bluuebunny/crossref_metadata_2025 huggingface dataset:
Blocking Double-Stranded RNA-Activated Protein Kinase PKR by Japanese Encephalitis Virus Nonstructural Protein 2A
Authors: Tu Yu-Chun , Yu Chia-Yi , Liang Jian-Jong , Lin Elong , Liao Ching-Len , Lin Yi-Ling
Published: July 2012

Japanese encephalitis virus (JEV) is an enveloped flavivirus with a single-stranded, positive-sense RNA genome encoding three structural and seven nonstructural proteins. To date, the role of JEV nonstructural protein 2A (NS2A) in the viral life cycle is largely unknown. The interferon (IFN)-induced double-stranded RNA (dsRNA)-activated protein kinase (PKR) phosphorylates the eukaryotic translation initiation factor 2α subunit (eIF2α) after sensing viral RNA and results in global translation arrest as an important host antiviral defense response. In this study, we found that JEV NS2A could antagonize PKR-mediated growth inhibition in a galactose-inducible PKR-expressing yeast system. In human cells, PKR activation, eIF2α phosphorylation, and the subsequent translational inhibition and cell death triggered by dsRNA and IFN-α were also repressed by JEV NS2A. Moreover, among the four eIF2α kinases, NS2A specifically blocked the eIF2α phosphorylation mediated by PKR and attenuated the PKR-promoted cell death induced by the chemotherapeutic drug doxorubicin. A single point mutation of NS2A residue 33 from Thr to Ile (T33I) abolished the anti-PKR potential of JEV NS2A. The recombinant JEV mutant carrying the NS2A-T33I mutation showed reduced
in vitro
growth and
in vivo
virulence phenotypes. Thus, JEV NS2A has a novel function in blocking the host antiviral response of PKR during JEV infection.
Mutations That Hamper Dimerization of Foot-and-Mouth Disease Virus 3A Protein Are Detrimental for Infectivity
Authors: González-Magaldi Mónica , Postigo Raúl , de la Torre Beatriz G. , Vieira Yuri A. , Rodríguez-Pulido Miguel , López-Viñas Eduardo , Gómez-Puertas Paulino , Andreu David , Kremer Leonor , Rosas María F. , Sobrino Francisco
Published: July 2012

Foot-and-mouth disease virus (FMDV) nonstructural protein 3A plays important roles in virus replication, virulence, and host range. In other picornaviruses, homodimerization of 3A has been shown to be relevant for its biological activity. In this work, FMDV 3A homodimerization was evidenced by an
in situ
protein fluorescent ligation assay. A molecular model of the FMDV 3A protein, derived from the nuclear magnetic resonance (NMR) structure of the poliovirus 3A protein, predicted a hydrophobic interface spanning residues 25 to 44 as the main determinant for 3A dimerization. Replacements L38E and L41E, involving charge acquisition at residues predicted to contribute to the hydrophobic interface, reduced the dimerization signal in the protein ligation assay and prevented the detection of dimer/multimer species in both transiently expressed 3A proteins and in synthetic peptides reproducing the N terminus of 3A. These replacements also led to production of infective viruses that replaced the acidic residues introduced (E) by nonpolar amino acids, indicating that preservation of the hydrophobic interface is essential for virus replication. Replacements that favored (Q44R) or impaired (Q44D) the polar interactions predicted between residues Q44 and D32 did not abolish dimer formation of transiently expressed 3A, indicating that these interactions are not critical for 3A dimerization. Nevertheless, while Q44R led to recovery of viruses that maintained the mutation, Q44D resulted in selection of infective viruses with substitution D44E with acidic charge but with structural features similar to those of the parental virus, suggesting that Q44 is involved in functions other than 3A dimerization.
An
ac34
Deletion Mutant of Autographa californica Nucleopolyhedrovirus Exhibits Delayed Late Gene Expression and a Lack of Virulence
In Vivo
Authors: Cai Yi , Long Zhao , Qiu Jianxiang , Yuan Meijin , Li Guanghong , Yang Kai
Published: July 2012

Ac34 and its homologs are highly conserved in all sequenced alphabaculoviruses. In this paper, we show that
ac34
transcripts were detected from 6 to 48 h postinfection (p.i.) in
Autographa californica nucleopolyhedrovirus
(AcMNPV)-infected Sf9 cells. Ac34 localized to both the cytoplasm and the nuclei of infected cells but was not a viral structural protein. To determine the function of
ac34
in the viral life cycle, an
ac34
knockout AcMNPV (vAc34KO) was constructed. Compared with wild-type and repair viruses, vAc34KO exhibited an approximately 100-fold reduction in infectious virus production. Further investigations showed that the
ac34
deletion did not affect the replication of viral DNA, polyhedron formation, or nucleocapsid assembly but delayed the expression of late genes, such as
vp39
,
38k
, and
p6.9
. Bioassays revealed that vAc34KO was unable to establish a fatal infection in
Trichoplusia ni
larvae via
per os
inoculation. Few infectious progeny viruses were detected in the hemolymph of the infected larvae, indicating that the replication of vAc34KO was attenuated. These results suggest that Ac34 is an activator protein that promotes late gene expression and is essential for the pathogenicity of AcMNPV.
Identification of Human Astrovirus Genome-Linked Protein (VPg) Essential for Virus Infectivity
Authors: Fuentes Cristina , Bosch Albert , Pintó Rosa M. , Guix Susana
Published: July 2012

Viral genome-linked proteins (VPgs) have been identified in several single-stranded positive-sense RNA virus families. The presence of such protein in the family
Astroviridae
has not been fully elucidated, although a putative VPg coding region in open reading frame 1a (ORF1a) of astrovirus with high amino acid sequence similarity to the VPg coding region of
Caliciviridae
has been previously identified. In this work we present several experimental findings that show that human astrovirus (HAstV) RNA encodes a VPg essential for viral infectivity: (i) RNase treatment of RNA purified from astrovirus-infected cells results in a single protein of 13 to 15 kDa, compatible with the predicted astrovirus VPg size; (ii) the antibody used to detect this 13- to 15-kDa protein is specifically directed against a region that includes the putative VPg coding region; (iii) the 13- to 15-kDa protein detected has been partially sequenced and the sequence obtained is contained in the computationally predicted VPg; (iv) the protein resulting from this putative VPg coding region is a highly disordered protein, resembling the VPg of sobemo-, calici- and potyviruses; (v) proteolytic treatment of the genomic RNA leads to loss of infectivity; and (vi) mutagenesis of Tyr-693 included in the putative VPg protein is lethal for HAstV replication, which strongly supports its functional role in the covalent link with the viral RNA.
A Multivalent Adsorption Apparatus Explains the Broad Host Range of Phage phi92: a Comprehensive Genomic and Structural Analysis
Authors: Schwarzer David , Buettner Falk F. R. , Browning Christopher , Nazarov Sergey , Rabsch Wolfgang , Bethe Andrea , Oberbeck Astrid , Bowman Valorie D. , Stummeyer Katharina , Mühlenhoff Martina , Leiman Petr G. , Gerardy-Schahn Rita
Published: July 2012

Bacteriophage phi92 is a large, lytic myovirus isolated in 1983 from pathogenic
Escherichia coli
strains that carry a polysialic acid capsule. Here we report the genome organization of phi92, the cryoelectron microscopy reconstruction of its virion, and the reinvestigation of its host specificity. The genome consists of a linear, double-stranded 148,612-bp DNA sequence containing 248 potential open reading frames and 11 putative tRNA genes. Orthologs were found for 130 of the predicted proteins. Most of the virion proteins showed significant sequence similarities to proteins of myoviruses rv5 and PVP-SE1, indicating that phi92 is a new member of the novel genus of rv5-like phages. Reinvestigation of phi92 host specificity showed that the host range is not limited to polysialic acid-encapsulated
Escherichia coli
but includes most laboratory strains of
Escherichia coli
and many
Salmonella
strains. Structure analysis of the phi92 virion demonstrated the presence of four different types of tail fibers and/or tailspikes, which enable the phage to use attachment sites on encapsulated and nonencapsulated bacteria. With this report, we provide the first detailed description of a multivalent, multispecies phage armed with a host cell adsorption apparatus resembling a nanosized Swiss army knife. The genome, structure, and, in particular, the organization of the baseplate of phi92 demonstrate how a bacteriophage can evolve into a multi-pathogen-killing agent.
Carbonyl J Acid Derivatives Block Protein Priming of Hepadnaviral P Protein and DNA-Dependent DNA Synthesis Activity of Hepadnaviral Nucleocapsids
Authors: Wang Yong-Xiang , Wen Yu-Mei , Nassal Michael
Published: July 2012

Current treatments for chronic hepatitis B are effective in only a fraction of patients. All approved directly antiviral agents are nucleos(t)ide analogs (NAs) that target the DNA polymerase activity of the hepatitis B virus (HBV) P protein; resistance and cross-resistance may limit their long-term applicability. P protein is an unusual reverse transcriptase that initiates reverse transcription by protein priming, by which a Tyr residue in the unique terminal protein domain acts as an acceptor of the first DNA nucleotide. Priming requires P protein binding to the ε stem-loop on the pregenomic RNA (pgRNA) template. This interaction also mediates pgRNA encapsidation and thus provides a particularly attractive target for intervention. Exploitingin vitropriming systems available for duck HBV (DHBV) but not HBV, we demonstrate that naphthylureas of the carbonyl J acid family, in particular KM-1, potently suppress protein priming by targeting P protein and interfering with the formation of P-DHBV ε initiation complexes. Quantitative evaluation revealed a significant increase in complex stability during maturation, yet even primed complexes remained sensitive to KM-1 concentrations below 10 μM. Furthermore, KM-1 inhibited the DNA-dependent DNA polymerase activity of both DHBV and HBV nucleocapsids, including from a lamivudine-resistant variant, directly demonstrating the sensitivity of human HBV to the compound. Activity against viral replication in cells was low, likely due to low intracellular availability. KM-1 is thus not yet a drug candidate, but its distinct mechanism of action suggests that it is a highly useful lead for developing improved, therapeutically applicable derivatives.
Hepatitis C Virus Infection Modulates Expression of Interferon Stimulatory Gene IFITM1 by Upregulating miR-130A
Authors: Bhanja Chowdhury Joydip , Shrivastava Shubham , Steele Robert , Di Bisceglie Adrian M. , Ray Ranjit , Ray Ratna B.
Published: July 2012

We have examined the underlying mechanism of hepatitis C virus (HCV)-mediated IFITM1 regulation. IFITM1 is a potential target of miR-130a. Our results demonstrated that miR-130a expression was significantly higher in HCV-infected hepatocytes and liver biopsy specimens than in controls. Introduction of anti-miR-130a in hepatocytes increased IFITM1 expression. Hepatocytes stably expressing IFITM1 reduced HCV replication. Together, these results suggested that HCV infection of hepatocytes upregulates miR-130a and that use of anti-miR-130a may have potential for restriction of HCV replication.
Herpes Simplex Virus Is Equipped with RNA- and Protein-Based Mechanisms To Repress Expression of ATRX, an Effector of Intrinsic Immunity
Authors: Jurak Igor , Silverstein Leah B. , Sharma Mayuri , Coen Donald M.
Published: July 2012

Intrinsic immunity is a first-line intracellular defense against virus infection, and viruses have evolved mechanisms to counteract it. During herpes simplex virus (HSV) infection, nuclear domain 10 (ND10) components localize adjacent to incoming viral genomes and generate a repressive environment for viral gene expression. Here, we found that the ND10 component, alpha-thalassemia/mental retardation syndrome X-linked (ATRX) protein, is predicted to be a target of HSV-1 miR-H1 and HSV-2 miR-H6. These microRNAs (miRNAs) share a seed sequence and are abundant during lytic infection. Mimics of both miRNAs could deplete endogenous ATRX, and an miR-H1 mimic could repress the expression of a reporter linked to the 3′ untranslated region of ATRX mRNA, identifying a cellular mRNA targeted by an HSV miRNA. Interestingly, ATRX protein and its mRNA were depleted in cells lytically infected with HSV, and ATRX protein was also depleted in cells infected with human cytomegalovirus. However, infection with an HSV-1 mutant lacking miR-H1 still resulted in ATRX depletion. This depletion was sensitive to a proteasome inhibitor and was largely ablated by a deletion of the gene encoding the immediate-early ICP0 protein. Additionally, a deletion of the gene encoding the tegument protein Vhs ablated most of the depletion of ATRX mRNA. Thus, HSV is equipped with multiple mechanisms to limit the expression of ATRX. As ATRX is implicated in repression of lytic viral gene expression, our results suggest roles for these different mechanisms during various phases of HSV infection.
Expression and Functional Characterization of the First Bacteriophage-Encoded Chaperonin
Authors: Kurochkina Lidia P. , Semenyuk Pavel I. , Orlov Victor N. , Robben Johan , Sykilinda Nina N. , Mesyanzhinov Vadim V.
Published: July 2012

Chaperonins promote protein folding
in vivo
and are ubiquitously found in bacteria, archaea, and eukaryotes. The first viral chaperonin GroEL ortholog, gene product 146 (gp146), whose gene was earlier identified in the genome of bacteriophage EL, has been shown to be synthesized during phage propagation in
Pseudomonas aeruginosa
cells. The recombinant gp146 has been expressed in
Escherichia coli
and characterized by different physicochemical methods for the first time. Using serum against the recombinant protein, gp146's native substrate, the phage endolysin gp188, has been immunoprecipitated from the lysate of EL-infected bacteria and identified by mass spectrometry.
In vitro
experiments have shown that gp146 has a protective effect against endolysin thermal inactivation and aggregation, providing evidence of its chaperonin function. The phage chaperonin has been found to have the architecture and some properties similar to those of GroEL but not to require cochaperonin for its functional activity.
Severe Acute Respiratory Syndrome Coronavirus Replication Is Severely Impaired by MG132 due to Proteasome-Independent Inhibition of M-Calpain
Authors: Schneider Martha , Ackermann Kerstin , Stuart Melissa , Wex Claudia , Protzer Ulrike , Schätzl Hermann M. , Gilch Sabine
Published: July 2012

The ubiquitin-proteasome system (UPS) is involved in the replication of a broad range of viruses. Since replication of the murine hepatitis virus (MHV) is impaired upon proteasomal inhibition, the relevance of the UPS for the replication of the severe acute respiratory syndrome coronavirus (SARS-CoV) was investigated in this study. We demonstrate that the proteasomal inhibitor MG132 strongly inhibits SARS-CoV replication by interfering with early steps of the viral life cycle. Surprisingly, other proteasomal inhibitors (e.g., lactacystin and bortezomib) only marginally affected viral replication, indicating that the effect of MG132 is independent of proteasomal impairment. Induction of autophagy by MG132 treatment was excluded from playing a role, and no changes in SARS-CoV titers were observed during infection of wild-type or autophagy-deficient ATG5
−/−
mouse embryonic fibroblasts overexpressing the human SARS-CoV receptor, angiotensin-converting enzyme 2 (ACE2). Since MG132 also inhibits the cysteine protease m-calpain, we addressed the role of calpains in the early SARS-CoV life cycle using calpain inhibitors III (MDL28170) and VI (SJA6017). In fact, m-calpain inhibition with MDL28170 resulted in an even more pronounced inhibition of SARS-CoV replication (>7 orders of magnitude) than did MG132. Additional m-calpain knockdown experiments confirmed the dependence of SARS-CoV replication on the activity of the cysteine protease m-calpain. Taken together, we provide strong experimental evidence that SARS-CoV has unique replication requirements which are independent of functional UPS or autophagy pathways compared to other coronaviruses. Additionally, this work highlights an important role for m-calpain during early steps of the SARS-CoV life cycle.
Differential HIV-1 Endocytosis and Susceptibility to Virus Infection in Human Macrophages Correlate with Cell Activation Status
Authors: Gobeil Lise-Andrée , Lodge Robert , Tremblay Michel J.
Published: July 2012

HIV-1 is an enveloped virus that enters target cells by fusion either directly at the plasma membrane or at the endosomal membrane. The latter mechanism follows a rapid engulfment of HIV-1 after its receptor engagement at the cell surface, and its scale depends on cellular endocytosis/degradation rates and virus fusion kinetics. HIV-1 has recently been shown to exploit a novel Pak1-dependent macropinocytosis mechanism as a way to productively infect macrophages. However, macrophages are highly heterogeneous cells that can adapt functionally to their changing environment, and their endosomal/lysosomal pathway is highly regulated upon cell activation. These changes might impact the ability of HIV-1 to exploit endocytosis as a way to productively infect macrophages. In this study, we compared HIV-1 endocytosis/degradation rates in nonactivated, M1-activated, and M2a-activated monocyte-derived macrophages (MDMs). We found that the rate of HIV-1 endocytosis was increased in M1-activated but decreased in M2a-activated MDMs. However, both M1 and M2a activations of MDMs led specifically to a greater clathrin-mediated endocytosis of HIV-1, which was independent of CD4 and CCR5 binding. Furthermore, clathrin-mediated endocytosis is unlikely to result in productive HIV-1 infection, given that it leads to increased viral degradation. Therefore, we suggest that viral fusion following endocytosis is restricted in activated macrophages.
Cell-Free Transmission of Human Adenovirus by Passive Mass Transfer in Cell Culture Simulated in a Computer Model
Authors: Yakimovich Artur , Gumpert Heidi , Burckhardt Christoph J. , Lütschg Verena A. , Jurgeit Andreas , Sbalzarini Ivo F. , Greber Urs F.
Published: July 2012

Viruses spread between cells, tissues, and organisms by cell-free and cell-cell transmissions. Both mechanisms enhance disease development, but it is difficult to distinguish between them. Here, we analyzed the transmission mode of human adenovirus (HAdV) in monolayers of epithelial cells by wet laboratory experimentation and a computer simulation. Using live-cell fluorescence microscopy and replication-competent HAdV2 expressing green fluorescent protein, we found that the spread of infection invariably occurred after cell lysis. It was affected by convection and blocked by neutralizing antibodies but was independent of second-round infections. If cells were overlaid with agarose, convection was blocked and round plaques developed around lytic infected cells. Infected cells that did not lyse did not give rise to plaques, highlighting the importance of cell-free transmission. Key parameters for cell-free virus transmission were the time from infection to lysis, the dose of free viruses determining infection probability, and the diffusion of single HAdV particles in aqueous medium. With these parameters, we developed an
in silico
model using multiscale hybrid dynamics, cellular automata, and particle strength exchange. This so-called white box model is based on experimentally determined parameters and reproduces viral infection spreading as a function of the local concentration of free viruses. These analyses imply that the extent of lytic infections can be determined by either direct plaque assays or can be predicted by calculations of virus diffusion constants and modeling.
NS1-Truncated Live Attenuated Virus Vaccine Provides Robust Protection to Aged Mice from Viral Challenge
Authors: Pica Natalie , Langlois Ryan A. , Krammer Florian , Margine Irina , Palese Peter
Published: July 2012

Immunological changes associated with age contribute to the high rates of influenza virus morbidity and mortality in the elderly. Compounding this problem, aged individuals do not respond to vaccination as well as younger, healthy adults. Efforts to increase protection to this demographic group are of utmost importance, as the proportion of the population above the age of 65 is projected to increase in the coming decade. Using a live influenza virus with a truncated nonstructural protein 1 (NS1), we are able to stimulate cellular and humoral immune responses of aged mice comparable to levels seen in young mice. Impressively, a single vaccination provided protection following stringent lethal challenge in aged mice.
Phosphorylation Regulates Binding of the Human Papillomavirus Type 8 E2 Protein to Host Chromosomes
Authors: Sekhar Vandana , McBride Alison A.
Published: July 2012

The papillomavirus E2 proteins are indispensable for the viral life cycle, and their functions are subject to tight regulation. The E2 proteins undergo posttranslational modifications that regulate their properties and roles in viral transcription, replication, and genome maintenance. During persistent infection, the E2 proteins from many papillomaviruses act as molecular bridges that tether the viral genomes to host chromosomes to retain them within the host nucleus and to partition them to daughter cells. The betapapillomavirus E2 proteins bind to pericentromeric regions of host mitotic chromosomes, including the ribosomal DNA loci. We recently reported that two residues (arginine 250 and serine 253) within the chromosome binding region of the human papillomavirus type 8 (HPV8) E2 protein are required for this binding. In this study, we show that serine 253 is phosphorylated, most likely by protein kinase A, and this modulates the interaction of the E2 protein with cellular chromatin. Furthermore, we show that this phosphorylation occurs in S phase, increases the half-life of the E2 protein, and promotes chromatin binding from S phase through mitosis.
Imiquimod Suppresses Propagation of Herpes Simplex Virus 1 by Upregulation of Cystatin A via the Adenosine Receptor A1Pathway
Authors: Kan Yuji , Okabayashi Tamaki , Yokota Shin-ichi , Yamamoto Soh , Fujii Nobuhiro , Yamashita Toshiharu
Published: July 2012

Imiquimod is recognized as an agonist for Toll-like receptor 7 (TLR7) in immunocompetent cells. TLR7, as well as TLR3 and TLR8, triggers the immune responses, such as the production of type I interferons (IFNs) and proinflammatory cytokines via recognition of viral nucleic acids in the infected cells. In this study, we proposed that imiquimod has an IFN-independent antiviral effect in nonimmune cells. Imiquimod, but not resiquimod, suppressed replication of human herpes simplex virus 1 (HSV-1) in FL cells. We analyzed alternation of gene expression by treatment with imiquimod using microarray analysis. Neither type I IFNs, nor TLRs, nor IFN-inducible antiviral genes were induced in imiquimod-treated FL cells. Cystatin A, a host cysteine protease inhibitor, was strongly upregulated by imiquimod and took a major part in the anti-HSV-1 activity deduced by the suppression experiment using its small interfering RNA. Upregulation of cystatin A was suggested to be mediated by antagonizing adenosine receptor A1and activating the protein kinase A pathway. Imiquimod, but not resiquimod, was shown to interact with adenosine receptor A1. Imiquimod-induced anti-HSV-1 activity was observed in other cells, such as HeLa, SiHa, and CaSki cells, in a manner consistent with the cystatin A induction by imiquimod. These results indicated that imiquimod acted as an antagonist for adenosine receptor A1and induced a host antiviral protein, cystatin A. The process occurred independently of TLR7 and type I IFNs.
Norovirus RNA Synthesis Is Modulated by an Interaction between the Viral RNA-Dependent RNA Polymerase and the Major Capsid Protein, VP1
Authors: Subba-Reddy Chennareddy V. , Yunus Muhammad Amir , Goodfellow Ian G. , Kao C. Cheng
Published: July 2012

Using a cell-based assay for RNA synthesis by the RNA-dependent RNA polymerase (RdRp) of noroviruses, we previously observed that VP1, the major structural protein of the human GII.4 norovirus, enhanced the GII.4 RdRp activity but not that of the related murine norovirus (MNV) or other unrelated RNA viruses (C. V. Subba-Reddy, I. Goodfellow, and C. C. Kao, J. Virol. 85:13027–13037, 2011). Here, we examine the mechanism of VP1 enhancement of RdRp activity and the mechanism of mouse norovirus replication. We determined that the GII.4 and MNV VP1 proteins can enhance cognate RdRp activities in a concentration-dependent manner. The VP1 proteins coimmunoprecipitated with their cognate RdRps. Coexpression of individual domains of VP1 with the viral RdRps showed that the VP1 shell domain (SD) was sufficient to enhance polymerase activity. Using SD chimeras from GII.4 and MNV, three loops connecting the central β-barrel structure were found to be responsible for the species-specific enhancement of RdRp activity. A differential scanning fluorimetry assay showed that recombinant SDs can bind to the purified RdRps
in vitro
. An MNV replicon with a frameshift mutation in open reading frame 2 (ORF2) that disrupts VP1 expression was defective for RNA replication, as quantified by luciferase reporter assay and real-time quantitative reverse transcription-PCR (qRT-PCR).
Trans
-complementation of VP1 or its SD significantly recovered the VP1 knockout MNV replicon replication, and the presence or absence of VP1 affected the kinetics of viral RNA synthesis. The results document a regulatory role for VP1 in the norovirus replication cycle, further highlighting the paradigm of viral structural proteins playing additional functional roles in the virus life cycle.
A Majority of Infectious Newcastle Disease Virus Particles Contain a Single Genome, while a Minority Contain Multiple Genomes
Authors: Goff Peter H. , Gao Qinshan , Palese Peter
Published: July 2012

Paramyxoviruses produce pleiomorphic particles containing variable amounts of genetic material that correlate with virion diameter by electron microscopy. However, the infectious nature of these particles is unknown, and functional genomes are indistinguishable from defective RNA. A quantitative approach to paramyxovirus packaging revealed a majority of infectious Newcastle disease viruses contain one functional genome. Virions encapsidating two or three genomes (approximately 25% of total) were also observed by utilizing three different recombinant viruses expressing unique fluorescent reporters.
Early Cytokine Elevation, PrPres Deposition, and Gliosis in Mouse Scrapie: No Effect on Disease by Deletion of Cytokine Genes IL-12p40 and IL-12p35
Authors: Tribouillard-Tanvier Déborah , Race Brent , Striebel James F. , Carroll James A. , Phillips Katie , Chesebro Bruce
Published: July 2012

Neurodegenerative diseases are typically associated with an activation of glia and an increased level of cytokines. In our previous studies of prion disease, the cytokine response in the brains of clinically sick scrapie-infected mice was restricted to a small group of cytokines, of which IL-12p40, CCL2, and CXCL10 were present at the highest levels. The goal of our current research was to determine the relationship between cytokine responses, gliosis, and neuropathology during prion disease. Here, in time course studies of C57BL/10 mice intracerebrally inoculated with 22L scrapie, abnormal protease-resistant prion protein (PrPres), astrogliosis, and microgliosis were first detected at 40 days after intracerebral scrapie inoculation. In cytokine studies, IL-12p40 was first elevated by 60 days; CCL3, IL-1β, and CXCL1 were elevated by 80 days; and CCL2 and CCL5 were elevated by 115 days. IL-12p40 showed the most extensive increase throughout disease and was 30-fold above control levels at the terminal stage. Because of the early onset and dramatic elevation of IL-12p40 during scrapie, we investigated whether IL-12p40 contributed to the development of prion disease neuropathogenesis by using three different scrapie strains (22L, RML, 79A) to infect knockout mice in which the gene encoding IL-12p40 was deleted. We also studied knockout mice lacking IL-12p35, which combines with IL-12p40 to form active IL-12 heterodimers. In all instances, knockout mice did not differ from control mice in survival time, clinical tempo, or levels of spongiosis, gliosis, or PrPres in the brain. Thus, neither IL-12p40 nor IL-12p35 molecules were required for prion disease-associated neurodegeneration or neuroinflammation.
Protective Capacity of Virus-Specific T Cell Receptor-Transduced CD8 T Cells
In Vivo
Authors: Mueller Katja , von Mässenhausen Anne , Aichele Ulrike , Stärck Lilian , Leisegang Matthias , Uckert Wolfgang , Pircher Hanspeter
Published: July 2012

The transfer of T cell receptor (TCR) genes by viral vectors represents a promising technique to generate antigen-specific T cells for adoptive immunotherapy. TCR-transduced T cells specific for infectious pathogens have been described, but their protective function
in vivo
has not yet been examined. Here, we demonstrate that CD8 T cells transduced with the P14 TCR specific for the gp33 epitope of lymphocytic choriomeningitis virus exhibit protective activities in both viral and bacterial infection models in mice.
HIV-1 Induces Telomerase Activity in Monocyte-Derived Macrophages, Possibly Safeguarding One of Its Reservoirs
Authors: Reynoso Rita , Wieser Matthias , Ojeda Diego , Bönisch Maximilian , Kühnel Harald , Bolcic Federico , Quendler Heribert , Grillari Johannes , Grillari-Voglauer Regina , Quarleri Jorge
Published: July 2012

Monocyte-derived macrophages (MDM) are widely distributed in all tissues and organs, including the central nervous system, where they represent the main part of HIV-infected cells. In contrast to activated CD4+T lymphocytes, MDM are resistant to cytopathic effects and survive HIV infection for a long period of time. The molecular mechanisms of how HIV is able to persist in macrophages are not fully elucidated yet. In this context, we have studied the effect ofin vitroHIV-1 infection on telomerase activity (TA), telomere length, and DNA damage. Infection resulted in a significant induction of TA. This increase was directly proportional to the efficacy of HIV infection and was found in both nuclear and cytoplasmic extracts, while neither UV light-inactivated HIV nor exogenous addition of the viral protein Tat or gp120 affected TA. Furthermore, TA was not modified during monocyte-macrophage differentiation, MDM activation, or infection with vaccinia virus. HIV infection did not affect telomere length. However, HIV-infected MDM showed less DNA damage after oxidative stress than noninfected MDM, and this resistance was also increased by overexpressing telomerase alone. Taken together, our results suggest that HIV induces TA in MDM and that this induction might contribute to cellular protection against oxidative stress, which could be considered a viral strategy to make macrophages better suited as longer-lived, more resistant viral reservoirs. In the light of the clinical development of telomerase inhibitors as anticancer therapeutics, inhibition of TA in HIV-infected macrophages might also represent a novel therapeutic target against viral reservoirs.
ORF45 of Kaposi's Sarcoma-Associated Herpesvirus Inhibits Phosphorylation of Interferon Regulatory Factor 7 by IKKε and TBK1 as an Alternative Substrate
Authors: Liang Qiming , Fu Bishi , Wu Fayi , Li Xiaojuan , Yuan Yan , Zhu Fanxiu
Published: July 2012

Open reading frame 45 (ORF45) of Kaposi's sarcoma-associated herpesvirus (KSHV) is an immediate-early and tegument protein that plays critical roles in antagonizing host antiviral responses. We have previously shown (Zhu et al, Proc. Natl. Acad. Sci. U. S. A., 99:5573–5578, 2002) that ORF45 suppresses activation of interferon regulatory factor 7 (IRF7), a crucial regulator of type I interferon gene expression, by blocking its virus-induced phosphorylation and nuclear accumulation. We report here further characterization of the mechanisms by which ORF45 inhibits IRF7 phosphorylation. In most cell types, IRF7 is phosphorylated and activated by IKKε and TBK1 after viral infection. We found that phosphorylation of IRF7 on Ser477 and Ser479 by IKKε or TBK1 is inhibited by ORF45. The inhibition is specific to IRF7 because phosphorylation of its close relative IRF3 is not affected by ORF45, implying that ORF45 does not inactivate the kinases directly. In fact, we found that ORF45 is phosphorylated efficiently on Ser41 and Ser162 by IKKε and TBK1. We demonstrated that ORF45 competes with the associated IRF7 and inhibits its phosphorylation by IKKε or TBK1 by acting as an alternative substrate.
Signatures of Host mRNA 5′ Terminus for Efficient Hantavirus Cap Snatching
Authors: Cheng Erdong , Mir Mohammad A.
Published: July 2012

Hantaviruses, similarly to other negative-strand segmented RNA viruses, initiate the synthesis of translation-competent capped mRNAs by a unique cap-snatching mechanism. Hantavirus nucleocapsid protein (N) binds to host mRNA caps and requires four nucleotides adjacent to the 5′ cap for high-affinity binding. N protects the 5′ caps of cellular transcripts from degradation by the cellular decapping machinery. The rescued 5′ capped mRNA fragments are stored in cellular P bodies by N, which are later efficiently used as primers by the hantaviral RNA-dependent RNA polymerase (RdRp) for transcription initiation. We showed that N also protects the host mRNA caps in P-body-deficient cells. However, the rescued caps were not effectively used by the hantavirus RdRp during transcription initiation, suggesting that caps stored in cellular P bodies by N are preferred for cap snatching. We examined the characteristics of the 5′ terminus of a capped test mRNA to delineate the minimum requirements for a capped transcript to serve as an efficient cap donor during hantavirus cap snatching. We showed that hantavirus RdRp preferentially snatches caps from the nonsense mRNAs compared to mRNAs engaged in translation. Hantavirus RdRp preferentially cleaves the cap donor mRNA at a G residue located 14 nucleotides downstream of the 5′ cap. The sequence complementarity between the 3′ terminus of viral genomic RNA and the nucleotides located in the vicinity of the cleavage site of the cap donor mRNA favors cap snatching. Our results show that hantavirus RdRp snatches caps from viral mRNAs. However, the negligible cap-donating efficiency of wild-type mRNAs in comparison to nonsense mRNAs suggests that viral mRNAs will not be efficiently used for cap snatching during viral infection due to their continuous engagement in protein synthesis. Our results suggest that efficiency of an mRNA to donate caps for viral mRNA synthesis is primarily regulated at the translational level.
Novel Cell Culture-Adapted Genotype 2a Hepatitis C Virus Infectious Clone
Authors: Date Tomoko , Kato Takanobu , Kato Junko , Takahashi Hitoshi , Morikawa Kenichi , Akazawa Daisuke , Murayama Asako , Tanaka-Kaneko Keiko , Sata Tetsutaro , Tanaka Yasuhito , Mizokami Masashi , Wakita Takaji
Published: July 2012

Although the recently developed infectious hepatitis C virus system that uses the JFH-1 clone enables the study of whole HCV viral life cycles, limited particular HCV strains have been available with the system. In this study, we isolated another genotype 2a HCV cDNA, the JFH-2 strain, from a patient with fulminant hepatitis. JFH-2 subgenomic replicons were constructed. HuH-7 cells transfected with
in vitro
transcribed replicon RNAs were cultured with G418, and selected colonies were isolated and expanded. From sequencing analysis of the replicon genome, several mutations were found. Some of the mutations enhanced JFH-2 replication; the 2217AS mutation in the NS5A interferon sensitivity-determining region exhibited the strongest adaptive effect. Interestingly, a full-length chimeric or wild-type JFH-2 genome with the adaptive mutation could replicate in Huh-7.5.1 cells and produce infectious virus after extensive passages of the virus genome-replicating cells. Virus infection efficiency was sufficient for autonomous virus propagation in cultured cells. Additional mutations were identified in the infectious virus genome. Interestingly, full-length viral RNA synthesized from the cDNA clone with these adaptive mutations was infectious for cultured cells. This approach may be applicable for the establishment of new infectious HCV clones.
The short Synacthen test: a questionnaire survey of current usage
Authors: Elder Charlotte Jane , Sachdev Pooja , Wright Neil Peter
Published: July 2012

Background

Supported by meta-analyses, the low-dose Synacthen test (LDST) has gained in popularity, with many believing it to be more sensitive than the supraphysiological standard (250 µg) short ST (SSST), particularly when assessing children prescribed high-dose inhaled corticosteroids (HDICS). However, consensus is lacking about its specific clinical application, what is considered ‘low dose’ and how that dose is made up.

Methods

To ascertain current use of the short Synacthen test (SST), a questionnaire was emailed to members of the British Society of Paediatric Endocrinology and Diabetes in the UK and Ireland (N=257), requesting a response from each department (N=92). A reminder was sent a month later to members of departments which had not responded.

Results

The authors received 39 replies, giving a response rate of 42%. All departments use the SST: 82% use an LDST, 87% use the SSST and 69% use both. The 1 µg dose was used by 44% of hospitals, with the other 56% using seven different doses based on age, weight and body surface area. There were 14 different methods of preparing the low dose test. Additionally, variations in the timings of cortisol sampling and the diagnostic cut-offs for adrenal insufficiency were found. Increased requests for SSTs in children with asthma prescribed HDICS were noted by 44% of respondents, with 67% reporting the detection of adrenal suppression in this group.

Conclusion

Standardisation of the SST is required to address the considerable variation in the methodology and application of this test in the UK and Ireland.
Long-term prophylaxis in hereditary angio-oedema: a systematic review
Authors: Costantino Giorgio , Casazza Giovanni , Bossi Ilaria , Duca Piergiorgio , Cicardi Marco
Published: July 2012

Objective

To systematically review the evidence regarding long-term prophylaxis in the prevention or reduction of attacks in hereditary angio-oedema (HAE).

Design

Systematic review and meta-analysis.

Data sources

Electronic databases were searched up to April 2011. Two reviewers selected the studies and extracted the study data, patient characteristics and outcomes of interest.

Eligibility criteria for selected studies

Controlled trials for HAE prophylaxis.

Results

7 studies were included, for a total of 73 patients and 587 HAE attacks. Due to the paucity of studies, a meta-analysis was not possible. Since two studies did not report the number of HAE attacks, five studies (52 patients) were finally included in the summary analysis. Four classes of drugs with at least one controlled trial have been proposed for HAE prophylaxis. All those drugs, except heparin, were found to be more effective than placebo. In the absence of direct comparisons, the relative efficacies of these drugs were determined by calculating a RR of attacks (drug vs placebo). The results were as follows: danazol (RR=0.023, 95% CI 0.003 to 0.162), methyltestosterone (RR=0.054, 95% CI 0.013 to 0.163), ɛ-aminocaproic acid (RR=0.095, 95% CI 0.025 to 0.356), tranexamic acid (RR=0.308, 95% CI 0.195 to 0.479) and C1-INH 0.491 (95% CI 0.395 to 0.607).

Conclusions

Few trials have evaluated the benefits of HAE prophylaxis, and all drugs but heparin seem to be effective in this setting. Since there are no direct comparisons of HAE drugs, it was not possible to draw definitive conclusions on the most effective one. Thus, to accumulate evidence for HAE prophylaxis, further studies are needed that consider the dose–efficacy relationship and include a head-to-head comparison between drugs, with the active group, rather than placebo, as the control.
Variation in physical development in schoolboy rugby players: can maturity testing reduce mismatch?
Authors: Nutton Richard W , Hamilton David F , Hutchison James D , Mitchell Martin J , Simpson A Hamish RW , MacLean James G B
Published: July 2012

Objectives

This study set out to pursue means of reducing mismatch in schoolboy rugby players. The primary objective was to determine whether application of previously reported thresholds of height and grip strength could be used to distinguish those 15-year-old boys appropriate to play under-18 school rugby from their peers. A secondary objective was to obtain normative data for height, weight and grip strength and to assess the variation within that data of current schoolboy rugby players.

Design

Cross-sectional cohort study.

Setting

3 Scottish schools and ‘Regional Assessment Centres’ organised by the Scottish Rugby Union.

Participants

472 rugby playing youths aged 15 years (Regional Assessment Centres) and 382 schoolboys aged between 12 and 18 years (three schools).

Outcome measures

Height, weight and grip strength.

Results

97% of 15-year-olds achieved the height and grip strength thresholds based on previous reported values. Larger mean values and wide variation of height, weight and grip strength were recorded in the schoolboy cohort. However, using the mean values of the cohort of 17-year-olds as a new threshold, only 7.7% of 15-year-olds would pass these thresholds.

Conclusions

Large morphological variation was observed in schoolboy rugby players of the same age. Physical maturity tests described in earlier literature as pre-participation screening for contact sports were not applicable to current day 15-year-old rugby players. New criteria were measured and found to be better at identifying those 15-year-old players who had sufficient physical development to play senior school rugby.
Continuous palliative sedation until death: practice after introduction of the Dutch national guideline
Authors: Swart Siebe J , van der Heide Agnes , Brinkkemper Tijn , van Zuylen Lia , Perez Roberto , Rietjens Judith
Published: July 2012

Background

In 2005, a national palliative guideline was launched in The Netherlands. The authors describe the practice of continuous palliative sedation until death (CPS) after the introduction of this guideline.

Methods

In 2008, a random sample of physicians (n=1580) were asked to fill out a questionnaire regarding the last patient in whom they had provided CPS until death.

Results

The response was 38%. In all, 82% of the respondents were aware of the existence of the national guideline. Dyspnoea, pain and physical exhaustion were most often mentioned as decisive indications for continuous sedation. The decision to use sedation was discussed with all competent patients, but in 18% this merely involved informing the patient. Life expectancy at the start of continuous sedation was estimated to be less than 2 weeks in 97% of the cases. In 14%, the physicians had felt pressure to start the sedation, predominantly from patients and relatives. Physicians were present at the start of the sedation in 81% of the cases. Midazolam was used to induce the sedation in 92%. Overall, 41% of the physicians estimated that continuous sedation had hastened death to some extent. Most physicians thought that patients' complaints were adequately relieved by continuous sedation, that relatives were satisfied and that a good quality of dying was achieved.

Interpretation

Continuous palliative sedation practice in The Netherlands largely reflects the recommendations from the national guideline. Issues needing further attention are the pressure felt by physicians to start continuous sedation and the potential life-shortening effect as mentioned by the physicians.
Spiritual care: how to do it
Authors: Sinclair Shane , Bouchal Shelley Raffin , Chochinov Harvey , Hagen Neil , McClement Susan
Published: July 2012

Objective

This study explores the provision of spiritual care by healthcare professionals working at the end of life.

Design

Qualitative–ethnographic inquiry.

Setting

Phase 1: five Canadian sites; phase 2: a residential hospice in Alberta, Canada.

Participants

Phase 1: six palliative care leaders; phase 2: 24 frontline palliative care clinicians.

Results

Data were collected over a 12-month period with analysis of findings occurring concurrently. Using semistructured interviews and participant observation, 11 themes, organised under five overarching categories, emerged from the data. Five bedside skills were identified as essential to spiritual care: hearing, sight, speech, touch and presence. The integration of these bedside skills with the intrinsic qualities of healthcare professionals, including their values and spiritual beliefs, appeared to be essential to their application in spiritual care. Spiritual care primarily involved the tacit qualities of healthcare professionals and their effect on patient's spiritual well-being, rather than their explicit technical skill set or expert knowledge base.

Conclusion

Participants identified spiritual care as both a specialised care domain and as a philosophy of care that informs and is embedded within physical and psychosocial care. Hearing, sight, speech, touch and presence were identified as the means by which healthcare professionals impacted patients' spiritual well-being regardless of clinician's awareness or intent. An empirical framework is presented providing clinicians with a pragmatic way of incorporating spiritual care into clinical practice.
Associations between childhood obesity and upper and lower extremity injuries
Authors: Adams Annette L , Kessler Jeffrey I , Deramerian Krikor , Smith Ning , Black Mary Helen , Porter Amy H , Jacobsen Steven J , Koebnick Corinna
Published: July 2012

Objectives

To estimate the overall and age-specific associations between obesity and extremity musculoskeletal injuries and pain in children.

Methods

This cross-sectional study used information from electronic medical records of 913 178 patients aged 2–19 years enrolled in an integrated health plan in the period 2007–2009. Children were classified as underweight, normal weight, overweight, or moderately/extremely obese and, using multivariable logistic regression methods, the associations between weight class and diagnosis of upper or lower extremity fractures, sprains, dislocations and pain were calculated.

Results

Overweight (OR 1.18, 95% CI 1.15 to 1.20), moderately obese (OR 1.24, 95% CI 1.20 to 1.27) and extremely obese (OR 1.34, 95% CI 1.30 to 1.39) children had statistically significantly higher odds of lower extremity injuries/pain compared to normal weight, adjusted for sex, age, race/ethnicity and insurance status. Age-stratified analyses yielded similar results. No consistent association was observed between body mass index and injuries/pain of the upper extremities.

Conclusions

Greater body mass index is associated with increased odds of lower extremity injuries and pain issues. Because the benefits of physical activity may still outweigh the risk of injury, attention should be paid to injury prevention strategies for these children at greater risk for lower extremity injuries.