bluuebunny/crossref_metadata_2025 huggingface dataset:
Dissociation of Rabies Virus Matrix Protein Functions in Regulation of Viral RNA Synthesis and Virus Assembly
Authors: Finke Stefan , Conzelmann Karl-Klaus
Published: October 2003

Recently, we have shown that the rabies virus (RV) matrix (M) protein regulates the balance of virus RNA synthesis by shifting synthesis activity from transcription to replication (S. Finke, R. Mueller-Waldeck, and K. K. Conzelmann, J. Gen. Virol. 84:1613-1621, 2003). Here we describe the identification of an M residue critical for regulation of RV RNA synthesis. By analyzing the phenotype of heterotypic RV M proteins with respect to RNA synthesis of RV SAD L16, we identified the M proteins of the RV ERA and PV strains as deficient. Comparison of M sequences suggested that a single residue, arginine 58, was critical. A recombinant virus having this amino acid exchanged with a glycine, SAD M(R58G), has lost the abilities to downregulate RV transcription and to stimulate replication. This resulted in an increase in the transcription rate of more than 15-fold, as previously observed for M deletion mutants. Most importantly, the efficiencies of virus assembly and budding were equal for wild-type M and M(R58G), as determined in assays studying the transient complementation of an M- and G-deficient RV construct, NPgrL. In addition, virus particle density, protein composition, and specific infectivity of SAD L16 and SAD M(R58G) viruses were identical. Thus, we have identified mutations that affect the function of M only in regulation of RNA synthesis, but not in assembly and budding, providing evidence that these functions are genetically separable.
Comprehensive Analysis of Class I and Class II HLA Antigens and Chronic Hepatitis B Virus Infection
Authors: Thio Chloe L. , Thomas David L. , Karacki Peter , Gao Xiaojiang , Marti Darlene , Kaslow Richard A. , Goedert James J. , Hilgartner Margaret , Strathdee Steffanie A. , Duggal Priya , O'Brien Stephen J. , Astemborski Jacquie , Carrington Mary
Published: October 2003

Following an acute hepatitis B virus (HBV) infection, clearance or persistence is determined in part by the vigor and breadth of the host immune response. Since the human leukocyte antigen system (HLA) is an integral component of the immune response, we hypothesized that the highly polymorphic
HLA
genes are key determinants of viral clearance.
HLA
class I and II genes were molecularly typed in 194 Caucasian individuals with viral persistence and 342 matched controls who had cleared the virus. A single class I allele,
A*0301
(odds ratio [OR], 0.47; 95% confidence interval [CI], 0.30 to 0.72;
P
= 0.0005) was associated with viral clearance. The class II allele
DRB1*1302
was also associated with clearance (OR, 0.42; 95% CI, 0.19 to 0.93;
P
= 0.03), but its significance decreased in a multivariate model that included other alleles associated with disease outcome as covariates.
B*08
was associated with viral persistence both independently (OR, 1.59; 95% CI, 1.04 to 2.43;
P
= 0.03) and as part of the conserved Caucasian haplotype
A*01-B*08-DRB1*03
. The
B*44-Cw*1601
(OR, 2.23; 95% CI, 1.13 to 4.42;
P
= 0.02) and
B*44-Cw*0501
(OR, 1.99; 95% CI, 1.22 to 3.24;
P
= 0.006) haplotypes were also associated with viral persistence. Interestingly, both the
B*08
haplotype and
DR7
, which forms a haplotype with
B*44-Cw*1601
, have been associated with nonresponse to the HBV vaccine. The associations with class I alleles are consistent with a previously implicated role for CD8-mediated cytolytic-T-cell response in determining the outcome of an acute HBV infection.
Subpopulations of Equine Infectious Anemia Virus Rev Coexist In Vivo and Differ in Phenotype
Authors: Baccam Prasith , Thompson Robert J. , Li Yuxing , Sparks Wendy O. , Belshan Michael , Dorman Karin S. , Wannemuehler Yvonne , Oaks J. Lindsay , Cornette James L. , Carpenter Susan
Published: October 2003

Lentiviruses exist in vivo as a population of related, nonidentical genotypes, commonly referred to as quasispecies. The quasispecies structure is characteristic of complex adaptive systems and contributes to the high rate of evolution in lentiviruses that confounds efforts to develop effective vaccines and antiviral therapies. Here, we describe analyses of genetic data from longitudinal studies of genetic variation in a lentivirus regulatory protein, Rev, over the course of disease in ponies experimentally infected with equine infectious anemia virus. As observed with other lentivirus data, the Rev variants exhibited a quasispecies character. Phylogenetic and partition analyses suggested that the Rev quasispecies comprised two distinct subpopulations that coexisted during infection. One subpopulation appeared to accumulate changes in a linear, time-dependent manner, while the other evolved radially from a common variant. Over time, the two subpopulations cycled in predominance coincident with changes in the disease state, suggesting that the two groups differed in selective advantage. Transient expression assays indicated the two populations differed significantly in Rev nuclear export activity. Chimeric proviral clones containing Rev genotypes representative of each population differed in rate and overall level of virus replication in vitro. The coexistence of genetically distinct viral subpopulations that differ in phenotype provides great adaptability to environmental changes within the infected host. A quasispecies model with multiple subpopulations may provide additional insight into the nature of lentivirus reservoirs and the evolution of antigenic and drug-resistant variants.
Human Immunodeficiency Virus Type 1 Enters Primary Human Brain Microvascular Endothelial Cells by a Mechanism Involving Cell Surface Proteoglycans Independent of Lipid Rafts
Authors: Argyris Elias G. , Acheampong Edward , Nunnari Giuseppe , Mukhtar Muhammad , Williams Kevin Jon , Pomerantz Roger J.
Published: October 2003

Several studies have reported a crucial role for cholesterol-enriched membrane lipid rafts and cell-associated heparan sulfate proteoglycans (HSPGs), a class of molecules that can localize in lipid rafts, in the entry of human immunodeficiency virus type 1 (HIV-1) into permissive cells. For the present study, we examined the role of these cell surface moieties in HIV-1 entry into primary human brain microvascular endothelial cells (BMVECs), which represent an important HIV-1 central nervous system-based cell reservoir and a portal for neuroinvasion. Cellular cholesterol was depleted by exposure to β-cyclodextrins and 3-hydroxy-3-methylglutaryl (HMG)-coenzyme A reductase inhibitors (statins), the loss of cholesterol was quantitated, and disruption of membrane rafts was verified by immunofluorescence. Nevertheless, these treatments did not affect binding of several strains of HIV-1 virions to BMVECs at 4°C or their infectivities at 37°C. In contrast, we confirmed that cholesterol depletion and raft disruption strongly inhibited HIV-1 binding and infection of Jurkat T cells. Enzymatic digestion of cell-associated HSPGs on human BMVECs dramatically inhibited HIV-1 infection, and our data from quantitative HIV-1 DNA PCR analysis strongly suggest that cell-associated chondroitin sulfate proteoglycans greatly facilitate infective entry of HIV-1 into human BMVECs. These findings, in combination with our earlier work showing that human BMVECs lack CD4, indicate that the molecular mechanisms for HIV-1 entry into BMVECs are fundamentally different from that of viral entry into T cells, in which lipid rafts, CD4, and probably HSPGs play important roles.
Human T-Cell Leukemia Virus Type 1 Tax Oncoprotein Suppression of Multilineage Hematopoiesis of CD34+Cells In Vitro
Authors: Tripp Adam , Liu Yingxian , Sieburg Michelle , Montalbano Joanne , Wrzesinski Stephen , Feuer Gerold
Published: October 2003

Human T-cell leukemia virus type 1 (HTLV-1) and HTLV-2 are highly related viruses that differ in disease manifestation. HTLV-1 is the etiologic agent of adult T-cell leukemia and lymphoma, an aggressive clonal malignancy of human CD4-bearing T lymphocytes. Infection with HTLV-2 has not been conclusively linked to lymphoproliferative disorders. We previously showed that human hematopoietic progenitor (CD34+) cells can be infected by HTLV-1 and that proviral sequences were maintained after differentiation of infected CD34+cells in vitro and in vivo. To investigate the role of the Tax oncoprotein of HTLV on hematopoiesis, bicistronic lentiviral vectors were constructed encoding the HTLV-1 or HTLV-2taxgenes (Tax1 and Tax2, respectively) and the green fluorescent protein marker gene. Human hematopoietic progenitor (CD34+) cells were infected with lentivirus vectors, and transduced cells were cultured in a semisolid medium permissive for the development of erythroid, myeloid, and primitive progenitor colonies. Tax1-transduced CD34+cells displayed a two- to fivefold reduction in the total number of hematopoietic clonogenic colonies that arose in vitro, in contrast to Tax2-transduced cells, which showed no perturbation of hematopoiesis. The ratio of colony types that developed from Tax1-transduced CD34+cells remained unaffected, suggesting that Tax1 inhibited the maturation of relatively early, uncommitted hematopoietic stem cells. Since previous reports have linked Tax1 expression with initiation of apoptosis, lentiviral vector-mediated transduction of Tax1 or Tax2 was investigated in CEM and Jurkat T-cell lines. Ectopic expression of either Tax1 or Tax2 failed to induce apoptosis in T-cell lines. These data demonstrate that Tax1 expression perturbs development and maturation of pluripotent hematopoietic progenitor cells, an activity that is not displayed by Tax2, and that the suppression of hematopoiesis is not attributable to induction of apoptosis. Since hematopoietic progenitor cells may serve as a latently infected reservoir for HTLV infection in vivo, the different abilities of HTLV-1 and -2 Tax to suppress hematopoiesis may play a role in the respective clinical outcomes after infection with HTLV-1 or -2.
Viral Blip Dynamics during Highly Active Antiretroviral Therapy
Authors: Di Mascio Michele , Markowitz Martin , Louie Michael , Hogan Christine , Hurley Arlene , Chung Chris , Ho David D. , Perelson Alan S.
Published: October 2003

Although intermittent episodes of low-level viremia are often observed in well-suppressed highly active antiretroviral therapy (HAART)-treated patients, the timing and amplitude of viral blips have never been examined in detail. We analyze here the dynamics of viral blips, i.e., plasma VL measurements of >50 copies/ml, in 123 HAART-treated patients monitored for a mean of 2.6 years (range, 5 months to 5.3 years). The mean (± the standard deviation) blip frequency was 0.09 ± 0.11/sample, with about one-third of patients showing no viral blips. The mean viral blip amplitude was 158 ± 132 human immunodeficiency virus type 1 (HIV-1) RNA copies/ml. Analysis of the blip frequency and amplitude distributions suggest that two blips less than 22 days apart have a significant chance of being part of the same episode of viremia. The data are consistent with a hypothetical model in which each episode of viremia consists of a phase of VL rise, followed by two-phase exponential decay. Thus, the term “viral blip” may be a misnomer, since viral replication appears to be occurring over an extended period. Neither the frequency nor the amplitude of viral blips increases with longer periods of observation, but the frequency is inversely correlated with the CD4
+
-T-cell count at the start of therapy, suggesting that host-specific factors but not treatment fatigue are determinants of blip frequency.
Colocalization and Interaction of the Porcine Arterivirus Nucleocapsid Protein with the Small Nucleolar RNA-Associated Protein Fibrillarin
Authors: Yoo Dongwan , Wootton Sarah K. , Li Gang , Song Cheng , Rowland Raymond R.
Published: October 2003

Porcine reproductive and respiratory syndrome virus (PRRSV) replicates in the cytoplasm of infected cells, but its nucleocapsid (N) protein localizes specifically to the nucleus and nucleolus. The mechanism of nuclear translocation and whether N associates with particular nucleolar components are unknown. In the present study, we show by confocal microscopy that the PRRSV N protein colocalizes with the small nucleolar RNA (snoRNA)-associated protein fibrillarin. Direct and specific interaction of N with fibrillarin was demonstrated in vivo by the mammalian two-hybrid assay in cells cotransfected with the N and fibrillarin genes and in vitro by the glutathione
S
-transferase pull-down assay using the expressed fibrillarin protein. Using a series of deletion mutants, the interactive domain of N with fibrillarin was mapped to a region of amino acids 30 to 37. For fibrillarin, the first 80 amino acids, which contain the glycine-arginine-rich region (the GAR domain), was determined to be the domain interactive with N. The N protein was able to bind to the full-length genomic RNA of PRRSV, and the RNA binding domain was identified as the region overlapping with the nuclear localization signal situated at positions 41 to 47. These results suggest that the N protein nuclear transport may be controlled by the binding of RNA to N. The PRRSV N protein was also able to bind to both 28S and 18S ribosomal RNAs. The protein-protein interaction between N and fibrillarin was RNA dependent but independent of N protein phosphorylation. Taken together, our studies demonstrate a specific interaction of the PRRSV nucleocapsid protein with the host cell protein fibrillarin in the nucleolus, and they imply a potential linkage of viral strategies for the modulation of host cell functions, possibly through rRNA precursor processing and ribosome biogenesis.
The N- and C-Terminal Regions of Rotavirus NSP5 Are the Critical Determinants for the Formation of Viroplasm-Like Structures Independent of NSP2
Authors: Mohan K. V. K. , Muller J. , Atreya C. D.
Published: October 2003

Molecular events and the interdependence of the two rotavirus nonstructural proteins, NSP5 and NSP2, in producing viroplasm-like structures (VLS) were previously evaluated by using transient cellular coexpression of the genes for the two proteins, and VLS domains as well as the NSP2-binding region of NSP5 were mapped in the context of NSP2. Review of the previous studies led us to postulate that NSP2 binding of NSP5 may block the N terminus of NSP5 or render it inaccessible and that any similar N-terminal blockage may render NSP5 alone capable of producing VLS independent of NSP2. This possibility was addressed in this report by using two forms of NSP5-green fluorescent protein (GFP) chimeras wherein GFP is fused at either the N or the C terminus of NSP5 (GFP-NSP5 and NSP5-GFP) and evaluating their VLS-forming capability (by light and electron microscopy) and phosphorylation and multimerization potential independent of NSP2. Our results demonstrate that NSP5 alone can form VLS when the N terminus is blocked by fusion with a nonrotavirus protein (GFP-NSP5) but the C terminus is unmodified. Only GFP-NSP5 was able to undergo hyperphosphorylation and multimerization with the native form of NSP5, emphasizing the importance of an unmodified C terminus for these events. Deletion analysis of NSP5 mapped the essential signals for VLS formation to the C terminus and clearly suggested that hyperphosphorylation of NSP5 is not required for VLS formation. The present study emphasizes in general that when fusion proteins are used for functional studies, constructs that represent fusions at both the N and the C termini of the protein should be evaluated.
Engineered Retargeting of Viral RNA Replication Complexes to an Alternative Intracellular Membrane
Authors: Miller David J. , Schwartz Michael D. , Dye Billy T. , Ahlquist Paul
Published: October 2003

Positive-strand RNA virus replication complexes are universally associated with intracellular membranes, although different viruses use membranes derived from diverse and sometimes multiple organelles. We investigated whether unique intracellular membranes are required for viral RNA replication complex formation and function in yeast by retargeting protein A, the Flock House virus (FHV) RNA-dependent RNA polymerase. Protein A, the only viral protein required for FHV RNA replication, targets and anchors replication complexes to outer mitochondrial membranes in part via an N-proximal sequence that contains a transmembrane domain. We replaced the FHV protein A mitochondrial outer membrane-targeting sequence with the N-terminal endoplasmic reticulum (ER)-targeting sequence from the yeast NADP cytochrome P450 oxidoreductase or inverted C-terminal ER-targeting sequences from the hepatitis C virus NS5B polymerase or the yeast t-SNARE Ufe1p. Confocal immunofluorescence microscopy confirmed that protein A chimeras retargeted to the ER. FHV subgenomic and genomic RNA accumulation in yeast expressing ER-targeted protein A increased 2- to 13-fold over that in yeast expressing wild-type protein A, despite similar protein A levels. Density gradient flotation assays demonstrated that ER-targeted protein A remained membrane associated, and in vitro RNA-dependent RNA polymerase assays demonstrated an eightfold increase in the in vitro RNA synthesis activity of the ER-targeted FHV RNA replication complexes. Electron microscopy showed a change in the intracellular membrane alterations from a clustered mitochondrial distribution with wild-type protein A to the formation of perinuclear layers with ER-targeted protein A. We conclude that specific intracellular membranes are not required for FHV RNA replication complex formation and function.
Generation of a Recombinant Cytomegalovirus for Expression of a Hantavirus Glycoprotein
Authors: Rizvanov Albert A. , van Geelen Albert G. M. , Morzunov Sergey , Otteson Elmer W. , Bohlman Charlotte , Pari Gregory S. , St. Jeor Stephen C.
Published: October 2003

A cytomegalovirus (CMV) was isolated from its natural host,
Peromyscus maniculatus
, and was designated Peromyscus CMV (PCMV). A recombinant PCMV was constructed that contained Sin Nombre virus glycoprotein G1 (SNV-G1) fused in frame to the enhanced green fluorescent protein (EGFP) gene inserted into a site homologous to the human CMV UL33 (P33) gene. The recombinant CMV was used for expression and immunization of deer mice against SNV-G1. The results of the study indicate that
P. maniculatus
could be infected with as few as 10 virus particles of recombinant virus. Challenge of
P. maniculatus
with either recombinant or wild-type PCMV produced no overt pathology in infected animals.
P. maniculatus
immunized with recombinant virus developed an antibody response to SNV and EGFP. When rechallenged with recombinant virus, animals exhibited an anamnestic response against SNV. Interestingly, a preexisting immune response against PCMV did not prevent reinfection with recombinant PCMV.
Structures of Host Range-Controlling Regions of the Capsids of Canine and Feline Parvoviruses and Mutants
Authors: Govindasamy Lakshmanan , Hueffer Karsten , Parrish Colin R. , Agbandje-McKenna Mavis
Published: October 2003

Canine parvovirus (CPV) and feline panleukopenia virus (FPV) differ in their ability to infect dogs and dog cells. Canine cell infection is a specific property of CPV and depends on the ability of the virus to bind the canine transferrin receptor (TfR), as well as other unidentified factors. Three regions in the capsid structure, located around VP2 residues 93, 300, and 323, can all influence canine TfR binding and canine cell infection. These regions were compared in the CPV and FPV capsid structures that have been determined, as well as in two new structures of CPV capsids that contain substitutions of the VP2 Asn-93 to Asp and Arg, respectively. The new structures, determined by X-ray crystallography to 3.2 and 3.3 Å resolutions, respectively, clearly showed differences in the interactions of residue 93 with an adjacent loop on the capsid surface. Each of the three regions show small differences in structure, but each appears to be structurally independent of the others, and the changes likely act together to affect the ability of the capsid to bind the canine TfR and to infect canine cells. This emphasizes the complex nature of capsid alterations that change the virus-cell interaction to allow infection of cells from different hosts.
Borna Disease Virus Phosphoprotein Represses p53-Mediated Transcriptional Activity by Interference with HMGB1
Authors: Zhang Guoqi , Kobayashi Takeshi , Kamitani Wataru , Komoto Satoshi , Yamashita Makiko , Baba Satoko , Yanai Hideyuki , Ikuta Kazuyoshi , Tomonaga Keizo
Published: October 2003

Borna disease virus(BDV) is a noncytolytic, neurotropic RNA virus that has a broad host range in warm-blooded animals, probably including humans. Recently, it was demonstrated that a 24-kDa phosphoprotein (P) of BDV directly binds to a multifunctional protein, amphoterin-HMGB1, and inhibits its function in cultured neural cells (W. Kamitani, Y. Shoya, T. Kobayashi, M. Watanabe, B. J. Lee, G. Zhang, K. Tomonaga, and K. Ikuta, J. Virol. 75:8742-8751, 2001). This observation suggested that expression of BDV P may cause deleterious effects in cellular functions by interference with HMGB1. In this study, we further investigated the significance of the binding between P and HMGB1. We demonstrated that P directly binds to the A-box domain on HMGB1, which is also responsible for interaction with a tumor suppression factor, p53. Recent works have demonstrated that binding between HMGB1 and p53 enhances p53-mediated transcriptional activity. Thus, we examined whether BDV P affects the transcriptional activity of p53 by interference with HMGB1. Mammalian two-hybrid analysis revealed that p53 and P competitively interfere with the binding of each protein to HMGB1 in a p53-deficient cell line, NCI-H1299. In addition, P was able to significantly decrease p53-mediated transcriptional activation of the cyclin G promoter. Furthermore, we showed that activation of p21waf1expression was repressed in cyclosporine-treated BDV-infected cells, as well as p53-transduced NCI-H1299 cells. These results suggested that BDV P may be a unique inhibitor of p53 activity via binding to HMGB1.
The Pseudorabies Virus Us2 Protein, a Virion Tegument Component, Is Prenylated in Infected Cells
Authors: Clase Amanda C. , Lyman Mathew G. , del Rio T. , Randall Jessica A. , Calton Christine M. , Enquist L. W. , Banfield Bruce W.
Published: October 2003

The Us2 gene is conserved among alphaherpesviruses, but its function is not known. We demonstrate here that the pseudorabies virus (PRV) Us2 protein is synthesized early after infection and localizes to cytoplasmic vesicles and to the plasma membrane, despite the lack of a recognizable signal sequence or membrane-spanning domain. Us2 protein is also packaged as part of the tegument of mature virions. The Us2 carboxy-terminal four amino acids comprise a CAAX motif, a well-characterized signal for protein prenylation. Treatment of infected cells with lovastatin, a drug that disrupts protein prenylation, changed the relative electrophoretic mobility of Us2 in sodium dodecyl sulfate-polyacrylamide gels. In addition, lovastatin treatment caused a dramatic relocalization of Us2 to cytoplasmic punctate structures associated with microtubules, which appeared to concentrate over the microtubule organizing center. When the CAAX motif was changed to GAAX and the mutant protein was synthesized from an expression plasmid, it concentrated in punctate cytoplasmic structures reminiscent of Us2 localization in infected cells treated with lovastatin. We suggest that prenylation of PRV Us2 protein is required for proper membrane association. Curiously, the Us2 protein isolated from purified virions does not appear to be prenylated. This is the first report to describe the prenylation of an alphaherpesvirus protein.
Structure and Function of CC-Chemokine Receptor 5 Homologues Derived from Representative Primate Species and Subspecies of the Taxonomic Suborders Prosimii and Anthropoidea
Authors: Kunstman Kevin J. , Puffer Bridget , Korber Bette T. , Kuiken Carla , Smith Una R. , Kunstman Jennifer , Stanton Jennifer , Agy Michael , Shibata Riri , Yoder Anne D. , Pillai Satish , Doms Robert W. , Marx Preston , Wolinsky Steven M.
Published: October 2003

A chemokine receptor from the seven-transmembrane-domain G-protein-coupled receptor superfamily is an essential coreceptor for the cellular entry of human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) strains. To investigate nonhuman primate CC-chemokine receptor 5 (CCR5) homologue structure and function, we amplified CCR5 DNA sequences from peripheral blood cells obtained from 24 representative species and subspecies of the primate suborders Prosimii (family Lemuridae) and Anthropoidea (families Cebidae, Callitrichidae, Cercopithecidae, Hylobatidae, and Pongidae) by PCR with primers flanking the coding region of the gene. Full-length CCR5 was inserted into pCDNA3.1, and multiple clones were sequenced to permit discrimination of both alleles. Compared to the human CCR5 sequence, the CCR5 sequences of the Lemuridae, Cebidae, and Cercopithecidae shared 87, 91 to 92, and 96 to 99% amino acid sequence homology, respectively. Amino acid substitutions tended to cluster in the amino and carboxy termini, the first transmembrane domain, and the second extracellular loop, with a pattern of species-specific changes that characterized CCR5 homologues from primates within a given family. At variance with humans, all primate species examined from the suborder Anthropoidea had amino acid substitutions at positions 13 (N to D) and 129 (V to I); the former change is critical for CD4-independent binding of SIV to CCR5. Within the Cebidae, Cercopithecidae, and Pongidae (including humans), CCR5 nucleotide similarities were 95.2 to 97.4, 98.0 to 99.5, and 98.3 to 99.3%, respectively. Despite this low genetic diversity, the phylogeny of the selected primate CCR5 homologue sequences agrees with present primate systematics, apart from some intermingling of species of the Cebidae and Cercopithecidae. Constructed HOS.CD4 cell lines expressing the entire CCR5 homologue protein from each of the Anthropoidea species and subspecies were tested for their ability to support HIV-1 and SIV entry and membrane fusion. Other than that of
Cercopithecus
pygerythrus
, all CCR5 homologues tested were able to support both SIV and HIV-1 entry. Our results suggest that the shared structure and function of primate CCR5 homologue proteins would not impede the movement of primate immunodeficiency viruses between species.
Human Papillomavirus Type 16 E7 Oncoprotein Can Induce Abnormal Centrosome Duplication through a Mechanism Independent of Inactivation of Retinoblastoma Protein Family Members
Authors: Duensing Stefan , Münger Karl
Published: October 2003

The human papillomavirus type 16 (HPV-16) E7 oncoprotein rapidly induces centrosome duplication errors in primary human cells, thereby increasing the propensity for multipolar mitoses, which can lead to chromosome missegregation and aneuploidy. We analyzed a series of HPV-16 E7 mutants and demonstrate that this biological activity of the E7 oncoprotein is mediated by sequences encompassing the core pRB binding site but is independent of its ability to inactivate the retinoblastoma tumor suppressor protein pRB and the related pocket proteins p107 and p130. In addition, interaction of E7 with the S4 subunit of the 26S proteasome and dysregulation of cdc25A transcription are also dispensable for the induction of centrosome duplication errors. Consistent with these results, expression of HPV-16 E7 induces abnormal centrosome duplication in a cell line that lacks functional pRB and in mouse embryo fibroblasts that are deficient for pRB, p107, and p130. These results demonstrate that the molecular mechanism whereby HPV-16 E7 induces centrosome duplication errors is independent of its ability to inactivate pRB, p107, and p130 or to interact with the S4 proteasome subunit.
High Frequency of Virus-Specific CD8+T Cells in the Central Nervous System of Macaques Chronically Infected with Simian Immunodeficiency Virus SIVmac251
Authors: Moniuszko Marcin , Brown Charlie , Pal Ranajit , Tryniszewska Elzbieta , Tsai Wen-Po , Hirsch Vanessa M. , Franchini Genoveffa
Published: October 2003

Infection with human immunodeficiency virus or simian immunodeficiency virus (SIV) induces virus-specific CD8+T cells that traffic to lymphoid and nonlymphoid tissues. In this study, we used Gag-specific tetramer staining to investigate the frequency of CD8+T cells in peripheral blood and the central nervous system of Mamu-A*01-positive SIV-infected rhesus macaques. Most of these infected macaques were vaccinated prior to SIVmac251 exposure. The frequency of Gag181-189CM9 tetramer-positive cells was consistently higher in the cerebrospinal fluid and the brain than in the blood of all animals studied and did not correlate with either plasma viremia or CD4+-T-cell level. Little or no infection in the brain was documented for most animals by nucleic acid sequence-based amplification or in situ hybridization. These data suggest that this Gag-specific response may contribute to the containment of viral replication in this locale.
RNA Helicase Domain of Tobamovirus Replicase Executes Cell-to-Cell Movement Possibly through Collaboration with Its Nonconserved Region
Authors: Hirashima Kyotaro , Watanabe Yuichiro
Published: October 2003

UR-hel, a chimeric virus obtained by replacement of the RNA helicase domain of tobacco mosaic virus (TMV)-U1 replicase with that from the TMV-R strain, could replicate similarly to TMV-U1 in protoplasts but could not move from cell to cell (K. Hirashima and Y. Watanabe, J. Virol. 75:8831-8836, 2001). It was suggested that TMV recruited both the movement protein (MP) and replicase for cell-to-cell movement by unknown mechanisms. Here, we found that a recombinant, UR-hel/V, in which the nonconserved region was derived from TMV-R in addition to the RNA helicase domain of replicase, could move from cell to cell. We also analyzed revertants isolated from UR-hel, which recovered cell-to-cell movement by their own abilities. We found amino acid substitutions responsible for phenotypic reversion only in the nonconserved region and/or RNA helicase domain but never in MP. Together, these data show that both the nonconserved region and the RNA helicase domain of replicase are involved in cell-to-cell movement. The RNA helicase domain of tobamovirus replicase possibly does not interact directly with MP but interacts with its nonconserved region to execute cell-to-cell movement.
A Bipartite Late-Budding Domain in Human Immunodeficiency Virus Type 1
Authors: Martin-Serrano Juan , Bieniasz Paul D.
Published: October 2003

Human immunodeficiency virus type 1 (HIV-1) encodes a PTAP motif within the p6 domain of Gag that recruits Tsg101 and associated factors to facilitate virion budding. In this study, we use
trans
-complementation assays to demonstrate that the PTAP motif acts synergistically with additional p6 sequences to mediate the formation of infectious extracellular HIV-1 virions. These studies suggest that Tsg101 recruitment is necessary but not sufficient to account for late-budding activity exhibited by HIV-1 p6.
Role of Macrophage Inflammatory Protein-1α in T-Cell-Mediated Immunity to Viral Infection
Authors: Madsen Andreas N. , Nansen Anneline , Christensen Jan P. , Thomsen Allan R.
Published: October 2003

The immune response to lymphocytic choriomeningitis virus in mice lacking macrophage inflammatory protein-1α (MIP-1α) was evaluated. Generation of virus-specific effector T cells is unimpaired in MIP-1α-deficient mice. Furthermore, MIP-1α is not required for T-cell-mediated virus control or virus-induced T-cell-dependent inflammation. Thus, MIP-1α is not mandatory for T-cell-mediated antiviral immunity.
Octadecylrhodamine B as a Specific Micelle-Binding Fluorescent Tag for Fluorescence Correlation Spectroscopy Studies of Amphiphilic Water-Soluble Block Copolymer Micelles. Spectroscopic Behavior in Aqueous Media
Authors: Humpolíčková Jana , Procházka Karel , Hof Martin
Published: October 2003

Fluorescence behavior of octadecylrhodamine B (ORB) and the association-dependent changes in UV-VIS absorption and fluorescence spectra in aqueous solutions were studied by UV-VIS absorption spectroscopy and steady-state and time-resolved fluorometry. Spectral changes are interpreted in terms of the hydrophobically driven formation of H- and J-dimers and higher aggregates. The knowledge gained serves as a basis for the interpretation of results of the fluorescence correlation spectroscopy study of polymeric micelles labeled with ORB.
Light Scattering, Atomic Force Microscopy and Fluorescence Correlation Spectroscopy Studies of Polystyrene-block-poly(2-vinylpyridine)-block-poly(ethylene oxide) Micelles
Authors: Štěpánek Miroslav , Humpolíčková Jana , Procházka Karel , Hof Martin , Tuzar Zdeněk , Špírková Milena , Wolff Thomas
Published: October 2003

Polymeric nanoparticles formed by triblock copolymer polystyrene-block-poly(2-vinylpyridine)-block-poly(ethylene oxide), PS-PVP-PEO, in aqueous media were studied by a combination of fluorescence correlation spectroscopy with other fluorescence techniques, light scattering and atomic force microscopy. The studied polymeric nanoparticles exist in the form of (i) core/shell micelles in acid solution at pH lower than 4.8 and (ii) three-layer onion micelles at higher pH. Since water is a very strong precipitant for PS, both types of micelles have kinetically frozen spherical PS cores. The cores of micelles in acid media are surrounded by soluble shells formed by partly protonated PVP and PEO, while the cores of micelles in alkaline media are surrounded by compact insoluble layers of deprotonated PVP and soluble PEO shells. The micellization behavior of PS-PVP-PEO micelles is accompanied by secondary aggregation of micelles, which is provoked by stirring, shaking and also by filtration of micellar solutions. Therefore fluorescence correlation spectroscopy (FCS), which, in contrast to light scattering techniques, does not require filtration, was used as the main experimental technique for the characterization of non-aggregated micelles. The binding of a fluorescence probe, octadecylrhodamine B (ORB), to polymeric micelles, was studied before the FCS study of micelles.
New 2-Alkynyl Derivatives of the Acyclic Nucleoside 9-(2,3-Dihydroxypropyl)adenine and Their 6-Guanidinopurine Counterparts as Potential Effectors of Adenosine Receptors
Authors: Česnek Michal , Holý Antonín , Masojídková Milena
Published: October 2003

A series of the new 2-alkynyl derivatives of the acyclic nucleoside 9-(2,3-dihydroxypropyl)- adenine and their 6-guanidinopurine analogues were prepared by the Sonogashira coupling. The effect of the prepared compounds on A1 and A2A receptors was examined.
Spatial distribution of catch and effort in a fishery for snow crab (Chionoecetes opilio): tests of predictions of the ideal free distribution
Authors: Swain Douglas P , Wade Elmer J
Published: October 2003

The ideal free distribution (IFD), a hypothesis from behavioural ecology, predicts that fishery effort should map resource distribution better than catch-per-unit-effort (CPUE) when interference competition occurs in the fishery. We tested this prediction using data from the fishery and annual research survey for snow crab (Chionoecetes opilio) in the southern Gulf of St. Lawrence. Effort was positively correlated with the local abundance of crabs in all years. Correlations between CPUE and local crab abundance were also positive in some years, but negative in others. In the latter cases, CPUE and effort were also negatively correlated, suggesting intense competition in the fishery. In most years, CPUE tended to be equalized among areas compared with the distributions of effort and local crab abundance, as predicted by the IFD. In most years, differences in spatial distribution were more significant between CPUE and crab abundance than between effort and crab abundance. Although effort was the more reliable indicator of resource distribution, even it provided a distorted view of this distribution, as predicted given expected violations of IFD assumptions. For example, effort tended to be higher than expected on fishing grounds near home ports and lower than expected on distant grounds.
Intraspecific density dependence in the dynamics of zooplankton under hypertrophic conditions
Authors: Declerck Steven , Geenens Vanessa , Podoor Nicole , Porcuna José Maria Conde , Meester Luc De
Published: October 2003

Intraspecific interactions may limit population growth of small cladoceran taxa under food-rich, hypertrophic conditions. Multiple-regression models significantly explained a large proportion of the variation in the body size adjusted fecundity and population growth rate of crustacean zooplankton taxa in a shallow, hypertrophic lake. The results of partial correlation analyses suggested exploitative competition to have only limited significance in determining the zooplankton dynamics. The analyses also revealed strong negative relationships between biomass and both body size adjusted fecundity and population growth rate within most taxa. Most of these relationships cannot be explained by food shortage or predation and suggest alternative mechanisms such as chemically mediated, intraspecific interference competition or life history shifts.
Differential response of marine populations to climate forcing
Authors: McCann Kevin S , Botsford Louis W , Hasting Alan
Published: October 2003

In searching for causes of fluctuations in marine populations, investigators often assume that populations respond on the same time scale as the environmental forcing period, but this may not hold true. Here we show how the response of populations to variable recruitment changes with the degree of overcompensation using models of two species with similar age structure but different density-dependent recruitment, chinook salmon (Oncorhynchus tshawytscha) and Dungeness crab (Cancer magister). For compensatory density dependence, as in chinook salmon, variability in recruitment tends to follow the period in environmental variability over all time scales. For overcompensatory density dependence, as in Dungeness crab, variability in recruitment follows the environmental variability only for periods much greater than the maximum age of the population. For periods in environmental variability less than the maximum age, the dominant period of the population response is slightly larger than the length of the age structure. Here, strong overcompensatory recruitment acts to filter out potentially good recruitment years, resulting in dominant periodicities slightly larger than the length of the age structure. These mechanisms appear to explain the differences between observed spectra of Dungeness crab and chinook salmon.
Energy losses due to routine and feeding metabolism in young-of-the-year juvenile Atlantic cod (Gadus morhua)
Authors: Peck Myron A , Buckley Lawrence J , Bengtson David A
Published: October 2003

We examined the effects of body size (313 cm total length) and temperature (4.5, 8.0, 12.0, and 15.5 °C) on routine (RR) and feeding (RSDA) energy losses by laboratory-reared, young-of-year juvenile Atlantic cod (Gadus morhua). The magnitude of the effect of temperature on RR, expressed via the Q10, was nonlinear. Q10 values were greatest at temperatures between 4.5 and 8.0 °C and were lowest between 8.0 and 15.5 °C, with larger fish tending to exhibit the greatest change in RR irrespective of the temperature combination. Energy losses resulting from RSDA were ~4% of consumed energy, a value less than half that estimated for larger, year-1+ juvenile cod fed similar-sized rations. Data from this and other studies were combined to generate an equation estimating routine energy loss at different temperatures and body sizes for cod. The equation describes RR over the eight orders of magnitude difference in body size from young larvae to adults within a range of environmental temperatures experienced by this species on Georges Bank and other areas in the North Atlantic.
Stress and saltwater-entry behavior of juvenile chinook salmon (Oncorhynchus tshawytscha): conflicts in physiological motivation
Authors: Price Carol Seals , Schreck Carl B
Published: October 2003

Two experiments were conducted to determine the effects of a mild or severe stressor on the saltwater preference of juvenile spring chinook salmon (Oncorhynchus tshawytscha). To observe the response of fish to an overhead threat, we presented stressed fish with an avian predator model in the second experiment. Experiments were conducted in 757-L tanks containing a stable vertical salinity gradient. Only 69% of fish stressed by being chased for 2 min before saltwater introduction (mild stressor) held in salt water, whereas 95% of unchased control fish preferred the saltwater layer. After the more severe handling and confinement stressor, only 20% of fish entered and remained in salt water compared with 100% of unstressed controls. After the presentation of the avian model, stressed fish holding in fresh water moved into the saltwater layer, but this behavioral response was transient. Fish began returning to fresh water within 10 min, and after 1 h, only 26% of stressed fish remained in the saltwater layer. Stress significantly decreases the saltwater preference of chinook salmon that would otherwise select full-strength salt water and may affect behavior in the estuary. Although smoltification primes these fish for seawater residence, stress apparently induced a conflicting physiological motivation.
Among-taxon congruence in biodiversity patterns: can stream insect diversity be predicted using single taxonomic groups?
Authors: Heino Jani , Muotka Timo , Paavola Riku , Paasivirta Lauri
Published: October 2003

The utility of single taxonomic groups as indicators of biodiversity variation in other taxa has recently gained increasing attention, but such studies on stream organisms are lacking. We studied the diversity patterns of mayflies, stoneflies, caddisflies, and chironomid midges across 110 headwater streams in Finland. Specifically, we examined if species richness and assemblage composition showed similar variation among the taxonomic groups across environmental gradients and if a single taxon could be used as a predictor of diversity in the other groups. Species richness and composition in different taxa exhibited slightly different relationships to environmental gradients, leading to low degrees of concordance. The diversity of mayflies and chironomids showed strongest relationships with stream acidity and water colour, whereas stoneflies and caddisflies exhibited more complex correlations with geographical location and local environmental variables. The overall assemblage composition, however, exhibited significant among-taxon congruence, as shown by Mantel tests. However, even these correlations remained rather low, thus limiting their potential for conservation purposes. Overall, our results do not support the use of single taxonomic groups as indicators of insect biodiversity in headwater stream ecosystems. Alternative approaches for lotic biodiversity assessment (e.g., morphospecies, higher-taxon richness, and environmental diversity) should thus be examined in future studies.
Predicting the location of optimal habitat boundaries for lake trout (Salvelinus namaycush) in Canadian Shield lakes
Authors: Dillon P J , Clark B J , Molot L A , Evans H E
Published: October 2003

The optimal habitat boundary for lake trout (Salvelinus namaycush Walbaum) has been defined as that portion of the lake having both more than 6 mg·L1 oxygen and temperature less than 10 °C. Here we use an existing hypolimnetic oxygen model to define the lower boundary of the optimal habitat by identifying the depth at which 6 mg·L1 oxygen occurs at the end-of-summer stratification period. Then we develop a new model to predict the 10 °C depth or upper habitat boundary for the same date. These two boundaries can be used in conjunction with measured individual strata volumes to calculate the optimal habitat volume. Parameters needed for the models include lake morphometry, total phosphorus (TP) concentration, and Secchi depth or dissolved organic carbon (DOC) concentration. Thus, the optimal habitat model can be used to predict the effects on optimal habitat volumes of changes in trophic status (increases or decreases in TP), or changes in light transmission (caused by changes in DOC or its surrogate in these lakes, i.e., Secchi depth) that result from changes in climate, land use, acidbase status, or incident ultraviolet radiation levels.
Quantifying migratory delay: a new application of survival analysis methods
Authors: Castro-Santos Theodore , Haro Alex
Published: October 2003

Statistical techniques commonly used in fish passage research fail to adequately quantify delays incurred at obstacles, or the effects of modifications to those obstacles on passage rates. Analyses of telemetry data describing these effects can be misleading, particularly when passage route of some individuals is not established (e.g., because of mortality, tag failure, passage through unmonitored or alternate routes, etc.). Here, we demonstrate how event-time analysis, better known as survival analysis, can be used to quantify passage rates for any study that allows tracking of individuals through time, even when some individuals fail to pass the route or obstacle in question. We review two of the primary methods of event-time analysis (parametric and Cox's proportional hazards regression analyses) and use them in combination with logistic regression to provide unbiased estimates of delay incurred at a hydroelectric facility, as well as insights on factors affecting both rates of passage and route selection. Passage rate increased with increased depth of a surface bypass sluice gate and, among fish that passed through the turbines, with turbine flow. The data further indicate that risk of turbine passage increased with both delay and turbine flow.
Bimodal size distributions in Arctic char, Salvelinus alpinus: artefacts of biased sampling
Authors: Finstad Anders Gravbrøt , Jansen Peder Andreas , Hirvonen Heikki
Published: October 2003

Bimodal population size and age distributions in Arctic char (Salvelinus alpinus (L.)) and hypotheses on growth patterns generating bimodality have drawn considerable attention during the last decade. However, such bimodality has also been suggested to be an artefact of biased sampling. We examined published data sets reporting bimodal size distributions in gill-net samples of Arctic char in order to confront hypotheses on growth patterns generating bimodal population size distributions. Growth patterns were derived from published length-at-age data. Simulations revealed that the observed growth patterns evidently could not generate a bimodal population size distribution. The basic reason for this was that growth did not stagnate strongly enough in the largest size classes of Arctic char. The reliability of growth approximations from length-at-age data was supported by empirical data on back-calculated growth trajectories. Furthermore, differences in year-class strength cannot explain all of the observed bimodal size and age distributions in gill-net samples, as they have been reported to persist over time. Thus, bias in the sampling procedure, which overestimates the frequency of old and large fish, is retained as the only plausible explanation for stable bimodal size distributions often observed in Arctic char gill-net samples.