 3AB and VPg in the viral RNA replication complex. Using a membrane-based pull-down assay, we have identified a binding "hot-spot" spanning residues 100 to 104 in the 3B (VPg) region of 3AB which plays a critical role in mediating the interaction of 3AB with the polymerase. Isothermal titration calorimetry shows that the interaction of VPg with 3D is enthalpically driven, with a dissociation constant of 11 microM. Mutational analyses of VPg indicate that a subset of the residues important for 3AB-3D binding are also important for VPg-3D binding. Two residues in particular, P14 and R17, were shown to be absolutely critical for the binding interaction. This work provides the direct characterization of two binding interactions critical for the replication of this important class of viruses and identifies a conserved polymerase binding sequence responsible for targeting the polymerase.

Infection of cells with picornaviruses can lead to a block in protein secretion. For poliovirus this is achieved by the 3A protein, and the consequent reduction in secretion of proinflammatory cytokines and surface expression of major histocompatibility complex class I proteins may inhibit host immune responses in vivo. Foot-and-mouth disease virus (FMDV), another picornavirus, can cause persistent infection of ruminants, suggesting it too may inhibit immune responses. Endoplasmic reticulum (ER)-to-Golgi apparatus transport of proteins is blocked by the FMDV 2BC protein. The observation that 2BC is processed to 2B and 2C during infection and that individual 2B and 2C proteins are unable to block secretion stimulated us to study the effects of 2BC processing on the secretory pathway. Even though 2BC was processed rapidly to 2B and 2C, protein transport to the plasma membrane was still blocked in FMDV-infected cells. The block could be reconstituted by coexpression of 2B and 2C, showing that processing of 2BC did not compromise the ability of FMDV to slow secretion. Under these conditions, 2C was located to the Golgi apparatus, and the block in transport also occurred in the Golgi apparatus. Interestingly, the block in transport could be redirected to the ER when 2B was coexpressed with a 2C protein fused to an ER retention element. Thus, for FMDV a block in secretion is dependent on both 2B and 2C, with the latter determining the site of the block.

The family Picornaviridae contains well-known human pathogens (e.g., poliovirus, coxsackievirus, rhinovirus, and parechovirus). In addition, this family contains a number of viruses that infect animals, including members of the genus Cardiovirus such as Encephalomyocarditis virus (EMCV) and Theiler's murine encephalomyelits virus (TMEV). The latter are important murine pathogens that cause myocarditis, type 1 diabetes and chronic inflammation in the brains, mimicking multiple sclerosis. Recently, a new picornavirus was isolated from humans, named Saffold virus (SAFV). The virus is genetically related to Theiler's virus and classified as a new species in the genus Cardiovirus, which until the discovery of SAFV did not contain human viruses. By analogy with the rodent cardioviruses, SAFV may be a relevant new human pathogen. Thus far, SAFVs have sporadically been detected by molecular techniques in respiratory and fecal specimens, but the epidemiology and clinical significance remained unclear. Here we describe the first cultivated SAFV type 3 (SAFV-3) isolate, its growth characteristics, full-length sequence, and epidemiology. Unlike the previously isolated SAFV-1 and -2 viruses, SAFV-3 showed efficient growth in several cell lines with a clear cytopathic effect. The latter allowed us to conduct a large-scale serological survey by a virus-neutralization assay. This survey showed that infection by SAFV-3 occurs early in life (>75% positive at 24 months) and that the seroprevalence reaches >90% in older children and adults. Neutralizing antibodies were found in serum samples collected in several countries in Europe, Africa, and Asia. In conclusion, this study describes the first cultivated SAFV-3 isolate, its full-length sequence, and epidemiology. SAFV-3 is a highly common and widespread human virus causing infection in early childhood. This finding has important implications for understanding the impact of these ubiquitous viruses and their possible role in acute and/or chronic disease.

The family Picornaviridae consists of a large group of plus-strand RNA viruses that share a similar genome organization. The nomenclature of the picornavirus proteins is based on their position in the viral RNA genome but does not necessarily imply a conserved function of proteins of different genera. The enterovirus 2B protein is a small hydrophobic protein that, upon individual expression, is localized to the endoplasmic reticulum (ER) and the Golgi complex, reduces ER and Golgi complex Ca(2+) levels, most likely by forming transmembrane pores, and inhibits protein trafficking through the Golgi complex. At present, little is known about the function of the other picornavirus 2B proteins. Here we show that rhinovirus 2B, which is phylogenetically closely related to enterovirus 2B, shows a similar subcellular localization and function to those of enterovirus 2B. In contrast, 2B proteins of hepatitis A virus, foot-and-mouth disease virus, and encephalomyocarditis virus, all of which are more distantly related to enteroviruses, show a different localization and have little, if any, effects on Ca(2+) homeostasis and intracellular protein trafficking. Our data suggest that the 2B proteins of enterovirus and rhinovirus share the same function in virus replication, while the other picornavirus 2B proteins support the viral life cycle in a different manner. Moreover, we show that an enterovirus 2B protein that is retained in the ER is unable to modify Ca(2+) homeostasis and inhibit protein trafficking, demonstrating the importance of Golgi complex localization for its functioning.

During enterovirus infection, host cell membranes are rigorously rearranged and modified. One ubiquitously expressed lipid-modifying enzyme that might contribute to these alterations is phospholipase D (PLD). Here, we investigated PLD activity in coxsackievirus-infected cells. We show that PLD activity is not required for efficient coxsackievirus RNA replication. Instead, PLD activity rapidly decreased upon infection and upon ectopic expression of the viral 3A protein, which inhibits the PLD activator ADP-ribosylation factor 1. However, similar decreases were observed during infection with coxsackieviruses carrying defective mutant 3A proteins. Possible causes for the reduction of PLD activity and the biological consequences are discussed.

The 3A protein of coxsackievirus B3 (CVB3), a small membrane protein that forms homodimers, inhibits endoplasmic reticulum (ER)-to-Golgi transport. Recently, we described the underlying mechanism by showing that the CVB3 3A protein binds to and inhibits the function of GBF1, a guanine-nucleotide exchange factor for ADP-ribosylation factor 1 (Arf1), thereby interfering with Arf1-mediated COP-I recruitment. This study was undertaken to gain more insight into the molecular determinants underlying the interaction between 3A and GBF1. Here, we show that 3A mutants that have lost the ability to dimerize are no longer able to bind to GBF1 and trap it on membranes. Moreover, we identify a conserved region in the N terminus of 3A that is crucial for GBF1 binding but not for 3A dimerization. Analysis of the binding domain in GBF1 showed that the extreme N terminus, the dimerization/cyclophilin binding (DCB) domain, as well as the homology upstream of Sec7 (HUS) domain are required for the interaction with 3A. In contrast to full-length GBF1, overexpression of a GBF1 mutant lacking its extreme N terminus failed to rescue the effects of 3A. Together, these data provide insight into the molecular requirements of the interaction between 3A and GBF1.

The 3A protein of the coxsackievirus B3 (CVB3), an enterovirus that belongs to the family of the picornaviruses, inhibits endoplasmic reticulum-to-Golgi transport. Recently, we elucidated the underlying mechanism by showing that CVB3 3A interferes with ADP-ribosylation factor 1 (Arf1)-dependent COP-I recruitment to membranes by binding and inhibiting the function of GBF1, a guanine nucleotide exchange factor that is required for the activation of Arf1 (E. Wessels et al., Dev. Cell 11:191-201, 2006). Here, we show that the 3A protein of poliovirus, another enterovirus, is also able to interfere with COP-I recruitment through the same mechanism. No interference with protein transport or COP-I recruitment was observed for the 3A proteins of any of the other picornaviruses tested here (human rhinovirus [HRV], encephalomyocarditis virus, foot-and-mouth disease virus, and hepatitis A virus). We show that the 3A proteins of HRV, which are the most closely related to the enteroviruses, are unable to inhibit COP-I recruitment, due to a reduced ability to bind GBF1. When the N-terminal residues of the HRV 3A proteins are replaced by those of CVB3 3A, chimeric proteins are produced that have gained the ability to bind GBF1 and, by consequence, to inhibit protein transport. These results show that the N terminus of the CVB3 3A protein is important for binding of GBF1 and its transport-inhibiting function. Taken together, our data demonstrate that the activity of the enterovirus 3A protein to inhibit GBF1-dependent COP-I recruitment is unique among the picornaviruses.

Many viruses modify cellular processes for their own benefit. The enterovirus 3A protein inhibits endoplasmic reticulum (ER)-to-Golgi transport, a function previously suggested to be important for viral suppression of immune responses. Here, we show that a virus carrying a 3A protein defective in inhibiting ER-to-Golgi transport is indeed less virulent in mice, and we unravel the mechanism by which 3A inhibits this trafficking step. Evidence is provided that 3A inhibits the activation of the GTPase ADP-ribosylation factor 1 (Arf1), which regulates the recruitment of the COP-I coat complex to membranes. 3A specifically inhibits the function of GBF1, a guanine nucleotide exchange factor for Arf1, by interacting with its N terminus. By specifically interfering with GBF1-mediated Arf1 activation, 3A may prove a valuable tool in dissecting the early steps of the secretory pathway.

The ability of the 3A protein of coxsackievirus B (CVB) to inhibit protein secretion was investigated for this study. Here we show that the ectopic expression of CVB 3A blocked the transport of both the glycoprotein of vesicular stomatitis virus, a membrane-bound secretory marker, and the alpha-1 protease inhibitor, a luminal secretory protein, at a step between the endoplasmic reticulum (ER) and the Golgi complex. CVB 3A contains a conserved proline-rich region in its N terminus. The importance of this proline-rich region was investigated by introducing Pro-to-Ala substitutions. The mutation of Pro19 completely abolished the ability of 3A to inhibit ER-to-Golgi transport. The mutation of Pro14, Pro17, or Pro20 also impaired this ability, but to a lesser extent. The mutation of Pro18 had no effect. We also investigated the possible importance of this proline-rich region for the function of 3A in viral RNA replication. To this end, we introduced the Pro-to-Ala mutations into an infectious cDNA clone of CVB3. The transfection of cells with in vitro-transcribed RNAs of these clones gave rise to mutant viruses that replicated with wild-type characteristics. We concluded that the proline-rich region in CVB 3A is required for its ability to inhibit ER-to-Golgi transport, but not for its function in viral RNA replication. The functional relevance of the proline-rich region is discussed in light of the proposed structural model of 3A.

The 2B protein of enterovirus is responsible for the alterations in the permeability of secretory membranes and the plasma membrane in infected cells. The structural requirements for the membrane association and the subcellular localization of this essential virus protein, however, have not been defined. Here, we provide evidence that the 2B protein is an integral membrane protein in vivo that is predominantly localized at the Golgi complex upon individual expression. Addition of organelle-specific targeting signals to the 2B protein revealed that the Golgi localization is an absolute prerequisite for the ability of the protein to modify plasma membrane permeability. Expression of deletion mutants and heterologous proteins containing specific domains of the 2B protein demonstrated that each of the two hydrophobic regions could mediate membrane binding individually. However, the presence of both hydrophobic regions was required for the correct membrane association, efficient Golgi targeting, and the membrane-permeabilizing activity of the 2B protein, suggesting that the two hydrophobic regions are cooperatively involved in the formation of a membrane-integral complex. The formation of membrane-integral pores by the 2B protein in the Golgi complex and the possible mechanism by which a Golgi-localized virus protein modifies plasma membrane permeability are discussed.

Picornaviruses contain stable RNA structures at the 5' and 3' ends of the RNA genome, OriL and OriR involved in viral RNA replication. The OriL RNA element found at the 5' end of the enterovirus genome folds into a cloverleaf-like configuration. In vivo SELEX experiments revealed that functioning of the poliovirus cloverleaf depends on a specific structure in this RNA element. Little is known about the OriL of cardioviruses. Here, we investigated structural aspects and requirements of the apical loop of proximal stem-loop SL-A of mengovirus, a strain of EMCV. Using NMR spectroscopy, we showed that the mengovirus SL-A apical loop consists of an octaloop. In vivo SELEX experiments demonstrated that a large number of random sequences are tolerated in the apical octaloop that support virus replication. Mutants in which the SL-A loop size and the length of the upper part of the stem were varied showed that both stem-length and stability of the octaloop are important determinants for viral RNA replication and virus reproduction. Together, these data show that stem-loop A plays an important role in virus replication. The high degree of sequence flexibility and the lack of selective pressure on the octaloop argue against a role in sequence specific RNA-protein or RNA-RNA interactions in which octaloop nucleotides are involved.

OBJECTIVE: The presence of the retrovirus xenotropic murine leukaemia virus-related virus (XMRV) has been reported in peripheral blood mononuclear cells of patients with chronic fatigue syndrome. Considering the potentially great medical and social relevance of such a discovery, we investigated whether this finding could be confirmed in an independent European cohort of patients with chronic fatigue syndrome. DESIGN: Analysis of a well defined cohort of patients and matched neighbourhood controls by polymerase chain reaction. SETTING: Certified (ISO 15189) laboratory of clinical virology in a university hospital in the Netherlands. Population Between December 1991 and April 1992, peripheral blood mononuclear cells were isolated from 76 patients and 69 matched neighbourhood controls. In this study we tested cells from 32 patients and 43 controls from whom original cryopreserved phials were still available. MAIN OUTCOME MEASURES: Detection of XMRV in peripheral blood mononuclear cells by real time polymerase chain reaction assay targeting the XMRV integrase gene and/or a nested polymerase chain reaction assay targeting the XMRV gag gene. RESULTS: We detected no XMRV sequences in any of the patients or controls in either of the assays, in which relevant positive and negative isolation controls and polymerase chain reaction controls were included. Spiking experiments showed that we were able to detect at least 10 copies of XMRV sequences per 10(5) peripheral blood mononuclear cells by real time as well as by nested polymerase chain reaction, demonstrating high sensitivity of both assays. CONCLUSIONS: This study failed to show the presence of XMRV in peripheral blood mononuclear cells of patients with chronic fatigue syndrome from a Dutch cohort. These data cast doubt on the claim that XMRV is associated with chronic fatigue syndrome in the majority of patients.

We report the use of threedimensional computational analysis of chloride channel 5 (ClC-5) based on a novel mutation, L266V, identified in a 15-year-old Japanese boy with Dent's disease. Since both leucine and valine are branched-chain amino acids, it has not been proved conclusively whether L266V mutation is actually responsible for the development of Dent's disease. In the present study using molecular analysis, we investigated the mechanism for loss of function of the ClC-5 protein resulting from the L266V mutation. Structural analysis of the normal ClC-5 transmembrane region using molecular modeling showed that the two respective Leu266 residues were located at the interface of the dimer formed by the aligned ClC-5 monomers. The Leu266 side-chains were positioned close to each other through hydrophobic interaction, resembling two interconnecting hooks. When Leu266 was replaced by a valine residue, the hydrophobic interaction between the CLC-5 monomers was reduced, and dimer formation was impaired. This computer simulation analysis has thus provided strong evidence for the important role of Leu266 in the dimerization of human ClC-5 in membranes. Conclusion: The finding of the present study suggest that computational modeling and molecular analysis could be an alternative to labor-intensive in vitro functional studies.

The arenavirus Z is a zinc-binding RING protein that has been implicated in multiple functions during the viral life cycle. These roles of Z involve interactions with viral and cellular proteins that remain incompletely understood. In this regard, Z inhibits viral RNA transcription and replication through direct interaction with the viral L polymerase. Here, we defined the L-binding domain of Tacaribe virus (TCRV) Z protein and the structural requirements mediating Z homo-oligomerization. By using site-directed mutagenesis, coimmunoprecipitation, and functional assays, we showed that residues R37, N39, W44, L50, and Y57, located around the zinc coordination site I, play a critical role in the Z-L interaction. We also found that Z protein from either TCRV or the pathogenic Junin virus (JUNV) self-associates into oligomeric forms in mammalian cells. Importantly, mutation of the myristoylation site, the strictly conserved residue G at position 2, severely impaired the ability of both TCRV Z and JUNV Z to self-interact as well as their capacity to accumulate at the plasma membrane, strongly suggesting that Z homo-oligomerization is associated with its myristoylation and cell membrane targeting. In contrast, disruption of the RING structure or substitution of W44 or N39, which are critical for L protein recognition, did not affect Z self-binding. Overall, the data presented here indicate that homo-oligomerization is not a requirement for Z-L interaction or Z-mediated polymerase activity inhibition.

C-terminal-binding protein (CtBP) is a well-characterized transcriptional co-repressor that requires homo-dimerization for its activity. CtBP can both repress and activate Wingless nuclear targets in Drosophila. Here, we examine the role of CtBP dimerization in these opposing processes. CtBP mutants that cannot dimerize are able to promote Wingless signalling, but are defective in repressing Wingless targets. To further test the role of dimerization in repression, the positions of basic and acidic residues that form inter-molecular salt bridges in the CtBP dimerization interface were swapped. These mutants cannot homo-dimerize and are compromised for repression. However, their co-expression leads to hetero-dimerization and consequent repression of Wingless targets. Our results support a model where CtBP is a gene-specific regulator of Wingless signalling, with some targets requiring CtBP dimers for inhibition while other targets utilize CtBP monomers for activation of their expression. Functional interactions between CtBP and Pygopus, a nuclear protein required for Wingless signalling, support a model where monomeric CtBP acts downstream of Pygopus in activating some Wingless targets.

Many gram-negative bacteria secrete specific proteins via the type II secretion systems (T2SS). These complex machineries share with the related archaeal flagella and type IV pilus (T4P) biogenesis systems the ability to assemble thin, flexible filaments composed of small, initially inner membrane-localized proteins called "pilins." In the T2SS from Klebsiella oxytoca, periplasmic pseudopili that are essential for pullulanase (PulA) secretion extend beyond the bacterial surface and form pili when the major pilin PulG is overproduced. Here, we describe the detailed, experimentally validated structure of the PulG pilus generated from crystallographic and electron microscopy data by a molecular modeling approach. Two intermolecular salt bridges crucial for function were demonstrated using single and complementary charge inversions. Double-cysteine substitutions in the transmembrane segment of PulG led to position-specific cross-linking of protomers in assembled pili. These biochemical data provided information on residue distances in the filament that were used to derive a refined model of the T2SS pilus at pseudoatomic resolution. PulG is organized as a right-handed helix of subunits, consistent with protomer organization in gonococcal T4P. The conserved character of residues involved in key hydrophobic and electrostatic interactions within the major pseudopilin family supports the general relevance of this model for T2SS pseudopilus structure.

ICP27 is a multifunctional protein that is required for herpes simplex virus 1 mRNA export. ICP27 interacts with the mRNA export receptor TAP/NXF1 and binds RNA through an RGG box motif. Unlike other RGG box proteins, ICP27 does not bind G-quartet structures but instead binds GC-rich sequences that are flexible in structure. To determine the contribution of arginines within the RGG box, we performed in vitro binding assays with N-terminal proteins encoding amino acids 1 to 160 of wild-type ICP27 or arginine-to-lysine substitution mutants. The R138,148,150K triple mutant bound weakly to sequences that were bound by the wild-type protein and single and double mutants. Furthermore, during infection with the R138,148,150K mutant, poly(A)(+) RNA and newly transcribed RNA accumulated in the nucleus, indicating that viral RNA export was impaired. To determine if structural changes had occurred, nuclear magnetic resonance (NMR) analysis was performed on N-terminal proteins consisting of amino acids 1 to 160 from wild-type ICP27 and the R138,148,150K mutant. This region of ICP27 was found to be highly flexible, and there were no apparent differences in the spectra seen with wild-type ICP27 and the R138,148,150K mutant. Furthermore, NMR analysis with the wild-type protein bound to GC-rich sequences did not show any discernible folding. We conclude that arginines at positions 138, 148, and 150 within the RGG box of ICP27 are required for binding to GC-rich sequences and that the N-terminal portion of ICP27 is highly flexible in structure, which may account for its preference for binding flexible sequences.

Substantial evidence has accumulated indicating a significant role for oligomerization in the function of E3 ubiquitin ligases. Among the many characterized E3 ligases, the yeast U-box protein Ufd2 and its mammalian homologue E4B appear to be unique in functioning as monomers. An E4B U-box domain construct (E4BU) has been subcloned, overexpressed in Escherichia coli, and purified, which enabled determination of a high-resolution NMR solution structure and detailed biophysical analysis. E4BU is a stable monomeric protein that folds into the same structure observed for other structurally characterized U-box domain homodimers. Multiple sequence alignment combined with comparative structural analysis reveals substitutions in the sequence that inhibit dimerization. The interaction between E4BU and the E2 conjugating enzyme UbcH5c has been mapped using NMR, and these data have been used to generate a structural model for the complex. The E2 binding site is found to be similar to that observed for dimeric U-box and RING domain E3 ligases. Despite the inability to dimerize, E4BU was found to be active in a standard autoubiquitination assay. The structure of E4BU and its ability to function as a monomer are discussed in light of the ubiquitous observation of U-box and RING domain oligomerization.

Membrane proteins account for nearly a quarter of all genes, but their structure and function remain incompletely understood. Most membrane proteins have transmembrane (TM) domains made up of bundles of hydrophobic alpha-helices. The lateral association of TM helices within the lipid bilayer is a key stage in the folding of membrane proteins. It may also play a role in signaling across cell membranes. Dimerization of TM helices is a simple example of such lateral association. Molecular dynamics (MD) simulations have been used for over a decade to study membrane proteins in a lipid bilayer environment. However, direct atomistic (AT) MD simulation of self-assembly of a TM helix bundle remains challenging. AT-MD may be complemented by coarse-grained (CG) simulations, in which small numbers of atoms are grouped together into particles. In this Account, we demonstrate how CG-MD may be used to simulate formation of dimers of TM helices. We also show how a serial combination of CG and AT simulation provides a multiscale approach for generating and refining models of TM helix dimers. The glycophorin A (GpA) TM helix dimer represents a paradigm for helix-helix packing, mediated by a GxxxG sequence motif. It is well characterized experimentally and so is a good test case for evaluating computational methods. CG-MD simulations in which two separate TM helices are inserted in a lipid bilayer result in spontaneous formation of a right-handed GpA dimer, in agreement with NMR structures. CG-MD models were evaluated via comparison with data on destabilizing mutants of GpA. Such mutants increased the conformational flexibility and the dissociation constants of helix dimers. GpA dimers have been used to evaluate a multiscale approach: A CG model is converted to an AT model, which is used as the basis of an AT-MD simulation. Comparison of three AT-MD simulations of GpA, one starting from a CG model and two starting from NMR structures, leads to convergence to a common refined structure for the dimer. CG-MD self-assembly has also been used to model dimerization of the TM domain of the syndecan-2 receptor protein. This TM helix contains a GxxxG motif, which mediates right-handed helix packing comparable to that of the GxxxG motif in GpA. The multiscale approach has been applied to a more complex system, the heterodimeric alphaIIb/beta3 integrin TM helix dimer. In CG-MD, both right-handed and left-handed structures were formed. Subsequent AT-MD simulations showed that the right-handed structure was more stable, yielding a dimer in which the GxxxG motif of the alphaIIb TM helix packed against a hydrophobic surface of the beta3 helix in a manner comparable to that observed in two recent NMR studies. This work demonstrates that the multiscale simulation approach can be used to model simple membrane proteins. The method may be applied to more complex proteins, such as the influenza M2 channel protein. Future refinements, such as extending the multiscale approach to a wider range of scales (from CG through QM/MM simulations, for example), will expand the range of applications and the accuracy of the resultant models.

Bile acid binding proteins (BABPs) are cytosolic lipid chaperones contributing to the maintenance of bile acid homeostasis and functional distribution within the cell. Liver BABPs act in parallel with ileal transporters to ensure vectorial transport of bile salts in hepatocytes and enterocytes, respectively. We describe the investigation of ligand binding to liver BABP, an essential step in the understanding of intracellular bile salt transport. Binding site occupancies were monitored in NMR titration experiments using (15)N-labelled ligand, while the relative populations of differently bound BABP forms were assessed by mass spectrometry. This site-specific information allowed the determination of intrinsic thermodynamic parameters and the identification of an extremely high cooperativity between two binding sites. Protein-observed NMR experiments revealed a global structural rearrangement which suggests an allosteric mechanism at the basis of the observed cooperativity. The view of a molecular tool capable of buffering against significant concentrations of free bile salts in a large range of solution conditions emerges from the observed pH-dependence of binding. We set to determine the molecular determinants of cooperativity by analysing the binding properties of a protein containing a mutated internal histidine. Both mass spectrometry and NMR experiments are consistent with an overall decreased binding affinity of the mutant, while the measured diffusion coefficients of ligand species reveal that the affinity loss concerns essentially one of the two binding sites. We therefore identified a mutation able to disrupt energetic communication functional to efficient binding and conclude that the buried histidine establishes contacts that stabilize the ternary complex. Proteins 2009.(c) 2009 Wiley-Liss, Inc.

The mechanistic understanding of how membrane-embedded sensor kinases recognize signals and regulate kinase activity is currently limited. Here we report structure-function relationships of the multidomain membrane sensor kinase DcuS using solid-state NMR, structural modeling and mutagenesis. Experimental data of an individual cytoplasmic Per-Arnt-Sim (PAS) domain were compared to structural models generated in silico. These studies, together with previous NMR work on the periplasmic PAS domain, enabled structural investigations of a membrane-embedded 40-kDa construct by solid-state NMR, comprising both PAS segments and the membrane domain. Structural alterations are largely limited to protein regions close to the transmembrane segment. Data from isolated and multidomain constructs favor a disordered N-terminal helix in the cytoplasmic domain. Mutations of residues in this region strongly influence function, suggesting that protein flexibility is related to signal transduction toward the kinase domain and regulation of kinase activity.

The second WW domain in mammalian Salvador protein (SAV1 WW2) is quite atypical, as it forms a beta-clam-like homodimer. The second WW domain in human MAGI1 (membrane associated guanylate kinase, WW and PDZ domain containing 1)(MAGI1 WW2) shares high sequence similarity with SAV1 WW2, suggesting comparable dimerization. However, an analytical ultracentrifugation study revealed that MAGI1 WW2 (Leu355-Pro390) chiefly exists as a monomer at low protein concentrations, with an association constant of 1.3 x 10(2) M(-1). We determined its solution structure, and a structural comparison with the dimeric SAV1 WW2 suggested that an Asp residue is crucial for the inhibition of the dimerization. The substitution of this acidic residue with Ser resulted in the dimerization of MAGI1 WW2. The spin-relaxation data suggested that the MAGI1 WW2 undergoes a dynamic process of transient dimerization that is limited by the charge repulsion. Additionally, we characterized a longer construct of this WW domain with a C-terminal extension (Leu355-Glu401), as the formation of an extra alpha-helix was predicted. An NMR structural determination confirmed the formation of an alpha-helix in the extended C-terminal region, which appears to be independent from the dimerization regulation. A thermal denaturation study revealed that the dimerized MAGI1 WW2 with the Asp-to-Ser mutation gained apparent stability in a protein concentration-dependent manner. A structural comparison between the two constructs with different lengths suggested that the formation of the C-terminal alpha-helix stabilized the global fold by facilitating contacts between the N-terminal linker region and the main body of the WW domain.