bluuebunny/crossref_metadata_2025_split huggingface dataset:
Association of Mumps Virus V Protein with RACK1 Results in Dissociation of STAT-1 from the Alpha Interferon Receptor Complex
Authors: Kubota Toru , Yokosawa Noriko , Yokota Shin-ichi , Fujii Nobuhiro 
Published: November 2002

ABSTRACT

It has been reported that mumps virus protein V or the C-terminal Cys-rich region of protein V (Vsp) is associated with blocking of the interferon (IFN) signal transduction pathway through a decrease in STAT-1 production. The intracellular target of the V protein was investigated by using a two-hybrid screening system with Vsp as bait. Full-length V protein and Vsp were able to bind to RACK1, and the interaction did not require two WD domains, WD1 and WD2, in RACK1. A significant interaction between V protein and RACK1 was also demonstrated in cells persistently infected with mumps virus (FLMT cells), and the formation of the complex was not affected by treatment with IFN. On the other hand, in uninfected cells, STAT-1 was associated with the long form of the β subunit of the alpha IFN receptor, and this association was mediated by the function of RACK1 as an adaptor protein. Immunoprecipitation and glutathione
S
-transferase pull-down experiments revealed that the association of RACK1 or mumps virus V protein with the IFN receptor was undetectable in mumps virus-infected cells. Furthermore, RACK1 interacted with mumps virus V protein with a higher affinity than STAT-1 did. Therefore, it is suggested that mumps virus V protein has the ability to interact strongly with RACK1 and consequently to bring about the disruption of the complex formed from STAT-1, RACK1, and the IFN receptor.
C-Terminal Region of STAT-1α Is Not Necessary for Its Ubiquitination and Degradation Caused by Mumps Virus V Protein
Authors: Yokosawa Noriko , Yokota Shin-ichi , Kubota Toru , Fujii Nobuhiro 
Published: November 2002

ABSTRACT
Constitutive levels of production of STAT-1 were reduced by 10 h postinfection (p.i.) and significantly lost by 24 h p.i. in FL cells acutely infected with mumps virus (MuV). This result was consistent with that observed in previous studies and experiments with cells persistently infected with MuV (FLMT cells). There was a marked decrease in the amount of STAT-1 in cells expressing MuV accessory protein V (MuV-V). Furthermore, single amino acid substitutions in the Cys-rich region of V protein (Vc189a, Vc207a, and Vc214a) showed that each cysteine residue plays an important role in the decrease in STAT-1 production, but substitution of a histidine residue at amino acid position 203 had no effect. These events and the resultant suppression of the alpha interferon (IFN-α) response were confirmed by a luciferase reporter gene assay with five tandem repeats of the IFN-α-stimulated response element as an enhancer element of the firely luciferase gene. STAT-1 production was restored and detectable in FLMT cells treated with a proteosome inhibitor, such as MG132 or lactacystin. In the presence of MG132, ubiquitination of STAT-1 and the interaction of MuV-V with STAT-1 were demonstrated in FLMT cells by immunoprecipitation with anti-STAT-1 antibody. The same results for the interaction and ubiquitination were obtained in experiments with an expression vector for a C-terminal deletion mutant of STAT-1. The truncated STAT-1 molecules were degraded in the presence of MuV-V. Therefore, the C-terminal region (transcriptional activation and Src homology 2 domains) of STAT-1 is not necessary for its degradation caused by MuV-V. Our data suggest that MuV-V promotes ubiquitination and degradation of STAT-1.
Effects of Membrane Potential and Sphingolipid Structures on Fusion of Semliki Forest Virus
Authors: Samsonov Andrey V. , Chatterjee Prodyot K. , Razinkov Vladimir I. , Eng Christina H. , Kielian Margaret , Cohen Fredric S. 
Published: November 2002

ABSTRACT

Cells expressing the E1 and E2 envelope proteins of Semliki Forest virus (SFV) were fused to voltage-clamped planar lipid bilayer membranes at low pH. Formation and evolution of fusion pores were electrically monitored by capacitance measurements, and membrane continuity was tracked by video fluorescence microscopy by including rhodamine-phosphatidylethanolamine in the bilayer. Fusion occurred without leakage for a negative potential applied to the
trans
side of the planar membrane. When a positive potential was applied, leakage was severe, obscuring the observation of any fusion. E1-mediated cell-cell fusion occurred without leakage for negative intracellular potentials but with substantial leakage for zero membrane potential. Thus, negative membrane potentials are generally required for nonleaky fusion. With planar bilayers as the target, the first fusion pore that formed almost always enlarged; pore flickering was a rare event. Similar to other target membranes, fusion required cholesterol and sphingolipids in the planar membrane. Sphingosine did not support fusion, but both ceramide, with even a minimal acyl chain (C
2
-ceramide), and lysosphingomyelin (lyso-SM) promoted fusion with the same kinetics. Thus, unrelated modifications to different parts of sphingosine yielded sphingolipids that supported fusion to the same degree. Fusion studies of pyrene-labeled SFV with cholesterol-containing liposomes showed that C
2
-ceramide supported fusion while lyso-SM did not, apparently due to its positive curvature effects. A model is proposed in which the hydroxyls of C-1 and C-3 as well as N of C-2 of the sphingosine backbone must orient so as to form multiple hydrogen bonds to amino acids of SFV E1 for fusion to proceed.
Detection of the Potyviral Genome-Linked Protein VPg in Virions and Its Phosphorylation by Host Kinases
Authors: Puustinen Pietri , Rajamäki Minna-Liisa , Ivanov Konstantin I. , Valkonen Jari P. T. , Mäkinen Kristiina 
Published: November 2002

ABSTRACT

The multifunctional genome-linked protein (VPg) of
Potato virus A
(PVA; genus
Potyvirus
) was found to be phosphorylated as a part of the virus particle by a cellular kinase activity from tobacco. Immunoprecipitation, immunolabeling, and immunoelectron microscopy experiments showed that VPg is exposed at one end of the virion and it is accessible to protein-protein interactions. Substitution Ser185Leu at the C-proximal part of VPg reduces accumulation of PVA in inoculated leaves of the wild potato species
Solanum commersonii
and delays systemic infection, which is not observed in tobacco plants. Our data show that kinases of
S. commersonii
differentially recognize the VPg containing Ser or Leu at position 185, whereas both forms of VPg are similarly recognized by tobacco kinases. Taken together, our data imply that the virion-bound VPg may interact with host proteins and that phosphorylation of VPg may play a role in the VPg-mediated functions during the infection cycle of potyviruses.
Novel Mutations That Control the Sphingolipid and Cholesterol Dependence of the Semliki Forest Virus Fusion Protein
Authors: Chatterjee Prodyot K. , Eng Christina H. , Kielian Margaret 
Published: November 2002

ABSTRACT

The enveloped alphavirus Semliki Forest virus (SFV) infects cells via a membrane fusion reaction mediated by the E1 membrane protein. Efficient SFV-membrane fusion requires the presence of cholesterol and sphingolipid in the target membrane. Here we report on two mutants,
srf-4
and
srf-5
, selected for growth in cholesterol-depleted cells. Like the previously isolated
srf-3
mutant (E1 proline 226 to serine), the phenotypes of the
srf-4
and
srf-5
mutants were conferred by single-amino-acid changes in the E1 protein: leucine 44 to phenylalanine and valine 178 to alanine, respectively. Like
srf-3
,
srf-4
and
srf-5
show striking increases in the cholesterol independence of growth, infection, membrane fusion, and exit. Unexpectedly, and unlike
srf-3
,
srf-4
and
srf-5
showed highly efficient fusion with sphingolipid-free membranes in both lipid- and content-mixing assays. Both
srf-4
and
srf-5
formed E1 homotrimers of decreased stability compared to the homotrimers of the wild type and the
srf-3
mutant. All three
srf
mutations lie in the same domain of E1, but the
srf-4
and
srf-5
mutations are spatially separated from
srf-3
. When expressed together, the three mutations could interact to produce increased sterol independence and to cause temperature-sensitive E1 transport. Thus, the
srf-4
and
srf-5
mutations identify novel regions of E1 that are distinct from the fusion peptide and
srf-3
region and modulate the requirements for both sphingolipid and cholesterol in virus-membrane fusion.
Similar Regulation of Cell Surface Human T-Cell Leukemia Virus Type 1 (HTLV-1) Surface Binding Proteins in Cells Highly and Poorly Transduced by HTLV-1-Pseudotyped Virions
Authors: Jones Kathryn S. , Nath Manisha , Petrow-Sadowski Cari , Baines Andrea C. , Dambach Megan , Huang Ying , Ruscetti Francis W. 
Published: November 2002

ABSTRACTLittle is known about the requirements for human T-cell leukemia virus type 1 (HTLV-1) entry, including the identity of the cellular receptor(s). Previous studies have shown that although the HTLV receptor(s) are widely expressed on cell lines of various cell types from different species, cell lines differ dramatically in their susceptibility to HTLV-Env-mediated fusion. Human cells (293, HeLa, and primary CD4+T cells) showed higher levels of binding at saturation than rodent (NIH 3T3 and NRK) cells to an HTLV-1 SU immunoadhesin. A direct comparison of the binding of the HTLV-1 surface glycoprotein (SU) immunoadhesin and transduction by HTLV-1 pseudotyped virus revealed parallels between the level of binding and the titer for various cell lines. When cells were treated with phorbol myristate acetate (PMA), which down-modulates a number of cell surface molecules, the level of SU binding was markedly reduced. However, PMA treatment only slightly reduced the titer of murine leukemia virus(HTLV-1) on both highly susceptible and poorly susceptible cells. Treatment of target cells with trypsin greatly reduced binding, indicating that the majority of HTLV SU binding is to proteins. Polycations, which enhance the infectivity of several other retroviruses, inhibited HTLV-1 Env-mediated binding and entry on both human and rodent cells. These results suggest that factors other than the number of primary binding receptors are responsible for the differences in the titers of HTLV-1 pseudotypes between highly susceptible cells and poorly susceptible cells.
Comparative Vaccine Studies in HLA-A2.1-Transgenic Mice Reveal a Clustered Organization of Epitopes Presented in Hepatitis C Virus Natural Infection
Authors: Himoudi Nourredine , Abraham Jean-Daniel , Fournillier Anne , Lone Yu Chun , Joubert Aurélie , De Beeck Anne Op , Freida Delphine , Lemonnier François , Kieny Marie Paule , Inchauspé Geneviève 
Published: November 2002

ABSTRACT

A polyepitopic CD8
+
-T-cell response is thought to be critical for control of hepatitis C virus (HCV) infection. Using transgenic mice, we analyzed the immunogenicity and dominance of most known HLA-A2.1 epitopes presented during infection by using vaccines that carry the potential to enter clinical trials: peptides, DNA, and recombinant adenoviruses. The vaccines capacity to induce specific cytotoxic T lymphocytes and interferon gamma-producing cells revealed that immunogenic epitopes are clustered in specific antigens. For two key antigens, flanking regions were shown to greatly enhance the scope of epitope recognition, whereas a DNA-adenovirus prime-boost vaccination strategy augmented epitope immunogenicity, even that of subdominant ones. The present study reveals a clustered organization of HCV immunogenic HLA.A2.1 epitopes and strategies to modulate their dominance.
Tomato Bushy Stunt Virus
Genomic RNA Accumulation Is Regulated by Interdependent
cis
-Acting Elements within the Movement Protein Open Reading Frames
Authors: Park Jong-Won , Desvoyes Bénédicte , Scholthof Herman B. 
Published: November 2002

ABSTRACT

This study on
Tomato bushy stunt virus
identified and defined three previously unknown regulatory sequences involved in RNA accumulation that are located within the 3′-proximal nested movement protein genes
p22
and
p19
. The first is a 16-nucleotide (nt) element termed III-A that is positioned at the very 3′ end of
p22
and is essential for RNA accumulation. Approximately 300 nt upstream of III-A resides an ∼80-nt inhibitory element (IE) that is obstructive to replication only in the absence of a third regulatory element of ∼30 nt (SUR-III) that is positioned immediately upstream of III-A. Inspection of the nucleotide sequences predicted that III-A and SUR-III can form looped hairpins. A comparison of different tombusviruses showed, in each case, conservation for potential base pairing between the two predicted hairpin-loops. Insertion of a spacer adjacent to the predicted hairpins had no or a minimal effect on RNA accumulation, whereas an insertion in the putative III-A loop abolished genomic RNA multiplication. We conclude that the sequences composing the predicted III-A and SUR-III hairpin-loops are crucial for optimal RNA accumulation and that the inhibitory effect of IE surfaces when the alleged interaction between SUR-III and III-A is disturbed.
General and Specific Alterations in Programming of Global Viral Gene Expression during Infection by VP16 Activation-Deficient Mutants of Herpes Simplex Virus Type 1
Authors: Yang William C. , Devi-Rao G. V. , Ghazal Peter , Wagner Edward K. , Triezenberg Steven J. 
Published: November 2002

ABSTRACT
During productive infection by herpes simplex virus 1 (HSV-1), viral gene expression occurs in a temporally regulated cascade in which transcription of the viral immediate-early (IE) genes is strongly stimulated by the virion protein VP16. We have employed an oligonucleotide microarray to examine the effect of VP16 mutations on the overall pattern of viral gene expression following infection of HeLa cells. This microarray detects essentially all HSV-1 transcripts with relative and absolute levels correlating well with known kinetics of expression. This analysis revealed that deletion of the VP16 activation domain sharply reduced overall viral gene expression; moreover, the pattern of this reduced expression varied greatly from the pattern of a wild-type (wt) infection. However, when this mutant virus was delivered at a high multiplicity of infection or in the presence of the cellular stress inducer hexamethylene bisacetamide, expression was largely restored to the wt levels and pattern. Infection with virions that deliver wt VP16 protein at the start of infection but synthesize only truncated VP16 resulted in a normal kinetic cascade. This suggests that newly synthesized VP16 does not play a significant role in the expression of later classes of transcripts. The VP16 activation domain comprises two subregions. Deletion of the C-terminal subregion resulted in minimal changes in the level and profile of gene expression compared to a normal (wt) cascade. In contrast, deletion of the N-terminal subregion reduced the overall expression levels and skewed the relative levels of IE transcripts but did not significantly alter the kinetic pattern of early and late transcript expression. We conclude that the general activation of IE gene transcription by VP16, but not the specific ratios of IE transcripts, is necessary for the subsequent ordered expression of viral genes. Moreover, this report establishes the feasibility of microarray analysis for globally assessing viral gene expression programs as a function of the conditions of infection.
Construction and Characterization of Adenovirus Serotype 5 Packaged by Serotype 3 Hexon
Authors: Wu Hongju , Dmitriev Igor , Kashentseva Elena , Seki Toshiro , Wang Minghui , Curiel David T. 
Published: November 2002

ABSTRACT
Adenovirus serotype 5 (Ad5) has great potential for gene therapy applications. A major limitation, however, is the host immune response against Ad5 infection that often prevents the readministration of Ad5 vectors. In this regard, the most abundant capsid protein, hexon, has been implicated as the major target for neutralizing antibodies. In this study, we sought to escape the host neutralization response against Ad5 via hexon replacement. We constructed a chimeric adenovirus vector, Ad5/H3, by replacing the Ad5 hexon gene with the hexon gene of Ad3. The chimeric viruses were successfully rescued in 293 cells. Compared to that for the control Ad5/H5, the growth rate of Ad5/H3 was significantly slower and the final yield was about 1 log order less. These data indicate that the Ad3 hexon can encapsidate the Ad5 genome, but with less efficiency than the Ad5 hexon. The gene transfer efficacy of Ad5/H3 in HeLa cells was also lower than that of Ad5/H5. Furthermore, we tested the host neutralization responses against the two viruses by using C57BL/6 mice. The neutralizing antibodies against Ad5/H3 and Ad5/H5 generated by the immunized mice did not cross-neutralize each other in the context of in vitro infection of HeLa cells. Preimmunization of C57BL/6 mice with one of the two types of viruses also did not prevent subsequent infection of the other type. These data suggest that replacing the Ad5 hexon with the Ad3 hexon can circumvent the host neutralization response to Ad5. This strategy may therefore be used to achieve the repeated administration of Ad5 in gene therapy applications.
Tissue-Specific Transcriptional Targeting of a Replication-Competent Retroviral Vector
Authors: Logg Christopher R. , Logg Aki , Matusik Robert J. , Bochner Bernard H. , Kasahara Noriyuki 
Published: November 2002

ABSTRACT
The inability of replication-defective viral vectors to efficiently transduce tumor cells in vivo has prevented the successful application of such vectors in gene therapy of cancer. To address the need for more efficient gene delivery systems, we have developed replication-competent retroviral (RCR) vectors based on murine leukemia virus (MLV). We have previously shown that such vectors are capable of transducing solid tumors in vivo with very high efficiency. While the natural requirement of MLV infection for cell division imparts a certain degree of specificity for tumor cells, additional means for confining RCR vector replication to tumor cells are desirable. Here, we investigated the parameters critical for successful tissue-specific transcriptional control of RCR vector replication by replacing various lengths of the MLV enhancer/promoter with sequences derived either from the highly prostate-specific probasin (PB) promoter or from a more potent synthetic variant of the PB promoter. We assessed the transcriptional specificity of the resulting hybrid long terminal repeats (LTRs) and the cell type specificity and efficiency of replication of vectors containing these LTRs. Incorporation of PB promoter sequences effectively restricted transcription from the LTR to prostate-derived cells and imparted prostate-specific RCR vector replication but required the stronger synthetic promoter and retention of native MLV sequences in the vicinity of the TATA box for optimal replicative efficiency and specificity. Our results have thus identified promoter strength and positioning within the LTR as important determinants for achieving both high transduction efficiency and strict cell type specificity in transcriptionally targeted RCR vectors.
A
cis
-Acting Element That Directs Circular Adeno-Associated Virus Replication and Packaging
Authors: Musatov Sergei , Roberts Jill , Pfaff Donald , Kaplitt Michael 
Published: November 2002

ABSTRACT

A novel pathway of adeno-associated virus (AAV) replication marked by the assembly of circular monomer duplex intermediates (cAAV) has been recently discovered. In the present report we identify a single AD domain of the inverted terminal repeat as a minimal origin of cAAV replication. A small internal palindrome (BB′), necessary for optimal Rep-inverted terminal repeat interaction, does not contribute to the efficiency of cAAV replication, while the terminal resolution site is an essential
cis
-acting element. Furthermore, recombinant cAAV vectors that encompass only the AD domain replicate exclusively in a circular form and no detectable linear duplex replicative intermediates are generated, suggesting that both pathways of AAV replication are independent and can be separated. In addition, we show that cAAVs are efficient templates for encapsidation of single-stranded DNA genomes, an observation that assigns a biological role for these novel replication species. Together, these findings shed new light on the current model of AAV replication and packaging.
Upregulation of CD40 Expression on Endothelial Cells Infected with Human Cytomegalovirus
Authors: Maisch Tim , Kropff Barbara , Sinzger Christian , Mach Michael 
Published: November 2002

ABSTRACT
CD40 has been identified as an important molecule for a number of processes, such as immune responses, inflammation, and the activation of endothelia. We investigated CD40 in endothelial cells (EC) following infection with an endotheliotropic strain of human cytomegalovirus (HCMV). Between 8 and 72 h postinfection, we observed a significant increase in CD40 levels on the surface of infected EC, as measured by fluorescence-activated cell sorting analysis. As a consequence of CD40 upregulation, increased levels of E-selectin were found on infected EC after stimulation with CD154-expressing T cells. Enhanced expression of CD40 was specific for EC, since infection of fibroblasts did not result in the upregulation of CD40. The addition of neutralizing antibodies as well as UV inactivation of virus completely prevented the upregulation of CD40 on EC. Also, laboratory-adapted HCMV strain AD169 was not able to induce CD40 on EC. De novo protein synthesis was necessary for the increased surface expression. At early times (4 to 24 h) postinfection, this change was not accompanied by increased levels of CD40 protein or mRNA. At late times (48 to 96 h) postinfection, increased amounts of CD40 protein and mRNA were detected. Immunohistochemical analysis of infected tissues demonstrated elevated levels of CD40 on HCMV-infected EC in vivo. Thus, infection of EC by HCMV may result in the activation of endothelia and in the augmentation of inflammatory processes.
The Complementary Strand of the Human T-Cell Leukemia Virus Type 1 RNA Genome Encodes a bZIP Transcription Factor That Down-Regulates Viral Transcription
Authors: Gaudray Gilles , Gachon Frederic , Basbous Jihane , Biard-Piechaczyk Martine , Devaux Christian , Mesnard Jean-Michel 
Published: November 2002

ABSTRACT

The RNA genome of the human T-cell leukemia virus type 1 (HTLV-1) codes for proteins involved in infectivity, replication, and transformation. We report in this study the characterization of a novel viral protein encoded by the complementary strand of the HTLV-1 RNA genome. This protein, designated HBZ (for HTLV-1 bZIP factor), contains a N-terminal transcriptional activation domain and a leucine zipper motif in its C terminus. We show here that HBZ is able to interact with the bZIP transcription factor CREB-2 (also called ATF-4), known to activate the HTLV-1 transcription by recruiting the viral
trans
-activator Tax on the Tax-responsive elements (TxREs). However, we demonstrate that the HBZ/CREB-2 heterodimers are no more able to bind to the TxRE and cyclic AMP response element sites. Taking these findings together, the functional inactivation of CREB-2 by HBZ is suggested to contribute to regulation of the HTLV-1 transcription. Moreover, the characterization of a minus-strand gene protein encoded by HTLV-1 has never been reported until now.