bluuebunny/crossref_metadata_2025_split huggingface dataset:
Multilocus Microsatellite Markers for Molecular Typing of
Candida glabrata
: Application to Analysis of Genetic Relationships between Bloodstream and Digestive System Isolates
Authors: Enache-Angoulvant A. , Bourget M. , Brisse S. , Stockman-Pannier C. , Diancourt L. , François N. , Rimek D. , Fairhead C. , Poulain Daniel , Hennequin C. 
Published: September 2010

ABSTRACT

Candida glabrata
has emerged as the second most common etiologic agent, after
Candida albicans
, of superficial and invasive candidiasis in adults. Strain typing is essential for epidemiological investigation, but easy-to-use and reliable typing methods are still lacking. We report the use of a multilocus microsatellite typing method with a set of eight markers on a panel of 180 strains, including 136 blood isolates from hospitalized patients and 34 digestive tract isolates from nonhospitalized patients. A total of 44 different alleles were observed, generating 87 distinct genotypes. In addition to perfect reproducibility, typing ability, and stability, the method had a discriminatory power calculated at 0.97 when all 8 markers were associated, making it suitable for tracing strains. In addition, it is shown that digestive tract isolates differed from blood culture isolates by exhibiting a higher genotypic diversity associated with different allelic frequencies and preferentially did not group in clonal complexes (CCs). The demonstration of the occurrence of microevolution in digestive strains supports the idea that
C. glabrata
can be a persistent commensal of the human gut.
Respiratory Syncytial Virus Induces Host RNA Stress Granules To Facilitate Viral Replication
Authors: Lindquist Michael E. , Lifland Aaron W. , Utley Thomas J. , Santangelo Philip J. , Crowe James E. 
Published: September 2010

ABSTRACT
Mammalian cell cytoplasmic RNA stress granules are induced during various conditions of stress and are strongly associated with regulation of host mRNA translation. Several viruses induce stress granules during the course of infection, but the exact function of these structures during virus replication is not well understood. In this study, we showed that respiratory syncytial virus (RSV) induced host stress granules in epithelial cells during the course of infection. We also showed that stress granules are distinct from cytoplasmic viral inclusion bodies and that the RNA binding protein HuR, normally found in stress granules, also localized to viral inclusion bodies during infection. Interestingly, we demonstrated that infected cells containing stress granules also contained more RSV protein than infected cells that did not form inclusion bodies. To address the role of stress granule formation in RSV infection, we generated a stable epithelial cell line with reduced expression of the Ras-GAP SH3 domain-binding protein (G3BP) that displayed an inhibited stress granule response. Surprisingly, RSV replication was impaired in these cells compared to its replication in cells with intact G3BP expression. In contrast, knockdown of HuR by RNA interference did not affect stress granule formation or RSV replication. Finally, using RNA probes specific for RSV genomic RNA, we found that viral RNA predominantly localized to viral inclusion bodies but a small percentage also interacted with stress granules during infection. These results suggest that RSV induces a host stress granule response and preferentially replicates in host cells that have committed to a stress response.
Sphingomyelin Activates Hepatitis C Virus RNA Polymerase in a Genotype-Specific Manner
Authors: Weng Leiyun , Hirata Yuichi , Arai Masaaki , Kohara Michinori , Wakita Takaji , Watashi Koichi , Shimotohno Kunitada , He Ying , Zhong Jin , Toyoda Tetsuya 
Published: September 2010

ABSTRACT
Hepatitis C virus (HCV) replication and infection depend on the lipid components of the cell, and replication is inhibited by inhibitors of sphingomyelin biosynthesis. We found that sphingomyelin bound to and activated genotype 1b RNA-dependent RNA polymerase (RdRp) by enhancing its template binding activity. Sphingomyelin also bound to 1a and JFH1 (genotype 2a) RdRps but did not activate them. Sphingomyelin did not bind to or activate J6CF (2a) RdRp. The sphingomyelin binding domain (SBD) of HCV RdRp was mapped to the helix-turn-helix structure (residues 231 to 260), which was essential for sphingomyelin binding and activation. Helix structures (residues 231 to 241 and 247 to 260) are important for RdRp activation, and 238S and 248E are important for maintaining the helix structures for template binding and RdRp activation by sphingomyelin. 241Q in helix 1 and the negatively charged 244D at the apex of the turn are important for sphingomyelin binding. Both amino acids are on the surface of the RdRp molecule. The polarity of the phosphocholine of sphingomyelin is important for HCV RdRp activation. However, phosphocholine did not activate RdRp. Twenty sphingomyelin molecules activated one RdRp molecule. The biochemical effect of sphingomyelin on HCV RdRp activity was virologically confirmed by the HCV replicon system. We also found that the SBD was the lipid raft membrane localization domain of HCV NS5B because JFH1 (2a) replicon cells harboring NS5B with the mutation A242C/S244D moved to the lipid raft while the wild type did not localize there. This agreed with the myriocin sensitivity of the mutant replicon. This sphingomyelin interaction is a target for HCV infection because most HCV RdRps have 241Q.
The HIV-1 Central Polypurine Tract Functions as a Second Line of Defense against APOBEC3G/F
Authors: Hu Chunling , Saenz Dyana T. , Fadel Hind J. , Walker William , Peretz Mary , Poeschla Eric M. 
Published: September 2010

ABSTRACT

HIV-1 and certain other retroviruses initiate plus-strand synthesis in the center of the genome as well as at the standard retroviral 3′ polypurine tract. This peculiarity of reverse transcription results in a central DNA “flap” structure that has been of controversial functional significance. We mutated both HIV-1 flap-generating elements, the central polypurine tract (cPPT) and the central termination sequence (CTS). To avoid an ambiguity of previous studies, we did so without affecting integrase coding. DNA flap formation was disrupted but single-cycle infection was unaffected in all target cells tested, regardless of cell cycle status. Spreading HIV-1 infection was also normal in most T cell lines, and flap mutant viruses replicated equivalently to the wild type in nondividing cells, including macrophages. However, spreading infection of flap mutant HIV-1 was impaired in non-
vif
-permissive cells (HuT78, H9, and primary human peripheral blood mononuclear cells [PBMCs]), suggesting APOBEC3G (A3G) restriction. Single-cycle infections confirmed that
vif
-intact flap mutant HIV-1 is restricted by producer cell A3G/F. Combining the Δ
vif
and cPPT-CTS mutations increased A3G restriction synergistically. Moreover, RNA interference knockdown of A3G in HuT78 cells released the block to flap mutant HIV-1 replication. Flap mutant HIV-1 also accrued markedly increased A3G-mediated G→A hypermutation compared to that of wild-type HIV-1 (a full log
10
in the 0.36 kb downstream of the mutant cPPT). We suggest that the triple-stranded DNA structure, the flap, is not the consequential outcome. The salient functional feature is central plus-strand initiation, which functions as a second line of defense against single-stranded DNA editing by A3 proteins that survive producer cell degradation by Vif.
The Varicella-Zoster Virus ORFS/L (ORF0) Gene Is Required for Efficient Viral Replication and Contains an Element Involved in DNA Cleavage
Authors: Kaufer Benedikt B. , Smejkal Benjamin , Osterrieder Nikolaus 
Published: September 2010

ABSTRACT

The genome of varicella-zoster virus (VZV), a human alphaherpesvirus, consists of two unique regions, unique long (U
L
) and unique short (U
S
), each of which is flanked by inverted repeats. During replication, four isomers of the viral DNA are generated which are distinguished by the relative orientations of U
L
and U
S
. VZV virions predominantly package two isomeric forms of the genome that have a fixed orientation of U
L
. An open reading frame (ORF) of unknown function, ORFS/L, also referred to as ORF0, is located at the extreme terminus of U
L
, directly adjacent to the
a
-like sequences, which are known to be involved in cleavage and packaging of viral DNA. We demonstrate here that the ORFS/L protein localizes to the Golgi network in infected and transfected cells. Furthermore, we were able to demonstrate that deletion of the predicted ORFS/L gene is lethal, while retention of the N-terminal 28 amino acid residues resulted in viable yet replication-impaired virus. The growth defect was only partially attributable to the expression of the ORFS/L product, suggesting that the 5′ region of ORFS/L contains a sequence element crucial for cleavage/packaging of viral DNA. Consequently, mutations introduced into the extreme 5′ terminus of ORFS/L resulted in a defect in DNA cleavage, indicating that the region is indeed involved in the processing of viral DNA. Since the sequence element has no counterpart at the other end of U
L
, we concluded that our results can provide an explanation for the almost exclusive orientation of the U
L
seen in packaged VZV DNA.
Extracellular 2′-5′ Oligoadenylate Synthetase Stimulates RNase L-Independent Antiviral Activity: a Novel Mechanism of Virus-Induced Innate Immunity
Authors: Kristiansen Helle , Scherer Christina A. , McVean Maralee , Iadonato Shawn P. , Vends Susanne , Thavachelvam Karthiga , Steffensen Thomas B. , Horan Kristy A. , Kuri Thomas , Weber Friedemann , Paludan Søren R. , Hartmann Rune 
Published: September 2010

ABSTRACTThe 2′-5′ oligoadenylate synthetase (OAS) proteins are traditionally considered intracellular antiviral proteins. However, several studies demonstrate a correlation between the concentration of freely circulating OAS protein in sera from hepatitis C patients and their clinical prognosis. Here we demonstrate that extracellular OAS1 enters into cells and possesses a strong antiviral activity, bothin vitroandin vivo, which is independent of RNase L. The OAS protein directly inhibits viral proliferation and does not require the activation of known antiviral signaling pathways. We propose that OAS produced by cells infected with viruses is released to the extracellular space, where it acts as a paracrine antiviral agent. Thus, the OAS protein represents the first direct antiviral compound released by virus-infected cells.
Intracellular Distribution of NS1 Correlates with the Infectivity and Interferon Antagonism of an Avian Influenza Virus (H7N1)
Authors: Keiner Bjoern , Maenz Benjamin , Wagner Ralf , Cattoli Giovanni , Capua Ilaria , Klenk Hans-Dieter 
Published: September 2010

ABSTRACT
Highly pathogenic avian influenza viruses of subtype H7N1 that emerged during an outbreak in 1999 and 2000 in Italy differ from their low-pathogenicity precursor viruses by changes in several genes, including three mutations in the NS1 protein. Two of them involve amino acid exchanges located within or closely adjacent to the nuclear export signal of NS1. The third mutation resulted in a new stop codon and thereby a C-terminal truncation of the NS1 protein of the highly pathogenic viruses. To find out whether these mutations contribute to the phenotypic differences between the highly pathogenic and low pathogenic viruses, we generated recombinants of the highly pathogenic A/ostrich/Italy/984/00 strain that contained the nuclear export signal and/or the extended C terminus of NS1 of a low pathogenic virus (A/chicken/Italy/1082/99). Using these recombinants we could demonstrate that replication rate and spread of infection in chicken fibroblast cultures, as well as infectivity for chicken embryos is reduced, whereas the mean death time for chicken embryos is increased, when the highly pathogenic virus acquires the NS1 motifs of the low pathogenic virus. Analysis of beta interferon transcription in chicken fibroblasts infected with the recombinants revealed that the mutations observed in the nuclear export signal of the highly pathogenic viruses were responsible for the enhanced interferon antagonism of these viruses. Cell fractionation and immunofluorescence studies in chicken fibroblasts showed that the nuclear export signal of the highly pathogenic viruses is responsible for cytoplasmic accumulation of NS1, whereas the C-terminal truncation promotes transport into the nucleoli. Comparative analysis in human A549 cells indicated that intracellular distribution of NS1 is host specific. Taken together, these observations support the concept that compartmentalization of NS1 within the cell contributes to the pathogenicity of avian influenza viruses.
Virulence-Associated Substitution D222G in the Hemagglutinin of 2009 Pandemic Influenza A(H1N1) Virus Affects Receptor Binding
Authors: Chutinimitkul Salin , Herfst Sander , Steel John , Lowen Anice C. , Ye Jianqiang , van Riel Debby , Schrauwen Eefje J. A. , Bestebroer Theo M. , Koel Björn , Burke David F. , Sutherland-Cash Kyle H. , Whittleston Chris S. , Russell Colin A. , Wales David J. , Smith Derek J. , Jonges Marcel , Meijer Adam , Koopmans Marion , Rimmelzwaan Guus F. , Kuiken Thijs , Osterhaus Albert D. M. E. , García-Sastre Adolfo , Perez Daniel R. , Fouchier Ron A. M. 
Published: September 2010

ABSTRACT

The clinical impact of the 2009 pandemic influenza A(H1N1) virus (pdmH1N1) has been relatively low. However, amino acid substitution D222G in the hemagglutinin of pdmH1N1 has been associated with cases of severe disease and fatalities. D222G was introduced in a prototype pdmH1N1 by reverse genetics, and the effect on virus receptor binding, replication, antigenic properties, and pathogenesis and transmission in animal models was investigated. pdmH1N1 with D222G caused ocular disease in mice without further indications of enhanced virulence in mice and ferrets. pdmH1N1 with D222G retained transmissibility via aerosols or respiratory droplets in ferrets and guinea pigs. The virus displayed changes in attachment to human respiratory tissues
in vitro
, in particular increased binding to macrophages and type II pneumocytes in the alveoli and to tracheal and bronchial submucosal glands. Virus attachment studies further indicated that pdmH1N1 with D222G acquired dual receptor specificity for complex α2,3- and α2,6-linked sialic acids. Molecular dynamics modeling of the hemagglutinin structure provided an explanation for the retention of α2,6 binding. Altered receptor specificity of the virus with D222G thus affected interaction with cells of the human lower respiratory tract, possibly explaining the observed association with enhanced disease in humans.
Virally Induced Cellular MicroRNA miR-155 Plays a Key Role in B-Cell Immortalization by Epstein-Barr Virus
Authors: Linnstaedt Sarah D. , Gottwein Eva , Skalsky Rebecca L. , Luftig Micah A. , Cullen Bryan R. 
Published: September 2010

ABSTRACT

Infection of resting primary human B cells by Epstein-Barr virus (EBV) results in their transformation into indefinitely proliferating lymphoblastoid cell lines (LCLs). LCL formation serves as a model for lymphomagenesis, and LCLs are phenotypically similar to EBV-positive diffuse large B-cell lymphomas (DLBCLs), which represent a common AIDS-associated malignancy. B-cell infection by EBV induces the expression of several cellular microRNAs (miRNAs), most notably miR-155, which is overexpressed in many tumors and can induce B-cell lymphomas when overexpressed in animals. Here, we demonstrate that miR-155 is the most highly expressed miRNA in LCLs and that the selective inhibition of miR-155 function specifically inhibits the growth of both LCLs and the DLBCL cell line IBL-1. Cells lacking miR-155 are inefficient in progressing through S phase and spontaneously undergo apoptosis. In contrast, three other B-cell lymphoma lines, including two EBV-positive Burkitt's lymphoma cell lines, grew normally in the absence of miR-155 function. These data identify the induction of cellular miR-155 expression by EBV as critical for the growth of both laboratory-generated LCLs and naturally occurring DLBCLs and suggest that targeted inhibition of miR-155 function could represent a novel approach to the treatment of DLBCL
in vivo
.
Dendritic Cell Activation by Recombinant Hemagglutinin Proteins of H1N1 and H5N1 Influenza A Viruses
Authors: Liu Wen-Chun , Lin Shih-Chang , Yu Yen-Ling , Chu Ching-Liang , Wu Suh-Chin 
Published: September 2010

ABSTRACT
Since dendritic cells may play a key role in defense against influenza virus infection, we examined the effects of recombinant hemagglutinin (HA) proteins derived from mouse-adapted H1N1 (A/WSN/1933), swine-origin 2009 pandemic H1N1 (A/Texas/05/2009), and highly pathogenic avian influenza H5N1 (A/Thailand/KAN-1/2004) viruses on mouse myeloid dendritic cells (mDCs). The results reveal that tumor necrosis factor alpha (TNF-α), interleukin-12 (IL-12) p70, and major histocompatibility complex class II (MHC-II) expression was increased in mDCs after treatment with recombinant HA proteins of H1N1 and H5N1. The specificity of recombinant HA treatments for mDC activation was diminished after proteinase K digestion. HA apparently promotes mDC maturation by enhancing CD40 and CD86 expression and suppressing endocytosis. No significant differences in mDC activation were observed among recombinant proteins of H1N1 and H5N1. The stimulation of mDCs by HA proteins of H1N1 and H5N1 was completely MyD88 dependent. These findings may provide useful information for the development of more-effective influenza vaccines.
High-Throughput, Sensitive Quantification of Repopulating Hematopoietic Stem Cell Clones
Authors: Kim Sanggu , Kim Namshin , Presson Angela P. , An Dong Sung , Mao Si Hua , Bonifacino Aylin C. , Donahue Robert E. , Chow Samson A. , Chen Irvin S. Y. 
Published: September 2010

ABSTRACT
Retroviral vector-mediated gene therapy has been successfully used to correct genetic diseases. However, a number of studies have shown a subsequent risk of cancer development or aberrant clonal growths due to vector insertion near or within proto-oncogenes. Recent advances in the sequencing technology enable high-throughput clonality analysis via vector integration site (VIS) sequencing, which is particularly useful for studying complex polyclonal hematopoietic progenitor/stem cell (HPSC) repopulation. However, clonal repopulation analysis using the current methods is typically semiquantitative. Here, we present a novel system and standards for accurate clonality analysis using 454 pyrosequencing. We developed a bidirectional VIS PCR method to improve VIS detection by concurrently analyzing both the 5′ and the 3′ vector-host junctions and optimized the conditions for the quantitative VIS sequencing. The assay was validated by quantifying the relative frequencies of hundreds of repopulating HPSC clones in a nonhuman primate. The reliability and sensitivity of the assay were assessed using clone-specific real-time PCR. The majority of tested clones showed a strong correlation between the two methods. This assay permits high-throughput and sensitive assessment of clonal populations and hence will be useful for a broad range of gene therapy, stem cell, and cancer research applications.
Role of N-Linked Glycans in the Functions of Hepatitis C Virus Envelope Proteins Incorporated into Infectious Virions
Authors: Helle François , Vieyres Gabrielle , Elkrief Laure , Popescu Costin-Ioan , Wychowski Czeslaw , Descamps Véronique , Castelain Sandrine , Roingeard Philippe , Duverlie Gilles , Dubuisson Jean 
Published: September 2010

ABSTRACT
Hepatitis C virus (HCV) envelope glycoproteins are highly glycosylated, with generally 4 and 11 N-linked glycans on E1 and E2, respectively. Studies using mutated recombinant HCV envelope glycoproteins incorporated into retroviral pseudoparticles (HCVpp) suggest that some glycans play a role in protein folding, virus entry, and protection against neutralization. The development of a cell culture system producing infectious particles (HCVcc) in hepatoma cells provides an opportunity to characterize the role of these glycans in the context of authentic infectious virions. Here, we used HCVcc in which point mutations were engineered at N-linked glycosylation sites to determine the role of these glycans in the functions of HCV envelope proteins. The mutants were characterized for their effects on virus replication and envelope protein expression as well as on viral particle secretion, infectivity, and sensitivity to neutralizing antibodies. Our results indicate that several glycans play an important role in HCVcc assembly and/or infectivity. Furthermore, our data demonstrate that at least five glycans on E2 (denoted E2N1, E2N2, E2N4, E2N6, and E2N11) strongly reduce the sensitivity of HCVcc to antibody neutralization, with four of them surrounding the CD81 binding site. Altogether, these data indicate that the glycans associated with HCV envelope glycoproteins play roles at different steps of the viral life cycle. They also highlight differences in the effects of glycosylation mutations between the HCVpp and HCVcc systems. Furthermore, these carbohydrates form a “glycan shield” at the surface of the virion, which contributes to the evasion of HCV from the humoral immune response.
Evolution of Functional and Sequence Variants of the Mammalian XPR1 Receptor for Mouse Xenotropic Gammaretroviruses and the Human-Derived Retrovirus XMRV
Authors: Yan Yuhe , Liu Qingping , Wollenberg Kurt , Martin Carrie , Buckler-White Alicia , Kozak Christine A. 
Published: September 2010

ABSTRACT

Genetic conflicts between retroviruses and their receptors result in the evolution of novel host entry restrictions and novel virus envelopes, and such variants can influence trans-species transmission. We screened rodents and other mammals for sequence variation in the
Xpr1
receptor for the mouse xenotropic or polytropic mouse leukemia viruses (X-MLVs or P-MLVs, respectively) of the gammaretrovirus family and for susceptibility to mouse-derived X/P-MLVs and to XMRV (xenotropic murine leukemia virus-related virus), an X-MLV-like virus isolated from humans with prostate cancer and chronic fatigue syndrome. We identified multiple distinct susceptibility phenotypes; these include the four known
Xpr1
variants in
Mus
and a novel fifth
Xpr1
gene found in
Mus molossinus
and
Mus musculus
. We describe the geographic and species distribution of the
Mus Xpr1
variants but failed to find the X-MLV-restrictive laboratory mouse allele in any wild mouse. We used mutagenesis and phylogenetic analysis to evaluate the functional contributions made by constrained, variable, and deleted residues. Rodent
Xpr1
is under positive selection, indicating a history of host-pathogen conflicts; several codons under selection have known roles in virus entry. All non-
Mus
mammals are susceptible to mouse X-MLVs, but some restrict other members of the X/P-MLV family, and the resistance of hamster and gerbil cells to XMRV indicates that XMRV has unique receptor requirements. We show that the hypervariable fourth extracellular XPR1 loop (ECL4) contains three evolutionarily constrained residues that do not contribute to receptor function, we identify two novel residues important for virus entry (I579 and T583), and we describe a unique pattern of ECL4 variation in the three virus-restrictive
Xpr1
variants found in MLV-infected house mice; these mice carry different deletions in ECL4, suggesting either that these sites or loop size affects receptor function.