bluuebunny/crossref_metadata_2025_split huggingface dataset:
Kaposi's Sarcoma-Associated Herpesvirus ORF18 and ORF30 Are Essential for Late Gene Expression during Lytic Replication
Authors: Gong Danyang , Wu Nicholas C. , Xie Yafang , Feng Jun , Tong Leming , Brulois Kevin F. , Luan Harding , Du Yushen , Jung Jae U. , Wang Cun-yu , Kang Mo Kwan , Park No-Hee , Sun Ren , Wu Ting-Ting 
Published: July 2014

ABSTRACT

Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with several human malignances. As saliva is likely the major vehicle for KSHV transmission, we studied
in vitro
KSHV infection of oral epithelial cells. Through infection of two types of oral epithelial cells, normal human oral keratinocytes (NHOKs) and papilloma-immortalized human oral keratinocyte (HOK16B) cells, we found that KSHV can undergo robust lytic replication in oral epithelial cells. By employing
de novo
lytic infection of HOK16B cells, we studied the functions of two previously uncharacterized genes, ORF18 and ORF30, during the KSHV lytic cycle. For this purpose, an ORF18-deficient virus and an ORF30-deficient virus were generated using a mutagenesis strategy based on bacterial artificial chromosome (BAC) technology. We found that neither ORF18 nor ORF30 is required for immediately early or early gene expression or viral DNA replication, but each is essential for late gene expression during both
de novo
lytic replication and reactivation. This critical role of ORF18 and ORF30 in late gene expression was also observed during KSHV reactivation. In addition, global analysis of viral transcripts by RNA sequencing indicated that ORF18 and ORF30 control the same set of viral genes. Therefore, we suggest that these two viral ORFs are involved in the same mechanism or pathway that coregulates the viral late genes as a group.

IMPORTANCE
While KSHV can infect multiple cell types
in vitro
, only a few can support a full lytic replication cycle with progeny virions produced. Consequently, KSHV lytic replication is mostly studied through reactivation, which requires chemicals to induce the lytic cycle or overexpression of the viral transcriptional activator, RTA. In this study, we present a robust
de novo
lytic infection system based on oral epithelial cells. Using this system, we demonstrate the role of two viral ORFs, ORF18 and ORF30, in regulating viral gene expression during KSHV lytic replication. As the major route of KSHV transmission is thought to be via saliva, this new KSHV lytic replication system will have important utility in the field.
New Insights into the Nonconserved Noncoding Region of the Subtype-Determinant Hemagglutinin and Neuraminidase Segments of Influenza A Viruses
Authors: Zhao Lili , Peng Yousong , Zhou Kai , Cao Mengmeng , Wang Jingfeng , Wang Xue , Jiang Taijiao , Deng Tao 
Published: July 2014

ABSTRACT
The noncoding regions (NCRs) of the eight-segmented viral RNAs (vRNAs) of influenza A virus consist of the highly conserved promoter region and the nonconserved segment-specific NCRs at both the 3′ and 5′ ends. The roles of the segment-specific NCRs of the eight segments have been extensively studied. However, the diversities in the same region of the two subtype-determinant hemagglutinin (HA) and neuraminidase (NA) segments have received little attention. In this study, we bioinformatically analyzed all available NCRs of HA and NA vRNAs of influenza A viruses and found that nucleotides in the segment-specific NCRs of HA and NA vRNAs are subtype specific and vary significantly in sequence and length at both the 3′ and 5′ ends among different subtypes. We then systematically studied the biological significance of the HA subtype-specific NCRs (HA ssNCRs) of the common HA subtypes (H1 to H7 and H9) in the context of the WSN (H1N1) reverse genetics system. We found that the HA ssNCRs play a critical role in HA vRNA virion incorporation. Upon HA vRNA incorporation, the 3′-end HA ssNCR plays a more critical role than the 5′-end HA ssNCR, and no stringent compatibility between the two ends is required. Furthermore, our data imply that, in addition to a particular nucleotide(s), the length of the HA ssNCR is involved in regulating HA vRNA incorporation efficiency. These results provide new insights into the HA segment virion incorporation that is critical for the emergence of epidemic and pandemic influenza A virus strains.

IMPORTANCE
The nonconserved noncoding regions (NCRs) of the vRNAs of influenza A virus have been extensively studied, whereas the diversities in the nonconserved NCRs of the two subtype-determinant segments hemagglutinin (HA) and neuraminidase (NA) have received little attention. In this study, we bioinformatically analyzed all available NCRs of HA and NA vRNAs and discovered that the HA and NA vRNAs contain key subtype signatures in the NCRs. Our functional studies of the HA subtype-specific NCRs (HA ssNCRs) of the common HA subtypes in the context of WSN virus (H1N1) demonstrated that the HA ssNCR modulates virus replication efficiency by influencing HA segment virion incorporation. Moreover, we revealed important features of the HA ssNCR in determining HA vRNA incorporation efficiency. These data not only show new genetic characteristics of influenza A viruses, but also provide further evidence for understanding the selective genome packaging of influenza virus required for the emergence of epidemic and pandemic influenza virus strains.
Downregulation of MicroRNA miR-526a by Enterovirus Inhibits RIG-I-Dependent Innate Immune Response
Authors: Xu Changzhi , He Xiang , Zheng Zirui , Zhang Zhe , Wei Congwen , Guan Kai , Hou Lihua , Zhang Buchang , Zhu Lin , Cao Yuan , Zhang Yanhong , Cao Ye , Ma Shengli , Wang Penghao , Zhang Pingping , Xu Quanbin , Ling Youguo , Yang Xiao , Zhong Hui 
Published: July 2014

ABSTRACT
Retinoic acid-inducible gene I (RIG-I) is an intracellular RNA virus sensor that induces type I interferon-mediated host-protective innate immunity against viral infection. Although cylindromatosis (CYLD) has been shown to negatively regulate innate antiviral response by removing K-63-linked polyubiquitin from RIG-I, the regulation of its expression and the underlying regulatory mechanisms are still incompletely understood. Here we show that RIG-I activity is regulated by inhibition of CYLD expression mediated by the microRNA miR-526a. We found that viral infection specifically upregulates miR-526a expression in macrophages via interferon regulatory factor (IRF)-dependent mechanisms. In turn, miR-526a positively regulates virus-triggered type I interferon (IFN-I) production, thus suppressing viral replication, the underlying mechanism of which is the enhancement of RIG-I K63-linked ubiquitination by miR-526a via suppression of the expression of CYLD. Remarkably, virus-induced miR-526a upregulation and CYLD downregulation are blocked by enterovirus 71 (EV71) 3C protein, while ectopic miR-526a expression inhibits the replication of EV71 virus. The collective results of this study suggest a novel mechanism of the regulation of RIG-I activity during RNA virus infection by miR-526a and suggest a novel mechanism for the evasion of the innate immune response controlled by EV71.

IMPORTANCE
RNA virus infection upregulates the expression of miR-526a in macrophages through IRF-dependent pathways. In turn, miR-526a positively regulates virus-triggered type I IFN production and inhibits viral replication, the underlying mechanism of which is the enhancement of RIG-I K-63 ubiquitination by miR-526a via suppression of the expression of CYLD. Remarkably, virus-induced miR-526a upregulation and CYLD downregulation are blocked by enterovirus 71 (EV71) 3C protein; cells with overexpressed miR-526a were highly resistant to EV71 infection. The collective results of this study suggest a novel mechanism of the regulation of RIG-I activity during RNA virus infection by miR-526a and propose a novel mechanism for the evasion of the innate immune response controlled by EV71.
Antiviral Regulation in Porcine Monocytic Cells at Different Activation States
Authors: Sang Yongming , Rowland Raymond R. R. , Blecha Frank 
Published: July 2014

ABSTRACT

Monocytic cells, including macrophages and dendritic cells, exist in different activation states that are critical to the regulation of antimicrobial immunity. Many pandemic viruses are monocytotropic, including porcine reproductive and respiratory syndrome virus (PRRSV), which directly infects subsets of monocytic cells and interferes with antiviral responses. To study antiviral responses in PRRSV-infected monocytic cells, we characterized inflammatory cytokine responses and genome-wide profiled signature genes to investigate response pathways in uninfected and PRRSV-infected monocytic cells at different activation states. Our findings showed suppressed interferon (IFN) production in macrophages in non-antiviral states and an arrest of lipid metabolic pathways in macrophages at antiviral states. Importantly, porcine monocytic cells at different activation states were susceptible to PRRSV and responded differently to viral infection. Based on Gene Ontology (GO) analysis, two approaches were used to potentiate antiviral activity: (i) pharmaceutical modulation of cellular lipid metabolism and (ii)
in situ
PRRSV replication-competent expression of interferon alpha (IFN-α). Both approaches significantly suppressed exogenous viral infection in monocytic cells. In particular, the engineered IFN-expressing PRRSV strain eliminated exogenous virus infection and sustained cell viability at 4 days postinfection in macrophages. These findings suggest an intricate interaction of viral infection with the activation status of porcine monocytic cells. An understanding and integration of antiviral infection with activation status of monocytic cells may provide a means of potentiating antiviral immunity.

IMPORTANCE
Activation statuses of monocytic cells, including monocytes, macrophages (Mϕs), and dendritic cells (DCs), are critically important for antiviral immunity. Unfortunately, the activation status of porcine monocytic cells or how cell activation status functionally interacts with antiviral immunity remains largely unknown. This is a significant omission because many economically important porcine viruses are monocytotropic, including our focus, PRRSV, which alone causes nearly $800 million economic loss annually in the U.S. swine industries. PRRSV is ideal for deciphering how monocytic cell activation statuses interact with antiviral immunity, because it directly infects subsets of monocytic cells and subverts overall immune responses. In this study, we systematically investigate the activation status of porcine monocytic cells to determine the intricate interaction of viral infection with activation statuses and functionally regulate antiviral immunity within the framework of the activation paradigm. Our findings may provide a means of potentiating antiviral immunity and leading to novel vaccines for PRRS prevention.
Characterization of the Mode of Action of a Potent Dengue Virus Capsid Inhibitor
Authors: Scaturro Pietro , Trist Iuni Margaret Laura , Paul David , Kumar Anil , Acosta Eliana G. , Byrd Chelsea M. , Jordan Robert , Brancale Andrea , Bartenschlager Ralf 
Published: July 2014

ABSTRACT

Dengue viruses (DV) represent a significant global health burden, with up to 400 million infections every year and around 500,000 infected individuals developing life-threatening disease. In spite of attempts to develop vaccine candidates and antiviral drugs, there is a lack of approved therapeutics for the treatment of DV infection. We have previously reported the identification of ST-148, a small-molecule inhibitor exhibiting broad and potent antiviral activity against DV
in vitro
and
in vivo
(C. M. Byrd et al., Antimicrob. Agents Chemother. 57:15–25, 2013, doi:10
.1128/AAC.01429-12). In the present study, we investigated the mode of action of this promising compound by using a combination of biochemical, virological, and imaging-based techniques. We confirmed that ST-148 targets the capsid protein and obtained evidence of bimodal antiviral activity affecting both assembly/release and entry of infectious DV particles. Importantly, by using a robust bioluminescence resonance energy transfer-based assay, we observed an ST-148-dependent increase of capsid self-interaction. These results were corroborated by molecular modeling studies that also revealed a plausible model for compound binding to capsid protein and inhibition by a distinct resistance mutation. These results suggest that ST-148-enhanced capsid protein self-interaction perturbs assembly and disassembly of DV nucleocapsids, probably by inducing structural rigidity. Thus, as previously reported for other enveloped viruses, stabilization of capsid protein structure is an attractive therapeutic concept that also is applicable to flaviviruses.

IMPORTANCE
Dengue viruses are arthropod-borne viruses representing a significant global health burden. They infect up to 400 million people and are endemic to subtropical and tropical areas of the world. Currently, there are neither vaccines nor approved therapeutics for the prophylaxis or treatment of DV infections, respectively. This study reports the characterization of the mode of action of ST-148, a small-molecule capsid inhibitor with potent antiviral activity against all DV serotypes. Our results demonstrate that ST-148 stabilizes capsid protein self-interaction, thereby likely perturbing assembly and disassembly of viral nucleocapsids by inducing structural rigidity. This, in turn, might interfere with the release of viral RNA from incoming nucleocapsids (uncoating) as well as assembly of progeny virus particles. As previously reported for other enveloped viruses, we propose the capsid as a novel tractable target for flavivirus inhibitors.
Comparison of Genotypes I and III in Japanese Encephalitis Virus Reveals Distinct Differences in Their Genetic and Host Diversity
Authors: Han Na , Adams James , Chen Ping , Guo Zhen-yang , Zhong Xiang-fu , Fang Wei , Li Na , Wen Lei , Tao Xiao-yan , Yuan Zhi-ming , Rayner Simon 
Published: July 2014

ABSTRACTJapanese encephalitis (JE) is an arthropod-borne disease associated with the majority of viral encephalitis cases in the Asia-Pacific region. The causative agent, Japanese encephalitis virus (JEV), has been phylogenetically divided into five genotypes. Recent surveillance data indicate that genotype I (GI) is gradually replacing genotype III (GIII) as the dominant genotype. To investigate the mechanism behind the genotype shift and the potential consequences in terms of vaccine efficacy, human cases, and virus dissemination, we collected (i) all full-length and partial JEV molecular sequences and (ii) associated genotype and host information comprising a data set of 873 sequences. We then examined differences between the two genotypes at the genetic and epidemiological level by investigating amino acid mutations, positive selection, and host range.IMPORTANCEWe found that although GI is dominant, it has fewer sites predicted to be under positive selection, a narrower host range, and significantly fewer human isolates. For the E protein, the sites under positive selection define a haplotype set for each genotype that shows striking differences in their composition and diversity, with GIII showing significantly more variety than GI. Our results suggest that GI has displaced GIII by achieving a replication cycle that is more efficient but is also more restricted in its host range.Japanese encephalitis is an arthropod-borne disease associated with the majority of viral encephalitis cases in the Asia-Pacific region. The causative agent, Japanese encephalitis virus (JEV), has been divided into five genotypes based on sequence similarity. Recent data indicate that genotype I (GI) is gradually replacing genotype III (GIII) as the dominant genotype. Understanding the reasons behind this shift and the potential consequences in terms of vaccine efficacy, human cases, and virus dissemination is important for controlling the spread of the virus and reducing human fatalities. We collected all available full-length and partial JEV molecular sequences and associated genotype and host information. We then examined differences between the two genotypes at the genetic and epidemiological levels by investigating amino acid mutations, positive selection, and host range. Our results suggest that GI has displaced GIII by achieving a replication cycle that is more efficient but more restricted in host range.
Undiagnosed diabetes from cross-sectional GP practice data: an approach to identify communities with high likelihood of undiagnosed diabetes
Authors: Bagheri Nasser , McRae Ian , Konings Paul , Butler Danielle , Douglas Kirsty , Del Fante Peter , Adams Robert 
Published: July 2014

ObjectivesTo estimate undiagnosed diabetes prevalence from general practitioner (GP) practice data and identify areas with high levels of undiagnosed and diagnosed diabetes.DesignData from the North-West Adelaide Health Survey (NWAHS) were used to develop a model which predicts total diabetes at a small area. This model was then applied to cross-sectional data from general practices to predict the total level of expected diabetes. The difference between total expected and already diagnosed diabetes was defined as undiagnosed diabetes prevalence and was estimated for each small area. The patterns of diagnosed and undiagnosed diabetes were mapped to highlight the areas of high prevalence.SettingNorth-West Adelaide, Australia.ParticipantsThis study used two population samples—one from the de-identified GP practice data (n=9327 active patients, aged 18 years and over) and another from NWAHS (n=4056, aged 18 years and over).Main outcome measuresTotal diabetes prevalence, diagnosed and undiagnosed diabetes prevalence at GP practice and Statistical Area Level 1.ResultsOverall, it was estimated that there was one case of undiagnosed diabetes for every 3–4 diagnosed cases among the 9327 active patients analysed. The highest prevalence of diagnosed diabetes was seen in areas of lower socioeconomic status. However, the prevalence of undiagnosed diabetes was substantially higher in the least disadvantaged areas.ConclusionsThe method can be used to estimate population prevalence of diabetes from general practices wherever these data are available. This approach both flags the possibility that undiagnosed diabetes may be a problem of less disadvantaged social groups, and provides a tool to identify areas with high levels of unmet need for diabetes care which would enable policy makers to apply geographic targeting of effective interventions.
‘I'm not a druggie, I'm just a diabetic’: a qualitative study of stigma from the perspective of adults with type 1 diabetes
Authors: Browne Jessica L , Ventura Adriana , Mosely Kylie , Speight Jane 
Published: July 2014

ObjectivesWhile health-related stigma has been the subject of considerable research in other conditions (eg, HIV/AIDS, obesity), it has not received substantial attention in diabetes. Our aim was to explore perceptions and experiences of diabetes-related stigma from the perspective of adults with type 1 diabetes mellitus (T1DM).DesignA qualitative study using semistructured interviews, which were audio recorded, transcribed and subject to thematic analysis.SettingAll interviews were conducted in non-clinical settings in metropolitan areas of Victoria, Australia.ParticipantsAdults aged ≥18 years with T1DM living in Victoria were eligible to take part. Participants were recruited primarily through the state consumer organisation representing people with diabetes. A total of 27 adults with T1DM took part: 15 (56%) were women; median IQR age was 42 (23) years and diabetes duration was 15 (20) years).ResultsAustralian adults with T1DM perceive and experience T1DM-specific stigma as well as stigma-by-association with type 2 diabetes. Such stigma is characterised by blame, negative social judgement, stereotyping, exclusion, rejection and discrimination. Participants identified the media, family and friends, healthcare professionals and school teachers as sources of stigma. The negative consequences of this stigma span numerous life domains, including impact on relationships and social identity, emotional well-being and behavioural management of T1DM. This stigma also led to reluctance to disclose the condition in various environments. Adults with T1DM can be both the target and the source of diabetes-related stigma.ConclusionsStigmatisation is part of the social experience of living with T1DM for Australian adults. Strategies and interventions to address and mitigate this diabetes-related stigma need to be developed and evaluated.
RB Family Tumor Suppressor Activity May Not Relate to Active Silencing of E2F Target Genes
Authors: Vormer Tinke L. , Wojciechowicz Kamila , Dekker Marleen , de Vries Sandra , van der Wal Anja , Delzenne-Goette Elly , Naik Sjalin H. , Song Ji-Ying , Dannenberg Jan-Hermen , Hansen Jacob B. , te Riele Hein 
Published: July 2014

Abstract
The retinoblastoma protein pRB and its two homologs p130 and p107 form the family of pocket proteins and play a major role in cell-cycle regulation and suppression of human and mouse tumorigenesis. Pocket proteins regulate the activity of E2F transcription factors during G1–S transition. Two mechanisms have been described: (i) pocket protein binding blocks the transactivation domain of activator E2Fs, inhibiting E2F-dependent transcription and (ii) E2F-bound pocket proteins can recruit chromatin remodeling proteins containing an LxCxE motif (x encoding any amino acid), resulting in active repression of E2F target genes. To investigate the importance of pRB's LxCxE-interacting motif in cell-cycle control and tumor suppression, we generated mouse embryonic fibroblasts and mice expressing a mutant pRB protein carrying an asparagine for phenylalanine substitution at position 750, abrogating LxCxE binding. Because p130 may compensate for loss of pRB, we studied pRBN750F activity in the presence and absence of p130. The pRB–LxCxE interaction was not required for cell-cycle arrest upon mitogen deprivation and cell-cell contact, but did contribute to RASV12- and radiation-induced cell-cycle arrest. Remarkably, the pRB–LxCxE interaction was not required for suppression of in vitro and in vivo transformation, even in the absence of p130. These results indicate that pRB's tumor suppressor activity is not effectuated by active silencing of E2F target genes, but rather by regulation of activator E2Fs or another unidentified mechanism. Furthermore, the in vitro response of pocket protein–perturbed cells to mitogen deprivation and cell–cell contact seems a better predictor of tumor development than the response to ectopic RASV12 expression. Cancer Res; 74(18); 5266–76. ©2014 AACR.