bluuebunny/crossref_metadata_2025_split huggingface dataset:
Nucleotide and Amino Acid Complexity of Hepatitis C Virus Quasispecies in Serum and Liver
Authors: Cabot Beatriz , Martell María , Esteban Juan I. , Sauleda Sílvia , Otero Teresa , Esteban Rafael , Guàrdia Jaime , Gómez Jordi 
Published: July 2002

ABSTRACT

The quasispecies nature of the hepatitis C virus (HCV) is thought to play a central role in maintaining and modulating viral replication. Several studies have tried to unravel, through the parameters that characterize HCV circulating quasispecies, prognostic markers of the disease. In a previous work we demonstrated that the parameters of circulating viral quasispecies do not always reflect those of the intrahepatic virus. Here, we have analyzed paired serum and liver quasispecies from 39 genotype 1b-infected patients with different degrees of liver damage, ranging from minimal changes to cirrhosis. Viral level was quantified by real-time reverse transcription-PCR, and viral heterogeneity was characterized through the cloning and sequencing of 540 HCV variants of a genomic fragment encompassing the E2-NS2 junction. Although in 95% of patients, serum and liver consensus HCV amino acid sequences were identical, quasispecies complexity varied considerably between the viruses isolated from each compartment. Patients with HCV quasispecies in serum more complex (26%) than, less complex (28%) than, or similarly complex (41%) to those in liver were found. Among the last, a significant correlation between fibrosis and all the parameters that measure the viral amino acid complexity was found. Correlation between fibrosis and serum viral load was found as well (
R
= 0.7). With regard to the origin of the differences in quasispecies complexity between serum and liver populations, sequence analysis argued against extrahepatic replication as a quantitatively important contributing factor and supported the idea of a differential effect or different selective forces on the virus depending on whether it is circulating in serum or replicating in the liver.
A New Primary Effusion Lymphoma-Derived Cell Line Yields a Highly Infectious Kaposi's Sarcoma Herpesvirus-Containing Supernatant
Authors: Cannon Jennifer S. , Ciufo Dolores , Hawkins Anita L. , Griffin Constance A. , Borowitz Michael J. , Hayward Gary S. , Ambinder Richard F. 
Published: July 2002

ABSTRACT
A primary effusion lymphoma (PEL) cell line, JSC-1, that yields highly infectious Kaposi's sarcoma herpesvirus (KSHV) supernatants was established from the ascitic fluid of a human immunodeficiency virus-positive patient. Flow cytometry showed strong expression of CD45 and lambda light-chain restriction. Southern blot hybridization showed immunoglobulin heavy-chain gene rearrangements in the tumor and the resultant cell line consistent with B-cell lineage. Expression of viral genes was assessed by reverse transcription-PCR and immunohistochemistry. Only latent Epstein-Barr virus (EBV) gene expression was detected, and this was at a low level. In contrast, lytic and latent KSHV gene expression were detected. Tetradecanoyl phorbol acetate and butyrate upregulated KSHV lytic expression, but not EBV lytic expression. Viral supernatant from JSC-1 was much more efficient at infecting primary human dermal microvascular endothelial cells (DMVECs) with KSHV than supernatants from BC-3 or BCP-1 PEL cell lines. Quantitation of viral yields produced by the PEL lines showed at least 2 orders of magnitude more DNase I-resistant KSHV DNA in the JSC-1 supernatant compared to BC-3 or BCP-1 supernatants. KSHV infection in DMVECs was associated with a change from a cobblestone to a spindle shape, LANA expression, and an increased number of mitoses.
A Significant Number of Human Immunodeficiency Virus Epitope-Specific Cytotoxic T Lymphocytes Detected by Tetramer Binding Do Not Produce Gamma Interferon
Authors: Goepfert Paul A. , Bansal Anju , Edwards Bradley H. , Ritter G. Douglas , Tellez Ildefonso , McPherson Sylvia A. , Sabbaj Steffanie , Mulligan Mark J. 
Published: July 2002

ABSTRACTDespite the seemingly important role of cytotoxic T-lymphocyte (CTL) responses in human immunodeficiency virus (HIV) disease pathogenesis, their measurement has relied on a variety of different techniques. We utilized three separate methodologies for the detection of CTLs in a cohort of HIV-infected individuals who were also human leukocyte antigen A2 (HLA-A2) positive. Among the different CTL assays, a correlation was seen only when the Gag epitope-specific HLA A*0201-restricted tetramer assay was compared with the ELISPOT assay performed after stimulation with the Gag epitope; however, this correlation was of borderline statistical significance. On average, the tetramer reagent detected a 10-fold-higher number of cells than were seen to produce gamma interferon by the ELISPOT assay. The implications of this CTL assay comparison and the possibility of phenotypic differences in HIV-specific CD8+T lymphocytes are discussed.
In Vivo Selection of Protease Cleavage Sites by Using Chimeric Sindbis Virus Libraries
Authors: Pacini Laura , Vitelli Alessandra , Filocamo Gessica , Bartholomew Linda , Brunetti Mirko , Tramontano Anna , Steinkühler Christian , Migliaccio Giovanni 
Published: July 2002

ABSTRACT
Identifying protease cleavage sites contributes to our understanding of their specificity and biochemical properties and can help in designing specific inhibitors. One route to this end is the generation and screening of random libraries of cleavage sites. Both synthetic and phage-displayed libraries have been extensively used in vitro. We describe a novel system based on recombinant Sindbis virus which can be used to identify cleavage sites in vivo, thus eliminating the need for a purified enzyme and overcoming the problem of choosing the correct in vitro conditions. As a model we used the serine protease of the hepatitis C virus (HCV). We engineered the gene coding for this enzyme and two specific cleavage sites in the Sindbis virus structural gene and constructed libraries of viral genomes with a random sequence at either of the cleavage sites. The system was designed so that only viral genomes coding for sequences cleaved by the protease would produce viable viruses. With this system we selected viruses containing sequences mirroring those of the natural HCV protease substrates which were cleaved with comparable efficiencies.
Tegument-Specific, Virus-Reactive CD4 T Cells Localize to the Cornea in Herpes Simplex Virus Interstitial Keratitis in Humans
Authors: Koelle David M. , Reymond Sigrid N. , Chen Hongbo , Kwok William W. , McClurkan Christopher , Gyaltsong Tashi , Petersdorf Effie W. , Rotkis Walter , Talley Audrey R. , Harrison Devin A. 
Published: July 2002

ABSTRACTHerpes stromal keratitis (HSK) is a prevalent and frequently vision-threatening disease associated with herpes simplex virus type 1 (HSV-1) infection. In mice, HSK progression occurs after viral clearance and requires T cells and neutrophils. One model implicates Th1-like CD4 T cells with cross-reactivity between the HSV-1 protein UL6 and a corneal autoantigen. HSK can be prevented by establishing specific immunological tolerance. However, HSK can also occur in T-cell receptor-transgenic X SCID mice lacking HSV-specific T cells. To study the pathogenesis of HSK in the natural host species, we measured local HSV-specific T-cell responses in HSK corneas removed at transplant surgery (n= 5) or control corneas (n= 2). HSV-1 DNA was detected by PCR in two specimens. HSV-specific CD4 T cells were enriched in three of the five HSK specimens and were not detectable in the control specimens. Reactivity with peptide epitopes within the tegument proteins UL21 and UL49 was documented. Responses to HSV-1 UL6 were not detected. Diverse HLA DR and DP alleles restricted these local responses. Most clones secreted gamma interferon, but not interleukin-5, in response to antigen. HSV-specific CD8 cells were also recovered. Some clones had cytotoxic-T-lymphocyte activity. The diverse specificities and HLA-restricting alleles of local virus-specific T cells in HSK are consistent with their contribution to HSK by a proinflammatory effect.
Rous Sarcoma Virus Translation Revisited: Characterization of an Internal Ribosome Entry Segment in the 5′ Leader of the Genomic RNA
Authors: Deffaud Clarence , Darlix Jean-Luc 
Published: July 2002

ABSTRACT
The 5′ leader of Rous sarcoma virus (RSV) genomic RNA and of retroviruses in general is long and contains stable secondary structures that are critical in the early and late steps of virus replication such as RNA dimerization and packaging and in the process of reverse transcription. The initiation of RSV Gag translation has been reported to be 5′ cap dependent and controlled by three short open reading frames located in the 380-nucleotide leader upstream of the Gag start codon. Translation of RSV Gag would thus differ from that prevailing in other retroviruses such as murine leukemia virus, reticuloendotheliosis virus type A, and simian immunodeficiency virus, in which an internal ribosome entry segment (IRES) in the 5′ end of the genomic RNA directs efficient Gag expression despite stable 5′ secondary structures. This prompted us to investigate whether RSV Gag translation might be controlled by an IRES-dependent mechanism. The results show that the 5′ leaders of RSV and v-Src RNA exhibit IRES properties, since these viral elements can promote efficient translation of monocistronic RNAs in conditions inhibiting 5′ cap-dependent translation. When inserted between two cistrons in a canonical bicistronic construct, both the RSV and v-Src leaders promote expression of the 3′ cistron. A genetic analysis of the RSV leader allowed the identification of two nonoverlapping 5′ and 3′ leader domains with IRES activity. In addition, the v-Src leader was found to contain unique 3′ sequences promoting an efficient reinitiation of translation. Taken together, these data lead us to propose a new model for RSV translation.
Genetic Studies with the Fission Yeast
Schizosaccharomyces pombe
Suggest Involvement of Wee1, Ppa2, and Rad24 in Induction of Cell Cycle Arrest by Human Immunodeficiency Virus Type 1 Vpr
Authors: Masuda Michiaki , Nagai Yukiko , Oshima Norihito , Tanaka Koichi , Murakami Hiroshi , Igarashi Hiroko , Okayama Hiroto 
Published: July 2002

ABSTRACT

Accessory protein Vpr of human immunodeficiency virus type 1 (HIV-1) arrests cell cycling at G
2
/M phase in human and simian cells. Recently, it has been shown that Vpr also causes cell cycle arrest in the fission yeast
Schizosaccharomyces pombe
, which shares the cell cycle regulatory mechanisms with higher eukaryotes including humans. In this study, in order to identify host cellular factors involved in Vpr-induced cell cycle arrest, the ability of Vpr to cause elongated cellular morphology (
cdc
phenotype) typical of G
2
/M cell cycle arrest in wild-type and various mutant strains of
S. pombe
was examined. Our results indicated that Vpr caused the
cdc
phenotype in wild-type
S. pombe
as well as in strains carrying mutations, such as the
cdc2-3w
, Δ
cdc25
,
rad1-1
, Δ
chk1
, Δ
mik1
, and Δ
ppa1
strains. However, other mutants, such as the
cdc2-1w
, Δ
wee1
, Δ
ppa2
, and Δ
rad24
strains, failed to show a distinct
cdc
phenotype in response to Vpr expression. Results of these genetic studies suggested that Wee1, Ppa2, and Rad24 might be required for induction of cell cycle arrest by HIV-1 Vpr. Cell proliferation was inhibited by Vpr expression in all of the strains examined including the ones that did not show the
cdc
phenotype. The results supported the previously suggested possibility that Vpr affects the cell cycle and cell proliferation through different pathways.
Identification of Amino Acid Residues in CD81 Critical for Interaction with Hepatitis C Virus Envelope Glycoprotein E2
Authors: Higginbottom Adrian , Quinn Elizabeth R. , Kuo Chiung-Chi , Flint Mike , Wilson Louise H. , Bianchi Elisabetta , Nicosia Alfredo , Monk Peter N. , McKeating Jane A. , Levy Shoshana 
Published: July 2002

ABSTRACTHuman CD81 has been previously identified as the putative receptor for the hepatitis C virus envelope glycoprotein E2. The large extracellular loop (LEL) of human CD81 differs in four amino acid residues from that of the African green monkey (AGM), which does not bind E2. We mutated each of the four positions in human CD81 to the corresponding AGM residues and expressed them as soluble fusion LEL proteins in bacteria or as complete membrane proteins in mammalian cells. We found human amino acid 186 to be critical for the interaction with the viral envelope glycoprotein. This residue was also important for binding of certain anti-CD81 monoclonal antibodies. Mutating residues 188 and 196 did not affect E2 or antibody binding. Interestingly, mutation of residue 163 increased both E2 and antibody binding, suggesting that this amino acid contributes to the tertiary structure of CD81 and its ligand-binding ability. These observations have implications for the design of soluble high-affinity molecules that could target the CD81-E2 interaction site(s).
Mutation Analysis of the GDD Sequence Motif of a Calicivirus RNA-Dependent RNA Polymerase
Authors: Vázquez Ana López , Alonso José M. Martín , Parra Francisco 
Published: July 2002

ABSTRACT

The RNA-dependent RNA polymerase from
rabbit hemorrhagic disease virus
, a calicivirus, is known to have a conserved GDD amino acid motif and several additional regions of sequence homology with all types of polymerases. To test whether both aspartic acid residues are in fact involved in the catalytic activity and metal ion coordination of the enzyme, several defined mutations have been made in order to replace them by glutamate, asparagine, or glycine. All six mutant enzymes were produced in
Escherichia coli
, and their in vitro poly(U) polymerase activity was characterized. The results demonstrated that the first aspartate residue was absolutely required for enzyme function and that some flexibility existed with respect to the second, which could be replaced by glutamate.
Reciprocal Interactions between Human T-Lymphotropic Virus Type 1 and Prostaglandins: Implications for Viral Transmission
Authors: Moriuchi Masako , Inoue Hiroyasu , Moriuchi Hiroyuki 
Published: July 2002

ABSTRACTHuman T-lymphotropic virus type 1 (HTLV-1), the etiologic agent of adult T-cell leukemia/lymphoma, is transmitted through breast milk and seminal fluid, which are rich in prostaglandins (PGs). We demonstrate that PGE2upregulates the HTLV-1 long terminal repeat promoter through the protein kinase A pathway, induces replication of HTLV-1 in peripheral blood mononuclear cells (PBMC) derived from asymptomatic carriers, and enhances transmission of HTLV-1 to cord blood mononuclear cells (CBMC). Furthermore, HTLV-1 Tax transactivates a promoter for cyclooxygenase 2, a PG synthetase, and induces PGE2expression in PBMC or CBMC. Thus, HTLV-1 interacts with and benefits from PGs, constituents of its own vehicle for transmission.
Characterization of Adenovirus-Induced Inverted Terminal Repeat-Independent Amplification of Integrated Adeno-Associated Virus
rep-cap
Sequences
Authors: Tessier Jacques , Chadeuf Gilliane , Nony Pascale , Avet-Loiseau Hervé , Moullier Philippe , Salvetti Anna 
Published: July 2002

ABSTRACT

Stable packaging cell lines expressing the
rep
and
cap
genes for recombinant adeno-associated virus type 2 (rAAV-2) assembly constitute an attractive alternative to transient transfection protocols. We recently characterized a stable HeLa
rep-cap
cell clone (HeRC32) and demonstrated that upon vector transfection and adenovirus infection, efficient rAAV assembly correlated with a 100-fold amplification of the integrated
rep-cap
sequence with the inverted terminal repeats (ITRs) deleted. We now report a more detailed analysis of this phenomenon and highlight the key cellular and viral factors involved. Determination of the
rep-cap
copy number of HeRC32 cells indicated that maximum
rep-cap
amplification occurred between 24 and 48 h following adenovirus infection. Analysis by pulsed-field gel electrophoresis of adenovirus-infected HeRC32 cells indicated that amplified
rep-cap
sequences were found in an extrachromosomal form. Amplification of the
rep-cap
sequence with the ITRs deleted was not dependent on adenovirus replication and still occurred when the highly specific adenovirus polymerase was inactivated. In contrast, amplification was inhibited in the presence of aphidicolin, indicating that cellular polymerases were needed. Our study also documented that among the adenovirus gene products, the DNA-binding protein (DBP) was essential, since
rep-cap
amplification was severely abrogated when HeRC32 cells were infected at a nonpermissive temperature with an adenovirus mutant encoding a thermosensitive DBP. Furthermore, expression of DBP alone in HeRC32 cells was sufficient to induce a sustained level of
rep-cap
amplification. Finally, immunofluorescence analysis showed that HeRC32 cells expressing the DBP also simultaneously expressed the Rep proteins, suggesting a possible involvement of the latter in
rep-cap
amplification. Indeed, the lack of detectable amplification in an adenovirus-infected stable
rep-cap
HeLa cell clone unable to produce Rep proteins further supported that, among the viral gene products, both the DBP and Rep proteins are necessary to induce the targeted amplification of the integrated
rep-cap
sequences in the absence of the AAV ITRs.
Induction of Polyomavirus-Specific CD8+T Lymphocytes by Distinct Dendritic Cell Subpopulations
Authors: Drake Donald R. , Shawver Mandy L. , Hadley Annette , Butz Eric , Maliszewski Charles , Lukacher Aron E. 
Published: July 2002

ABSTRACTDendritic cells are pivotal antigen-presenting cells for generating adaptive T-cell responses. Here, we show that dendritic cells belonging to either the myeloid-related or lymphoid-related subset are permissive for infection by mouse polyomavirus and, when loaded with a peptide corresponding to the immunodominant anti-polyomavirus CD8+T-cell epitope or infected by polyomavirus, are each capable of driving expansion of primary polyomavirus-specific CD8+T-cell responses in vivo.
Conservation of the Conformation and Positive Charges of Hepatitis C Virus E2 Envelope Glycoprotein Hypervariable Region 1 Points to a Role in Cell Attachment
Authors: Penin François , Combet Christophe , Germanidis Georgios , Frainais Pierre-Olivier , Deléage Gilbert , Pawlotsky Jean-Michel 
Published: July 2002

ABSTRACT
Chronic hepatitis C virus (HCV) infection is a major cause of liver disease. The HCV polyprotein contains a hypervariable region (HVR1) located at the N terminus of the second envelope glycoprotein E2. The strong variability of this 27-amino-acid region is due to its apparent tolerance of amino acid substitutions together with strong selection pressures exerted by anti-HCV immune responses. No specific function has so far been attributed to HVR1. However, its presence at the surface of the viral particle suggests that it might be involved in viral entry. This would imply that HVR1 is not randomly variable. We sequenced 460 HVR1 clones isolated at various times from six HCV-infected patients receiving alpha interferon therapy (which exerts strong pressure towards quasispecies genetic evolution) and analyzed their amino acid sequences together with those of 1,382 nonredundant HVR1 sequences collected from the EMBL database. We found that (i) despite strong amino acid sequence variability related to strong pressures towards change, the chemicophysical properties and conformation of HVR1 were highly conserved, and (ii) HVR1 is a globally basic stretch, with the basic residues located at specific sequence positions. This conservation of positively charged residues indicates that HVR1 is involved in interactions with negatively charged molecules such as lipids, proteins, or glycosaminoglycans (GAGs). As with many other viruses, possible interaction with GAGs probably plays a role in host cell recognition and attachment.
Hantavirus Infection Induces the Expression of RANTES and IP-10 without Causing Increased Permeability in Human Lung Microvascular Endothelial Cells
Authors: Sundstrom J. Bruce , McMullan Laura K. , Spiropoulou Christina F. , Hooper W. Craig , Ansari Aftab A. , Peters Clarence J. , Rollin Pierre E. 
Published: July 2002

ABSTRACTSin Nombre virus (SNV) and Hantaan virus (HTN) infect endothelial cells and are associated with different patterns of increased vascular permeability during human disease. It is thought that such patterns of increased vascular permeability are a consequence of endothelial activation and subsequent dysfunction mediated by differential immune responses to hantavirus infection. In this study, the ability of hantavirus to directly induce activation of human lung microvascular endothelial cells (HMVEC-Ls) was examined. No virus-specific modulation in the constitutive or cytokine-induced expression of cellular adhesion molecules (CD40, CD54, CD61, CD62E, CD62P, CD106, and major histocompatibility complex classes I and II) or in cytokines and chemokines (eotaxin, tumor necrosis factor alpha, interleukin 1β [IL-1β], IL-6, IL-8, MCP-1, MIP-1α, and MIP-1β) was detected at either the protein or message level in hantavirus-infected HMVEC-Ls. Furthermore, no virus-specific enhancement of paracellular or transcellular permeability or changes in the organization and distribution of endothelial intercellular junctional proteins was observed. However, infection with either HTN or SNV resulted in detectable levels of the chemokines RANTES and IP-10 (the 10-kDa interferon-inducible protein) in HMVEC-Ls within 72 h and was associated with nuclear translocation of interferon regulatory factor 3 (IRF-3) and IRF-7. Gamma interferon (IFN-γ)-induced expression of RANTES and IP-10 could also be detected in uninfected HMVEC-Ls and was associated with nuclear translocation of IRF-1 and IRF-3. Treatment of hantavirus-infected HMVEC-Ls with IFN-γ for 24 h resulted in a synergistic enhancement in the expression of both RANTES and IP-10 and was associated with nuclear translocation of IRF-1, IRF-3, IRF-7, and NF-κB p65. These results reveal a possible mechanism by which hantavirus infection and a TH1 immune response can cooperate to synergistically enhance chemokine expression by HMVEC-Ls and trigger immune-mediated increases in vascular permeability.
Kaposi's Sarcoma-Associated Herpesvirus Open Reading Frame 50 Represses p53-Induced Transcriptional Activity and Apoptosis
Authors: Gwack Yousang , Hwang Seungmin , Byun Hyewon , Lim Chunghun , Kim Jin Woo , Choi Eui-Ju , Choe Joonho 
Published: July 2002

ABSTRACT
Kaposi's sarcoma-associated herpesvirus (KSHV) open reading frame 50 (ORF50) encodes a viral transcriptional activator which stimulates the transcription of viral early and late genes of KSHV. Here we show that ORF50 represses transcriptional activity of p53 and p53-induced apoptosis through interaction with CREB binding protein (CBP). This inhibitory effect of ORF50 on the transcriptional activity of p53 was relieved by the addition of CBP. ORF50 mutants, which are defective in interaction with CBP, lost the inhibitory effects on p53. Our data provide a framework for delineating the regulatory mechanisms used by KSHV to modulate cellular transcription and the cell cycle.