pinecone/core-2020-05-10-deduplication huggingface dataset:
Melanoplus sanguinipes Entomopoxvirus DNA Topoisomerase: Site-Specific DNA Transesterification and Effects of 5′-Bridging Phosphorothiolates

AbstractMelanoplus sanguinipes entomopoxvirus (MsEPV) encodes a 328 amino acid polypeptide related to the type I topoisomerases of six other genera of vertebrate and insect poxviruses. The gene encoding MsEPV topoisomerase was expressed in bacteria, and the recombinant protein was purified by ion-exchange chromatography and glycerol gradient sedimentation. MsEPV topoisomerase, a monomeric protein, catalyzed the relaxation of supercoiled plasmid DNA at ∼0.6 supercoils/s. Like other poxvirus topoisomerases, the MsEPV enzyme formed a covalent adduct with duplex DNA at the target sequence CCCTT↓. The kinetic and equilibrium parameters of the DNA transesterification reaction of MsEPV topoisomerase were kcl = 0.3 s−1 and Kcl = 0.25. The introduction of a 5′-bridging phosphorothiolate at the scissile phosphate increased the cleavage equilibrium constant from 0.25 to ≥30. Similar phosphorothiolate effects were observed with vaccinia topoisomerase. Kinetic analysis of single-turnover cleavage and religation reactions established that the altered equilibrium was the result of a ∼10−4 decrement in the rate of topoisomerase-catalyzed attack of 5′-SH DNA on the DNA-(3′-phosphotyrosyl)-enzyme intermediate. 5′-bridging phosphorothiolates at the scissile phosphate and other positions within the CCCTT element had no significant effect on kcl
The Effects of Targeting the Vaccinia Virus B5R Protein to the Endoplasmic Reticulum on Virus Morphogenesis and Dissemination

AbstractThe consequence of redirecting the vaccinia virus (VV) B5R protein to the endoplasmic reticulum (ER) has been investigated by the addition of an ER retrieval signal KKSL (K2X2) to the B5R C-terminus. This mutant B5R gene and a version of the gene with the inactive ER retrieval sequence KKSLAL (K2X4) were inserted into the thymidine kinase locus of a VV mutant lacking the B5R gene, vΔB5R. Similar levels of B5R protein were made by each virus, but the B5R-K2X2 protein remained sensitive to endoglycosidase H and colocalised with protein disulphide isomerase in the ER. In contrast, the B5R-K2X4 protein colocalised with 1,4-galactosyltransferase in the trans-Golgi network. Electron microscopy revealed that even when the B5R protein was redirected to the ER, intracellular mature virus particles were wrapped by cellular membranes to form intracellular enveloped virus particles, although more incompletely wrapped particles were evident compared with wild type. These intracellular enveloped virus particles were, however, unable to efficiently induce the polymerisation of actin and the plaque size formed by vB5R-K2X2 was small. Nevertheless, the amount and specific infectivity of EEV produced by vB5R-K2X2 were similar to those of wild type, despite the dramatic reduction in the amount of B5R protein present in vB5R-K2X2 EEV
The Nucleocapsid Protein of Coronavirus Mouse Hepatitis Virus Interacts with the Cellular Heterogeneous Nuclear Ribonucleoprotein A1 in Vitro and in Vivo

AbstractThe nucleocapsid (N) protein of mouse hepatitis virus (MHV) and the cellular heterogeneous nuclear ribonucleoprotein A1 (hnRNP-A1) are RNA-binding proteins, binding to the leader RNA and the intergenic sequence of MHV negative-strand template RNAs, respectively. Previous studies have suggested a role for both N and hnRNP-A1 proteins in MHV RNA synthesis. However, it is not known whether the two proteins can interact with each other. Here we employed a series of methods to determine their interactions both in vitro and in vivo. Both N and hnRNP-A1 genes were cloned and expressed in Escherichia coli as glutathione S-transferase (GST) fusion proteins, and their interactions were determined with a GST-binding assay. Results showed that N protein directly and specifically interacted with hnRNP-A1 in vitro. To dissect the protein-binding domain on the N protein, 15 deletion constructs were made by PCR and expressed as GST fusion proteins. Two hnRNP-A1-binding sites were identified on N protein: site A is located at amino acids 1 to 292 and site B at amino acids 392 to 455. In addition, we found that N protein interacted with itself and that the self-interacting domain coincided with site A but not with site B. Using a fluorescence double-staining technique, we showed that N protein colocalized with hnRNP-A1 in the cytoplasm, particularly in the perinuclear region, of MHV-infected cells, where viral RNA replication/transcription occurs. The N protein and hnRNP-A1 were coimmunoprecipitated from the lysates of MHV-infected cells either by an N- or by an hnRNP-A1-specific monoclonal antibody, indicating a physical interaction between N and hnRNP-A1 proteins. Furthermore, using the yeast two-hybrid system, we showed that N protein interacted with hnRNP-A1 in vivo. These results thus establish that MHV N protein interacts with hnRNP-A1 both in vitro and in vivo
Mutagenesis of the RGD Motif in the Yellow Fever Virus 17D Envelope Protein

AbstractThe envelope protein of yellow fever virus 17D (YFV-17D) contains a solvent-exposed RGD motif, which has led to the suggestion that integrins may function as cellular receptors for YFV-17D. We found that mutating the RGD motif to RGE had no effect on viral titers, whereas changing RGD to TGD, TGE, TAD, TAE, or RGS led to reduced titers. Substitution of RGD by RAD or RAE yielded RNA genomes that replicated in mammalian cells but could not spread to neighboring cells at 37°C. These mutants did spread through the cell monolayer at 30°C (both in mosquito cells and in SW13 cells) and viruses grown at this temperature were capable of infecting mammalian cells at 37°C. These results strongly suggest that RGD-mediated integrin binding does not play a major role in YFV-17D entry, since the RGD to RAD mutation, as well as many or all of the other mutations studied, should disrupt all RGD-dependent integrin binding. However, the RGD to RAD or RAE mutations (as well as TAD and TAE) severely destabilized the envelope protein at 37°C, providing an explanation for the observed phenotype. Implications of these findings are discussed in light of the fact that mutations that alter tropism or virulence in different flaviviruses are often found within the loop containing the RGD motif
Restoration of the 3′ End of Potyvirus RNA Derived from Poly(A)-Deficient Infectious cDNA Clones

AbstractPoly(A)-deficient full-length cDNA clones of clover yellow vein virus (ClYVV), a member of the genus Potyvirus, were found to be infectious when expressed from the CaMV 35S promoter. The poly(A) tail was replaced with different short sequences and the infectivities of the cDNA constructs were examined. Although the infectivity of the plasmid varied depending on the sequences introduced, all the constructs were infectious. In all cases, progeny viral RNAs from the cDNA clones had an authentic viral sequence at their 3′ regions with poly(A) tails and the downstream nonviral sequences were completely lost. However, two minor mutations, a two-nucleotide deletion at the 3′ end and a single-nucleotide addition at the second nucleotide position downstream of the poly(A) site, were also observed. The clones of the viral (−) strand RNAs had poly(U) tracts at their 5′ ends, suggesting that their synthesis is primed by the poly(U) sequence. It furthermore suggests that the mutations were introduced during or after primary transcription from the cDNA and were maintained during authentic viral replication. Although the mechanism involved is not known, recovery of the poly(A) tail is an essential step for maintaining the infectivity of the viral cDNAs
The Hypersensitive Response to Cucumber Mosaic Virus in Chenopodium amaranticolor Requires Virus Movement Outside the Initially Infected Cell

AbstractCucumber mosaic virus (CMV) expressing the green fluorescent protein (GFP), and lacking either the 3a movement protein or the coat protein (CP), failed to induce a hypersensitive response producing local lesions in inoculated leaves of Chenopodium amaranticolor. Cytological analysis showed that both viral-encoded proteins are required for cell-to-cell movement of the virus and the simultaneous appearance of cellular necrosis. In the absence of either or both proteins, infection was confined to single, non-necrotized, epidermal cells. CMV with a mutation in the 3a protein (M8 CMV) could infect tobacco systemically but did not induce necrotic lesions in C. amaranticolor. In this host, the mutated 3a protein was unable to promote viral movement out of the initially infected epidermal cell. Movement-deficient CMV expressing wild-type (WT) 3a protein as a fusion to the GFP, as well as WT CP, also failed to induce necrosis. Finally, single epidermal cells infected with a movement-deficient CMV expressing WT 3a protein, WT CP, and free GFP did not show necrosis. These data indicate that viral movement out of the initially infected epidermal cell, and not the simultaneous expression in this cell of the 3a protein and the CP, is required for the induction of cell death
The Distribution and Kinetics of Polyomavirus in Lungs of Intranasally Infected Newborn Mice

AbstractThe primary cell types that sustain polyomavirus (Py) replication following intranasal infection as well as the nature of the host cellular response to Py were unknown. As this is an essential and specific site for virus entry, it seems likely that viral gene function must be adapted to these mucosal tissues. Using immunohistochemistry and in situ hybridization, we determined the cell types in the lung that support Py gene expression and replication following intranasal inoculation of newborn mice within 24 h of birth. Lungs were collected daily from days 1 to 10 postinfection for Py DNA and early T antigen analysis and for histological examination by H&E staining, using methods that preserve the delicate newborn lung architecture. Viral DNA was present in increasing quantities from 2 to 6 dpi in a subset of the Clara cells lining the inner lumen of the bronchi and bronchioles, while T antigen expression was present in a majority of the cells in the bronchi and bronchiole lumen. A distinct and transient pattern of hyperplasia was observed among the cells expressing T antigen and was present from 3 through 6 dpi. Py DNA-containing cells exfoliated into the bronchiole lumen and alveolar ducts, but Py T antigen was not detected in these cells. Py DNA was first detected at 2 dpi, increased through 6 dpi, and abruptly declined through 9 dpi at which time there was no sign of viral DNA in the lungs by in situ hybridization. An unusual infiltration of neutrophils began before the presence of exfoliated cells or Py replication and continued for 2–3 days and was followed by a lymphocytic infiltration at 8–10 dpi lasting 2–3 days. Neither the hyperplasia nor the neutrophil infiltration occurred following infection with the MOP1033 MT-Ag or RB1 LT-Ag mutants of Py. In addition, both the neutrophil infiltration and the transient hyperplasia are in stark contrast to the heavy macrophage infiltration that follows infection of lungs with mouse adenovirus. Thus it appears that Py elicits a distinct host response pattern not seen with other DNA viral infections
Development of Bivalent (B/E) Vaccines Able to Neutralize CCR5-Dependent Viruses from the United States and Thailand

AbstractRecombinant envelope glycoproteins prepared from a subtype B (MN) strain and a subtype E (CM244) strain of HIV-1 were combined to create a bivalent vaccine (B/E) effective against viruses circulating in the United States and Asia. Combining the two antigens resulted in formulations that increased the breadth and potency of the inter-subtype neutralizing response. Antibodies to the bivalent vaccine formulation neutralized viruses possessing diverse phenotypes, including syncytia-inducing and non-syncytia-inducing primary isolates, viruses using either the CCR5 or the CXCR4 chemokine receptors, and viruses differing in their sensitivity to soluble CD4. These studies demonstrate for the first time that the magnitude and quality of the immune response to HIV-1 can be improved by combining recombinant envelope glycoproteins from different genetic subtypes
Particle-Mediated DNA Immunization of Cattle Confers Long-Lasting Immunity against Bovine Herpesvirus-1

AbstractParticle-mediated delivery was used as a method to vaccinate ruminants with a DNA vaccine. The optimal conditions for gene gun-based delivery of gold particles into the epidermal layer of the skin were determined. After delivery of the gold particles, an inflammatory response was observed. This response occurred regardless of the presence of plasmid and therefore was a result of the physical disturbance of the skin by the gold particles. To identify transfected cells, a plasmid expressing a green fluorescent protein was delivered into the skin. Fluorescent cells were located primarily in the outermost layers of the epidermis and outside the core of gold particles deposited by the gene gun. Cattle were immunized by gene gun with a plasmid expressing a truncated, secreted form of bovine herpesvirus-1 glycoprotein D. Serum antibody responses, antigen-specific proliferation, and interferon-γ secretion by peripheral blood lymphocytes were demonstrated. These immune responses were found to be of long duration and sufficient magnitude to protect cattle against challenge with bovine herpesvirus-1, which demonstrates the efficacy of gene gun-based delivery of DNA vaccines to target species
Glycans and Proteoglycans Are Involved in the Interactions of Human Immunodeficiency Virus Type 1 Envelope Glycoprotein and of SDF-1α with Membrane Ligands of CD4+ CXCR4+ Cells

AbstractWe demonstrate that human immunodeficiency virus HIV-1LAI envelope glycoprotein 120 (gp120LAi) specifically interacts with several membrane ligands on lymphoid CEM or monocytic U937 cells in addition to its previously identified receptor, CD4, and CXCR4, its coreceptor. In its native state, gp120LAI is able to elicit specific multimolecular complexes with these membrane ligands at the surface of the cells; most of the interactions are abolished by mannan or heparin but not by dextran. Similarly, stromal cell-derived factor (SDF)-1α interacts not only with CXCR4 expressed by CXCR4+ CD4+ U937, CEM, and HOS-CD4+ CXCR4+ cells but also with CD4 expressed by intact U937, CEM, and HOS-CD4+ CXCR4+/− cells or electroblotted onto Immobilon. SDF-1α binding to CD4+ CXCR4+/− cells, or soluble CD4 electroblotted onto Immobilon, is significantly inhibited by sCD4, whereas truncated sCD4 lacking D3 and D4 domains had no significant effect, which indicates that SDF-1 binds to CD4 but at regions different from the HIV-gp120-binding site. Heparin and mannan also inhibit SDF-1α binding to intact CD4+ CXCR4+/− cells, and electroblotted soluble CD4. Heparitinase treatment of such cells reduced SDF-1α binding. These data demonstrate that glycans and glycosaminoglycans are directly or indirectly involved in the interactions of HIV-1 gp120LAI and of SDF-1α with membrane ligands of CD4+ CXCR4+ cells and thus could play a role both in HIV-1 infection and in the physiology of SDF-1α
Delta-Peptide Is the Carboxy-Terminal Cleavage Fragment of the Nonstructural Small Glycoprotein sGP of Ebola Virus

AbstractIn the present study we have investigated processing and maturation of the nonstructural small glycoprotein (sGP) of Ebola virus. When sGP expressed from vaccinia virus vectors was analyzed by pulse-chase experiments using SDS–PAGE under reducing conditions, the mature form and two different precursors have been identified. First, the endoplasmic reticulum form sGPer, full-length sGP with oligomannosidic N-glycans, was detected, sGPer was then replaced by the Golgi-specific precursor pre-sGP, full-length sGP containing complex N-glycans. This precursor was finally converted by proteolysis into mature sGP and a smaller cleavage fragment, Δ-peptide. Studies employing site-directed mutagenesis revealed that sGP was cleaved at a multibasic amino acid motif at positions 321 to 324 of the open reading frame. Cleavage was blocked by RVKR-chloromethyl ketone. Uncleaved pre-sGP forms a disulfide-linked homodimer and is secreted into the culture medium in the presence of the inhibitor as efficiently as proteolytically processed sGP. In vitro treatment of pre-sGP by purified recombinant furin resulted in efficient cleavage, confirming the importance of this proprotein convertase for the processing and maturation of sGP. Δ-peptide is also secreted into the culture medium and therefore represents a novel nonstructural expression product of the GP gene of Ebola virus. Both cleavage fragments contain sialic acid, but only Δ-peptide is highly O-glycosylated
Cell Cycle-Dependent Stimulation of the HIV-1 Promoter by Tat-Associated CAK Activator

AbstractActivation of the HIV-1 promoter by the virally encoded Tat protein is characterized by efficient processive transcription, mediated by host cell factors that are tethered to the promoter with the Tat–TAR RNA complex. Importantly, viral gene activation has been shown to be stimulated in mitogenically induced cells, although the link between cell cycle regulation and viral gene activation is unclear. We reported a Tat-associated CAK/CTD kinase from mitogenically induced primary human T-cells (TTK) (S. Nekhai et al., 1997, J. Virol. 71, 7436–7441). Here, biological activity of the kinase has been studied by direct microinjection at the individual-cell level. The TTK-dependent Tat response is maximal during G1 phase as shown by co-injection with Tat protein in cells synchronized at the various stages of the cell cycle. The cell cycle dependence of the Tat response was confirmed by inhibiting G0 → G1 progression with the expression of dominant negative mutant RasAsn17 or the cyclin-dependent kinase CDK4. The results support a mechanism whereby transactivation of the HIV promoter is regulated by cell growth signal transduction pathways that target the Tat cofactor
Construction of Infectious Feline Foamy Virus Genomes: Cat Antisera Do Not Cross-Neutralize Feline Foamy Virus Chimera with Serotype-Specific Env Sequences

AbstractFull-length genomes of the feline foamy virus (FFV or FeFV) isolate FUV were constructed. DNA clone pFeFV-7 stably directed the expression of infectious FFV progeny virus indistinguishable from wild-type, uncloned FFV isolate FUV. The env and bel 1 genes of pFeFV-7 were substituted for by corresponding sequences of the FFV serotype 951 since previous studies implicated a defined part of FFV Env protein as responsible for serotype-specific differences in serum neutralization (I. G. Winkler, R. M. Flügel, M. Löchelt, and R. L. P. Flower, 1998. Virology 247: 144–151). Recombinant virus derived from chimeric plasmid pFeFV-7/951 containing the hybrid env gene and the parental clone pFeFV-7 were used for neutralization studies. By means of a rapid titration assay for FFV infectivity, we show that progeny virus derived from plasmid pFeFV-7 was neutralized by FUV- but not by 951-specific antisera, whereas pFeFV-7/951-derived chimeric virus was neutralized by 951-specific antisera only. Both recombinant proviruses will be useful for repeated delivery of foreign genes for therapeutic gene applications into cats
The Refined Crystal Structure of Cowpea Mosaic Virus at 2.8 Å Resolution

AbstractComoviruses are a group of plant viruses in the picornavirus superfamily. The type member of comoviruses, cowpea mosaic virus (CPMV), was crystallized in the cubic space group I23, a = 317 Å and the hexagonal space group P6122, a = 451 Å, c = 1038 Å. Structures of three closely similar nucleoprotein particles were determined in the cubic form. The roughly 300-Å capsid was similar to the picornavirus capsid displaying a pseudo T = 3 (P = 3) surface lattice. The three β-sandwich domains adopt two orientations, one with the long axis radial and the other two with the long axes tangential in reference to the capsid sphere. T = 3 viruses display one or the other of these two orientations. The CPMV capsid was permeable to cesium ions, leading to a disturbance of the β-annulus inside a channel-like structure, suggesting an ion channel. The hexagonal crystal form diffracted X rays to 3 Å resolution, despite the large unit cell. The large (∼200 Å) solvent channels in the lattice allow exchange of CPMV cognate Fab fragments. As an initial step in the structure determination of the CPMV/Fab complex, the P6122 crystal structure was solved by molecular replacement with the CPMV model determined in the cubic cell
Cryphonectria hypovirus 3, a Virus Species in the Family Hypoviridae with a Single Open Reading Frame

AbstractIsolate Grand Haven (GH) 2 is a naturally occurring isolate of the chestnut blight fungus, Cryphonectria parasitica, that is greatly reduced in virulence due to the presence of a double-stranded RNA virus. Unlike many other virus-infected, hypovirulent isolates, GH2 is not substantially reduced in pigmentation, conidiation, or laccase expression compared to its virus-free counterpart. The dsRNA genome of the GH2 virus was cloned, sequenced, and compared to hypovirulence-associated viruses of the family Hypoviridae. GH2 dsRNA is considerably smaller than previously characterized members of the family, 9.8 kb compared to 12.5–12.7 kb for other members. The genome organization of GH2 dsRNA reflected the substantial difference in genome size. Like other members of the family, one strand contained a poly(A)+ tail at the 3′ end and a long sequence with several minicistrons at the 5′ end of the same strand. Only a single open reading frame (ORF) of 8622 nucleotides was predicted from deduced translations of the poly(A)+-containing strand, however. This contrasts with the two-ORF structures of previously characterized members. Analysis of the deduced ORF of GH2 dsRNA revealed putative proteinase, RNA polymerase, and helicase domains similar to those previously identified in confirmed members of the virus family Hypoviridae. GH2 dsRNA was more distantly related to Cryphonectria hypovirus (CHV) 1-EP713 and CHV2-NB58 than the latter two were to each other but has features in common with each of those viruses. We propose that the GH2 virus be included in this taxon as a member of the genus Hypovirus, representing a strain of a new species, CHV3