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PubMed1990

Biochemical characterization of porcine enteric calicivirus: analysis of structural and nonstructural viral proteins.

In this report, the molecular weight and antigenicity of the proteins of a porcine enteric calicivirus (PEC) were characterized. The PEC virions were purified from intestinal contents of infected pigs and from infected cell culture lysates. The average buoyant density of the purified virus was 1.37 gm/cm3 in cesium chloride. One major structural protein with a molecular weight of approximately 58 k was found in the gut and cell culture-passaged PEC using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Using immunoblotting techniques only one immunoreactive protein (58 k) ws identified. The PEC and a prototype calicivirus, feline calicivirus (FCV) were propagated in pig kidney and feline kidney (Crandell) cell lines, respectively and intrinsically labeled using [35S]methionine at various times post-inoculation (PI). SDS-PAGE of the radiolabeled proteins indicated the presence of the major structural protein (58 k) and one probable nonstructural protein (28 k) synthesized in the PEC-infected cell lysates by 12 h PI. Other minor protein bands were also evident by 24 h PI (32 k and 82 k). Only the 58 k major protein was detected by radioimmunoprecipitation (RIP) analysis using hyperimmune anti-PEC serum. SDS-PAGE and RIP analysis of FCV-infected cell lysates using hyperimmune anti-FCV serum identified a single major protein of approximately 64 k. No antigenic relationship between PEC and FCV proteins was detected by RIP analysis. The single major structural protein of PEC, the morphological appearance and size of the virus, and its average density of 1.37 gm/cm3 in cesium chloride are consistent with properties of other members of the family Caliciviridae.

Animals↗

PubMed2002

Expansion of pre-terminally differentiated CD8 T cells in chronic HIV-positive patients presenting a rapid viral rebound during structured treatment interruption.

OBJECTIVE: The influence of structured treatment interruption on effector/memory CD8 T cell dynamics was analysed in chronic HIV-infected patients showing a rapid or delayed viral rebound. DESIGN: Structured treatment interruption consisted of at least one month of discontinuation, followed by highly active antiretroviral therapy (HAART) resumption. Two groups of HIV structured treatment interruption patients were selected on the basis of plasma viral HIV-RNA value (> 30 000 copies/ml, branched DNA): group A (n = 14), patients with a rapid viral rebound (within one month) and group B (n = 6), patients with a delayed or no viral rebound (after a minimum of 4 months). METHODS: A clinical and immunological follow-up was performed at HAART suspension (t 0), one month from suspension (t 1), at HAART resumption (t 2), and 30 days from resumption (t 3). RESULTS: A sustained viral rebound was observed in group A patients, showing a rapid expansion of circulating CD8 T lymphocytes. In this group, the frequencies of CD8 T cells releasing IFN-gamma after mitogen-induced or Gag-specific stimulation were highly increased after HAART discontinuation. Nevertheless, these CD8 T lymphocytes were mainly composed of pre-terminally differentiated cytotoxic T lymphocytes (CTL) expressing a CCR7 CD27 CD45RA phenotype and a reduced amount of perforin. In contrast, group B patients showed no significant changes in immunological parameters during a prolonged drug-free period. CONCLUSION: These data indicate that monitoring CD8 T cell dynamics during structured treatment interruption could be clinically relevant, and new therapeutic strategies should aim qualitatively to restore CTL effector functions.

Adult↗

PubMed2001

Detecting structural changes in viral capsids by hydrogen exchange and mass spectrometry.

Amide hydrogen exchange and mass spectrometry have been used to study the pH-induced structural changes in the capsid of brome mosaic virus (BMV). Capsid protein was labeled in a structurally sensitive way by incubating intact viral particles in D(2)O at pH 5.4 and 7.3. Deuterium levels in the intact coat protein and its proteolytic fragments were determined by mass spectrometry. The largest deuterium increases induced by structural alteration occurred in the regions around the quasi-threefold axes, which are located at the center of the asymmetric unit. The increased levels of deuterium indicate loosening of structure in these regions. This observation confirms the previously proposed swelling model for BMV and cowpea chlorotic mottle virus (CCMV) and is consistent with the structure of swollen CCMV recently determined by cryo-electron microscopy and image reconstruction. Structural changes in the extended N- and C-terminal arms were also detected and compared with the results obtained with other swollen plant viruses. This study demonstrates that protein fragmentation/amide hydrogen exchange is a useful tool for probing structural changes in viral capsids.

Amino Acid Sequence↗

PubMed2005

The oligomeric structure of vaccinia viral envelope protein A27L is essential for binding to heparin and heparan sulfates on cell surfaces: a structural and functional approach using site-specific mutagenesis.

The soluble domain of the self-assembly vaccinia virus envelope protein A27L, sA27L-aa, consists of a flexible extended coil at the N terminus and a rigid hydrophobic coiled-coil region at the C terminus. In the former, a basic strip of 12 residues is responsible for binding to cell-surface heparan sulfates. Although the latter is believed to mediate self-assembly, its biological role is unclear. However, an in vitro bioassay showed that peptides comprising the 12 residue basic region alone failed to interact with heparin, suggesting that the C-terminal coiled-coil region might serve an indispensable role in biological function. To explore this structural and functional relationship, we performed site-specific mutagenesis in an attempt to specifically disrupt the hydrophobic core of the coiled coil. Three single mutants, L47A, L51A, and L54A, and one triple mutant, L47,51,54A, were expressed and purified from Escherichia coli. The physical properties of the mutants were carefully examined by gel-filtration chromatography, CD, and NMR spectroscopy, and the biological activities were assessed by an in vitro SPR bioassay and three in vivo bioassays: binding to cells, blocking virus infection and blocking cell fusion. We showed that the L47A mutant, which is similar to the parental sA27L-aa in forming a hexamer, is biologically active. L51A and L54A mutants form tetramers and are less active. Notably, in the triple mutant, the self-assembly hydrophobic core structure is uncoiled; as a consequence, the tetrameric structure is biologically inactive. Thus, we conclude that the leucine residues, in particular Leu51 and Leu54, sustain the hydrophobic core structure that is essential for the biological function of vaccinia virus envelope protein A27L, binding to cell-surface heparan sulfate.

Cell Fusion↗

PubMed1997

Structure of a viral procapsid with molecular scaffolding.

The assembly of a macromolecular structure proceeds along an ordered morphogenetic pathway, and is accomplished by the switching of proteins between discrete conformations as they are added to the nascent assembly. Scaffolding proteins often play a catalytic role in the assembly process, rather like molecular chaperones. Although macromolecular assembly processes are fundamental to all biological systems, they have been characterized most thoroughly in viral systems, such as the icosahedral Escherichia coli bacteriophage phiX174. The phiX174 virion contains the proteins F, G, H and J. During assembly, two scaffoldingproteins B and D are required for the formation of a 108S, 360-A-diameter procapsid from pentameric precursors containing the F, G and H proteins. The procapsid contains 240 copies of protein D, forming an external scaffold, and 60 copies each of the internal scaffolding protein B, the capsid protein F, and the spike protein G. Maturation involves packaging of DNA and J proteins and loss of protein B, producing a 132S intermediate. Subsequent removal of the external scaffold yields the mature virion. Both the F and G proteins have the eight-stranded antiparallel beta-sandwich motif common to many plant and animal viruses. Here we describe the structure of a procapsid-like particle at 3.5-A resolution, showing how the scaffolding proteins coordinate assembly of the virus by interactions with the F and G proteins, and showing that the F protein undergoes conformational changes during capsid maturation.

Bacteriophage phi X 174↗

PubMed2006

Role of invariant Thr80 in human immunodeficiency virus type 1 protease structure, function, and viral infectivity.

Sequence variability associated with human immunodeficiency virus type 1 (HIV-1) is useful for inferring structural and/or functional constraints at specific residues within the viral protease. Positions that are invariant even in the presence of drug selection define critically important residues for protease function. While the importance of conserved active-site residues is easily understood, the role of other invariant residues is not. This work focuses on invariant Thr80 at the apex of the P1 loop of HIV-1, HIV-2, and simian immunodeficiency virus protease. In a previous study, we postulated, on the basis of a molecular dynamics simulation of the unliganded protease, that Thr80 may play a role in the mobility of the flaps of protease. In the present study, both experimental and computational methods were used to study the role of Thr80 in HIV protease. Three protease variants (T80V, T80N, and T80S) were examined for changes in structure, dynamics, enzymatic activity, affinity for protease inhibitors, and viral infectivity. While all three variants were structurally similar to the wild type, only T80S was functionally similar. Both T80V and T80N had decreased the affinity for saquinavir. T80V significantly decreased the ability of the enzyme to cleave a peptide substrate but maintained infectivity, while T80N abolished both activity and viral infectivity. Additionally, T80N decreased the conformational flexibility of the flap region, as observed by simulations of molecular dynamics. Taken together, these data indicate that HIV-1 protease functions best when residue 80 is a small polar residue and that mutations to other amino acids significantly impair enzyme function, possibly by affecting the flexibility of the flap domain.

Amino Acid Substitution↗

PubMed2001

Solution structure of a viral DNA polymerase X and evidence for a mutagenic function.

The African swine fever virus DNA polymerase X (ASFV Pol X or Pol X), the smallest known nucleotide polymerase, has recently been reported to be an extremely low fidelity polymerase that may be involved in strategic mutagenesis of the viral genome. Here we report the solution structure of Pol X. The structure, unique within the realm of nucleotide polymerases, consists of only palm and fingers subdomains. Despite the absence of a thumb subdomain, which is important for DNA binding in other polymerases, we show that Pol X binds DNA with very high affinity. Further structural analyses suggest a novel mode of DNA binding that may contribute to low fidelity synthesis. We also demonstrate that the ASFV DNA ligase is a low fidelity ligase capable of sealing a nick that contains a G-G mismatch. This supports the hypothesis of a virus-encoded, mutagenic base excision repair pathway consisting of a tandem Pol X/ligase mutator.

African Swine Fever Virus↗

PubMed2004

Genetic population structure of marine viral haemorrhagic septicaemia virus (VHSV).

The nucleotide sequences of a specific region of the nucleoprotein gene were compared in order to investigate the genetic population structure of marine viral haemorrhagic septicaemia virus (VHSV). Analysis of the sequence from 128 isolates of diverse geographic and host origin renders this the most comprehensive molecular epidemiological study of marine VHSV conducted to date. Phylogenetic analysis of nucleoprotein gene sequences confirmed the existence of the 4 major genotypes previously identified based on N- and subsequent G-gene based analyses. The range of Genotype I included subgroups of isolates associated with rainbow trout aquaculture (Genotype Ia) and those from the Baltic marine environment (Genotype Ib) to emphasise the relatively close genetic relationship between these isolates. The existence of an additional genotype circulating within the Baltic Sea (Genotype II) was also confirmed. Genotype III included marine isolates from around the British Isles in addition to those associated with turbot mariculture, highlighting a continued risk to the development of this industry. Genotype IV consisted of isolates from the marine environment in North America. Taken together, these findings suggest a marine origin of VHSV in rainbow trout aquaculture. The implications of these findings with respect to the future control of VHSV are discussed. The capacity for molecular phylogenetic analysis to resolve complex epidemiological problems is also demonstrated and its likely future importance to disease management issues highlighted.

Animals↗

PubMed1980

Unifected avian cells contain structurally unrelated progenitors of viral sarcoma genes.

A single gene, src of Rous sarcoma virus (RSV) coding for a protein of pp60src, is responsible for the transformation of fibroblasts. DNA sequences homologous to src (the endogenous sarc) are presented in uninfected cells of chickens and other vertebrates. Endogenous nucleotide sequences have also been found in the putative transforming genes of MC29 (ref. 6) and avian erythroblastosis virus. These viruses, however, induce different spectra of tumours in animals. From analysis of a new avian sarcoma virus, Y73, we present here evidence suggesting that multiforms of viral sarcoma genes originated from cellular genetic sequences, and that these viral genes are structurally unrelated, but have similar pathogenicities.

Alpharetrovirus↗

PubMed2001

Psychological impact of structured treatment interruptions in patients with prolonged undetectable HIV-1 viral loads.

Structured treatment interruption strategies may help overcome problems of highly active antiretroviral therapy, but might also represent a cause of stress. We present data that indicate a psychological benefit from structured treatment interruption. Although some disturbances appear at the resumption of therapy, no definitive problems are found that preclude such therapeutic approaches from a psychological perspective. However, a close follow-up of patients during interruption periods is advisable to avoid difficulties reported at treatment resumption presenting a risk to patients' health.

Antiretroviral Therapy, Highly Active↗

PubMed2006

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

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.

Amino Acid Sequence↗

PubMed1983

tRNA-like structures of plant viral RNAs: conformational requirements for adenylation and aminoacylation.

Bromo- and cucumovirus RNAs contain a tRNA-like structure as an integral part of their genome. This structure is located at the 3' end of the viral RNA and is an acceptor of tyrosine. The 3' regions of representative viral RNAs have been sequenced and quite unorthodox secondary foldings have been proposed for these 3' ends. The question therefore remained as to how these structures could be recognized by tRNA-specific enzymes. We have established the minimum number of nucleotides from the 3' end of the brome mosaic virus and broad bean mottle virus RNAs required for the formation of structures recognized by the tyrosyl-tRNA synthetase and/or the tRNA nucleotidyltransferase. The results obtained delineate the length of the tRNA-like region, and indicate that the 5' region of the tRNA-like structure participates in the formation of the amino acid stem. This has led us to propose an 'L'-shaped secondary structure for these tRNA-like regions.

Anticodon↗

PubMed2006

Structural analysis of viral nucleocapsids by subtraction of partial projections.

The nucleocapsid of flavivirus particles does not have a recognizable capsid structure when using icosahedral averaging for cryo-electron microscopy structure determinations. The apparent absence of a definitive capsid structure could be due to a lack of synchronization of the symmetry elements of the external glycoprotein layer with those of the core or because the nucleocapsid does not have the same structure within each particle. A technique has been developed to determine the structure of the capsid, and possibly also of the genome, for icosahedral viruses, such as flaviviruses, using cryo-electron microscopy. The method is applicable not only to the analyses of viral cores, but also to the missing structure of multi-component complexes due to symmetry mismatches. The density contributed by external glycoprotein and membrane layers, derived from previously determined three-dimensional icosahedrally averaged reconstructions, was subtracted from the raw images of the virus particles. The resultant difference images were then used for a three-dimensional reconstruction. After appropriate test data sets were constructed and tested, the procedure was applied to examine the nucleocapsids of flaviviruses, which showed that there is no distinct protein density surrounding the genome. Furthermore, there was no evidence of any icosahedral symmetry within the nucleocapsid core.

Flavivirus↗

PubMed1991

The minute virus of mice capsid specifically recognizes the 3' hairpin structure of the viral replicative-form DNA: mapping of the binding site by hydroxyl radical footprinting.

The terminal hairpin structures of the DNA of minute virus of mice (MVM) are essential for viral replication. Here we show that the hairpin 3' terminus of MVM replicative-form DNA binds specifically to empty MVM capsids. Binding of the same terminal DNA sequence in its linear double-stranded (extended) conformation was not observed. After heat denaturation and quick cooling of 3'-terminal extended-form fragments, not only the virion strand but also the complementary strand was found to bind to the capsid, presumably because each strand re-formed a similar hairpin structure. No binding affinity for the capsid was found to be associated with hairpin or extended 5' termini or with any other region of the viral DNA. Hydroxyl radical footprinting analyses revealed three protected nucleotide stretches forming a binding site at the branch point of the two 3'-terminal hairpin arms looping out from the DNA stem (T structure). Single base changes within this site did not affect the binding. In band shift experiments, specific binding to the T structure was demonstrated for VPI but not for VP2.

Base Sequence↗

PubMedDate not supplied

Review: conformation and folding of novel beta-structural elements in viral fiber proteins: the triple beta-spiral and triple beta-helix.

Apart from alpha-helical coiled coils and the collagen triple helices, fibrous proteins can contain beta-structure in various conformations. Elongated enzymes such as pectate lyase and the bacteriophage P22 tailspike protein contain single-stranded beta-helices. Virus and bacteriophage fibers, which are often trimeric, have been shown to contain novel triple-stranded beta-structures such as the triple beta-spiral and the triple beta-helix. The conformation and folding of viral fibers containing beta-structure are discussed.

Amino Acid Sequence↗

PubMed1987

What does the molecular structure of viruses tell us about viral functions?

The structures of the protein shell of a number of simple icosahedral RNA plant and animal viruses are now known at atomic resolution. In addition, components of viral capsids such as the haemagglutinin and neuraminidase spikes of influenza virus and the hexon unit of adenovirus are known in similar detail. These structures have provided a wealth of information on viral assembly, viral disassembly, the antigenic surface on viruses available to neutralizing antibodies, the host cell receptor attachment site, fusion of viral particles with the host cell, processing of polyproteins during maturation and the manner in which antiviral agents can interfere with the function of a viral capsid.

Antibodies, Viral↗

PubMed1992

Crystal structures of two viral peptides in complex with murine MHC class I H-2Kb.

The x-ray structures of a murine MHC class I molecule (H-2Kb) were determined in complex with two different viral peptides, derived from the vesicular stomatitis virus nucleoprotein (52-59), VSV-8, and the Sendai virus nucleoprotein (324-332), SEV-9. The H-2Kb complexes were refined at 2.3 A for VSV-8 and 2.5 A for SEV-9. The structure of H-2Kb exhibits a high degree of similarity with human HLA class I, although the individual domains can have slightly altered dispositions. Both peptides bind in extended conformations with most of their surfaces buried in the H-2Kb binding groove. The nonamer peptide maintains the same amino- and carboxyl-terminal interactions as the octamer primarily by the insertion of a bulge in the center of an otherwise beta conformation. Most of the specific interactions are between side-chain atoms of H-2Kb and main-chain atoms of peptide. This binding scheme accounts in large part for the enormous diversity of peptide sequences that bind with high affinity to class I molecules. Small but significant conformational changes in H-2Kb are associated with peptide binding, and these synergistic movements may be an integral part of the T cell receptor recognition process.

Amino Acid Sequence↗

PubMed1996

Structure-based design of peptide presentation on a viral surface: the crystal structure of a plant/animal virus chimera at 2.8 A resolution.

BACKGROUND: We employed a genetically engineered icosahedral plant virus, cowpea mosaic virus (CPMV), as an expression and presentation system to display a 14 amino acid linear antigenic epitope found in a capsid protein of human rhinovirus 14 (HRV14). RESULT: Gram quantities of the CPMV/HRV 14 chimera were made in plants and purified particles were crystallized in a form isomorphous with wild-type CPMV. The 2.8 A resolution structure of the chimera shows that the inserted loop is well ordered and that if the loop were intact, a phenylalanine residue of CPMV would be placed in a hydrophilic environment. The resultant strain may make the loop an attractive substrate for endogenous plant proteases, as roughly 80% of the inserted polypeptides are cleaved, allowing the phenylalanine to be partially buried. Altering the phenylalanine to an arginine could relieve the stress, reducing the propensity for cleavage and increasing the likelihood that the peptide will assume a structure closely similar to its structure in HRV14. CONCLUSIONS: Successful crystallization of other CPMV chimeras in forms isomorphous with the native virus suggests that this is a viable system for structure-based design of peptide presentation.

Amino Acid Sequence↗