bluuebunny/crossref_metadata_2025_split huggingface dataset:
Borna Disease Virus Nucleoprotein Requires both Nuclear Localization and Export Activities for Viral Nucleocytoplasmic Shuttling
Authors: Kobayashi Takeshi , Kamitani Wataru , Zhang Guoqi , Watanabe Makiko , Tomonaga Keizo , Ikuta Kazuyoshi 
Published: July 2002

ABSTRACT

Nuclear transport of viral nucleic acids is crucial to the life cycle of many viruses. Borna disease virus (BDV) belongs to the order
Mononegavirales
and replicates its RNA genome in the nucleus. Previous studies have suggested that BDV nucleoprotein (N) and phosphoprotein (P) have important functions in the nuclear import of the viral ribonucleoprotein (RNP) complexes via their nuclear targeting activity. Here, we showed that BDV N has cytoplasmic localization activity, which is mediated by a nuclear export signal (NES) within the sequence. Our analysis using deletion and substitution mutants of N revealed that NES of BDV N consists of a canonical leucine-rich motif and that the nuclear export activity of the protein is mediated through the chromosome region maintenance protein-dependent pathway. Interspecies heterokaryon assay indicated that BDV N shuttles between the nucleus and cytoplasm as a nucleocytoplasmic shuttling protein. Furthermore, interestingly, the NES region overlaps a binding site to the BDV P protein, and nuclear export of a 38-kDa form of BDV N is prevented by coexpression of P. These results suggested that BDV N has two contrary activities, nuclear localization and export activity, and plays a critical role in the nucleocytoplasmic transport of BDV RNP by interaction with other viral proteins.
Replication of the Human Hepatitis Delta Virus Genome Is Initiated in Mouse Hepatocytes following Intravenous Injection of Naked DNA or RNA Sequences
Authors: Chang Jinhong , Sigal Luis J. , Lerro Anthony , Taylor John 
Published: July 2002

ABSTRACT

As early as 5 days after DNA copies of the hepatitis delta virus (HDV) genome or even
in vitro
-transcribed HDV RNA sequences were injected into the mouse tail vein using the hydrodynamics-based transfection procedure of F. Liu et al. (Gene Ther. 6:1258–1266, 1999), it was possible to detect in the liver by Northern analyses of RNA, immunoblots of protein, and immunostaining of liver sections what were considered typical features of HDV genome replication. This transfection strategy should have valuable applications for in vivo studies of HDV replication and pathogenesis and may also be useful for studies of other hepatotropic viruses.
Activation of Membrane Fusion by Murine Leukemia Viruses Is Controlled in
cis
or in
trans
by Interactions between the Receptor-Binding Domain and a Conserved Disulfide Loop of the Carboxy Terminus of the Surface Glycoprotein
Authors: Lavillette Dimitri , Boson Bertrand , Russell Stephen J. , Cosset François-Loı̈c 
Published: July 2002

ABSTRACT

Cell entry of retroviruses is initiated by the recognition of cellular receptors and the subsequent membrane fusion between viral and cellular membranes. These two steps are mediated by the surface (SU) and transmembrane (TM) subunits of the retroviral envelope glycoprotein (Env), respectively. Determinants regulating membrane fusion have been described throughout SU and TM, but the processes coupling receptor recognition to fusion are still elusive. Here we establish that a critical interaction is formed between the receptor-binding domain (RBD) and the major disulfide loop of the carboxy-terminal domain (C domain) of the murine leukemia virus SU. Receptor binding causes an alteration of this interaction and, in turn, promotes further events of Env fusion activation. We characterize mutations which, by lowering this interaction and reducing the compatibility between the RBD and C domains of Env glycoprotein chimeras, affect both Env fusogenicity and sensitivity to receptor interference. Additionally, we demonstrate that suboptimal interactions in such mutant Env proteins can be compensated in
trans
by soluble RBDs in a manner that depends on their compatibility with the C domain. Our results therefore indicate that RBD/C domain interactions may occur in
cis
, via the proper RBD of the viral Env itself, or in
trans
, via a distinct RBD expressed by virion-free Env glycoproteins expressed endogenously by the infected cells or provided by neighboring Env trimers.
Requirements for Assembly of Poliovirus Replication Complexes and Negative-Strand RNA Synthesis
Authors: Teterina Natalya L. , Egger Denise , Bienz Kurt , Brown David M. , Semler Bert L. , Ehrenfeld Ellie 
Published: July 2002

ABSTRACT

HeLa cells were transfected with several plasmids that encoded all poliovirus (PV) nonstructural proteins. Viral RNAs were transcribed by T7 RNA polymerase expressed from recombinant vaccinia virus. All plasmids produced similar amounts of viral proteins that were processed identically; however, RNAs were designed either to serve as templates for replication or to contain mutations predicted to prevent RNA replication. The mutations included substitution of the entire PV 5′ noncoding region (NCR) with the encephalomyocarditis virus (EMCV) internal ribosomal entry site, thereby deleting the 5′-terminal cloverleaf-like structure, or insertion of three nucleotides in the 3D
pol
coding sequence. Production of viral proteins was sufficient to induce the characteristic reorganization of intracellular membranes into heterogeneous-sized vesicles, independent of RNA replication. The vesicles were stably associated with viral RNA only when RNA replication could occur. Nonreplicating RNAs localized to distinct, nonoverlapping regions in the cell, excluded from the viral protein-membrane complexes. The absence of accumulation of positive-strand RNA from both mutated RNAs in transfected cells was documented. In addition, no minus-strand RNA was produced from the EMCV chimeric template RNA in vitro. These data show that the 5′-terminal sequences of PV RNA are essential for initiation of minus-strand RNA synthesis at its 3′ end.
In Vivo Accumulation of Cyclin A and Cellular Replication Factors in Autonomous Parvovirus Minute Virus of Mice-Associated Replication Bodies
Authors: Bashir Tarig , Rommelaere Jean , Cziepluch Celina 
Published: July 2002

ABSTRACT
Autonomous parvovirus minute virus of mice (MVM) DNA replication is strictly dependent on cellular factors expressed during the S phase of the cell cycle. Here we report that MVM DNA replication proceeds in specific nuclear structures termed autonomous parvovirus-associated replication bodies, where components of the basic cellular replication machinery accumulate. The presence of DNA polymerases α and δ in these bodies suggests that MVM utilizes partially preformed cellular replication complexes for its replication. The recruitment of cyclin A points to a role for this cell cycle factor in MVM DNA replication beyond its involvement in activating the conversion of virion single-stranded DNA to the duplex replicative form.
Infection of Cells with Human Cytomegalovirus during S Phase Results in a Blockade to Immediate-Early Gene Expression That Can Be Overcome by Inhibition of the Proteasome
Authors: Fortunato Elizabeth A. , Sanchez Veronica , Yen Judy Y. , Spector Deborah H. 
Published: July 2002

ABSTRACT

Cells infected with human cytomegalovirus (HCMV) after commencing DNA replication do not initiate viral immediate-early (IE) gene expression and divide before arresting. To determine the nature of this blockade, we examined cells that were infected 24 h after release from G
0
using immunofluorescence, laser scanning cytometry, and fluorescence-activated cell sorting (FACS) analysis. Approximately 40 to 50% of the cells had 2N DNA content, became IE
+
in the first 12 h, and arrested. Most but not all of the cells with >2N DNA content did not express IE antigens until after mitosis. To define the small population of IE
+
cells that gradually accumulated within the S and G
2
/M compartments, cells were pulsed with bromodeoxyuridine (BrdU) just prior to S-phase infection and analyzed at 12 h postinfection for IE gene expression, BrdU positivity, and cell cycle position. Most of the BrdU
+
cells were IE
−
and had progressed into G
2
/M or back to G
1
. The majority of the IE
+
cells in S and G
2
/M were BrdU
−
. Only a few cells were IE
+
BrdU
+
, and they resided in G
2
/M. Multipoint BrdU pulse-labeling revealed that, compared to cells actively synthesizing DNA at the beginning of the infection, a greater percentage of the cells that initiated DNA replication 4 h later could express IE antigens and proceed into S. Synchronization of the cells with aphidicolin also indicated that the blockade to the activation of IE gene expression was established in cells soon after initiation of DNA replication. It appears that a short-lived protein in S-phase cells may be required for IE gene expression, as it is partially restored by treatment with the proteasome inhibitor MG132.
Preexisting Immunity to Adenovirus in Rhesus Monkeys Fails To Prevent Vector-Induced Toxicity
Authors: Varnavski Andrei N. , Zhang Yi , Schnell Michael , Tazelaar John , Louboutin Jean-Pierre , Yu Qian-Chun , Bagg Adam , Gao Guang-ping , Wilson James M. 
Published: July 2002

ABSTRACTIn an earlier study we evaluated innate immune responses to a first-generation adenoviral vector infused into the portal vein of rhesus monkeys who had never been exposed to adenovirus previously. In these animals, the systemic administration of E1/E3-deleted adenoviral vectors resulted in immediate activation of innate immunity and serious toxicity caused by targeting of vector to antigen-presenting cells and systemic inflammation. We analyze here how these responses are affected by vector-specific preexisting immunity that was induced by intramuscular immunization 6 months prior to evaluation. Our results show that preexposure to the vector substantially diminishes the transgene expression in most tissues but has little effect on gene transfer. Significantly, preimmunization does not eliminate systemic vector-induced toxicity. These conclusions are based on the presence of clinical features of coagulopathy and elevated levels of proinflammatory cytokine interleukin-6 in the serum of animals treated with vector after intramuscular immunization. Furthermore, preexisting immunity appears to induce a vector-specific inhibitory effect on erythroid progenitor development in the bone marrow that is not found when naive animals are challenged with vector.
Virus-Like Particles of a Fish Nodavirus Display a Capsid Subunit Domain Organization Different from That of Insect Nodaviruses
Authors: Tang Liang , Lin Chan-Shing , Krishna Neel K. , Yeager Mark , Schneemann Anette , Johnson John E. 
Published: July 2002

ABSTRACT

The structure of recombinant virus-like particles of malabaricus grouper nervous necrosis virus (MGNNV), a fish nodavirus isolated from the grouper
Epinephelus malabaricus
, was determined by electron cryomicroscopy (cryoEM) and three-dimensional reconstruction at 23-Å resolution. The cryoEM structure, sequence comparison, and protein fold recognition analysis indicate that the coat protein of MGNNV has two domains resembling those of tomato bushy stunt virus and Norwalk virus, rather than the expected single-domain coat protein of insect nodaviruses. The analysis implies that residues 83 to 216 fold as a β-sandwich which forms the inner shell of the T=3 capsid and residues 217 to 308 form the trimeric surface protrusions observed in the cryoEM map. The structural similarities between fish nodaviruses and members of the tombusvirus and calicivirus groups provide significant new data for understanding the evolution of the nodavirus family.
γδ T Cells Promote a Th1 Response during Coxsackievirus B3 Infection In Vivo: Role of Fas and Fas Ligand
Authors: Huber Sally , Shi Cuixia , Budd Ralph C. 
Published: July 2002

ABSTRACTFas/Fas ligand (FasL) interactions regulate disease outcome in coxsackievirus B3 (CVB3)-induced myocarditis. MRL+/+mice infected with CVB3 develop severe myocarditis, a dominant CD4+Th1 (gamma interferon [IFN-γ+]) response to the virus, and a predominance of γδ T cells in the myocardial infiltrates. MRLlpr/lprand MRLgld/gldmice, which lack normal expression of Fas and express a mutated FasL, respectively, have minimal myocarditis and show a dominant CD4+Th2 (interleukin-4 [IL-4+]) phenotype to CVB3. Spleen cells from virus-infected wild-type,lpr, andgldanimals proliferate equally to virus in vitro. Adoptive transfer of γδ T cells from hearts of CVB3-infected MRL+/+mice (FasL+) into infected MRLgld/gldrecipients (FasL−/Fas+) restores both disease susceptibility and Th1 cell phenotype. However, transfer of these cells into MRLlpr/lprrecipients (FasL+/Fas−) did not promote myocarditis and the viral response remained Th2 biased. This paralleled the expression of very high surface levels of FasL by myocardial γδ T cells, as well as their propensity to selectively lyse Th2 virus-specific CD4+T cells. These results demonstrate that Fas/FasL interactions conferred by γδ Τ cells on lymphocyte subpopulations may regulate the cytokine response to CVB3 infection and pathogenicity.
Vaccinia Virus G4L Glutaredoxin Is an Essential Intermediate of a Cytoplasmic Disulfide Bond Pathway Required for Virion Assembly
Authors: White Christine L. , Senkevich Tatiana G. , Moss Bernard 
Published: July 2002

ABSTRACT

Our previous studies provided evidence that E10R, a vaccinia virus protein belonging to the ERV1/ALR family, has a redox function and is required for virion assembly. Repression of E10R prevented the formation of intramolecular disulfide bonds of the G4L glutaredoxin, the L1R membrane protein, and the structurally related F9L protein. Here, we demonstrate an oxidation pathway (E10R
SS
→ G4L
SS
→ L1R
SS
, F9L
SS
) in which G4L occupies an intermediate position. By reacting free thiols with 4-acetamido-4′-malemideylstilbene-2,2′-disulfonic acid, alkylated and nonalkylated disulfide-bonded forms of G4L could be resolved from each other by polyacrylamide gel electrophoresis. The cysteines of intracellular G4L were in both disulfide and reduced forms, whereas those of E10R, L1R, and F9L and virion-associated G4L were mostly disulfide bonded. Repression of G4L expression prevented the formation of disulfide bonds in both L1R and F9L but not E10R. Both cysteines of G4L were required for L1R and F9L disulfide bond formation or for
trans
-complementation of virus infectivity when G4L expression was repressed. No role in the E10R-G4L redox pathway was found for O2L, a nonessential glutaredoxin encoded by vaccinia virus. We suggest that cytoplasmic G4L is a redox shuttle between membrane-associated E10R and L1R or F9L.
Paired Charge-to-Alanine Mutagenesis of Dengue Virus Type 4 NS5 Generates Mutants with Temperature-Sensitive, Host Range, and Mouse Attenuation Phenotypes
Authors: Hanley Kathryn A. , Lee Jay J. , Blaney Joseph E. , Murphy Brian R. , Whitehead Stephen S. 
Published: July 2002

ABSTRACT

Charge-to-alanine mutagenesis of dengue virus type 4 (DEN4) NS5 gene generated a collection of attenuating mutations for potential use in a recombinant live attenuated DEN vaccine. Codons for 80 contiguous pairs of charged amino acids in NS5 were individually mutagenized to create uncharged pairs of alanine residues, and 32 recombinant mutant viruses were recovered from the 80 full-length mutant DEN4 cDNA constructs. These mutant viruses were tested for temperature-sensitive (ts) replication in both Vero cells and HuH-7 human hepatoma cells. Of the 32 mutants, 13 were temperature sensitive (ts) in both cell lines, 11 were not ts in either cell line, and 8 exhibited a host range (
tshr
) phenotype. One
tshr
mutant was ts only in Vero cells, and seven were ts only in HuH-7 cells. Nineteen of the 32 mutants were 10-fold or more restricted in replication in the brains of suckling mice compared to that of wild-type DEN4, and three mutants were approximately 10,000-fold restricted in replication. The level of temperature sensitivity of replication in vitro did not correlate with attenuation in vivo. A virus bearing two pairs of charge-to-alanine mutations was constructed and demonstrated increased temperature sensitivity and attenuation relative to either parent virus. This large set of charge-to-alanine mutations specifying a wide range of attenuation for mouse brain should prove useful in fine-tuning recombinant live attenuated DEN vaccines.
Human Papillomavirus Type 16 E7 Maintains Elevated Levels of the cdc25A Tyrosine Phosphatase during Deregulation of Cell Cycle Arrest
Authors: Nguyen Don X. , Westbrook Thomas F. , McCance Dennis J. 
Published: July 2002

ABSTRACT

Essential to the oncogenic properties of human papillomavirus type 16 (HPV-16) are the activities encoded by the early gene product E7. HPV-16 E7 (E7.16) binds to cellular factors involved in cell cycle regulation and differentiation. These include the retinoblastoma tumor suppressor protein (Rb) and histone deacetylase (HDAC) complexes. While the biological significance of these interactions remains unclear, E7 is believed to help maintain cells in a proliferative state, thus establishing an environment that is conducive to viral replication. Most pathways that govern cell growth converge on downstream effectors. Among these is the cdc25A tyrosine phosphatase. cdc25A is required for G
1
/S transition, and its deregulation is associated with carcinogenesis. Considering the importance of cdc25A in cell cycle progression, it represents a relevant target for viral oncoproteins. Accordingly, the present study focuses on the putative deregulation of cdc25A by E7.16. Our results indicate that E7.16 can impede growth arrest induced during serum starvation and keratinocyte differentiation. Importantly, these E7-specific phenotypes correlate with elevated cdc25A steady-state levels. Reporter assays performed with NIH 3T3 cell lines and human keratinocytes indicate that E7 can transactivate the
cdc25A
promoter. In addition, transcriptional activation by E7.16 requires the distal E2F site within the
cdc25A
promoter. We further demonstrate that the ability of E7 to abrogate cell cycle arrest, activate
cdc25A
transcription, and increase cdc25A protein levels requires intact Rb and HDAC-1 binding domains. Finally, by using the cdk inhibitor roscovitine, we reveal that E7 activates the
cdc25A
promoter independently of cell cycle progression and cdk activity. Consequently, we propose that E7.16 can directly target
cdc25A
transcription and maintains
cdc25A
gene expression by disrupting Rb/E2F/HDAC-1 repressor complexes.
Intracellular Distribution of Human T-Cell Leukemia Virus Type 1 Gag Proteins Is Independent of Interaction with Intracellular Membranes
Authors: Le Blanc Isabelle , Blot Vincent , Bouchaert Isabelle , Salamero Jean , Goud Bruno , Rosenberg Arielle R. , Dokhélar Marie-Christine 
Published: July 2002

ABSTRACT
Retrovirus Gag proteins are synthesized on free ribosomes, and are sufficient to govern the assembly and release of virus particles. Like type C retroviruses, human T-cell leukemia virus type 1 (HTLV-1) assembles and buds at the plasma membrane. After immunofluorescence staining, HTLV-1 Gag proteins appear as punctuated intracellular clusters, which suggests that they are associated either with intracellular membranes or with the plasma membrane. However, colocalization experiments using a panel of markers demonstrated that Gag proteins were not associated with the membranes involved in the secretory or endocytosis pathway. Small amounts of Gag proteins were detected at the plasma membrane and colocalized with the envelope glycoproteins. Moreover, Gag proteins were excluded from streptolysin-O permeabilized cells and in this respect behaved like cytoplasmic proteins. This suggests that the trafficking of HTLV-1 Gag proteins through the cytoplasm of the host cell is independent of any cell membrane system.
CDP Binding to Multiple Sites in the Mouse Mammary Tumor Virus Long Terminal Repeat Suppresses Basal and Glucocorticoid-Induced Transcription
Authors: Zhu Quan , Dudley Jaquelin P. 
Published: July 2002

ABSTRACTMouse mammary tumor virus (MMTV) is transcribed at high levels in the lactating mammary gland to ensure transmission of virus from the milk of infected female mice to susceptible offspring. We previously have shown that the transcription factor CCAAT displacement protein (CDP) is expressed in high amounts in virgin mammary gland, yet DNA-binding activity for the MMTV long terminal repeat (LTR) disappears as mammary tissue differentiates during lactation. CDP is a repressor of MMTV expression and, therefore, MMTV expression is suppressed during early mammary gland development. In this study, we have shown using DNase I footprinting and electrophoretic mobility shift assays that there are at least five CDP-binding sites in the MMTV LTR upstream of those previously described in the promoter-proximal negative regulatory element (NRE). Single mutations in two of these upstream sites (+691 or +692 and +735 relative to the first base of the LTR) reduced CDP binding to the cognate sites and elevated reporter gene expression from the full-length MMTV LTR. Combination of a mutation in the promoter-distal NRE with a mutation in the proximal NRE gave approximately additive increases in LTR-reporter gene activity, suggesting that these binding sites act independently. Mutations in several different CDP-binding sites allowed elevation of reporter gene activity from the MMTV promoter in the absence and presence of glucocorticoids, hormones that contribute to high levels of MMTV transcription during lactation by activation of hormone receptor binding to the LTR. In addition, overexpression of CDP in transient-transfection assays suppressed both basal and glucocorticoid-induced LTR-mediated transcription in a dose-dependent manner. These data suggest that multiple CDP-binding sites contribute independently to regulate binding of positive factors, including glucocorticoid receptor, to the MMTV LTR during mammary gland development.
Role of an Intact Splenic Microarchitecture in Early Lymphocytic Choriomeningitis Virus Production
Authors: Müller Stefan , Hunziker Lukas , Enzler Susanne , Bühler-Jungo Myriam , Di Santo James P. , Zinkernagel Rolf M. , Mueller C. 
Published: July 2002

ABSTRACTAn acute infection with lymphocytic choriomeningitis virus (LCMV) is efficiently controlled by the cytotoxic-T-cell (CTL) response of the host, and LCMV titers in the spleen and peripheral solid organs usually fall sharply after day 4 to 6 postinfection. Surprisingly, infection of immunodeficient recombination-activating gene 2-deficient (RAG2−/−) mice with 5 × 102PFU of LCMV-WE causes about 80-fold-lower LCMV titers in the spleen on day 4 postinfection compared with C57BL/6 control mice. This could not be attributed to NK cell activity, since common gamma-chain-deficient RAG2−/−mice lacking NK cells show low LCMV titers comparable to those for RAG2−/−mice. Furthermore, the reduced early LCMV production in spleens could not be explained by an enhanced gamma interferon production in RAG2−/−mice. Analysis of mutant mice exhibiting various defects in the splenic microarchitecture, including (i) tumor necrosis factor alpha-negative (TNF-α−/−), lymphotoxin alpha-negative (LTα−/−), B-cell-deficient μMT mice, (ii) immunoglobulin M-negative mice, and (iii) RAG−/−mice reconstituted with wild-type versus TNF-α−/−LTα−/−B cells, revealed a clear correlation between an intact splenic marginal zone, rapid early replication of LCMV in the spleen, and efficient CTL induction. These results suggest that by the preferential infection of the highly organized splenic microarchitecture, LCMV seems to successfully exploit one of the key elements in the chain of the adaptive immune system. Not only does the early tropism of LCMV for the splenic marginal zone trigger a potent immune response, but at the same time the marginal zone may also become a target of early CTL-mediated immunopathology that impairs immune responsiveness.