bluuebunny/crossref_metadata_2025_split huggingface dataset:
Neutralizing IgG at the Portal of Infection Mediates Protection against Vaginal Simian/Human Immunodeficiency Virus Challenge
Authors: Klein Katja , Veazey Ronald S. , Warrier Ranjit , Hraber Peter , Doyle-Meyers Lara A. , Buffa Viviana , Liao Hua-Xin , Haynes Barton F. , Shaw George M. , Shattock Robin J. 
Published: August 2013

ABSTRACT
Neutralizing antibodies may have critical importance in immunity against human immunodeficiency virus type 1 (HIV-1) infection. However, the amount of protective antibody needed at mucosal surfaces has not been fully established. Here, we evaluated systemic and mucosal pharmacokinetics (PK) and pharmacodynamics (PD) of 2F5 IgG and 2F5 Fab fragments with respect to protection against vaginal challenge with simian-human immunodeficiency virus-BaL in macaques. Antibody assessment demonstrated that 2F5 IgG was more potent than polymeric forms (IgM and IgA) across a range of cellular and tissue models. Vaginal challenge studies demonstrated a dose-dependent protection for 2F5 IgG and no protection with 2F5 Fab despite higher vaginal Fab levels at the time of challenge. Animals receiving 50 or 25 mg/kg of body weight 2F5 IgG were completely protected, while 3/5 animals receiving 5 mg/kg were protected. In the control animals, infection was established by a minimum of 1 to 4 transmitted/founder (T/F) variants, similar to natural human infection by this mucosal route; in the two infected animals that had received 5 mg 2F5 IgG, infection was established by a single T/F variant. Serum levels of 2F5 IgG were more predictive of sterilizing protection than measured vaginal levels. Fc-mediated antiviral activity did not appear to influence infection of primary target cells in cervical explants. However, PK studies highlighted the importance of the Fc portion in tissue biodistribution. Data presented in this study may be important in modeling serum levels of neutralizing antibodies that need to be achieved by either vaccination or passive infusion to prevent mucosal acquisition of HIV-1 infection in humans.
KLRG1 Negatively Regulates Natural Killer Cell Functions through the Akt Pathway in Individuals with Chronic Hepatitis C Virus Infection
Authors: Wang Jia M. , Cheng Yong Q. , Shi Lei , Ying Ruo S. , Wu Xiao Y. , Li Guang Y. , Moorman Jonathan P. , Yao Zhi Q. 
Published: August 2013

ABSTRACT

In this study, we demonstrate that killer cell lectin-like receptor subfamily G member 1 (KLRG1), a transmembrane protein preferentially expressed on T cells, is highly expressed on CD56
+
NK cells, which are significantly reduced in their numbers and functions in the peripheral blood of patients with chronic hepatitis C virus (HCV) infection compared to subjects without infection. KLRG1 expression is also upregulated on healthy NK cells exposed to Huh-7 hepatocytes infected with HCV
in vitro
. Importantly, the expression levels of KLRG1 are inversely associated with the capacity of NK cells to proliferate and to produce gamma interferon (IFN-γ) but positively associated with apoptosis of NK cells in response to inflammatory cytokine stimulation. KLRG1
+
NK cells, including CD56
bright
and CD56
dim
subsets, exhibit impaired cell activation and IFN-γ production but increased apoptosis compared to KLRG1
−
NK cells, particularly in HCV-infected individuals. Importantly, blockade of KLRG1 signaling significantly recovered the impaired IFN-γ production by NK cells from HCV-infected subjects. Blockade of KLRG1 also enhanced the impaired phosphorylation of Akt (Ser473) in NK cells from HCV-infected subjects. Taken together, these results indicate that KLRG1 negatively regulates NK cell numbers and functions via the Akt pathway, thus providing a novel marker and therapeutic target for HCV infection.
Global RNA Structure Analysis of Poliovirus Identifies a Conserved RNA Structure Involved in Viral Replication and Infectivity
Authors: Burrill Cecily P. , Westesson Oscar , Schulte Michael B. , Strings Vanessa R. , Segal Mark , Andino Raul 
Published: August 2013

ABSTRACT

The genomes of RNA viruses often contain RNA structures that are crucial for translation and RNA replication and may play additional, uncharacterized roles during the viral replication cycle. For the picornavirus family member poliovirus, a number of functional RNA structures have been identified, but much of its genome, especially the open reading frame, has remained uncharacterized. We have now generated a global RNA structure map of the poliovirus genome using a chemical probing approach that interrogates RNA structure with single-nucleotide resolution. In combination with orthogonal evolutionary analyses, we uncover several conserved RNA structures in the open reading frame of the viral genome. To validate the ability of our global analyses to identify functionally important RNA structures, we further characterized one of the newly identified structures, located in the region encoding the RNA-dependent RNA polymerase, 3D
pol
, by site-directed mutagenesis. Our results reveal that the structure is required for viral replication and infectivity, since synonymous mutants are defective in these processes. Furthermore, these defects can be partially suppressed by mutations in the viral protein 3C
pro
, which suggests the existence of a novel functional interaction between an RNA structure in the 3D
pol
-coding region and the viral protein(s) 3C
pro
and/or its precursor 3CD
pro
.
Uptake of Rabies Virus into Epithelial Cells by Clathrin-Mediated Endocytosis Depends upon Actin
Authors: Piccinotti Silvia , Kirchhausen Tomas , Whelan Sean P. J. 
Published: August 2013

ABSTRACT
Rabies virus (RABV) causes a fatal zoonotic encephalitis. Disease symptoms require replication and spread of the virus within neuronal cells; however, in infected animals as well as in cell culture the virus replicates in a broad range of cell types. Here we use a single-cycle RABV and a recombinant vesicular stomatitis virus (rVSV) in which the glycoprotein (G) was replaced with that of RABV (rVSV RABV G) to examine RABV uptake into the African green monkey kidney cell line BS-C-1. Combining biochemical studies and real-time spinning-disk confocal fluorescence microscopy, we show that the predominant entry pathway of RABV particles into BS-C-1 cells is clathrin dependent. Viral particles enter cells in pits with elongated structures and incomplete clathrin coats which depend upon actin to complete the internalization process. By measuring the time of internalization and the abundance of the clathrin adaptor protein AP2, we further show that the pits that internalize RABV particles are similar to those that internalize VSV particles. Pharmacological perturbations of dynamin or of actin polymerization inhibit productive infection, linking our observations on particle uptake with viral infectivity. This work extends to RABV particles the finding that clathrin-mediated endocytosis of rhabdoviruses proceeds through incompletely coated pits which depend upon actin.
Gag-Dependent Enrichment of HIV-1 RNA near the Uropod Membrane of Polarized T Cells
Authors: Hatch Steven C. , Sardo Luca , Chen Jianbo , Burdick Ryan , Gorelick Robert , Fivash Matthew J. , Pathak Vinay K. , Hu Wei-Shau 
Published: August 2013

ABSTRACT
The enrichment of HIV-1 macromolecules at the uropod of polarized T cells can significantly promote virus assembly and cell-mediated infection. Using live-cell fluorescence microscopy, we demonstrate that full-length HIV-1 RNA is enriched at the uropod membrane; furthermore, the presence of HIV-1 Gag containing a functional nucleocapsid domain is necessary for this HIV-1 RNA enrichment. The results from these studies provide novel insights into the mechanism of HIV-1 replication in polarized T cells.
A Functional Interaction between gp41 and gp120 Is Observed for Monomeric but Not Oligomeric, Uncleaved HIV-1 Env gp140
Authors: Guttman Miklos , Lee Kelly K. 
Published: August 2013

ABSTRACT
The envelope glycoprotein (Env) is the sole antigenic feature on the surface of HIV and the target for the humoral immune system. Soluble, uncleaved gp140 Env constructs truncated at the transmembrane domain are being investigated intensively as potential vaccine immunogens by many groups, and understanding their structural properties is essential. We used hydrogen/deuterium-exchange mass spectrometry and small-angle X-ray scattering to probe structural order in a panel of commonly used gp140 constructs and matched gp120 monomers. We observed that oligomeric forms of uncleaved gp140, generally presumed to be trimeric, contain a protease-resistant form of gp41 akin to the postfusion, helical bundle conformation and appear to lack specific interactions between gp120 and gp41. In contrast, the monomeric form of gp140 shows significant stabilization of the gp120 inner domain imparted by the gp41 region, demonstrating excellent agreement with past mutagenesis studies. Moreover, the gp140 monomers respond to CD4 binding in manner that is consistent with the initial stages of Env activation: CD4 binding induces structural ordering throughout gp120 while loosening its association with gp41. The results indicate that uncleaved gp140 oligomers do not represent an authentic prefusion form of Env, whereas gp140 monomers isolated from the same glycoprotein preparations in many ways exhibit function and internal structural order that are consistent with expectations for certain aspects of native Env. gp140 monomers may thus be a useful reagent for advancing structural and functional studies.
Multiple Heparan Sulfate Binding Site Engagements Are Required for the Infectious Entry of Human Papillomavirus Type 16
Authors: Richards Kathleen F. , Bienkowska-Haba Malgorzata , Dasgupta Jhimli , Chen Xiaojiang S. , Sapp Martin 
Published: August 2013

ABSTRACT
Human papillomavirus (HPV) entry is accompanied by multiple receptor-induced conformational changes (CCs) affecting both the major and minor capsid proteins, L1 and L2. Interaction of heparan sulfate (HS) with L1 is essential for successful HPV16 entry. Recently, cocrystallization of HPV16 with heparin revealed four distinct binding sites. Here we characterize mutant HPV16 to delineate the role of engagement with HS binding sites during infectious internalization. Site 1 (Lys278, Lys361), which mediates primary binding, is sufficient to trigger an L2 CC, exposing the amino terminus. Site 2 (Lys54, Lys356) and site 3 (Asn57, Lys59, Lys442, Lys443) are engaged following primary attachment and are required for infectious entry. Site 2 mutant particles are efficiently internalized but fail to undergo an L1 CC on the cell surface and subsequent uncoating in the endocytic compartment. After initial attachment to the cell, site 3 mutants undergo L1 and L2 CCs and then accumulate on the extracellular matrix (ECM). We conclude that the induction of CCs following site 1 and site 2 interactions results in reduced affinity for the primary HS binding site(s) on the cell surface, which allows engagement with site 3. Taken together, our findings suggest that HS binding site engagement induces CCs that prepare the virus for downstream events, such as the exposure of secondary binding sites, CCs, transfer to the uptake receptor, and uncoating.
Processing of the VP1/2A Junction Is Not Necessary for Production of Foot-and-Mouth Disease Virus Empty Capsids and Infectious Viruses: Characterization of “Self-Tagged” Particles
Authors: Gullberg Maria , Polacek Charlotta , Bøtner Anette , Belsham Graham J. 
Published: August 2013

ABSTRACT

The foot-and-mouth disease virus (FMDV) capsid protein precursor, P1-2A, is cleaved by 3C
pro
to generate VP0, VP3, VP1, and the peptide 2A. The capsid proteins self-assemble into empty capsid particles or viruses which do not contain 2A. In a cell culture-adapted strain of FMDV (O1 Manisa [Lindholm]), three different amino acid substitutions (E83K, S134C, and K210E) were identified within the VP1 region of the P1-2A precursor compared to the field strain (wild type [wt]). Expression of the O1 Manisa P1-2A (wt or with the S134C substitution in VP1) plus 3C
pro
, using a transient expression system, resulted in efficient capsid protein production and self-assembly of empty capsid particles. Removal of the 2A peptide from the capsid protein precursor had no effect on capsid protein processing or particle assembly. However, modification of E83K alone abrogated particle assembly with no apparent effect on protein processing. Interestingly, the K210E substitution, close to the VP1/2A junction, completely blocked processing by 3C
pro
at this cleavage site, but efficient assembly of “self-tagged” empty capsid particles, containing the uncleaved VP1-2A, was observed. These self-tagged particles behaved like the unmodified empty capsids in antigen enzyme-linked immunosorbent assays and integrin receptor binding assays. Furthermore, mutant viruses with uncleaved VP1-2A could be rescued in cells from full-length FMDV RNA transcripts encoding the K210E substitution in VP1. Thus, cleavage of the VP1/2A junction is not essential for virus viability. The production of such engineered self-tagged empty capsid particles may facilitate their purification for use as diagnostic reagents and vaccines.
Maturation-Associated Destabilization of Hepatitis B Virus Nucleocapsid
Authors: Cui Xiuji , Ludgate Laurie , Ning Xiaojun , Hu Jianming 
Published: August 2013

ABSTRACT

The mature nucleocapsid (NC) of hepatitis B virus containing the relaxed circular (RC) DNA genome can be secreted extracellularly as virions after envelopment with the viral surface proteins or, alternatively, can be disassembled to release RC DNA (i.e., uncoating) into the host cell nucleus to form the covalently closed circular (CCC) DNA, which sustains viral replication and persistence. In contrast, immature NCs containing the viral single-stranded DNA or the pregenomic RNA are incompetent for either envelopment or uncoating. Little is currently known about how mature NCs, and not the immature ones, are specifically selected for these processes. Here, we have carried out a biochemical analysis of the different NC populations upon their separation through sucrose gradient centrifugation. We have found that the maturation of NCs is associated with their destabilization, manifested as increased protease and nuclease sensitivity, altered sedimentation during sucrose gradient centrifugation, and retarded mobility during native agarose gel electrophoresis. Also, three distinct populations of intracellular mature NCs could be differentiated based on these characteristics. Furthermore, mature NCs generated
in vitro
under cell-free conditions acquired similar properties. These results have thus revealed significant structural changes associated with NC maturation that likely play a role in the selective uncoating of the mature NC for CCC DNA formation and/or its preferential envelopment for virion secretion.
The Human Adenovirus 5 L4 Promoter Is Activated by Cellular Stress Response Protein p53
Authors: Wright Jordan , Leppard Keith N. 
Published: August 2013

ABSTRACT
During adenovirus infection, the emphasis of gene expression switches from early genes to late genes in a highly regulated manner. Two gene products, L4-22K and L4-33K, contribute to this switch by activating the major late transcription unit (MLTU) and regulating the splicing of its transcript. L4-22K and L4-33K expression is driven initially by a recently described L4 promoter (L4P) embedded within the MLTU that is activated by early and intermediate viral factors: E1A, E4 Orf3, and IVa2. Here we show that this promoter is also significantly activated by the cellular stress response regulator, p53. Exogenous expression of p53 activated L4P in reporter assays, while depletion of endogenous p53 inhibited the induction of L4P by viral activators. Chromatin immunoprecipitation studies showed that p53 associates with L4P and that during adenovirus type 5 (Ad5) infection, this association peaks at 12 h postinfection, coinciding with the phase of the infectious cycle when L4P is active, and is then lost as MLP activation commences. p53 activation of L4P is significant during Ad5 infection, since depletion of p53 prior to infection of either immortalized or normal cells led to severely reduced late gene expression. The association of p53 with L4P is transient due to the action of products of L4P activity (L4-22K/33K), which establish a negative feedback loop that ensures the transient activity of L4P at the start of the late phase and contributes to an efficient switch from early- to late-phase virus gene expression.
How active are our children? Findings from the Millennium Cohort Study
Authors: Griffiths Lucy J , Cortina-Borja Mario , Sera Francesco , Pouliou Theodora , Geraci Marco , Rich Carly , Cole Tim J , Law Catherine , Joshi Heather , Ness Andrew R , Jebb Susan A , Dezateux Carol 
Published: August 2013

ObjectiveTo describe levels of physical activity, sedentary time and adherence to Chief Medical Officers (CMO) physical activity guidelines among primary school-aged children across the UK using objective accelerometer-based measurements.DesignNationally representative prospective cohort study.SettingChildren born across the UK, between 2000 and 2002.Participants6497 7-year-old to 8-year-old singleton children for whom reliable accelerometer data were available for at least 10 h a day for at least 2 days.Main outcome measuresPhysical activity in counts per minute (cpm); time spent in sedentary and moderate-to-vigorous intensity physical activity (MVPA); proportion of children meeting CMO guidelines (≥60 min/day MVPA); average daily steps.Explanatory measuresGender, ethnicity, maternal current/most recent occupation, lone parenthood status, number of children in the household and country/region of residence.ResultsThe median daily physical activity level was 595 cpm (IQR 507, 697). Children spent a median of 60 min (IQR 47–76) in MVPA/day and were sedentary for a median of 6.4 h/day (IQR 6–7). Only 51% met CMO guidelines, with girls (38%) less active than boys (63%). Children took an average of 10 229 (95% CI (8777 to 11 775)) steps each day. Children of Indian ethnicity were significantly less active overall than all other ethnic groups. Children of Bangladeshi origin and those living in Northern Ireland were least likely to meet CMO guidelines.ConclusionsOnly half of 7-year-old children in the UK achieve recommended levels of physical activity, with significant gender, ethnic and geographic variations. Longitudinal studies are needed to better understand the relevance of these (in)activity patterns for long-term health and well-being. In the meantime population-wide efforts to boost physical activity among young people are needed which are likely to require a broad range of policy interventions.
Extending the use of PROMs in the NHS—using the Oxford Knee Score in patients undergoing non-operative management for knee osteoarthritis: a validation study
Authors: Harris Kristina K , Dawson Jill , Jones Luke D , Beard David J , Price Andrew J 
Published: August 2013

ObjectivesTo assess the validity of the Oxford Knee Score (OKS) for use in patients undergoing non-operative management for their knee osteoarthritis (OA) within the National Health Service (NHS).DesignObservational cohort study.SettingSingle orthopaedic centre in England.Participants134 patients undergoing non-operative management for knee OA.Main outcome measuresOKS, the Intermittent and Constant Osteoarthritis Pain (ICOAP), the Knee Injury and Osteoarthritis Score-Physical Function Short Form (KOOS-PS), at baseline and 3-month follow-up, transition item of change at 3 months.ResultsThe OKS summary scale and its pain and functional component subscales demonstrated good test–retest reliability (intraclass correlation coefficient 0.93, 0.91 and 0.92, respectively) and measurement precision which, allows its use with groups of patients with knee OA (research/audit) and with individuals (clinical practice). The results in this study were consistent with a priori set hypotheses about the relationship of OKS with other validated measures (KOOS-PS, ICOAP and short form 12 (SF-12)), which provided evidence of its construct validity and responsiveness. Confirmatory factor analysis confirmed the structural validity of OKS. However, there was a lack of satisfactory evidence of structural validity for ICOAP and KOOS. The minimum detectable change (MDC90) was ±6 for OKS (±16 for the Pain Component Score (OKS-PCS) and ±15 for the Functional Component Score (OKS-FCS)). Minimal important changes were ≈7 for OKS (≈17 for OKS-PCS and ≈11 for OKS-FCS) and minimal important differences were ≈6 for OKS (≈14 for OKS-PCS and ≈10 for OKS-FCS). These values were also calculated for ICOAP and KOOS-PS.ConclusionsThe OKS summary scale, together with its pain and functional component subscales, has excellent measurement properties when used with patients with knee OA undergoing non-operative treatment and is superior to ICOAP and KOOS-PS for this purpose. This evidence provides support for the validity of the use of OKS when used across the spectrum of knee OA disease severity, both in research and clinical practice.
Does comorbidity increase the risk of mortality among children under 3 years of age?
Authors: Fischer Walker Christa L , Perin Jamie , Liu Jodi L , Katz Joanne , Tielsch James M , Black Robert 
Published: August 2013

ObjectivesDiarrhoea and pneumonia remain leading causes of morbidity and mortality in children under 5 years of age. Little data is available to quantify the burden of comorbidity and the relationship between comorbid diarrhoea and pneumonia infections and mortality. We sought to quantify the relationship between comorbidity and risk of mortality among young children in two community-based studies conducted among South Asian children.DesignSecondary data analysis of two cohort studies.ParticipantsWe identified two cohort studies of children under 3 years of age with prospective morbidity at least once every 2 weeks and ongoing mortality surveillance.Outcome measuresWe calculated the mortality risk for diarrhoea and acute lower respiratory infection (ALRI) episodes and further quantified the risk of mortality when both diseases occur at the same time using a semiparametric additive model.ResultsAmong Nepali children, the estimated additional risk of mortality for comorbid diarrhoea and ALRI was 0.0014 (−0.0033, 0.0060). Among South Indian children, the estimated additional risk of mortality for comorbid diarrhoea and ALRI was 0.0032 (−0.0098, 0.0162). This risk is in addition to the single infection risk of mortality observed among these children.ConclusionsWe observed an additional risk of mortality in children who experienced simultaneous diarrhoea and ALRI episodes though the CI was wide indicating low statistical support. Additional studies with adequate power to detect the increased risk of comorbidity on mortality are needed to improve confidence around the effect size estimate.