bluuebunny/crossref_metadata_2025_split huggingface dataset:
Effects of Amino Acid Substitutions at Position 115 on the Fidelity of Human Immunodeficiency Virus Type 1 Reverse Transcriptase
Authors: Boyer Paul L. , Hughes Stephen H. 
Published: July 2002

ABSTRACT
We compared the fidelity of wild-type human immunodeficiency virus type-1 (HIV-1) reverse transcriptase (RT) and two RT mutants, Y115F and Y115V. Although neither mutation had a large effect on the overall fidelity of the enzyme, both mutations altered the spectrum of mutations and the precise nature of the mutational hot spots. The effects of Y115V were greater than those of Y115F. When we compared the behavior of the wild-type enzyme with published data, we found that, in contrast to what has been published, misalignment/slippage could account for only a small fraction of the mutations we observed. We also found that a preponderance of the mutations (both transitions and transversions) resulted in the insertion of an A. Because we were measuring DNA-dependent DNA synthesis (plus-strand synthesis), this bias could contribute to the A-rich nature of the HIV-1 genome.
Wild-Type and YMDD Mutant Murine Leukemia Virus Reverse Transcriptases Are Resistant to 2′,3′-Dideoxy-3′-Thiacytidine
Authors: Halvas Elias K. , Svarovskaia Evguenia S. , Freed Eric O. , Pathak Vinay K. 
Published: July 2002

ABSTRACT
The antiretroviral nucleoside analog 2′,3′-dideoxy-3′-thiacytidine (3TC) is a potent inhibitor of wild-type human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT). A methionine-to-valine or methionine-to-isoleucine substitution at residue 184 in the HIV-1 YMDD motif, which is located at the RT active site, leads to a high level of resistance to 3TC. We sought to determine whether 3TC can inhibit the replication of wild-type murine leukemia virus (MLV), which contains V223 at the YVDD active site motif of the MLV RT, and of the V223M, V223I, V223A, and V223S mutant RTs. Surprisingly, the wild type and all four of the V223 mutants of MLV RT were highly resistant to 3TC. These results indicate that determinants outside the YVDD motif of MLV RT confer a high level of resistance to 3TC. Therefore, structural differences among similar RTs might result in widely divergent sensitivities to antiretroviral nucleoside analogs.
Lymphomas and High-Level Expression of Murine Leukemia Viruses in CFW Mice
Authors: Taddesse-Heath Lekidelu , Chattopadhyay Sisir K. , Dillehay Dirck L. , Lander Marilyn R. , Nagashfar Zohreh , Morse Herbert C. , Hartley Janet W. 
Published: July 2002

ABSTRACT
Historically, Swiss Webster mice of the CFW subline, both inbred and random-bred stocks, have been considered to have a low spontaneous occurrence of hematopoietic system tumors, and previous reports of infectious expression of murine leukemia viruses (MuLVs) have been rare and unremarkable. In marked contrast, in the present study of CFW mice from one source observed by two laboratories over a 2-year period, nearly 60% developed tumors, 85% of which were lymphomas, the majority of B-cell origin. All tumors tested expressed ecotropic MuLVs, and most expressed mink cell focus-inducing (MCF) MuLVs. Among normal mice of weanling to advanced age, over one-half were positive for ecotropic virus in tail or lymphoid tissues, and MCF virus was frequently present in lymphoid tissue, less often in tail. Patterns of ecotropic proviral integration indicated that natural infection occurred by both genetic and exogenous routes. Lymphomas were induced in NIH Swiss mice infected as neonates with tissue culture-propagated MuLVs isolated from normal and tumor tissue of CFW mice.
Structural Determinants of Murine Leukemia Virus Reverse Transcriptase That Affect the Frequency of Template Switching
Authors: Svarovskaia Evguenia S. , Delviks Krista A. , Hwang Carey K. , Pathak Vinay K. 
Published: July 2002

ABSTRACT
Retroviral reverse transcriptases (RTs) frequently switch templates within the same RNA or between copackaged viral RNAs to generate mutations and recombination. To identify structural elements of murine leukemia virus RT important for template switching, we developed an in vivo assay in which RT template switching within direct repeats functionally reconstituted the green fluorescent protein gene. We quantified the effect of mutations in the YXDD motif, the deoxynucleoside triphosphate binding site, the thumb domain, and the RNase H domain of RT and hydroxyurea treatment on the frequencies of template switching. Hydroxyurea treatment and some mutations in RT increased the frequency of RT template switching up to fivefold, while all of the mutations tested in the RNase H domain decreased the frequency of template switching by twofold. Based on these results, we propose a dynamic copy choice model in which both the rate of DNA polymerization and the rate of RNA degradation influence the frequency of RT template switching.
Murine Model of Interstitial Cytomegalovirus Pneumonia in Syngeneic Bone Marrow Transplantation: Persistence of Protective Pulmonary CD8-T-Cell Infiltrates after Clearance of Acute Infection
Authors: Podlech Jürgen , Holtappels Rafaela , Pahl-Seibert Marcus-Folker , Steffens Hans-Peter , Reddehase Matthias J. 
Published: July 2002

ABSTRACTInterstitial pneumonia (IP) is a severe organ manifestation of cytomegalovirus (CMV) disease in the immunocompromised host, in particular in recipients of bone marrow transplantation (BMT). Diagnostic criteria for the definition of CMV-IP include clinical evidence of pneumonia together with CMV detected in bronchoalveolar lavage or lung biopsy. We have used the model of syngeneic BMT and simultaneous infection of BALB/c mice with murine CMV for studying the pathogenesis of CMV-IP by controlled longitudinal analysis. A disseminated cytopathic infection of the lungs with fatal outcome was observed only when reconstituting CD8 T cells were depleted. Neither CD8 nor CD4 T cells mediated an immunopathogenesis of acute CMV-IP. By contrast, after efficient hematolymphopoietic reconstitution, viral replication in the lungs was moderate and focal. The histopathological picture was dominated by preferential infiltration of CD8 T cells confining viral replication to inflammatory foci. Notably, after clearance of acute infection, CD62Lloand CD62Lhisubsets of CD44+memory CD8 T cells were found to persist in lung tissue. One can thus operationally distinguish an early CMV-positive IP (phase 1) and a late CMV-negative IP (phase 2). According to the definition, phase 2 histopathology would not be diagnosed as a CMV-IP and could instead be misinterpreted as a CMV-induced immunopathology. We document here that phase 1 as well as phase 2 pulmonary CD8 T cells are capable of exerting effector functions and are effectual in protecting against productive infection. We propose that antiviral “stand-by” memory-effector T cells persist in the lungs to prevent virus recurrence from latency.
Intranasal Administration of 2/6-Rotavirus-Like Particles with MutantEscherichia coliHeat-Labile Toxin (LT-R192G) Induces Antibody-Secreting Cell Responses but Not Protective Immunity in Gnotobiotic Pigs
Authors: Yuan Lijuan , Geyer Annelise , Hodgins Douglas C. , Fan Zhiqian , Qian Yuan , Chang Kyeong-Ok , Crawford Sue E. , Parreño Viviana , Ward Lucy A. , Estes Mary K. , Conner Margaret E. , Saif Linda J. 
Published: July 2002

ABSTRACTWe investigated the immunogenicity of recombinant double-layered rotavirus-like particle (2/6-VLPs) vaccines derived from simian SA11 or human (VP6) Wa and bovine RF (VP2) rotavirus strains. The 2/6-VLPs were administered to gnotobiotic pigs intranasally (i.n.) with a mutantEscherichia coliheat-labile toxin, LT-R192G (mLT), as mucosal adjuvant. Pigs were challenged with virulent Wa (P1A[8],G1) human rotavirus at postinoculation day (PID) 21 (two-dose VLP regimen) or 28 (three-dose VLP regimen). In vivo antigen-activated antibody-secreting cells (ASC) (effector B cells) and in vitro antigen-reactivated ASC (derived from memory B cells) from intestinal and systemic lymphoid tissues (duodenum, ileum, mesenteric lymph nodes [MLN], spleen, peripheral blood lymphocytes [PBL], and bone marrow lymphocytes) collected at selected times were quantitated by enzyme-linked immunospot assays. Rotavirus-specific immunoglobulin M (IgM), IgA, and IgG ASC and memory B-cell responses were detected by PID 21 or 28 in intestinal and systemic lymphoid tissues after i.n. inoculation with two or three doses of 2/6-VLPs with or without mLT. Greater mean numbers of virus-specific ASC and memory B cells in all tissues prechallenge were induced in pigs inoculated with two doses of SA11 2/6-VLPs plus mLT compared to SA11 2/6-VLPs without mLT. After challenge, anamnestic IgA and IgG ASC and memory B-cell responses were detected in intestinal lymphoid tissues of all VLP-inoculated groups, but serum virus-neutralizing antibody titers were not significantly enhanced compared to the challenged controls. Pigs inoculated with Wa-RF 2/6-VLPs (with or without mLT) developed higher anamnestic IgA and IgG ASC responses in ileum after challenge compared to pigs inoculated with SA11 2/6-VLPs (with or without mLT). Three doses of SA 11 2/6-VLP plus mLT induced the highest mean numbers of IgG memory B cells in MLN, spleen, and PBL among all groups postchallenge. However, no significant protection against diarrhea or virus shedding was evident in any of the 2/6-VLP (with or without mLT)-inoculated pigs after challenge with virulent Wa human rotavirus. These results indicate that 2/6-VLP vaccines are immunogenic in gnotobiotic pigs when inoculated i.n. and that the adjuvant mLT enhanced their immunogenicity. However, i.n. inoculation of gnotobiotic pigs with 2/6-VLPs did not confer protection against human rotavirus challenge.
Template/Primer Requirements and Single Nucleotide Incorporation by Hepatitis C Virus Nonstructural Protein 5B Polymerase
Authors: Zhong Weidong , Ferrari Eric , Lesburg Charles A. , Maag David , Ghosh Saikat Kumar B. , Cameron Craig E. , Lau Johnson Y. N. , Hong Zhi 
Published: July 2002

ABSTRACT
Nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) possesses an RNA-dependent RNA polymerase activity responsible for viral genome RNA replication. Despite several reports on the characterization of this essential viral enzyme, little is known about the reaction pathway of NS5B-catalyzed nucleotide incorporation due to the lack of a kinetic system offering efficient assembly of a catalytically competent polymerase/template/primer/nucleotide quaternary complex. In this report, specific template/primer requirements for efficient RNA synthesis by HCV NS5B were investigated. For intramolecular copy-back RNA synthesis, NS5B utilizes templates with an unstable stem-loop at the 3′ terminus which exists as a single-stranded molecule in solution. A template with a stable tetraloop at the 3′ terminus failed to support RNA synthesis by HCV NS5B. Based on these observations, a number of single-stranded RNA templates were synthesized and tested along with short RNA primers ranging from two to five nucleotides. It was found that HCV NS5B utilized di- or trinucleotides efficiently to initiate RNA replication. Furthermore, the polymerase, template, and primer assembled initiation-competent complexes at the 3′ terminus of the template RNA where the template and primer base paired within the active site cavity of the polymerase. The minimum length of the template is five nucleotides, consistent with a structural model of the NS5B/RNA complex in which a pentanucleotide single-stranded RNA template occupies a groove located along the fingers subdomain of the polymerase. This observation suggests that the initial docking of RNA on NS5B polymerase requires a single-stranded RNA molecule. A unique β-hairpin loop in the thumb subdomain may play an important role in properly positioning the single-stranded template for initiation of RNA synthesis. Identification of the template/primer requirements will facilitate the mechanistic characterization of HCV NS5B and its inhibitors.
Phosphatidylserine-Mediated Phagocytosis of Influenza A Virus-Infected Cells by Mouse Peritoneal Macrophages
Authors: Shiratsuchi Akiko , Kaido Masako , Takizawa Takenori , Nakanishi Yoshinobu 
Published: July 2002

ABSTRACTInfluenza virus induces apoptosis in cultured cell lines as well as in animal tissues. HeLa cells were infected with influenza virus A/Udon/72 (H3N2) under conditions resulting in almost 100% infection. Such cells underwent typical caspase-dependent apoptosis and were efficiently phagocytosed by macrophages prepared from peritoneal fluids of thioglycolate-treated mice. The membrane phospholipid phosphatidylserine appeared on the surfaces of virus-infected cells at around the time efficient phagocytosis became detectable. In fact, the phagocytosis was almost completely inhibited in the presence of liposomes containing phosphatidylserine, which did not influence the antibody-dependent uptake of zymosan particles by the same macrophages. These results indicate that macrophages phagocytose influenza virus-infected HeLa cells in a manner mediated by phosphatidylserine that appears on the surfaces of infected cells during the process of apoptosis.
Recognition of
N
-Glycolylneuraminic Acid Linked to Galactose by the α2,3 Linkage Is Associated with Intestinal Replication of Influenza A Virus in Ducks
Authors: Ito Toshihiro , Suzuki Yasuo , Suzuki Takashi , Takada Ayato , Horimoto Taisuke , Wells Krisna , Kida Hiroshi , Otsuki Koichi , Kiso Makoto , Ishida Hideharu , Kawaoka Yoshihiro 
Published: July 2002

ABSTRACT

The hemagglutinin (HA) of H3 human influenza viruses does not support viral replication in duck intestine despite its avian origin. A Leu-to-Gln mutation at position 226 and a Ser-to-Gly mutation at position 228 in the HA of human A/Udorn/307/72 (H3N2) permit a reassortant virus [human Udorn HA, with all other genes from A/mallard/New York/6750/78 (H2N2)] to replicate in ducks. To understand the molecular basis of this change in host range restriction, we investigated the receptor specificity of duck influenza viruses as well as of human-duck virus reassortants. The results indicate that the recognition of a glycoconjugate moiety possessing
N
-glycolneuramic acid (NeuGc) linked to galactose by the α2,3 linkage (NeuGcα2,3Gal) is associated with viral replication in duck intestine. Immunofluorescence assays with NeuGcα2,3Gal-specific antiserum detected this moiety primarily on the crypt epithelial cells of duck colon. Such recognition, together with biochemical evidence of NeuGc in crypt cells, correlated exactly with the ability of the virus to replicate in duck colon. These results suggest that recognition of the NeuGcα2,3-Gal moiety plays an important role in the enterotropism of avian influenza viruses.
Site-Specific Integration of an Adeno-Associated Virus Vector Plasmid Mediated by Regulated Expression of Rep Based on Cre-
loxP
Recombination
Authors: Satoh Wataru , Hirai Yukihiko , Tamayose Kenji , Shimada Takashi 
Published: July 2002

ABSTRACT

Recombinant adeno-associated virus (AAV) type 2 has attracted attention because it appears to have the potential to serve as a vector for human gene therapy. An interesting feature of wild-type AAV is its site-specific integration into AAVS1, a defined locus on chromosome 19. This reaction requires the presence of two viral elements: inverted terminal repeats and Rep78/68. Accordingly, current AAV vectors lacking the
rep
gene lack the capacity for site-specific integration. In this report, we describe the use of Cre-
loxP
recombination in a novel system for the regulated, transient expression of Rep78, which is potentially cytotoxic when synthesized constitutively. We constructed a plasmid in which the p5 promoter was situated downstream of the
rep
coding sequence; in this configuration,
rep
expression is silent. However, Cre circularizes the
rep
expression unit, directly joining the p5 promoter to the 5′ end of the
rep
78 coding sequence, resulting in expression of Rep78. Such structural and functional changes were confirmed by detailed molecular analysis. A key feature of this system is that Rep expression was terminated when the circular molecule was linearized and integrated into the chromosome. Using this regulated expression system, we attempted site-specific integration of AAV vector plasmids. A PCR-based assay and analysis of fluorescence in situ hybridization showed that the AAV vector sequence was integrated into chromosome 19. Sequence analysis also confirmed that transient expression of Rep78 was sufficient for site-specific integration at the AAVS1 locus, as is observed with integration of wild-type AAV.
Analysis of Individual Human Trigeminal Ganglia for Latent Herpes Simplex Virus Type 1 and Varicella-Zoster Virus Nucleic Acids Using Real-Time PCR
Authors: Cohrs Randall J. , Randall Jessica , Smith John , Gilden Donald H. , Dabrowski Christine , van der Keyl Harjeet , Tal-Singer Ruth 
Published: July 2002

ABSTRACT
Herpes simplex virus type 1 (HSV-1) and varicella-zoster virus (VZV) establish latent infections in the peripheral nervous system following primary infection. During latency both virus genomes exhibit limited transcription, with the HSV-1 LATs and at least four VZV transcripts consistently detected in latently infected human ganglia. In this study we used real-time PCR quantitation to determine the viral DNA copy number in individual trigeminal ganglia (TG) from 17 subjects. The number of HSV-1 genomes was not significantly different between the left and right TG from the same individual and varied per subject from 42.9 to 677.9 copies per 100 ng of DNA. The number of VZV genomes was also not significantly different between left and right TG from the same individual and varied per subject from 37.0 to 3,560.5 copies per 100 ng of DNA. HSV-1 LAT transcripts were consistently detected in ganglia containing latent HSV-1 and varied in relative expression by >500-fold. Of the three VZV transcripts analyzed, only transcripts mapping to gene 63 were consistently detected in latently infected ganglia and varied in relative expression by >2,000-fold. Thus, it appears that, similar to LAT transcription in HSV-1 latently infected ganglia, VZV gene 63 transcription is a hallmark of VZV latency.
Association of Structural Changes in the V2 and V3 Loops of the gp120 Envelope Glycoprotein with Acquisition of Neutralization Resistance in a Simian-Human Immunodeficiency Virus Passaged In Vivo
Authors: Ye Yongjun , Si Zhi Hai , Moore John P. , Sodroski Joseph 
Published: July 2002

ABSTRACT
The in vivo passage of a neutralization-sensitive, laboratory-adapted simian-human immunodeficiency virus (SHIV-HXBc2) generated a pathogenic, neutralization-resistant virus, SHIV-HXBc2P 3.2. SHIV-HXBc2P 3.2 differs from SHIV-HXBc2 only in 13 amino acid residues of the viral envelope glycoproteins. Here we used antibody competition analysis to examine the structural changes that occurred in the SHIV-HXBc2P 3.2 gp120 exterior envelope glycoprotein. The relationships among the antibody epitopes on the conserved gp120 core of SHIV-HXBc2 and SHIV-HXBc2P 3.2 were similar. The third variable (V3) loop was more closely associated with the fourth conserved (C4) region and CD4-induced epitopes on the gp120 core in the HXBc2P 3.2 gp120 glycoprotein compared with the HXBc2 gp120 glycoprotein. Rearrangements of the second variable (V2) loop with respect to the CD4 binding site and associated epitopes were evident in comparisons of the two gp120 glycoproteins. Thus, the in vivo evolution of a neutralization-resistant virus involves conformational adjustments of the V2 and V3 variable loops with respect to the conserved receptor-binding regions of the gp120 core.
Cloning the Antibody Response in Humans with Chronic Inflammatory Disease: Immunopanning of Subacute Sclerosing Panencephalitis (SSPE) Brain Sections with Antibody Phage Libraries Prepared from SSPE Brain Enriches for Antibody Recognizing Measles Virus Antigens In Situ
Authors: Owens Gregory P. , Williamson R. Anthony , Burgoon Mark P. , Ghausi Omar , Burton Dennis R. , Gilden Donald H. 
Published: July 2002

ABSTRACTIn central nervous system (CNS) infectious and inflammatory diseases of known cause, oligoclonal bands represent antibody directed against the causative agent. To determine whether disease-relevant antibodies can be cloned from diseased brain, we prepared an antibody phage display library from the brain of a human with subacute sclerosing panencephalitis (SSPE), a chronic encephalitis caused by measles virus, and selected the library against SSPE brain sections. Antibodies that were retrieved reacted strongly with measles virus cell extracts by enzyme-linked immunosorbent assay and were specific for the measles virus nucleocapsid protein. These antibodies immunostained cells in different SSPE brains but not in control brain. Our data provide the first demonstration that diseased brain can be used to select in situ for antibodies directed against the causative agent of disease and point to the potential usefulness of this approach in identifying relevant antibodies in chronic CNS or systemic inflammatory diseases of unknown cause.
Hepatitis C Virus Core Protein Interacts with 14-3-3 Protein and Activates the Kinase Raf-1
Authors: Aoki Hiroshi , Hayashi Junpei , Moriyama Mitsuhiko , Arakawa Yasuyuki , Hino Okio 
Published: July 2002

ABSTRACT
Persistent hepatitis C virus (HCV) infection is a major cause of chronic liver dysfunction in humans and is epidemiologically closely associated with the development of human hepatocellular carcinoma. Among HCV components, core protein has been reported to be implicated in cell growth regulation both in vitro and in vivo, although mechanisms explaining those effects are still unclear. In the present study, we identified that members of the 14-3-3 protein family associate with HCV core protein. 14-3-3 protein bound to HCV core protein in a phosphoserine-dependent manner. Introduction of HCV core protein caused a substantial increase in Raf-1 kinase activity in HepG2 cells and in a yeast genetic assay. Furthermore, the HCV core–14-3-3 interaction was essential for Raf-1 kinase activation by HCV core protein. These results suggest that HCV core protein may represent a novel type of Raf-1 kinase-activating protein through its interaction with 14-3-3 protein and may contribute to hepatocyte growth regulation.