bluuebunny/crossref_metadata_2025_split huggingface dataset:
Ordered Processing of the Human Immunodeficiency Virus Type 1 GagPol Precursor Is Influenced by the Context of the Embedded Viral Protease
Authors: Pettit Steven C. , Clemente Jose C. , Jeung Jennifer A. , Dunn Ben M. , Kaplan Andrew H. 
Published: July 2005

ABSTRACT

Ordered and accurate processing of the human immunodeficiency virus type 1 (HIV-1) GagPol polyprotein precursor by a virally encoded protease is an indispensable step in the appropriate assembly of infectious viral particles. The HIV-1 protease (PR) is a 99-amino-acid enzyme that is translated as part of the GagPol precursor. Previously, we have demonstrated that the initial events in precursor processing are accomplished by the PR domain within GagPol in
cis
, before it is released from the polyprotein. Despite the critical role that ordered processing of the precursor plays in viral replication, the forces that define the order of cleavage remain poorly understood. Using an in vitro assay in which the full-length HIV-1 GagPol is processed by the embedded PR, we examined the effect of PR context (embedded within GagPol versus the mature 99-amino-acid enzyme) on precursor processing. Our data demonstrate that the PR domain within GagPol is constrained in its ability to cleave some of the processing sites in the precursor. Further, we find that this constraint is dependent upon the presence of a proline as the initial amino acid in the embedded PR; substitution of an alanine at this position produces enhanced cleavage at additional sites when the precursor is processed by the embedded, but not the mature, PR. Overall, our data support a model in which the selection of processing sites and the order of precursor processing are defined, at least in part, by the structure of GagPol itself.
CD8+T-Cell Dysfunction due to Cytolytic Granule Deficiency in Persistent Friend Retrovirus Infection
Authors: Zelinskyy Gennadiy , Robertson Shelly J. , Schimmer Simone , Messer Ronald J. , Hasenkrug Kim J. , Dittmer Ulf 
Published: July 2005

ABSTRACTVirus-specific CD8+T cells are critical for the control of acute Friend virus (FV) infections, but are rendered impotent by CD4+regulatory T cells during the chronic phase of infection. The current study examines this CD8+T-cell dysfunction by analyzing the production and release of cytolytic molecules by CD8+T cells. CD8+T cells with an activated phenotype (CD43+) from acutely infected mice produced all three key components of lytic granules: perforin, granzyme A, and granzyme B. Furthermore, they displayed evidence of recent degranulation and in vivo cytotoxicity. In contrast, activated CD8+T cells from chronically infected mice were deficient in cytolytic molecules and showed little evidence of recent degranulation and poor in vivo cytotoxicity. Evidence from tetramer-positive CD8+T cells with known virus specificity confirmed the findings from the activated subset of CD8+T cells. Interestingly, perforin and granzyme A mRNA levels were not significantly reduced during chronic infection, indicating control at a posttranscriptional level. Granzyme B deficiency was associated with a significant decrease in mRNA levels, but posttranscriptional control also appeared to contribute to deficiency. These results demonstrate a broad impairment of cytotoxic CD8+T-cell effector function during chronic retroviral infection and explain the inability of virus-specific CD8+T cells to eliminate persistent virus.
Evolution of Human Immunodeficiency Virus Type 1 Protease Genotypes and Phenotypes In Vivo under Selective Pressure of the Protease Inhibitor Ritonavir
Authors: Resch Wolfgang , Parkin Neil , Watkins Terri , Harris Janera , Swanstrom Ronald 
Published: July 2005

ABSTRACT

We examined the population dynamics of human immunodeficiency virus type 1
pro
variants during the evolution of resistance to the protease inhibitor ritonavir (RTV) in vivo.
pro
variants were followed in subjects who had added RTV to their previously failed reverse transcriptase inhibitor therapy using a heteroduplex tracking assay designed to detect common resistance-associated mutations. In most cases the initial variant appeared rapidly within 2 to 3 months followed by one or more subsequent population turnovers. Some of the subsequent transitions between variants were rapid, and some were prolonged with the coexistence of multiple variants. In several cases variants without resistance mutations persisted despite the emergence of new variants with an increasing number of resistance-associated mutations. Based on the rate of turnover of
pro
variants in the RTV-treated subjects we estimated that the mean fitness of newly emerging variants was increased 1.2-fold (range, 1.02 to 1.8) relative to their predecessors. A subset of
pro
genes was introduced into infectious molecular clones. The corresponding viruses displayed impaired replication capacity and reduced susceptibility to RTV. A subset of these clones also showed increased susceptibility to two nonnucleoside reverse transcriptase inhibitors and the protease inhibitor saquinavir. Finally, a significant correlation between the reduced replication capacity and reduced processing at the
gag
NC-p1 processing site was noted. Our results reveal a complexity of patterns in the evolution of resistance to a protease inhibitor. In addition, these results suggest that selection for resistance to one protease inhibitor can have pleiotropic effects that can affect fitness and susceptibility to other drugs.
Nef Proteins from Diverse Groups of Primate Lentiviruses Downmodulate CXCR4 To Inhibit Migration to the Chemokine Stromal Derived Factor 1
Authors: Hrecka Kasia , Swigut Tomek , Schindler Michael , Kirchhoff Frank , Skowronski Jacek 
Published: July 2005

ABSTRACTNef proteins of primate lentiviruses promote viral replication, virion infectivity, and evasion of antiviral immune responses by modulating signal transduction pathways and downregulating expression of receptors at the cell surface that are important for efficient antigen-specific responses, such as CD4, CD28, T-cell antigen receptor, and class I and class II major histocompatibility complex. Here we show that Nef proteins from diverse groups of primate lentiviruses which do not require the chemokine receptor CXCR4 for entry into target cells strongly downmodulate the cell surface expression of CXCR4. In contrast, all human immunodeficiency virus type 1 (HIV-1) and the majority of HIV-2 Nef proteins tested did not have such strong effects. SIVmac239 Nef strongly inhibited lymphocyte migration to CXCR4 ligand, the chemokine stromal derived factor 1 (SDF-1). SIVmac239 Nef downregulated CXCR4 by accelerating the rate of its endocytosis. Downmodulation of CXCR4 was abolished by mutations that disrupt the constitutively strong AP-2 clathrin adaptor binding element located in the N-terminal region of the Nef molecule, suggesting that Nef accelerates CXCR4 endocytosis via an AP-2-dependent pathway. Together, these results point to CXCR4 as playing an important role in simian immunodeficiency virus and possibly also HIV-2 persistence in vivo that is unrelated to viral entry into target cells. We speculate that Nef targets CXCR4 to disrupt ordered trafficking of infected leukocytes between local microenvironments in order to facilitate their dissemination and/or impair the antiviral immune response.
An S101P Substitution in the Putative Cleavage Motif of the Human Metapneumovirus Fusion Protein Is a Major Determinant for Trypsin-Independent Growth in Vero Cells and Does Not Alter Tissue Tropism in Hamsters
Authors: Schickli Jeanne H. , Kaur Jasmine , Ulbrandt Nancy , Spaete Richard R. , Tang Roderick S. 
Published: July 2005

ABSTRACT

Human metapneumovirus (hMPV), a recently described paramyxovirus, is a major etiological agent for lower respiratory tract disease in young children that can manifest with severe cough, bronchiolitis, and pneumonia. The hMPV fusion glycoprotein (F) shares conserved functional domains with other paramyxovirus F proteins that are important for virus entry and spread. For other paramyxovirus F proteins, cleavage of a precursor protein (F
0
) into F
1
and F
2
exposes a fusion peptide at the N terminus of the F
1
fragment, a likely prerequisite for fusion activity. Many hMPV strains have been reported to require trypsin for growth in tissue culture. The majority of these strains contain RQSR at the putative cleavage site. However, strains hMPV/NL/1/00 and hMPV/NL/1/99 expanded in our laboratory contain the sequence RQPR and do not require trypsin for growth in Vero cells. The contribution of this single amino acid change was verified directly by generating recombinant virus (rhMPV/NL/1/00) with either proline or serine at position 101 in F. These results suggested that cleavage of F protein in Vero cells could be achieved by trypsin or S101P amino acid substitution in the putative cleavage site motif. Moreover, trypsin-independent cleavage of hMPV F containing 101P was enhanced by the amino acid substitution E93K. In hamsters, rhMPV/93K/101S and rhMPV/93K/101P grew to equivalent titers in the respiratory tract and replication was restricted to respiratory tissues. The ability of these hMPV strains to replicate efficiently in the absence of trypsin should greatly facilitate the generation, preclinical testing, and manufacturing of attenuated hMPV vaccine candidates.
High Sequence Conservation of Human Immunodeficiency Virus Type 1 Reverse Transcriptase under Drug Pressure despite the Continuous Appearance of Mutations
Authors: Ceccherini-Silberstein Francesca , Gago Federico , Santoro Maria , Gori Caterina , Svicher Valentina , Rodríguez-Barrios Fátima , d'Arrigo Roberta , Ciccozzi Massimo , Bertoli Ada , Monforte Antonella d'Arminio , Balzarini Jan , Antinori Andrea , Perno Carlo-Federico 
Published: July 2005

ABSTRACT
To define the extent of sequence conservation in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) in vivo, the first 320 amino acids of RT obtained from 2,236 plasma-derived samples from a well-defined cohort of 1,704 HIV-1-infected individuals (457 drug naïve and 1,247 drug treated) were analyzed and examined in structural terms. In naïve patients, 233 out of these 320 residues (73%) were conserved (<1% variability). The majority of invariant amino acids clustered into defined regions comprising between 5 and 29 consecutive residues. Of the nine longest invariant regions identified, some contained residues and domains critical for enzyme stability and function. In patients treated with RT inhibitors, despite profound drug pressure and the appearance of mutations primarily associated with resistance, 202 amino acids (63%) remained highly conserved and appeared mostly distributed in regions of variable length. This finding suggests that participation of consecutive residues in structural domains is strictly required for cooperative functions and sustainability of HIV-1 RT activity. Besides confirming the conservation of amino acids that are already known to be important for catalytic activity, stability of the heterodimer interface, and/or primer/template binding, the other 62 new invariable residues are now identified and mapped onto the three-dimensional structure of the enzyme. This new knowledge could be of help in the structure-based design of novel resistance-evading drugs.
A Begomovirus DNAβ-Encoded Protein Binds DNA, Functions as a Suppressor of RNA Silencing, and Targets the Cell Nucleus
Authors: Cui Xiaofeng , Li Guixin , Wang Daowen , Hu Dongwei , Zhou Xueping 
Published: July 2005

ABSTRACT

Our previous results demonstrated that the DNAβ satellite (Y10β) associated with
Tomato yellow leaf curl China virus
Y10 isolate (TYLCCNV-Y10) is essential for induction of leaf curl symptoms in plants and that transgenic expression of its
βC1
gene in
Nicotiana
plants induces virus-like symptoms. In the present study, in vitro DNA binding activity of the βC1 proteins of Y10β and DNAβ (Y35β) found in the
Tobacco curly shoot virus
Y35 isolate (TbCSV-Y35) were studied following their expression as six-His fusion proteins in
Escherichia coli
. Electrophoretic mobility shift assays and UV cross-linking experiments revealed that βC1 proteins could bind both single-stranded and double-stranded DNA without size or sequence specificity. Suppression of green fluorescent protein (GFP) transgene silencing was observed with the new leaves of GFP-expressing
Nicotiana benthamiana
plants coinoculated by TYLCCNV-Y10 plus Y10β or by TbCSV-Y35 plus Y35β. In a patch agroinfiltration assay, the transiently expressed
βC1
gene of Y10β or Y35β was able to suppress host RNA silencing activities and permitted the accumulation of high levels of GFP mRNA in the infiltrated leaf patches of GFP transgenic
N. benthamiana
plants. The βC1 protein of Y10β accumulated primarily in the nuclei of plant and insect cells when fused with β-glucuronidase or GFP and immunogold labeling showed that the βC1 protein is present in the nuclei of infected
N. benthamiana
plants. A mutant version of Y10β carrying the mutations within the putative nuclear localization sequence of the Y10 βC1 protein failed to induce disease symptoms, suppress RNA silencing, or accumulate in the nucleus, suggesting that nuclear localization of the βC1 protein is a key requirement for symptom induction and silencing suppression.
Caveola-Dependent Endocytic Entry of Amphotropic Murine Leukemia Virus
Authors: Beer Christiane , Andersen Ditte S. , Rojek Aleksandra , Pedersen Lene 
Published: July 2005

ABSTRACT

Early results suggested that the amphotropic murine leukemia virus (A-MLV) does not enter cells via endocytosis through clathrin-coated pits and this gammaretrovirus has therefore been anticipated to fuse directly with the plasma membrane. However, here we present data implicating a caveola-mediated endocytic entry route for A-MLV via its receptor Pit2. Caveolae belong to the cholesterol-rich microdomains characterized by resistance to nonionic detergents such as Triton X-100. Extraction of murine fibroblastic NIH 3T3 cells in cold Triton X-100 showed the presence of the A-MLV receptor Pit2 in detergent-insoluble microdomains. Using coimmunoprecipitation of cell extracts, we were able to demonstrate direct association of Pit2 with caveolin-1, the structural protein of caveolae. Other investigations revealed that A-MLV infection in contrast to vesicular stomatitis virus infection is a slow process (
t
≈5 h), which is dependent on plasma membrane cholesterol but independent of NH
4
Cl treatment of cells; NH
4
Cl impairs entry via clathrin-coated pits. Furthermore, expression of dominant-negative caveolin-1 decreased the susceptibility to infection via Pit2 by approximately 70%. These results show that A-MLV can enter cells via a caveola-dependent entry route. Moreover, increase in A-MLV infection by treatment with okadaic acid as well as entry of fusion-defective fluorescent A-MLV virions in NIH 3T3 cells further confirmed our findings and show that A-MLV can enter mouse fibroblasts via an endocytic entry route involving caveolae. Finally, we also found colocalization of fusion-defective fluorescent A-MLV virions with caveolin-1 in NIH 3T3 cells. This is the first time substantial evidence has been presented implicating the existence of a caveola-dependent endocytic entry pathway for a retrovirus.
Interactions between Rubella Virus Capsid and Host Protein p32 Are Important for Virus Replication
Authors: Beatch Martin D. , Everitt Jason C. , Law LokMan J. , Hobman Tom C. 
Published: July 2005

ABSTRACTThe distribution and morphology of mitochondria are dramatically affected during infection with rubella virus (RV). Expression of the capsid, in the absence of other viral proteins, was found to induce both perinuclear clustering of mitochondria and the formation of electron-dense intermitochondrial plaques, both hallmarks of RV-infected cells. We previously identified p32, a host cell mitochondrial matrix protein, as a capsid-binding protein. Here, we show that two clusters of arginine residues within capsid are required for stable binding to p32. Mutagenic ablation of the p32-binding site in capsid resulted in decreased mitochondrial clustering, indicating that interactions with this cellular protein are required for capsid-dependent reorganization of mitochondria. Recombinant viruses encoding arginine-to-alanine mutations in the p32-binding region of capsid exhibited altered plaque morphology and replicated to lower titers. Further analysis indicated that disruption of stable interactions between capsid and p32 was associated with decreased production of subgenomic RNA and, consequently, infected cells produced significantly lower amounts of viral structural proteins under these conditions. Together, these results suggest that capsid-p32 interactions are important for nonstructural functions of capsid that include regulation of virus RNA replication and reorganization of mitochondria during infection.
Bovine Viral Diarrhea Virus Entry Is Dependent on Clathrin-Mediated Endocytosis
Authors: Lecot Steve , Belouzard Sandrine , Dubuisson Jean , Rouillé Yves 
Published: July 2005

ABSTRACT
Cellular mechanisms of bovine viral diarrhea virus (BVDV) entry in MDBK cells were investigated. Chloroquine, bafilomycin A1, or ammonium chloride inhibited BVDV infection, indicating that an acidic endosomal pH is required for BVDV entry. The tyrosine kinase inhibitor genistein partially inhibited BVDV infection at a postentry step, whereas BVDV entry was strongly inhibited by chlorpromazine or by the overexpression of a dominant-negative form of EPS15, a protein essential for the formation of clathrin-coated vesicles at the plasma membrane. Together, these data indicate that BVDV infection requires an active clathrin-dependent endocytic pathway.
Information theory-based algorithm for in silico prediction of PCR products with whole genomic sequences as templates
Authors: Cao Youfang , Wang Lianjie , Xu Kexue , Kou Chunhai , Zhang Yulei , Wei Guifang , He Junjian , Wang Yunfang , Zhao Liping 
Published: July 2005

Abstract
Background
A new algorithm for assessing similarity between primer and template has been developed based on the hypothesis that annealing of primer to template is an information transfer process.

Results
Primer sequence is converted to a vector of the full potential hydrogen numbers (3 for G or C, 2 for A or T), while template sequence is converted to a vector of the actual hydrogen bond numbers formed after primer annealing. The former is considered as source information and the latter destination information. An information coefficient is calculated as a measure for fidelity of this information transfer process and thus a measure of similarity between primer and potential annealing site on template.

Conclusion
Successful prediction of PCR products from whole genomic sequences with a computer program based on the algorithm demonstrated the potential of this new algorithm in areas like in silico PCR and gene finding.
Computational tradeoffs in multiplex PCR assay design for SNP genotyping
Authors: Rachlin John , Ding Chunming , Cantor Charles , Kasif Simon 
Published: July 2005

Abstract

Background
Multiplex PCR is a key technology for detecting infectious microorganisms, whole-genome sequencing, forensic analysis, and for enabling flexible yet low-cost genotyping. However, the design of a multiplex PCR assays requires the consideration of multiple competing objectives and physical constraints, and extensive computational analysis must be performed in order to identify the possible formation of primer-dimers that can negatively impact product yield.

Results
This paper examines the computational design limits of multiplex PCR in the context of SNP genotyping and examines tradeoffs associated with several key design factors including multiplexing level (the number of primer pairs per tube), coverage (the % of SNP whose associated primers are actually assigned to one of several available tube), and tube-size uniformity. We also examine how design performance depends on the total number of available SNPs from which to choose, and primer stringency criterial. We show that finding high-multiplexing/high-coverage designs is subject to a computational phase transition, becoming dramatically more difficult when the probability of primer pair interaction exceeds a critical threshold. The precise location of this critical transition point depends on the number of available SNPs and the level of multiplexing required. We also demonstrate how coverage performance is impacted by the number of available snps, primer selection criteria, and target multiplexing levels.

Conclusion
The presence of a phase transition suggests limits to scaling Multiplex PCR performance for high-throughput genomics applications. Achieving broad SNP coverage rapidly transitions from being very easy to very hard as the target multiplexing level (# of primer pairs per tube) increases. The onset of a phase transition can be "delayed" by having a larger pool of SNPs, or loosening primer selection constraints so as to increase the number of candidate primer pairs per SNP, though the latter may produce other adverse effects. The resulting design performance tradeoffs define a benchmark that can serve as the basis for comparing competing multiplex PCR design optimization algorithms and can also provide general rules-of-thumb to experimentalists seeking to understand the performance limits of standard multiplex PCR.
Genotyping of Mycobacterium tuberculosis clinical isolates in two cities of Turkey: Description of a new family of genotypes that is phylogeographically specific for Asia Minor
Authors: Zozio Thierry , Allix Caroline , Gunal Selami , Saribas Zeynep , Alp Alpaslan , Durmaz Riza , Fauville-Dufaux Maryse , Rastogi Nalin , Sola Christophe 
Published: July 2005

AbstractBackgroundPopulation-based bacterial genetics using repeated DNA loci is an efficient approach to study the biodiversity and phylogeographical structure of human pathogens, such asMycobacterium tuberculosis, the agent of tuberculosis. Indeed large genetic diversity databases are available for this pathogen and are regularly updated. No population-based polymorphism data were yet available forM. tuberculosisin Turkey, at the crossroads of Eurasia.ResultsA total of 245 DNAs fromMycobacterium tuberculosisclinical isolates from tuberculosis patients residing in Turkey (Malatya n = 147 or Ankara n = 98) were genotyped by spoligotyping, a high-throughput genotyping method based on the polymorphism of the Direct Repeat locus. Thirty-three spoligotyping-defined clusters including 206 patients and 39 unique patterns were found. The ST41 cluster, as designated according to the international SpolDB3 database project, represented one fourth and when gathered to three genotypes, ST53, ST50 and ST284, one half of all the isolates. Out of 34 clinical isolates harboring ST41 which were further genotyped by IS6110and by MIRU-VNTR typing, a typical 2-copy IS6110-RFLP pattern and a "215125113322" MIRU-VNTR pattern were observed among 21 clinical isolates. Further search in various databases confirms the likely Turkish-phylogeographical specificity of this clonal complex.ConclusionWe described a new phylogeographically-specific clone ofM. tuberculosis, designated LAM7-TUR. Further investigations to assess its frequency within all regions of Turkey and its phylogeographical origin and phylogenetic position within the globalM. tuberculosisphylogenetic tree will shed new light on its endemicity in Asia Minor.