bluuebunny/crossref_metadata_2025_split huggingface dataset:
Promoter-Proximal Regulatory Elements Involved in
oriP
-EBNA1-Independent and -Dependent Activation of the Epstein-Barr Virus C Promoter in B-Lymphoid Cell Lines
Authors: Nilsson Tina , Zetterberg Henrik , Wang Yuyan Camilla , Rymo Lars 
Published: July 2002

ABSTRACT

The identification of the cellular factors that control the transcription regulatory activity of the Epstein-Barr virus C promoter (Cp) is fundamental to the understanding of the molecular mechanisms that control virus latent gene expression. Using transient transfection of reporter plasmids in group I phenotype B-lymphoid cells, we have previously shown that the −248 to −55 region (−248/−55 region) of Cp contains elements that are essential for
oriPI
-EBNA1-dependent as well as
oriPI
-EBNA1-independent activation of the promoter. We now establish the importance of this region by a detailed mutational analysis of reporter plasmids carrying Cp regulatory sequences together with or without
oriPI
. The reporter plasmids were transfected into group I phenotype Rael cells and group III phenotype cbc-Rael cells, and the Cp activity measured was correlated with the binding of candidate transcription factors in electrophoretic mobility shift assays and further assessed in cotransfection experiments. We show that the NF-Y transcription factor interacts with the previously identified CCAAT box in the −71/−63 Cp region (M. T. Puglielli, M. Woisetschlaeger, and S. H. Speck, J. Virol. 70:5758–5768, 1996). We also show that members of the C/EBP transcription factor family interact with a C/EBP consensus sequence in the −119/−112 region of Cp and that this interaction is important for promoter activity. A central finding is the identification of a GC-rich sequence in the −99/−91 Cp region that is essential for
oriPI
-EBNA1-independent as well as
oriPI
-EBNA1-dependent activity of the promoter. This region contains overlapping binding sites for Sp1 and Egr-1, and our results suggest that Sp1 is a positive and Egr-1 is a negative regulator of Cp activity. Furthermore, we demonstrate that a reporter plasmid that in addition to
oriPI
contains only the −111/+76 region of Cp still retains the ability to be activated by EBNA1.
Expression and Functional Characterization of Bluetongue Virus VP5 Protein: Role in Cellular Permeabilization
Authors: Hassan S. H. , Wirblich C. , Forzan M. , Roy P. 
Published: July 2002

ABSTRACT

Segment 5 of bluetongue virus (BTV) serotype 10, which encodes the outer capsid protein VP5, was tagged with glutathione
S
-transferase and expressed by a recombinant baculovirus. The recombinant protein was subsequently purified to homogeneity, and its possible biological role in virus infection was investigated. Purified VP5 was able to bind mammalian cells but was not internalized, which indicates it is not involved in receptor-mediated endocytosis. The purified VP5 protein was shown to be able to permeabilize mammalian and
Culicoides
insect cells, inducing cytotoxicity. Sequence analysis revealed that VP5 possesses characteristic structural features (including two amino-terminal amphipathic helices) compatible with virus penetration activity. To assess the role of each feature in the observed cytotoxicity, a series of deleted VP5 molecules were generated, and their expression and biological activity was compared with the parental molecule. VP5 derivatives that included the two amphipathic helices exhibited cytotoxicity, while those that omitted these sequences did not. To confirm their role in membrane destabilization two synthetic peptides (amino acids [aa] 1 to 20 and aa 22 to 41) encompassing the two helices and an additional peptide representing the adjacent downstream sequences were also assessed for their effect on the cell membrane. Both helices, but not the downstream VP5 sequence, exhibited cytotoxicity with the most-amino-terminal helix (aa 1 to 20) showing a higher activity than the adjacent peptide (aa 22 to 41). Purified VP5 was shown to readily form trimers in solution, a feature of many proteins involved in membrane penetration. Taken together, these data support a role for VP5 in virus-cell penetration consistent with its revelation in the entry vesicle subsequent to cell binding and endocytosis.
Vaccinia Virus Telomeres: Interaction with the Viral I1, I6, and K4 Proteins
Authors: DeMasi Joseph , Du Shan , Lennon David , Traktman Paula 
Published: July 2002

ABSTRACT
The 192-kb linear DNA genome of vaccinia virus has covalently closed hairpin termini that are extremely AT rich and contain 12 extrahelical bases. Vaccinia virus telomeres have previously been implicated in the initiation of viral genome replication; therefore, we sought to determine whether the telomeres form specific protein-DNA complexes. Using an electrophoretic mobility shift assay, we found that extracts prepared from virions and from the cytoplasm of infected cells contain telomere binding activity. Four shifted complexes were detected using hairpin probes representing the viral termini, two of which represent an interaction with the “flip” isoform and two with the “flop” isoform. All of the specificity for protein binding lies within the terminal 65-bp hairpin sequence. Viral hairpins lacking extrahelical bases cannot form the shifted complexes, suggesting that DNA structure is crucial for complex formation. Using an affinity purification protocol, we purified the proteins responsible for hairpin-protein complex formation. The vaccinia virus I1 protein was identified as being necessary and sufficient for the formation of the upper doublet of shifted complexes, and the vaccinia virus I6 protein was shown to form the lower doublet of shifted complexes. Competition and challenge experiments confirmed that the previously uncharacterized I6 protein binds tightly and with great specificity to the hairpin form of the viral telomeric sequence. Incubation of viral hairpins with extracts from infected cells also generates a smaller DNA fragment that is likely to reflect specific nicking at the apex of the hairpin; we show that the vaccinia virus K4 protein is necessary and sufficient for this reaction. We hypothesize that these telomere binding proteins may play a role in the initiation of vaccinia virus genome replication and/or genome encapsidation.
Role of Tachykinins in the Host Response to Murine Gammaherpesvirus Infection
Authors: Payne Catherine M. , Heggie Caroline J. , Brownstein David G. , Stewart James P. , Quinn John P. 
Published: July 2002

ABSTRACTTachykinins function not only as neurotransmitters but also as immunological mediators. We used infection of tachykinin-deficient (PPT-A−/−) mice and wild-type controls with murine gammaherpesvirus to assess the role of tachykinins in the host response to a virus infection. Although infection was ultimately controlled inPPT-A−/−mice, there were higher titers of infectious virus in the lungs, accompanied by a more rapid influx of inflammatory cells. Clearance of latently infected cells from the spleen was also delayed. This is the first report of the direct influence of tachykinins in the host response to a virus infection.
A Chimeric Human-Bovine Parainfluenza Virus Type 3 Expressing Measles Virus Hemagglutinin Is Attenuated for Replication but Is Still Immunogenic in Rhesus Monkeys
Authors: Skiadopoulos Mario H. , Surman Sonja R. , Riggs Jeffrey M. , Collins Peter L. , Murphy Brian R. 
Published: July 2002

ABSTRACT

The chimeric recombinant virus rHPIV3-N
B
, a version of human parainfluenza virus type 3 (HPIV3) that is attenuated due to the presence of the bovine PIV3 nucleocapsid (N) protein open reading frame (ORF) in place of the HPIV3 ORF, was modified to encode the measles virus hemagglutinin (HA) inserted as an additional, supernumerary gene between the HPIV3 P and M genes. This recombinant, designated rHPIV3-N
B
HA, replicated like its attenuated rHPIV3-N
B
parent virus in vitro and in the upper and lower respiratory tracts of rhesus monkeys, indicating that the insertion of the measles virus HA did not further attenuate rHPIV3-N
B
in vitro or in vivo. Monkeys immunized with rHPIV3-N
B
HA developed a vigorous immune response to both measles virus and HPIV3, with serum antibody titers to both measles virus (neutralizing antibody) and HPIV3 (hemagglutination inhibiting antibody) of over 1:500. An attenuated HPIV3 expressing a major protective antigen of measles virus provides a method for immunization against measles by the intranasal route, a route that has been shown with HPIV3 and respiratory syncytial virus vaccines to be relatively refractory to the neutralizing and immunosuppressive effects of maternally derived virus-specific serum antibodies. It should now be possible to induce a protective immune response against measles virus in 6-month-old infants, an age group that in developing areas of the world is not responsive to the current measles virus vaccine.
Glycoprotein K Specified by Herpes Simplex Virus Type 1 Is Expressed on Virions as a Golgi Complex-Dependent Glycosylated Species and Functions in Virion Entry
Authors: Foster Timothy P. , Rybachuk Galena V. , Kousoulas Konstantin G. 
Published: July 2002

ABSTRACT

To facilitate detection of glycoprotein K (gK) specified by herpes simplex virus, a 12-amino-acid epitope tag was inserted within gK domain III. Recombinant virus gKprotC-DIII, expressing the tagged gK, was isolated. This virus formed wild-type plaques and replicated as efficiently as the wild-type KOS virus in Vero cells. Anti-protein C MAb detected high-mannose and Golgi complex-dependent glycosylated gK within cells as well as on purified virions. The gK-null virus ΔgK (gK
−/−
) entered Vero cells substantially more slowly than the wild-type KOS (gK
+/+
), while ΔgK virus grown in complementing VK302 cells (gK
−/+
) entered with entry kinetics similar to those of the KOS virus.
Improved Adenovirus Vectors for Infection of Cardiovascular Tissues
Authors: Havenga M. J. E. , Lemckert A. A. C. , Grimbergen J. M. , Vogels R. , Huisman L. G. M. , Valerio D. , Bout A. , Quax P. H. A. 
Published: July 2002

ABSTRACT

To identify improved adenovirus vectors for cardiovascular gene therapy, a library of adenovirus vectors based on adenovirus serotype 5 (Ad5) but carrying fiber molecules of other human serotypes, was generated. This library was tested for efficiency of infection of human primary vascular endothelial cells (ECs) and smooth muscle cells (SMCs). Based on luciferase, LacZ, or green fluorescent protein (GFP) marker gene expression, several fiber chimeric vectors were identified that displayed improved infection of these cell types. One of the viruses that performed particularly well is an Ad5 carrying the fiber of Ad16 (Ad5.Fib16), a subgroup B virus. This virus showed, on average, 8- and 64-fold-increased luciferase activities on umbilical vein ECs and SMCs, respectively, compared to the parent vector. GFP and
lacZ
markers showed that approximately 3-fold (ECs) and 10-fold (SMCs) more cells were transduced. Experiments performed with both cultured SMCs and organ cultures derived from different vascular origins (saphenous vein, iliac artery, left interior mammary artery, and aorta) and from different species demonstrated that Ad5.Fib16 consistently displays improved infection in primates (humans and rhesus monkeys). SMCs of the same vessels of rodents and pigs were less infectable with Ad5.Fib16 than with Ad5. This suggests that either the receptor for human Ad16 is not conserved between different species or that differences in the expression levels of the putative receptor exist. In conclusion, our results show that an Ad5-based virus carrying the fiber of Ad16 is a potent vector for the transduction of primate cardiovascular cells and tissues.
Essential Roles for CD8+T Cells and Gamma Interferon in Protection of Mice against Retrovirus-Induced Immunosuppression
Authors: Dittmer Ulf , Race Brent , Peterson Karin E. , Stromnes Ingunn M. , Messer Ronald J. , Hasenkrug Kim J. 
Published: July 2002

ABSTRACTIt is known that both animal and human retroviruses typically cause immunosuppression in their respective hosts, but the mechanisms by which this occurs are poorly understood. The present study uses Friend virus (FV) infections of mice as a model to determine how major histocompatibility complex (MHC) genes influence immunosuppression. Previously, MHC-I genes were shown to influence antibody responses to potent antigenic challenges given during acute FV infection. The mapping of an immune response to an MHC-I gene implicated CD8+T cells in the mechanism, so we directly tested for their role by using in vivo CD8+T-cell depletions. Mice resistant to FV-induced immunosuppression became susceptible when they were depleted of CD8+T cells. Resistance also required gamma interferon (IFN-γ), as in vivo neutralization of IFN-γ converted mice from a resistant to susceptible phenotype. On the other hand, susceptibility to FV-induced immunosuppression was dependent on the immunosuppressive cytokine, interleukin-10 (IL-10), as antibody responses were restored in susceptible mice when IL-10 function was blocked in vivo. Thus, FV-induced immunosuppression of antibody responses involves complex mechanisms controlled at least in part by CD8+T cells.
Pivotal Role of the Non-
hr
Origin of DNA Replication in the Genesis of Defective Interfering Baculoviruses
Authors: Pijlman Gorben P. , Dortmans Jos C. F. M. , Vermeesch Angela M. G. , Yang Kai , Martens Dirk E. , Goldbach Rob W. , Vlak Just M. 
Published: July 2002

ABSTRACT

The generation of deletion mutants, including defective interfering viruses, upon serial passage of
Spodoptera exigua
multicapsid nucleopolyhedrovirus (Se
M
NPV) in insect cell culture has been studied. Sequences containing the non-homologous region origin of DNA replication (non-
hr ori
) became hypermolar in intracellular viral DNA within 10 passages in Se301 insect cells, concurrent with a dramatic drop in budded virus and polyhedron production. These predominant non-
hr ori
-containing sequences accumulated in larger concatenated forms and were generated de novo as demonstrated by their appearance and accumulation upon infection with a genetically homogenous bacterial clone of Se
M
NPV (bacmid). Sequences were identified at the junctions of the non-
hr ori
units within the concatemers, which may be potentially involved in recombination events. Deletion of the Se
M
NPV non-
hr ori
using RecE/RecT-mediated homologous ET recombination in
Escherichia coli
resulted in a recombinant bacmid with strongly enhanced stability of virus and polyhedron production upon serial passage in insect cells. This suggests that the accumulation of non-
hr ori
s upon passage is due to the replication advantage of these sequences. The non-
hr ori
deletion mutant Se
M
NPV bacmid can be exploited as a stable eukaryotic heterologous protein expression vector in insect cells.
Binding of FADD and Caspase-8 to Molluscum Contagiosum Virus MC159 v-FLIP Is Not Sufficient for Its Antiapoptotic Function
Authors: Garvey Tara L. , Bertin John , Siegel Richard M. , Wang Guang-hua , Lenardo Michael J. , Cohen Jeffrey I. 
Published: July 2002

ABSTRACT
Molluscum contagiosum virus (MCV), a member of the human poxvirus family, encodes the MC159 protein that inhibits Fas-, tumor necrosis factor (TNF)-, and TNF-related apoptosis-inducing ligant (TRAIL)-induced apoptosis. We used site-directed mutagenesis to change charged or hydrophobic amino acid residues to alanines to identify regions of MC159 that are critical for protection from apoptosis and for protein-protein interactions. Surprisingly, while MC159 is thought to block apoptosis by binding to Fas-associated death domain (FADD) or caspase-8, several mutants that lost apoptosis blocking activity still bound to both FADD and caspase-8. Mutations in the predicted hydrophobic patch 1 and α2 regions of both death effector domains (DEDs) within MC159 resulted in loss of the ability to bind to FADD or caspase-8 and to block apoptosis. Amino acid substitutions in the RXDL motif located in the α6 region of either DED resulted in loss of protection from apoptosis induced by Fas, TNF, and TRAIL and abolished the ability of MC159 to block death effector filament formation. Thus, charged or hydrophobic amino acids in three regions of the MC159 DEDs (hydrophobic patch 1, α2, and α6) are critical for the protein’s ability to interact with cellular proteins and to block apoptosis.
High Prevalence of Simian T-Lymphotropic Virus Type L in Wild Ethiopian Baboons
Authors: Takemura Taichiro , Yamashita Masahiro , Shimada Makoto K. , Ohkura Sadayuki , Shotake Takayoshi , Ikeda Mikio , Miura Tomoyuki , Hayami Masanori 
Published: July 2002

ABSTRACT
Simian T-cell leukemia viruses (STLVs) are the simian counterparts of human T-cell leukemia viruses (HTLVs). A novel, divergent type of STLV (STLV-L) from captive baboons was reported in 1994, but its natural prevalence remained unclear. We investigated the prevalence of STLV-L in 519 blood samples from wild-living nonhuman primates in Ethiopia. Seropositive monkeys having cross-reactive antibodies against HTLV were found among 22 out of 40 hamadryas baboons, 8 of 96 anubis baboons, 24 of 50 baboons that are hybrids between hamadryas and anubis baboons, and 41 of 177 grivet monkeys, but not in 156 gelada baboons. A Western blotting assay showed that sera obtained from seropositive hamadryas and hybrid baboons exhibited STLV-L-like reactivity. A PCR assay successfully amplified STLV sequences, which were subsequently sequenced and confirmed as being closely related to STLV-L. Surprisingly, further PCR showed that nearly half of the hamadryas (20 out of 40) and hybrid (19 out of 50) baboons had STLV-L DNA sequences. In contrast, most of the seropositive anubis baboons and grivet monkeys carried typical STLV-1 but not STLV-L. These observations demonstrate that STLV-L naturally prevails among hamadryas and hybrid baboons at significantly high rates. STLV-1 and -2, the close relative of STLV-L, are believed to have jumped across simian-human barriers, which resulted in widespread infection of HTLV-1 and -2. Further studies are required to know if STLV-L is spreading into human populations.
De Novo Infection and Serial Transmission of Kaposi's Sarcoma-Associated Herpesvirus in Cultured Endothelial Cells
Authors: Lagunoff Michael , Bechtel Jill , Venetsanakos Eleni , Roy Anne-Marie , Abbey Nancy , Herndier Brian , McMahon Martin , Ganem Don 
Published: July 2002

ABSTRACT
Infection by Kaposi's sarcoma-associated herpesvirus (KSHV) is central to the pathogenesis of the endothelial neoplasm Kaposi's sarcoma (KS) and is also linked to the rare B-cell tumor known as primary effusion lymphoma (PEL). Latently infected PEL cell lines can be induced to enter the lytic cycle and produce KSHV virions. However, such cells do not support de novo infection or serial propagation of KSHV. These limitations have prevented the development of systems for the genetic analysis of KSHV and have impeded a deeper understanding of KS pathogenesis. Here we show that human dermal microvascular endothelial cells immortalized by expression of telomerase can be readily infected by KSHV virions produced by PEL cells. Infection is predominantly latent, but a small subpopulation enters the lytic cycle spontaneously. Phorbol ester (tetradecanoyl phorbol acetate [TPA]) treatment of latently infected cells leads to enhanced induction of lytic KSHV replication, resulting in foci of cytopathic effect. There is no cytopathic effect or viral DNA expansion when infected TIME cells (telomerase-immortalized microvascular endothelial cells) are TPA induced in the presence of phosphonoacetic acid (PAA), an inhibitor of herpesvirus replication. Supernatants from phorbol-induced cultures transfer latent KSHV infection to uninfected cells, which can likewise be induced to undergo lytic replication by TPA treatment, and the virus can be further serially transmitted. Serial passage of the virus in TIME cells is completely inhibited when TPA treatment is done in the presence of PAA. Latently infected endothelial cells do not undergo major morphological changes or growth transformation, and infection is lost from the culture upon serial passage. This behavior faithfully recapitulates the behavior of spindle cells explanted from primary KS biopsies, strongly supporting the biological relevance of this culture system. These findings suggest that either the stability or the growth-deregulatory potential of the KSHV latency program in endothelial cells is more limited than might be predicted by analogy with other oncogenic viruses.
Mapping the Rubella Virus Subgenomic Promoter
Authors: Tzeng Wen-Pin , Frey Teryl K. 
Published: July 2002

ABSTRACT

Rubella virus
(RUB), the sole member of the
Rubivirus
genus in the
Togaviridae
family of positive-strand RNA viruses, synthesizes a single subgenomic (SG) RNA containing sequences from the 3′ end of the genomic RNA including the open reading frame (ORF) that encodes the virion proteins. The synthesis of SG RNA is initiated internally on a negative-strand, genome-length template at a site known as the SG promoter (SGP). Mapping the RUB SGP was initiated by using an infectious cDNA vector, dsRobo402/GFP, in which the region containing the SGP was duplicated (K. V. Pugachev, W.-P. Tzeng, and T. K. Frey, J. Virol. 74:10811-10815, 2000). In dsRobo402/GFP, the 5′-proximal nonstructural protein ORF (NS-ORF) is followed by the first SGP (SGP-1), the green fluorescent protein (GFP) gene, the second SGP (SGP-2), and the structural protein ORF. The duplicated SGP, SGP-2, contained nucleotides (nt) −175 to +76 relative to the SG start site, including the 3′ 127 nt of the NS-ORF and 47 nt between the NS-ORF and the SG start site. 5′ Deletions of SGP-2 to nt −40 (9 nt beyond the 3′ end of the NS-ORF) resulted in a wild-type (wt) phenotype in terms of virus replication and RNA synthesis. Deletions beyond this point impaired viability; however, the analysis was complicated by homologous recombination between SGP-1 and SGP-2 that resulted in deletion of the GFP gene and resurrection of viable virus with one SGP. Since the NS-ORF region was not necessary for SGP activity, subsequent mapping was done by using both replicon vectors, RUBrep/GFP and RUBrep/CAT, in which the SP-ORF is replaced with the reporter GFP and chloramphenical acetyltransferase genes, respectively, and the wt infectious clone, Robo402. In the replicon vectors, 5′ deletions to nt −26 resulted in the synthesis of SG RNA. In the infectious clone, deletions through nt −28 gave rise to viable virus. A series of short internal deletions confirmed that the region between nt −28 and the SG start site was essential for viability and showed that the repeated UCA triplet at the 5′ end of SG RNA was also required. Thus, the minimal SGP maps from nt −26 through the SG start site and appears to extend to at least nt +6, although a larger region is required for the generation of virus with a wt phenotype. Interestingly, while the positioning of the RUB SGP immediately adjacent the SG start site is thus similar to that of members of the genus
Alphavirus
, the other genus in the
Togaviridae
family, it does not include a region of nucleotide sequence homology with the alphavirus SGP that is located between nt −48 and nt −23 with respect to the SG start site in the RUB genome.