Increased association between Epstein-Barr virus EBNA2 from type 2 strains and the transcriptional repressor BS69 restricts EBNA2 activity.

Natural variation separates Epstein-Barr virus (EBV) into type 1 and type 2 strains. Type 2 EBV is less transforming in vitro due to sequence differences in the EBV transcription factor EBNA2. This correlates with reduced activation of the EBV oncogene LMP1 and some cell genes. Transcriptional activation by type 1 EBNA2 can be suppressed through the binding of two PXLXP motifs in its transactivation domain (TAD) to the dimeric coiled-coil MYND domain (CC-MYND) of the BS69 repressor protein (ZMYND11). We identified a third conserved PXLXP motif in type 2 EBNA2. We found that type 2 EBNA2 peptides containing this motif bound BS69CC-MYND efficiently and that the type 2 EBNA2TAD bound an additional BS69CC-MYND molecule. Full-length type 2 EBNA2 also bound BS69 more efficiently in pull-down assays. Molecular weight analysis and low-resolution structures obtained using small-angle X-ray scattering showed that three BS69CC-MYND dimers bound two molecules of type 2 EBNA2TAD, in line with the dimeric state of full-length EBNA2 in vivo. Importantly, mutation of the third BS69 binding motif in type 2 EBNA2 improved B-cell growth maintenance and the transcriptional activation of the LMP1 and CXCR7 genes. Our data indicate that increased association with BS69 restricts the function of type 2 EBNA2 as a transcriptional activator and driver of B cell growth and may contribute to reduced B-cell transformation by type 2 EBV.

Cross-species conservation of episome maintenance provides a basis for in vivo investigation of Kaposi's sarcoma herpesvirus LANA.

Many pathogens, including Kaposi's sarcoma herpesvirus (KSHV), lack tractable small animal models. KSHV persists as a multi-copy, nuclear episome in latently infected cells. KSHV latency-associated nuclear antigen (kLANA) binds viral terminal repeat (kTR) DNA to mediate episome persistence. Model pathogen murine gammaherpesvirus 68 (MHV68) mLANA acts analogously on mTR DNA. kLANA and mLANA differ substantially in size and kTR and mTR show little sequence conservation. Here, we find kLANA and mLANA act reciprocally to mediate episome persistence of TR DNA. Further, kLANA rescued mLANA deficient MHV68, enabling a chimeric virus to establish latent infection in vivo in germinal center B cells. The level of chimeric virus in vivo latency was moderately reduced compared to WT infection, but WT or chimeric MHV68 infected cells had similar viral genome copy numbers as assessed by immunofluorescence of LANA intranuclear dots or qPCR. Thus, despite more than 60 Ma of evolutionary divergence, mLANA and kLANA act reciprocally on TR DNA, and kLANA functionally substitutes for mLANA, allowing kLANA investigation in vivo. Analogous chimeras may allow in vivo investigation of genes of other human pathogens.

The Kaposi Sarcoma Herpesvirus Latency-associated Nuclear Antigen DNA Binding Domain Dorsal Positive Electrostatic Patch Facilitates DNA Replication and Episome Persistence.

Kaposi sarcoma-associated herpesvirus (KSHV) has a causative role in several human malignancies. KSHV latency-associated nuclear antigen (LANA) mediates persistence of viral episomes in latently infected cells. LANA mediates KSHV DNA replication and segregates episomes to progeny nuclei. The structure of the LANA DNA binding domain was recently solved, revealing a positive electrostatic patch opposite the DNA binding surface, which is the site of BET protein binding. Here we investigate the functional role of the positive patch in LANA-mediated episome persistence. As expected, LANA mutants with alanine or glutamate substitutions in the central, peripheral, or lateral portions of the positive patch maintained the ability to bind DNA by EMSA. However, all of the substitution mutants were deficient for LANA DNA replication and episome maintenance. Mutation of the peripheral region generated the largest deficiencies. Despite these deficiencies, all positive patch mutants concentrated to dots along mitotic chromosomes in cells containing episomes, similar to LANA. The central and peripheral mutants, but not the lateral mutants, were reduced for BET protein interaction as assessed by co-immunoprecipitation. However, defects in BET protein binding were independent of episome maintenance function. Overall, the reductions in episome maintenance closely correlated with DNA replication deficiencies, suggesting that the replication defects account for the reduced episome persistence. Therefore, the electrostatic patch exerts a key role in LANA-mediated DNA replication and episome persistence and may act through a host cell partner(s) other than a BET protein or by inducing specific structures or complexes.

KSHV but not MHV-68 LANA induces a strong bend upon binding to terminal repeat viral DNA.

Latency-associated nuclear antigen (LANA) is central to episomal tethering, replication and transcriptional regulation of γ2-herpesviruses. LANA binds cooperatively to the terminal repeat (TR) region of the viral episome via adjacent LANA binding sites (LBS), but the molecular mechanism by which LANA assembles on the TR remains elusive. We show that KSHV LANA and MHV-68 LANA proteins bind LBS DNA using strikingly different modes. Solution structure of LANA complexes revealed that while kLANA tetramer is intrinsically bent both in the free and bound state to LBS1-2 DNA, mLANA oligomers instead adopt a rigid linear conformation. In addition, we report a novel non-ring kLANA structure that displays more flexibility at its assembly interface than previously demonstrated. We identified a hydrophobic pivot point located at the dimer-dimer assembly interface, which gives rotational freedom for kLANA to adopt variable conformations to accommodate both LBS1-2 and LBS2-1-3 DNA. Alterations in the arrangement of LBS within TR or at the tetramer assembly interface have a drastic effect on the ability of kLANA binding. We also show kLANA and mLANA DNA binding functions can be reciprocated. Although KSHV and MHV-68 are closely related, the findings provide new insights into how the structure, oligomerization, and DNA binding of LANA have evolved differently to assemble on the TR DNA.

Crystal structure of human CRMP-4: correction of intensities for lattice-translocation disorder.

Collapsin response mediator proteins (CRMPs) are cytosolic phosphoproteins that are mainly involved in neuronal cell development. In humans, the CRMP family comprises five members. Here, crystal structures of human CRMP-4 in a truncated and a full-length version are presented. The latter was determined from two types of crystals, which were either twinned or partially disordered. The crystal disorder was coupled with translational NCS in ordered domains and manifested itself with a rather sophisticated modulation of intensities. The data were demodulated using either the two-lattice treatment of lattice-translocation effects or a novel method in which demodulation was achieved by independent scaling of several groups of intensities. This iterative protocol does not rely on any particular parameterization of the modulation coefficients, but uses the current refined structure as a reference. The best results in terms of R factors and map correlation coefficients were obtained using this new method. The determined structures of CRMP-4 are similar to those of other CRMPs. Structural comparison allowed the confirmation of known residues, as well as the identification of new residues, that are important for the homo- and hetero-oligomerization of these proteins, which are critical to nerve-cell development. The structures provide further insight into the effects of medically relevant mutations of the DPYSL-3 gene encoding CRMP-4 and the putative enzymatic activities of CRMPs.

Crystal structure of the gamma-2 herpesvirus LANA DNA binding domain identifies charged surface residues which impact viral latency.

Latency-associated nuclear antigen (LANA) mediates γ2-herpesvirus genome persistence and regulates transcription. We describe the crystal structure of the murine gammaherpesvirus-68 LANA C-terminal domain at 2.2 Šresolution. The structure reveals an alpha-beta fold that assembles as a dimer, reminiscent of Epstein-Barr virus EBNA1. A predicted DNA binding surface is present and opposite this interface is a positive electrostatic patch. Targeted DNA recognition substitutions eliminated DNA binding, while certain charged patch mutations reduced bromodomain protein, BRD4, binding. Virus containing LANA abolished for DNA binding was incapable of viable latent infection in mice. Virus with mutations at the charged patch periphery exhibited substantial deficiency in expansion of latent infection, while central region substitutions had little effect. This deficiency was independent of BRD4. These results elucidate the LANA DNA binding domain structure and reveal a unique charged region that exerts a critical role in viral latent infection, likely acting through a host cell protein(s).

Insights into the oligomerization of CRMPs: crystal structure of human collapsin response mediator protein 5.

Collapsin response mediator protein-5 (CRMP-5) is the latest identified member of the CRMP cytosolic phosphoprotein family, which is crucial for neuronal development and repair. CRMPs exist as homo- and/or hetero-tetramers in vivo and participate in signaling transduction, cytoskeleton rearrangements, and endocytosis. CRMP-5 antagonizes many of the other CRMPs' functions either by directly interacting with them or by competing for their binding partners. We determined the crystal structures of a full length and a truncated version of human CRMP-5, both of which form a homo-tetramer similar to those observed in CRMP-1 and CRMP-2. However, solution studies indicate that CRMP-5 and CRMP-1 form weaker homo-tetramers compared with CRMP-2, and that divalent cations, Ca(2+) and Mg(2+), destabilize oligomers of CRMP-5 and CRMP-1, but promote CRMP-2 oligomerization. On the basis of comparative analysis of the CRMP-5 crystal structure, we identified residues that are crucial for determining the preference for hetero-oligomer or homo-oligomer formation. We also show that in spite of being the CRMP family member most closely related to dihydropyrimidinase, CRMP-5 does not have any detectable amidohydrolase activity. The presented findings provide new detailed insights into the structure, oligomerization, and regulation of CRMPs.

Nonstructural proteins 7 and 8 of feline coronavirus form a 2:1 heterotrimer that exhibits primer-independent RNA polymerase activity.

Nonstructural proteins 7 and 8 of severe acute respiratory syndrome coronavirus (SARS-CoV) have previously been shown by X-ray crystallography to form an 8:8 hexadecamer. In addition, it has been demonstrated that N-terminally His(6)-tagged SARS-CoV Nsp8 is a primase able to synthesize RNA oligonucleotides with a length of up to 6 nucleotides. We present here the 2.6-Å crystal structure of the feline coronavirus (FCoV) Nsp7:Nsp8 complex, which is a 2:1 heterotrimer containing two copies of the α-helical Nsp7 with conformational differences between them, and one copy of Nsp8 that consists of an α/ß domain and a long-α-helix domain. The same stoichiometry is found for the Nsp7:Nsp8 complex in solution, as demonstrated by chemical cross-linking, size exclusion chromatography, and small-angle X-ray scattering. Furthermore, we show that FCoV Nsp8, like its SARS-CoV counterpart, is able to synthesize short oligoribonucleotides of up to 6 nucleotides in length when carrying an N-terminal His(6) tag. Remarkably, the same protein harboring the sequence GPLG instead of the His(6) tag at its N terminus exhibits a substantially increased, primer-independent RNA polymerase activity. Upon addition of Nsp7, the RNA polymerase activity is further enhanced so that RNA up to template length (67 nucleotides) can be synthesized. Further, we show that the unprocessed intermediate polyprotein Nsp7-10 of human coronavirus (HCoV) 229E is also capable of synthesizing oligoribonucleotides up to a chain length of six. These results indicate that in case of FCoV as well as of HCoV 229E, the formation of a hexadecameric Nsp7:Nsp8 complex is not necessary for RNA polymerase activity. Further, the FCoV Nsp7:Nsp8 complex functions as a noncanonical RNA polymerase capable of synthesizing RNA of up to template length.

X-ray structure of the SH3 domain of the phosphoinositide 3-kinase p85ß subunit.

Src-homology 3 (SH3) domains are involved in extensive protein-protein interactions and constitute key elements of intracellular signal transduction. Three-dimensional structures have been reported for SH3 domains of various proteins, including the 85 kDa regulatory subunit (p85) of phosphoinositide 3-kinase. However, all of the latter structures are of p85 isoform α and no crystal structure of the SH3 domain of the equally important isoform ß has been reported to date. In this structural communication, the recombinant production, crystallization and X-ray structure determination at 2.0 Å resolution of the SH3 domain of human p85ß is described. The structure reveals a compact ß-barrel fold very similar to that of p85α. However, binding studies with two classes of proline-rich ligand peptides demonstrate that the ligand-binding specificity differs slightly between the SH3 domains of human p85ß and p85α, despite their high structural similarity.

Non-invasive predictors of esophageal varices.

BACKGROUND/AIM: Current guidelines recommend screening cirrhotic patients with an endoscopy to detect esophageal varices and to institute prophylactic measures in patients with large esophageal varices. In this study, we aimed at identifying non-endoscopic parameters that could predict the presence and grades of esophageal varices. PATIENTS AND METHODS: In a prospective study, 229 newly diagnosed patients with liver cirrhosis, without a history of variceal bleeding, were included. Demographic, clinical, biochemical and ultrasonographic parameters were recorded. Esophageal varices were classified as small and large, at endoscopy. Univariate analysis and multivariate logistic regression analysis were done to identify independent predictors for the presence and grades of varices. RESULTS: Of the 229 patients (141 males; median age 42 years; range 17-73 years) with liver cirrhosis, 97 (42.3%) had small and 81 (35.4%) had large varices. On multivariate analysis, low platelet count (Odd's Ratio [OR], 4.3; 95% confidence interval [CI], 1.2-14.9), Child Pugh class B/C (OR, 3.3; 95% CI, 1.8-6.3), spleen diameter (OR, 4.3; 95% CI, 1.6-11.9) and portal vein diameter (OR, 2.4; 95% CI, 1.1-5.3) were independent predictors for the presence of varices. Likewise, for the presence of large esophageal varices, low platelet count (OR, 2.7; 95% CI, 1.4-5.2), Child Pugh class B/C (OR, 3.8; 95% CI, 2.3-6.5) and spleen diameter (OR, 3.1; 95% CI, 1.6-6.0) were the independent risk factors. CONCLUSION: The presence and higher grades of varices can be predicted by a low platelet count, Child-Pugh class B/C and spleen diameter. These may be considered as non-endoscopic predictors for the diagnosis and management of large grade varices.

Is balloon sweeping detrimental in portal biliopathy? A report of 3 cases.

BACKGROUND: Portal biliopathy is characterized by biliary obstruction and is commonly caused by dilated vascular channels in the pericholedochal plexus causing choledochal varices, leading to the formation of stones in the common bile duct. Endoscopic treatment leads to severe bleeding complications because of the rupture of these vascular channels. OBJECTIVE: We report on our management of the unique bleeding complications we faced when performing endotherapy on these patients. DESIGN: A case series. SETTING: Single-center experience in a tertiary care unit. PATIENTS AND INTERVENTIONS: Patients with portal hypertension and choledochal varices, presenting with symptoms of biliary obstruction caused by gallstones, were subjected to ERCP and stone removal by use of the balloon sweeping and basket extraction techniques. LIMITATION: Small volume case series; there is need for a randomized controlled study. CONCLUSION: During stone removal, when compared to basket extraction, excessive bleeding was observed when balloon sweeping was used because this technique could cause squeezing of the choledochal varices.

Carbon-nanotube-based materials for protein crystallization.

We report on the first use of carbon-nanotube-based films to produce crystals of proteins. The crystals nucleate on the surface of the film. The difficulty of crystallizing proteins is a major bottleneck in the determination of the structure and function of biological molecules. The crystallization of two model proteins and two medically relevant proteins was studied. Quantitative data on the crystallization times of the model protein lysozyme are also presented. Two types of nanotube films, one made with the surfactant Triton X-100 (TX-100) and one with gelatin, were tested. Both induce nucleation of the crystal phase at supersaturations at which the protein solution would otherwise remain clear; however, the gelatin-based film induced nucleation down to much lower supersaturations for the two model proteins with which it was used. It appears that the interactions of gelatin with the protein molecules are particularly favorable to nucleation. Crystals of the C1 domain of the human cardiac myosin-binding protein-C that diffracted to a resolution of 1.6 A were obtained on the TX-100 film. This is far superior to the best crystals obtained using standard techniques, which only diffracted to 3.0 A. Thus, both of our nanotube-based films are very promising candidates for future work on crystallizing difficult-to-crystallize target proteins.

Production of coronavirus nonstructural proteins in soluble form for crystallization.

For biophysical investigations on viral proteins, in particular for structure determination by X-ray crystallography, relatively large quantities of purified protein are necessary. However, expression of cDNAs coding for viral proteins in prokaryotic or eukaryotic systems is often not straightforward, and frequently the amount and/or the solubility of the protein obtained are not sufficient. Here, we describe a number of protocols for production of nonstructural proteins of coronaviruses that have proven to be efficient in increasing expression yields or solubilities.

Variable oligomerization modes in coronavirus non-structural protein 9.

Non-structural protein 9 (Nsp9) of coronaviruses is believed to bind single-stranded RNA in the viral replication complex. The crystal structure of Nsp9 of human coronavirus (HCoV) 229E reveals a novel disulfide-linked homodimer, which is very different from the previously reported Nsp9 dimer of SARS coronavirus. In contrast, the structure of the Cys69Ala mutant of HCoV-229E Nsp9 shows the same dimer organization as the SARS-CoV protein. In the crystal, the wild-type HCoV-229E protein forms a trimer of dimers, whereas the mutant and SARS-CoV Nsp9 are organized in rod-like polymers. Chemical cross-linking suggests similar modes of aggregation in solution. In zone-interference gel electrophoresis assays and surface plasmon resonance experiments, the HCoV-229E wild-type protein is found to bind oligonucleotides with relatively high affinity, whereas binding by the Cys69Ala and Cys69Ser mutants is observed only for the longest oligonucleotides. The corresponding mutations in SARS-CoV Nsp9 do not hamper nucleic acid binding. From the crystal structures, a model for single-stranded RNA binding by Nsp9 is deduced. We propose that both forms of the Nsp9 dimer are biologically relevant; the occurrence of the disulfide-bonded form may be correlated with oxidative stress induced in the host cell by the viral infection.

Peripancreatic tuberculous lymphadenopathy. An impostor posing diagnostic difficulty.

CONTEXT: Involvement of the peripancreatic lymph nodes by tuberculosis is rare and the clinical presentation varies. It can mimic cystic neoplasms of the pancreas. CASE REPORT: We report the case of a patient having peripancreatic tuberculosis who presented with fever, jaundice, supraclavicular lymphadenopathy and weight loss. Imaging and tumor markers also suggested a pancreatic malignancy. MRI helped to identify the peripancreatic lymph node involvement and biopsy of the supraclavicular lymph node clinched the diagnosis. CONCLUSION: Pancreatic and peripancreatic tuberculosis should be considered in the differential diagnosis of cystic lesions of the head of the pancreas.