bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cross-Clade Ultrasensitive PCR-Based Assays To Measure HIV Persistence in Large-Cohort Studies
Authors: Vandergeeten Claire , Fromentin Rémi , Merlini Esther , Lawani Mariam B. , DaFonseca Sandrina , Bakeman Wendy , McNulty Amanda , Ramgopal Moti , Michael Nelson , Kim Jerome H. , Ananworanich Jintanat , Chomont Nicolas 
Published: August 2014

ABSTRACTA small pool of infected cells persists in HIV-infected individuals receiving antiretroviral therapy (ART). Here, we developed ultrasensitive assays to precisely measure the frequency of cells harboring total HIV DNA, integrated HIV DNA, and two long terminal repeat (2-LTR) circles. These assays are performed on cell lysates, which circumvents the labor-intensive step of DNA extraction, and rely on the coquantification of each HIV molecular form together with CD3 gene sequences to precisely measure cell input. Using primary isolates from HIV subtypes A, B, C, D, and CRF01_A/E, we demonstrate that these assays can efficiently quantify low target copy numbers from diverse HIV subtypes. We further used these assays to measure total HIV DNA, integrated HIV DNA, and 2-LTR circles in CD4+T cells from HIV-infected subjects infected with subtype B. All samples obtained from ART-naive subjects were positive for the three HIV molecular forms (n= 15). Total HIV DNA, integrated HIV DNA, and 2-LTR circles were detected in, respectively, 100%, 94%, and 77% of the samples from individuals in which HIV was suppressed by ART. Higher levels of total HIV DNA and 2-LTR circles were detected in untreated subjects than individuals on ART (P= 0.0003 andP= 0.0004, respectively), while the frequency of CD4+T cells harboring integrated HIV DNA did not differ between the two groups. These results demonstrate that these novel assays have the ability to quantify very low levels of HIV DNA of multiple HIV subtypes without the need for nucleic acid extraction, making them well suited for the monitoring of viral persistence in large populations of HIV-infected individuals.IMPORTANCESince the discovery of viral reservoirs in HIV-infected subjects receiving suppressive ART, measuring the degree of viral persistence has been one of the greatest challenges in the field of HIV research. Here, we report the development and validation of ultrasensitive assays to measure HIV persistence in HIV-infected individuals from multiple geographical regions. These assays are relatively inexpensive, do not require DNA extraction, and can be completed in a single day. Therefore, they are perfectly adapted to monitor HIV persistence in large cohorts of HIV-infected individuals and, given their sensitivity, can be used to monitor the efficacy of therapeutic strategies aimed at interfering with HIV persistence after prolonged ART.
Modulation of Hepatitis C Virus Genome Replication by Glycosphingolipids and Four-Phosphate Adaptor Protein 2
Authors: Khan Irfan , Katikaneni Divya S. , Han Qingxia , Sanchez-Felipe Lorena , Hanada Kentaro , Ambrose Rebecca L. , Mackenzie Jason M. , Konan Kouacou V. 
Published: August 2014

ABSTRACT
Hepatitis C virus (HCV) assembles its replication complex on cytosolic membrane vesicles often clustered in a membranous web (MW). During infection, HCV NS5A protein activates PI4KIIIα enzyme, causing massive production and redistribution of phosphatidylinositol 4-phosphate (PI4P) lipid to the replication complex. However, the role of PI4P in the HCV life cycle is not well understood. We postulated that PI4P recruits host effectors to modulate HCV genome replication or virus particle production. To test this hypothesis, we generated cell lines for doxycycline-inducible expression of short hairpin RNAs (shRNAs) targeting the PI4P effector, four-phosphate adaptor protein 2 (FAPP2). FAPP2 depletion attenuated HCV infectivity and impeded HCV RNA synthesis. Indeed, FAPP2 has two functional lipid-binding domains specific for PI4P and glycosphingolipids. While expression of the PI4P-binding mutant protein was expected to inhibit HCV replication, a marked drop in replication efficiency was observed unexpectedly with the glycosphingolipid-binding mutant protein. These data suggest that both domains are crucial for the role of FAPP2 in HCV genome replication. We also found that HCV significantly increases the level of some glycosphingolipids, whereas adding these lipids to FAPP2-depleted cells partially rescued replication, further arguing for the importance of glycosphingolipids in HCV RNA synthesis. Interestingly, FAPP2 is redistributed to the replication complex (RC) characterized by HCV NS5A, NS4B, or double-stranded RNA (dsRNA) foci. Additionally, FAPP2 depletion disrupts the RC and alters the colocalization of HCV replicase proteins. Altogether, our study implies that HCV coopts FAPP2 for virus genome replication via PI4P binding and glycosphingolipid transport to the HCV RC.

IMPORTANCE
Like most viruses with a positive-sense RNA genome, HCV replicates its RNA on remodeled host membranes composed of lipids hijacked from various internal membrane compartments. During infection, HCV induces massive production and retargeting of the PI4P lipid to its replication complex. However, the role of PI4P in HCV replication is not well understood. In this study, we have shown that FAPP2, a PI4P effector and glycosphingolipid-binding protein, is recruited to the HCV replication complex and is required for HCV genome replication and replication complex formation. More importantly, this study demonstrates, for the first time, the crucial role of glycosphingolipids in the HCV life cycle and suggests a link between PI4P and glycosphingolipids in HCV genome replication.
Polyomavirus Small t Antigen Interacts with Yes-Associated Protein To Regulate Cell Survival and Differentiation
Authors: Hwang Justin H. , Pores Fernando Arun T. , Faure Nathalie , Andrabi Shaida , Hahn William C. , Schaffhausen Brian S. , Roberts Thomas M. 
Published: August 2014

ABSTRACT

Murine polyomavirus small t antigen (PyST) regulates cell cycle, cell survival, apoptosis, and differentiation and cooperates with middle T antigen (MT) to transform primary cells
in vitro
and
in vivo
. Like all polyomavirus T antigens, PyST functions largely via its interactions with host cell proteins. Here, we show that PyST binds both Yes-associated protein 1 (YAP1) and YAP2, integral parts of the Hippo signaling pathway, which is a subject of increasing interest in human cancer. The transcription factor TEAD, which is a known target of YAP, is also found in PyST complexes. PyST enhanced YAP association with protein phosphatase 2A (PP2A), leading to decreased YAP phosphorylation. PyST increased YAP levels by decreasing its degradation. This effect was mediated by a reduction in YAP association with β-transducin repeat protein (βTRCP), which is known to regulate YAP turnover in a phosphorylation-dependent manner. Genetic analysis has identified PyST mutants defective in YAP binding. These mutants demonstrated that YAP binding is important for PyST to block myoblast differentiation and to synergize with the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) to promote cell death in 3T3-L1 preadipocytes placed under differentiation conditions. In addition to YAP binding, both of these phenotypes require PyST binding to PP2A.

IMPORTANCE
The Hippo/YAP pathway is a highly conserved cascade important for tissue development and homeostasis. Defects in this pathway are increasingly being associated with cancer. Polyomavirus small t antigen is a viral oncogene that cooperates with middle T antigen in transformation. On its own, small t antigen controls cell survival and differentiation. By binding YAP, small t antigen brings it together with protein phosphatase 2A. This work shows how this association of small t antigen with YAP is important for its effects on cell phenotype. It also suggests that PyST can be used to characterize cellular processes that are regulated by YAP.
Visualization of Retroviral Envelope Spikes in Complex with the V3 Loop Antibody 447-52D on Intact Viruses by Cryo-Electron Tomography
Authors: Dutta Moumita , Liu Jun , Roux Kenneth H. , Taylor Kenneth A. 
Published: August 2014

ABSTRACTThe gp120 portion of the envelope spike on human immunodeficiency virus type 1 (HIV-1) plays a critical role in viral entry into host cells and is a key target for the humoral immune response, and yet many structural details remain elusive. We have used cryoelectron tomography to visualize the binding of the broadly neutralizing monoclonal antibody (MAb) 447-52D to intact envelope spikes on virions of HIV-1 MN strain. Antibody 447-52D has previously been shown to bind to the tip of the V3 loop. Our results show antibody arms radiating from the sides of the gp120 protomers at a range of angles and place the antibody-bound V3 loop in an orientation that differs from that predicted by most current models but consistent with the idea that antibody binding dislodges the V3 loop from its location in the Env spike, making it flexible and disordered. These data reveal information on the position of the V3 loop and its relative flexibility and suggest that 447-52D neutralizes HIV-1 MN by capturing the V3 loop, blocking its interaction with the coreceptor and altering the structure of the envelope spike.IMPORTANCEAntibody neutralization is one of the primary ways that the body fights infection with HIV. Because HIV is a highly mutable virus, the body must constantly produce new antibodies to counter new strains of HIV that the body itself is producing. Consequently, antibodies capable of neutralizing multiple HIV strains are comparatively few. An improved understanding of the mechanism of antibody neutralization might advance the development of immunogens. Most neutralizing antibodies target the Env glycoprotein spikes found on the virus surface. The broadly neutralizing antibody 447-52D targets the highly conserved β-turn of variable loop 3 (V3) of gp120. The importance of V3 lies in its contribution to the coreceptor binding site on the target cell. We show here that 447-52D binding to V3 converts the Env conformation from closed to open and makes the V3 loop highly flexible, implying disruption of coreceptor binding and attachment to the target cell.
Core-Binding Factor Subunit Beta Is Not Required for Non-Primate Lentiviral Vif-Mediated APOBEC3 Degradation
Authors: Ai Youwei , Zhu Dantong , Wang Cuihui , Su Chao , Ma Jian , Ma Jianzhang , Wang Xiaojun 
Published: August 2014

ABSTRACT
Viral infectivity factor (Vif) is required for lentivirus fitness and pathogenicity, except in equine infectious anemia virus (EIAV). Vif enhances viral infectivity by a Cullin5-Elongin B/C E3 complex to inactivate the host restriction factor APOBEC3. Core-binding factor subunit beta (CBF-β) is a cell factor that was recently shown to be important for the primate lentiviral Vif function. Non-primate lentiviral Vif also degrades APOBEC3 through the proteasome pathway. However, it is unclear whether CBF-β is required for the non-primate lentiviral Vif function. In this study, we demonstrated that the Vifs of non-primate lentiviruses, including feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), caprine arthritis encephalitis virus (CAEV), and maedi-visna virus (MVV), do not interact with CBF-β. In addition, CBF-β did not promote the stability of FIV, BIV, CAEV, and MVV Vifs. Furthermore, CBF-β silencing or overexpression did not affect non-primate lentiviral Vif-mediated APOBEC3 degradation. Our results suggest that non-primate lentiviral Vif induces APOBEC3 degradation through a different mechanism than primate lentiviral Vif.

IMPORTANCE
The APOBEC3 protein family members are host restriction factors that block retrovirus replication. Vif, an accessory protein of lentivirus, degrades APOBEC3 to rescue viral infectivity by forming Cullin5-Elongin B/C-based E3 complex. CBF-β was proved to be a novel regulator of primate lentiviral Vif function. In this study, we found that CBF-β knockdown or overexpression did not affect FIV Vif's function, which induced polyubiquitination and degradation of APOBEC3 by recruiting the E3 complex in a manner similar to that of HIV-1 Vif. We also showed that other non-primate lentiviral Vifs did not require CBF-β to degrade APOBEC3. CBF-β did not interact with non-primate lentiviral Vifs or promote their stability. These results suggest that a different mechanism exists for the Vif-APOBEC interaction and that non-primates are not suitable animal models for exploring pharmacological interventions that disrupt Vif–CBF-β interaction.
Regulation of Autophagic Activation by Rta of Epstein-Barr Virus via the Extracellular Signal-Regulated Kinase Pathway
Authors: Hung Chien-Hui , Chen Lee-Wen , Wang Wen-Hung , Chang Pey-Jium , Chiu Ya-Fang , Hung Chen-Chia , Lin Ying-Ju , Liou Jieh-Yuan , Tsai Wan-Ju , Hung Chia-Ling , Liu Shih-Tung 
Published: August 2014

ABSTRACTAutophagy is an intracellular degradation pathway that provides a host defense mechanism against intracellular pathogens. However, many viruses exploit this mechanism to promote their replication. This study shows that lytic induction of Epstein-Barr virus (EBV) increases the membrane-bound form of LC3 (LC3-II) and LC3-containing punctate structures in EBV-positive cells. Transfecting 293T cells with a plasmid that expresses Rta also induces autophagy, revealing that Rta is responsible for autophagic activation. The activation involves Atg5, a key component of autophagy, but not the mTOR pathway. The expression of Rta also activates the transcription of the genes that participate in the formation of autophagosomes, includingLC3A, LC3B, andATG9Bgenes, as well as those that are involved in the regulation of autophagy, including the genesTNF,IRGM, andTRAIL. Additionally, treatment with U0126 inhibits the Rta-induced autophagy and the expression of autophagy genes, indicating that the autophagic activation is caused by the activation of extracellular signal-regulated kinase (ERK) signaling by Rta. Finally, the inhibition of autophagic activity by an autophagy inhibitor, 3-methyladenine, or Atg5 small interfering RNA, reduces the expression of EBV lytic proteins and the production of viral particles, revealing that autophagy is critical to EBV lytic progression. This investigation reveals how an EBV-encoded transcription factor promotes autophagy to affect viral lytic development.
Commentary: HIV Vaccine Trial Exploits a Dual and Central Role for Innate Immunity
Authors: Fuller Deborah Heydenburg , Richert-Spuhler Laura E. , Klatt Nichole R. 
Published: August 2014

ABSTRACT

Limited understanding of correlates of protection from HIV transmission hinders development of an efficacious vaccine. D. J. M. Lewis and colleagues (J. Virol. 88:11648–11657, 2014,
doi:10.1128/JVI.01621-14
)
now report that vaginal immunization with an HIVgp140 vaccine linked to the 70-kDa heat shock protein downregulated the human immunodeficiency virus (HIV) coreceptor CCR5 (chemokine [C-C motif] receptor 5) and increased expression of the HIV resistance factor APOBEC3G (apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like 3G), in women. These effects correlated with HIV suppression
ex vivo
. Thus, vaccine-induced innate responses not only facilitate adaptive immunity–they may prove to be critical for preventing HIV transmission.
Kinetic and Phenotypic Analysis of CD8
+
T Cell Responses after Priming with Alphavirus Replicons and Homologous or Heterologous Booster Immunizations
Authors: Knudsen Maria Lisa , Ljungberg Karl , Kakoulidou Maria , Kostic Linda , Hallengärd David , García-Arriaza Juan , Merits Andres , Esteban Mariano , Liljeström Peter 
Published: August 2014

ABSTRACT

Alphavirus replicons are potent inducers of CD8
+
T cell responses and thus constitute an attractive vaccine vector platform for developing novel vaccines. However, the kinetics and memory phenotype of CD8
+
T cell responses induced by alphavirus replicons are not well characterized. Furthermore, little is known how priming with alphavirus replicons affects booster immune responses induced by other vaccine modalities. We demonstrate here that a single immunization with an alphavirus replicon, administered as viral particles or naked DNA, induced an antigen-specific CD8
+
T cell response that had a sharp peak, followed by a rapid contraction. Administering a homologous boost before contraction had occurred did not further increase the response. In contrast, boosting after contraction when CD8
+
T cells had obtained a memory phenotype (based on CD127/CD62L expression), resulted in maintenance of CD8
+
T cells with a high recall capacity (based on CD27/CD43 expression). Increasing the dose of replicon particles promoted T effector memory (Tem) and inhibited T central memory development. Moreover, infection with a replicating alphavirus induced a similar distribution of CD8
+
T cells as the replicon vector. Lastly, the distribution of T cell subpopulations induced by a DNA-launched alphavirus replicon could be altered by heterologous boosts. For instance, boosting with a poxvirus vector (MVA) favored expansion of the Tem compartment. In summary, we have characterized the antigen-specific CD8
+
T cell response induced by alphavirus replicon vectors and demonstrated how it can be altered by homologous and heterologous boost immunizations.

IMPORTANCE
Alphavirus replicons are promising vaccine candidates against a number of diseases and are by themselves developed as vaccines against, for example, Chikungunya virus infection. Replicons are also considered to be used for priming, followed by booster immunization using different vaccine modalities. In order to rationally design prime-boost immunization schedules with these vectors, characterization of the magnitude and phenotype of CD8
+
T cell responses induced by alphavirus replicons is needed. Here, we demonstrate how factors such as timing and dose affect the phenotypes of memory T cell populations induced by immunization with alphavirus replicons. These findings are important for designing future clinical trials with alphaviruses, since they can be used to tailor vaccination regimens in order to induce a CD8
+
T cell response that is optimal for control and/or clearance of a specific pathogen.
Rates and predictors of ACE inhibitor discontinuation subsequent to elevated serum creatinine: a retrospective cohort study
Authors: Jackevicius Cynthia A , Wong Joyce , Aroustamian Irina , Gee Manyee , Mody Freny Vaghaiwalla 
Published: August 2014

ObjectivesACE inhibitors (ACEI) are underutilised despite cardiovascular benefits, in part due to concerns of known transient elevations in serum creatinine (SCr) after initiation. Our objectives were to evaluate rates and predictors of ACEI discontinuation after SCr elevation post-ACEI initiation since limited data are available that examine this issue.SettingPrimary and tertiary Veterans healthcare system in Los Angeles, California, USAParticipants3039 outpatients initiating an ACEI with a SCr measured within 6 months prior to and approximately 3 months after initiating an ACEI. Patients were divided into three groups (SCr <1.5, 1.5–2 and >2).Primary and secondary outcome measuresRates and factors associated with ACEI discontinuation subsequent to SCr elevation after ACEI initiation and for patients with baseline SCr >2 mg/dL, the change in SCr associated with chronic use. Predictors were identified using multivariate logistic regression modelling.ResultsAt 3 months follow-up, for those with an increase in SCr, the mean increase post-ACEI initiation was 26%, ranging from −0.01 mg/dL to 0.42 mg/dL varying according to a level of baseline renal function. ACEI discontinuation was higher in patients with elevated baseline SCr (19/165, 11.5%) compared with those with SCr <1.5 (135/2497, 5.4%), and those with SCr 1.5–2.0 (28/377, 7.4%). Male patients, and those with heart failure were less likely to discontinue ACEI after an elevation of SCr post-ACEI initiation, while those taking non-steroidal anti-inflammatory drugs, diuretics and β-blockers were more likely to discontinue ACEI.ConclusionsSCr increases <30% on average within 3 months of ACEI initiation, with subsequent discontinuation rates varying by baseline SCr. Elevation in SCr was not associated with ACEI discontinuation rates. In patients with SCr >2 mg/dL at baseline, despite an acute increase in SCr after ACEI initiation, chronic ACEI use was associated with a decrease in SCr in most patients.
WHAT IS THE RETURN ON YOUR UNPLANNED RETURNS?
Authors: Burch Greg , Walters Steven , Jessop Vanessa , Somers Lisa 
Published: August 2014

Objectives & BackgroundUnplanned emergency re-attendance within seven days is a national quality indicator set at 1–5% by the Department of Health. Newham University Hospital (NUH), one of three hospitals forming Barts Health NHS Trust, recorded rates exceeded this target in October 2012, with a spike at 10% in April 2013 prompting investigation of the root cause. A review was carried out to determine the potential causes and provide solutions.MethodsRecords of all patients who attended twice or more within seven days with the same problem were analysed retrospectively over a two week period in October 2012. Spot check analyses were also made over the following two months. Over 250 re-attendances were reviewed. All patients presenting to the common front door of the ED (ED, Urgent Care Centre and GP co-operative) were included, as per DoH definition. Clinical notes were reviewed to establish the initial presenting complaint and reason for subsequent re-attendance.ResultsThe total number of recorded unplanned returns was 119. However, the true number was 90 (24% less than the recorded figure); including five missed unplanned returns.16 patients were wrongly coded and 18 patients left without treatment on at least one occasion.One in five re-attenders were ‘frequent flyers’, presenting repeatedly with minor chronic conditions, intoxication, homelessness and mental health issues. Reasons for re-attendance given by patients included being unable to get primary care appointments, lack of GP registration and dissatisfaction with GP or Urgent Care Centre advice.ConclusionWe discovered that different definitions across the three Barts Health ED sites were being applied. Following presentation of results a unified definition was drawn up. Coding error was the main reason for our blip of 10% in April 2013 and was rectified by education of reception staff. We have now instigated bimonthly review of ‘frequent fliers’ so that case-management plans can be implemented with relevant Trust and community teams. This review has highlighted several issues contributing to inaccurate figures which are likely to apply to many emergency departments and has shown how useful it is to qualify as well as quantify unplanned returns.