bluuebunny/crossref_metadata_2025 huggingface dataset:
Isomerization of a Uniquely Designed Amplicon during Herpes Simplex Virus-Mediated Replication
Authors: Wang Hua , Fu Xinping , Zhang Xiaoliu
Published: July 2002

Herpes simplex virus (HSV) type 1 DNA isomerization was studied using a uniquely designed amplicon that mimics the viral genomic structure. The results revealed that amplicon concatemers frequently contain adjacent amplicon units with their segments in opposed orientations. These unusual concatemers were generated through homologous recombination, which does not require HSV DNA as the source of homology.
CD4-Independent Use of Rhesus CCR5 by Human Immunodeficiency Virus Type 2 Implicates an Electrostatic Interaction between the CCR5 N Terminus and the gp120 C4 Domain
Authors: Lin George , Lee Benhur , Haggarty Beth S. , Doms Robert W. , Hoxie James A.
Published: July 2002

Envelope glycoproteins (Envs) of human immunodeficiency virus type 2 (HIV-2) are frequently able to use chemokine receptors, CXCR4 or CCR5, in the absence of CD4. However, while these Envs are commonly dual-tropic, no isolate has been described to date that is CD4 independent on both CXCR4 and CCR5. In this report we show that a variant of HIV-2/NIHz, termed HIV-2/vcp, previously shown to utilize CXCR4 without CD4, is also CD4 independent on rhesus (rh) CCR5, but requires CD4 to fuse with human (hu) CCR5. The critical determinant for this effect was an acidic amino acid at position 13 in the CCR5 N terminus, which is an asparagine in huCCR5 and an aspartic acid in rhCCR5. Transferring the huCCR5 N terminus with an N13D substitution to CCR2b or CXCR2 was sufficient to render these heterologous chemokine receptors permissive for CD4-independent fusion. Chimeric Envs between HIV-2/vcp and a CD4-dependent clone of HIV-2/NIHz as well as site-directed Env mutations implicated a positively charged amino acid (lysine or arginine) at position 427 in the C4 region of the HIV-2/vcp
env
gene product (VCP) gp120 as a key determinant for this phenotype. Because CD4-independent use of CCR5 mapped to a negatively charged amino acid in the CCR5 N terminus and a positively charged amino acid in the gp120 C4 domain, an electrostatic interaction between these residues or domains is likely. Although not required for CD4-dependent fusion, this interaction may serve to increase the binding affinity of Env and CCR5 and/or to facilitate subsequent conformational changes that are required for fusion. Because the structural requirements for chemokine receptor use by HIV are likely to be more stringent in the absence of CD4, CD4-independent viruses should be particularly useful in dissecting molecular events that are critical for viral entry.
Rotavirus 2/6 Virus-Like Particles Administered Intranasally in Mice, with or without the Mucosal Adjuvants Cholera Toxin andEscherichia coliHeat-Labile Toxin, Induce a Th1/Th2-Like Immune Response
Authors: Fromantin Catherine , Jamot Béatrice , Cohen Jean , Piroth Lionel , Pothier Pierre , Kohli Evelyne
Published: July 2002

We investigated the rotavirus-specific lymphocyte responses induced by intranasal immunization of adult BALB/c mice with rotavirus 2/6 virus-like particles (2/6-VLPs) of the bovine RF strain, by assessing the profile of cytokines produced after in vitro restimulation and serum and fecal antibody responses. The cytokines produced by splenic cells were first evaluated. Intranasal immunization with 50 μg of 2/6-VLPs induced a high serum antibody response, including immunoglobulin G1 (IgG1) and IgG2a, a weak fecal antibody response, and a mixed Th1/Th2-like profile of cytokines characterized by gamma interferon and interleukin 10 (IL-10) production and very low levels of IL-2, IL-4, and IL-5. Intranasal immunization with 10 μg of 2/6-VLPs coadministered with the mucosal adjuvants cholera toxin andEscherichia coliheat-labile toxin (LT) considerably enhanced the Th1/Th2-like response; notably, significant levels of IL-2, IL-4, and IL-5 were observed. Since rotavirus is an enteric pathogen, we next investigated the production of IL-2 and IL-5, as being representative of Th1 and Th2 responses, by Peyer's patch and mesenteric lymph node cells from mice immunized intranasally with 2/6-VLPs and LT. The results were compared to those obtained from splenic and cervical lymph node cells. We found that both cytokines were produced by cells from each of these lymphoid tissues. These results confirm the Th1/Th2-like response observed at the systemic level and show, on the assumption that T cells are the primary cells producing the cytokines after in vitro restimulation, that rotavirus-specific T lymphocytes are present in the intestine after intranasal immunization with 2/6-VLPs and LT.
Structure and Assembly of Intracellular Mature Vaccinia Virus: Thin-Section Analyses
Authors: Griffiths Gareth , Roos Norbert , Schleich Sybille , Locker Jacomine Krijnse
Published: July 2002

In the preceding study (see accompanying paper), we showed by a variety of different techniques that intracellular mature vaccinia virus (vaccinia IMV) is unexpectedly complex in its structural organization and that this complexity also extends to the underlying viral core, which is highly folded. With that analysis as a foundation, we now present different thin-section electron microscopy approaches for analyzing the IMV and the processes by which it is assembled in infected HeLa cells. We focus on conventional epoxy resin thin sections as well as cryosections to describe key intermediates in the assembly process. We took advantage of streptolysin O's ability to selectively permeabilize the plasma membrane of infected cells to improve membrane contrast, and we used antibodies against bone fide integral membrane proteins of the virus to unequivocally identify membrane profiles in thin sections. All of the images presented here can be rationalized with respect to the model put forward for the assembly of the IMV in the accompanying paper.
Role of the UL25 Gene Product in Packaging DNA into the Herpes Simplex Virus Capsid: Location of UL25 Product in the Capsid and Demonstration that It Binds DNA
Authors: Ogasawara Masahiro , Suzutani Tatsuo , Yoshida Itsuro , Azuma Masanobu
Published: July 2002

Recent studies have suggested that the herpes simplex type 1 (HSV-1) UL25 gene product, a minor capsid protein, is required for encapsidation but not cleavage of replicated viral DNA. This study set out to investigate the potential interactions of UL25 protein with other virus proteins and determine what properties it has for playing a role in DNA encapsidation. The UL25 protein is found in 42 ± 17 copies per B capsid and is present in both pentons and hexons. We introduced green fluorescent protein (GFP) as a fluorescent tag into the N terminus of UL25 protein to identify its location in HSV-1-infected cells and demonstrated the relocation of UL25 protein from the cytoplasm into the nucleus at the late stage of HSV-1 infection. To clarify the cause of this relocation, we analyzed the interactions of UL25 protein with other virus proteins. The UL25 protein associates with VP5 and VP19C of virus capsids, especially of the penton structures, and the association with VP19C causes its relocation into the nucleus. Gel mobility shift analysis shows that UL25 protein has the potential to bind DNA. Moreover, the amino-terminal one-third of the UL25 protein is particularly important in DNA binding and forms a homo-oligomer. In conclusion, the UL25 gene product forms a tight connection with the capsid being linked with VP5 and VP19C, and it may play a role in anchoring the genomic DNA.
Different Regions of Primase Subunit p48 Control Mouse Polyomavirus and Simian Virus 40 DNA Replication In Vitro
Authors: Kautz Armin R. , Schneider Annerose , Weisshart Klaus , Geiger Christiane , Nasheuer Heinz-Peter
Published: July 2002

DNA polymerase α-primase (pol-prim), a complex consisting of four subunits, is the major species-specific factor for mouse polyomavirus (PyV) and simian virus 40 (SV40) DNA replication. Although p48 is the most conserved subunit of pol-prim, it is required for in vitro PyV DNA replication but can inhibit cell-free SV40 DNA replication. Production of chimeric human-mouse p48 revealed that different regions of p48 are involved in supporting PyV DNA replication and inhibiting SV40 DNA replication. The N and C-terminal parts of p48 do not have species-specific functions in cell-free PyV DNA replication, but the central part (amino acids [aa] 129 to 320) controls PyV DNA replication in vitro. However, PyV T antigen physically binds to mouse, human, and chimeric pol-prim complexes independently, whether they support PyV DNA replication or not. In contrast to the PyV system, the inhibitory effects of mouse p48 on SV40 DNA replication are mediated by N- and C-terminal regions of p48. Thus, a chimeric p48 containing human aa 1 to 128, mouse aa 129 to 320, and human aa 321 to 418 is active in both PyV and SV40 DNA replication in vitro.
CREB/ATF-Dependent Repression of Cyclin A by Human T-Cell Leukemia Virus Type 1 Tax Protein
Authors: Kibler Karen V. , Jeang Kuan-Teh
Published: July 2002

Expression of the human T-cell leukemia virus type 1 (HTLV-1) oncoprotein Tax is correlated with cellular transformation contributing to the development of adult T-cell leukemia. Tax has been shown to modulate the activities of several cellular promoters. Existing evidence suggests that Tax need not directly bind to DNA to accomplish these effects but rather that it can act through binding to cellular factors, including members of the CREB/ATF family. Exact mechanisms of HTLV-1 transformation of cells have yet to be fully defined, but the process is likely to include both activation of cellular-growth-promoting factors and repression of cellular tumor-suppressing functions. While transcriptional activation has been well studied, transcriptional repression by Tax, reported recently from several studies, remains less well understood. Here, we show that Tax represses the TATA-less cyclin A promoter. Repression of the cyclin A promoter was seen in both ts13 adherent cells and Jurkat T lymphocytes. Two other TATA-less promoters, cyclin D3 and DNA polymerase α, were also found to be repressed by Tax. Interestingly, all three promoters share a common feature of at least one conserved upstream CREB/ATF binding site. In electrophoretic mobility shift assays, we observed that Tax altered the formation of a complex(es) at the cyclin A promoter-derived ATF site. Functionally, we correlated removal of the CREB/ATF site from the promoter with loss of repression by Tax. Furthermore, since a Tax mutant protein which binds CREB repressed the cyclin A promoter while another mutant protein which does not bind CREB did not, we propose that this Tax repression occurs through protein-protein contact with CREB/ATF.
Role of Single-Stranded DNA Binding Activity of T Antigen in Simian Virus 40 DNA Replication
Authors: Wu Chunxiao , Roy Rupa , Simmons Daniel T.
Published: July 2002

We have previously mapped the single-stranded DNA binding domain of large T antigen to amino acid residues 259 to 627. By using internal deletion mutants, we show that this domain most likely begins after residue 301 and that the region between residues 501 and 550 is not required. To study the function of this binding activity, a series of single-point substitutions were introduced in this domain, and the mutants were tested for their ability to support simian virus 40 (SV40) replication and to bind to single-stranded DNA. Two replication-defective mutants (429DA and 460EA) were grossly impaired in single-stranded DNA binding. These two mutants were further tested for other biochemical activities needed for viral DNA replication. They bound to origin DNA and formed double hexamers in the presence of ATP. Their ability to unwind origin DNA and a helicase substrate was severely reduced, although they still had ATPase activity. These results suggest that the single-stranded DNA binding activity is involved in DNA unwinding. The two mutants were also very defective in structural distortion of origin DNA, making it likely that single-stranded DNA binding is also required for this process. These data show that single-stranded DNA binding is needed for at least two steps during SV40 DNA replication.
Disturbance of Tumor Necrosis Factor Alpha-Mediated Beta Interferon Signaling in Cervical Carcinoma Cells
Authors: Bachmann Anastasia , Hanke Brigitte , Zawatzky Rainer , Soto Ubaldo , van Riggelen Jan , zur Hausen Harald , Rösl Frank
Published: July 2002

In the present study we show that malignant human papillomavirus (HPV)-positive cells lost their ability to synthesize endogenous beta interferon (IFN-β) upon tumor necrosis factor alpha (TNF-α) treatment. IFN-β transcription, however, was reinducible in nonmalignant HPV-positive cells, which was confirmed in functional protection assays against encephalomyocarditis virus or vesicular stomatitis virus infections. Addition of neutralizing antibodies against IFN-β blocked the antiviral effect, excluding the possibility that other IFN types were involved. Conversely, both malignant and immortalized cells could be protected against viral cytolysis when either IFN-β, IFN-α, or IFN-γ was added exogenously. This indicates that only the cross talk between TNF-α and the IFN-β pathways, and not IFN-α/β and IFN-γ signaling in general, is perturbed in cervical carcinoma cells. Notably, full virus protection was restricted exclusively to nonmalignant cells, indicating that the antiviral effect correlates with the growth-inhibitory and virus-suppressive properties of TNF-α. The IFN-regulatory factors IRF-1 and p48 (ISGF3γ) emerged as key regulatory molecules in the differential IFN-β response, since their transcription was either absent or only inefficiently enhanced in tumorigenic cells upon treatment with TNF-α. Inducibility of both genes, however, became reestablished in cervical carcinoma cells, which were complemented to nontumorigenicity after somatic cell hybridization. Complementation was paralleled by the entire reconstitution of cytokine-mediated IFN-β expression and the ability of TNF-α to exert an antiviral state. In contrast, under conditions where tumor suppression was not accomplished upon somatic cell hybridization, neither expression of IRF-1, p48, and IFN-β nor antiviral activity could be restored.
Wild-Type Puumala Hantavirus Infection Induces Cytokines, C-Reactive Protein, Creatinine, and Nitric Oxide in Cynomolgus Macaques
Authors: Klingström J. , Plyusnin A. , Vaheri A. , Lundkvist Å.
Published: July 2002

Hantaviruses cause two severe human diseases: hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). Approximately 200,000 cases are reported annually, and there is to date no specific treatment available. A major obstacle in studying the medical aspects of HFRS and HPS has been the lack of an adequate animal model. Here we show that infection of cynomolgus macaques by wild-type Puumala hantavirus resulted in typical signs of HFRS including lethargy, anorexia, proteinuria, and/or hematuria, in addition to cytokine (interleukin 6 [IL-6], IL-10, and tumor necrosis factor alpha), C-reactive protein, creatinine, and nitric oxide responses. Viral RNA was detected in plasma from days 3 to 7 postinoculation until days 24 to 28 postinoculation, infectious virus was recovered, and the virus-specific immune responses (immunoglobulin M [IgM], IgG, and neutralizing antibodies) mimicked those seen in humans. The results indicated that the monkey model will provide a valuable tool for studies of pathogenesis, candidate vaccines, and antivirals for hantavirus disease.
Mutational Analysis of Simian Virus 40 T-Antigen Primosome Activities in Viral DNA Replication
Authors: Ott Robert D. , Wang Yingda , Fanning Ellen
Published: July 2002

The recruitment of DNA polymerase α-primase (pol-prim) is a crucial step in the establishment of a functional replication complex in eukaryotic cells, but the mechanism of pol-prim loading and the composition of the eukaryotic primosome are poorly understood. In the model system for simian virus 40 (SV40) DNA replication in vitro, synthesis of RNA primers at the origin of replication requires only the viral tumor (T) antigen, replication protein A (RPA), pol-prim, and topoisomerase I. On RPA-coated single-stranded DNA (ssDNA), T antigen alone mediates priming by pol-prim, constituting a relatively simple primosome. T-antigen activities proposed to participate in its primosome function include DNA helicase and protein-protein interactions with RPA and pol-prim. To test the role of these activities of T antigen in mediating priming by pol-prim, three replication-defective T antigens with mutations in the ATPase or helicase domain have been characterized. All three mutant proteins interacted physically and functionally with RPA and pol-prim and bound ssDNA, and two of them displayed some helicase activity. However, only one of these, 5030, mediated primer synthesis and elongation by pol-prim on RPA-coated ssDNA. The results suggest that a novel activity, present in 5030 T antigen and absent in the other two mutants, is required for T-antigen primosome function.
Molecular Evolution of Human Immunodeficiency Virus
env
in Humans and Monkeys: Similar Patterns Occur during Natural Disease Progression or Rapid Virus Passage
Authors: Hofmann-Lehmann Regina , Vlasak Josef , Chenine Agnès-Laurence , Li Pei-Lin , Baba Timothy W. , Montefiori David C. , McClure Harold M. , Anderson Daniel C. , Ruprecht Ruth M.
Published: July 2002

Neonatal rhesus macaque 95-3 was inoculated with nonpassaged simian-human immunodeficiency virus strain SHIV-vpu
+
, which encodes
env
of the laboratory-adapted human immunodeficiency virus (HIV) strain IIIB and is considered nonpathogenic. CD4
+
T-cell counts dropped to <200 cells/μl within 4.6 years, and monkey 95-3 died with opportunistic infections 5.9 years postinoculation. Transfer of blood from 95-3 to two naive adult macaques resulted in high peak viral loads and rapid, persistent T-cell depletion. Progeny virus evolved in 95-3 despite high SHIV-vpu
+
neutralizing antibody titers and still used CXCR4 but, in contrast to parental SHIV-vpu
+
, productively infected macrophages and resisted neutralization. Sequence analysis revealed three new potential glycosylation sites in gp120; another two were lost. Strikingly similar mutations were detected in a laboratory worker who progressed to AIDS after accidental HIV-IIIB infection (T. Beaumont et al., J. Virol. 75:2246-2252, 2001), thus supporting the SHIV-vpu
+
/rhesus macaque system as a relevant model. Similar mutations were also described after rapid passage of chimeric viruses encoding IIIB
env
in rhesus and pig-tailed macaques (M. Cayabyab et al., J. Virol. 73:976-984, 1999; Z. Q. Liu et al., Virology 260:295-307, 1999; S. V. Narayan et al., Virology 256:54-63, 1999; R. Raghavan et al., Brain Pathol. 7:851-861, 1997; E. B. Stephens et al., Virology 231:313-321, 1997). Thus, HIV-IIIB
env
evolved similarly in three different species; this selection occurred in chronically infected individuals during disease progression as well as after rapid virus passage. We postulate that evolutionary pressure led to the outgrowth of more aggressive viral variants in all three species.
Polarized Release of Human Cytomegalovirus from Placental Trophoblasts
Authors: Hemmings D. G. , Guilbert L. J.
Published: July 2002

Human cytomegalovirus (HCMV) is a ubiquitous infectious pathogen that, when transmitted to the fetus in utero,can result in numerous sequelae, including late-onset sensorineural damage. The villous trophoblast, the cellular barrier between maternal blood and fetal tissue in the human placenta, is infected by HCMV in vivo. Primary trophoblasts cultured on impermeable surfaces can be infected by HCMV, but release of progeny virus is delayed and minimal. It is not known whether these epithelial cells when fully polarized can release HCMV and, if so, if release is from the basal membrane surface toward the fetus. We therefore ask whether, and in which direction, progeny virus release occurs from HCMV-infected trophoblasts cultured on semipermeable (3.0-μm-pore-size) membranes that allow functional polarization. We show that infectious HCMV readily diffuses across cell-free 3.0-μm-pore-size membranes and that apical infection of confluent and multilayered trophoblasts cultured on these membranes reaches cells at the membrane surface. Using two different infection and culture protocols, we found that up to 20% of progeny virus is released but that <1% of released virus is detected in the basal culture chamber. These results suggest that very little, if any, HCMV is released from an infected villous trophoblast into the villous stroma where the virus could ultimately infect the fetus.
Adenovirus E3-6.7K Maintains Calcium Homeostasis and Prevents Apoptosis and Arachidonic Acid Release
Authors: Moise Alexander R. , Grant Jason R. , Vitalis Timothy Z. , Jefferies Wilfred A.
Published: July 2002

E3-6.7K is a small and hydrophobic membrane glycoprotein encoded by the E3 region of subgroup C adenovirus. Recently, E3-6.7K has been shown to be required for the downregulation of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) receptors by the adenovirus E3/10.4K and E3/14.5K complex of proteins. We demonstrate here that E3-6.7K has additional protective roles, independent of other virus proteins. In transfected Jurkat T-cell lymphoma cells, E3-6.7K was found to maintain endoplasmic reticulum-Ca2+homeostasis and inhibit the induction of apoptosis by thapsigargin. The presence of E3-6.7K also lead to a reduction in the TNF-induced release of arachidonic acid from transfected U937 human histiocytic lymphoma cells. In addition, E3-6.7K protected cells against apoptosis induced through Fas, TNF receptor, and TRAIL receptors. Therefore, E3-6.7K confers a wide range of protective effects against both Ca2+flux-induced and death receptor-mediated apoptosis.
Frequency of Missense Mutations in the Coding Region of a Eukaryotic Gene Transferred by Retroviral Vectors
Authors: De Angioletti Maria , Rovira Ana , Sadelain Michel , Luzzatto Lucio , Notaro Rosario
Published: July 2002

A relatively high mutation rate is probably a major factor in the evolutionary success of retroviruses, because it generates the genetic diversity that helps them to cope with changes in the environment. When using recombinant retroviruses as vectors for gene transfer and gene therapy, it is important to consider the implications of this biological characteristic. Until now, the mutation rate has been studied by using noneukaryotic genes as reporters. Here we report point mutations in the human glucose-6-phosphate dehydrogenase (hG6PD) gene transferred by Moloney murine leukemia virus-based vectors into murine bone marrow cells and NIH 3T3 murine fibroblasts. After bone marrow transplantation, we observed an hG6PD with abnormal electrophoretic mobility for 2 out of 34 mice. Next, we studied this phenomenon quantitatively and found 1 electrophoretically abnormal hG6PD variant among 93 independently isolated NIH 3T3 clones, from which we estimate a mutation rate of 1.4 × 10
−5
per base pair per replication cycle. Mutations in the transferred gene can thus contribute to the impairment of the effectiveness of retrovirus-mediated gene transfer.
Recruitment of the Crm1 Nuclear Export Factor Is Sufficient To Induce Cytoplasmic Expression of Incompletely Spliced Human Immunodeficiency Virus mRNAs
Authors: Yi Rui , Bogerd Hal P. , Cullen Bryan R.
Published: July 2002

Cytoplasmic expression of the incompletely spliced RNA transcripts that encode the late, structural proteins of human immunodeficiency virus type 1 (HIV-1) is dependent on the viral Rev regulatory protein. General agreement exists that Rev acts, at least in part, by recruiting the cellular Crm1 nuclear export factor to HIV-1 transcripts bearing the Rev response element RNA target, and thereby inducing their nuclear egress. However, several groups have argued that Crm1 recruitment may not be sufficient for Rev function. Thus, several additional candidate cofactors for Rev have been proposed, and Rev has also been suggested to also inhibit the nuclear splicing of HIV-1 transcripts and/or to directly enhance their cytoplasmic translation. To examine whether Crm1 recruitment is, instead, sufficient to activate the nuclear export of viral mRNAs, we targeted a leucine-rich Crm1 binding domain, derived from a heterologous protein that normally plays no role in RNA metabolism, to HIV-1 RNAs and showed that this tethered Crm1 binding domain is sufficient to induce the nuclear export and cytoplasmic translation of late HIV-1 mRNA species. More importantly, we show that direct tethering of the Crm1 nuclear export factor to target mRNAs, by fusion to a heterologous RNA binding domain, is in and of itself sufficient to induce the nuclear export and cytoplasmic expression of the unspliced HIV-1 mRNAs that encode the viral Gag proteins.
Altering the Expression Kinetics of VP5 Results in Altered Virulence and Pathogenesis of Herpes Simplex Virus Type 1 in Mice
Authors: Tran Robert K. , Lieu Pauline T. , Aguilar Santiago , Wagner Edward K. , Bloom David C.
Published: July 2002

While many herpes simplex virus (HSV) structural proteins are expressed with strict-late kinetics, the HSV virion protein 5 (VP5) is expressed as a “leaky-late” protein, such that appreciable amounts of VP5 are made prior to DNA replication. Our goal has been to determine if leaky-late expression of VP5 is a requirement for a normal HSV infection. It had been shown previously that recombinant viruses in which the VP5 promoter was replaced with promoters of other kinetic classes (including a strict late promoter) exhibited no alterations in replication kinetics or virus yields in vitro. In contrast, here we report that alterations in pathogenesis were observed when these recombinants were analyzed by experimental infection of mice. Following intracranial inoculation, a recombinant expressing VP5 from a strict-late promoter (U
L
38) exhibited an increased 50% lethal dose and a 10-fold decrease in virus yields in the central nervous system, while a recombinant expressing VP5 from an early (dUTPase) or another leaky-late (VP16) promoter exhibited wild-type neurovirulence. Moreover, following infection of the footpad, changing the expression kinetics of VP5 from leaky-late to strict-late resulted in 100-fold-less virus in the spinal ganglia during the acute infection than produced by either the parent virus or the rescued virus. These data indicate that the precise timing of appearance of the major capsid protein plays a role in the pathogenesis of HSV infections and that changing the expression kinetics has different effects in different cell types and tissues.
Effects of Herpes Simplex Virus on Structure and Function of Nectin-1/HveC
Authors: Krummenacher Claude , Baribaud Isabelle , Sanzo James F. , Cohen Gary H. , Eisenberg Roselyn J.
Published: July 2002

Herpes simplex virus (HSV) entry requires the interaction between the envelope glycoprotein D (gD) and a cellular receptor such as nectin-1 (also named herpesvirus entry mediator C [HveC]) or HveA/HVEM. Nectin-1 is a cell adhesion molecule found at adherens junctions associated with the cytoplasmic actin-binding protein afadin. Nectin-1 can act as its own ligand in a homotypic interaction to bridge cells together. We used a cell aggregation assay to map an adhesive functional site on nectin-1 and identify the effects of gD binding and HSV early infection on nectin-1 function. Soluble forms of nectin-1 and anti-nectin-1 monoclonal antibodies were used to map a functional adhesive site within the first immunoglobulin-like domain (V domain) of nectin-1. This domain also contains the gD-binding site, which appeared to overlap the adhesive site. Thus, soluble forms of gD were able to prevent nectin-1-mediated cell aggregation and to disrupt cell clumps in an affinity-dependent manner. HSV also prevented nectin-1-mediated cell aggregation by occupying the receptor. Early in infection, nectin-1 was not downregulated from the cell surface. Rather, detection of nectin-1 changed gradually over a 30-min period of infection, as reflected by a decrease in the CK41 epitope and an increase in the CK35 epitope. The level of detection of virion gD on the cell surface increased within 5 min of infection in a receptor-dependent manner. These observations suggest that cell surface nectin-1 and gD may undergo conformational changes during HSV entry as part of an evolving interaction between the viral envelope and the cell plasma membrane.
Decreased Frequency of Cytomegalovirus (CMV)-Specific CD4+T Lymphocytes in Simian Immunodeficiency Virus-Infected Rhesus Macaques: Inverse Relationship with CMV Viremia
Authors: Kaur Amitinder , Hale Corrina L. , Noren Bradley , Kassis Nadine , Simon Meredith A. , Johnson R. Paul
Published: July 2002

The frequency of cytomegalovirus (CMV)-specific CD4+T lymphocytes was determined in CMV-seropositive rhesus macaques with or without simian immunodeficiency virus (SIV) infection by using the sensitive assays of intracellular cytokine staining and gamma interferon ELISPOT. Both techniques yielded 3- to 1,000-fold-higher frequencies of CMV-specific CD4+T lymphocytes than traditional proliferative limiting dilution assays. The median frequency of CMV-specific CD4+T lymphocytes in 23 CMV-seropositive SIV-negative macaques was 0.63% (range, 0.16 to 5.8%). The majority of CMV-specific CD4+T lymphocytes were CD95posand CD27lobut expressed variable levels of CD45RA. A significant reduction (P< 0.05) in the frequency of CMV-specific CD4+T lymphocytes was observed in pathogenic SIV-infected macaques but not in macaques infected with live attenuated strains of SIV. CMV-specific CD4+T lymphocytes were not detected in six of nine pathogenic SIV-infected rhesus macaques. CMV DNA was detected in the plasma of four of six of these macaques but in no animal with detectable CMV-specific CD4+T lymphocytes. In pathogenic SIV-infected macaques, loss of CMV-specific CD4+T lymphocytes was not predicted by the severity of CD4+T lymphocytopenia. Neither was it predicted by the pre-SIV infection frequencies of CD45RAnegor CCR5posCMV-specific CD4+T lymphocytes. However, the magnitude of activation, as evidenced by the intensity of CD40L expression on CMV-specific CD4+T lymphocytes pre-SIV infection, was three- to sevenfold greater in the two macaques that subsequently lost these cells after SIV infection than in the two macaques that retained CMV-specific CD4+T lymphocytes post-SIV infection. Future longitudinal studies with these techniques will facilitate the study of CMV pathogenesis in AIDS.
Human Immunodeficiency Virus Type 1 Vpr Protein Does Not Modulate Surface Expression of the CD4 Receptor
Authors: Argañaraz Enrique , Cortés María José , Leibel Sydney , Lama Juan
Published: July 2002

The CD4 receptor is required for the entry of human immunodeficiency virus (HIV) into target cells. It has long been known that Nef, Env, and Vpu participate in the removal of the viral receptor from the cell surface. Recently, it has been proposed that the HIV type 1 (HIV-1) Vpr protein may also play a role in the downmodulation of CD4 from the surfaces of infected cells (L. Conti, B. Varano, M. C. Gauzzi, P. Matarrese, M. Federico, W. Malorani, F. Belardelli, and S. Gessani, J. Virol. 74:10207-10211, 2000). To investigate the possible role of Vpr in the downregulation of the viral receptor Vpr alleles from HIV-1 and simian immunodeficiency virus were transiently expressed in transformed T cells and in 293T fibroblasts, and their ability to modulate surface CD4 was evaluated. All Vpr alleles efficiently arrested cells in the G
2
stage of the cell cycle. However, none of the tested Vpr proteins altered the expression of CD4 on the cell surface. In comparison, HIV-1 Nef efficiently downmodulated surface CD4 in all the experimental settings. Transformed T cells and primary lymphocytes were challenged with wild-type, Nef-defective, and Vpr-defective viruses. A significant reduction in the HIV-induced downmodulation of surface CD4 was observed in viruses lacking Nef. However, Vpr-deletion-containing viruses showed no defect in their ability to remove CD4 from the surfaces of infected cells. Our results indicate that Vpr does not play a role in the HIV-induced downmodulation of the CD4 receptor.
The Conformation of the Mature Dimeric Human Immunodeficiency Virus Type 1 RNA Genome Requires Packaging of Pol Protein
Authors: Shehu-Xhilaga M. , Hill M. , Marshall J. A. , Kappes J. , Crowe S. M. , Mak J.
Published: July 2002

The packaging of a mature dimeric RNA genome is an essential step in human immunodeficiency virus type 1 (HIV-1) replication. We have previously shown that overexpression of a protease (PR)-inactive HIV-1 Gag-Pro-Pol precursor protein generates noninfectious virions that contain mainly monomeric RNA (M. Shehu-Xhilaga, S. M. Crowe, and J. Mak, J. Virol. 75:1834-1841, 2001). To further define the contribution of HIV-1 Gag and Gag-Pro-Pol to RNA maturation, we analyzed virion RNA dimers derived from Gag particles in the absence of Gag-Pro-Pol. Compared to wild-type (WT) dimeric RNAs, these RNA dimers have altered mobility and low stability under electrophoresis conditions, suggesting that the HIV-1 Gag precursor protein alone is not sufficient to stabilize the dimeric virion RNA structure. The inclusion of an active viral PR, without reverse transcriptase (RT) and integrase (IN), rescued the stability of the virion RNA dimers in the Gag particles but did not restore the mobility of the RNAs, suggesting that RT and IN are also required for virion RNA dimer maturation. Thin-section electron microscopy showed that viral particles deficient in RT and IN contain empty cone-shaped cores. The abnormal core structure indicates a requirement for Gag-Pro-Pol packaging during core maturation. Supplementing viral particles with either RT or IN via Vpr-RT or Vpr-IN alone did not correct the conformation of the dimer RNAs, whereas expression of both RT and IN in
trans
as a Vpr-RT-IN fusion restored RNA dimer conformation to that of the WT virus and also restored the electron-dense, cone-shaped virion core characteristic of WT virus. Our data suggest a role for RT-IN in RNA dimer conformation and the formation of the electron-dense viral core.
Pathogenesis of Avian Influenza A (H5N1) Viruses in Ferrets
Authors: Zitzow Lois A. , Rowe Thomas , Morken Timothy , Shieh Wun-Ju , Zaki Sherif , Katz Jacqueline M.
Published: July 2002

Highly pathogenic avian influenza A H5N1 viruses caused outbreaks of disease in domestic poultry and humans in Hong Kong in 1997. Direct transmission of the H5N1 viruses from birds to humans resulted in 18 documented cases of respiratory illness, including six deaths. Here we evaluated two of the avian H5N1 viruses isolated from humans for their ability to replicate and cause disease in outbred ferrets. A/Hong Kong/483/97 virus was isolated from a fatal case and was highly pathogenic in the BALB/c mouse model, whereas A/Hong Kong/486/97 virus was isolated from a case with mild illness and exhibited a low-pathogenicity phenotype in mice. Ferrets infected intranasally with 10750% egg infectious doses (EID50) of either H5N1 virus exhibited severe lethargy, fever, weight loss, transient lymphopenia, and replication in the upper and lower respiratory tract, as well as multiple systemic organs, including the brain. Gastrointestinal symptoms were seen in some animals. In contrast, weight loss and severe lethargy were not noted in ferrets infected with 107EID50of two recent human H3N2 viruses, although these viruses were also isolated from the brains, but not other extrapulmonary organs, of infected animals. The results demonstrate that both H5N1 viruses were highly virulent in the outbred ferret model, unlike the differential pathogenicity documented in inbred BALB/c mice. We propose the ferret as an alternative model system for the study of these highly pathogenic avian viruses.
Polarographic Determination of 4'-Substituted Derivatives of 3-Carboxy-4-hydroxy-6-acetylaminoazobenzene
Authors: Barek Jiří , Savarino Piero , Stuchlíková Lucie , Zima Jiří
Published: July 2002

Tast polarography, differential pulse polarography and cyclic voltammetry were applied to the examination of 7 derivatives of 3-carboxy-4-hydroxy-6-acetylaminoazobenzene with H, CH3, OCH3, Cl, COCH3, NO2 and NHCOCH3 substituents in position 4'. A general reduction mechanism was suggested for the substances. The half-wave potentials obtained from tast polarographic measurements were correlated with the Hammett σ constants of the substituents in the para-position. The plot of this dependence is linear, which suggests that the reduction mechanism is identical within the entire series. The optimum conditions for quantitation of the substances were found within the concentration regions of 1 .10-4 to 1 .10-6 mol l-1 for tast polarography and 1 .10-4 to 1 .10-7 mol l-1 for differential pulse polarography.
Hydration of Cyclohexene Catalyzed by Zeolites
Authors: Kučera Milan , Králik Milan , Maťaš Michal , Hudec Pavol , Hronec Milan
Published: July 2002

Liquid phase hydration of cyclohexene over faujasite, mordenite and ZSM-5 catalysts at 10 MPa and temperatures 150 - 210 °C in a batch system was studied. Influence of the type and Si/Al ratio of the zeolite on the conversion of cyclohexene and the selectivity to cyclohexanol formation was determined. Performance of the catalyst was also investigated through adsorption measurements of water and cyclohexene. Mordenite with the Si/Al ratio 70 provided the best results. The selectivity to cyclohexanol was 99% at the conversion of cyclohexene 27% at 200 °C after 4 h. All the catalysts were regenerated by calcination at 450 °C.
Properties of Dilute Solutions of Ethylene-Propylene-Diene Terpolymers in Benzene and Toluene
Authors: Bohdanecký Miloslav , Sikora Antonín , Horský Jiří , Petrus Vladimír
Published: July 2002

Commercial samples of ethylene-propylene-diene terpolymers (EPDM) and their fractions were characterized by osmometry (number-average molecular weight, second virial coefficient), viscometry (intrisic viscosity) and 1H NMR spectrometry (mole fraction of propylene monomeric units). The results were analyzed in terms of the two-parameter theory of polymer solutions. Theta temperatures and polymer-solvent interaction parameters were estimated and correlated with composition. Existence of organized structures is indicated by high values of the reduced partial molar entropy parameter y. The increase in y with increasing content of ethylene units is assigned to their association. The results lend support to the previous study on the Huggins viscosity coefficient of EPDM.
Crystal and Molecular Structure of cis-2,2',3',2"-Tetraacetoxy-1,1':4',1"-ternaphthyl
Authors: Podlaha Jaroslav , Císařová Ivana , Bělohradský Martin , Závada Jiří
Published: July 2002

The configuration and conformation of the title compound as a representative of conformationally locked ternaphthyls was determined by single-crystal X-ray diffraction. The arrangement of the mean planes of naphthyl and acetoxy groups results from intramolecular van der Waals forces.
Restricted Intramolecular Energy Flow in Large Molecules. Heterogeneous and Enzyme Catalysis
Authors: Šolc Milan
Published: July 2002

The free intramolecular energy flow can be restricted by the presence of a heavy atom in the molecule. As a result of this restriction, adsorbed molecules bonded on the metal surface and/or substrate molecules in the enzyme-substrate complex with a metal atom near the binding site can have a higher vibrational energy than the surroundings. The reaction rate is then enhanced by this energy localization.
2,4-Dihydroxyacetophenone Derivatives as Antileukotrienics with a Multiple Activity Mechanism
Authors: Kuchař Miroslav , Čulíková Kateřina , Panajotovová Vladimíra , Brunová Bohumila , Jandera Antonín , Kmoníček Vojtěch
Published: July 2002

A series of 2,4-dihydroxy-3-propylacetophenone derivatives containing alkylthiobenzoic or arylacetic acid moiety was prepared. The antileukotrienic activity of the substances was assessed in terms of inhibition of the biosynthesis of LTB4 and bonding to LTB4 and LTD4 receptors. Some of the substances were found to exert a complex antileukotrienic effect in all of the three tests. The compounds also displayed an antiinflammatory effect, as manifested by a significant inhibition of LTD4-induced bronchospasm.
Structural Studies of Chelirubine and Chelilutine Free Bases
Authors: Dostál Jiří , Slavík Jiří , Potáček Milan , Marek Radek , Humpa Otakar , Sklenář Vladimír , Toušek Jaromír , Hoffmann Edmond de , Rozenberg Raoul
Published: July 2002

The structures of chelirubine and chelilutine free bases have been examined by 2D NMR spectroscopy and mass spectrometry. Chelirubine chloride (1a) upon treatment with Na2CO3 yielded free base which possessed the constitution of bis(5,6-dihydrochelirubin-6-yl) ether (2a). The free base of chelilutine (1b) was determined to be bis(5,6-dihydrochelilutin-6-yl) ether (2b). The aqueous NH3 treatment of chelilutine (1b) produced bis(5,6-dihydrochelilutin-6-yl)amine (3b). 6-Hydroxy-5,6-dihydrochelirubine (4a) and 6-hydroxy-5,6-dihydrochelilutine (4b) were detected only in CDCl3 solution by NMR spectroscopy. In CDCl3, the compound 2b underwent hydrolysis to 4b that was immediately followed by the reverse condensation to a diastereomer of 2b. Pseudokinetics of this reaction was followed by NMR spectroscopy and quantum chemical calculations were carried out to support the suggested type of molecular symmetry alteration. The known alkaloid dihydrochelirubine (5) was isolated for the first time from Sanguinaria canadensis.
New Approach to the Theory of Circular Dichroism
Authors: Zamastil Jaroslav , Skála Lubomír , Pančoška Petr , Bílek Oldřich
Published: July 2002

Using the semiclassical approach for the description of the propagation of the electromagnetic waves in optically active isotropic media we derive a new formula for the circular dichroism parameter. The theory is based on the idea of the time damped electromagnetic wave interacting with the molecules of the sample. In this theory, the Lambert-Beer law need not be taken as an empirical law, however, it follows naturally from the requirement that the electromagnetic wave obeys the Maxwell equations.
Search for the Most Stable Structures on Potential Energy Surfaces
Authors: Piela Lucjan
Published: July 2002

Smoothing techniques for global optimization in search for the most stable structures (clusters or conformers) have been a novel possibility for the last decade. The techniques turned out to be related to a variety of fundamental laws: Fick's diffusion equation, time-dependent and time-independent Schrodinger equations, Smoluchowski dynamics equation, Bloch equation of canonical ensemble evolution with temperature, Gibbs free-energy principle. The progress indicator of global optimization in those methods takes different physical meanings: time, imaginary time, Planck constant, or the inverse absolute temperature. Despite this large spectrum of physical phenomena, the resulting global optimization procedures have a remarkable common feature. In the case of the Gaussian Ansatz for the wave function or density distribution, the underlying differential equations of motion for the Gaussian position and width are similar for all these phenomena. In all techniques the smoothed potential energy function plays a central role rather than the potential energy function itself. The smoothed potential results from a Gaussian convolution or filtering out high frequency Fourier components of the original potential energy function. During the minimization, the Gaussian position moves according to the negative gradient of the smoothed potential energy function. The Gaussian width is position dependent through the curvature of the potential energy function, and evolves according to the following rule. For sufficiently positive curvatures (close to minima of the smoothed potential) the width decreases, thus leading to a smoothed potential approaching the original potential energy function, while for negative curvatures (close to maxima) the width increases leading eventually to the disappearance of humps of the original potential energy function. This allows for crossing barriers separating the energy basins. Some methods result in an additional term, which increases the width, when the potential becomes flat. This may be described as a feature allowing hunting for distant minima.
Balancing the risk of injury to gymnasts: how effective are the counter measures?
Authors: Daly R M , Bass S L , Finch C F
Published: July 2002

Background—To minimise injury risk and maximise gymnastics performance, coaches, parents, and health professionals working with young gymnasts need to understand and practise safe gymnastics.

Aims—To (a) identify the various injury counter measures specific to gymnastics, (b) critically review the literature describing each injury prevention measure, and (c) assess, using available risk factor and injury data, the weight of evidence to support each of these counter measures. Specific recommendations for further research and implementation strategies to prevent injury and improve safety are also given.

Methods—The relevant literature was identified through the use of Medline (1966 to May 1998) and SPORT Discus (1975 to May 1998) searches, hand searching of journals and reference lists, and discussions with key Australian gymnastics organisations.

Results—The key gymnastics injury counter measures identified in this review include coaching (physical preparation, education, spotting, and performance technique), equipment, and the health support system (medical screening, treatment, and rehabilitation). Categorisation of the type of evidence for the effectiveness of each of these counter measures in preventing injury showed that most of it is based on informal opinion/anecdotal evidence, uncontrolled data based studies, and several prospective epidemiological studies. There is no evidence from formally controlled trials or specific evaluation studies of counter measures for gymnastics.

Conclusions—Although gymnastics is a sport associated with young participants and frequent high volume, high impact training, there is a paucity of information on injury risk factors and the effectiveness of injury practices. Further controlled trials are needed to examine the extent to which injury prevention counter measures can prevent or reduce the occurrence of injury and re-injury. Particular attention should be devoted to improving training facilities, the design and testing of apparatus and personal equipment used by gymnasts, and coaching and the role of spotting in preventing injury.
Disparities in parenting criteria: an exploration of the issues, focusing on adoption and embryo donation
Authors: Widdows H , MacCallum F
Published: July 2002

This paper examines the consistency of parent selection procedures, focusing on adoption and embryo donation. It outlines the current methods of selection and their disparities, and considers reasons for these disparities; namely, the intentionality of the parents, the gestational experience, and the technological imperative. This discussion is followed by an analysis of the ethical validity of these reasons, in terms of their consistency and how well they meet standards of equity and justice. The paper concludes that current approaches to parent selection are unsystematic and inadequate, and discusses the implications of this assessment.