bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Cellular TATA Binding Protein Is Required for Rep-Dependent Replication of a Minimal Adeno-Associated Virus Type 2 p5 Element
Authors: François Achille , Guilbaud Mickaël , Awedikian Rafi , Chadeuf Gilliane , Moullier Philippe , Salvetti Anna 
Published: August 2005

ABSTRACT

The p5 promoter region of adeno-associated virus type 2 (AAV-2) is a multifunctional element involved in
rep
gene expression, Rep-dependent replication, and site-specific integration. We initially characterized a 350-bp p5 region by its ability to behave like a
cis
-acting replication element in the presence of Rep proteins and adenoviral factors. The objective of this study was to define the minimal elements within the p5 region required for Rep-dependent replication. Assays performed in transfected cells (in vivo) indicated that the minimal p5 element was composed by a 55-bp sequence (nucleotides 250 to 304 of wild-type AAV-2) containing the TATA box, the Rep binding site, the terminal resolution site present at the transcription initiation site (trs
+1
), and a downstream 17-bp region that could potentially form a hairpin structure localizing the trs
+1
at the top of the loop. Interestingly, the TATA box was absolutely required for in vivo but dispensable for in vitro, i.e., cell-free, replication. We also demonstrated that Rep binding and nicking at the trs
+1
was enhanced in the presence of the cellular TATA binding protein, and that overexpression of this cellular factor increased in vivo replication of the minimal p5 element. Together, these studies identified the minimal replication origin present within the AAV-2 p5 promoter region and demonstrated for the first time the involvement of the TATA box, in
cis
, and of the TATA binding protein, in
trans
, for Rep-dependent replication of this viral element.
Down-Regulation of p53 by Double-Stranded RNA Modulates the Antiviral Response
Authors: Marques Joao T. , Rebouillat Dominique , Ramana Chilakamarti V. , Murakami Junko , Hill Jason E. , Gudkov Andrei , Silverman Robert H. , Stark George R. , Williams Bryan R. G. 
Published: August 2005

ABSTRACT

p53 has been well characterized as a tumor suppressor gene, but its role in antiviral defense remains unclear. A recent report has demonstrated that p53 can be induced by interferons and is activated after vesicular stomatitis virus (VSV) infection. We observed that different nononcogenic viruses, including encephalomyocarditis virus (EMCV) and human parainfluenza virus type 3 (HPIV3), induced down-regulation of p53 in infected cells. Double-stranded RNA (dsRNA) and a mutant vaccinia virus lacking the dsRNA binding protein E3L can also induce this effect, indicating that dsRNA formed during viral infection is likely the trigger for down-regulation of p53. The mechanism of down-regulation of p53 by dsRNA relies on translation inhibition mediated by the PKR and RNase L pathways. In the absence of p53, the replication of both EMCV and HPIV3 was retarded, whereas, conversely, VSV replication was enhanced. Cell cycle analysis indicated that wild-type (WT) but not p53 knockout (KO) fibroblasts undergo an early-G
1
arrest following dsRNA treatment. Moreover, in WT cells the onset of dsRNA-induced apoptosis begins after p53 levels are down-regulated, whereas p53 KO cells, which lack the early-G
1
arrest, rapidly undergo apoptosis. Hence, our data suggest that the down-regulation of p53 facilitates apoptosis, thereby limiting viral replication.
Impact of 2-Bromo-5,6-Dichloro-1-β-
d
-Ribofuranosyl Benzimidazole Riboside and Inhibitors of DNA, RNA, and Protein Synthesis on Human Cytomegalovirus Genome Maturation
Authors: McVoy Michael A. , Nixon Daniel E. 
Published: August 2005

ABSTRACT

Herpesvirus genome maturation is a complex process in which concatemeric DNA molecules are translocated into capsids and cleaved at specific sequences to produce encapsidated-unit genomes. Bacteriophage studies further suggest that important ancillary processes, such as RNA transcription and DNA synthesis, concerned with repeat duplication, recombination, branch resolution, or damage repair may also be involved with the genome maturation process. To gain insight into the biochemical activities needed for herpesvirus genome maturation, 2-bromo-5,6-dichloro-1-β-
d
-ribofuranosyl benzimidazole riboside (BDCRB) was used to allow the accumulation of human cytomegalovirus concatemeric DNA while the formation of new genomes was being blocked. Genome formation was restored upon BDCRB removal, and addition of various inhibitors during this time window permitted evaluation of their effects on genome maturation. Inhibitors of protein synthesis, RNA transcription, and the viral DNA polymerase only modestly reduced genome formation, demonstrating that these activities are not required for genome maturation. In contrast, drugs that inhibit both viral and host DNA polymerases potently blocked genome formation. Radioisotope incorporation in the presence of a viral DNA polymerase inhibitor further suggested that significant host-mediated DNA synthesis occurs throughout the viral genome. These results indicate a role for host DNA polymerases in genome maturation and are consistent with a need for terminal repeat duplication, debranching, or damage repair concomitant with DNA packaging or cleavage. Similarities to previously reported effects of BDCRB on guinea pig cytomegalovirus were also noted; however, BDCRB induced low-level formation of a supergenomic species called monomer+ DNA that is unique to human cytomegalovirus. Analysis of monomer+ DNA suggested a model for its formation in which BDCRB permits limited packaging of concatemeric DNA but induces skipping of cleavage sites.
Epstein-Barr Virus Can Establish Infection in the Absence of a Classical Memory B-Cell Population
Authors: Conacher Margaret , Callard Robin , McAulay Karen , Chapel Helen , Webster David , Kumararatne Dinakantha , Chandra Anita , Spickett Gavin , Hopwood Paul A. , Crawford Dorothy H. 
Published: August 2005

ABSTRACT

Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus that persists in the body for life after primary infection. The primary site of EBV persistence is the memory B lymphocyte, but whether the virus initially infects naïve or memory B cells is still disputed. We have analyzed EBV infection in nine cases of X-linked hyper-immunoglobulin M (hyper-IgM) syndrome who, due to a mutation in CD40 ligand gene, do not have a classical, class-switched memory B-cell population (IgD
−
CD27
+
). We found evidence of EBV infection in 67% of cases, which is similar to the infection rate found in the general United Kingdom population (60 to 70% for the relevant age range). We detected EBV DNA in peripheral blood B cells and showed in one case that the infection was restricted to the small population of nonclassical, germinal center-independent memory B cells (IgD
+
CD27
+
). Detection of EBV small RNAs, latent membrane protein 2, and EBV nuclear antigen 3C expression in peripheral blood suggests full latent viral gene expression in this population. Analysis of EBV DNA in serial samples showed variability over time, suggesting cycles of infection and loss. Our results demonstrate that short-term EBV persistence can occur in the absence of a germinal center reaction and a classical memory B-cell population.
Ubiquitylation of Cdk9 by Skp2 Facilitates Optimal Tat Transactivation
Authors: Barboric Matjaz , Zhang Fan , Besenicar Mojca , Plemenitas Ana , Peterlin B. Matija 
Published: August 2005

ABSTRACT
By recruiting the positive transcriptional elongation factor b (P-TEFb) to paused RNA polymerase II, the transactivator Tat stimulates transcriptional elongation of the human immunodeficiency virus type 1 (HIV-1) genome. We found that cyclin-dependent kinase 9 (Cdk9), the catalytic subunit of P-TEFb, is ubiquitylated in vivo. This ubiquitylation depended on the Skp1/Cul1/F-box protein E3 ubiquitin ligase Skp2. Likewise, Tat required Skp2 since its transactivation of the HIV-1 long terminal repeat decreased in primary mouse embryonic fibroblasts, which lacked Skp2. The ubiquitylation of Cdk9 by Skp2 facilitated the formation of the ternary complex between P-TEFb, Tat, and transactivation response element. Thus, our findings underscore the requirement of ubiquitylation for the coactivator function in regulating HIV-1 transcriptional elongation.
Ternary Complex Formation of Human Immunodeficiency Virus Type 1 Env, CD4, and Chemokine Receptor Captured as an Intermediate of Membrane Fusion
Authors: Mkrtchyan Samvel R. , Markosyan Ruben M. , Eadon Michael T. , Moore John P. , Melikyan Gregory B. , Cohen Fredric S. 
Published: August 2005

ABSTRACT
Human immunodeficiency virus (HIV) Env-induced fusion is highly temperature dependent. When effector and target cells were coincubated at 37°C, there was a kinetic delay before fusion commenced. When effector and target cells were coincubated for varied times at 23°C, a temperature that does not permit fusion, a temperature-arrested stage was created. Raising temperature to 37°C from the 23°C intermediate eliminated the kinetic delay. Inhibitors (T22, AMD3100, and Sch-C) that block fusion by binding chemokine receptors were added after creating the intermediate so as to assess the extent of engagement between gp120 and chemokine receptors at that stage. For both CXCR4 and CCR5 as coreceptors, increasingly long times of coincubation at 23°C reduced the efficacy of the coreceptor-binding inhibitors in blocking fusion. This implies that an increasing number of ternary Env/CD4/coreceptor complexes form over time at 23°C. It also shows that ternary complex formation has a lower temperature threshold than the downstream steps that include Env folding into a six-helix bundle; this provides an experimental means to separate coreceptor binding by gp120 from the subsequent refolding of gp41 into a six-helix bundle structure. As the time of cell coincubation at 23°C was prolonged, more cells quickly fused upon the raising of the temperature to 37°C, and the increase quantitatively correlated with the greater percentage of fusion that was resistant to drugs. Therefore the pronounced kinetic delay in HIV Env-induced fusion is caused predominantly by the time needed for ternary complexes to form.
Cross-Reactivity of T Lymphocytes Recognizing a Human Cytotoxic T-Lymphocyte Epitope within BK and JC Virus VP1 Polypeptides
Authors: Krymskaya Ludmila , Sharma Madeva C. , Martinez Joy , Haq Wahajul , Huang Eric C. , Limaye Ajit P. , Diamond Don J. , Lacey Simon F. 
Published: August 2005

ABSTRACT

A transgenic mouse model was used to identify an HLA-A*02-restricted epitope within the VP1 polypeptide of a human polyomavirus, BK virus (BKV), which is associated with polyomavirus-associated nephropathy in kidney transplant patients. Peptide stimulation of splenocytes from mice immunized with recombinant modified vaccinia virus Ankara expressing BKV VP1 resulted in expansion of cytotoxic T lymphocytes (CTLs) recognizing the sequence LLMWEAVTV corresponding to amino acid residues 108 to 116 (BKV VP1p108). These effector T-cell populations represented functional CTLs as assessed by cytotoxicity and cytokine production and were cross-reactive against antigen-presenting cells pulsed with a peptide corresponding to the previously described JC virus (JCV) VP1 homolog sequence ILMWEAVTL (JCV VP1p100) (I. J. Koralnik et al., J. Immunol.
168:
499-504, 2002). A panel of 10 healthy HLA-A*02 human volunteers and two kidney transplant recipients were screened for T-cell immunity to this BK virus VP1 epitope by in vitro stimulation of their peripheral blood mononuclear cells (PBMC) with the BKV VP1p108 peptide, followed by tetramer labeling combined with simultaneous assays to detect intracellular cytokine production and degranulation. PBMC from 4/10 subjects harbored CTL populations that recognized both the BKV VP1p108 and the JCV VP1p100 peptides with comparable efficiencies as measured by tetramer binding, gamma interferon production, and degranulation. CTL responses to the JCV VP1p100 epitope have been associated with prolonged survival in progressive multifocal leukoencephalopathy patients (R. A. Du Pasquier et al., Brain
127:
1970-1978, 2004; I. J. Koralnik et al., J. Immunol.
168:
499-504, 2002). Given that both human polyomaviruses are resident in a high proportion of healthy individuals and that coinfection occurs (W. A. Knowles et al., J. Med. Virol.
71:
115-123, 2003), our findings suggest a reinterpretation of this protective T-cell immunity, suggesting that the same VP1 epitope is recognized in HLA-A*02 persons in response to either BK or JC virus infection.
Glycosylation, Disulfide Bond Formation, and the Presence of a WSXWS-Like Motif in the Orf Virus GIF Protein Are Critical for Maintaining the Integrity of Binding to Ovine Granulocyte-Macrophage Colony-Stimulating Factor and Interleukin-2
Authors: McInnes C. J. , Deane D. , Haig D. , Percival A. , Thomson J. , Wood A. R. 
Published: August 2005

ABSTRACTOrf virus (ORFV), the type species of the familyParapoxviridae, encodes a protein (GIF) that binds and inhibits the ovine cytokines granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-2 (IL-2). There is no obvious sequence homology between the ORFV protein and any known mammalian GM-CSF- or IL-2-binding proteins. We demonstrate here that many of the biochemical properties of mammalian GM-CSF receptors that are required for efficient binding of GM-CSF are also critical to the GIF protein for binding to ovine GM-CSF (ovGM-CSF). Site-directed mutagenesis of the GIF protein demonstrated, first, the importance of disulfide bonds, and second, that a sequence motif (WDPWV), related to the WSXWS motif of the type 1 cytokine receptor superfamily, was necessary for biological activity. Finally, glycosylation of the GIF protein was also critical for binding to GM-CSF.
Human Immunodeficiency Virus Type 1 Subtype B Ancestral Envelope Protein Is Functional and Elicits Neutralizing Antibodies in Rabbits Similar to Those Elicited by a Circulating Subtype B Envelope
Authors: Doria-Rose N. A. , Learn G. H. , Rodrigo A. G. , Nickle D. C. , Li F. , Mahalanabis M. , Hensel M. T. , McLaughlin S. , Edmonson P. F. , Montefiori D. , Barnett S. W. , Haigwood N. L. , Mullins J. I. 
Published: August 2005

ABSTRACTHuman immunodeficiency virus type 1 (HIV-1) is a difficult target for vaccine development, in part because of its ever-expanding genetic diversity and attendant capacity to escape immunologic recognition. Vaccine efficacy might be improved by maximizing immunogen antigenic similarity to viruses likely to be encountered by vaccinees. To this end, we designed a prototype HIV-1 envelope vaccine using a deduced ancestral state for theenvgene. The ancestral state reconstruction method was shown to be >95% accurate by computer simulation and 99.8% accurate when estimating the known inoculum used in an experimental infection study in rhesus macaques. Furthermore, the deduced ancestor gene differed from the set of sequences used to derive the ancestor by an average of 12.3%, while these latter sequences were an average of 17.3% different from each other. A full-length ancestral subtype B HIV-1envgene was constructed and shown to produce a glycoprotein of 160 kDa that bound and fused with cells expressing the HIV-1 coreceptor CCR5. This Env was also functional in a virus pseudotype assay. When either gp160- or gp140-expressing plasmids and recombinant gp120 were used to immunize rabbits in a DNA prime-protein boost regimen, the artificial gene induced immunoglobulin G antibodies capable of weakly neutralizing heterologous primary HIV-1 strains. The results were similar for rabbits immunized in parallel with a natural isolate, HIV-1 SF162. Further design efforts to better present conserved neutralization determinants are warranted.
Broad Cross-Clade T-Cell Responses to Gag in Individuals Infected with Human Immunodeficiency Virus Type 1 Non-B Clades (A to G): Importance of HLA Anchor Residue Conservation
Authors: Geels Mark J. , Dubey Sheri A. , Anderson Kiersten , Baan Elly , Bakker Margreet , Pollakis Georgios , Paxton William A. , Shiver John W. , Goudsmit Jaap 
Published: August 2005

ABSTRACT
We aimed to identify cross-clade human immunodeficiency virus type 1 (HIV-1) specific T-cell responses among 10 HLA-typed individuals who were infected with non-B HIV-1 strains (A, AG, C, D, G, or F) and to correlate these responses with genetic variation in documented T-cell epitopes. T-cell reactivity was tested against peptide pools spanning clade B Gag, Pol, Nef, Rev, and Tat consensus, with Gag and Nef providing the highest responses. Nine individuals who responded to clade B Gag demonstrated cross-reactive T-cell responses against clade A and C Gag pools, while six of seven responders to Nef-B reacted to clade A and C Nef pools. An inverse correlation between the height of the T-cell responses and the sequence divergence of the HLA class I-restricted epitopes was identified when we compared autologous Gag and Nef sequences with the reactive consensus pools. This could be explained for the Gag sequences through observed variations in the HLA anchor residues. Through mapping of 30 amino acid cross-clade-reactive regions using Gag-B pools, we were able to link 58% (14/24) of the T-cell responses to regions containing previously described HLA class I-restricted epitopes. Forty-two percent (10/24) of the responses were directed to regions containing new epitopes, for which predicted HLA class I motifs could be recognized in 70% (7/10) of individuals. We demonstrate here that cross-clade T-cell responses are frequently induced in individuals infected with distinct HIV-1 clades, suggesting that interclade variation outside of HLA anchor residues may have less impact on vaccine-induced T-cell reactivity than previously thought.
Are Ducks Contributing to the Endemicity of Highly Pathogenic H5N1 Influenza Virus in Asia?
Authors: Sturm-Ramirez K. M. , Hulse-Post D. J. , Govorkova E. A. , Humberd J. , Seiler P. , Puthavathana P. , Buranathai C. , Nguyen T. D. , Chaisingh A. , Long H. T. , Naipospos T. S. P. , Chen H. , Ellis T. M. , Guan Y. , Peiris J. S. M. , Webster R. G. 
Published: August 2005

ABSTRACT
Wild waterfowl are the natural reservoir of all influenza A viruses, and these viruses are usually nonpathogenic in these birds. However, since late 2002, H5N1 outbreaks in Asia have resulted in mortality among waterfowl in recreational parks, domestic flocks, and wild migratory birds. The evolutionary stasis between influenza virus and its natural host may have been disrupted, prompting us to ask whether waterfowl are resistant to H5N1 influenza virus disease and whether they can still act as a reservoir for these viruses. To better understand the biology of H5N1 viruses in ducks and attempt to answer this question, we inoculated juvenile mallards with 23 different H5N1 influenza viruses isolated in Asia between 2003 and 2004. All virus isolates replicated efficiently in inoculated ducks, and 22 were transmitted to susceptible contacts. Viruses replicated to higher levels in the trachea than in the cloaca of both inoculated and contact birds, suggesting that the digestive tract is not the main site of H5N1 influenza virus replication in ducks and that the fecal-oral route may no longer be the main transmission path. The virus isolates' pathogenicities varied from completely nonpathogenic to highly lethal and were positively correlated with tracheal virus titers. Nevertheless, the eight virus isolates that were nonpathogenic in ducks replicated and transmitted efficiently to naïve contacts, suggesting that highly pathogenic H5N1 viruses causing minimal signs of disease in ducks can propagate silently and efficiently among domestic and wild ducks in Asia and that they represent a serious threat to human and veterinary public health.
Virulent Variants Emerging in Mice Infected with the Apathogenic Prototype Strain of the Parvovirus Minute Virus of Mice Exhibit a Capsid with Low Avidity for a Primary Receptor
Authors: Rubio Mari-Paz , López-Bueno Alberto , Almendral José M. 
Published: August 2005

ABSTRACT
The mechanisms involved in the emergence of virulent mammalian viruses were investigated in the adult immunodeficient SCID mouse infected by the attenuated prototype strain of the parvovirus Minute Virus of Mice (MVMp). Cloned MVMp intravenously inoculated in mice consistently evolved during weeks of subclinical infection to variants showing altered plaque phenotypes. All the isolated large-plaque variants spread systemically from the oronasal cavity and replicated in major organs (brain, kidney, liver), in sharp contrast to the absolute inability of the MVMp and small-plaque variants to productively invade SCID organs by this natural route of infection. The virulent variants retained the MVMp capacity to infect mouse fibroblasts, consistent with the lack of genetic changes across the 220-to-335 amino acid sequence of VP2, a capsid domain containing main determinants of MVM tropism. However, the capsid of the virulent variants shared a lower affinity than the wild type for a primary receptor used in the cytotoxic infection. The capsid gene of a virulent variant engineered in the MVMp background endowed the recombinant virus with a large-plaque phenotype, lower affinity for the receptor, and productive invasiveness by the oronasal route in SCID mice, eventually leading to 100% mortality. In the analysis of virulence in mice, both MVMp and the recombinant virus similarly gained the bloodstream 1 to 2 days postoronasal inoculation and remained infectious when adsorbed to blood cells in vitro. However, the wild-type MVMp was cleared from circulation a few days afterwards, in contrast to the viremia of the recombinant virus, which was sustained for life. Significantly, attachment to an abundant receptor of primary mouse kidney epithelial cells by both viruses could be quantitatively competed by wild-type MVMp capsids, indicating that virulence is not due to an extended receptor usage in target tissues. We conclude that the selection of capsid-receptor interactions of low affinity, which favors systemic infection, is a major evolutionary process in the adaptation of parvoviruses to new hosts and in the cause of disease.
The UL7 Gene of Pseudorabies Virus Encodes a Nonessential Structural Protein Which Is Involved in Virion Formation and Egress
Authors: Fuchs Walter , Granzow Harald , Klopfleisch Robert , Klupp Barbara G. , Rosenkranz Daniela , Mettenleiter Thomas C. 
Published: August 2005

ABSTRACT

Homologues of the UL7 gene of herpes simplex virus type 1 are conserved in alpha-, beta-, and gammaherpesviruses. However, little is known about their functions. Using a monospecific rabbit antiserum raised against a bacterial fusion protein, we identified the UL7 gene product of the neurotropic alphaherpesvirus pseudorabies virus (PrV). In Western blot analyses of infected cells and purified PrV particles the serum specifically detected a 29-kDa protein, which matches the calculated mass of the 266-amino-acid translation product of PrV UL7. For functional analysis, UL7 was deleted by mutagenesis of an infectious full-length clone of the PrV genome in
Escherichia coli
. The obtained recombinant PrV-ΔUL7F was replication competent in rabbit kidney cells, but maximum virus titers were decreased nearly 10-fold and plaque diameters were reduced by ca. 60% compared to wild-type PrV. Electron microscopy of infected cells revealed that in the absence of UL7, formation and nuclear egress of nucleocapsids were not affected, whereas secondary envelopment of cytoplasmic nucleocapsids appeared to be delayed and release of mature virions was less efficient. The observed replication defects were corrected by repair of the viral UL7 gene or by propagation of PrV-ΔUL7F in UL7-expressing cells. PrV-ΔUL7F was moderately attenuated in mice. Compared to wild-type virus, mean survival times were prolonged from 2 to 3 days after intranasal infection. However, neuroinvasion and transneuronal spread of PrV were not abolished in the absence of UL7. Thus, UL7 encodes a virion protein of PrV, which plays a role during virion maturation and egress both in vitro and in vivo.