bluuebunny/crossref_metadata_2025 huggingface dataset:
Evidence of Different
Enterocytozoon bieneusi
Genotypes in Patients with and without Human Immunodeficiency Virus Infection
Authors: Liguory Olivier , Sarfati Claudine , Derouin Francis , Molina Jean-Michel
Published: July 2002

We classified 100
Enterocytozoon bieneusi
isolates into five genotypes by a PCR-restriction fragment length polymorphism method. Type I strains were encountered only in human immunodeficiency virus (HIV)-infected patients, whereas type II strains were more frequently found in non-HIV-infected patients (75 versus 10%, respectively;
P
< 10
−4
), suggesting differences in the epidemiology of
E. bieneusi
among these patients.
Genetic Diversity of
Pneumocystis carinii
f. sp.
hominis
Based on Variations in Nucleotide Sequences of Internal Transcribed Spacers of rRNA Genes
Authors: Nimri Laila F. , Moura Iaci N. S. , Huang Laurence , del Rio Carlos , Rimland David , Duchin Jeffrey S. , Dotson Ellen M. , Beard Charles B.
Published: July 2002

A variety of genes have been used to type
Pneumocystis carinii.
In the present study, nucleotide sequence variations in the ITS1 and ITS2 internal transcribed spacer (ITS) regions of the rRNA genes were used to type
Pneumocystis carinii
f. sp.
hominis
DNA obtained from the lungs of 60 human immunodeficiency virus-infected individuals. These regions were amplified by PCR, cloned, and sequenced. Multibase polymorphisms were identified among samples. Several new genotypes are reported on the basis of the nucleotide sequence variations at previously unreported positions of both the ITS1 and the ITS2 regions. Twelve new ITS1 sequences were observed, in addition to the nine sequence types reported previously. The most common was type E, which was observed in 60.5% of the samples. The sequence variations in the ITS1 region were mainly located at positions 5, 12, 23, 24, 45, 53, and 54. Sixteen new ITS2 types were also identified, in addition to the 13 types reported previously. The most common was type g (26.6%). The sequences of the ITS2 regions in most specimens were different from the previously published sequence at bases 120 and 166 through 183. The most common variations observed were deletions at positions 177 through 183. The presence of more than one sequence type in some patients (60%) suggested the occurrence of coinfection with multiple
P. carinii
strains. The genetic polymorphism observed demonstrates the degree of diversity of
Pneumocystis
strains that infect humans. Furthermore, the high degree of polymorphism suggests that these genes are evolving faster than other genes. Consequently, the sequence information derived is useful for purposes such as examination of the potential of person-to-person transmission and recurrent infections but perhaps not for other genotyping applications that rely on more stable genetic loci.
Molecular Identification of
Staphylococcus lugdunensis
in a Patient with Meningitis
Authors: Kaabia N. , Scauarda D. , Lena G. , Drancourt M.
Published: July 2002

A 12-year-old child developed meningitis 6 days after a third ventriculostomy by endoscopy. A coagulase-negative
Staphylococcus
sp. was isolated in pure culture from the cerebrospinal fluid and was definitely identified as
Staphylococcus lugdunensis
after the 16S ribosomal DNA gene and
rpoB
gene were sequenced. This report describes the first case of
S. lugdunensis
meningitis.
Direct Detection of
Helicobacter pylori
Mutations Associated with Macrolide Resistance in Gastric Biopsy Material Taken from Human Immunodeficiency Virus-Infected Subjects
Authors: Scarpellini Paolo , Carrera Paola , Cavallero Annalisa , Cernuschi Massimo , Mezzi Gianni , Testoni Pier Alberto , Zingale Anna , Lazzarin Adriano
Published: July 2002

One hundred forty gastric biopsies were tested by microbiological methods and by amplifying a sequence of 23S rRNA and identifying mutations associated to clarithromycin resistance. Seventy-six specimens were positive for
Helicobacter pylori
. Mutational analysis revealed alterations in 18 (39.1%) of 46 and 2 (8.7%) of 23 samples from human immunodeficiency virus-seropositive and -seronegative persons, respectively. The results of the mutational analysis fully correlated with those of the susceptibility tests.
Infection of Polarized Cultures of Human Intestinal Epithelial Cells with Hepatitis A Virus: Vectorial Release of Progeny Virions through Apical Cellular Membranes
Authors: Blank Christian A. , Anderson David A. , Beard Michael , Lemon Stanley M.
Published: July 2002

Although hepatitis A virus (HAV) is typically transmitted by the fecal-oral route, little is known of its interactions with cells of the gastrointestinal tract. We studied the replication of HAV in polarized cultures of Caco-2 cells, a human cell line which retains many differentiated functions of small intestinal epithelial cells. Virus uptake was 30- to 40-fold more efficient when the inoculum was placed on the apical rather than the basolateral surface of these cells, suggesting a greater abundance of the cellular receptor for HAV on the apical surface. Infection proceeded without cytopathic effect and did not influence transepithelial resistance or the diffusion of inulin across cell monolayers. Nonetheless, there was extensive release of progeny virus, which occurred almost exclusively into apical supernatant fluids (36.4% ± 12.5% of the total virus yield compared with 0.23% ± 0.13% release into basolateral fluids). Brefeldin A caused a profound inhibition of HAV replication, but also selectively reduced apical release of virus. These results indicate that polarized human epithelial cell cultures undergo vectorial infection with HAV and that virus release is largely restricted to the apical membrane. Virus release occurs in the absence of cytopathic effect and may involve cellular vesicular transport mechanisms.
Functional Analysis of the CD4
+
T-Cell Response to Epstein-Barr Virus: T-Cell-Mediated Activation of Resting B Cells and Induction of Viral BZLF1 Expression
Authors: Fu Zheng , Cannon Martin J.
Published: July 2002

In contrast to the major role played by Epstein-Barr virus (EBV)-specific CD8
+
cytotoxic T-cell responses in immunosurveillance, recent studies have offered the apparently paradoxical suggestion that development of EBV-driven human B-cell lymphoproliferative disorders and tumors in SCID/hu mice is dependent on the presence of T cells, in particular CD4
+
T cells. This study presents a functional analysis of the CD4
+
T-cell response to EBV and shows that while CD4
+
T cells may be cytotoxic, they also express Th2 cytokines and CD40 ligand (gp39) and possess B-cell helper function. We show that EBV-specific CD4
+
T cells can provide non-HLA-restricted help for activation of resting B cells via a gp39-CD40-dependent pathway and are able to induce expression of BZLF1, a viral lytic cycle transactivator in latently infected resting B cells, ultimately resulting in rapid outgrowth of transformed B-cell colonies. These results support the proposal that CD4
+
T cells may play a key role in reactivation of latent EBV infection and may thus contribute to the pathogenesis of EBV-driven lymphoproliferative disorders.
The Large Delta Antigen of Hepatitis Delta Virus Potently Inhibits Genomic but Not Antigenomic RNA Synthesis: a Mechanism Enabling Initiation of Viral Replication
Authors: Modahl Lucy E. , Lai Michael M. C.
Published: July 2002

Hepatitis delta virus (HDV) contains two types of hepatitis delta antigens (HDAg) in the virion. The small form (S-HDAg) is required for HDV RNA replication, whereas the large form (L-HDAg) potently inhibits it by a dominant-negative inhibitory mechanism. The sequential appearance of these two forms in the infected cells regulates HDV RNA synthesis during the viral life cycle. However, the presence of almost equal amounts of S-HDAg and L-HDAg in the virion raised a puzzling question concerning how HDV can escape the inhibitory effects of L-HDAg and initiate RNA replication after infection. In this study, we examined the inhibitory effects of L-HDAg on the synthesis of various HDV RNA species. Using an HDV RNA-based transfection approach devoid of any artificial DNA intermediates, we showed that a small amount of L-HDAg is sufficient to inhibit HDV genomic RNA synthesis from the antigenomic RNA template. However, the synthesis of antigenomic RNA, including both the 1.7-kb HDV RNA and the 0.8-kb HDAg mRNA, from the genomic-sense RNA was surprisingly resistant to inhibition by L-HDAg. The synthesis of these RNAs was inhibited only when L-HDAg was in vast excess over S-HDAg. These results explain why HDV genomic RNA can initiate replication after infection even though the incoming viral genome is complexed with equal amounts of L-HDAg and S-HDAg. These results also suggest that the mechanisms of synthesis of genomic versus antigenomic RNA are different. This study thus resolves a puzzling question about the early events of the HDV life cycle.
Attenuated Modified Vaccinia Virus Ankara Can Be Used as an Immunizing Agent under Conditions of Preexisting Immunity to the Vector
Authors: Ramírez Juan C. , Gherardi M. Magdalena , Rodríguez Dolores , Esteban Mariano
Published: July 2002

A problem associated with the use of vaccinia virus recombinants as vaccines is the existence of a large human population with preexisting immunity to the vector. Here we showed that after a booster with attenuated recombinant modified vaccinia virus Ankara (rMVA), higher humoral and cellular immune responses to foreign antigens (human immunodeficiency virus type 1 Env and β-galactosidase) were found in mice preimmunized with rMVA than in mice primed with the virulent Western Reserve strain and boosted with rMVA. This enhancement correlated with higher levels of expression of foreign antigens after the booster.
Functional Characterization of Intracellular and Secreted Forms of a Truncated Hepatitis C Virus E2 Glycoprotein
Authors: Flint Mike , Dubuisson Jean , Maidens Catherine , Harrop Richard , Guile Geoffrey R. , Borrow Persephone , McKeating Jane A.
Published: July 2002

The E2 protein of hepatitis C virus (HCV) is believed to be a virion surface glycoprotein that is a candidate for inclusion in an antiviral vaccine. A truncated soluble version of E2 has recently been shown to interact with CD81, suggesting that this protein may be a component of the receptor for HCV. When expressed in eukaryotic cells, a significant proportion of E2 forms misfolded aggregates. To analyze the specificity of interaction between E2 and CD81, the aggregated and monomeric forms of a truncated E2 glycoprotein (E2
661
) were separated by high-pressure liquid chromatography and analyzed for CD81 binding. Nonaggregated forms of E2 preferentially bound CD81 and a number of conformation-dependent monoclonal antibodies (MAbs). Furthermore, intracellular forms of E2
661
were found to bind CD81 with greater affinity than the extracellular forms. Intracellular and secreted forms of E2
661
were also found to differ in reactivity with MAbs and human sera, consistent with differences in antigenicity. Together, these data indicate that proper folding of E2 is important for its interaction with CD81 and that modifications of glycans can modulate this interaction. Identification of the biologically active forms of E2 will assist in the future design of vaccines to protect against HCV infection.
Identification of gp120 Regions Targeted by a Highly Potent Neutralizing Antiserum Elicited in a Chimpanzee Inoculated with a Primary Human Immunodeficiency Virus Type 1 Isolate
Authors: Cho Michael W. , Lee Myung K. , Chen Chin H. , Matthews Tom , Martin Malcolm A.
Published: July 2002

We have previously reported that a chimpanzee infected with a primary human immunodeficiency virus type 1 (HIV-1) isolate (HIV-1DH12) developed an extremely potent virus-neutralizing antibody. Immunoglobulin G purified from this animal conferred sterilizing immunity following passive transfer to macaques which were subsequently challenged with simian immunodeficiency virus/HIV-1 chimeric virus strain DH12. In addition to being highly strain specific, the chimpanzee antiserum did not bind to the V3 loop peptide of HIV-1DH12, nor did it block the interaction of gp120 with the CD4 receptor. When neutralization was examined in the context of virus particles carrying chimeric envelope glycoproteins, the presence of all five hypervariable regions (V1 to V5) was required for optimal neutralization. Virions bearing chimeric gp120 containing the V1–V2 and V4 regions of HIV-1DH12could also be neutralized, but larger quantities of the chimpanzee antiserum were needed to block infection. These results indicate that the HIV-1 gp120 epitope(s) targeted by the chimpanzee antiserum is highly conformational, involving surface elements contributed by all of the hypervariable domains of the envelope glycoprotein.
Characterization of Hepatitis C Virus (HCV) and HCV E2 Interactions with CD81 and the Low-Density Lipoprotein Receptor
Authors: Wünschmann Sabina , Medh Jheem D. , Klinzmann Donna , Schmidt Warren N. , Stapleton Jack T.
Published: July 2002

Hepatitis C virus (HCV) or HCV–low-density lipoprotein (LDL) complexes interact with the LDL receptor (LDLr) and the HCV envelope glycoprotein E2 interacts with CD81 in vitro. However, E2 interactions with LDLr and HCV interactions with CD81 have not been clearly described. Using sucrose gradient-purified low-density particles (1.03 to 1.07 g/cm
3
), intermediate-density particles (1.12 to 1.18 g/cm
3
), recombinant E2 protein, or control proteins, we assessed binding to MOLT-4 cells, foreskin fibroblasts, or LDLr-deficient foreskin fibroblasts at 4°C by flow cytometry and confocal microscopy. Viral entry was determined by measuring the coentry of α-sarcin, a protein synthesis inhibitor. We found that low-density HCV particles, but not intermediate-density HCV or controls bound to MOLT-4 cells and fibroblasts expressing the LDLr. Binding correlated with the extent of cellular LDLr expression and was inhibited by LDL but not by soluble CD81. In contrast, E2 binding was independent of LDLr expression and was inhibited by human soluble CD81 but not mouse soluble CD81 or LDL. Based on confocal microscopy, we found that low-density HCV particles and LDL colocalized on the cell surface. The addition of low-density HCV but not intermediate-density HCV particles to MOLT-4 cells allowed coentry of α-sarcin, indicating viral entry. The amount of viral entry also correlated with LDLr expression and was independent of the CD81 expression. Using a solid-phase immunoassay, recombinant E2 protein did not interact with LDL. Our data indicate that E2 binds CD81; however, virus particles utilize LDLr for binding and entry. The specific mechanism by which HCV particles interact with LDL or the LDLr remains unclear.
Comparison between Human Cytomegalovirus pUL97 and Murine Cytomegalovirus (MCMV) pM97 Expressed by MCMV and Vaccinia Virus: pM97 Does Not Confer Ganciclovir Sensitivity
Authors: Wagner Markus , Michel Detlef , Schaarschmidt Peter , Vaida Bianca , Jonjic Stipan , Messerle Martin , Mertens Thomas , Koszinowski Ulrich
Published: July 2002

The UL97 protein (pUL97) of human cytomegalovirus (HCMV) is a protein kinase that also phosphorylates ganciclovir (GCV), but its biological function is not yet clear. The M97 protein (pM97) of mouse cytomegalovirus (MCMV) is the homolog of pUL97. First, we studied the consequences of genetic replacement of M97 by UL97. Using the infectious bacterial plasmid clone of the full-length MCMV genome (M. Wagner, S. Jonjic, U. H. Koszinowski, and M. Messerle, J. Virol. 73:7056–7060, 1999), we replaced the M97 gene with the UL97 gene and constructed an MCMV M97 deletion mutant and a revertant virus. In addition, pUL97 and pM97 were expressed by recombinant vaccinia virus to compare both for known functions. Remarkably, pM97 proved not to be the reason for the GCV sensitivity of MCMV. When expressed by the recombinant MCMV, however, pUL97 was phosphorylated and endowed MCMV with the capacity to phosphorylate GCV, thereby rendering MCMV more susceptible to GCV. We found that deletion of pM97, although it is not essential for MCMV replication, severely affected virus growth. This growth deficit was only partially amended by pUL97 expression. When expressed by recombinant vaccinia viruses, both proteins were phosphorylated and supported phosphorylation of GCV, but pUL97 was about 10 times more effective than pM97. One hint of the functional differences between the proteins was provided by the finding that pUL97 accumulates in the nucleus, whereas pM97 is predominantly located in the cytoplasm of infected cells. In vivo testing revealed that the UL97-MCMV recombinant should allow evaluation of novel antiviral drugs targeted to the UL97 protein of HCMV in mice.
Repositioning Basic Residues in the M Domain of the Rous Sarcoma Virus Gag Protein
Authors: Callahan Eric M. , Wills John W.
Published: July 2002

The first 86 residues of the Rous sarcoma virus (RSV) Gag protein form a membrane-binding (M) domain that directs Gag to the plasma membrane during budding. Unlike other retroviral Gag proteins, RSV Gag is not myristylated; however, the RSV M domain does contain 11 basic residues that could potentially interact with acidic phospholipids in the plasma membrane. To investigate this possibility, we analyzed mutants in which basic residues in the M domain were replaced with asparagines or glutamines. The data show that neutralizing as few as two basic residues in the M domain blocked particle release and prevented Gag from localizing to the plasma membrane. Though not as severe, single neutralizations also diminished budding and, when expressed in the context of proviral clones, reduced the ability of RSV to spread in cell cultures. To further explore the role of basic residues in particle production, we added lysines to new positions in the M domain. Using this approach, we found that the budding efficiency of RSV Gag can be improved by adding pairs of lysines and that the basic residues in the M domain can be repositioned without affecting particle release. These data provide the first gain-of-function evidence for the importance of basic residues in a retroviral M domain and support a model in which RSV Gag binds to the plasma membrane via electrostatic interactions.
Vaccinia Virus F12L Protein Is Required for Actin Tail Formation, Normal Plaque Size, and Virulence
Authors: Zhang Wei-Hong , Wilcock Diane , Smith Geoffrey L.
Published: July 2002

Vaccinia virus gene F12L is shown to encode a 65-kDa protein that is synthesized early and late during infection and that is not modified by glycosylation. Computational sequence comparison revealed that related proteins are encoded by all sequenced chordopoxviruses. A virus deletion mutant lacking the F12L gene (vΔF12L) and a revertant virus with the F12L gene reinserted into the deletion mutant (vF12L-rev) were constructed and analyzed. A version of the F12L gene with a C-terminal amino acid tag derived from the influenza virus hemagglutinin and that is recognized by a monoclonal antibody was also inserted into the F12L locus of vΔF12L. Loss of the F12L protein reduced the formation of IMV 2-fold, but there was a dramatic (99.5%) reduction in actin tail formation, and the levels of cell-associated enveloped virus and extracellular enveloped virus were reduced 8- to 11-fold and 7-fold, respectively. Consistent with the lack of actin tail formation, vΔF12L produced a very small plaque. The vΔF12L virus was severely attenuated in vivo, such that a dose of vΔF12L 10,000-fold greater than the dose of wild-type virus that induced severe disease was unable to induce disease in mice infected intranasally.
Contributions of Fas-Fas Ligand Interactions to the Pathogenesis of Mouse Hepatitis Virus in the Central Nervous System
Authors: Parra Beatriz , Lin Mark T. , Stohlman Stephen A. , Bergmann Cornelia C. , Atkinson Roscoe , Hinton David R.
Published: July 2002

The pathogenesis of the neurotropic strain of mouse hepatitis virus in Fas-deficient mice suggested that Fas-mediated cytotoxicity may be required during viral clearance after the loss of perforin-mediated cytotoxicity. The absence of both Fas- and perforin-mediated cytolysis resulted in an uncontrolled infection, suggesting a redundancy of cytolytic pathways to control virus replication.
The Epstein-Barr Virus Pol Catalytic Subunit Physically Interacts with the BBLF4-BSLF1-BBLF2/3 Complex
Authors: Fujii Ken , Yokoyama Naoaki , Kiyono Tohru , Kuzushima Kiyotaka , Homma Michio , Nishiyama Yukihiro , Fujita Masatoshi , Tsurumi Tatsuya
Published: July 2002

The Epstein-Barr virus (EBV)-encoded replication proteins that account for the basic reactions at the replication fork are thought to be the EBV Pol holoenzyme, consisting of the BALF5 Pol catalytic and the BMRF1 Pol accessory subunits, the putative helicase-primase complex, comprising the BBLF4, BSLF1, and BBLF2/3 proteins, and the BALF2 single-stranded DNA-binding protein. Immunoprecipitation analyses using anti-BSLF1 or anti-BBLF2/3 protein-specific antibody with clarified lysates of B95-8 cells in a viral productive cycle suggested that the EBV Pol holoenzyme physically interacts with the BBLF4-BSLF1-BBLF2/3 complex to form a large complex. Although the complex was stable in 500 mM NaCl and 1% NP-40, the BALF5 protein became dissociated in the presence of 0.1% sodium dodecyl sulfate. Experiments using lysates from insect cells superinfected with combinations of recombinant baculoviruses capable of expressing each of viral replication proteins showed that not the BMRF1 Pol accessory subunit but rather the BALF5 Pol catalytic subunit directly interacts with the BBLF4-BSLF1-BBLF2/3 complex. Furthermore, double infection with pairs of recombinant viruses revealed that each component of the BBLF4-BSLF1-BBLF2/3 complex makes contact with the BALF5 Pol catalytic subunit. The interactions of the EBV DNA polymerase with the EBV putative helicase-primase complex warrant particular attention because they are thought to coordinate leading- and lagging-strand DNA synthesis at the replication fork.
Dominant Role of Host Selective Pressure in Driving Hepatitis C Virus Evolution in Perinatal Infection
Authors: Manzin Aldo , Solforosi Laura , Debiaggi Maurizia , Zara Francesca , Tanzi Elisabetta , Romanò Luisa , Zanetti Alessandro R. , Clementi Massimo
Published: July 2002

The dynamics of the genetic diversification of hepatitis C virus (HCV) populations was addressed in perinatal infection. Clonal sequences of hypervariable region 1 of the putative E2 envelope protein of HCV were obtained from four HCV-infected newborns (sequential samples spanning a period of 6 to 13 months after birth) and from their mothers (all samples collected at delivery). The data show that the variants detected between birth and the third month of life in samples from the four newborns were present in the HCV populations of their mothers at delivery. In the newborns, a unique viral variant (or a small group of closely related variants) remained stable for weeks despite active viral replication. Diversification of the intrahost HCV population was observed 6 to 13 months after birth and was substantially higher in two of the four subjects, as documented by the intersample genetic distance (GD) (
P
= 0.007). Importantly, a significant correlation between increasing GD and high values for the intersample

K
a
/K
s

ratio (the ratio between antonymous and synonymous substitutions; an index of the action of selective forces) was observed, as documented by the increase of both parameters over time (
P
= 0.01). These data argue for a dominant role of positive selection for amino acid changes in driving the pattern of genetic diversification of HCV populations, indicate that the intrahost evolution of HCV populations is compatible with a Darwinian model system, and may have implications in the designing of future antiviral strategies.
Interferon Regulatory Factor 7 Mediates Activation of Tap-2 by Epstein-Barr Virus Latent Membrane Protein 1
Authors: Zhang Luwen , Pagano Joseph S.
Published: July 2002

Transporter associated with antigen processing 2 (Tap-2) is responsible for ATP-dependent transport of peptides from the cytosol to the endoplasmic reticulum, where peptides bind to newly synthesized human leukocyte antigen (HLA) class I molecules, which are essential for cellular immune responses. Epstein-Barr virus (EBV) latent membrane protein 1 (LMP-1) has been shown to induce the expression of Tap-2. In this study, the induction of endogenous Tap-2 by LMP-1 is shown to be associated with and requires the expression of interferon regulatory factor 7 (IRF-7). In DG75 Burkitt's lymphoma (BL) cells, in which LMP-1 induces the expression of IRF-7, LMP-1 induced endogenous Tap-2, and ectopic expression of IRF-7 could enhance the induction. In Akata BL cells, in which LMP-1 could not induce IRF-7, LMP-1 could not induce Tap-2. Addition of IRF-7, which complements the defect in Akata cells, could stimulate the expression of Tap-2. Furthermore, LMP-1 and IRF-7A but not other IRF-7 splicing variants could activate endogenous Tap-2. A Tap-2 promoter reporter construct could be activated by the overexpression of IRF-7A. The activation could be specifically enhanced by LMP-1 and was dependent on an intact interferon-stimulated response element (ISRE) present in the Tap-2 promoter. Also, IRF-7 can bind to the Tap-2 promoter under physiological conditions in vivo, as shown by formaldehyde cross-linking, as well as to the Tap-2 ISRE in vitro, as shown by gel mobility shift assays. Furthermore, LMP-1 facilitates the phosphorylation and nuclear translocation of IRF-7. These data point to the role of IRF-7 as a secondary mediator of LMP-1-activated signal transduction for Tap-2 as follows: LMP-1 stimulates the expression of IRF-7 and facilitates its phosphorylation and nuclear translocation, and then the activated IRF-7 mediates the activation of the cellular Tap-2 gene. The induction of Tap-2 by IRF-7 and LMP-1 may have an important implication for the immune response to EBV and its persistence in vivo.
Maintenance of the Gag/Gag-Pol Ratio Is Important for Human Immunodeficiency Virus Type 1 RNA Dimerization and Viral Infectivity
Authors: Shehu-Xhilaga Miranda , Crowe Suzanne M. , Mak Johnson
Published: July 2002

Production of the human immunodeficiency virus type 1 (HIV-1) Gag-Pol precursor protein results from a −1 ribosomal frameshifting event. In infected cells, this generates Gag and Gag-Pol in a ratio that is estimated to be 20:1, a ratio that is conserved among retroviruses. To examine the impact of this ratio on HIV-1 replication and viral assembly, we altered the Gag/Gag-Pol ratio in virus-producing cells by cotransfecting HIV-1 proviral DNA with an HIV-1 Gag-Pol expression vector. Two versions of the Gag-Pol expression vector were used; one contains an active protease [PR(+)], and the other contains an inactive protease [PR(−)]. In an attempt to produce viral particles with Gag/Gag-Pol ratios ranging from 20:21 to 20:1 (wild type), 293T cells were cotransfected with various ratios of wild-type proviral DNA and proviral DNA from either Gag-Pol expression vector. Viral particles derived from cells with altered Gag/Gag-Pol ratios via overexpression of PR(−) Gag-Pol showed a ratio-dependent defect in their virion protein profiles. However, the defects in virion infectivity were independent of the nature of the Gag-Pol expression vector, i.e., PR(+) or PR(−). Based on equivalent input of reverse transcriptase activity, we estimated that HIV-1 infectivity was reduced 250- to 1,000-fold when the Gag/Gag-Pol ratio in the virion-producing cells was altered from 20:1 to 20:21. Although virion RNA packaging was not affected by altering Gag/Gag-Pol ratios, changing the ratio from 20:1 to 20:21 progressively reduced virion RNA dimer stability. The impact of the Gag/Gag-Pol ratio on virion RNA dimerization was amplified when the Gag-Pol PR(−) expression vector was expressed in virion-producing cells. Virions produced from cells expressing Gag and Gag-Pol PR(−) in a 20:21 ratio contained mainly monomeric RNA. Our observations provide the first direct evidence that, in addition to proteolytic processing, the ratio of Gag/Gag-Pol proteins is also important for RNA dimerization and that stable RNA dimers are not required for encapsidation of genomic RNA in HIV-1.
Exposure to Low pH Is Not Required for Penetration of Mosquito Cells by Sindbis Virus
Authors: Hernandez Raquel , Luo Tianci , Brown Dennis T.
Published: July 2002

It is widely held that the penetration of cells by alphaviruses is dependent on exposure to the acid environment of an endosome. The alphavirus Sindbis virus replicates in both vertebrate and invertebrate cell cultures. We have found that exposure to an acid environment may not be required for infection of cells of the insect host. In this work, we investigated the effects of two agents (NH
4
Cl and chloroquine), which raise the pH of intracellular compartments (lysosomotropic weak bases) on the infection and replication of Sindbis virus in cells of the insect host
Aedes albopictus
. The results show that both of these agents increase the pH of endosomes, as indicated by protection against diphtheria toxin intoxication. NH
4
Cl blocked the production of infectious virus and blocked virus RNA synthesis when added prior to infection. Chloroquine, in contrast to its effect on vertebrate cells, had no inhibitory effect on infectious virus production in mosquito cells even when added prior to infection. Treatment with NH
4
Cl did not prevent the penetration of virus RNA into the cell cytoplasm or translation of the RNA to produce a precursor to virus nonstructural proteins. These data suggest that while these two drugs raise the pH of endosomes, they do not block insect cell penetration. These data support previous results published by our laboratory suggesting that exposure to an acid environment within the cell may not be an obligatory step in the process of infection of cells by alphaviruses.
Single-Chain Antibody Displayed on a Recombinant Measles Virus Confers Entry through the Tumor-Associated Carcinoembryonic Antigen
Authors: Hammond Anthea L. , Plemper Richard K. , Zhang Jie , Schneider Urs , Russell Stephen J. , Cattaneo Roberto
Published: July 2002

To redirect the tropism of the vaccine strain of measles virus (MV), Edmonston B, to a targeted cell population, we displayed on the viral hemagglutinin (H) a single-chain antibody (scAb) specific for the tumor-associated carcinoembryonic antigen (CEA). We generated H fusion proteins with three forms of the scAb appended, differing in the lengths of the linkers separating the V
H
and V
L
domains and thus in the oligomerization states of the scAbs. All proteins were stable, appeared properly folded, and were transported to the cell surface, but only H displaying the long-linker form of scAb was functional in supporting cell-cell fusion. This protein induced extensive syncytia in cells expressing the normal virus receptor CD46 and also in CD46-negative cells expressing the targeted receptor, human CEA. Replication-competent MV with H replaced by H displaying the long-linker form of scAb was recovered and replicated efficiently in both CD46-positive and CD46-negative, CEA-positive cells. Thus, MV not only tolerates the addition of a scAb on its H protein but also infects cells via a novel interaction between the scAb and its targeted receptor.
STAT2 Acts as a Host Range Determinant for Species-Specific Paramyxovirus Interferon Antagonism and Simian Virus 5 Replication
Authors: Parisien Jean-Patrick , Lau Joe F. , Horvath Curt M.
Published: July 2002

The antiviral state induced by alpha/beta interferon (IFN-α/β) is a powerful selective pressure for virus evolution of evasive strategies. The paramyxoviruses simian virus 5 (SV5) and human parainfluenza virus 2 (HPIV2) overcome IFN-α/β responses through the actions of their V proteins, which induce proteasomal degradation of cellular IFN-α/β-activated signal transducers and activators of transcription STAT1 and STAT2. SV5 infection induces STAT1 degradation and IFN-α/β inhibition efficiently in human cells but not in mouse cells, effectively restricting SV5 host range. Here, the cellular basis for this species specificity is demonstrated to result from differences between human and murine STAT2. Expression in mouse cells of full-length or truncated human STAT2 cDNA is sufficient to permit antagonism of endogenous murine IFN-α/β signaling by SV5 and HPIV2 V proteins. Furthermore, virus-induced STAT protein degradation is observed in mouse cells only in the presence of ectopically expressed human STAT2. The results indicate that STAT2 acts as an intracellular determinant of paramyxovirus host range restriction, which contributes to the species specificity of virus replication, and that human STAT2 can confer a growth advantage for SV5 in the murine host.
Human Herpesvirus 6 Variant A but Not Variant B Induces Fusion from Without in a Variety of Human Cells through a Human Herpesvirus 6 Entry Receptor, CD46
Authors: Mori Yasuko , Seya Tsukasa , Huang Hong Lan , Akkapaiboon Pilailuk , Dhepakson Panadda , Yamanishi Koichi
Published: July 2002

Human herpesvirus 6 (HHV-6) is a lymphotropic betaherpesvirus that productively infects T cells and monocytes. HHV-6 isolates can be differentiated into two groups, variants A and B (HHV-6A and HHV-6B). Here, we show a functional difference between HHV-6A and -6B in that HHV-6A induced syncytium formation of diverse human cells but HHV-6B did not. The syncytium formation induced by HHV-6A was observed 2 h after infection; moreover, it was found in the presence of cycloheximide, indicating that HHV-6A induced fusion from without (FFWO) in the target cells. Furthermore, the fusion event was dependent on the expression of the HHV-6 entry receptor, CD46, on the target cell membrane. In addition, we determined that short consensus repeat 2 (SCR2), -3, and -4 of the CD46 ectodomain were essential for the formation of the virus-induced syncytia. Monoclonal antibodies against glycoproteins B and H of HHV-6A inhibited the fusion event, indicating that the syncytium formation induced by HHV-6A required glycoproteins H and B. These findings suggest that FFWO, which HHV-6A induced in a variety of cell lines, may play an important role in the pathogenesis of HHV-6A, not only in lymphocytes but also in various tissues, because CD46 is expressed ubiquitously in human tissues.
Antiviral Effects of an Iminosugar Derivative on Flavivirus Infections
Authors: Wu Shu-Fen , Lee Chyan-Jang , Liao Ching-Len , Dwek Raymond A. , Zitzmann Nicole , Lin Yi-Ling
Published: July 2002

Endoplasmic reticulum (ER) α-glucosidase inhibitors, which block the trimming step of N-linked glycosylation, have been shown to eliminate the production of several ER-budding viruses. Here we investigated the effects of one such inhibitor,
N
-nonyl-deoxynojirimycin (
N
N-DNJ), a 9-carbon alkyl iminosugar derivative, on infection by Japanese encephalitis virus (JEV) and dengue virus serotype 2 (DEN-2). In the presence of
N
N-DNJ, JEV and DEN-2 infections were suppressed in a dose-dependent manner. This inhibitory effect appeared to influence DEN-2 infection more than JEV infection, since lower concentrations of
N
N-DNJ substantially blocked DEN-2 replication. Secretion of the flaviviral glycoproteins E and NS1 was greatly reduced, and levels of DEN-2 viral RNA replication measured by fluorogenic reverse transcription-PCR were also decreased, by
N
N-DNJ. Notably, the viral glycoproteins, prM, E, and NS1 were found to associate transiently with the ER chaperone calnexin, and this interaction was affected by
N
N-DNJ, suggesting a potential role of calnexin in the folding of flaviviral glycoproteins. Additionally, in a mouse model of lethal challenge by JEV infection, oral delivery of
N
N-DNJ reduced the mortality rate. These findings show that
N
N-DNJ has an antiviral effect on flavivirus infection, likely through interference with virus replication at the posttranslational modification level, occurring mainly in the ER.
Ty1 Defect in Proteolysis at High Temperature
Authors: Lawler Joseph F. , Haeusser Daniel P. , Dull Angie , Boeke Jef D. , Keeney Jill B.
Published: July 2002

Retrotransposition of the Ty1 element of
Saccharomyces cerevisiae
is temperature sensitive. Transposition activity of Ty1 is abolished at temperatures above 34°C. In this report, we show that the major block to transposition at high temperature is the inhibition of processing of the Gag-Pol-p199 polyprotein and the concomitant reduction of reverse transcriptase (RT) activity. Expression of a Ty1 protease construct in
Escherichia coli
shows that protease enzymatic activity is inherently temperature sensitive. In yeast, Gag processing is only partially inhibited at high temperature, while cleavage of the Gag-Pol polyprotein is completely inhibited. Sites of proteolytic processing are differentially susceptible to cleavage during growth at high temperature. Overall levels of the Gag-Pol polyprotein are reduced at high temperature, although the efficiency of the requisite +1 frameshifting event appears to be increased. RT activity is inherently relatively temperature resistant, yet no cDNA is made at high temperature and the amount of RT activity is greatly reduced in virus-like particles formed at high temperature. Taken together, these results suggest that alterations in Ty1 proteins that occur at high temperature affect both protease activity and RT activity, such that Ty1 transposition is abolished.
The Carboxy Terminus of Simian Virus 40 Large T Antigen Is Required To Disrupt the Yeast Cell Cycle
Authors: Fewell Sheara W. , Markle Dena M. , Brodsky Jeffrey L.
Published: July 2002

Wild-type and J domain mutant simian virus 40 large T antigens alter the cell cycle and bud morphology of
Saccharomyces cerevisiae
. In contrast, yeast cells expressing mutant T antigen lacking the carboxy-terminal 150 aa exhibit normal morphology, indicating that this region of T antigen is required for cell cycle disruption.
Essential Arginine Residues in Lactate Dehydrogenase from Germinating Soybean
Authors: Barthová Jana , Hulová Irena , Birčáková Miroslava
Published: July 2002

The lactate dehydrogenase was isolated from soybean (Glycine max. L.) by a procedure that employed biospecific chromatography on a column of Blue-Sepharose CL-6B. The participation of the guanidine group of arginine residues in the mechanism of enzyme action was determined through kinetic and chemical modification studies. The dependence of enzyme activity on pH was followed in the alkaline region (pH 8.6 - 12.8). The pK values found were 12.4 for the enzyme substrate complex and 11.1 for the free enzyme. The enzyme was inactivated by phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione and p-hydroxyphenylglyoxal reagents used in modification experiments. Kinetic analysis of the modification indicated that one arginine residue is modified when inactivation occurs. No effect was observed on the rate of inactivation upon addition of coenzyme. The extent of enzyme modification by p-hydroxyphenylglyoxal was determined. It appears there are at least two arginine residues in the active site of the enzyme.
Certain Problems with the Application of Stochastic Diffusion Processes for the Description of Chemical Engineering Phenomena. Dynamic Stochastic Model of Flow Mixer
Authors: Kudrna Vladimír , Vejmola Libor , Hasal Pavel
Published: July 2002

The one-dimensional stochastic diffusion model of a continuous flow mixer is proposed incorporating (contrary to commonly used diffusion models) a distribution of velocities of diffusing particles. Simplifying assumptions enabled us to derive an analytical expression for the liquid residence time distribution and concentration profile inside the mixer. For extreme values of parameters, the model becomes identical with the common idealized models usually adopted in chemical engineering.
Preconcentration and Determination of Cadmium and Lead by Ion-Pair Solid Phase Extraction on Silica Gel Followed by Flame Atomic Absorption Spectrometry
Authors: Rychlovský Petr , Bílý Jiří , Bazakasová Eva
Published: July 2002

The ion-pair formation between the metal complex with 4-(4'-nitrophenylazo)-1-naphthol and cetyltrimethylammonium bromide and its sorption on silica gel form the basis for preconcentration of cadmium and lead, which are subsequently determined on-line by flame AAS. The method was applied to the quantitation of cadmium and lead in potable and surface waters, with limits of determination (10s) of 0.5 and 2 μg l-1, respectively.
Synthesis of 3-(4-Pyridinyl)-, 3-(2-Chloro-4-pyridinyl)- and 3-(2-Amino-4-pyridinyl)propoxymethanephosphonic Acid
Authors: Krečmerová Marcela , Holý Antonín , Masojídková Milena
Published: July 2002

Reaction of sodium salt of 4-(3-hydroxypropyl)pyridine (I) with diisopropyl p-toluenesulfonyloxymethanephosphonate (II) afforded diisopropyl ester of the corresponding phosphonomethyl derivative (III), together with 4-cyclopropylpyridine (IV). The ester III was converted into free 3-(4-pyridinyl)propoxymethanephosphonic acid (V). The synthesis of 3-(2-amino-4-pyridinyl)propoxymethanephosphonic acid (XVI) started from 4-(3-benzoyloxypropyl)pyridine (VII) via the N-oxide VIII which on heating with phosphoryl chloride afforded the 2-chloro derivative IX. Compound IX was debenzoylated with sodium methoxide to give 2-chloro-4-(3-hydroxypropyl)pyridine (XI). Condensation of sodium salt of XI with tosylate II afforded diisopropyl 3-(2-chloro-4-pyridinyl)propoxymethanephosphonate (XIII) and 2-chloro-4-cyclopropylpyridine (XIV). Deprotection of the ester groups in XIII with bromotrimethylsilane gave 3-(2-chloro-4-pyridinyl)propoxymethanephosphonic acid (XV). The chlorine atom in position 2 was replaced by reaction of compound XV with aqueous ammonia at 200 °C under catalysis with copper(II) sulfate. This gave 3-(2-amino-4-pyridinyl)propoxymethanephosphonic acid (XVI) as the final product.
1H, 13C, and 15N NMR Spectra of Ni(II) Complexes of Schiff Bases of (S)-2-(N-Benzylprolyl)aminobenzophenone and α-Monosubstituted Glycine and Determination of Configuration of the Complexes by 2D NOESY Spectra
Authors: Jirman Josef , Popkov Alexander
Published: July 2002

1H, 13C, and 15N NMR spectra have been measured of substituted Ni(II) complexes of Schiff bases of (S)-2-(N-benzylprolyl)aminobenzophenone and glycine. The absolute configuration at C19 of the substituted glycine can be determined from 2D NOESY spectra using the NOESY interactions with the proton of the second chiral centre of the complex. It is possible to determine the rate of rotation of phenyl group of benzophenone unless its rotation is prevented by requatorialr orientation of dimethylamino group as it is the case with the Ni(II) complex of Schiff base of (S)-2-(N-benzylprolyl)aminobenzophenone and (S)-α-dimethylaminoglycine.
Study of UV Laser Ablation of Nitrided Steels Using Inductively Coupled Plasma Atomic Emission Spectrometry
Authors: Kanický Viktor , Musil Jan , Benda Marcel , Mermet Jean-Michel
Published: July 2002

A depth profile study of nitrided steels containing Ti or V sputtered in the surface layer was performed by means of laser ablation inductively coupled plasma atomic emission spectrometry. An Nd:YAG laser was used in the Q-switched mode and operated at 355 and 266 nm. The laser ablation patterns were obtained on the material surface by moving the ablation cell relative to the laser beam by means of an XY-translator. Depth resolution was obtained by using successive limited numbers of ablation cycles. The corresponding transient signals of Ti or V and Fe were measured simultaneously with a dual monochromator. The effect of the number of cycles, the laser power and laser wavelength on the time-dependent behaviour of the Ti or V and Fe line intensities was studied along with the erosion rate. It was found that the erosion rate was lower than 0.6 μm per cycle and depended on the depth of penetration. Emission of sputtered Ti and V was mainly observed in an external layer of less than 2 μm. The laser wavelength did not modify the shape of the crater but significantly changed the line intensity of Fe. The Fe line intensity was also enhanced during the ablation of the nitrided steel compared with the ablation of untreated steel. This study contributes to the knowledge of behaviour of the modified metal surface during interaction with the laser radiation, which is important for development of the method for determination of composition of nitrided surface layers.
Syntheses and Crystal Structures of Bis[(trimethylsilyl)tetramethylcyclopentadienyl]titanium Dichloride and Monochloride
Authors: Horáček Michal , Gyepes Róbert , Císařová Ivana , Polášek Miroslav , Varga Vojtech , Mach Karel
Published: July 2002

(Trimethylsilyl)tetramethylcyclopentadiene (Cp'H) was obtained from the lithium salt of tetramethylcyclopentadiene and trimethylsilyl chloride. Bis[(trimethylsilyl)tetramethylcyclopentadienyl]titanium dichloride (1) and monochloride (2) were prepared by the generally known procedures. The X-ray crystal structures of 1 and 2 showed the placement of the bulky trimethylsilyl groups in side positions on opposite sides of the CE-Ti-CE planes. The steric congestion between the two chlorine atoms and two trimethylsilyl groups in 1 resulted in the nearly eclipsed conformation of the rings. Consequently, the steric hindrance between the methyl groups at the hinge position of the Cp' ligands led to a decrease in the angle between the Cp' ring planes by 4.8°, compared with the value of 44.6° in (C5Me5)2TiCl2. The titanocene skeleton in 2 is virtually the same as in (C5Me5)2TiCl. The electronic effect of the trimethylsilyl group upon various properties of 1 and 2 appeared to be ambiguous. Based on the position of λmax of electronic absorption bands of 1 the trimethylsilyl group exerted a stronger electron donation effect than the methyl group, however, the affinity of 2 to 2-methyltetrahydrofuran put the effect of the trimethylsilyl group close to that of hydrogen and the anisotropy of the EPR g-tensor of 2 close to that of the methyl group.
Er-pH and Er-mKOH Diagrams for Nickel at 25 °C
Authors: Balej Jan , Divisek Jiri
Published: July 2002

Based on the conclusion that no Ni(III) oxo compounds can exist in oxidized nickel oxide electrodes with the total oxidation number 2.0 < zNi < approx. 3.6 and that non-ideal solid solutions with a formal composition (1 - X2) Ni(OH)2.X2NiO2. xH2O are involved, Er-pH and Er-mKOH diagrams at 25 °C were calculated and graphically represented using the thermodynamic standard data determined for hypothetical NiO2.x H2O. The calculations were performed assuming aH2O = 1 in the entire pH range (Er-pH diagram) and considering the true water activity in KOH solutions of various concentrations (Er-mKOH diagram).
Hydrophilicity Quantification of Highly Hydrophobic Decadodecasil 3R
Authors: Exter Marcel J. den , Jansen Jacobus C. , Bekkum Herman van , Zikánová Arlette , Dubský Josef , Kočiřík Milan
Published: July 2002

Sorption properties of the clathrasil Decadodecasil 3R (DD3R) have been investigated by measuring equilibrium sorption isotherms of water. The dependence of the adsorbed amount on the relative water pressure has been found almost invariant of temperature. This behaviour has been explained by a scarce population of hydrophilic sorption sites isolated sufficiently from each other and by a polymolecular adsorption on them. Such a behaviour is consistent with a simple BET model. Both the low concentration of hydrophilic sorption sites and their weak strength confirm the generally accepted assumption on weakly hydrophilic character of DD3R.
Cobaltacarboranes with Intramolecular Monophosphorus Bridges 8,8'-μ-Me2P(1,2-C2B9H10)2-3-Co, 6,6'-μ-Me2P(1,7-C2B9H10)2-2-Co and the Respective Non-Bridged Trimethylphosphine Derivatives (8-Me3P-1,2-C2B9H10)-3-Co-(1,2-C2B9H11) and (6-Me3P-1,7-C2B9H10)-2-Co-(1,7-C2B9H11)
Authors: Franken Andreas , Plešek Jaromír , Fusek Jiří , Semrau Markus
Published: July 2002

The first two cobaltacarboranes with intramolecular phoshorus bridges and two related non-bridged trimethylphosphine-cobaltacarborane derivatives are described and characterized by mass spectrometry and multinuclear NMR; all four compounds are zwitterions: 8,8'-μ-Me2P(1,2-C2B9H10)2-3-Co (1a), 6,6'-μ-Me2P(1,7-C2B9H10)2-2-Co (2a), (8-Me3P-1,2-C2B9H10)-3-Co-(1,2-C2B9H11) (1b) and (6-Me3P-1,7-C2B9H10)-2-Co-(1,7-C2B9H11) (2b). X-Ray structures of the first two and of the last compound has been established.