bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification of Key Residues in Subgroup A Avian Leukosis Virus Envelope Determining Receptor Binding Affinity and Infectivity of Cells Expressing Chicken or Quail Tva Receptor
Authors: Holmen Sheri L. , Melder Deborah C. , Federspiel Mark J. 
Published: July 2002

ABSTRACT
To better understand retroviral entry, we have characterized the interactions between subgroup A avian leukosis virus [ALV(A)] envelope glycoproteins and Tva, the receptor for ALV(A), that result in receptor interference. We have recently shown that soluble forms of the chicken and quail Tva receptor (sTva), expressed from genes delivered by retroviral vectors, block ALV(A) infection of cultured chicken cells (∼200-fold antiviral effect) and chickens (>98% of the birds were not infected). We hypothesized that inhibition of viral replication by sTva would select virus variants with mutations in the surface glycoprotein (SU) that altered the binding affinity of the subgroup A SU for the sTva protein and/or altered the normal receptor usage of the virus. Virus propagation in the presence of quail sTva-mIgG, the quail Tva extracellular region fused to the constant region of the mouse immunoglobulin G (IgG) protein, identified viruses with three mutations in the subgroup A hr1 region of SU, E149K, Y142N, and Y142N/E149K. These mutations reduced the binding affinity of the subgroup A envelope glycoproteins for quail sTva-mIgG (32-, 324-, and 4,739-fold, respectively) but did not alter their binding affinity for chicken sTva-mIgG. The ALV(A) mutants efficiently infected cells expressing the chicken Tva receptor but were 2-fold (E149K), 10-fold (Y142N), and 600-fold (Y142N/E149K) less efficient at infecting cells expressing the quail Tva receptor. These mutations identify key determinants of the interaction between the ALV(A) glycoproteins and the Tva receptor. We also conclude from these results that, at least for the wild-type and variant ALV(A)s tested, the receptor binding affinity was directly related to infection efficiency.
Chromosomal Integration Pattern of a Helper-Dependent Minimal Adenovirus Vector with a Selectable Marker Inserted into a 27.4-Kilobase Genomic Stuffer
Authors: Hillgenberg Moritz , Tönnies Holger , Strauss Michael 
Published: July 2002

ABSTRACT

Helper-dependent minimal adenovirus vectors are promising tools for gene transfer and therapy because of their high capacity and the absence of immunostimulatory or cytotoxic viral genes. In order to characterize this new vector system with respect to its integrative properties, the integration pattern of a minimal adenovirus vector with a
neo
r
gene inserted centrally into a noncoding 27.4-kb genomic stuffer element derived from the human X chromosome after infection of a sex chromosome aneuploid (X0) human glioblastoma cell line was studied. Our results indicate that even extensive homologies and abundant chromosomal repeat elements present in the vector did not lead to integration of the vector via homologous or homology-mediated mechanisms. Instead, integration occurred primarily by insertion of a monomer with no or little loss of sequences at the vector ends, apparently at random sites, which is very similar to E1 deletion adenovirus vectors. It is therefore unlikely that the incorporation of stuffer elements derived from human genomic DNA, which were shown to allow long-term transgene expression in vivo in a number of studies, leads to an enhanced risk of insertional mutagenesis. Furthermore, our findings indicate that the potential of minimal adenovirus vectors as tools for targeted insertion and gene targeting is limited despite the possibility of incorporating long stretches of homologous sequences. However, we found an enhanced efficiency of stable
neo
r
transduction of the minimal adenovirus vector compared to an E1 deletion adenovirus vector, possibly caused by the absence of potential growth-inhibitory viral genes. Complete integration of the vector and tolerance of the integrated vector sequences by the cell might indicate a potential use of these vectors as tools for stable transfer of (large) genes.
Long-Term Subclinical Carrier State Precedes Scrapie Replication and Adaptation in a Resistant Species: Analogies to Bovine Spongiform Encephalopathy and Variant Creutzfeldt-Jakob Disease in Humans
Authors: Race Richard , Raines Anne , Raymond Gregory J. , Caughey Byron , Chesebro Bruce 
Published: July 2002

ABSTRACT
Cattle infected with bovine spongiform encephalopathy (BSE) appear to be a reservoir for transmission of variant Creutzfeldt-Jakob disease (vCJD) to humans. Although just over 100 people have developed clinical vCJD, millions have probably been exposed to the infectivity by consumption of BSE-infected beef. It is currently not known whether some of these individuals will develop disease themselves or act as asymptomatic carriers of infectivity which might infect others in the future. We have studied agent persistence and adaptation after cross-species infection using a model of mice inoculated with hamster scrapie strain 263K. Although mice inoculated with hamster scrapie do not develop clinical disease after inoculation with 10 million hamster infectious doses, hamster scrapie infectivity persists in brain and spleen for the life span of the mice. In the present study, we were surprised to find a 1-year period postinfection with hamster scrapie where there was no evidence for replication of infectivity in mouse brain. In contrast, this period of inactive persistence was followed by a period of active replication of infectivity as well as adaptation of new strains of agent capable of causing disease in mice. In most mice, neither the early persistent phase nor the later replicative phase could be detected by immunoblot assay for protease-resistant prion protein (PrP). If similar asymptomatic carriers of infection arise after exposure of humans or animals to BSE, this could markedly increase the danger of additional spread of BSE or vCJD infection by contaminated blood, surgical instruments, or meat. If such subclinical carriers were negative for protease-resistant PrP, similar to our mice, then the recently proposed screening of brain, tonsils, or other tissues of animals and humans by present methods such as immunoblotting or immunohistochemistry might be too insensitive to identify these individuals.
Structure and Temporal Dynamics of Populations within Wheat Streak Mosaic Virus Isolates
Authors: Hall Jeffrey S. , French Roy , Morris T. Jack , Stenger Drake C. 
Published: July 2002

ABSTRACT

Variation within the Type and Sidney 81 strains of wheat streak mosaic virus was assessed by single-strand conformation polymorphism (SSCP) analysis and confirmed by nucleotide sequencing. Limiting-dilution subisolates (LDSIs) of each strain were evaluated for polymorphism in the P1, P3, NIa, and CP cistrons. Different SSCP patterns among LDSIs of a strain were associated with single-nucleotide substitutions. Sidney 81 LDSI-S10 was used as founding inoculum to establish three lineages each in wheat, corn, and barley. The P1, HC-Pro, P3, CI, NIa, NIb, and CP cistrons of LDSI-S10 and each lineage at passages 1, 3, 6, and 9 were evaluated for polymorphism. By passage 9, each lineage differed in consensus sequence from LDSI-S10. The majority of substitutions occurred within NIa and CP, although at least one change occurred in each cistron except HC-Pro and P3. Most consensus sequence changes among lineages were independent, with substitutions accumulating over time. However, LDSI-S10 bore a variant nucleotide (G
6016
) in NIa that was restored to A
6016
in eight of nine lineages by passage 6. This near-global reversion is most easily explained by selection. Examination of nonconsensus variation revealed a pool of unique substitutions (singletons) that remained constant in frequency during passage, regardless of the host species examined. These results suggest that mutations arising by viral polymerase error are generated at a constant rate but that most newly generated mutants are sequestered in virions and do not serve as replication templates. Thus, a substantial fraction of variation generated is static and has yet to be tested for relative fitness. In contrast, nonsingleton variation increased upon passage, suggesting that some mutants do serve as replication templates and may become established in a population. Replicated mutants may or may not rise to prominence to become the consensus sequence in a lineage, with the fate of any particular mutant subject to selection and stochastic processes such as genetic drift and population growth factors.
Variability of Viral Load in Plasma of Rhesus Monkeys Inoculated with Simian Immunodeficiency Virus or Simian-Human Immunodeficiency Virus: Implications for Using Nonhuman Primate AIDS Models To Test Vaccines and Therapeutics
Authors: Parker Robert A. , Regan Meredith M. , Reimann Keith A. 
Published: July 2002

ABSTRACTViral RNA level in plasma is a sensitive experimental endpoint for evaluating the efficacy of AIDS vaccines or therapies in nonhuman primates. By quantifying viral RNA in the plasma of 77 rhesus monkeys for 10 weeks after inoculation with simian-human immunodeficiency virus 89.6P (SHIV-89.6P) or simian immunodeficiency virus mac 251 (SIVmac 251), we estimated variability in three viral load (VL) measures: peak VL, the postacute set point VL, and VL decline from peak. Such estimates of biological variability are essential for determining the number of animals needed per group and may be helpful for selecting the most appropriate measure to use as the experimental endpoint. Peak VL was positively correlated with set point VL for both viruses. Variability (standard deviation) was substantially higher in monkeys infected with SIVmac 251 than in those infected with SHIV-89.6P for set point VL and VL decline. The variability of peak VL was less than one-half that of set point VL variability and only about two-thirds of that of VL decline, implying that the same treatment-related difference in peak VL could be detected with fewer animals than set point VL or VL decline. Thus, differences in VL variability over the course of infection and between viruses need to be considered when designing studies using the nonhuman primate AIDS models.
Early and Persistent Bone Marrow Hematopoiesis Defect in Simian/Human Immunodeficiency Virus-Infected Macaques despite Efficient Reduction of Viremia by Highly Active Antiretroviral Therapy during Primary Infection
Authors: Thiebot Hugues , Louache Fawzia , Vaslin Bruno , de Revel Thierry , Neildez Olivier , Larghero Jérome , Vainchenker William , Dormont Dominique , Le Grand Roger 
Published: July 2002

ABSTRACTThe hematological abnormalities observed in human immunodeficiency virus (HIV)-infected patients appear to be mainly due to bone marrow dysfunction. A macaque models of AIDS could greatly facilitate an in vivo approach to the pathogenesis of such dysfunction. Here, we evaluated in this model the impact of infection with a pathogenic simian/human immunodeficiency virus (SHIV) on bone marrow hematopoiesis. Three groups of macaques were inoculated with 50 50% median infective doses of pathogenic SHIV 89.P, which expressesenvof dual-tropic HIV type 1 (HIV-1) 89.6 primary isolate. During the primary phase of infection, animals were treated with either a placebo or highly active antiretroviral therapy (HAART) combining zidovudine, lamivudine, and indinavir, initiated 4 or 72 h postinfection (p.i.) and administered twice a day until day 28 p.i. In both placebo-treated and HAART-treated animals, bone marrow colony-forming cells (CFC) progressively decreased quite early, during the first month p.i. One year p.i., both placebo- and HAART-treated animals displayed decreases in CFC to about 56% of preinfection values. At the same time, a dramatic decrease (greater than 77%) of bone marrow CD34+long-term culture-initiating cells was noted in all animals were found. No statistically significant differences between placebo- and HAART-treated monkeys were found. These data argue for an early and profound alteration of myelopoiesis at the level of the most primitive CD34+progenitor cells during SHIV infection, independently of the level of viremia, circulating CD4+cell counts, or antiviral treatment.
Respiratory Syncytial Virus M2-1 Protein Requires Phosphorylation for Efficient Function and Binds Viral RNA during Infection
Authors: Cartee Tara L. , Wertz Gail W. 
Published: July 2002

ABSTRACT

The M2-1 protein of respiratory syncytial (RS) virus is a transcriptional processivity and antitermination factor. The M2-1 protein has a Cys3His1 zinc binding motif which is essential for function, is phosphorylated, and has been shown to interact with the RS virus nucleocapsid (N) protein. In the work reported here, we determined the sites at which the M2-1 protein was phosphorylated and investigated the importance of these phosphorylated residues for M2-1 function in transcription. By combining protease digestion, matrix-assisted laser desorption ionization–time of flight mass spectrometry, and site-directed mutagenesis, we identified the phosphorylated residues as serines 58 and 61, not threonine 56 and serine 58 as previously reported. Serines 58 and 61 and the surrounding amino acids are in a consensus sequence for phosphorylation by casein kinase I. Consistent with this, we showed that the unphosphorylated M2-1 protein synthesized in
Escherichia coli
could be phosphorylated in vitro by casein kinase I. The effect of eliminating phosphorylation by site-specific mutagenesis of serines 58 and 61 on the function of the M2-1 protein in transcription of RS virus subgenomic replicons was assayed. The activities of the M2-1 protein phosphorylation mutants in transcriptional antitermination were tested over a range of concentrations and were found to be substantially inhibited at all concentrations. The data show that phosphorylation is important for the M2-1 protein function in transcription. However, mutation of the M2-1 phosphorylation sites did not interfere with the ability of the M2-1 protein to interact with the N protein in transfected cells. The interaction of the M2-1 and N proteins in cotransfected cells was found to be sensitive to RNase A, indicating that the M2-1–N protein interaction was mediated via RNA. Furthermore, the M2-1 protein was shown to bind monocistronic and polycistronic RS virus mRNAs during infection.
Genetic and Functional Diversity of Human Immunodeficiency Virus Type 1 Subtype B Nef Primary Isolates
Authors: Foster John L. , Molina Rene P. , Luo Tianci , Arora Vivek K. , Huang Yaoxing , Ho David D. , Garcia J. Victor 
Published: July 2002

ABSTRACT

We have characterized the functional integrity of seven primary Nef isolates: five from a long-term nonprogressing human immunodeficiency virus (HIV)-infected individual and one each from two patients with AIDS. One of the seven Nefs was defective for CD4 downregulation, two others were defective for PAK-2 activation, and one Nef was defective for PAK-2 activation and major histocompatibility complex (MHC) class I downregulation. Five of the Nefs were tested and found to be functional for the enhancement of virus particle infectivity. The structural basis for each of the functional defects has been analyzed by constructing a consensus
nef
, followed by mutational analysis of the variant amino acid residues. Mutations A29V and F193I were deleterious to CD4 downregulation and PAK-2 activation, respectively, while S189R rendered Nef defective for both MHC class I downregulation and PAK-2 activation. A search of the literature identified HIVs from five patients with Nefs predominantly mutated at F193 and from one patient with Nefs predominantly mutated at A29. A29 is highly conserved in all HIV subtypes except for subtype E. F193 is conserved in subtype B (and possibly in the closely related subtype D), but none of the other HIV group M subtypes. Our results suggest that functional distinctions may exist between HIV subtypes.
Activated Notch1 Can Transiently Substitute for EBNA2 in the Maintenance of Proliferation of LMP1-Expressing Immortalized B Cells
Authors: Höfelmayr Heike , Strobl Lothar J. , Marschall Gabriele , Bornkamm Georg W. , Zimber-Strobl Ursula 
Published: July 2002

ABSTRACTEpstein-Barr virus (EBV) nuclear antigen 2 (EBNA2) and latent membrane protein 1 (LMP1) are essential for immortalization of human B cells by EBV. EBNA2 and activated Notch transactivate genes by interacting with the cellular transcription factor RBP-Jκ/CBF1. Therefore, EBNA2 can be regarded as a functional homologue of activated Notch. We have shown previously that the intracellular domain of Notch1 (Notch1-IC) is able to transactivate EBNA2-regulated viral promoters and to induce phenotypic changes in B cells similar to those caused by EBNA2. Here we investigated whether Notch1-IC can substitute for EBNA2 in the maintenance of B-cell proliferation. Using an EBV-immortalized lymphoblastoid cell line in which EBNA2 function can be regulated by estrogen, we demonstrate that murine Notch1-IC, in the absence of functional EBNA2, is unable to maintain LMP1 expression and to maintain cell proliferation. However, in a lymphoblastoid cell line expressing LMP1 independently of EBNA2, murine Notch1-IC can transiently maintain proliferation after EBNA2 inactivation. After 4 days, cell numbers do not increase further, and cells in the G2phase of the cell cycle start to die. In contrast to EBNA2, murine Notch1-IC is unable to upregulate the expression of the c-mycgene in these cells.
Phenotypic Characterization of Three Phylogenetically Conserved Stem-Loop Motifs in the Mengovirus 3′ Untranslated Region
Authors: Duque Hernando , Palmenberg Ann C. 
Published: July 2002

ABSTRACT

An alignment of cardiovirus sequences led to the prediction of three conserved stem-loops in the 3′ untranslated region (UTR) of mengovirus. Deletions of each stem were engineered in mengovirus cDNAs and also in mengovirus replicons, in which part of the viral capsid sequences were replaced with the firefly luciferase gene. The effect of deletion on RNA infectivity and plaque phenotype was evaluated after transfection of viral transcripts into HeLa cells or by luciferase assays of cellular extracts after transfection with RNA replicons. Stem I (mengovirus bases 7666 to 7687) was found to be dispensable for viral growth or exponential luciferase expression. Deletion of stem III (bases 7711 to 7721) was lethal to the virus, and the replicons were incapable of RNA synthesis. Deletion of stem II (ΔII; bases 7692 to 7705) produced an intermediate phenotype, in that replicons had marginal RNA synthesis activity but transfection with genomic RNA usually failed to produce plaques after normal incubation times (31 h, 37°C). In a few of the ΔII transfections, however, plaques were observed after long incubation, especially if the cells received large amounts of RNA (3 μg per 3 × 10
6
cells). Viruses from two ΔII-derived plaques were isolated and amplified. Their RNAs were converted into cDNA, sequenced, and mapped for genotype. Each maintained the ΔII deletion and, in addition, had one or two reversion mutations, which were characterized by reverse genetics as responsible for the phenotypes. One reversion caused an amino acid change in the polymerase (3D
pol
), and the other was localized to the 3′ UTR, upstream of stem I.
Rhabdovirus-Based Vectors with Human Immunodeficiency Virus Type 1 (HIV-1) Envelopes Display HIV-1-Like Tropism and Target Human Dendritic Cells
Authors: Foley Heather D. , Otero Miguel , Orenstein Jan M. , Pomerantz Roger J. , Schnell Matthias J. 
Published: July 2002

ABSTRACTWe describe replication-competent, vaccine strain-based rabies viruses (RVs) that lack their own single glycoprotein and express, instead, a chimeric RV-human immunodeficiency virus type 1 (HIV-1) envelope protein composed of the ectodomain and transmembrane domains of HIV-1 gp160 and the cytoplasmic domain of RV G. The envelope proteins from both X4 (NL4-3)- and R5X4 (89.6)-tropic HIV-1 strains were utilized. These recombinant viruses very closely mimicked an HIV-1- like tropism, as indicated by blocking experiments. Infection was inhibited by SDF-1 on cells expressing CD4 and CXCR4 for both viruses, whereas RANTES abolished infection of cells expressing CCR5 in addition to CD4 in studies of the RV expressing HIV-189.6Env. In addition, preincubation with soluble CD4 or monoclonal antibodies directed against HIV-1 gp160 blocked the infectivity of both G-deficient viruses but did not affect the G-containing RVs. Our results also indicated that the G-deficient viruses expressing HIV-1 envelope protein, in contrast to wild-type RV but similar to HIV-1, enter cells by a pH-independent pathway. As observed for HIV-1, the surrogate viruses were able to target human peripheral blood mononuclear cells, macrophages, and immature and mature human dendritic cells (DC). Moreover, G-containing RV-based vectors also infected mature human DC, indicating that infection of these cells is also supported by RV G. The ability of RV-based vectors to infect professional antigen-presenting cells efficiently further emphasizes the potential use of recombinant RVs as vaccines.
Molecular Epidemiology of Echovirus 30: Temporal Circulation and Prevalence of Single Lineages
Authors: Palacios G. , Casas I. , Cisterna D. , Trallero G. , Tenorio A. , Freire C. 
Published: July 2002

ABSTRACT
Echovirus 30 (EV30) is one of the most frequently isolated EVs, causing extensive outbreaks of EV30 aseptic meningitis in temperate climates. EV30 is antigenically heterogeneous, and three major antigenic groups have been defined, although the basis for the antigenic differences is unknown. A reverse transcription-nested PCR which amplifies the 3′-terminal region of the VP1 gene directly from clinical samples was selected for studying EV30 molecular epidemiology, since the major antigenic sites in this region reflect the serotypic pattern of this virus. The different previous approaches to the genetic classification of EV30 were analyzed. A complete study of the EV30 strains was performed by analyzing the sequences from the 112 EV30 strains amplified in this work and the complete set of EV30 strains previously published. A total of 318 strains of EV30 were divided into two broad genotypes (I and II). This classification was supported by the phylogenetic trees obtained from amino acid sequences, and it correlated with the antigenic heterogeneity of the reference strains described in earlier studies. The genotypes could be further divided into subgroups, and these subgroups could be divided into lineages based on their nucleotide distances and levels of bootstrapping. On the other hand, the subgroups and lineages did not result in the same correlation between amino acid and nucleotide differentiation. The molecular epidemiology of EV30 can be compared to influenza virus epidemiology, where prevailing lineages displace the less established lineages on the basis of immune escape. This pattern of evolution is clearly different from that of other enteroviruses. A single lineage at a time appears to be circulating worldwide. This behavior may be related to the epidemic activity of EV30.