bluuebunny/crossref_metadata_2025_split huggingface dataset:
Kaposi's Sarcoma-Associated Herpesvirus Encodes a Viral Deubiquitinase
Authors: González Carlos M. , Wang Ling , Damania Blossom 
Published: July 2009

ABSTRACT
Kaposi's sarcoma-associated herpesvirus (KSHV) is etiologically linked to Kaposi's sarcoma, primary effusion lymphomas, and multicentric Castleman's disease. Like other herpesviruses, KSHV can exist in either a lytic or a latent phase during its life cycle. We report that the lytic protein encoded by KSHV open reading frame 64 (Orf64) is a viral deubiquitinase (DUB) enzyme capable of deubiquitinating cellular proteins in vitro and in vivo. Orf64 DUB activity is effective against lysine 48 (K48)- and lysine 63 (K63)-linked ubiquitin chains. Thus, KSHV Orf64 is a viral DUB that does not show specificity toward K48 or K63 ubiquitin linkages. Orf64 DUB activity lies within the first 205 residues of the protein, and deubiquitination is dependent on a cysteine at position 29, since mutation of this residue ablated this activity. Cell fractionation studies revealed that the N terminus and the full-length protein localized to both the nuclear and cytoplasmic compartments. The function of Orf64 was tested by short interfering RNA (siRNA) knockdown studies on latently infected cells that were induced into lytic replication. We found that depletion of Orf64 by siRNA resulted in decreased viral lytic transcription and lytic protein expression. These experiments indicate that Orf64 plays a role in KSHV lytic replication.
Tomato Bushy Stunt Virus
Recombination Guided by Introduced MicroRNA Target Sequences
Authors: Kumar Pavan , Uratsu Sandra , Dandekar Abhaya , Falk Bryce W. 
Published: July 2009

ABSTRACT

Previously we described
Tomato bushy stunt virus
(TBSV) vectors, which retained their capsid protein gene and were engineered with magnesium chelatase (ChlH) and phytoene desaturase (PDS) gene sequences from
Nicotiana benthamiana
. Upon plant infection, these vectors eventually lost the inserted sequences, presumably as a result of recombination. Here, we modified the same vectors to also contain the plant miR171 or miR159 target sequences immediately 3′ of the silencing inserts. We inoculated
N. benthamiana
plants and sequenced recombinant RNAs recovered from noninoculated upper leaves. We found that while some of the recombinant RNAs retained the microRNA (miRNA) target sites, most retained only the 3′ 10 and 13 nucleotides of the two original plant miRNA target sequences, indicating in planta miRNA-guided RNA-induced silencing complex cleavage of the recombinant TBSV RNAs. In addition, recovered RNAs also contained various fragments of the original sequence (ChlH and PDS) upstream of the miRNA cleavage site, suggesting that the 3′ portion of the miRNA-cleaved TBSV RNAs served as a template for negative-strand RNA synthesis by the TBSV RNA-dependent RNA polymerase (RdRp), followed by template switching by the RdRp and continued RNA synthesis resulting in loss of nonessential nucleotides.
Diverse Cross-Reactive Potential and Vβ Gene Usage of an Epitope-Specific Cytotoxic T-Lymphocyte Population in Monkeys Immunized with Diverse Human Immunodeficiency Virus Type 1 Env Immunogens
Authors: Hulot Sandrine L. , Seaman Michael S. , Sen Pritha , Autissier Patrick A. , Manuel Edwin R. , Letvin Norman L. 
Published: July 2009

ABSTRACTAn ideal human immunodeficiency virus type 1 (HIV-1) vaccine would elicit potent cellular and humoral immune responses that recognize diverse strains of the virus. In the present study, combined methodologies (flow cytometry, Vβ repertoire analysis, and complementarity-determining region 3 sequencing) were used to determine the clonality of CD8+T lymphocytes taking part in the recognition of variant epitope peptides elicited in Mamu-A*01-positive rhesus monkeys immunized with vaccines encoding diverse HIV-1 envelopes (Envs). Monkeys immunized with clade B Envs generated CD8+T lymphocytes that cross-recognized both clade B- and clade C-p41A epitope peptides using a large degree of diversity in Vβ gene usage. However, with two monkeys immunized with clade C Env, one monkey exhibited p41A-specific cytotoxic T-lymphocytes (CTL) with the capacity for cross-recognition of variant epitopes, while the other monkey did not. These studies demonstrate that the cross-reactive potential of variant p41A epitope peptide-specific CTL populations can differ between monkeys that share the same restricting major histocompatibility complex class I molecule and receive the same vaccine immunogens.
Severe Acute Respiratory Syndrome Coronavirus Nonstructural Protein 2 Interacts with a Host Protein Complex Involved in Mitochondrial Biogenesis and Intracellular Signaling
Authors: Cornillez-Ty Cromwell T. , Liao Lujian , Yates John R. , Kuhn Peter , Buchmeier Michael J. 
Published: July 2009

ABSTRACT
The severe acute respiratory syndrome coronavirus (SARS-CoV) generates 16 nonstructural proteins (nsp's) through proteolytic cleavage of a large precursor protein. Although several nsp's exhibit catalytic activities that are important for viral replication and transcription, other nsp's have less clearly defined roles during an infection. In order to gain a better understanding of their functions, we attempted to identify host proteins that interact with nsp's during SARS-CoV infections. For nsp2, we identified an interaction with two host proteins, prohibitin 1 (PHB1) and PHB2. Our results suggest that nsp2 may be involved in the disruption of intracellular host signaling during SARS-CoV infections.
Growth-Promoting Properties of Epstein-Barr Virus EBER-1 RNA Correlate with Ribosomal Protein L22 Binding
Authors: Houmani Jennifer L. , Davis Christopher I. , Ruf Ingrid K. 
Published: July 2009

ABSTRACT
The Epstein-Barr virus (EBV)-encoded RNAs, EBER-1 and EBER-2, are highly abundant noncoding nuclear RNAs expressed during all forms of EBV latency. The EBERs have been shown to impart significant tumorigenic potential upon EBV-negative Burkitt lymphoma (BL) cells and to contribute to the growth potential of other B-cell lymphoma-, gastric carcinoma-, and nasopharyngeal carcinoma-derived cell lines. However, the mechanisms underlying this EBER-dependent enhancement of cell growth potential remain to be elucidated. Here we focused on the known interaction between EBER-1 and the cellular ribosomal protein L22 and the consequences of this interaction with respect to the growth-promoting properties of the EBERs. L22, a component of 60S ribosomal subunits, binds three sites on EBER-1, and a substantial fraction of available L22 is relocalized from nucleoli to the nucleoplasm in EBV-infected cells. To investigate the hypothesis that EBER-1-mediated relocalization of L22 in EBV-infected cells is critical for EBER-dependent functions, we investigated whether EBER-1 expression is necessary and sufficient for nucleoplasmic retention of L22. Following demonstration of this, we utilized RNA-protein binding assays and fluorescence localization studies to demonstrate that mutation of the L22 binding sites on EBER-1 prevents L22 binding and inhibits EBER-1-dependent L22 relocalization. Finally, the in vivo consequence of preventing L22 relocalization in EBER-expressing cells was examined in soft agar colony formation assays. We demonstrate that BL cells expressing mutated EBER-1 RNAs rendered incapable of binding L22 have significantly reduced capacity to enhance cell growth potential relative to BL cells expressing wild-type EBERs.
Dominant CD8+T-Lymphocyte Responses Suppress Expansion of Vaccine-Elicited Subdominant T Lymphocytes in Rhesus Monkeys Challenged with Pathogenic Simian-Human Immunodeficiency Virus
Authors: Manuel Edwin R. , Yeh Wendy W. , Seaman Michael S. , Furr Kathryn , Lifton Michelle A. , Hulot Sandrine L. , Autissier Patrick , Letvin Norman L. 
Published: July 2009

ABSTRACTEmerging data suggest that a cytotoxic T-lymphocyte response against a diversity of epitopes confers greater protection against a human immunodeficiency virus/simian immunodeficiency virus infection than does a more focused response. To facilitate the creation of vaccine strategies that will generate cellular immune responses with the greatest breadth, it will be important to understand the mechanisms employed by the immune response to regulate the relative magnitudes of dominant and nondominant epitope-specific cellular immune responses. In this study, we generated dominant Gag p11C- and subdominant Env p41A-specific CD8+T-lymphocyte responses inMamu-A*01+rhesus monkeys through vaccination with plasmid DNA and recombinant adenovirus encoding simian-human immunodeficiency virus (SHIV) proteins. Infection of vaccinatedMamu-A*01+rhesus monkeys with a SHIV Gag Δp11C mutant virus generated a significantly increased expansion of the Env p41A-specific CD8+T-lymphocyte response in the absence of secondary Gag p11C-specific CD8+T-lymphocyte responses. These results indicate that the presence of the Gag p11C-specific CD8+T-lymphocyte response following virus challenge may exert suppressive effects on primed Env p41A-specific CD8+T-lymphocyte responses. These findings suggest that immunodomination exerted by dominant responses during SHIV infection may diminish the breadth of recall responses primed during vaccination.
Role of the Cytoplasmic Tail Amino Acid Sequences of Newcastle Disease Virus Hemagglutinin-Neuraminidase Protein in Virion Incorporation, Cell Fusion, and Pathogenicity
Authors: Kim Shin-Hee , Yan Yongqi , Samal Siba K. 
Published: July 2009

ABSTRACT
To determine the role of amino acid sequences of the hemagglutinin-neuraminidase (HN) cytoplasmic tail in Newcastle disease virus (NDV) replication and pathogenicity, we generated recombinant NDVs with a deletion or point mutation in the N-terminal cytoplasmic tail. The first 2-amino-acid deletion in the cytoplasmic tail did not affect the biological characteristics of NDV. However, a 4-amino-acid deletion and the substitution of alanine for serine at position 6 affected cell fusion, pathogenicity, and colocalization of the HN and M proteins of NDV, indicating that these residues of the HN cytoplasmic tail are critical for its specific incorporation into virions.
Intracellular Localization of the Pseudorabies Virus Large Tegument Protein pUL36
Authors: Möhl Britta S. , Böttcher Sindy , Granzow Harald , Kuhn Jana , Klupp Barbara G. , Mettenleiter Thomas C. 
Published: July 2009

ABSTRACT

Homologs of the essential large tegument protein pUL36 of herpes simplex virus 1 are conserved throughout the
Herpesviridae
, complex with pUL37, and form part of the capsid-associated “inner” tegument. pUL36 is crucial for transport of the incoming capsid to and docking at the nuclear pore early after infection as well as for virion maturation in the cytoplasm. Its extreme C terminus is essential for pUL36 function interacting with pUL25 on nucleocapsids to start tegumentation (K. Coller, J. Lee, A. Ueda, and G. Smith, J. Virol. 81:11790-11797, 2007). However, controversy exists about the cellular compartment in which pUL36 is added to the nascent virus particle. We generated monospecific rabbit antisera against four different regions spanning most of pUL36 of the alphaherpesvirus pseudorabies virus (PrV). By immunofluorescence and immunoelectron microscopy, we then analyzed the intracellular location of pUL36 after transient expression and during PrV infection. While reactivities of all four sera were comparable, none of them showed specific intranuclear staining during PrV infection. In immunoelectron microscopy, neither of the sera stained primary enveloped virions in the perinuclear cleft, whereas extracellular mature virus particles were extensively labeled. However, transient expression of pUL36 alone resulted in partial localization to the nucleus, presumably mediated by nuclear localization signals (NLS) whose functionality was demonstrated by fusion of the putative NLS to green fluorescent protein (GFP) and GFP-tagged pUL25. Since PrV pUL36 can enter the nucleus when expressed in isolation, the NLS may be masked during infection. Thus, our studies show that during PrV infection pUL36 is not detectable in the nucleus or on primary enveloped virions, correlating with the notion that the tegument of mature virus particles, including pUL36, is acquired in the cytosol.
Chimeric L1-L2 Virus-Like Particles as Potential Broad-Spectrum Human Papillomavirus Vaccines
Authors: Schellenbacher Christina , Roden Richard , Kirnbauer Reinhard 
Published: July 2009

ABSTRACT
The amino (N) terminus of the human papillomavirus (HPV) minor capsid protein L2 can induce low-titer, cross-neutralizing antibodies. The aim of this study was to improve immunogenicity of L2 peptides by surface display on highly ordered, self-assembled virus-like particles (VLP) of major capsid protein L1, and to more completely characterize neutralization epitopes of L2. Overlapping peptides comprising amino acids (aa) 2 to 22 (hereafter, chimera or peptide 2-22), 13 to 107, 18 to 31, 17 to 36, 35 to 75, 75 to 112, 115 to 154, 149 to 175, and 172 to 200 of HPV type 16 (HPV16) L2 were genetically engineered into the DE surface loop of bovine papillomavirus type 1 L1 VLP. Except for chimeras 35-75 and 13-107, recombinant fusion proteins assembled into VLP. Vaccination of rabbits with Freund's adjuvanted native VLP induced higher L2-specific antibody titers than vaccination with corresponding sodium dodecyl sulfate-denatured proteins. Immune sera to epitopes within residues 13 to 154 neutralized HPV16 in pseudovirion neutralization assays, whereas chimera 17-36 induced additional cross-neutralization to divergent high-risk HPV18, -31, -45, -52, and -58; low-risk HPV11; and beta-type HPV5 (titers of 50 to 10,000). Aluminum hydroxide-monophosphoryl lipid A (Alum-MPL)-adjuvanted VLP induced similar patterns of neutralization in both rabbits and mice, albeit with 100-fold-lower titers than Freund's adjuvant. Importantly, Alum-MPL-adjuvanted immunization with chimeric HPV16L1-HPV16L2 (peptide 17-36) VLP induced neutralization or cross-neutralization of HPV16, -18, -31, -45, -52, and -58; HPV6 and -11; and HPV5 (titers of 50 to 100,000). Immunization with HPV16 L1-HPV16 L2 (chimera 17-36) VLP in adjuvant applicable for human use induces broad-spectrum neutralizing antibodies against HPV types evolutionarily divergent to HPV16 and thus may protect against infection with mucosal high-risk, low-risk, and beta HPV types and associated disease.
Genetic and Pharmacologic Alteration of Cathepsin Expression Influences Reovirus Pathogenesis
Authors: Johnson Elizabeth M. , Doyle Joshua D. , Wetzel J. Denise , McClung R. Paul , Katunuma Nobuhiko , Chappell James D. , Washington M. Kay , Dermody Terence S. 
Published: July 2009

ABSTRACTThe cathepsin family of endosomal proteases is required for proteolytic processing of several viruses during entry into host cells. Mammalian reoviruses utilize cathepsins B (Ctsb), L (Ctsl), and S (Ctss) for disassembly of the virus outer capsid and activation of the membrane penetration machinery. To determine whether cathepsins contribute to reovirus tropism, spread, and disease outcome, we infected 3-day-old wild-type (wt),Ctsb−/−,Ctsl−/−, andCtss−/−mice with the virulent reovirus strain T3SA+. The survival rate ofCtsb−/−mice was enhanced in comparison to that of wt mice, whereas the survival rates ofCtsl−/−andCtss−/−mice were diminished. Peak titers at sites of secondary replication in all strains of cathepsin-deficient mice were lower than those in wt mice. Clearance of the virus was delayed inCtsl−/−andCtss−/−mice in comparison to the levels for wt andCtsb−/−mice, consistent with a defect in cell-mediated immunity in mice lacking cathepsin L or S. Cathepsin expression was dispensable for establishment of viremia, but cathepsin L was required for maximal reovirus growth in the brain. Treatment of wt mice with an inhibitor of cathepsin L led to amelioration of reovirus infection. Collectively, these data indicate that cathepsins B, L, and S influence reovirus pathogenesis and suggest that pharmacologic modulation of cathepsin activity diminishes reovirus disease severity.
Zanamivir-Resistant Influenza Viruses with a Novel Neuraminidase Mutation
Authors: Hurt Aeron C. , Holien Jessica K. , Parker Michael , Kelso Anne , Barr Ian G. 
Published: July 2009

ABSTRACT
The neuraminidase inhibitors zanamivir and oseltamivir are marketed for the treatment and prophylaxis of influenza and have been stockpiled by many countries for use in a pandemic. Although recent surveillance has identified a striking increase in the frequency of oseltamivir-resistant seasonal influenza A (H1N1) viruses in Europe, the United States, Oceania, and South Africa, to date there have been no reports of significant zanamivir resistance among influenza A (H1N1) viruses or any other human influenza viruses. We investigated the frequency of oseltamivir and zanamivir resistance in circulating seasonal influenza A (H1N1) viruses in Australasia and Southeast Asia. Analysis of 391 influenza A (H1N1) viruses isolated between 2006 and early 2008 from Australasia and Southeast Asia revealed nine viruses (2.3%) that demonstrated markedly reduced zanamivir susceptibility and contained a previously undescribed Gln136Lys (Q136K) neuraminidase mutation. The mutation had no effect on oseltamivir susceptibility but caused approximately a 300-fold and a 70-fold reduction in zanamivir and peramivir susceptibility, respectively. The role of the Q136K mutation in conferring zanamivir resistance was confirmed using reverse genetics. Interestingly, the mutation was not detected in the primary clinical specimens from which these mutant isolates were grown, suggesting that the resistant viruses either occurred in very low proportions in the primary clinical specimens or arose during MDCK cell culture passage. Compared to susceptible influenza A (H1N1) viruses, the Q136K mutant strains displayed greater viral fitness than the wild-type virus in MDCK cells but equivalent infectivity and transmissibility in a ferret model.
Similarities and Differences in Antagonism of Neuron Alpha/Beta Interferon Responses by Venezuelan Equine Encephalitis and Sindbis Alphaviruses
Authors: Yin Jun , Gardner Christina L. , Burke Crystal W. , Ryman Kate D. , Klimstra William B. 
Published: July 2009

ABSTRACT
Venezuelan equine encephalitis virus (VEEV) is highly virulent in adult laboratory mice, while Sindbis virus (SINV) is avirulent regardless of dose or inoculation route, dependent upon functioning alpha/beta interferon (IFN-α/β) responses. We have examined each virus' resistance to and/or antagonism of IFN-α/β responses in neurons, a cell type targeted by both viruses in mice, by infecting IFN-α/β-treated or untreated primary cultures with viruses or virus-derived replicons that lacked the structural proteins. Priming with IFN-α/β prior to infection revealed that VEEV replication and progeny virion production were resistant to an established antiviral state while those of SINV were more sensitive. Postinfection IFN-α/β treatment revealed that phosphorylation of STAT1 and STAT2 was partially blocked by infection with either virus, dependent upon expression of nonstructural proteins (nsP), but not structural proteins (sP). However, inhibition of STAT phosphorylation by VEEV replicons was not correlated with inhibition of IFN-stimulated gene (ISG) mRNA induction, yet ISG induction was inhibited when sP were present. Host translation was inhibited by VEEV nsP even when cells were pretreated with IFN-α/β. SINV blocked ISG induction and translation, associated with nsP-mediated shutoff of macromolecular synthesis, but both activities were sensitive to IFN-α/β pretreatment. We conclude that both VEEV and SINV limit ISG induction in infected neurons through shutoff of host transcription and translation but that inhibition by VEEV is more resistant to IFN-α/β priming. Likewise, both viruses inhibit IFN receptor-initiated signaling, although the effect upon host responses is not clear. Finally, VEEV appears to be more resistant to effectors of the preestablished antiviral state.
Small Molecule Targets Env for Endoplasmic Reticulum-Associated Protein Degradation and Inhibits Human Immunodeficiency Virus Type 1 Propagation
Authors: Jejcic Alenka , Daniels Robert , Goobar-Larsson Laura , Hebert Daniel N. , Vahlne Anders 
Published: July 2009

ABSTRACTHuman immunodeficiency virus type 1 (HIV-1) is dependent on its envelope glycoprotein (Env) to bind, fuse, and subsequently infect a cell. We show here that treatment of HIV-1-infected cells with glycyl-prolyl-glycine amide (GPG-NH2), dramatically reduced the infectivity of the released viral particles by decreasing their Env incorporation. The mechanism of GPG-NH2was uncovered by examining Env expression and maturation in treated cells. GPG-NH2treatment was found to affect Env by significantly decreasing its steady-state levels, its processing into gp120/gp41, and its mass by inducing glycan removal in a manner dependent on its native signal sequence and the proteasome. Therefore, GPG-NH2negatively impacts Env maturation, facilitating its targeting for endoplasmic reticulum-associated protein degradation, where Env is deglycosylated en route to its degradation. These findings illustrate that nontoxic drugs such as GPG-NH2, which can selectively target glycoproteins to existing cellular degradation pathways, may be useful for pathogen therapy.
Characterisation of organisational issues in paediatric clinical ethics consultation: a qualitative study
Authors: Opel D J , Wilfond B S , Brownstein D , Diekema D S , Pearlman R A 
Published: July 2009

Background:The traditional approach to resolving ethics concerns may not address underlying organisational issues involved in the evolution of these concerns. This represents a missed opportunity to improve quality of care “upstream”. The purpose of this study was to understand better which organisational issues may contribute to ethics concerns.Methods:Directed content analysis was used to review ethics consultation notes from an academic children’s hospital from 1996 to 2006 (N  =  71). The analysis utilised 18 categories of organisational issues derived and modified from published quality improvement protocols.Results:Organisational issues were identified in 68 of the 71 (96%) ethics consult notes across a range of patient settings and reasons for consultation. Thirteen of the 18 categories of organisational issues were identified and there was a median of two organisational issues per consult note. The most frequently identified organisational issues were informal organisational culture (eg, collective practices and approaches to situations with ethical dimensions that are not guided by policy), policies and procedures (eg, staff knows policy and/or procedural guidelines for an ethical concern but do not follow it) and communication (eg, communication about critical information, orders, or hand-offs repeatedly does not occur among services).Conclusions:Organisational issues contribute to ethical concerns that result in clinical ethics consults. Identifying and addressing organisational issues such as informal culture and communication may help decrease the recurrence of future similar ethics concerns.