bluuebunny/crossref_metadata_2025_split huggingface dataset:
Variant Upstream Regulatory Region Sequences Differentially Regulate Human Papillomavirus Type 16 DNA Replication throughout the Viral Life Cycle
Authors: Hubert Walter G. 
Published: April 2005

ABSTRACT

While the central role of the viral upstream regulatory region (URR) in the human papillomavirus (HPV) life cycle has been well established, its effects on viral replication factor expression and plasmid replication of HPV type 16 (HPV16) remain unclear. Some nonprototypic variants of HPV16 contain altered URR sequences and are considered to increase the oncogenic risk of infections. To determine the relationship between viral replication and variant URRs, hybrid viral genomes were constructed with the replication-competent HPV16 prototype W12 and analyzed in assays which recapitulate the different phases of normal viral replication. The establishment efficiencies of hybrid HPV16 genomes differed about 20-fold among European prototypes and variants from Africa and America. Generally, European and African genomes exhibited the lowest replication efficiencies. The high replication levels observed with American variants were primarily attributable to their efficient expression of the replication factors E1 and E2. The maintenance levels of these viral genomes varied about fivefold, which correlated with their respective establishment phenotypes and published P
97
activities. Vegetative DNA amplification could also be observed with replicating HPV16 genomes. These results indicate that efficient E1/E2 expression and elevated plasmid replication levels during the persistent stage of infection may comprise a risk factor in HPV16-mediated oncogenesis.
Identification of
cis
-Acting Elements That Mediate the Replication and Maintenance of Human Papillomavirus Type 16 Genomes in
Saccharomyces cerevisiae
Authors: Kim Kitai , Angeletti Peter C. , Hassebroek Elizabeth C. , Lambert Paul F. 
Published: April 2005

ABSTRACT

Papillomaviruses contain small double-stranded DNA genomes that are maintained in persistently infected mammalian host epithelia as nuclear plasmids and rely upon the host replication machinery for replication. Papillomaviruses encode a DNA helicase, E1, which can specifically bind to the viral genome and support DNA synthesis. Under some conditions in mammalian cells, E1 is not required for viral DNA synthesis, leading to the hypothesis that papillomavirus DNA can be replicated solely by the host replication machinery. This machinery is highly conserved among eukaryotes. We and others found that papillomavirus DNA could replicate in a simple eukaryote,
Saccharomyces cerevisiae
. Specifically, papillomavirus DNA could substitute for the function of the autonomously replicating sequence (ARS) and centromere (CEN) elements that are normally both required for the stable replication of extrachromosomal DNAs in yeast. Furthermore, this form of replication in yeast was E1 independent. In this study, we map the elements in the human papillomavirus type 16 (HPV16) genome that can substitute for yeast ARS and CEN elements. A single element, termed
rep
, was identified that can substitute for ARS, and multiple elements, termed
mtc
, could substitute for CEN. The location of one of these
mtc
elements overlaps the location of
rep
, and this approximately 1,000-bp region of HPV16 was sufficient to support stable replication of a bacterial-yeast shuttle plasmid deleted of both ARS and CEN elements.
Characterization of the Interaction of Lassa Fever Virus with Its Cellular Receptor α-Dystroglycan
Authors: Kunz Stefan , Rojek Jillian M. , Perez Mar , Spiropoulou Christina F. , Oldstone Michael B. A. 
Published: April 2005

ABSTRACTThe cellular receptor for the Old World arenaviruses Lassa fever virus (LFV) and lymphocytic choriomeningitis virus (LCMV) has recently been identified as α-dystroglycan (α-DG), a cell surface receptor that provides a molecular link between the extracellular matrix and the actin-based cytoskeleton. In the present study, we show that LFV binds to α-DG with high affinity in the low-nanomolar range. Recombinant vesicular stomatitis virus pseudotyped with LFV glycoprotein (GP) adopted the receptor binding characteristics of LFV and depended on α-DG for infection of cells. Mapping of the binding site of LFV on α-DG revealed that LFV binding required the same domains of α-DG that are involved in the binding of LCMV. Further, LFV was found to efficiently compete with laminin α1 and α2 chains for α-DG binding. Together with our previous studies on receptor binding of the prototypic immunosuppressive LCMV isolate LCMV clone 13, these findings indicate a high degree of conservation in the receptor binding characteristics between the highly human-pathogenic LFV and murine-immunosuppressive LCMV isolates.
Targeting Human Glioblastoma Cells: Comparison of Nine Viruses with Oncolytic Potential
Authors: Wollmann Guido , Tattersall Peter , van den Pol Anthony N. 
Published: April 2005

ABSTRACT
Brain tumors classified as glioblastomas have proven refractory to treatment and generally result in death within a year of diagnosis. We used seven in vitro tests and one in vivo trial to compare the efficacy of nine different viruses for targeting human glioblastoma. Green fluorescent protein (GFP)-expressing vesicular stomatitis (VSV), Sindbis virus, pseudorabies virus (PRV), adeno-associated virus (AAV), and minute virus of mice i-strain (MVMi) and MVMp all infected glioblastoma cells. Mouse and human cytomegalovirus, and simian virus 40 showed only low levels of infection or GFP expression. VSV and Sindbis virus showed strong cytolytic actions and high rates of replication and spread, leading to an elimination of glioblastoma. PRV and both MVM strains generated more modest lytic effects and replication capacity. VSV showed a similar oncolytic profile on U-87 MG and M059J glioblastoma. In contrast, Sindbis virus showed strong preference for U-87 MG, whereas MVMi and MVMp preferred M059J. Sindbis virus and both MVM strains showed highly tumor-selective actions in glioblastoma plus fibroblast coculture. VSV and Sindbis virus were serially passaged on glioblastoma cells; we isolated a variant, VSV-rp30, that had increased selectivity and lytic capacity in glioblastoma cells. VSV and Sindbis virus were very effective at replicating, spreading within, and selectively killing human glioblastoma in an in vivo mouse model, whereas PRV and AAV remained at the injection site with minimal spread. Together, these data suggest that four (VSV, Sindbis virus, MVMi, and MVMp) of the nine viruses studied merit further analysis for potential therapeutic actions on glioblastoma.
Biologic and Genetic Characterization of a Panel of 60 Human Immunodeficiency Virus Type 1 Isolates, Representing Clades A, B, C, D, CRF01_AE, and CRF02_AG, for the Development and Assessment of Candidate Vaccines
Authors: Brown Bruce K. , Darden Janice M. , Tovanabutra Sodsai , Oblander Tamara , Frost Julie , Sanders-Buell Eric , de Souza Mark S. , Birx Deborah L. , McCutchan Francine E. , Polonis Victoria R. 
Published: April 2005

ABSTRACT
A critical priority for human immunodeficiency virus type 1 (HIV-1) vaccine development is standardization of reagents and assays for evaluation of immune responses elicited by candidate vaccines. To provide a panel of viral reagents from multiple vaccine trial sites, 60 international HIV-1 isolates were expanded in peripheral blood mononuclear cells and characterized both genetically and biologically. Ten isolates each from clades A, B, C, and D and 10 isolates each from CRF01_AE and CRF02_AG were prepared from individuals whose HIV-1 infection was evaluated by complete genome sequencing. The main criterion for selection was that the candidate isolate was pure clade or pure circulating recombinant. After expansion in culture, the complete envelope (gp160) of each isolate was verified by sequencing. The 50% tissue culture infectious dose and p24 antigen concentration for each viral stock were determined; no correlation between these two biologic parameters was found. Syncytium formation in MT-2 cells and CCR5 or CXCR4 coreceptor usage were determined for all isolates. Isolates were also screened for neutralization by soluble CD4, a cocktail of monoclonal antibodies, and a pool of HIV-1-positive patient sera. The panel consists of 49 nonsyncytium-inducing isolates that use CCR5 as a major coreceptor and 11 syncytium-inducing isolates that use only CXCR4 or both coreceptors. Neutralization profiles suggest that the panel contains both neutralization-sensitive and -resistant isolates. This collection of HIV-1 isolates represents the six major globally prevalent strains, is exceptionally large and well characterized, and provides an important resource for standardization of immunogenicity assessment in HIV-1 vaccine trials.
Cellular Localization and Antigenic Characterization of Crimean-Congo Hemorrhagic Fever Virus Glycoproteins
Authors: Bertolotti-Ciarlet Andrea , Smith Jonathan , Strecker Karin , Paragas Jason , Altamura Louis A. , McFalls Jeanne M. , Frias-Stäheli Natalia , García-Sastre Adolfo , Schmaljohn Connie S. , Doms Robert W. 
Published: April 2005

ABSTRACT

Crimean-Congo hemorrhagic fever virus (CCHFV), a member of the genus
Nairovirus
of the family
Bunyaviridae
, causes severe disease with high rates of mortality in humans. The CCHFV M RNA segment encodes the virus glycoproteins G
N
and G
C
. To understand the processing and intracellular localization of the CCHFV glycoproteins as well as their neutralization and protection determinants, we produced and characterized monoclonal antibodies (MAbs) specific for both G
N
and G
C
. Using these MAbs, we found that G
N
predominantly colocalized with a Golgi marker when expressed alone or with G
C
, while G
C
was transported to the Golgi apparatus only in the presence of G
N
. Both proteins remained endo-β-
N
-acetylglucosaminidase H sensitive, indicating that the CCHFV glycoproteins are most likely targeted to the
cis
Golgi apparatus. Golgi targeting information partly resides within the G
N
ectodomain, because a soluble version of G
N
lacking its transmembrane and cytoplasmic domains also localized to the Golgi apparatus. Coexpression of soluble versions of G
N
and G
C
also resulted in localization of soluble G
C
to the Golgi apparatus, indicating that the ectodomains of these proteins are sufficient for the interactions needed for Golgi targeting. Finally, the mucin-like and P35 domains, located at the N terminus of the G
N
precursor protein and removed posttranslationally by endoproteolysis, were required for Golgi targeting of G
N
when it was expressed alone but were dispensable when G
C
was coexpressed. In neutralization assays on SW-13 cells, MAbs to G
C
, but not to G
N
, prevented CCHFV infection. However, only a subset of G
C
MAbs protected mice in passive-immunization experiments, while some nonneutralizing G
N
MAbs efficiently protected animals from a lethal CCHFV challenge. Thus, neutralization of CCHFV likely depends not only on the properties of the antibody, but on host cell factors as well. In addition, nonneutralizing antibody-dependent mechanisms, such as antibody-dependent cell-mediated cytotoxicity, may be involved in the in vivo protection seen with the MAbs to G
C
.
The Polypyrimidine Tract Binding Protein Is Required for Efficient Picornavirus Gene Expression and Propagation
Authors: Florez Paola M. , Sessions October M. , Wagner Eric J. , Gromeier Matthias , Garcia-Blanco Mariano A. 
Published: April 2005

ABSTRACT

Mammalian host factors required for efficient viral gene expression and propagation have been often recalcitrant to genetic analysis. A case in point is the function of cellular factors that
trans
-activate internal ribosomal entry site (IRES)-driven translation, which is operative in many positive-stranded RNA viruses, including all picornaviruses. These IRES
trans
-acting factors have been elegantly studied in vitro, but their in vivo importance for viral gene expression and propagation has not been widely confirmed experimentally. Here we use RNA interference to deplete mammalian cells of one such factor, the polypyrimidine tract binding protein, and test its requirement in picornavirus gene expression and propagation. Depletion of the polypyrimidine tract binding protein resulted in a marked delay of particle propagation and significantly decreased synthesis and accumulation of viral proteins of poliovirus and encephalomyocarditis virus. These effects could be partially restored by expression of an RNA interference-resistant exogenous polypyrimidine tract binding protein. These data indicate a critical role for the polypyrimidine tract binding protein in picornavirus gene expression and strongly suggest a requirement for efficient IRES-dependent translation.
Carboxyl-Proximal Regions of Reovirus Nonstructural Protein μNS Necessary and Sufficient for Forming Factory-Like Inclusions
Authors: Broering Teresa J. , Arnold Michelle M. , Miller Cathy L. , Hurt Jessica A. , Joyce Patricia L. , Nibert Max L. 
Published: April 2005

ABSTRACT

Mammalian orthoreoviruses are believed to replicate in distinctive, cytoplasmic inclusion bodies, commonly called viral factories or viroplasms. The viral nonstructural protein μNS has been implicated in forming the matrix of these structures, as well as in recruiting other components to them for putative roles in genome replication and particle assembly. In this study, we sought to identify the regions of μNS that are involved in forming factory-like inclusions in transfected cells in the absence of infection or other viral proteins. Sequences in the carboxyl-terminal one-third of the 721-residue μNS protein were linked to this activity. Deletion of as few as eight residues from the carboxyl terminus of μNS resulted in loss of inclusion formation, suggesting that some portion of these residues is required for the phenotype. A region spanning residues 471 to 721 of μNS was the smallest one shown to be sufficient for forming factory-like inclusions. The region from positions 471 to 721 (471-721 region) includes both of two previously predicted coiled-coil segments in μNS, suggesting that one or both of these segments may also be required for inclusion formation. Deletion of the more amino-terminal one of the two predicted coiled-coil segments from the 471-721 region resulted in loss of the phenotype, although replacement of this segment with
Aequorea victoria
green fluorescent protein, which is known to weakly dimerize, largely restored inclusion formation. Sequences between the two predicted coiled-coil segments were also required for forming factory-like inclusions, and mutation of either one His residue (His570) or one Cys residue (Cys572) within these sequences disrupted the phenotype. The His and Cys residues are part of a small consensus motif that is conserved across μNS homologs from avian orthoreoviruses and aquareoviruses, suggesting this motif may have a common function in these related viruses. The inclusion-forming 471-721 region of μNS was shown to provide a useful platform for the presentation of peptides for studies of protein-protein association through colocalization to factory-like inclusions in transfected cells.
Capsid Protein Synthesis from Replicating RNA Directs Specific Packaging of the Genome of a Multipartite, Positive-Strand RNA Virus
Authors: Venter P. Arno , Krishna Neel K. , Schneemann Anette 
Published: April 2005

ABSTRACT

Flock house virus
(FHV) is a bipartite, positive-strand RNA insect virus that encapsidates its two genomic RNAs in a single virion. It provides a convenient model system for studying the principles underlying the copackaging of multipartite viral RNA genomes. In this study, we used a baculovirus expression system to determine if the uncoupling of viral protein synthesis from RNA replication affected the packaging of FHV RNAs. We found that neither RNA1 (which encodes the viral replicase) nor RNA2 (which encodes the capsid protein) were packaged efficiently when capsid protein was supplied in
trans
from nonreplicating RNA. However, capsid protein synthesized in
cis
from replicating RNA2 packaged RNA2 efficiently in the presence and absence of RNA1. These results demonstrated that capsid protein translation from replicating RNA2 is required for specific packaging of the FHV genome. This type of coupling between genome replication and translation and RNA packaging has not been observed previously. We hypothesize that RNA2 replication and translation must be spatially coordinated in FHV-infected cells to facilitate retrieval of the viral RNAs for encapsidation by newly synthesized capsid protein. Spatial coordination of RNA and capsid protein synthesis may be key to specific genome packaging and assembly in other RNA viruses.
Induction of Long-Term Protective Antiviral Endogenous Immune Response by Short Neutralizing Monoclonal Antibody Treatment
Authors: Gros Laurent , Dreja Hanna , Fiser Anne Laure , Plays Marc , Pelegrin Mireia , Piechaczyk Marc 
Published: April 2005

ABSTRACTLong-term immune control of viral replication still remains a major challenge in retroviral diseases. Several monoclonal antibodies (MAbs) have already shown antiviral activities in vivo, including in the clinic but their effects on the immune system of treated individuals are essentially unknown. Using the lethal neurodegeneration induced in mice upon infection of neonates by the FrCasEretrovirus as a model, we report here that transient treatment by a neutralizing MAb shortly after infection can, after an immediate antiviral effect, favor the development of a strong protective host immune response containing viral propagation long after the MAb has disappeared. In vitro virus neutralization- and complement-mediated cell lysis assays, as well as in vivo viral challenges and serum transfer experiments, indicate a clear and essential contribution of the humoral response to antiviral protection. Our observation may have important therapeutic consequences as it suggests that short antibody-based therapies early after infection should be considered, at least in the case of maternally infected infants, as adjunctive treatment strategies against human immunodeficiency virus, not only for a direct effect on the viral load but also for favoring the emergence of an endogenous antiviral immune response.
Genetic Determinants of Cell Type-Specific Poliovirus Propagation in HEK 293 Cells
Authors: Campbell Stephanie A. , Lin Jennifer , Dobrikova Elena Y. , Gromeier Matthias 
Published: April 2005

ABSTRACT
The ability of poliovirus to propagate in neuronal cells can be reduced by introducing appropriate nucleotide substitutions into the viral genome. Specific mutations scattered throughout the poliovirus genome yielded the live attenuated vaccine strains of poliovirus. Neuron-specific propagation deficits of the Sabin strains are partially encrypted within a confined region of the internal ribosomal entry site (IRES), which carries attenuating point mutations in all three serotypes. Recently, high levels of neurovirulence attenuation were achieved with genetically engineered polioviruses containing heterologous IRES elements. This is exemplified with poliovirus recombinants replicating under control of a human rhinovirus type 2 (HRV2) IRES element. We have carried out experiments delineating the genetic basis for neuronal IRES function. Neuronal dysfunction of the HRV2 IRES is determined mainly by IRES stem-loop domain V, the locus for attenuating point mutations within the Sabin strains. Neuronal incompetence associated with HRV2 IRES domain V is substantially more pronounced than that observed with the attenuating IRES point mutation of the Sabin serotype 1 vaccine strain. Mix-and-match recombination of polio and HRV2 IRES domain V suggests that the attenuation phenotype correlates with overall structural features rather than primary sequence. Our experiments have identified HEK 293 cells as a novel system for the study of neuron-specific replication phenotypes of poliovirus. This cell line, originally derived from embryonic human kidney, has recently been described to display neuronal characteristics. We report propagation properties in HEK 293 cells for poliovirus recombinants with attenuated neurovirulence in experimental animals that corroborate this observation.
Distinct Mechanisms of CD4+and CD8+T-Cell Activation and Bystander Apoptosis Induced by Human Immunodeficiency Virus Type 1 Virions
Authors: Holm Geoffrey H. , Gabuzda Dana 
Published: April 2005

ABSTRACTApoptosis of uninfected bystander T cells contributes to T-cell depletion during human immunodeficiency virus type 1 (HIV-1) infection. HIV-1 envelope/receptor interactions and immune activation have been implicated as contributors to bystander apoptosis. To better understand the relationship between T-cell activation and bystander apoptosis during HIV-1 pathogenesis, we investigated the effects of the highly cytopathic CXCR4-tropic HIV-1 variant ELI6 on primary CD4+and CD8+T cells. Infection of primary T-cell cultures with ELI6 induced CD4+T-cell depletion by direct cell lysis and bystander apoptosis. Exposure of primary CD4+and CD8+T cells to nonreplicating ELI6 virions induced bystander apoptosis through a Fas-independent mechanism. Bystander apoptosis of CD4+T cells required direct contact with virions and Env/CXCR4 binding. In contrast, the apoptosis of CD8+T cells was triggered by a soluble factor(s) secreted by CD4+T cells. HIV-1 virions activated CD4+and CD8+T cells to express CD25 and HLA-DR and preferentially induced apoptosis in CD25+HLA-DR+T cells in a CXCR4-dependent manner. Maximal levels of binding, activation, and apoptosis were induced by virions that incorporated MHC class II and B7-2 into the viral membrane. These results suggest that nonreplicating HIV-1 virions contribute to chronic immune activation and T-cell depletion during HIV-1 pathogenesis by activating CD4+and CD8+T cells, which then proceed to die via apoptosis. This mechanism may represent a viral immune evasion strategy to increase viral replication by activating target cells while killing immune effector cells that are not productively infected.
An Adenovirus Type 5 (Ad5) Amplicon-Based Packaging Cell Line for Production of High-Capacity Helper-Independent ΔE1-E2-E3-E4 Ad5 Vectors
Authors: Catalucci Daniele , Sporeno Elisabetta , Cirillo Agostino , Ciliberto Gennaro , Nicosia Alfredo , Colloca Stefano 
Published: April 2005

ABSTRACT

Production of multiply deleted adenoviral (Ad) vectors with increased cloning capacity and reduced immunogenicity to adenovirus gene products requires the concomitant generation of efficient packaging cell lines. High expression levels of the complementing genes must be achieved in a coordinated fashion with viral replication. This is a particularly difficult task in light of the significant cytotoxicity displayed by adenoviral proteins. To this end, we developed a novel adenovirus-based amplicon with an Epstein-Barr virus origin of replication, Ad type 5 (Ad5) inverted terminal repeats, all Ad5 early region 2 (E2) genes, and the early region 4 (E4) open reading frame 6 (ORF6) under the control of a tetracycline-dependent promoter. The amplicon (pE2) was stably maintained in multiple copies in the nuclei of 293 cells stably expressing the Epstein-Barr virus nuclear antigen 1 (EBNA1) and allowed replication as a linear DNA upon induction of E2 and ORF6 gene expression. A stable cell line (2E2) was generated by introducing pE2 into 293EBNATet cells expressing the tetracycline-dependent transcriptional silencer and the reverse Tet transactivator (rtTA2). Upon induction with doxicycline, 2E2 cells produced higher levels of polymerase, precursor terminal protein (pTP), and DNA binding protein than noninduced 2E2 cells infected with first-generation Ad5 vector and supported efficient amplification of a multiply deleted Ad5 vector lacking E1, E2, E3, and E4 genes (Ad5ΔE
1-4
). The high cloning capacity of Ad5ΔE
1-4
(up to 12.6 kb) was exploited to construct a vector encoding the entire hepatitis C virus (HCV) polyprotein. Infection of HeLa cells by the resulting vector showed high levels of correctly processed HCV proteins.