VISP: Center for Viral Infection Structural Proteomics: VISPCollaboratorPublications

Togaviridae: the viruses and their replication.

Sophie Coon — Fri, 02 Mar 2007 01:32:33 GMT

Authors: Kuhn R, et al.

Abstract:

Journal: In Fields Virology. D. M. Knipe and P. M. Howley (eds), Lippincott Williams & Wilkins

Date: 12/30/2006

Reference:

Kuhn RJ. Togaviridae: the viruses and their replication. In Fields Virology. D. M. Knipe and P. M. Howley (eds), Lippincott Williams & Wilkins. (2006) in press.

Keyword: VISP,FLAVI

Structural changes of bacteriophage phi29 upon DNA packaging and release

Sophie Coon — Fri, 02 Mar 2007 01:32:33 GMT

Authors: Xiang Y, et al.

Abstract:

Cryo-electron microscopy three-dimensional reconstructions have been made of mature and of emptied bacteriophage phi29 particles without making symmetry assumptions. Comparisons of these structures with each other and with the phi29 prohead indicate how conformational changes might initiate successive steps of assembly and infection. The 12 adsorption capable 'appendages' were found to have a structure homologous to the bacteriophage P22 tailspikes. Two of the appendages are extended radially outwards, away from the long axis of the virus, whereas the others are around and parallel to the phage axis. The appendage orientations are correlated with the symmetry-mismatched positions of the five-fold related head fibers, suggesting a mechanism for partial cell wall digestion upon rotation of the head about the tail when initiating infection. The narrow end of the head-tail connector is expanded in the mature virus. Gene product 3, bound to the 5' ends of the genome, appears to be positioned within the expanded connector, which may potentiate the release of DNA-packaging machine components, creating a binding site for attachment of the tail.

Journal: EMBO

Date: 11/1/2006

Link: PMID: 17053784

Reference:

Xiang Y, Morais MC, Battisti AJ, Grimes S, Jardine PJ, Anderson DL, Rossmann MG. Structural changes of bacteriophage phi29 upon DNA packaging and release. EMBO J. 25(21):5229-39 (2006).

PMID: PMID: 17053784

Keyword: VISP

Evolution of bacteriophage tails: structure of T4 gene product 10.

Sophie Coon — Fri, 02 Mar 2007 01:32:33 GMT

Authors: Leiman PG

Abstract:

The success of tailed bacteriophages to infect cells far exceeds that of most other viruses on account of their specialized tail and associated baseplate structures. The baseplate protein gene product (gp) 10 of bacteriophage T4, whose structure was determined to 1.2 A resolution, was fitted into the cryo-electron microscopy structures of the pre and post-infection conformations of the virus. gp10 functions as a molecular lever that rotates and extends the hinged short tail fibers to facilitate cell attachment. The central folding motif of the gp10 trimer is similar to that of the baseplate protein gp11 and to the receptor-binding domain of the short tail fiber, gp12. The three proteins comprise the periphery of the baseplate and interact with each other. The structural and functional similarities of gp10, gp11, and gp12 and their sequential order in the T4 genome suggest that they evolved separately, subsequent to gene triplication from a common ancestor. Such events are usual in the evolution of complex organelles from a common primordial molecule.

Journal: J Mol Biol.

Date: 5/5/2006

Link: PMID: 16554069

Reference:

Leiman, P. G., M. M. Shneider, V. V. Mesyanzhinov, M. G. Rossmann. 2006. Evolution of bacteriophage tails: structure of T4 gene product 10. J. Mol. Biol. 358:912-921.

PMID: 16554069

Keyword: VISP

The structure of bovine viral diarrhea virus RNA-dependent RNA polymerase and its amino-terminal domain

Sophie Coon — Fri, 02 Mar 2007 01:32:33 GMT

Authors: Choi KH, et al.

Abstract:

Viral RNA-dependent RNA polymerases (RdRp) differ from DNA-dependent RNA polymerases, DNA-dependent DNA polymerases, and reverse transcriptases in that RdRps contain "fingertips" consisting of several polypeptide strands in the fingers domain interacting with the thumb domain. The crystal structure of bovine viral diarrhea virus (BVDV) RdRp containing an Asn438 duplication shows that the "N-terminal domain," which occurs only in pestiviruses such as BVDV, interacts with the polymerase component of the same polypeptide chain. This contrasts with the domain swapping observed in the previously determined structure of the BVDV NADL strain RdRp. By comparison with the NADL structure and through the use of biochemical data, it is possible that the N-terminal domain, in conjunction with the fingertips, is required to bind and assist the translocation of the RNA template. The partial disorder of the loop containing the additional Asn438 residue may explain the low replication rate of the recombinant compared with the wild-type virus.

Journal: Structure

Date: 7/1/2006

Link: PMID: 16843892

Reference:

Choi, K. H., A. Gallei, P. Becher, M. G. Rossmann. The structure of bovine viral diarrhea virus RNA-dependent RNA polymerase and its amino-terminal domain. Structure 14: 1107-1113. (2006)

PMID: 16843892

Keyword: VISP

Cryo-EM structure of a bacteriophage T4 gp24 bypass mutant: the evolution of pentameric vertex proteins in icosahedral viruses

Sophie Coon — Fri, 02 Mar 2007 01:32:33 GMT

Authors: Fokine A, et al.

Abstract:

Many large viral capsids require special pentameric proteins at their fivefold vertices. Nevertheless, deletion of the special vertex protein gene product 24 (gp24) in bacteriophage T4 can be compensated by mutations in the homologous major capsid protein gp23. The structure of such a mutant virus, determined by cryo-electron microscopy to 26 angstroms, shows that the gp24 pentamers are replaced by mutant major capsid protein (gp23) pentamers at the vertices, thus re-creating a viral capsid prior to the evolution of specialized major capsid proteins and vertex proteins. The mutant gp23* pentamer is structurally similar to the wild-type gp24* pentamer but the insertion domain is slightly more distant from the gp23* pentamer center. There are additional SOC molecules around the gp23* pentamers in the mutant virus that were not present around the gp24* pentamers in the wild-type virus.

Journal: J Struct Biol.

Date: 6/1/2006

Link: PMID: 16530424

Reference:

Fokine, A., A. J. Battisti, V. A. Kostyuchenko, L. W. Black, M. G. Rossmann. Cryo-EM structure of a bacteriophage T4 gp24 bypass mutant: the evolution of pentameric vertex proteins in icosahedral viruses. J. Struct. Biol. 154:255-259. (2006)

PMID: 16530424

Keyword: VISP

Human cytomegalovirus attenuates TLR/IL1 and TNF proinflammatory signaling by independent blockade of NF-kB activation.

Sophie Coon — Fri, 02 Mar 2007 01:32:33 GMT

Authors: Jarvis MA, et al.

Abstract:

Viral infection is associated with a vigorous inflammatory response characterized by cellular infiltration and release of the proinflammatory cytokines interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF-alpha). In the present study, we identified a novel function of human cytomegalovirus (HCMV) that results in inhibition of IL-1 and TNF-alpha signaling pathways. The effect on these pathways was limited to cells infected with the virus, occurred at late times of infection, and was independent of cell type or virus strain. IL-1 and TNF-alpha signaling pathways converge at a point upstream of NF-kappaB activation and involve phosphorylation and degradation of the NF-kappaB inhibitory molecule IkappaBalpha. The HCMV inhibition of IL-1 and TNF-alpha pathways corresponded to a suppression of NF-kappaB activation. Analysis of IkappaBalpha phosphorylation and degradation suggested that HCMV induced two independent blocks in NF-kappaB activation, which occurred upstream from the point of convergence of the IL-1 and TNF-alpha pathways. We believe that the ability of HCMV to inhibit these two major proinflammatory pathways reveals a critical aspect of HCMV biology, with possible importance for immune evasion, as well as establishment of infection in cell types persistently infected by this virus.

Journal: J. Virol.

Date: 6/1/2006

Link: PMID: 16699040

Reference:

Jarvis M.A., J.A. Borton, A.M. Keech, J. Wong, W. J. Britt, B. E. Magun, and J.A. Nelson. Human cytomegalovirus attenuates TLR/IL1 and TNFa proinflammatory signaling by independent blockade of NF-kB activation. J Virol. 80(11):5588-98, (2006).

PMID: 16699040

Keyword: VISP, KSHV

Interferon regulatory factor 3 is necessary for induction of antiviral genes during human cytomegalovirus infection.

Sophie Coon — Fri, 02 Mar 2007 01:32:33 GMT

Authors: DeFilippis VR, et al.

Abstract:

Viral infection activates interferon regulatory factor 3 (IRF3), a cofactor for the induction of interferon-stimulated genes (ISGs). The role of IRF3 in the activation of ISGs by human cytomegalovirus (HCMV) is controversial despite the fact that HCMV has consistently been shown to induce ISGs during infection of fibroblasts. To address the function of IRF3 in HCMV-mediated ISG induction, we monitored ISG expression and global gene expression in HCMV-infected cells in which IRF3 function had been depleted by small interfering RNA or blocked by dominant negative IRF3. A specific reduction of ISG induction was observed, whereas other transcripts were unaffected. We therefore conclude that IRF3 specifically regulates ISG induction during the initial phase of HCMV infection.

Journal: J Virol.

Date: 1/1/2006

Link: PMID: 16379004

Reference:

DeFillipis, V. R., B Robinson, T. M.Keck, S. G. Hansen, J. A. Nelson, K. J. Frueh. Interferon regulatory factor 3 is necessary for induction of antiviral genes during human cytomegalovirus infection. J Virol 80:1032-7. (2006)

PMID: 16379004

Keyword: VISP, KSHV

Human Cytomegaloviruses lacking UL128-150 cannot enter epithelial cells and have an additional defect in early stages of infecting endothelial cells

Sophie Coon — Fri, 02 Mar 2007 01:32:33 GMT

Authors: Ryckman M, et al.

Abstract:

Human cytomegalovirus (HCMV) replication in epithelial and endothelial cells appears to be important in virus spread, disease, and persistence. It has been difficult to study infection of these cell types because HCMV laboratory strains (e.g., AD169 and Towne) have lost their ability to infect cultured epithelial and endothelial cells during extensive propagation in fibroblasts. Clinical strains of HCMV (e.g., TR and FIX) possess a cluster of genes (UL128 to UL150) that are largely mutated in laboratory strains, and recent studies have indicated that these genes facilitate replication in epithelial and endothelial cells. The mechanisms by which these genes promote infection of these two cell types are unclear. We derived an HCMV UL128-to-UL150 deletion mutant from strain TR, TRdelta4, and studied early events in HCMV infection of epithelial and endothelial cells, and the role of genes UL128 to UL150. Analysis of wild-type TR indicated that HCMV enters epithelial and endothelial cells by endocytosis followed by low-pH-dependent fusion, which is different from the pH-independent fusion with the plasma membrane observed with human fibroblasts. TRdelta4 displayed a number of defects in early infection processes. Adsorption and entry of TRdelta4 on epithelial cells were poor compared with those of TR, but these defects could be overcome with higher doses of virus and the use of polyethylene glycol (PEG) to promote fusion between virion and cellular membranes. High multiplicity and PEG treatment did not promote infection of endothelial cells by TRdelta4, yet virus particles were internalized. Together, these data indicate that genes UL128 to UL150 are required for HCMV adsorption and penetration of epithelial cells and to promote some early stage of virus replication, subsequent to virus entry, in endothelial cells.

Journal: J. Virol.

Date: 1/1/2006

Link: PMID: 16378974

Reference:

Ryckman, M Jarvis, D. Drummond, J.A. Nelson, and D. C. Johnson. Human Cytomegaloviruses lacking UL128-150 cannot enter epithelial cells and have an additional defect in early stages of infecting endothelial cells. J Virol. 80:710-22 (2006)

PMID: 16378974

Keyword: VISP, KSHV

Structural biology: Antiviral drugs fit for a purpose

Sophie Coon — Fri, 02 Mar 2007 01:32:33 GMT

Authors: Luo M.

Abstract:

Journal: Nature

Date: 9/7/2006

Link: PMID: 16915238

Reference:

Luo M. Structural biology: Antiviral drugs fit for a purpose. Nature. 2006. 443:37-38 (2006)

PMID: 16915238

Keyword: VISP, POX

Structure of the vesicular stomatitis virus nucleoprotein-RNA complex.

Sophie Coon — Fri, 02 Mar 2007 01:32:33 GMT

Authors: Green TJ, et al.

Abstract:

Vesicular stomatitis virus is a negative-stranded RNA virus. Its nucleoprotein (N) binds the viral genomic RNA and is involved in multiple functions including transcription, replication, and assembly. We have determined a 2.9 angstrom structure of a complex containing 10 molecules of the N protein and 90 bases of RNA. The RNA is tightly sequestered in a cavity at the interface between two lobes of the N protein. This serves to protect the RNA in the absence of polynucleotide synthesis. For the RNA to be accessed, some conformational change in the N protein should be necessary.

Journal: Science

Date: 7/21/2006

Link: PMID: 16778022

Reference:

Green TJ, Zhang X, Wertz GW, Luo M. “Structure of the vesicular stomatitis virus nucleoprotein-RNA complex”. Science. 2006 Jul 21;313(5785):357-60 (2006)

PMID: 16778022

Keyword: VISP, POX

Purification, crystallization and preliminary X-ray crystallographic analysis of the nucleocapsid protein of Bunyamwera virus

Sophie Coon — Fri, 02 Mar 2007 01:32:33 GMT

Authors: Rogers JW, et al.

Abstract:

Bunyamwera virus (BUNV) is the prototypic member of the Bunyaviridae family of segmented negative-sense RNA viruses. The BUNV nucleocapsid protein has been cloned and expressed in Escherichia coli. The purified protein has been crystallized and a complete data set has been collected to 3.3 angstroms resolution at a synchrotron source. Crystals of the nucleocapsid protein belong to space group C2, with unit-cell parameters a = 384.7, b = 89.8, c = 89.2 angstroms, beta = 94.4 degrees . Self-rotation function analysis of the X-ray diffraction data has provided insight into the oligomeric state of the protein as well as the orientation of the oligomers in the asymmetric unit. The structure determination of the protein is ongoing.

Journal: Acta Crystallograph Sect F Struct Biol Cryst Commun

Date: 1/4/2006

Link: PMID: 16582485

Reference:

Rogers JW, Zhou Q, Green TJ, Barr JN, and Luo M. “Purification, crystallization and preliminary X-ray crystallographic analysis of the nucleocapsid protein of Bunyamwera virus”. Acta Cryst. 2006 F62:361-364. (2006)

PMID: 16582485

Keyword: VISP, POX

Chapter 17: Flaviviruses

Sophie Coon — Fri, 02 Mar 2007 01:32:33 GMT

Authors: Kuhn P

Abstract:

Journal: In Fundamentals of Molecular Virology. N. A. Acheson (ed.)

Date: 2/28/2007

Reference:

Kuhn RJ. Chapter 17: Flaviviruses. In Fundamentals of Molecular Virology. N. A. Acheson (ed.), pp. 181-190. John Wiley & Sons, Inc. (2006)

Keyword: VISP, FLAVI

Resolution improvement of X-ray diffraction data of crystals of a vesicular stomatitis virus nucleocapsid protein oligomer complexed with RNA

Sophie Coon — Fri, 02 Mar 2007 01:32:33 GMT

Authors: Green TJ, et al.

Abstract:

Vesicular stomatitis virus (VSV) is a non-segmented negative-stranded RNA virus. The nucleocapsid (N) protein of VSV is found tightly associated with the viral genomic RNA and this complex serves as the template for transcription and replication. A method for the soluble expression of the N protein in Escherichia coli has previously been reported. An N protein-RNA oligomer was isolated from this system, the stoichiometry of which was determined to be ten molecules of the N protein bound to approximately 90 nucleotides of RNA. Here, the crystallization of this protein-nucleic acid complex is presented. The crystals belong to space group P2(1)2(1)2, with unit-cell parameters a = 165.65, b = 235.35, c = 75.71 A and a diffraction limit of 6 A. Self-rotation function analysis has shown the oligomer to have tenfold rotational symmetry. In a search to identify heavy-atom derivatives, uranyl acetate was discovered to stabilize the crystals, giving them an increase in diffraction limits to beyond 2.9 A. Based on the internal symmetry of the oligomer, the size of the oligomer determined previously by negative-stained electron microscopy, the space-group symmetry and packing considerations, the packing arrangement in the crystal has been determined.

Journal: Acta Crystallogr D Biol Crystallogr.

Date: 5/1/2006

Link: PMID: 16627942

Reference:

Green TJ, Luo M. “Resolution improvement of X-ray diffraction data of crystals of a vesicular stomatitis virus nucleocapsid protein oligomer complexed with RNA”. Acta Ccryst. 2006 D62:498-504. (2006)

PMID: 16627942

Keyword: VISP, POX

Crystal structure of the oligomerization domain of the phosphoprotein of vesicular stomatitis virus

Sophie Coon — Fri, 02 Mar 2007 01:32:33 GMT

Authors: Ding H, et al.

Abstract:

In the replication cycle of nonsegmented negative-strand RNA viruses, the viral RNA-dependent RNA polymerase (L) recognizes a nucleoprotein (N)-enwrapped RNA template during the RNA polymerase reaction. The viral phosphoprotein (P) is a polymerase cofactor essential for this recognition. We report here the 2.3-angstroms-resolution crystal structure of the central domain (residues 107 to 177) of P from vesicular stomatitis virus. The fold of this domain consists of a beta hairpin, an alpha helix, and another beta hairpin. The alpha helix provides the stabilizing force for forming a homodimer, while the two beta hairpins add additional stabilization by forming a four-stranded beta sheet through domain swapping between two molecules. This central dimer positions the N- and C-terminal domains of P to interact with the N and L proteins, allowing the L protein to specifically recognize the nucleocapsid-RNA template and to progress along the template while concomitantly assembling N with nascent RNA. The interdimer interactions observed in the noncrystallographic packing may offer insight into the mechanism of the RNA polymerase processive reaction along the viral nucleocapsid-RNA template.

Journal: J. Virol.

Date: 3/1/2006

Link: PMID: 16501089

Reference:

Ding H, Green TJ, Lu S, and Luo M. “Crystal structure of the oligomerization domain of the phosphoprotein of vesicular stomatitis virus”. J. Virol. 80(6):2808-14 (2006)

PMID: 16501089

Keyword: VISP, POX

Production of a novel class of polyreactive pathogenic autoantibodies in BXD2 mice causes glomerulonephritis and arthritis

Sophie Coon — Fri, 02 Mar 2007 01:32:33 GMT

Authors: Hsu HC, et al.

Abstract:

OBJECTIVE: The BXD2 mouse strain spontaneously develops glomerulonephritis and erosive arthritis. The goal of this study was to identify the antigenic target proteins and epitopes and to unravel the mechanisms by which the related conditions arise in BXD2 mice. METHODS: Individual hybridomas isolated from the spleen of a 10-month-old BXD2 mouse were injected intraperitoneally into nonautoimmune mice for evaluation of pathogenicity of each autoantibody. Autoantigens were immunoprecipitated with the pathogenic autoantibody L3A4. Autoantigens were identified using enzyme-linked immunosorbent assay, Western blotting, 2-dimensional gel electrophoresis, and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MS) and tandem MS. Antigenic epitopes were determined using a high-throughput epitope mapping method. RESULTS: The production of autoantibodies in BXD2 mice occurred in an orderly progression, with peak levels of autoantibodies to nitrotyrosine (NT)-modified enolase, Ro, alpha-actin, and heat-shock proteins (HSPs) preceding peak levels of antihistone, anti-DNA, and rheumatoid factor. Two monoclonal autoantibodies, L3A4 and T56G10, were identified that could induce immune complexes, renal disease, and/or arthritis. Both L3A4 and T56G10 were polyreactive, and each reacted with separate sets of autoantigens. The antigenic targets of L3A4 consisted of NT-modified enolase, ATP5b, alpha-actin, and Hsp70 family proteins including Hspa5 and Hsp74. The antigenic epitopes of NT-modified enolase and Hspa5 exhibited sequence homology and cross-reactivity, suggesting that epitope spreading may occur through a molecular mimicry mechanism. CONCLUSION: The polyreactivity of autoantibodies that target a novel class of autoantigens may enable these autoantibodies to induce erosive arthritis or glomerulonephritis either by direct pathogenic mechanisms or indirectly via Fc or immune complex deposition.

Journal: Arthritis Rheum.

Date: 2/28/2007

Link: PMID: 16385526

Reference:

Hsu HC, Zhou T, Kim H, Barnes S, Yang P, Wu Q, Zhou J, Freeman BA, Luo M, Mountz JD. Production of a novel class of polyreactive pathogenic autoantibodies in BXD2 mice causes glomerulonephritis and arthritis. Arthritis Rheum. 54(1):343-55 (2006)

PMID: 16385526

Keyword: VISP, POX

SGCEdb: a flexible database and web interface integrating experimental results and analysis for structural genomics focusing on Caenorhabditis elegans

Sophie Coon — Fri, 02 Mar 2007 01:32:33 GMT

Authors: Johnson DH, et al.

Abstract:

The SGCEdb (http://sgce.cbse.uab.edu) database/interface serves the primary purpose of reporting progress of the Structural Genomics of Caenorhabditis elegans project at the University of Alabama at Birmingham. It stores and analyzes results of experiments ranging from solubility screening arrays to individual protein purification and structure solution. External databases and algorithms are referenced and evaluated for target selection in the human, C.elegans and Pneumocystis carinii genomes. The flexible and reusable design permits tracking of standard and custom experiment types in a scientist-defined sequence. The database coordinates efforts between collaborators and is adaptable to a wide range of biological applications.

Journal: Nucleic Acids Res.

Date: 1/1/2006

Link: PMID: 16381914

Reference:

Johnson DH, Tsao J, Luo M, Carson M. “SGCEdb: a flexible database and web interface integrating experimental results and analysis for structural genomics focusing on Caenorhabditis elegans”. Nucleic Acids Res. 1;34(Database issue):D471-4. (2006)

PMID: 16381914

Keyword: VISP, POX

Soluble expression, purification, and stabilization of a pro-apoptotic human protein, CARP.

Sophie Coon — Fri, 02 Mar 2007 01:32:33 GMT

Authors: Chen Q, et al.

Abstract:

CARP is a novel pro-apoptotic protein that has been cloned and characterized in our previous report. Previous studies showed that suppression of CARP expression results in cell proliferation in several mammalian cell lines and over-expression of CARP leads to apoptosis and inhibition of proliferation in seven tumor cell lines [Liu et al., CARP is a novel caspase recruitment domain containing pro-apoptotic protein, Biochem. Biophys. Res. Commun. 293 (2002) 1396]. To obtain soluble and active form of CARP protein for further functional and structural studies, we have expressed CARP in Escherichia coli by using Gateway cloning system. Optimal induction and expression conditions were also studied. Recombinant histidine-tagged CARP was expressed in E. coli when the carp gene was subcloned into a Gateway expression vector pET21-DEST. The partially soluble recombinant CARP protein was purified to near homogeneity by a two-step FPLC procedure, first by Ni2+ affinity chromatography followed by a gel-filtration chromatography, which yielded about 10 mg protein/L culture with at least 95% purity. Two peaks were detected in the analytical gel-filtration chromatograph while only one peak corresponding to monomer of the CARP protein was left after adding 2 mM dithiothreitol (DTT). The polymers observed are likely due to the formation of intermolecular disulfide bridges. These results suggest that adding DTT is a good solution to prevent the formation of disulfide bonds and to stabilize the protein. Successfully growing crystals of the purified CARP protein also proved that we can produce well folded CARP protein in E. coli.

Journal: Protein Expr Purif.

Date: 2/1/2006

Link: PMID: 16139514

Reference:

Chen Q, Hui R, Sun C, Gu X, Luo M, Zheng X. “Soluble expression, purification, and stabilization of a pro-apoptotic human protein, CARP”. Protein Expr Purif. 45:329-334. (2006)

PMID: 16139514

Keyword: VISP, POX

Catalytic core of alphavirus nonstructural protein nsP4 possesses terminal adenylytransferase activity.

Sophie Coon — Fri, 02 Mar 2007 01:32:33 GMT

Authors: Tomar S, et al.

Abstract:

The RNA-dependent RNA polymerase nsP4 is an integral part of the alphavirus replication complex. To define the role of nsP4 in viral RNA replication and for a structure-function analysis, we expressed Sindbis virus nsP4 in Escherichia coli. The core catalytic domain of nsP4 (Delta97nsP4, a deletion of the N-terminal 97 amino acids), which consists of the predicted polymerase domain containing the GDD amino acid motif required for viral RNA synthesis, was stable against proteolytic degradation during expression. Therefore, the recombinant core domain and selected mutants were expressed and purified to homogeneity. We determined that Delta97nsP4 possesses terminal adenylyltransferase (TATase) activity, as it specifically catalyzed the addition of adenine to the 3' end of an acceptor RNA in the presence of divalent cations. Furthermore, Delta97nsP4 is unable to transfer other nucleotides (UTP, CTP, GTP, and dATP) to the acceptor RNA in the absence or presence of other nucleotides. Delta97nsP4 possessing a GDD-to-GAA mutation completely inactivates the enzymatic activity. However, a GDD-to-SNN mutation did not inactivate the enzyme but reduced its activity to approximately 45% of that of the wild type in the presence of Mg(2+). Investigation of the TATase of the GDD-to-SNN mutant revealed that it had TATase equivalent to that of the wild type in the presence of Mn(2+). Identification of Delta97nsP4 TATase activity suggests a novel function of the alphavirus RNA-dependent RNA polymerase in the maintenance and repair of the poly(A) tail, an element required for replication of the viral genome.

Journal: J. Virol.

Date: 10/1/2006

Link: Pubmed: 17005674

Reference:

Tomar S, Hardy RW, Smith JL and Kuhn RJ. Catalytic core of alphavirus nonstructural protein nsP4 possesses terminal adenylytransferase activity. J. Virol. 80:9962-9969. (2006)

PMID: 17005674

Keyword: VISP, FLAVI

Development of novel antivirals against flaviviruses

Sophie Coon — Fri, 02 Mar 2007 01:32:33 GMT

Authors: Patkar CG, et al.

Abstract:

Dengue virus is responsible for a significant amount of human disease in predominantly tropical areas of the world. Much effort has focused on the development of vaccines against the four serotypes of dengue, and within the next few years a vaccine is anticipated. Less progress has been made at developing antivirals that might reduce disease severity. Recent advances in the structural biology of dengue virus and other flaviviruses have opened new possibilities for the rational design of small molecule inhibitors of virus replication. This chapter describes the structural attributes of the dengue virion and how knowledge of its structure, assembly, and entry mechanisms are guiding new strategies toward the development of compounds that will interfere with the viral replication process.

Journal: Novartis Found. Symp.

Date: 8/10/2006

Link: PMID: 17319153

Reference:

Patkar CG, Kuhn RJ. Development of novel antivirals against flaviviruses. Novartis Found Symp. (2006);277:41-52; discussion 52-6, 71-3, 251-3.

PMID: 17319153

Keyword: VISP, FLAVI

Alphavirus capsid protein Helix I controls a checkpoint in nucleocapsid core assembly

Sophie Coon — Fri, 02 Mar 2007 01:32:33 GMT

Authors: Hong EM

Abstract:

The assembly of the alphavirus nucleocapsid core has been investigated using an in vitro assembly system. The C-terminal two-thirds of capsid protein (CP), residues 81 to 264 in Sindbis virus (SINV), have been previously shown to have all the RNA-CP and CP-CP contacts required for core assembly in vitro. Helix I, which is located in the N-terminal dispensable region of the CP, has been proposed to stabilize the core by forming a coiled coil in the CP dimer formed by the interaction of residues 81 to 264. We examined the ability of heterologous alphavirus CPs to dimerize and form phenotypically mixed core-like particles (CLPs) using an in vitro assembly system. The CPs of SINV and Ross River virus (RRV) do not form phenotypically mixed CLPs, but SINV and Western equine encephalitis virus CPs do form mixed cores. In addition, CP dimers do not form between SINV and RRV in these assembly reactions. In contrast, an N-terminal truncated SINV CP (residues 81 to 264) forms phenotypically mixed CLPs when it is assembled with full-length heterologous CPs, suggesting that the region that controls the mixing is present in the N-terminal 80 residues. Furthermore, this result suggests that the dimeric interaction, which was absent between SINV and RRV CPs, can be restored by the removal of the N-terminal 80 residues of the SINV CP. We mapped the determinant that is responsible for phenotypic mixing onto helix I by using domain swapping experiments. Thus, discrimination of the CP partner in alphavirus core assembly appears to be dependent on helix I sequence compatibility. These results suggest that helix I provides one of the important interactions during nucleocapsid core formation and may play a regulatory role during the early steps of the assembly process.

Journal: J. Virol.

Date: 9/1/2006

Link: PMID: 16940497

Reference:

Hong EM, Perera R, Kuhn RJ. Alphavirus capsid protein Helix I controls a checkpoint in nucleocapsid core assembly. J. Virol. 80:8848-8855 (2006)

PMID: 16940497

Keyword: VISP, FLAVI

West Nile virus in complex with the Fab fragment of a neutralizing monoclonal antibody

Sophie Coon — Fri, 02 Mar 2007 01:32:33 GMT

Authors: Kaufmann B, et al.

Abstract:

, such as West Nile virus (WNV), are significant human pathogens. The humoral immune response plays an important role in the control of flavivirus infection and disease. The structure of WNV complexed with the Fab fragment of the strongly neutralizing mAb E16 was determined to 14.5-Angstrom resolution with cryo-electron microscopy. E16, an antibody with therapeutic potential, binds to domain III of the WNV envelope glycoprotein. Because of steric hindrance, Fab E16 binds to only 120 of the 180 possible binding sites on the viral surface. Fitting of the previously determined x-ray structure of the Fab-domain III complex into the cryo-electron microscopy density required a change of the elbow angle between the variable and constant domains of the Fab. The structure suggests that the E16 antibody neutralizes WNV by blocking the initial rearrangement of the E glycoprotein before fusion with a cellular membrane.

Journal: Proc Natl Acad Sci USA

Date: 8/15/2006

Link: PMID: 16895988

Reference:

Kaufmann B, Nybakken GE, Chipman PR, Zhang W, Diamond MS, Fremont DH, Kuhn RJ, Rossmann MG. West Nile virus in complex with the Fab fragment of a neutralizing monoclonal antibody. Proc. Natl. Acad. Sci. USA, 103:12400-12404. (2006)

PMID: 16895988

Keyword: VISP, FLAVI

Cryo-EM reconstruction of dengue virus in complex with the carbohydrate recognition domain of DC-SIGN

Sophie Coon — Fri, 02 Mar 2007 01:32:33 GMT

Authors: Pokidysheva EY, et al.

Abstract:

Dengue virus (DENV) is a significant human pathogen that causes millions of infections and results in about 24,000 deaths each year. Dendritic cell-specific ICAM3 grabbing nonintegrin (DC-SIGN), abundant in immature dendritic cells, was previously reported as being an ancillary receptor interacting with the surface of DENV. The structure of DENV in complex with the carbohydrate recognition domain (CRD) of DC-SIGN was determined by cryo-electron microscopy at 25 A resolution. One CRD monomer was found to bind to two glycosylation sites at Asn67 of two neighboring glycoproteins in each icosahedral asymmetric unit, leaving the third Asn67 residue vacant. The vacancy at the third Asn67 site is a result of the nonequivalence of the glycoprotein environments, leaving space for the primary receptor binding to domain III of E. The use of carbohydrate moieties for receptor binding sites suggests a mechanism for avoiding immune surveillance.

Journal: Cell

Date: 2/10/2006

Link: PMID: 16469696

Reference:

Pokidysheva E, Zhang Y, Battisti AJ, Bator-Kelly CM, Chipman PR, Xiao C, Gregorio GG, Hendrickson WA, Kuhn RJ, Rossmann MG. Cryo-EM reconstruction of dengue virus in complex with the carbohydrate recognition domain of DC-SIGN. Cell 124:485-493. (2006)

PMID: 16469696

Keyword: VISP, FLAVI

Mapping the structure and function of the E1 and E2 glycoproteins in alphaviruses

Sophie Coon — Fri, 02 Mar 2007 01:32:33 GMT

Authors: Mukhopadhyay S, et al.

Abstract:

The 9 A resolution cryo-electron microscopy map of Sindbis virus presented here provides structural information on the polypeptide topology of the E2 protein, on the interactions between the E1 and E2 glycoproteins in the formation of a heterodimer, on the difference in conformation of the two types of trimeric spikes, on the interaction between the transmembrane helices of the E1 and E2 proteins, and on the conformational changes that occur when fusing with a host cell. The positions of various markers on the E2 protein established the approximate topology of the E2 structure. The largest conformational differences between the icosahedral surface spikes at icosahedral 3-fold and quasi-3-fold positions are associated with the monomers closest to the 5-fold axes. The long E2 monomers, containing the cell receptor recognition motif at their extremities, are shown to rotate by about 180 degrees and to move away from the center of the spikes during fusion.

Journal: Structure

Date: 1/1/2006

Link: PMID: 16407066

Reference:

Mukhopadhyay S, Zhang W, Gabler S, Chipman PR, Strauss EG, Strauss JH, Baker TS, Kuhn RJ, Rossmann MG. Mapping the structure and function of the E1 and E2 glycoproteins in alphaviruses. Structure 14:63-73. (2006)

PMID: 16407066

Keyword: VISP, FLAVI

Determinants of bacteriophage phi29 head morphology.

Sophie Coon — Fri, 02 Mar 2007 01:32:33 GMT

Authors: Choi KH, et al.

Abstract:

Bacteriophage phi29 requires scaffolding protein to assemble the 450 x 540 A prolate prohead with T = 3 symmetry end caps. In infections with a temperature-sensitive mutant scaffolding protein, capsids assemble predominantly into 370 A diameter isometric particles with T = 3 symmetry that lack a head-tail connector. However, a few larger, 430 A diameter, particles are also assembled. Cryo-electron microscopy shows that these larger particles are icosahedral with T = 4 symmetry. The prolate prohead, as well as the two isometric capsids with T = 3 and T = 4 symmetry, are composed of similar pentamers and differently skewed hexamers. The skewing of the hexamers in the equatorial region of proheads and in the T = 4 isometric particles reflects their different environments. One of the functions of the scaffolding protein, present in the prohead, may be to stabilize skewed hexamers during assembly.

Journal: Structure

Date: 11/1/2006

Link: PMID: 17098197

Reference:

Choi KH, Morais MC, Anderson DL, Rossmann MG. Determinants of bacteriophage phi29 head morphology. Structure. 14:1723-1727. (2006)

PMID: 17098197

Keyword: VISP