bluuebunny/crossref_metadata_2025_split huggingface dataset:
Novel Method Based on “En Passant” Mutagenesis Coupled with a Gaussia Luciferase Reporter Assay for Studying the Combined Effects of Human Cytomegalovirus Mutations
Authors: Drouot Emilien , Piret Jocelyne , Boivin Guy 
Published: July 2013

ABSTRACT

Human cytomegalovirus (HCMV) resistance to antivirals is a major problem in immunocompromised patients. Drug resistance is characterized by phenotypic testing or genotypic analysis of the phosphotransferase (UL97) and DNA polymerase (UL54) genes. However, genotypic assays require further characterization of unknown mutations in the drug resistance phenotype. Here, we describe a novel method for generating single or multiple mutations anywhere in the HCMV genome and for studying their effects on drug susceptibility. This method is based on cloning of the reference AD169 strain in a bacterial artificial chromosome and the use of “en passant” mutagenesis in bacteria to introduce mutations in recombinant HCMV without scar sequences. We also used this methodology to introduce the
Gaussia
luciferase reporter gene into the genome of the recombinant virus. To validate our system, the well-characterized single mutants with UL97 A594V and UL54 E756K mutations as well as the undescribed A594V/E756K double mutant were generated and their drug susceptibility profiles were determined by measuring the luciferase activity in cell culture supernatants. Drug susceptibility phenotypes for the A594V (8.2-fold increase in ganciclovir 50% effective concentration [EC
50
]) and E756K (1.9-, 3.9-, and 3.0-fold increases in ganciclovir, foscarnet, and cidofovir EC
50
s, respectively) mutants were similar to those previously reported, while the double mutant exhibited 10.8-, 4.1-, and 2.0-fold increases in ganciclovir, foscarnet, and cidofovir EC
50
s, respectively. The combination of the
Gaussia
luciferase reporter-based assay with the markerless “en passant” mutagenesis methodology constitutes an efficient system for studying phenotypes with single or multiple HCMV mutations.
Virion Factors That Target Daxx To Overcome Intrinsic Immunity
Authors: Schreiner Sabrina , Wodrich Harald 
Published: July 2013

ABSTRACTPML nuclear bodies and their associated functions are part of an intrinsic cellular mechanism aimed at maintaining transcriptional control over viral gene expression and preventing replication of invading viruses. To overcome these barriers, many viruses express early nonstructural, multifunctional proteins to support the viral replication cycle or modulate host immune responses. Virion proteins constituting the invading particle are traditionally investigated for their role in transport during entry or egress and in the assembly of new virions. The additional functions of virion proteins have largely been ignored, in contrast to those of their nonstructural counterparts. A number of recent reports suggest that several virion proteins may also play vital roles in gene activation processes, in particular by counteracting intrinsic immune mechanisms mediated by the PML nuclear body-associated cellular factors Daxx, ATRX, and Sp100. These virion proteins share several features with their more potent nonstructural counterparts, and they may serve to bridge the gap in the early phase of an infection until immediate early viral gene expression is established. In this review, we discuss how virion proteins are an integral part of gene regulation among several viral families and to what extent structural proteins of incoming virions may contribute to species barrier, latency, and oncogenesis.
Uncoupling of Influenza A Virus Transcription and Replication through Mutation of the Unpaired Adenosine in the Viral RNA Promoter
Authors: te Velthuis Aartjan J. W. , Turrell Lauren , Vreede Frank T. , Fodor Ervin 
Published: July 2013

ABSTRACT
Transcription and replication of the influenza A virus RNA genome are mediated by the viral RNA polymerase from a promoter consisting of the partially base-paired 3′ and 5′ termini of viral genome segments. Here we show that transcription and replication can be uncoupled by mutation of an unpaired adenosine in the 5′ strand of the promoter. This residue is important for transcription but not replication by being essential for the cap-binding activity of the RNA polymerase.
The C-Terminal Domain of Chikungunya Virus nsP2 Independently Governs Viral RNA Replication, Cytopathicity, and Inhibition of Interferon Signaling
Authors: Fros Jelke J. , van der Maten Erika , Vlak Just M. , Pijlman Gorben P. 
Published: July 2013

ABSTRACT
Alphavirus nonstructural protein 2 (nsP2) has pivotal roles in viral RNA replication, host cell shutoff, and inhibition of antiviral responses. Mutations that individually rendered other alphaviruses noncytopathic were introduced into chikungunya virus nsP2. Results show that (i) nsP2 mutation P718S only in combination with KR649AA or adaptive mutation D711G allowed noncytopathic replicon RNA replication, (ii) prohibiting nsP2 nuclear localization abrogates inhibition of antiviral interferon-induced JAK-STAT signaling, and (iii) nsP2 independently affects RNA replication, cytopathicity, and JAK-STAT signaling.
The Short Stalk Length of Highly Pathogenic Avian Influenza H5N1 Virus Neuraminidase Limits Transmission of Pandemic H1N1 Virus in Ferrets
Authors: Blumenkrantz Deena , Roberts Kim L. , Shelton Holly , Lycett Samantha , Barclay Wendy S. 
Published: July 2013

ABSTRACT
H5N1 influenza viruses pose a pandemic threat but have not acquired the ability to support sustained transmission between mammals in nature. The restrictions to transmissibility of avian influenza viruses in mammals are multigenic, and overcoming them requires adaptations in hemagglutinin (HA) and PB2 genes. Here we propose that a further restriction to mammalian transmission of the majority of highly pathogenic avian influenza (HPAI) H5N1 viruses may be the short stalk length of the neuraminidase (NA) protein. This genetic feature is selected for when influenza viruses adapt to chickens. In our study, a recombinant virus with seven gene segments from a human isolate of the 2009 H1N1 pandemic combined with the NA gene from a typical chicken-adapted H5N1 virus with a short stalk did not support transmission by respiratory droplet between ferrets. This virus was also compromised in multicycle replication in cultures of human airway epithelial cells at 32°C. These defects correlated with a reduction in the ability of virus with a short-stalk NA to penetrate mucus and deaggregate virions. The deficiency in transmission and in cleavage of tethered substrates was overcome by increasing the stalk length of the NA protein. These observations suggest that H5N1 viruses that acquire a long-stalk NA through reassortment might be more likely to support transmission between humans. Phylogenetic analysis showed that reassortment with long-stalk NA occurred sporadically and as recently as 2011. However, all identified H5N1 viruses with a long-stalk NA lacked other mammalian adapting features and were thus several genetic steps away from becoming transmissible between humans.
The
HEX1
Gene of Fusarium graminearum Is Required for Fungal Asexual Reproduction and Pathogenesis and for Efficient Viral RNA Accumulation of Fusarium graminearum Virus 1
Authors: Son Moonil , Lee Kyung-Mi , Yu Jisuk , Kang Minji , Park Jin Man , Kwon Sun-Jung , Kim Kook-Hyung 
Published: July 2013

ABSTRACT

The accumulation of viral RNA depends on many host cellular factors. The hexagonal peroxisome (Hex1) protein is a fungal protein that is highly expressed when the DK21 strain of
Fusarium graminearum
virus 1 (FgV1) infects its host, and Hex1 affects the accumulation of FgV1 RNA. The Hex1 protein is the major constituent of the Woronin body (WB), which is a peroxisome-derived electron-dense core organelle that seals the septal pore in response to hyphal wounding. To clarify the role of Hex1 and the WB in the relationship between FgV1 and
Fusarium graminearum
, we generated targeted gene deletion and overexpression mutants. Although neither
HEX1
gene deletion nor overexpression substantially affected vegetative growth, both changes reduced the production of asexual spores and reduced virulence on wheat spikelets in the absence of FgV1 infection. However, the vegetative growth of deletion and overexpression mutants was increased and decreased, respectively, upon FgV1 infection compared to that of an FgV1-infected wild-type isolate. Viral RNA accumulation was significantly decreased in deletion mutants but was significantly increased in overexpression mutants compared to the viral RNA accumulation in the virus-infected wild-type control. Overall, these data indicate that the
HEX1
gene plays a direct role in the asexual reproduction and virulence of
F. graminearum
and facilitates viral RNA accumulation in the FgV1-infected host fungus.
Exosomes Derived from Epstein-Barr Virus-Infected Cells Are Internalized via Caveola-Dependent Endocytosis and Promote Phenotypic Modulation in Target Cells
Authors: Nanbo Asuka , Kawanishi Eri , Yoshida Ryuji , Yoshiyama Hironori 
Published: July 2013

ABSTRACT
Epstein-Barr virus (EBV), a human gammaherpesvirus, establishes a lifelong latent infection in B lymphocytes and epithelial cells following primary infection. Several lines of evidence suggest that exosomes derived from EBV-infected cells are internalized and transfer viral factors, including EBV-encoded latent membrane protein and microRNAs, to the recipient cells. However, the detailed mechanism by which exosomes are internalized and their physiological impact on the recipient cells are still poorly understood. In this study, we visualized the internalization of fluorescently labeled exosomes derived from EBV-uninfected and EBV-infected B cells of type I and type III latency into EBV-negative epithelial cells. In this way, we demonstrated that exosomes derived from all three cell types were internalized into the target cells in a similar fashion. Internalization of exosomes was significantly suppressed by treatment with an inhibitor of dynamin and also by the knockdown of caveolin-1. Labeled exosomes were colocalized with caveolae and subsequently trafficked through endocytic pathways. Moreover, we observed that exosomes derived from type III latency cells upregulated proliferation and expression of intercellular adhesion molecule 1 (ICAM-1) in the recipient cells more significantly than did those derived from EBV-negative and type I latency cells. We also identified the EBV latent membrane protein 1 (LMP1) gene as responsible for induction of ICAM-1 expression. Taken together, our data indicate that exosomes released from EBV-infected B cells are internalized via caveola-dependent endocytosis, which, in turn, contributes to phenotypic changes in the recipient cells through transferring one or more viral factors.
The Homologous Tripartite Viral RNA Polymerase of A/Swine/Korea/CT1204/2009(H1N2) Influenza Virus Synergistically Drives Efficient Replication and Promotes Respiratory Droplet Transmission in Ferrets
Authors: Pascua Philippe Noriel Q. , Song Min-Suk , Kwon Hyeok-Il , Lim Gyo-Jin , Kim Eun-Ha , Park Su-Jin , Lee Ok-Jun , Kim Chul-Joong , Webby Richard J. , Webster Robert G. , Choi Young-Ki 
Published: July 2013

ABSTRACT

We previously reported that influenza A/swine/Korea/1204/2009(H1N2) virus was virulent and transmissible in ferrets in which the respiratory-droplet-transmissible virus (CT-Sw/1204) had acquired simultaneous hemagglutinin (HA
D225G
) and neuraminidase (NA
S315N
) mutations. Incorporating these mutations into the nonpathogenic A/swine/Korea/1130/2009(H1N2, Sw/1130) virus consequently altered pathogenicity and growth in animal models but could not establish efficient transmission or noticeable disease. We therefore exploited various reassortants of these two viruses to better understand and identify other viral factors responsible for pathogenicity, transmissibility, or both. We found that possession of the CT-Sw/1204 tripartite viral polymerase enhanced replicative ability and pathogenicity in mice more significantly than did expression of individual polymerase subunit proteins. In ferrets, homologous expression of viral RNA polymerase complex genes in the context of the mutant Sw/1130 carrying the HA
225G
and NA
315N
modifications induced optimal replication in the upper nasal and lower respiratory tracts and also promoted efficient aerosol transmission to respiratory droplet contact ferrets. These data show that the synergistic function of the tripartite polymerase gene complex of CT-Sw/1204 is critically important for virulence and transmission independent of the surface glycoproteins. Sequence comparison results reveal putative differences that are likely to be responsible for variation in disease. Our findings may help elucidate previously undefined viral factors that could expand the host range and disease severity induced by triple-reassortant swine viruses, including the A(H1N1)pdm09 virus, and therefore further justify the ongoing development of novel antiviral drugs targeting the viral polymerase complex subunits.
The Template Specificity of Bacteriophage Φ6 RNA Polymerase
Authors: Qiao Jian , Mindich Leonard 
Published: July 2013

ABSTRACT

Bacteriophage Φ6 contains three double-stranded RNA (dsRNA) genomic segments, L, M, and S. The RNA is located inside a core particle composed of multiple copies of a major structural protein, an RNA-dependent RNA polymerase, a hexameric NTPase, and an auxiliary protein. The virion RNA polymerase in the core particle transcribes segments M and S
in vitro
. Segment L is transcribed poorly because its transcript starts with GU instead of GG found on segments S and M. Transcription
in vivo
is modified by the binding of host protein YajQ to the outside the core particle so that segment L is transcribed well. This mechanism is the determinant of the temporal control of gene expression in Φ6. Mutants of Φ6 have been isolated that are independent of YajQ for transcription of segment L. The mutations are found in the gene of the viral polymerase or the major capsid protein or both. These mutants are capable of transcribing segment L with the GU start or GA or GC. The same is found to be true when YajQ is added to wild-type particles. Minus-strand synthesis has restrictions that are different from that of plus-strand synthesis, and YajQ or mutations to independence do not modify minus-strand synthesis behavior. Purified polymerase P2 is able to transcribe dsRNA, but transcription behavior of segment L by both wild-type and mutant polymerases is different from that seen in capsid structures. Adding YajQ to purified polymerase does not change its transcription specificity.
Presentation Overrides Specificity: Probing the Plasticity of Alphaviral Proteolytic Activity through Mutational Analysis
Authors: Lulla Valeria , Karo-Astover Liis , Rausalu Kai , Merits Andres , Lulla Aleksei 
Published: July 2013

ABSTRACT

Semliki Forest virus (genus
Alphavirus
) is an important model for studying regulated nonstructural (ns) polyprotein processing. In this study, we evaluated the strictness of the previously outlined cleavage rules, accounting for the timing and outcome of each of three cleavages within the ns polyprotein P1234, and assessed the significance of residues P6 to P4 within the cleavage sites using an alanine scanning approach. The processing of the 1/2 and 3/4 sites was most strongly affected following changes in residues P5 and P4, respectively. However, none of the mutations had a detectable effect on the processing of the 2/3 site. An analysis of recombinant viruses bearing combinations of mutations in cleavage sites revealed tolerance toward the cooccurrence of native and mutated cleavage sites within the same polyprotein, suggesting a remarkable plasticity of the protease recognition pocket. Even in a virus in which all of the cleavage sequences were replaced with alanines in the P6, P5, and P4 positions, the processing pattern was largely preserved, without leading to reversion of cleavage site mutations. Instead, the emergence of second-site mutations was identified, among which Q706R/L in nsP2 was confirmed to be associated with the recognition of the P4 position within the modified cleavage sites. Our results imply that the spatial arrangement of the viral replication complex inherently contributes to scissile-site presentation for the protease, alleviating stringent sequence recognition requirements yet ensuring the precision and the correct order of processing events. Obtaining a proper understanding of the consequences of cleavage site manipulations may provide new tools for taming alphaviruses.
Interaction of Herpes Simplex Virus ICP0 with ND10 Bodies: a Sequential Process of Adhesion, Fusion, and Retention
Authors: Gu Haidong , Zheng Yi , Roizman Bernard 
Published: July 2013

ABSTRACT
On entry into the nucleus, herpes simplex virus 1 (HSV-1) DNA localizes to nuclear bodies known as ND10. Gene repression imposed by ND10 is released by a viral protein, ICP0, via degradation of the ND10 constituents promyelocytic leukemia protein (PML) and Sp100 and the subsequent dispersal of ND10 bodies. In order to understand the dynamic interaction between ICP0 and ND10, we carried out deletion mapping to identify the domains of ICP0 responsible for its association with ND10. Here, we report the following. (i) An ND10 entry signal (ND10-ES), located between residues 245 and 474, is required for ICP0 to penetrate and fuse with ND10. ICP0 lacking ND10-ES adheres to the surface of ND10 but fails to enter. (ii) In the absence of ND10-ES, the E3 ubiquitin ligase of ICP0 facilitates the transient adhesion of the truncated ICP0 to the ND10 surface, whereas the presence of ND10-ES in ICP0 renders ND10 fusion regardless of the E3 ligase activity. (iii) The C terminus of ICP0 is required for retention of ICP0 in ND10 but plays no role in the recruitment process. (iv) The adverse effects of an inactive RING domain on viral replication are partially reversed by deleting either ND10-ES or the C-terminal retention domain, suggesting that additional ICP0 functions require the release of ICP0 from ND10. Based on these results, we conclude that association of ICP0 and ND10 is a dynamic process, in which three sequential steps—adhesion, fusion, and retention—are adopted to stabilize the interaction. A faithful execution of these steps defines the ultimate productivity of the virus.
Association of the Vaccinia Virus A11 Protein with the Endoplasmic Reticulum and Crescent Precursors of Immature Virions
Authors: Maruri-Avidal Liliana , Weisberg Andrea S. , Moss Bernard 
Published: July 2013

ABSTRACT

The apparent
de novo
formation of viral membranes within cytoplasmic factories is a mysterious, poorly understood first step in poxvirus morphogenesis. Genetic studies identified several viral proteins essential for membrane formation and the assembly of immature virus particles. Their repression results in abortive replication with the accumulation of dense masses of viroplasm. In the present study, we further characterized one of these proteins, A11, and investigated its association with cellular and viral membranes under normal and abortive replication conditions. We discovered that A11 colocalized in cytoplasmic factories with the endoplasmic reticulum (ER) and L2, another viral protein required for morphogenesis. Confocal microscopy and subcellular fractionation indicated that A11 was not membrane associated in uninfected cells, whereas L2 still colocalized with the ER. Cell-free transcription and translation experiments indicated that both A11 and L2 are tail-anchored proteins that associate posttranslationally with membranes and likely require specific cytoplasmic targeting chaperones. Transmission electron microscopy indicated that A11, like L2, associated with crescent membranes and immature virions during normal infection and with vesicles and tubules near masses of dense viroplasm during abortive infection in the absence of the A17 or A14 protein component of viral membranes. When the synthesis of A11 was repressed, “empty” immature-virion-like structures formed in addition to masses of viroplasm. The immature-virion-like structures were labeled with antibodies to A17 and to the D13 scaffold protein and were closely associated with calnexin-labeled ER. These studies revealed similarities and differences between A11 and L2, both of which may be involved in the recruitment of the ER for virus assembly.
How do patients with end-stage ankle arthritis decide between two surgical treatments? A qualitative study
Authors: Zaidi Razi , Pfeil Michael , Macgregor Alexander J , Goldberg Andy 
Published: July 2013

ObjectiveTo examine how patients decide between ankle fusion and ankle replacement in end-stage ankle arthritis.DesignPurposive patient selection, semistructured interviews, thematic analysis.SettingRoyal National Orthopaedic Hospital, Stanmore, UK.Participants14 patients diagnosed with end-stage ankle osteoarthritis.ResultsWe interviewed 6 men and 8 women with a mean age of 58 years (range 41–83). All had opted for surgery after failure of at least 6 months of conservative management, sequentially trading-off daily activities to limit the evolving pain. To decide between two offered treatments of ankle fusion and total ankle replacement (TAR), three major sources informed the patients’ decision-making process: their surgeon, peers and the internet. The treating surgeon was viewed as the most reliable and influential source of information. Information gleaned from other patients was also important, but with questionable reliability, as was information from the internet, both of which invariably required validation by the surgeon and in some cases the general practitioner.ConclusionsPatients seek knowledge from a wealth of sources including the internet, web forums and other patients. While they leverage each of these sources to guide decision-making, the most important and influential factor in governing how patients decide on any particular surgical intervention is their surgeon. A high quality doctor–patient relationship, coupled with clear, balanced and complete information is essential to enable shared decision-making to become a standard model of care.