bluuebunny/crossref_metadata_2025_split huggingface dataset:
Tight Binding of Influenza Virus Hemagglutinin to Its Receptor Interferes with Fusion Pore Dilation
Authors: Ohuchi Masanobu , Ohuchi Reiko , Sakai Tatsuya , Matsumoto Akira 
Published: November 2002

ABSTRACT
Deletion of oligosaccharide side chains near the receptor binding site of influenza virus A/USSR/90/77 (H1N1) hemagglutinin (HA) enhanced the binding of HA to erythrocyte receptors, as was also observed with A/FPV/Rostock/34 (H7N1). Correlated with the enhancement of binding activity, the cell fusion activity of HA was reduced. A mutant HA in which three oligosaccharide side chains were deleted showed the highest level of binding and the lowest level of fusion among the HAs tested. The cell fusion activity of the oligosaccharide deletion mutant of HA, however, was drastically elevated when the binding activity was reduced by deletion of four amino acids adjacent to the receptor binding site. Thus, a reciprocal relationship was observed between the receptor binding and the cell fusion activities of H1/USSR HA. No difference was observed, however, in lipid mixing activity, so-called hemifusion, between wild-type (WT) and oligosaccharide deletion mutant HAs. Soluble dye transfer testing showed that even the HA with the lowest cell fusion activity was able to form fusion pores through which a small molecule such as calcein could pass. However, electron microscopic studies revealed that a large molecule such as hemoglobin hardly passed through the fusion pores formed by the mutant HA, whereas hemoglobin did efficiently pass through those formed by the WT HA. These results suggested that interference in the process of dilation of fusion pores occurs when the binding of HA to the receptor is too tight. Since the viral nucleocapsid is far larger than hemoglobin, appropriate receptor binding affinity is important for virus entry.
Frequencies of Ex Vivo-Activated Human Immunodeficiency Virus Type 1-Specific Gamma-Interferon-Producing CD8+T Cells in Infected Children Correlate Positively with Plasma Viral Load
Authors: Buseyne Florence , Scott-Algara Daniel , Porrot Françoise , Corre Béatrice , Bellal Nassima , Burgard Marianne , Rouzioux Christine , Blanche Stéphane , Rivière Yves 
Published: November 2002

ABSTRACTHIV-specific CD8+T cells are critical in controlling human immunodeficiency virus (HIV) replication. We present the evaluation of a gamma-interferon (IFN-γ)-based enzyme linked immunospot (ELISPOT) assay for the quantification of HIV-specific CD8+T cells from HIV-infected children. We studied 20 HLA-A∗0201-positive HIV-infected children. The IFN-γ production in response to stimulation with two HLA-A∗0201-restricted immunodominant CD8 epitopes (SLYNTVATL [SL9] in Gag and ILKEPVHGV [IV9] in Pol) was tested using the ELISPOT assay. The results were compared to labeling with the corresponding tetramers. Among the 20 children, 18 had detectable responses against the SL9 and/or the IV9 epitope using the ELISPOT assay (medians, 351 and 134 spot-forming cells/106peripheral blood mononuclear cells, respectively). Comparison of results from the tetramer and ELISPOT assays suggests that only a fraction of HIV-specific CD8+T cells were able to produce IFN-γ. Most importantly, we found that the frequencies of IFN-γ-producing CD8+T cells were positively correlated with the viral load whereas the frequencies of tetramer-binding CD8+T cells were not. The high sensitivity of the ELISPOT assay and the fact that this functional assay provided information different from that of tetramer labeling support its use for measurement of HIV-specific CD8+T cells. In conclusion, our results show that the ex vivo-activated IFN-γ-producing HIV-specific CD8+-T-cell subset is dependent upon continuous antigenic stimulation.
Virus-Specific CD8+Lymphocytes Share the Same Effector-Memory Phenotype but Exhibit Functional Differences in Acute Hepatitis B and C
Authors: Urbani Simona , Boni Carolina , Missale Gabriele , Elia Gianfranco , Cavallo Cristina , Massari Marco , Raimondo Giovanni , Ferrari Carlo 
Published: November 2002

ABSTRACTHepatitis B and hepatitis C viruses (HBV and HCV) are both noncytopathic and can cause acute and chronic infections of the liver. Although they share tropism for the same organ, development of chronic hepatitis is much more frequent following HCV infection, suggesting different mechanisms of viral persistence. In this study, we show that circulating HBV- and HCV-specific tetramer-positive CD8 cells during the acute phase of hepatitis B and C belong almost entirely to an effector-memory subset (CCR7−CD45RA−). Despite this phenotypic similarity, HBV- and HCV-specific CD8 cells show striking functional differences. HBV-specific tetramer-positive CD8 cells express high perforin content ex vivo, expand vigorously, and display efficient cytotoxic activity and gamma interferon (IFN-γ) production upon peptide stimulation. A comparable degree of functional efficiency is maintained after the resolution of hepatitis B. In contrast, HCV-specific CD8 cells in the acute phase of hepatitis C express significantly lower levels of perforin molecules ex vivo and show depressed CD8 function in terms of proliferation, lytic activity, and IFN-γ production, irrespective of the final outcome of the disease. This defect is transient, because HCV-specific CD8 cells can progressively improve their function in patients with self-limited hepatitis C, while the CD8 function remains persistently depressed in subjects with a chronic evolution.
Characterization of the Transcription Profile of Adeno-Associated Virus Type 5 Reveals a Number of Unique Features Compared to Previously Characterized Adeno-Associated Viruses
Authors: Qiu Jianming , Nayak Ramnath , Tullis Gregory E. , Pintel David J. 
Published: November 2002

ABSTRACT

We report the initial characterization of adeno-associated virus type 5 (AAV5) RNAs generated following viral infection and the construction of a replicating infectious clone of AAV5. While the basic transcription profile of AAV5 was similar to that of AAV2, there were also significant differences. Mapping of the AAV5 transcripts demonstrated an efficient transcription initiation site within the AAV5 inverted terminal repeat (ITR), and mapping of the AAV5 intron revealed that it is considerably smaller than that of AAV2. Furthermore, in contrast to the case for AAV2, neither the Rep protein nor additional adenovirus gene products were required to achieve efficient promoter activity and pre-mRNA splicing following transfection of an AAV5
rep/cap
plasmid clone lacking the ITRs into 293 cells. Perhaps most surprisingly, RNAs generated from both the AAV5 P7 and P19 promoters were efficiently polyadenylated at a site lying within the intronic region in the center of the genome. Because P7- and P19-generated transcripts are polyadenylated at this site and not spliced, Rep78 and Rep52 were the only Rep proteins detected during AAV5 infection.
Elevated Levels of Circulating Interleukin-18 in Human Immunodeficiency Virus-Infected Individuals: Role of Peripheral Blood Mononuclear Cells and Implications for AIDS Pathogenesis
Authors: Ahmad Rasheed , Sindhu Sardar T. A. , Toma Emil , Morisset Richard , Ahmad Ali 
Published: November 2002

ABSTRACTOriginally identified as the gamma interferon-inducing factor, interleukin-18 (IL-18) was rediscovered as a proinflammatory cytokine related to the IL-1 family of cytokines that plays an important role in both innate and adaptive immune responses against viruses and intracellular pathogens. Despite its importance in inducing and regulating immune responses, relatively little is known about its production in HIV infection. We report here significantly (P< 0.05) elevated levels of this cytokine in the sera of human immunodeficiency virus (HIV)-infected/AIDS patients compared to those of HIV-seronegative healthy persons. Surprisingly, the peripheral blood mononuclear cells (PBMC) from HIV-infected/AIDS patients were compromised in the ability to upregulate IL-18 gene expression and produce this cytokine with and without lipopolysaccharide (LPS) stimulation. A significant positive correlation (P< 0.05) existed between the concentration of IL-18 in serum and its production from PBMC of HIV-seronegative healthy individuals but not those of HIV-infected/AIDS patients. Furthermore, the patients' PBMC expressed relatively reduced levels of activated caspase-1 constitutively as well as in response to LPS stimulation. Our data suggest the involvement of transforming growth factor beta (TGF-β) in suppressing IL-18 production from the patients' PBMC for the following reasons. (i) In in vitro studies it suppressed the production of IL-18 from PBMC. (ii) Its levels were significantly higher in the plasma of patients compared to that of control subjects. (iii) A significant negative correlation existed between the concentrations of TGF-β in plasma and of IL-18 in serum of the patients. The elevated levels of IL-18 in the serum of HIV-infected individuals may contribute to AIDS pathogenesis, whereas its compromised production from their PBMC in response to stimuli may reduce their innate defense to opportunistic intracellular pathogens.
Cytoplasmic Expression of mRNAs Containing the Internal Ribosome Entry Site and 3′ Noncoding Region of Hepatitis C Virus: Effects of the 3′ Leader on mRNA Translation and mRNA Stability
Authors: Kong Li Kuo , Sarnow Peter 
Published: November 2002

ABSTRACT
Translation initiation in many eukaryotic mRNAs is modulated by an interaction between the cap binding protein complex, bound to the 5′ end of the mRNA, and the polyadenosine binding protein, bound to the 3′-terminal polyadenosine sequences. A few cellular and viral mRNAs, such as the hepatitis C virus (HCV) mRNA genome, lack 3′-terminal polyadenosine sequences. For such mRNAs, the question of whether their 3′-end sequences also regulate the initiation phase of protein synthesis via an interaction with their 5′ ends has received intense scrutiny. For HCV mRNA, various experimental designs have led to conflicting interpretations, that the 3′ end of the RNA can modulate translation initiation either in a positive or in a negative fashion. To examine the possibility of end-to-end communication in HCV in detail, mRNAs containing the HCV internal ribosome entry site linked to a luciferase coding region, followed by different 3′ noncoding regions, were expressed in the cytoplasm of cultured cells by T7 RNA polymerase. The intracellular translation efficiencies, steady-state levels, stabilities, and 3′-end sequences of these chimeric RNAs were examined. It was found that the HCV 3′ noncoding region modulates neither the translation nor the stability of the mRNAs. Thus, there is no detectable end-to-end communication in cytoplasmically expressed chimeric mRNAs containing the HCV noncoding regions. However, it remains an open question whether end-to-end communication occurs in full-length HCV mRNAs in the infected liver.
Covalent Modifications of the Ebola Virus Glycoprotein
Authors: Jeffers Scott A. , Sanders David Avram , Sanchez Anthony 
Published: November 2002

ABSTRACT

The role of covalent modifications of the Ebola virus glycoprotein (GP) and the significance of the sequence identity between filovirus and avian retrovirus GPs were investigated through biochemical and functional analyses of mutant GPs. The expression and processing of mutant GPs with altered N-linked glycosylation, substitutions for conserved cysteine residues, or a deletion in the region of O-linked glycosylation were analyzed, and virus entry capacities were assayed through the use of pseudotyped retroviruses. Cys-53 was the only GP
1
(∼130 kDa) cysteine residue whose replacement resulted in the efficient secretion of GP
1
, and it is therefore proposed that it participates in the formation of the only disulfide bond linking GP
1
to GP
2
(∼24 kDa). We propose a complete cystine bridge map for the filovirus GPs based upon our analysis of mutant Ebola virus GPs. The effect of replacement of the conserved cysteines in the membrane-spanning region of GP
2
was found to depend on the nature of the substitution. Mutations in conserved N-linked glycosylation sites proved generally, with a few exceptions, innocuous. Deletion of the O-linked glycosylation region increased GP processing, incorporation into retrovirus particles, and viral transduction. Our data support a common evolutionary origin for the GPs of Ebola virus and avian retroviruses and have implications for gene transfer mediated by Ebola virus GP-pseudotyped retroviruses.
African Swine Fever Virus Polyproteins pp220 and pp62 Assemble into the Core Shell
Authors: Andrés Germán , Alejo Alí , Salas José , Salas María L. 
Published: November 2002

ABSTRACT
African swine fever virus (ASFV), a complex enveloped DNA virus, expresses two polyprotein precursors, pp220 and pp62, which after proteolytic processing give rise to several major components of the virus particle. We have analyzed the structural role of polyprotein pp62, the precursor form of mature products p35 and p15, in virus morphogenesis. Densitometric analysis of one- and two-dimensional gels of purified virions showed that proteins p35 and p15, as well as the pp220-derived products, are present in equimolecular amounts in the virus particle. Immunoelectron microscopy revealed that the pp62-derived products localize at the core shell, a matrix-like domain placed between the DNA-containing nucleoid and the inner envelope, where the pp220-derived products are also localized. Pulse-chase experiments indicated that the processing of both polyprotein precursors is concomitant with virus assembly. Furthermore, using inducible ASFV recombinants, we show that pp62 processing requires the expression of the pp220 core precursor, whereas the processing of both precursors pp220 and pp62 is dependent on expression of the major capsid protein p72. Interestingly, when p72 expression is blocked, unprocessed pp220 and pp62 polyproteins assemble into aberrant zipper-like elements consisting of an elongated membrane-bound protein structure reminiscent of the core shell. Moreover, the two polyproteins, when coexpressed in COS cells, interact with each other to form zipper-like structures. Together, these findings indicate that the mature products derived from both polyproteins, which collectively account for about 30% of the virion protein mass, are the basic components of the core shell and that polyprotein processing represents a maturational process related to ASFV morphogenesis.
Evidence that Replication of the Antitumor Adenovirus ONYX-015 Is Not Controlled by the p53 and p14
ARF
Tumor Suppressor Genes
Authors: Edwards Sara J. , Dix Brett R. , Myers Colleen J. , Dobson-Le Deirdre , Huschtscha Lily , Hibma Merilyn , Royds Janice , Braithwaite Antony W. 
Published: November 2002

ABSTRACT

The adenovirus mutant ONYX-015 is in phase III clinical trials as a novel antitumor therapy. Its apparent efficacy is thought to be due to its ability to replicate selectively in tumor cells defective in the signaling pathway for p53. Recent data have shown that p14
ARF
, a positive regulator of p53, inhibits ONYX-015 replication in cells with a wild-type p53, a phenotype that characterizes normal cells. We, however, found that ONYX-015 activates p53 in tumor cells and in normal cells and that this can occur without p14
ARF
induction. We also show that ONYX-015 is not attenuated in cells with functional p53, whether or not p14
ARF
is expressed, and that where attenuation does occur, it is cell type specific.
Coronaviruses Maintain Viability despite Dramatic Rearrangements of the Strictly Conserved Genome Organization
Authors: de Haan Cornelis A. M. , Volders Haukeline , Koetzner Cheri A. , Masters Paul S. , Rottier Peter J. M. 
Published: November 2002

ABSTRACT

Despite their high frequency of RNA recombination, the plus-strand coronaviruses have a characteristic, strictly conserved genome organization with the essential genes occurring in the order 5′-polymerase (
pol
)-S-E-M-N-3′. We have investigated the significance of this remarkable conservation by rearrangement of the murine coronavirus genome through targeted recombination. Thus, viruses were prepared with the following gene order: 5′-
pol
-S-M-E-N-3′, 5′-
pol
-S-N-E-M-3′, 5′-
pol
-M-S-E-N-3′, and 5′-
pol
-E-M-S-N-3′. All of these viruses were surprisingly viable, and most viruses replicated in cell culture with growth characteristics similar to those of the parental virus. The recombinant virus with the gene order 5′-
pol
-E-M-S-N-3′ was also tested for the ability to replicate in the natural host, the mouse. The results indicate that the canonical coronavirus genome organization is not essential for replication in vitro and in vivo. Deliberate rearrangement of the viral genes may be useful in the generation of attenuated coronaviruses, which due to their reduced risk of generating viable viruses by recombination with circulating field viruses, would make safer vaccines.
The Epstein-Barr Virus Immediate-Early Protein BZLF1 Regulates p53 Function through Multiple Mechanisms
Authors: Mauser Amy , Saito Shin'ichi , Appella Ettore , Anderson Carl W. , Seaman William T. , Kenney Shannon 
Published: November 2002

ABSTRACT
The Epstein-Barr virus (EBV) immediate-early protein BZLF1 is a transcriptional activator that mediates the switch between the latent and the lytic forms of EBV infection. It was previously reported that BZLF1 inhibits p53 transcriptional function in reporter gene assays. Here we further examined the effects of BZLF1 on p53 function by using a BZLF1-expressing adenovirus vector (AdBZLF1). Infection of cells with the AdBZLF1 vector increased the level of cellular p53 but prevented the induction of p53-dependent cellular target genes, such as p21 and MDM2. BZLF1-expressing cells had increased p53-specific DNA binding activity in electrophoretic mobility shift assays, increased p53 phosphorylation at multiple residues (including serines 6, 9, 15, 33, 46, 315, and 392), and increased acetylation at lysine 320 and lysine 382. Thus, the inhibitory effects of BZLF1 on p53 transcriptional function cannot be explained by its effects on p53 phosphorylation, acetylation, or DNA binding activity. BZLF1 substantially reduced the level of cellular TATA binding protein (TBP) in both normal human fibroblasts and A549 cells, and the inhibitory effects of BZLF1 on p53 transcriptional function could be partially rescued by the overexpression of TBP. Thus, BZLF1 has numerous effects on p53 posttranslational modification but may inhibit p53 transcriptional function in part through an indirect mechanism involving the suppression of TBP expression.
Mechanism of De Novo Initiation by the Hepatitis C Virus RNA-Dependent RNA Polymerase: Role of Divalent Metals
Authors: Ranjith-Kumar C. T. , Kim Young-Chan , Gutshall Les , Silverman Carol , Khandekar Sanjay , Sarisky Robert T. , Kao C. Cheng 
Published: November 2002

ABSTRACT

We functionally analyzed the role of metal ions in RNA-dependent RNA synthesis by three recombinant RNA-dependent RNA polymerases (RdRps) from GB virus-B (GBV), bovine viral diarrhea virus (BVDV), and hepatitis C virus (HCV), with emphasis on the HCV RdRp. Using templates capable of both de novo initiation and primer extension and RdRps purified in the absence of metal, we found that only reactions with exogenously provided Mg
2+
and Mn
2+
gave rise to significant amounts of synthesis. Mg
2+
and Mn
2+
affected the mode of RNA synthesis by the three RdRps. Both metals supported primer-dependent and de novo-initiated RNA by the GBV RdRp, while Mn
2+
significantly increased the amount of de novo-initiated products by the HCV and BVDV RdRps. For the HCV RdRp, Mn
2+
reduced the

K
m

for the initiation nucleotide, a GTP, from 103 to 3 μM. However, it increased de novo initiation even at GTP concentrations that are comparable to physiological levels. We hypothesize that a change in RdRp structure occurs upon GTP binding to prevent primer extension. Analysis of deleted proteins revealed that the C terminus of the HCV RdRp plays a role in Mn
2+
-induced de novo initiation and can contribute to the suppression of primer extension. Spectroscopy examining the intrinsic fluorescence of tyrosine and tryptophan residues in the HCV RdRp produced results consistent with the protein undergoing a conformational change in the presence of metal. These results document the fact that metal can affect de novo initiation or primer extension by flaviviral RdRps.
Requirements for De Novo Initiation of RNA Synthesis by Recombinant Flaviviral RNA-Dependent RNA Polymerases
Authors: Ranjith-Kumar C. T. , Gutshall Les , Kim Min-Ju , Sarisky Robert T. , Kao C. Cheng 
Published: November 2002

ABSTRACT

RNA-dependent RNA polymerases (RdRps) that initiate RNA synthesis by a de novo mechanism should specifically recognize the template initiation nucleotide, T1, and the substrate initiation nucleotide, the NTPi. The RdRps from hepatitis C virus (HCV), bovine viral diarrhea virus (BVDV), and GB virus-B all can initiate RNA synthesis by a de novo mechanism. We used RNAs and GTP analogs, respectively, to examine the use of the T1 nucleotide and the initiation nucleotide (NTPi) during de novo initiation of RNA synthesis. The effects of the metal ions Mg
2+
and Mn
2+
on initiation were also analyzed. All three viral RdRps require correct base pairing between the T1 and NTPi for efficient RNA synthesis. However, each RdRp had some distinct tolerances for modifications in the T1 and NTPi. For example, the HCV RdRp preferred an NTPi lacking one or more phosphates regardless of whether Mn
2+
was present or absent, while the BVDV RdRp efficiently used GDP and GMP for initiation of RNA synthesis only in the presence of Mn
2+
. These and other results indicate that although the three RdRps share a common mechanism of de novo initiation, each has distinct preferences.
Healthy Skin of Many Animal Species Harbors Papillomaviruses Which Are Closely Related to Their Human Counterparts
Authors: Antonsson Annika , Hansson Bengt Göran 
Published: November 2002

ABSTRACT
Papillomaviruses associated with clinical symptoms have been found in many vertebrate species. In this study, we have used an L1 gene consensus PCR test designed to detect a broad spectrum of human skin papillomaviruses to analyze swab samples from healthy skin of 111 animals belonging to 19 vertebrate species. In eight of the species, papillomavirus DNA was found with the following prevalences: chimpanzees, 9 of 11 samples positive; gorillas, 3 of 4; long-tailed macaques, 14 of 16; spider monkeys, 2 of 2; ruffed lemurs, 1 of 2; cows, 6 of 10; European elks, 4 of 4; aurochs, 1 of 1. In total, 53 new putative animal papillomavirus types were found. The results show that skin papillomaviruses can be detected in healthy skin from many different animal species and are sufficiently related genetically to their human counterparts to be identified by a human skin papillomavirus primer set (FAP59 and FAP64).