bluuebunny/crossref_metadata_2025_split huggingface dataset:
Targeting of Hepatitis C Virus Core Protein to Mitochondria through a Novel C-Terminal Localization Motif
Authors: Schwer Björn , Ren Shaotang , Pietschmann Thomas , Kartenbeck Jürgen , Kaehlcke Katrin , Bartenschlager Ralf , Yen T. S. Benedict , Ott Melanie 
Published: July 2004

ABSTRACT
The hepatitis C virus (HCV) core protein represents the first 191 amino acids of the viral precursor polyprotein and is cotranslationally inserted into the membrane of the endoplasmic reticulum (ER). Processing at position 179 by a recently identified intramembrane signal peptide peptidase leads to the generation and potential cytosolic release of a 179-amino-acid matured form of the core protein. Using confocal microscopy, we observed that a fraction of the mature core protein colocalized with mitochondrial markers in core-expressing HeLa cells and in Huh-7 cells containing the full-length HCV replicon. Subcellular fractionation confirmed this observation and showed that the core protein associates with purified mitochondrial fractions devoid of ER contaminants. The core protein also fractionated with mitochondrion-associated membranes, a site of physical contact between the ER and mitochondria. Using immunoelectron microscopy and in vitro mitochondrial import assays, we showed that the core protein is located on the mitochondrial outer membrane. A stretch of 10 amino acids within the hydrophobic C terminus of the processed core protein conferred mitochondrial localization when it was fused to green fluorescent protein. The location of the core protein in the outer mitochondrial membrane suggests that it could modulate apoptosis or lipid transfer, both of which are associated with this subcellular compartment, during HCV infection.
Intracellular Processing of Human Herpesvirus 6 Glycoproteins Q1 and Q2 into Tetrameric Complexes Expressed on the Viral Envelope
Authors: Akkapaiboon Pilailuk , Mori Yasuko , Sadaoka Tomohiko , Yonemoto Sayoko , Yamanishi Koichi 
Published: July 2004

ABSTRACT

Human herpesvirus 6 (HHV-6) glycoproteins H and L (gH and gL, respectively) and the 80-kDa form of glycoprotein Q (gQ-80K) form a heterotrimeric complex that is found on the viral envelope and that is a viral ligand for human CD46. Besides gQ-80K, the gQ gene encodes an additional product whose mature molecular mass is 37 kDa (gQ-37K) and which is derived from a different transcript. Therefore, we designated gQ-80K as gQ1 and gQ-37K as gQ2. We show here that gQ2 also interacts with the gH-gL-gQ1 complex in HHV-6-infected cells and in virions. To examine how these components interact in HHV-6-infected cells, we performed pulse-chase studies. The results demonstrated that gQ2-34K, which is endo-β-
N
-acetylglucosaminidase H sensitive and which is the precursor form of gQ2-37K, associates with gQ1-74K, which is the precursor form of gQ1-80K, within 30 min of the pulse period. After a 1-h chase, these precursor forms had associated with the gH-gL dimer. Interestingly, an anti-gH monoclonal antibody coimmunoprecipitated mainly gQ1-80K and gQ2-37K, with little gQ1-74K or gQ2-34K. These results indicate that although gQ2-34K and gQ1-74K interact in the endoplasmic reticulum, the gH-gL-gQ1-80K-gQ2-37K heterotetrameric complex arises in the post-endoplasmic reticulum compartment. The mature complex is subsequently incorporated into viral particles.
Functional Antagonism of Chemokine Receptor CCR1 Reduces Mortality in Acute Pneumovirus Infection In Vivo
Authors: Bonville Cynthia A. , Lau Vincent K. , DeLeon Jordana M. , Gao Ji-Liang , Easton Andrew J. , Rosenberg Helene F. , Domachowske Joseph B. 
Published: July 2004

ABSTRACT

We present an antiviral-immunomodulatory therapeutic strategy involving the chemokine receptor antagonist Met-RANTES, which yields significant survival in the setting of an otherwise fatal respiratory virus infection. In previous work, we demonstrated that infection with the natural rodent pathogen pneumonia virus of mice involves robust virus replication accompanied by cellular inflammation modulated by the CC chemokine macrophage inflammatory protein 1α (MIP-1α). We found that the antiviral agent ribavirin limited virus replication in vivo but had no impact on morbidity and mortality associated with this disease in the absence of immunomodulatory control. We show here that ribavirin reduces mortality, from 100% to 10 and 30%, respectively, in gene-deleted CCR1
−/−
mice and in wild-type mice treated with the small-molecule chemokine receptor antagonist, Met-RANTES. As MIP-1α-mediated inflammation is a common response to several distantly related respiratory virus pathogens, specific antiviral therapy in conjunction with blockade of the MIP-1α/CCR1 inflammatory cascade may ultimately prove to be a useful, generalized approach to severe respiratory virus infection and its pathological sequelae in human subjects.
Transport of African Swine Fever Virus from Assembly Sites to the Plasma Membrane Is Dependent on Microtubules and Conventional Kinesin
Authors: Jouvenet Nolwenn , Monaghan Paul , Way Michael , Wileman Thomas 
Published: July 2004

ABSTRACT
African swine fever virus (ASFV) is a large DNA virus that assembles in perinuclear viral factories located close to the microtubule organizing center. In this study, we have investigated the mechanism by which ASFV reaches the cell surface from the site of assembly. Immunofluorescence microscopy revealed that at 16 h postinfection, mature virions were aligned along microtubules. Furthermore, virus movement to the cell periphery was inhibited when microtubules were depolymerized by nocodazole. In addition, ASFV infection resulted in the increased acetylation of microtubules as well as their protection against depolymerization by nocodazole. Immunofluorescence microscopy showed that conventional kinesin was recruited to virus factories and to a large fraction of virus particles in the cytoplasm. Consistent with a role for conventional kinesin during ASFV egress to the cell periphery, overexpression of the cargo-binding domain of the kinesin light chain severely inhibited the movement of particles to the plasma membrane. Based on our observations, we propose that ASFV is recognized as cargo by conventional kinesin and uses this plus-end microtubule motor to move from perinuclear assembly sites to the plasma membrane.
Compartmentalization of VP16 in Cells Infected with Recombinant Herpes Simplex Virus Expressing VP16-Green Fluorescent Protein Fusion Proteins
Authors: La Boissière Sylvie , Izeta Ander , Malcomber Sophie , O'Hare Peter 
Published: July 2004

ABSTRACT
VP16 is an essential structural protein of herpes simplex virus. It plays important roles in immediate-early transcriptional regulation, in the modulation of the activities of other viral components, and in the pathway of assembly and egress of infectious virions. To gain further insight into the compartmentalization of this multifunctional protein we constructed and characterized recombinant viruses expressing VP16 linked to the green fluorescent protein (GFP). These viruses replicate with virtually normal kinetics and yields and incorporate the fusion protein into the virion, resulting in autofluorescent particles. De novo-synthesized VP16-GFP was first detected in a diffuse pattern within the nucleus. Nuclear VP16-GFP was progressively recruited to replication compartments, which coalesced into large globular domains. By 10 to 12 h after infection additional distinct foci containing VP16-GFP could be seen, almost exclusively located at the periphery of the replication compartments. At the same time pronounced accumulation was observed in the cytoplasm, first in a diffuse pattern and then accumulating in vesicle-like compartments which were concentrated in an asymmetric fashion reminiscent of the Golgi. Inhibition of DNA replication resulted in prolonged diffuse nuclear distribution with minimal cytoplasmic accumulation. Treatment with brefeldin disrupted the cytoplasm vesicular pattern, resulting in redistributed large foci. Time-lapse microscopy demonstrated various dynamic features of infection, including the active induction of very long cellular projections (up to 100 μM). Vesicular clusters containing VP16 were transported within projections to the termini, which developed bulbous ends and appeared to embed into the membranes of adjacent uninfected cells.
Coexpression of UL20p and gK Inhibits Cell-Cell Fusion Mediated by Herpes Simplex Virus Glycoproteins gD, gH-gL, and Wild-Type gB or an Endocytosis-Defective gB Mutant and Downmodulates Their Cell Surface Expression
Authors: Avitabile Elisa , Lombardi Giulia , Gianni Tatiana , Capri Miriam , Campadelli-Fiume Gabriella 
Published: July 2004

ABSTRACT

Syncytium formation in cells that express herpes simplex virus glycoprotein B (gB), gD, gH, and gL is blocked by gK (E. Avitabile, G. Lombardi, and G. Campadelli-Fiume, J. Virol.
77:
6836-6844, 2003). Here, we report the results of two series of experiments. First, UL20 protein (UL20p) expression weakly inhibited cell-cell fusion. Coexpression of UL20p and gK drastically reduced fusion in a cell-line-dependent manner, with the highest inhibition in BHK cells. Singly expressed UL20p and gK localized at the endoplasmic reticulum and nuclear membranes. When they were coexpressed, both proteins relocalized to the Golgi apparatus. Remarkably, in cells that coexpressed UL20p and gK, the antifusion activity correlated with a downmodulation of gD, gB, gH, and gL cell surface expression. Second, gB
Δ867
has a partial deletion in the cytoplasmic tail that removed endocytosis motifs. Whereas wild-type (wt) gB was internalized in vesicles lined with the endosomal marker Rab5, gB
Δ867
was not internalized, exhibited enhanced cell surface expression, and was more efficient in mediating cell-cell fusion than wt gB. The antifusion activity of UL20p and gK was also exerted when gB
Δ867
replaced wt gB in the cell fusion assay. These studies show that the gB C tail carries a functional endocytosis motif(s) and that the removal of the motif correlated with increased gB surface expression and increased fusion activity. We conclude that cell-cell fusion in wt-virus-infected cells is negatively controlled by at least two mechanisms. The novel mechanism described here involves the concerted action of UL20p and gK and correlates with a moderate but consistent reduction in the cell surface expression of the fusion glycoproteins. This mechanism is independent of the one exerted through endocytosis-mediated downmodulation of gB from the plasma membrane.
Degenerate In Vitro Genetic Selection Reveals Mutations That Diminish Alfalfa Mosaic Virus RNA Replication without Affecting Coat Protein Binding
Authors: Rocheleau Gail , Petrillo Jessica , Guogas Laura , Gehrke Lee 
Published: July 2004

ABSTRACTThe alfalfa mosaic virus (AMV) RNAs are infectious only in the presence of the viral coat protein; however, the mechanisms describing coat protein's role during replication are disputed. We reasoned that mechanistic details might be revealed by identifying RNA mutations in the 3′-terminal coat protein binding domain that increased or decreased RNA replication without affecting coat protein binding. Degenerate (doped) in vitro genetic selection, based on a pool of randomized 39-mers, was used to select 30 variant RNAs that bound coat protein with high affinity. AUGC sequences that are conserved among AMV and ilarvirus RNAs were among the invariant nucleotides in the selected RNAs. Five representative clones were analyzed in functional assays, revealing diminished viral RNA expression resulting from apparent defects in replication and/or translation. These data identify a set of mutations, including G-U wobble pairs and nucleotide mismatches in the 5′ hairpin, which affect viral RNA functions without significant impact on coat protein binding. Because the mutations associated with diminished function were scattered over the 3′-terminal nucleotides, we considered the possibility that RNA conformational changes rather than disruption of a precise motif might limit activity. Native polyacrylamide gel electrophoresis experiments showed that the 3′ RNA conformation was indeed altered by nucleotide substitutions. One interpretation of the data is that coat protein binding to the AUGC sequences determines the orientation of the 3′ hairpins relative to one another, while local structural features within these hairpins are also critical determinants of functional activity.
Cytokine-Mediated Downregulation of Coxsackievirus-Adenovirus Receptor in Endothelial Cells
Authors: Vincent Theresa , Pettersson Ralf F. , Crystal Ronald G. , Leopold Philip L. 
Published: July 2004

ABSTRACTEndothelial cells have the ability to change their complement of cell surface proteins in response to inflammatory cytokines. We hypothesized that the expression of the coxsackievirus-adenovirus receptor (CAR), a viral receptor and putative cell-cell adhesion molecule, may be altered during the response of endothelial cells to inflammation. To test this hypothesis, we evaluated CAR protein and mRNA levels in human umbilical vein endothelial cells after they were exposed to tumor necrosis factor alpha, gamma interferon, or a combination of the two cytokines. Flow cytometric and Western blot analyses indicated that cytokine treatment led to a synergistic decrease in CAR protein expression. A Western blot analysis showed that CAR levels decreased to 16% ± 4% or 1% ± 4% of the CAR protein levels in untreated cells with either 24 or 48 h of cytokine treatment, respectively. Quantitative reverse transcription-PCR demonstrated that the combination treatment caused CAR mRNA levels to decrease to 21% ± 12% or 5% ± 3% of the levels in untreated cells after a 24- or 48-h cytokine treatment, respectively. Reduced CAR expression led to a decrease in adenovirus (Ad) binding of 80% ± 3% (compared with untreated endothelial cells), with a subsequent decrease in Ad-mediated gene transfer that was dependent on the dose and duration of cytokine treatment but not on the dose of Ad. A similar decrease in CAR protein level and susceptibility to Ad infection was observed in human microvascular endothelial cells, while CAR expression on normal human bronchial epithelial cells or A549 lung epithelial cells was less affected by cytokine treatments. Taken together, the data demonstrate that inflammatory cytokines decrease CAR mRNA and protein expression with a concomitant decrease in Ad binding, reflecting the impact of cell physiology on the function of CAR and the potential effect of inflammation on the ability of Ad to transfer genes to endothelial cells.
Interactions between the Inner and Outer Capsids of Bluetongue Virus
Authors: Nason Emma L. , Rothagel Rosalba , Mukherjee Sharmila K. , Kar Alak Kanti , Forzan Mario , Prasad B. V. Venkataram , Roy Polly 
Published: July 2004

ABSTRACT
Bluetongue virus is a large and structurally complex virus composed of three concentric capsid layers that surround 10 segments of a double-stranded RNA genome. X-ray crystallographic analysis of the particles without the outer capsid layer has provided atomic structural details of VP3 and VP7, which form the inner two layers. However, limited structural information is available on the other five proteins in the virion—two of which are important for receptor recognition, hemagglutination, and membrane interaction—are in the outer layer, and the others, important for endogenous transcriptase activity are internal. Here we report the electron cryomicroscopy (cryo-EM) reconstruction of the mature particle, which shows that the outer layer has a unique non-T = 13 icosahedral organization consisting of two distinct triskelion and globular motifs interacting extensively with the underlying T = 13 layer. Comparative cryo-EM analysis of the recombinant corelike particles has shown that VP1 (viral polymerase) and VP4 (capping enzyme) together form a flower-shaped structure attached to the underside of VP3, directly beneath the fivefold axis. The structural data have been substantiated by biochemical studies demonstrating the interactions between the individual outer and inner capsid proteins.
Isolation of Capsid Protein Dimers from the Tick-Borne Encephalitis Flavivirus and In Vitro Assembly of Capsid-Like Particles
Authors: Kiermayr Stefan , Kofler Regina M. , Mandl Christian W. , Messner Paul , Heinz Franz X. 
Published: July 2004

ABSTRACT
Flaviviruses have a spherical capsid that is composed of multiple copies of a single capsid protein and, in contrast to the viral envelope, apparently does not have an icosahedral structure. So far, attempts to isolate distinct particulate capsids and soluble forms of the capsid protein from purified virions as well as to assemble capsid-like particles in vitro have been largely unsuccessful. Here we describe the isolation of nucleocapsids from tick-borne encephalitis (TBE) virus and their disintegration into a capsid protein dimer by high-salt treatment. Purified capsid protein dimers could be assembled in vitro into capsid-like particles when combined with in vitro transcribed viral RNA. Particulate structures could also be obtained when single-stranded DNA oligonucleotides were used. These data suggest that the dimeric capsid protein functions as a basic building block in the assembly process of flaviviruses.
Appearance of the Bona Fide Spiral Tubule of Orf Virus Is Dependent on an Intact 10-Kilodalton Viral Protein
Authors: Spehner D. , De Carlo S. , Drillien R. , Weiland F. , Mildner K. , Hanau D. , Rziha H.-J. 
Published: July 2004

ABSTRACT

Parapoxviruses can be morphologically distinguished from other poxviruses in conventional negative staining electron microscopy (EM) by their ovoid appearance and the spiral tubule surrounding the virion's surface. However, this technique may introduce artifacts. We have examined Orf virus (ORFV; the prototype species of the
Parapoxvirus
genus) by cryoelectron microscopy (cryo-EM) and cryo-negative staining EM. From these studies we suggest that the shape and unique spiral tubule are authentic features of the parapoxviruses. We also constructed an ORFV mutant deleted of a gene encoding a 10-kDa protein, which is an orthologue of the vaccinia virus (VACV) 14-kDa fusion protein, and investigated its ultrastructure. This mutant virus multiplied slowly in permissive cells and produced infectious but morphologically aberrant particles. Mutant virions lacked the spiral tubule but displayed short disorganized tubules similar to those observed on the surface of VACV. In addition, thin extensions or loop-like structures were appended to the ORFV mutant particles. We suggest that these appended structures arise from a failure of the mutant virus particles to properly seal and that the sealing activity is dependent on the 10-kDa protein.
Epitope Mapping of the Major Capsid Protein of Type 2 Porcine Circovirus (PCV2) by Using Chimeric PCV1 and PCV2
Authors: Lekcharoensuk Porntippa , Morozov Igor , Paul Prem S. , Thangthumniyom Nattarat , Wajjawalku Worawidh , Meng X. J. 
Published: July 2004

ABSTRACT
Type 2 porcine circovirus (PCV2) is associated with postweaning multisystemic wasting syndrome in pigs, whereas the genetically related type 1 PCV (PCV1) is nonpathogenic. In this study, seven monoclonal antibodies (MAbs) against PCV2-ORF2 capsid protein were generated, biologically characterized, and subsequently used to map the antigenic sites of PCV2 capsid protein by using infectious PCV DNA clones containing PCV1/PCV2-ORF2 chimeras. The PCV1/PCV2-ORF2 chimeras were constructed by serial deletions of PCV2-ORF2 and replacement with the corresponding sequences of the PCV1-ORF2. The reactivities of chimeric PCV1/PCV2 clones in transfected PK-15 cells with the seven MAbs were detected by an immunofluorescence assay (IFA). The chimera (r140) with a deletion of 47 amino acids at the N terminus of PCV2-ORF2 reacted strongly to all seven MAbs. Expanding the deletion of PCV2-ORF2 from residues 47 to 57 (r175) abolished the recognition of MAb 3B7, 3C11, 4A10, 6H2, or 8F6 to the chimera. Further deletion of PCV2-ORF2 to 62 residues disrupted the binding of this chimera to all seven MAbs. IFA reactivities with all MAbs were absent when residues 165 to 233 at the C terminus of PCV2-ORF2 was replaced with that of PCV1-ORF2. Extending the sequence of PCV2-ORF2 from residues 165 (r464) to 185 (r526), 200 (r588), or 224 (r652) restored the ability of the three chimeras to react with MAbs 3C11, 6H2, 9H7, and 12G3 but not with 8F6, 3B7, or 4A10. When the four amino acids at the C terminus of r588 were replaced with that of PCV2-ORF2, the resulting chimera (r588F) reacted with all seven MAbs. The results from this study suggest that these seven MAbs recognized at least five different but overlapping conformational epitopes within residues 47 to 63 and 165 to 200 and the last four amino acids at the C terminus of the PCV2 capsid protein.
Structure and Function of the 3′ Terminal Six Nucleotides of the West Nile Virus Genome in Viral Replication
Authors: Tilgner Mark , Shi Pei-Yong 
Published: July 2004

ABSTRACT

Using a self-replicating reporting replicon of West Nile (WN) virus, we performed a mutagenesis analysis to define the structure and function of the 3′-terminal 6 nucleotides (nt) (5′-GGAUCU
OH
-3′) of the WN virus genome in viral replication. We show that mutations of nucleotide sequence or base pair structure of any of the 3′-terminal 6 nt do not significantly affect viral translation, but exert discrete effects on RNA replication. (i) The flavivirus-conserved terminal 3′ U is optimal for WN virus replication. Replacement of the wild-type 3′ U with a purine A or G resulted in a substantial reduction in RNA replication, with a complete reversion to the wild-type sequence. In contrast, replacement with a pyrimidine C resulted in a replication level similar to that of the 3′ A or G mutants, with only partial reversion. (ii) The flavivirus-conserved 3′ penultimate C and two upstream nucleotides (positions 78 and 79), which potentially base pair with the 3′-terminal CU
OH
, are absolutely essential for viral replication. (iii) The base pair structures, but not the nucleotide sequences at the 3rd (U) and the 4th (A) positions, are critical for RNA replication. (iv) The nucleotide sequences of the 5th (G) position and its base pair nucleotide (C) are essential for viral replication. (v) Neither the sequence nor the base pair structure of the 6th nucleotide (G) is critical for WN virus replication. These results provide strong functional evidence for the existence of the 3′ flavivirus-conserved RNA structure, which may function as contact sites for specific assembly of the replication complex or for efficient initiation of minus-sense RNA synthesis.