bluuebunny/crossref_metadata_2025_split huggingface dataset:
Hepatitis Delta Antigen Requires a Flexible Quasi-Double-Stranded RNA Structure To Bind and Condense Hepatitis Delta Virus RNA in a Ribonucleoprotein Complex
Authors: Griffin Brittany L. , Chasovskikh Sergey , Dritschilo Anatoly , Casey John L. 
Published: April 2014

ABSTRACT

The circular genome and antigenome RNAs of hepatitis delta virus (HDV) form characteristic unbranched, quasi-double-stranded RNA secondary structures in which short double-stranded helical segments are interspersed with internal loops and bulges. The ribonucleoprotein complexes (RNPs) formed by these RNAs with the virus-encoded protein hepatitis delta antigen (HDAg) perform essential roles in the viral life cycle, including viral replication and virion formation. Little is understood about the formation and structure of these complexes and how they function in these key processes. Here, the specific RNA features required for HDAg binding and the topology of the complexes formed were investigated. Selective 2′OH acylation analyzed by primer extension (SHAPE) applied to free and HDAg-bound HDV RNAs indicated that the characteristic secondary structure of the RNA is preserved when bound to HDAg. Notably, the analysis indicated that predicted unpaired positions in the RNA remained dynamic in the RNP. Analysis of the
in vitro
binding activity of RNAs in which internal loops and bulges were mutated and of synthetically designed RNAs demonstrated that the distinctive secondary structure, not the primary RNA sequence, is the major determinant of HDAg RNA binding specificity. Atomic force microscopy analysis of RNPs formed
in vitro
revealed complexes in which the HDV RNA is substantially condensed by bending or wrapping. Our results support a model in which the internal loops and bulges in HDV RNA contribute flexibility to the quasi-double-stranded structure that allows RNA bending and condensing by HDAg.

IMPORTANCE
RNA-protein complexes (RNPs) formed by the hepatitis delta virus RNAs and protein, HDAg, perform critical roles in virus replication. Neither the structures of these RNPs nor the RNA features required to form them have been characterized. HDV RNA is unusual in that it forms an unbranched quasi-double-stranded structure in which short base-paired segments are interspersed with internal loops and bulges. We analyzed the role of the HDV RNA sequence and secondary structure in the formation of a minimal RNP and visualized the structure of this RNP using atomic force microscopy. Our results indicate that HDAg does not recognize the primary sequence of the RNA; rather, the principle contribution of unpaired bases in HDV RNA to HDAg binding is to allow flexibility in the unbranched quasi-double-stranded RNA structure. Visualization of RNPs by atomic force microscopy indicated that the RNA is significantly bent or condensed in the complex.
Cellular Transcriptome Analysis Reveals Differential Expression of Pro- and Antiapoptosis Genes by Varicella-Zoster Virus-Infected Neurons and Fibroblasts
Authors: Markus Amos , Waldman Ben-Asher Hiba , Kinchington Paul R. , Goldstein Ronald S. 
Published: April 2014

ABSTRACT
Transcriptional changes following varicella-zoster virus (VZV) infection of cultured human neurons derived from embryonic stem cells were compared to those in VZV-infected human foreskin fibroblasts. Transcription of 340 neuronal genes significantly altered by VZV infection included 223 transcript changes unique to neurons. Strikingly, genes inhibiting apoptosis were upregulated in neurons, while proapoptotic gene transcription was increased in fibroblasts. These data are a basis for discovery of differences in virus-host interactions between these VZV targets.
NS2 Proteins of GB Virus B and Hepatitis C Virus Share Common Protease Activities and Membrane Topologies
Authors: Boukadida Célia , Marnata Caroline , Montserret Roland , Cohen Lisette , Blumen Brigitte , Gouttenoire Jérôme , Moradpour Darius , Penin François , Martin Annette 
Published: April 2014

ABSTRACT

GB virus B (GBV-B), which is hepatotropic in experimentally infected small New World primates, is a member of the
Hepacivirus
genus but phylogenetically relatively distant from hepatitis C virus (HCV). To gain insights into the role and specificity of hepaciviral nonstructural protein 2 (NS2), which is required for HCV polyprotein processing and particle morphogenesis, we investigated whether NS2 structural and functional features are conserved between HCV and GBV-B. We found that GBV-B NS2, like HCV NS2, has cysteine protease activity responsible for cleavage at the NS2/NS3 junction, and we experimentally confirmed the location of this junction within the viral polyprotein. A model for GBV-B NS2 membrane topology was experimentally established by determining the membrane association properties of NS2 segments fused to green fluorescent protein (GFP) and their nuclear magnetic resonance structures using synthetic peptides as well as by applying an N-glycosylation scanning approach. Similar glycosylation studies confirmed the HCV NS2 organization. Together, our data show that despite limited amino acid sequence similarity, GBV-B and HCV NS2 proteins share a membrane topology with 3 N-terminal transmembrane segments, which is also predicted to apply to other recently discovered hepaciviruses. Based on these data and using
trans
-complementation systems, we found that intragenotypic hybrid NS2 proteins with heterologous N-terminal membrane segments were able to efficiently
trans
-complement an assembly-deficient HCV mutant with a point mutation in the NS2 C-terminal domain, while GBV-B/HCV or intergenotypic NS2 chimeras were not. These studies indicate that virus- and genotype-specific intramolecular interactions between N- and C-terminal domains of NS2 are critically involved in HCV morphogenesis.

IMPORTANCE
Nonstructural protein 2 (NS2) of hepatitis C virus (HCV) is a multifunctional protein critically involved in polyprotein processing and virion morphogenesis. To gain insights into NS2 mechanisms of action, we investigated whether NS2 structural and functional features are conserved between HCV and GB virus B (GBV-B), a phylogenetically relatively distant primate hepacivirus. We showed that GBV-B NS2, like HCV NS2, carries cysteine protease activity. We experimentally established a model for GBV-B NS2 membrane topology and demonstrated that despite limited sequence similarity, GBV-B and HCV NS2 share an organization with three N-terminal transmembrane segments. We found that the role of HCV NS2 in particle assembly is genotype specific and relies on critical interactions between its N- and C-terminal domains. This first comparative analysis of NS2 proteins from two hepaciviruses and our structural predictions of NS2 from other newly identified mammal hepaciviruses highlight conserved key features of the hepaciviral life cycle.
Prolonged Activity of the Pestiviral RNase E
rns
as an Interferon Antagonist after Uptake by Clathrin-Mediated Endocytosis
Authors: Zürcher Christoph , Sauter Kay-Sara , Mathys Veronika , Wyss Fabienne , Schweizer Matthias 
Published: April 2014

ABSTRACT

The RNase activity of the envelope glycoprotein E
rns
of the pestivirus bovine viral diarrhea virus (BVDV) is required to block type I interferon (IFN) synthesis induced by single-stranded RNA (ssRNA) and double-stranded RNA (dsRNA) in bovine cells. Due to the presence of an unusual membrane anchor at its C terminus, a significant portion of E
rns
is also secreted. In addition, a binding site for cell surface glycosaminoglycans is located within the C-terminal region of E
rns
. Here, we show that the activity of soluble E
rns
as an IFN antagonist is not restricted to bovine cells. Extracellularly applied E
rns
protein bound to cell surface glycosaminoglycans and was internalized into the cells within 1 h of incubation by an energy-dependent mechanism that could be blocked by inhibitors of clathrin-dependent endocytosis. E
rns
mutants that lacked the C-terminal membrane anchor retained RNase activity but lost most of their intracellular activity as an IFN antagonist. Surprisingly, once taken up into the cells, E
rns
remained active and blocked dsRNA-induced IFN synthesis for several days. Thus, we propose that E
rns
acts as an enzymatically active decoy receptor that degrades extracellularly added viral RNA mainly in endolysosomal compartments that might otherwise activate intracellular pattern recognition receptors (PRRs) in order to maintain a state of innate immunotolerance.

IMPORTANCE
The pestiviral RNase E
rns
was previously shown to inhibit viral ssRNA- and dsRNA-induced interferon (IFN) synthesis. However, the localization of E
rns
at or inside the cells, its species specificity, and its mechanism of interaction with cell membranes in order to block the host's innate immune response are still largely unknown. Here, we provide strong evidence that the pestiviral RNase E
rns
is taken up within minutes by clathrin-mediated endocytosis and that this uptake is mostly dependent on the glycosaminoglycan binding site located within the C-terminal end of the protein. Remarkably, the inhibitory activity of E
rns
remains for several days, indicating the very potent and prolonged effect of a viral IFN antagonist. This novel mechanism of an enzymatically active decoy receptor that degrades a major viral pathogen-associated molecular pattern (PAMP) might be required to efficiently maintain innate and, thus, also adaptive immunotolerance, and it might well be relevant beyond the bovine species.
Vaginal Myeloid Dendritic Cells Transmit Founder HIV-1
Authors: Shen Ruizhong , Kappes John C. , Smythies Lesley E. , Richter Holly E. , Novak Lea , Smith Phillip D. 
Published: April 2014

ABSTRACT

We report that primary human vaginal dendritic cells (DCs) display a myeloid phenotype and express CD4, CCR5, and CXCR4. Vaginal CD13
+
CD11c
+
DCs rapidly and efficiently bound transmitted/founder (T/F) CCR5-tropic (R5) viruses, transported them through explanted vaginal mucosa, and transmitted them in
trans
to vaginal and blood lymphocytes. Vaginal myeloid DCs may play a key role in capturing and disseminating T/F R5 HIV-1
in vivo
and are candidate “gatekeeper” cells in HIV-1 transmission.
CTCF Binding to the First Intron of the Major Immediate Early (MIE) Gene of Human Cytomegalovirus (HCMV) Negatively Regulates MIE Gene Expression and HCMV Replication
Authors: Martínez Francisco Puerta , Cruz Ruth , Lu Fang , Plasschaert Robert , Deng Zhong , Rivera-Molina Yisel A. , Bartolomei Marisa S. , Lieberman Paul M. , Tang Qiyi 
Published: April 2014

ABSTRACT
Human cytomegalovirus (HCMV) gene expression during infection is highly regulated, with sequential expression of immediate-early (IE), early (E), and late (L) gene transcripts. To explore the potential role of chromatin regulatory factors that may regulate HCMV gene expression and DNA replication, we investigated the interaction of HCMV with the cellular chromatin-organizing factor CTCF. Here, we show that HCMV-infected cells produce higher levels of CTCF mRNA and protein at early stages of infection. We also show that CTCF depletion by short hairpin RNA results in an increase in major IE (MIE) and E gene expression and an about 50-fold increase in HCMV particle production. We identified a DNA sequence (TTAACGGTGGAGGGCAGTGT) in the first intron (intron A) of the MIE gene that interacts directly with CTCF. Deletion of this CTCF-binding site led to an increase in MIE gene expression in both transient-transfection and infection assays. Deletion of the CTCF-binding site in the HCMV bacterial artificial chromosome plasmid genome resulted in an about 10-fold increase in the rate of viral replication relative to either wild-type or revertant HCMV. The CTCF-binding site deletion had no detectable effect on MIE gene-splicing regulation, nor did CTCF knockdown or overexpression of CTCF alter the ratio of IE1 to IE2. Therefore, CTCF binds to DNA within the MIE gene at the position of the first intron to affect RNA polymerase II function during the early stages of viral transcription. Finally, the CTCF-binding sequence in CMV is evolutionarily conserved, as a similar sequence in murine CMV (MCMV) intron A was found to interact with CTCF and similarly function in the repression of MCMV MIE gene expression mediated by CTCF.

IMPORTANCE
Our findings that CTCF binds to intron A of the cytomegalovirus (CMV) major immediate-early (MIE) gene and functions to repress MIE gene expression and viral replication are highly significant. For the first time, a chromatin-organizing factor, CTCF, has been found to facilitate human CMV gene expression, which affects viral replication. We also identified a CTCF-binding motif in the first intron (also called intron A) that directly binds to CTCF and is required for CTCF to repress MIE gene expression. Finally, we show that the CTCF-binding motif is conserved in CMV because a similar DNA sequence was found in murine CMV (MCMV) that is required for CTCF to bind to MCMV MIE gene to repress MCMV MIE gene expression.
Determinants of Moloney Murine Leukemia Virus Gag-Pol and Genomic RNA Proportions
Authors: Johnson Silas F. , Collins John T. , D'Souza Victoria M. , Telesnitsky Alice 
Published: April 2014

ABSTRACT
The Moloney murine leukemia virus (MoMLV) ribonucleoprotein complex is composed of an approximately 20:1 mixture of Gag and Gag-Pol polyproteins plus a single genomic RNA (gRNA) dimer. The mechanisms that regulate these proportions are unknown. Here, we examined whether virion proportions of Gag, Gag-Pol, and gRNA were determined by sampling (that is, if they reflected expression ratios or intracellular concentrations) or more specific recruitment. To this end, MoMLV Gag, Gag-Pol, and gRNA were expressed separately or together in various ratios. Varying the expression ratios of Gag and Gag-Pol revealed that Gag-Pol incorporation was stochastic and that the conserved 20:1 Gag/Gag-Pol ratio coincided with maximal particle production. When skewed expression ratios resulted in excess Gag-Pol, the released virions maintained the intracellular Gag/Gag-Pol ratios and the infectivity per virion was largely maintained, but virion production decreased sharply with high levels of Gag-Pol. The determinants of gRNA proportions were addressed by manipulating the amounts and contexts of functional nucleocapsid (NC) and the ratios of Gag to gRNA. The results showed that the NC domain of either Gag or Gag-Pol could provide gRNA packaging functions equally well. Unlike Gag-Pol, gRNA incorporation was saturable. An upper limit of gRNA incorporation was observed, and particle production was not disrupted by excess gRNA expression. These results indicate that the determinants of Gag/Gag-Pol proportions differ from those for Gag/gRNA. On the basis of the assumption that MoMLV evolved to produce virion components in optimal proportions, these data provide a means of estimating the proportion of unspliced MoMLV RNA that serves as genomic RNA.

IMPORTANCE
Viruses assemble their progeny from within the cells that they parasitize, where they must sort through a rich milieu of host proteins and nucleic acids to gather together their own building blocks, which are also proteins and nucleic acids. The research described here addresses whether or not the proportions of viral proteins and nucleic acids that are brought together to form a retroviral particle are determined by random sampling from the cell—and thus dictated by the components' availabilities within the cell—or if the amounts of each molecule are specified by the virus replication process. The results indicated that protein components of the murine retrovirus studied here are recruited by chance but that a specific counting mechanism defines the amount of nucleic acid incorporated into each progeny virion.
Mechanism and Significance of Cell Type-Dependent Neutralization of Flaviviruses
Authors: Mukherjee Swati , Dowd Kimberly A. , Manhart Carolyn J. , Ledgerwood Julie E. , Durbin Anna P. , Whitehead Stephen S. , Pierson Theodore C. 
Published: April 2014

ABSTRACT
The production of neutralizing antibodies (NAbs) is a correlate of protection for many human vaccines, including currently licensed vaccines against flaviviruses. NAbs are typically measured using a plaque reduction neutralization test (PRNT). Despite its extensive use, parameters that impact the performance of the PRNT have not been investigated from a mechanistic perspective. The results of a recent phase IIb clinical trial of a tetravalent dengue virus (DENV) vaccine suggest that NAbs, as measured using a PRNT performed with Vero cells, do not correlate with protection. This surprising finding highlights the importance of understanding how well the PRNT captures the complexity of the NAb response to DENV. In this study, we demonstrated that the structural heterogeneity of flaviviruses arising from inefficient virion maturation impacts the results of neutralization assays in a cell type-dependent manner. Neutralization titers of several monoclonal antibodies were significantly reduced when assayed on Vero cells compared to Raji cells expressing DC-SIGNR. This pattern can be explained by differences in the efficiency with which partially mature flaviviruses attach to each cell type, rather than a differential capacity of antibody to block infection. Vero cells are poorly permissive to the fraction of virions that are most sensitive to neutralization. Analysis of sera from recipients of live-attenuated monovalent DENV vaccine candidates revealed a strong correlation between the sensitivity of serum antibodies to the maturation state of DENV and cell type-dependent patterns of neutralization. Cross-reactive patterns of neutralization may be underrepresented by the “gold-standard” PRNT that employs Vero cells.

IMPORTANCE
Cell type-dependent patterns of neutralization describe a differential capacity of antibodies to inhibit virus infection when assayed on multiple cellular substrates. In this study, we established a link between antibodies that neutralize infection in a cell type-dependent fashion and those sensitive to the maturation state of the flavivirus virion. We demonstrated that cell type-dependent neutralization reflects a differential capacity to measure neutralization of viruses that are incompletely mature. Partially mature virions that most efficiently bind maturation state-sensitive antibodies are poorly represented by assays typically used in support of flavivirus vaccine development. The selection of cellular substrate for neutralization assays may significantly impact evaluation of the neutralization potency of the polyclonal response. These data suggest that current assays do not adequately capture the full complexity of the neutralizing antibody response and may hinder the identification of correlates of protection following flavivirus vaccination.
Transcription Factor Binding Site Mapping Using ChIP-Seq
Authors: Jaini Suma , Lyubetskaya Anna , Gomes Antonio , Peterson Matthew , Tae Park Sang , Raman Sahadevan , Schoolnik Gary , Galagan James 
Published: April 2014

ABSTRACT

Transcription factors (TFs) play a central role in regulating gene expression in all bacteria. Yet until recently, studies of TF binding were limited to a small number of factors at a few genomic locations. Chromatin immunoprecipitation followed by sequencing (ChIP-Seq) provides the ability to map binding sites globally for TFs, and the scalability of the technology enables the ability to map binding sites for every DNA binding protein in a prokaryotic organism. We have developed a protocol for ChIP-Seq tailored for use with mycobacteria and an analysis pipeline for processing the resulting data. The protocol and pipeline have been used to map over 100 TFs from
Mycobacterium tuberculosis
, as well as numerous TFs from related mycobacteria and other bacteria. The resulting data provide evidence that the long-accepted spatial relationship between TF binding site, promoter motif, and the corresponding regulated gene may be too simple a paradigm, failing to adequately capture the variety of TF binding sites found in prokaryotes. In this article we describe the protocol and analysis pipeline, the validation of these methods, and the results of applying these methods to
M. tuberculosis
.
Amyotrophic lateral sclerosis and parkinsonism in Papua, Indonesia: 2001–2012 survey results
Authors: Okumiya Kiyohito , Wada Taizo , Fujisawa Michiko , Ishine Masayuki , Garcia del Saz Eva , Hirata Yutaka , Kuzuhara Shigeki , Kokubo Yasumasa , Seguchi Harumichi , Sakamoto Ryota , Manuaba Indrajaya , Watofa Paulina , Rantetampang Andreas L , Matsubayashi Kozo 
Published: April 2014

ObjectiveOnly one previous follow-up study of amyotrophic lateral sclerosis (ALS) and parkinsonism in Papua, Indonesia has been carried out since a survey undertaken in 1962–1981 by Gajdusek and colleagues. Therefore, to clarify the clinical epidemiology of ALS and parkinsonism in the southern coastal region of Papua, the clinical characteristics and prevalence of the diseases in this region were examined and assessed.MethodsCases of ALS and parkinsonism were clinically examined during a 2001–2012 survey in Bade and other villages along the Ia, Edera, Dumut and Obaa rivers in Papua, Indonesia. Possible, probable and definite ALS was diagnosed clinically by certified neurologists based on El Escorial criteria. The criteria for a diagnosis of parkinsonism were the presence of at least two of the four following signs: tremor, rigidity, bradykinesia and postural impairment with a progressive course.ResultsDuring the survey, 46 cases of ALS and/or parkinsonism were diagnosed within a population range of 7000 (2001–2002) to 13 900 (2007–2012). The 46 cases consisted of 17 probable-definite cases of ALS, including three with cognitive impairment (CI), 13 cases of overlapping possible, probable or definite ALS and parkinsonism, including five with CI, and 16 cases of parkinsonism, including one with CI. The crude point prevalence rate of pure ALS was estimated to be at least 73 (95% CI 0 to 156) to 133 (27 to 240)/100 000 people and that of overlapping ALS and parkinsonism at least 53 (0 to 126) to 98 (2 to 193)/100 000 in 2007, or 2010 in some regions.ConclusionsWhile the prevalence of ALS in Papua has decreased over the past ∼30–35 years, it remains higher than the global average. There was a high prevalence of overlapping ALS, parkinsonism and CI, which has also been previously reported in Guam and Kii.
Mapping of Crohn's disease outcomes to faecal calprotectin levels in patients maintained on biologic therapy
Authors: Turvill James 
Published: April 2014

BackgroundFaecal calprotectin (FC) is a sensitive marker of intestinal mucosal inflammation. The gold standard in Crohn's disease management is mucosal healing. The role of FC to support clinical practice in Crohn's disease is not yet defined.AimsTo determine, in patients with Crohn's disease established on biologic therapy: (1) the correlation between disease activity and FC levels, (2) whether FC can predict for relapse and (3) the sensitivity and specificity of the C-reactive protein (CRP) when compared with FC.MethodsIn this retrospective, single-site study, Crohn's disease activity, clinical outcomes, FC and CRP of 76 patients established on biologic therapy were reviewed and mapped over time.ResultsPatients were mapped for a median of 34 months (IQR 21–57.5). FC levels were determined every 7 (4–13) months on average. Mean FC in quiescent disease was 105 μg/g (SEM 19); in mild disease, 282 (SEM 71); in moderate disease, 611 (SEM 80) and in severe disease, 1314 (SEM 224) (p<0.001). In asymptomatic patients who relapsed at 6 months, the optimal FC, with an area under the curve of 0.886 (p<0.001), was 357.5. In discriminating quiescent from active disease (FC>100 μg/g) the sensitivity and specificity of CRP were 0.48 (0.36–0.61) and 0.73 (0.6–0.85), and in mild from moderate or active disease (FC>250 μg/g), 0.60 (0.43–0.74) and (0.72 (0.60–0.82).ConclusionsFC is an accurate marker of Crohn's disease activity and predicts for relapse, thus providing the clinician time to optimise therapy. FC is a more sensitive marker of Crohn's disease activity than CRP.