bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Impact of Individual Human Immunodeficiency Virus Type 1 Protease Mutations on Drug Susceptibility Is Highly Influenced by Complex Interactions with the Background Protease Sequence
Authors: Van Marck H. , Dierynck I. , Kraus G. , Hallenberger S. , Pattery T. , Muyldermans G. , Geeraert L. , Borozdina L. , Bonesteel R. , Aston C. , Shaw E. , Chen Q. , Martinez C. , Koka V. , Lee J. , Chi E. , de Béthune M.-P. , Hertogs K. 
Published: July 2009

ABSTRACT
The requirement for multiple mutations for protease inhibitor (PI) resistance necessitates a better understanding of the molecular basis of resistance development. The novel bioinformatics resistance determination approach presented here elaborates on genetic profiles observed in clinical human immunodeficiency virus type 1 (HIV-1) isolates. Synthetic protease sequences were cloned in a wild-type HIV-1 background to generate a large number of close variants, covering 69 mutation clusters between multi-PI-resistant viruses and their corresponding genetically closely related, but PI-susceptible, counterparts. The vast number of mutants generated facilitates a profound and broad analysis of the influence of the background on the effect of individual PI resistance-associated mutations (PI-RAMs) on PI susceptibility. Within a set of viruses, all PI-RAMs that differed between susceptible and resistant viruses were varied while maintaining the background sequence from the resistant virus. The PI darunavir was used to evaluate PI susceptibility. Single sets allowed delineation of the impact of individual mutations on PI susceptibility, as well as the influence of PI-RAMs on one another. Comparing across sets, it could be inferred how the background influenced the interaction between two mutations, in some cases even changing antagonistic relationships into synergistic ones or vice versa. The approach elaborates on patient data and demonstrates how the specific mutational background greatly influences the impact of individual mutations on PI susceptibility in clinical patterns.
Mouse Norovirus Replication Is Associated with Virus-Induced Vesicle Clusters Originating from Membranes Derived from the Secretory Pathway
Authors: Hyde Jennifer L. , Sosnovtsev Stanislav V. , Green Kim Y. , Wobus Christiane , Virgin Herbert W. , Mackenzie Jason M. 
Published: July 2009

ABSTRACTHuman noroviruses (familyCaliciviridae) are the leading cause of nonbacterial gastroenteritis worldwide. Despite the prevalence of these viruses within the community, the study of human norovirus has largely been hindered due to the inability to cultivate the viruses ex vivo and the lack of a small-animal model. In 2003, the discovery of a novel murine norovirus (MNV-1) and the identification of the tropism of MNV-1 for cells of a mononuclear origin led to the establishment of the first norovirus tissue culture system. Like other positive-sense RNA viruses, MNV-1 replication is associated with host membranes, which undergo significant rearrangement during infection. We characterize here the subcellular localization of the MNV-1 open reading frame 1 proteins and viral double-stranded RNA (dsRNA). Over the course of infection, dsRNA and the MNV-1 RNA-dependent RNA polymerase (NS7) were observed to proliferate from punctate foci located in the perinuclear region. All of the MNV-1 open reading frame 1 proteins were observed to colocalize with dsRNA during the course of infection. The MNV-1 replication complex was immunolocalized to virus-induced vesicle clusters formed in the cytoplasm of infected cells. Both dsRNA and MNV-1 NS7 were observed to localize to the limiting membrane of the individual clusters by cryo-immunoelectron microscopy. We show that the MNV-1 replication complex initially associates with membranes derived from the endoplasmic reticulum,trans-Golgi apparatus, and endosomes. In addition, we show that MNV-1 replication is insensitive to the fungal metabolite brefeldin A and consistently does not appear to recruit coatomer protein complex I (COPI) or COPII component proteins during replication. These data provide preliminary insights into key aspects of replication of MNV-1, which will potentially further our understanding of the pathogenesis of noroviruses and aid in the identification of potential targets for drug development.
Disruption of LANA in Rhesus Rhadinovirus Generates a Highly Lytic Recombinant Virus
Authors: Wen Kwun Wah , Dittmer Dirk P. , Damania Blossom 
Published: July 2009

ABSTRACT
Rhesus monkey rhadinovirus (RRV) is a gammaherpesvirus that is closely related to human Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8). RRV is the closest relative to KSHV that has a fully sequenced genome and serves as an in vitro and an in vivo model system for KSHV. The latency-associated nuclear antigen (LANA) protein of both KSHV and RRV plays key roles in the establishment and maintenance of these herpesviruses. We have constructed a RRV recombinant virus (RRVΔLANA/GFP) in which the RRV LANA open reading frame has been disrupted with a green fluorescent protein (GFP) expression cassette generated by homologous recombination. The integrity of the recombinant virus was confirmed by diagnostic PCR, restriction digestion, Southern blot analysis, and whole-genome sequencing. We compared the single-step and multistep replication kinetics of RRVΔLANA/GFP, RRV-GFP, wild-type (WT) RRV H26-95, and a revertant virus using traditional plaque assays, as well as real-time quantitative PCR-based genome quantification assays. The RRVΔLANA/GFP recombinant virus exhibited significantly higher lytic replicative properties compared to RRV-GFP, WT RRV, or the revertant virus. This was observed upon de novo infection and in the absence of chemical inducers such as phorbol esters. In addition, by using a quantitative real-time PCR-based viral array, we are the first to report differences in global viral gene expression between WT and recombinant viruses. The RRVΔLANA/GFP virus displayed increased lytic gene transcription at all time points postinfection compared to RRV-GFP. Moreover, we also examined several cellular genes that are known to be repressed by KSHV LANA and report that these genes are derepressed during de novo lytic infection with the RRVΔLANA/GFP virus compared to RRV-GFP. Finally, we also demonstrate that the RRVΔLANA/GFP virus fails to establish latency in B cells, as measured by the loss of GFP-positive cells and intracellular viral genomes.
Influenza Virus Directly Infects Human Natural Killer Cells and Induces Cell Apoptosis
Authors: Mao Huawei , Tu Wenwei , Qin Gang , Law Helen Ka Wai , Sia Sin Fun , Chan Ping-Lung , Liu Yinping , Lam Kwok-Tai , Zheng Jian , Peiris Malik , Lau Yu-Lung 
Published: July 2009

ABSTRACT
Influenza is an acute respiratory viral disease that is transmitted in the first few days of infection. Evasion of host innate immune defenses, including natural killer (NK) cells, is important for the virus's success as a pathogen of humans and other animals. NK cells encounter influenza viruses within the microenvironment of infected cells and are important for host innate immunity during influenza virus infection. It is therefore important to investigate the direct effects of influenza virus on NK cells. In this study, we demonstrated for the first time that influenza virus directly infects and replicates in primary human NK cells. Viral entry into NK cells was mediated by both clathrin- and caveolin-dependent endocytosis rather than through macropinocytosis and was dependent on the sialic acids on cell surfaces. In addition, influenza virus infection induced a marked apoptosis of NK cells. Our findings suggest that influenza virus can directly target and kill NK cells, a potential novel strategy of influenza virus to evade the NK cell innate immune defense that is likely to facilitate viral transmission and may also contribute to virus pathogenesis.
Human Immunodeficiency Virus Type 1 Nucleocapsid Inhibitors Impede
trans
Infection in Cellular and Explant Models and Protect Nonhuman Primates from Infection
Authors: Wallace Gregory S. , Cheng-Mayer Cecilia , Schito Marco L. , Fletcher Patricia , Miller Jenkins Lisa M. , Hayashi Ryo , Neurath A. Robert , Appella Ettore , Shattock Robin J. 
Published: July 2009

ABSTRACT

Here, we report that the
S
-acyl-2-mercaptobenzamide thioester (SAMT) class of human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein (NCp7) inhibitors was able to prevent transmission of HIV-1 from infected cells, including primary cells. Furthermore, when SAMTs were introduced during an HIV-1 challenge of cervical explant tissue, inhibition of dissemination of infectious virus by cells emigrating from the tissue explants was observed. Preliminary studies using a rhesus macaque vaginal challenge model with mixed R5 and X4 simian-human immunodeficiency virus infection found that five of six monkeys were completely protected, with the remaining animal being partially protected, infected only by the R5 virus. These data suggest that SAMTs may be promising new drug candidates for further development in anti-HIV-1 topical microbicide applications.
The Porcine Reproductive and Respiratory Syndrome Virus nsp2 Cysteine Protease Domain Possesses bothtrans- andcis-Cleavage Activities
Authors: Han Jun , Rutherford Mark S. , Faaberg Kay S. 
Published: July 2009

ABSTRACTThe N terminus of the replicase nonstructural protein 2 (nsp2) of porcine reproductive and respiratory syndrome virus (PRRSV) contains a putative cysteine protease domain (PL2). Previously, we demonstrated that deletion of either the PL2 core domain (amino acids [aa] 47 to 180) or the immediate downstream region (aa 181 to 323) is lethal to the virus. In this study, the PL2 domain was found to encode an active enzyme that mediates efficient processing of nsp2-3 in CHO cells. The PL2 protease possessed bothtrans- andcis-cleavage activities, which were distinguished by individual point mutations in the protease domain. The minimal size required to maintain these two enzymatic activities included nsp2 aa 47 to 240 (Tyr47to Cys240) and aa 47 to 323 (Tyr47to Leu323), respectively. Introduction of targeted amino acid mutations in the protease domain confirmed the importance of the putative Cys55- His124catalytic motif for nsp2/3 proteolysis in vitro, as were three additional conserved cysteine residues (Cys111, Cys142, and Cys147). The conserved aspartic acids (e.g., Asp89) were essential for the PL2 proteasetrans-cleavage activity. Reverse genetics revealed that the PL2trans-cleavage activity played an important role in the PRRSV replication cycle in that mutations that impaired the PL2 proteasetransfunction, but not thecisactivity, were detrimental to viral viability. Lastly, the potential nsp2/3 cleavage site was probed. Mutations with the largest impact on in vitro cleavage were at or near the G1196|G1197dipeptide.
Clearance of Measles Virus from Persistently Infected Cells by Short Hairpin RNA
Authors: Zinke Michael , Kendl Sabine , Singethan Katrin , Fehrholz Markus , Reuter Dajana , Rennick Linda , Herold Marco J. , Schneider-Schaulies Jürgen 
Published: July 2009

ABSTRACT
Subacute sclerosing panencephalitis (SSPE) is a demyelinating central nervous system disease caused by a persistent measles virus (MV) infection of neurons and glial cells. There is still no specific therapy available, and in spite of an intact innate and adaptive immune response, SSPE leads inevitably to death. In order to select effective antiviral short interfering RNAs (siRNAs), we established a plasmid-based test system expressing the mRNA of DsRed2 fused with mRNA sequences of single viral genes, to which certain siRNAs were directed. siRNA sequences were expressed as short hairpin RNA (shRNA) from a lentiviral vector additionally expressing enhanced green fluorescent protein (EGFP) as an indicator. Evaluation by flow cytometry of the dual-color system (DsRed and EGFP) allowed us to find optimal shRNA sequences. Using the most active shRNA constructs, we transduced persistently infected human NT2 cells expressing virus-encoded HcRed (piNT2-HcRed) as an indicator of infection. shRNA against N, P, and L mRNAs of MV led to a reduction of the infection below detectable levels in a high percentage of transduced piNT2-HcRed cells within 1 week. The fraction of virus-negative cells in these cultures was constant over at least 3 weeks posttransduction in the presence of a fusion-inhibiting peptide (Z-Phe-Phe-Gly), preventing the cell fusion of potentially cured cells with persistently infected cells. Transduced piNT2 cells that lost HcRed did not fuse with underlying Vero/hSLAM cells, indicating that these cells do not express viral proteins any more and are “cured.” This demonstrates in tissue culture that NT2 cells persistently infected with MV can be cured by the transduction of lentiviral vectors mediating the long-lasting expression of anti-MV shRNA.
CXCR3 Deficiency Increases Susceptibility to Genital Herpes Simplex Virus Type 2 Infection: Uncoupling of CD8+T-Cell Effector Function but Not Migration
Authors: Thapa Manoj , Carr Daniel J. J. 
Published: July 2009

ABSTRACTCXCR3 is a G-protein-coupled receptor preferentially expressed by activated T cells, NK cells, and dendritic cells. Signaling through gamma interferon-regulated chemokines CXCL9, CXCL10, CXCL11, and CXCR3 plays a critical role in the immune response of many viral pathogens. However, the relevance of CXCR3 for optimal T-cell activation and the induction of regulatory transcription factors (i.e., T-bet and eomesodermin) relative to host immune defense against genital herpes simplex virus type 2 (HSV-2) infection have been poorly defined. In this study, we evaluated the requirement of CXCR3 expression during genital HSV-2 infection using mice deficient in CXCR3 (CXCR3−/−) along with wild-type (WT) controls, assessing the resistance of mice to viral infection and focusing on the cytokine/chemokine response, phenotypic analysis of recruited leukocytes, and functional analysis of CD8+T cells. CXCR3−/−mice showed a heightened sensitivity to infection compared to WT animals in terms of the viral burden in infected tissues as well as elevated mortality. The poor response of CXCR3−/−mice to viral infection was associated with reduced cytotoxic T-lymphocyte activity through the impairment of T-bet, perforin, and granzyme B expression by CD8+T cells. Corresponding with the defective cytolytic activity, a reduction in recruitment of plasmacytoid dendritic cells and CD80 expression in CD11c+dendritic cells in the draining lymph nodes of CXCR3−/−mice were detected. Collectively, the results provide a new perspective to CXCR3 signaling for the appropriate activation of CD8+T cells required for host defense against genital HSV-2 infection.
Nucleoside Monophosphate Complex Structures of the Endonuclease Domain from the Influenza Virus Polymerase PA Subunit Reveal the Substrate Binding Site inside the Catalytic Center
Authors: Zhao Cong , Lou Zhiyong , Guo Yu , Ma Ming , Chen Yutao , Liang Shuaiyi , Zhang Liang , Chen Shoudeng , Li Xuemei , Liu Yingfang , Bartlam Mark , Rao Zihe 
Published: July 2009

ABSTRACT

Highly pathogenic influenza virus strains currently in circulation pose a significant risk of a global pandemic. Following the reported crystal structure of the endonuclease domain from the avian influenza virus polymerase PA subunit, here we report the results of a systematic X-ray crystallographic analysis of its complex with adenosine, uridine, and thymidine nucleoside monophosphates (NMPs). Electron density corresponding to the monophosphate moiety of each nucleotide was apparent in each NMP complex and bound to the catalytic metal. A hydrophobic site was found to contribute to nucleoside binding. The NMP complex structures should represent the conformation of the bound product after nuclease cleavage. Moreover, one solvent molecule was found to occupy an equivalent position to the second reported Mn
2+
ion, where it mediates the interaction between bound NMPs and the N-terminal PA domain in the presence of the Mg
2+
ion. The results presented here indicate a possible cleavage mechanism and identify a distinct nucleotide binding pocket. The identification of this binding pocket opens a new avenue for anti-influenza drug discovery, targeting the cap-dependent endonuclease, in response to the worldwide threat of influenza.
Gag Determinants of Fitness and Drug Susceptibility in Protease Inhibitor-Resistant Human Immunodeficiency Virus Type 1
Authors: Parry Chris M. , Kohli Arinder , Boinett Christine J. , Towers Greg J. , McCormick Adele L. , Pillay Deenan 
Published: July 2009

ABSTRACT

Mutations can accumulate in the protease and
gag
genes of human immunodeficiency virus in patients who fail therapy with protease inhibitor drugs. Mutations within protease, the drug target, have been extensively studied. Mutations in
gag
have been less well studied, mostly concentrating on cleavage sites. A retroviral vector system has been adapted to study full-length
gag
, protease, and reverse transcriptase genes from patient-derived viruses. Patient plasma-derived mutant full-length
gag
, protease, and
gag
-protease from a multidrug-resistant virus were studied. Mutant protease alone led to a 95% drop in replication capacity that was completely rescued by coexpressing the full-length coevolved mutant
gag
gene. Cleavage site mutations have been shown to improve the replication capacity of mutated protease. Strikingly, in this study, the matrix region and part of the capsid region from the coevolved mutant
gag
gene were sufficient to achieve full recovery of replication capacity due to the mutant protease, without cleavage site mutations. The same region of
gag
from a second, unrelated, multidrug-resistant clinical isolate also rescued the replication capacity of the original mutant protease, suggesting a common mechanism that evolves with resistance to protease inhibitors. Mutant
gag
alone conferred reduced susceptibility to all protease inhibitors and acted synergistically when linked to mutant protease. The matrix region and partial capsid region of
gag
sufficient to rescue replication capacity also conferred resistance to protease inhibitors. Thus, the amino terminus of Gag has a previously unidentified and important function in protease inhibitor susceptibility and replication capacity.
Systematic sampling of discrete and continuous populations: sample selection and the choice of estimator
Authors: Valentine Harry T. , Affleck David L.R. , Gregoire Timothy G. 
Published: July 2009

Systematic sampling is easy, efficient, and widely used, though it is not generally recognized that a systematic sample may be drawn from the population of interest with or without restrictions on randomization. The restrictions or the lack of them determine which estimators are unbiased, when using the sampling design as the basis for inference. We describe the selection of a systematic sample, with and without restriction, from populations of discrete elements and from linear and areal continuums (continuous populations). We also provide unbiased estimators for both restricted and unrestricted selection. When the population size is known at the outset, systematic sampling with unrestricted selection is most likely the best choice. Restricted selection affords estimation of attribute totals for a population when the population size — for example, the area of an areal continuum — is unknown. Ratio estimation, however, is most likely a more precise option when the selection is restricted and the population size becomes known at the end of the sampling. There is no difference between restricted and unrestricted selection if the sampling interval or grid tessellates the frame in such a way that all samples contain an equal number of measurements. Moreover, all the estimators are unbiased and identical in this situation.
Contribution of atmospheric nitrogen deposition to net primary productivity in a northern hardwood forest
Authors: Nave Lucas E. , Vogel Christoph S. , Gough Christopher M. , Curtis Peter S. 
Published: July 2009

Net primary productivity (NPP) in northern temperate forests is an important part of the global carbon cycle. Because NPP often is limited by nitrogen (N), atmospheric N deposition (Ndep) may increase forest NPP. At a northern hardwood forest site in northern Lower Michigan, USA, we quantified rates of N supply by Ndep, canopy retention of Ndep (Ncr), and soil net N mineralization (Nmin); calculated the N requirement of NPP; and estimated the fraction of NPP that could be attributed to atmospheric N inputs. Net N mineralization supplied 42.6 kg N·ha–1·year–1 (84% of the NPP N requirement), and Ndep averaged 7.5 kg N·ha–1·year–1 (15%). The forest canopy retained 38% of Ndep (Ncr = 2.8 kg N·ha–1·year–1), primarily in the forms of organic N and NH4-N. Fine root (62%) and leaf (31%) N requirements dominated the NPP N requirement of 50.7 kg N·ha–1·year–1. Annual N supply by the processes we measured agreed very closely with the NPP N requirement, suggesting that internally cycled N and N of atmospheric origin contribute to the N nutrition of this forest. Our results indicate that up to 15% of the NPP at this site may be driven by atmospheric N inputs.
Soil greenhouse gas and nutrient dynamics in fertilized western Canadian plantation forests
Authors: Basiliko Nathan , Khan Amer , Prescott Cindy E. , Roy Réal , Grayston Sue J. 
Published: July 2009

We explored the impacts of fertilization on soil greenhouse gas fluxes and underlying soil nutrient transformations using short-term (up to 7 months) simulated operational fertilization with urea-nitrogen or nitrogen, phosphorus, potassium, and micronutrients in lodgepole pine ( Pinus contorta Dougl. ex Loud. var. latifolia Engelm.), western hemlock ( Tsuga heterophylla (Raf.) Sarge.), and Douglas-fir ( Pseudotsuga menziesii (Mirb.) Franco) forests in British Columbia. Urea appeared to be rapidly mineralized to ammonium, and nitrification (relative to controls) was only observed at the lodgepole pine site and represented only 0.5% of added nitrogen. All sites were small net sinks for atmospheric methane, and fertilization effects, both stimulatory and inhibitory, were short-lived. Across all sites and treatments, soils were as likely to consume as emit nitrous oxide, and among treatment replicates, rates were never significantly different from 0, with the exception of one efflux rate of 1.5 µg·m–2·h–1on the warmest day in the study. We conclude from this pilot study that in acidic, unpolluted (with regard to nitrogen deposition) upland conifer forest soils in western Canada fertilized once or infrequently with urea, ammonium, or a combination of nutrients, the dynamics of soil greenhouse-gas fluxes are generally not disturbed over the short-term, with soils remaining small sinks for atmospheric methane and neutral with regard to flux of nitrous oxide.
Chapter 26 CP and T Violation
Authors: Bigi Ikaros , Li Hai-Bo 
Published: July 2009

The following sections are included: Formalism and Review CP Violation in [Formula: see text] Mixing CP Violation in Decay CP Violation in the Interference Between Decays With and Without Mixing Rate of the CP Violation in the Coherent [Formula: see text] Pair T Violation