bluuebunny/crossref_metadata_2025_split huggingface dataset:
Reconstitution of a Functional Duck Hepatitis B Virus Replication Initiation Complex from Separate Reverse Transcriptase Domains Expressed in
Escherichia coli
Authors: Beck Jürgen , Nassal Michael 
Published: July 2002

ABSTRACT

Hepatitis B viruses replicate through reverse transcription of an RNA intermediate, the pregenomic RNA (pgRNA). Replication is initiated de novo and requires formation of a ribonucleoprotein complex comprising the viral reverse transcriptase (P protein), an RNA stem-loop structure (ɛ) on the pgRNA, and cellular proteins, including the heat shock protein Hsp90, the cochaperone p23, and additional, as yet unknown, factors. Functional complexes catalyze the synthesis of a short DNA primer that is templated by ɛ and covalently linked to the terminal protein (TP) domain of P protein. Currently, the only system for generating such complexes in the test tube is in vitro translation of duck hepatitis B virus (DHBV) P protein in rabbit reticulocyte lysate (RRL), which also provides the necessary factors. However, its limited translation capacity precludes a closer analysis of the complex. To overcome this restriction we sought to produce larger amounts of DHBV P protein by expression in
Escherichia coli
, followed by complex reconstitution in RRL. Because previous attempts to generate full-length P protein in bacteria have failed we investigated whether separate expression of the TP and reverse transcriptase-RNase H (RT-RH) domains would allow higher yields and whether these domains could
trans
complement each other. Indeed, TP and, after minor C-terminal modifications, also RT-RH could be expressed in substantial amounts, and when added to RRL, they were capable of ɛ-dependent DNA primer synthesis, demonstrating posttranslational activation. This reconstitution system should pave the way for a detailed understanding of the unique hepadnaviral replication initiation mechanism.
Coxsackievirus A9 VP1 Mutants with Enhanced or Hindered A Particle Formation and Decreased Infectivity
Authors: Airaksinen Antero , Roivainen Merja , Hovi Tapani 
Published: July 2002

ABSTRACT
We have studied coxsackievirus A9 (CAV9) mutants that each have a single amino acid substitution in the conserved 29-PALTAVETGHT-39 motif of VP1 and a reduced capacity to produce infectious progeny virus. After uncoating, all steps in the infection cycle occurred according to the same kinetics as and similar efficiency to the wild-type virus. However, the particle/infectious unit ratio in the progeny was significantly increased. The differences were apparently due to altered stability of the capsid: there were mutant viruses with enhanced or hindered uncoating, and both of these characteristics were found to reduce fitness under standard passaging conditions. At 32°C the instable mutants had an advantage, while the wild-type and the most stable mutant grew poorly. When comparing the newly published CAV9 structure and the other enterovirus structures, we found that the PALTAVETGHT motif is always in exactly the same position, in a cavity formed by the 3 other capsid proteins, with the C terminus of VP4 between this motif and the RNA. In the 7 enterovirus structures determined to date, the most conserved residues of the studied motif have identical contacts to neighboring residues of VP2, VP3, and VP4. We conclude that (i) the mutations affect the uncoating step necessary for infection, resulting in an untimely or hindered externalization of the VP1 N terminus together with the VP4, and (ii) the reason for the studied motif being evolutionarily conserved is its role in maintaining an optimal balance between the protective stability and the functional flexibility of the capsid.
Host RNA Polymerase Requirements for Transcription of the Human Hepatitis Delta Virus Genome
Authors: Moraleda Gloria , Taylor John 
Published: July 2002

ABSTRACT
Replication of the genome of hepatitis delta virus (HDV) requires RNA-directed RNA synthesis using a host polymerase(s). This manuscript reviews the relevant published evidence. It also provides two new studies, both of which made use of transiently transfected Huh7 cells undergoing HDV RNA-directed RNA synthesis. For the first study, RNA transcription inhibitors were added to the transfected cells for periods of 1 to 2 days, after which assays of the effects on the accumulation of processed unit-length genomic HDV RNA were performed. For the second study, nuclei were isolated at 6 days after transfection, and then in vitro runoff transcription was used to assay the effects of RNA transcription inhibitors. Overall, the data support the interpretation that HDV transcription does not require host polymerase I or III (pol I or III) but at least primarily involves an enzyme resembling pol II.
Cytopathic Effects of Non-Syncytium-Inducing and Syncytium-Inducing Human Immunodeficiency Virus Type 1 Variants on Different CD4+-T-Cell Subsets Are Determined Only by Coreceptor Expression
Authors: Kwa David , Vingerhoed Jose , Boeser-Nunnink Brigitte , Broersen Silvia , Schuitemaker Hanneke 
Published: July 2002

ABSTRACTIn peripheral blood mononuclear cells, syncytium-inducing (SI) human immunodeficiency virus type 1 (HIV-1) infected and depleted all CD4+T cells, including naive T cells. Non-SI HIV-1 infected and depleted only the CCR5-expressing T-cell subset. This may explain the accelerated CD4 cell loss after SI conversion in vivo.
Sequences Adjacent to
oriP
Improve the Persistence of Epstein-Barr Virus-Based Episomes in B Cells
Authors: White Robert E. , Wade-Martins Richard , James Michael R. 
Published: July 2002

ABSTRACT

Epstein-Barr virus (EBV)
oriP
and the EBV nuclear antigen 1 (EBNA-1) protein allow persistence of EBV-based episomes. A nuclear matrix attachment region (MAR) spans
oriP
and the adjacent region of the EBV genome containing the EBV-expressed RNAs. Here, we show that episomes with the MAR are retained significantly more efficiently in EBV-positive B cells than episomes containing
oriP
alone.
RNA-Binding Activity of the Rotavirus Phosphoprotein NSP5 Includes Affinity for Double-Stranded RNA
Authors: Vende Patrice , Taraporewala Zenobia F. , Patton John T. 
Published: July 2002

ABSTRACT
Phosphoprotein NSP5 is a component of replication intermediates that catalyze the synthesis of the segmented double-stranded RNA (dsRNA) rotavirus genome. To study the role of the protein in viral replication, His-tagged NSP5 was expressed in bacteria and purified by affinity chromatography. In vitro phosphorylation assays showed that NSP5 alone contains minimal autokinase activity but undergoes hyperphosphorylation when combined with the NTPase and helix-destabilizing protein NSP2. Hence, NSP2 mediates the hyperphosphorylation of NSP5 in the absence of other viral or cellular proteins. RNA-binding assays demonstrated that NSP5 has unique nonspecific RNA-binding activity, recognizing single-stranded RNA and dsRNA with similar affinities. The possible functions of the RNA-binding activities of NSP5 are to cooperate with NSP2 in the destabilization of RNA secondary structures and in the packaging of RNA and/or to prevent the interferon-induced dsRNA-dependent activation of the protein kinase PKR.
P0 of Beet Western Yellows Virus Is a Suppressor of Posttranscriptional Gene Silencing
Authors: Pfeffer S. , Dunoyer P. , Heim F. , Richards K. E. , Jonard G. , Ziegler-Graff V. 
Published: July 2002

ABSTRACTHigher plants employ a homology-dependent RNA-degradation system known as posttranscriptional gene silencing (PTGS) as a defense against virus infection. Several plant viruses are known to encode proteins that can suppress PTGS. Here we show that P0 of beet western yellows virus (BWYV) displays strong silencing suppressor activity in a transient expression assay based upon its ability to inhibit PTGS of green fluorescent protein (GFP) when expressed in agro-infiltrated leaves ofNicotiana benthamianacontaining a GFP transgene. PTGS suppressor activity was also observed for the P0s of two other poleroviruses, cucurbit aphid-borne yellows virus and potato leafroll virus. P0 is encoded by the 5′-proximal gene in BWYV RNA but does not accumulate to detectable levels when expressed from the genome-length RNA during infection. The low accumulation of P0 and the resulting low PTGS suppressor activity are in part a consequence of the suboptimal translation initiation context of the P0 start codon in viral RNA, although other factors, probably related to the viral replication process, also play a role. A mutation to optimize the P0 translation initiation efficiency in BWYV RNA was not stable during virus multiplication in planta. Instead, the P0 initiation codon in the progeny was frequently replaced by a less efficient initiation codon such as ACG, GTG, or ATA, indicating that there is selection against overexpression of P0 from the viral genome.
Visna Virus-Induced Activation of MAPK Is Required for Virus Replication and Correlates with Virus-Induced Neuropathology
Authors: Barber Sheila A. , Bruett Linda , Douglass Brian R. , Herbst David S. , Zink M. Christine , Clements Janice E. 
Published: July 2002

ABSTRACTIt is well accepted that viruses require access to specific intracellular environments in order to proliferate or, minimally, to secure future proliferative potential as latent reservoirs. Hence, identification of essential virus-cell interactions should both refine current models of virus replication and proffer alternative targets for therapeutic intervention. In the present study, we examined the activation states of mitogen-activated protein kinases (MAPKs), ERK-1/2, in primary cells susceptible to visna virus and report that virus infection induces and sustains activation of the ERK/MAPK pathway. Treatment of infected cells with PD98059, a specific inhibitor of the ERK/MAPK pathway, abolishes visna virus replication, as evidenced by extremely low levels of Gag protein expression and reverse transcriptase activity in culture supernatants. In addition, although visna virus-induced activation of MAPK is detectable within 15 min, early events of viral replication (i.e., reverse transcription, integration, and transcription) are largely unaffected by PD98059. Interestingly, further examination demonstrated that treatment with PD98059 results in decreased cytoplasmic expression ofgagandenv, but notrev, mRNA, highly suggestive of an ERK/MAPK-dependent defect in Rev function. In vivo analysis of ERK-1/2 activation in brains derived from visna virus-infected sheep demonstrates a strong correlation between ERK/MAPK activation and virus-associated encephalitis. Moreover, double-labeling experiments revealed that activation of MAPK occurs not only in cells classically infected by visna virus (i.e., macrophages and microglia), but also in astrocytes, cells not considered to be major targets of visna virus replication, suggesting that activation of the ERK/MAPK pathway may contribute to the virus-induced processes leading to neurodegenerative pathology.
Murine Retroviral Pseudotype Virus Containing Hepatitis B Virus Large and Small Surface Antigens Confers Specific Tropism for Primary Human Hepatocytes: a Potential Liver-Specific Targeting System
Authors: Sung Vicky M.-H. , Lai Michael M. C. 
Published: July 2002

ABSTRACT
We have developed a system for producing murine leukemia virus (MLV) pseudotyped with human hepatitis B virus (HBV) large (L) and small (S) surface antigens (HBsAg) for targeting primary human hepatocytes. Using the MLV(HBV) pseudotype virus containing a β-galactosidase reporter gene, we demonstrated that this pseudotype virus exhibits strict tropism for primary human hepatocytes, similar to the natural target cell specificity of HBV. It does not infect any of the established tissue culture cell lines, including human hepatoma cell lines (HepG2 and Huh-7), or rat primary hepatocytes. The infectivity of MLV(HBV) for human hepatocytes was inhibited by anti-HBs antibody. The L form of HBsAg was both necessary and sufficient for virus infectivity, but the presence of both L and S forms enhanced the surface expression of HBsAg and thus increased virus production. The middle form of HBsAg was not necessary. This pseudotype virus bypasses the requirement for the liver-specific transcription factors for HBV replication, enabling direct study of HBV tissue tropism conferred by the viral envelope proteins. This virus also offers a potential liver-specific targeting system for gene therapy.
Rotavirus NSP5: Mapping Phosphorylation Sites and Kinase Activation and Viroplasm Localization Domains
Authors: Eichwald Catherine , Vascotto Fulvia , Fabbretti Elsa , Burrone Oscar R. 
Published: July 2002

ABSTRACT

Rotavirus NSP5 is a nonstructural protein that localizes in cytoplasmic viroplasms of infected cells. NSP5 interacts with NSP2 and undergoes a complex posttranslational hyperphosphorylation, generating species with reduced polyacrylamide gel electrophoresis mobility. This process has been suggested to be due in part to autophosphorylation. We developed an in vitro phosphorylation assay using as a substrate an in vitro-translated NSP5 deletion mutant that was phosphorylated by extracts from MA104 cells transfected with NSP5 mutants but not by extracts from mock-transfected cells. The phosphorylated products obtained showed shifts in mobility similar to what occurs in vivo. From these and other experiments we concluded that NSP5 activates a cellular kinase(s) for its own phosphorylation. Three NSP5 regions were found to be essential for kinase(s) activation. Glutathione
S-
transferase-NSP5 mutants were produced in
Escherichia coli
and used to determine phosphoacceptor sites. These were mapped to four serines (Ser
153
, Ser
155
, Ser
163
, and Ser
165
) within an acidic region with homology to casein kinase II (CKII) phosphorylation sites. CKII was able to phosphorylate NSP5 in vitro. NSP5 and its mutants fused to enhanced green fluorescent protein were used in transfection experiments followed by virus infection and allowed the determination of the domains essential for viroplasm localization in the context of virus infection.
Rolling Circle Replication of Hepatitis Delta Virus RNA Is Carried Out by Two Different Cellular RNA Polymerases
Authors: Macnaughton Thomas B. , Shi Stephanie T. , Modahl Lucy E. , Lai Michael M. C. 
Published: July 2002

ABSTRACT

Hepatitis delta virus (HDV) contains a viroid-like circular RNA that is presumed to replicate via a rolling circle replication mechanism mediated by cellular RNA polymerases. However, the exact mechanism of rolling circle replication for HDV RNA and viroids is not clear. Using our recently described cDNA-free transfection system (L. E. Modahl and M. M. Lai, J. Virol. 72:5449-5456, 1998), we have succeeded in detecting HDV RNA replication by metabolic labeling with [
32
P]orthophosphate in vivo and obtained direct evidence that HDV RNA replication generates high-molecular-weight multimeric species of HDV RNA, which are processed into monomeric and dimeric forms. Thus, these multimeric RNAs are the true intermediates of HDV RNA replication. We also found that HDV RNA synthesis is highly temperature sensitive, occurring most efficiently at 37 to 40°C and becoming virtually undetectable at temperatures below 30°C. Moreover, genomic HDV RNA synthesis was found to occur at a rate roughly 30-fold higher than that of antigenomic RNA synthesis. Finally, in lysolecithin-permeabilized cells, the synthesis of full-length antigenomic HDV RNA was completely resistant to high concentrations (100 μg/ml) of α-amanitin. In contrast, synthesis of genomic HDV RNA was totally inhibited by α-amanitin at concentrations as low as 2.5 μg/ml. Thus, these results suggest that genomic and antigenomic HDV RNA syntheses are performed by two different host cell enzymes. This observation, combined with our previous finding that hepatitis delta antigen mRNA synthesis is likely performed by RNA polymerase II, suggests that the different HDV RNA species are synthesized by different cellular transcriptional machineries.
Feedback Controlled Chemical Reactor with Feed of Two Reaction Components with Spectrophotometric Indication
Authors: Skopal František , Dušek Václav 
Published: July 2002

Theoretical relationships and simplifying conditions have been derived for the feed of two reaction components into a nonisochoric reactor with ideal stirring. The feed of reaction components is controlled by the negative feedback at a constant absorbance of the reaction mixture. The theoretical relationships have been verified using model 2. order oxidation-reduction reactions of Ce(IV)/V(IV) and Fe(III)/V(III) in 1 M sulfuric acid at 20 °C.
Exercise in preventing falls and fall related injuries in older people: a review of randomised controlled trials
Authors: Gardner Melinda M , Robertson M Clare , Campbell A John 
Published: July 2002

Objective—To assess the effectiveness of exercise programmes in preventing falls (and/or lowering the risk of falls and fall related injuries) in older people.Design—A review of controlled clinical trials designed with the aim of lowering the risk of falling and/or fall injuries through an exercise only intervention or an intervention that included an exercise componentMain outcome measures—Falls, fall related injuries, time between falls, costs, cost effectiveness.Subjects—A total of 4933 men and women aged 60 years and older.Results—Eleven trials meeting the criteria for inclusion were reviewed. Eight of these trials had separate exercise interventions, and three used interventions with an exercise programme component. Five trials showed a significant reduction in the rate of falls or the risk of falling in the intervention group.Conclusions—Exercise is effective in lowering falls risk in selected groups and should form part of falls prevention programmes. Lowering fall related injuries will reduce health care costs but there is little available information on the costs associated with programme replication or the cost effectiveness of exercise programmes aimed at preventing falls in older people.
Effect of body position on measurements of diffusion capacity after exercise
Authors: Stewart Ian B , Potts Jim E , McKenzie Don C , Coutts Ken D 
Published: July 2002

Background—Pulmonary diffusing capacity for carbon monoxide (Dlco), alveolar capillary membrane diffusing capacity (Dm), and pulmonary capillary blood volume (Vc) are all significantly reduced after exercise.Objective—To investigate whether measurement position affects this impaired gas transfer.Methods—Before and one, two, and four hours after incremental cycle ergometer exercise to fatigue, single breath Dlco, Dm, and Vc measurements were obtained in 10 healthy men in a randomly assigned supine and upright seated position.Results—After exercise, Dlco, Dm, and Vc were significantly depressed compared with baseline in both positions. The supine position produced significantly higher values over time for Dlco (5.22 (0.13)v4.66 (0.15) ml/min/mm Hg/l, p = 0.022) and Dm (6.78 (0.19)v6.03 (0.19) ml/min/mm Hg/l, p = 0.016), but there was no significant position effect for Vc. There was a similar pattern of change over time for Dlco, Dm, and Vc in the two positions.Conclusions—The change in Dlco after exercise appears to be primarily due to a decrease in Vc. Although the mechanism for the reduction in Vc cannot be determined from these data, passive relocation of blood to the periphery as the result of gravity can be discounted, suggesting that active vasoconstriction of the pulmonary vasculature and/or peripheral vasodilatation is occurring after exercise.
Interferon γ inhibits growth of human pancreatic carcinoma cells via caspase-1 dependent induction of apoptosis
Authors: Detjen K M , Farwig K , Welzel M , Wiedenmann B , Rosewicz S 
Published: July 2002

BACKGROUND AND AIMSThe poor prognosis of pancreatic cancer is partly due to resistance to a broad spectrum of apoptotic stimuli. To identify intact proapoptotic pathways of potential clinical relevance, we characterised the effects of interferon γ (IFN-γ) on growth and survival in human pancreatic cancer cells.METHODSIFN-γ receptor expression and signal transduction were examined by reverse transcriptase-polymerase chain reaction (RT-PCR), immunoprecipitation, western blot analysis, and transactivation assays. Effects on cell growth and survival were evaluated in terms of cell numbers, colony formation, cell cycle analysis, DNA fragmentation, and poly(ADP ribose) polymerase (PARP) cleavage.RESULTSAll four pancreatic cancer cell lines examined expressed functional IFN-γ receptors and downstream effectors, including the putative tumour suppressor interferon regulatory factor 1 (IRF-1). IFN-γ treatment profoundly inhibited anchorage dependent and independent growth of pancreatic cancer cells. Cell cycle analyses revealed subdiploid cells suggesting apoptosis, which was confirmed by demonstration of DNA fragmentation and PARP cleavage. Time and dose dependency of apoptosis induction and growth inhibition correlated closely, identifying apoptosis as the main, if not exclusive, mechanism responsible for growth inhibition. Apoptosis was preceded by upregulation of procaspase-1 and accompanied by proteolytic activation. Furthermore, the caspase inhibitor z-vad-fmk completely prevented IFN-γ mediated apoptosis.CONCLUSIONSThese results identify an intact proapoptotic pathway in pancreatic cancer cells and suggest that IRF-1 and/or procaspase-1 may represent potential therapeutic targets to be further explored.
Audiometric notch as a sign of noise induced hearing loss
Authors: McBride D I , Williams S 
Published: July 2002

OBJECTIVESTo investigate the relation between different types of exposure to noise and a classic sign of noise induced hearing loss (NIHL), the audiometric notch.METHODSThe study sample had exposure to both continuous and impulse noise and was drawn from a population of electrical transmission workers. Audiograms, taken as part of a hearing conservation programme, were read by three clinicians experienced in the assessment of NIHL. Working independently and using their clinical judgment, they were asked to identify localised increases in the threshold of hearing (audiometric notches) which they would attribute to noise, had a suitable history of exposure been elicited. Prevalent cases of NIHL were identified by the presence of a notch in either ear. Risk factors for NIHL were assessed by a questionnaire which sought information about exposure to air blast circuit breaker noise; firearms; explosions, and continuous noise. The odds of exposure to these factors in those with and without hearing loss were calculated, and odds ratios (ORs) and 95% confidence intervals (95% CIs) were estimated by logistic regression.RESULTSOf the 648 questionnaires sent out 357 were returned, a response rate of 55%. Of these, at least two out of the three assessors identified 175 (49%) people with a notch at any audiometric frequency. There was no association between these cases and the NIHL risk factors identified by the questionnaire, but a further frequency specific analysis showed a small proportion of people (15 (4%)) with notches at 4 kHz who had the expected associations with exposure to noise and a significant OR for firearms of 4.25 (95% CI 1.28 to 14.1). The much larger proportion of people with 6 kHz notches (110 (31%)) did not show these associations.CONCLUSIONSTo diagnose NIHL it is important to elicit a detailed and accurate history of exposure to noise: although the notch at 4 kHz is a well established clinical sign and may be valuable in confirming the diagnosis, the 6 kHz notch is variable and of limited importance.