bluuebunny/crossref_metadata_2025 huggingface dataset:
Immunization of Rhesus Macaques with a DNA Prime/Modified Vaccinia Virus Ankara Boost Regimen Induces Broad Simian Immunodeficiency Virus (SIV)-Specific T-Cell Responses and Reduces Initial Viral Replication but Does Not Prevent Disease Progression following Challenge with Pathogenic SIVmac239
Authors: Horton Helen , Vogel Thorsten U. , Carter Donald K. , Vielhuber Kathy , Fuller Deborah H. , Shipley Tim , Fuller James T. , Kunstman Kevin J. , Sutter Gerd , Montefiori David C. , Erfle Volker , Desrosiers Ronald C. , Wilson Nancy , Picker Louis J. , Wolinsky Steven M. , Wang Chenxi , Allison David B. , Watkins David I.
Published: July 2002

Producing a prophylactic vaccine for human immunodeficiency virus (HIV) has proven to be a challenge. Most biological isolates of HIV are difficult to neutralize, so that conventional subunit-based antibody-inducing vaccines are unlikely to be very effective. In the rhesus macaque model, some protection was afforded by DNA/recombinant viral vector vaccines. However, these studies used as the challenge virus SHIV-89.6P, which is neutralizable, making it difficult to determine whether the observed protection was due to cellular immunity, humoral immunity, or a combination of both. In this study, we used a DNA prime/modified vaccinia virus Ankara boost regimen to immunize rhesus macaques against nearly all simian immunodeficiency virus (SIV) proteins. These animals were challenged intrarectally with pathogenic molecularly cloned SIVmac239, which is resistant to neutralization. The immunization regimen resulted in the induction of virus-specific CD8+and CD4+responses in all vaccinees. Although anamnestic neutralizing antibody responses against laboratory-adapted SIVmac251 developed after the challenge, no neutralizing antibodies against SIVmac239 were detectable. Vaccinated animals had significantly reduced peak viremia compared with controls (P< 0.01). However, despite the induction of virus-specific cellular immune responses and reduced peak viral loads, most animals still suffered from gradual CD4 depletion and progressed to disease.
Characterization of the Glycoproteins of Crimean-Congo Hemorrhagic Fever Virus
Authors: Sanchez Angela J. , Vincent Martin J. , Nichol Stuart T.
Published: July 2002

Crimean-Congo hemorrhagic fever (CCHF) virus is the cause of an important tick-borne disease of humans throughout regions of Africa, Europe, and Asia. Like other members of the genus
Nairovirus
, family
Bunyaviridae
, the CCHF virus M genome RNA segment encodes the virus glycoproteins. Sequence analysis of the CCHF virus (Matin strain) M RNA segment revealed one major open reading frame that potentially encodes a precursor polyprotein 1,689 amino acids (aa) in length. Comparison of the deduced amino acid sequences of the M-encoded polyproteins of Nigerian, Pakistani, and Chinese CCHF virus strains revealed two distinct protein regions. The carboxyl-terminal 1,441 aa are relatively highly conserved (up to 8.4% identity difference), whereas the amino-terminal 243 to 248 aa are highly variable (up to 56.4% identity difference) and have mucin-like features, including a high serine, threonine, and proline content (up to 47.3%) and a potential for extensive O-glycosylation. Analysis of released virus revealed two major structural glycoproteins, G2 (37 kDa) and G1 (75 kDa). Virus protein analysis by various techniques, including pulse-chase analysis and/or reactivity with CCHF virus-specific polyclonal and antipeptide antibodies, demonstrated that the 140-kDa (which contains the mucin-like region) and 85-kDa nonstructural proteins are the precursors of the mature G2 and G1 proteins, respectively. The amino termini of the CCHF virus (Matin strain) G2 and G1 proteins were established by microsequencing to be equivalent to aa 525 and 1046, respectively, of the encoded polyprotein precursor. The tetrapeptides RRLL and RKPL are immediately upstream of the cleavage site for mature G2 and G1, respectively. These are completely conserved among the predicted polyprotein sequences of all the CCHF virus strains and closely resemble the tetrapeptides that represent the major cleavage recognition sites present in the glycoprotein precursors of arenaviruses, such as Lassa fever virus (RRLL) and Pichinde virus (RKLL). These results strongly suggest that CCHF viruses (and other members of the genus
Nairovirus
) likely utilize the subtilase SKI-1/S1P-like cellular proteases for the major glycoprotein precursor cleavage events, as has recently been demonstrated for the arenaviruses.
Identification of the Neoplastically Transformed Cells in Marek's Disease Herpesvirus-Induced Lymphomas: Recognition by the Monoclonal Antibody AV37
Authors: Burgess Shane C. , Davison T. Fred
Published: July 2002

Understanding the interactions between herpesviruses and their host cells and also the interactions between neoplastically transformed cells and the host immune system is fundamental to understanding the mechanisms of herpesvirus oncology. However, this has been difficult as no animal models of herpesvirus-induced oncogenesis in the natural host exist in which neoplastically transformed cells are also definitively identified and may be studied in vivo. Marek's disease (MD) herpesvirus (MDV) of poultry, although a recognized natural oncogenic virus causing T-cell lymphomas, is no exception. In this work, we identify for the first time the neoplastically transformed cells in MD as the CD4+major histocompatibility complex (MHC) class Ihi, MHC class IIhi, interleukin-2 receptor α-chain-positive, CD28lo/−, phosphoprotein 38-negative (pp38−), glycoprotein B-negative (gB−), αβ T-cell-receptor-positive (TCR+) cells which uniquely overexpress a novel host-encoded extracellular antigen that is also expressed by MDV-transformed cell lines and recognized by the monoclonal antibody (MAb) AV37. Normal uninfected leukocytes and MD lymphoma cells were isolated directly ex vivo and examined by flow cytometry with MAb recognizing AV37, known leukocyte antigens, and MDV antigens pp38 and gB. CD28 mRNA was examined by PCR. Cell cycle distribution and in vitro survival were compared for each lymphoma cell population. We demonstrate for the first time that the antigen recognized by AV37 is expressed at very low levels by small minorities of uninfected leukocytes, whereas particular MD lymphoma cells uniquely express extremely high levels of the AV37 antigen; the AV37hiMD lymphoma cells fulfill the accepted criteria for neoplastic transformation in vivo (protection from cell death despite hyperproliferation, presence in all MD lymphomas, and not supportive of MDV production); the lymphoma environment is essential for AV37+MD lymphoma cell survival; pp38 is an antigen expressed during MDV-productive infection and is not expressed by neoplastically transformed cells in vivo; AV37+MD lymphoma cells have the putative immune evasion mechanism of CD28 down-regulation; AV37hiperipheral blood leukocytes appear early after MDV infection in both MD-resistant and -susceptible chickens; and analysis of TCR variable β chain gene family expression suggests that MD lymphomas have polyclonal origins. Identification of the neoplastically transformed cells in MD facilitates a detailed understanding of MD pathogenesis and also improves the utility of MD as a general model for herpesvirus oncology.
Evaluation of Tat-Encoding Bicistronic Human Immunodeficiency Virus Type 1 Gene Transfer Vectors in Primary Canine Bone Marrow Mononuclear Cells
Authors: Srinivasakumar Narasimhachar , Zaboikin Michail , Zaboikina Tatiana , Schuening Friedrich
Published: July 2002

Tat-encoding human immunodeficiency virus type 1 (HIV-1) gene transfer vectors were evaluated in primary canine bone marrow mononuclear cells. Tat vectors provided higher levels of gene expression than vectors with internal promoters. The HIV-1 vector was also more efficient than Moloney murine leukemia virus (MoMLV) vectors for transduction of canine bone marrow mononuclear cells in vitro. Transplantation experiments in dogs with transduced autologous marrow cells confirmed the superiority of HIV-1 vectors over MoMLV vectors for gene transfer into canine bone marrow cells. Tat vectors may be useful not only for providing high levels of therapeutic gene expression in hematopoietic cells but also for study of the biological effects of Tat in those tissues in the canine model.
PG13 Packaging Cells Produce Recombinant Retroviruses Carrying a Diphtheria Toxin Mutant Which Kills Cancer Cells
Authors: Qiao Jian , Caruso Manuel
Published: July 2002

The development of suicide gene therapy with gene products that are directly toxic to cells, such as the A subunit of diphtheria toxin (DT-A), has been hampered by the difficulty of engineering recombinant viruses. DT-A is a strong inhibitor of protein synthesis that acts by ADP-ribosylating elongation factor 2, and a low level of DT-A expression in virus producer cells prevents the production of recombinant virus. We analyzed here the natural resistance of packaging cells to DT-A toxicity, and we report that PG13 and PA317 packaging cell lines are resistant to H21G, a DT-A mutant. PG13 cells produce recombinant H21G virus that efficiently kills a variety of human tumor cells. Our finding indicates that PG13 packaging cells provide a new potential for the development of DT-A-based suicide gene therapy.
RNA Editing in Hepatitis Delta Virus Genotype III Requires a Branched Double-Hairpin RNA Structure
Authors: Casey John L.
Published: July 2002

RNA editing at the amber/W site plays a central role in the replication scheme of hepatitis delta virus (HDV), allowing the virus to produce two functionally distinct forms of the sole viral protein, hepatitis delta antigen (HDAg), from the same open reading frame. Editing is carried out by a cellular activity known as ADAR (adenosine deaminase), which acts on RNA substrates that are at least partially double stranded. In HDV genotype I, editing requires a highly conserved base-paired structure that occurs within the context of the unbranched rod structure characteristic of HDV RNA. This base-paired structure is disrupted in the unbranched rod of HDV genotype III, which is the most distantly related of the three known HDV genotypes and is associated with the most severe disease. Here I show that RNA editing in HDV genotype III requires a branched double-hairpin structure that deviates substantially from the unbranched rod structure, involving the rearrangement of nearly 80 bp. The structure includes a UNCG RNA tetraloop, a highly stable structural motif frequently involved in the folding of large RNAs such as rRNA. The double-hairpin structure is required for editing, and hence for virion formation, but not for HDV RNA replication, which requires the unbranched rod structure. HDV genotype III thus relies on a dynamic conformational switch between the two different RNA structures: the unbranched rod characteristic of HDV RNA and a branched double-hairpin structure that is required for RNA editing. The different mechanisms of editing in genotypes I and III underscore their functional differences and may be related to pathogenic differences as well.
Robust Recall and Long-Term Memory T-Cell Responses Induced by Prime-Boost Regimens with Heterologous Live Viral Vectors Expressing Human Immunodeficiency Virus Type 1 Gag and Env Proteins
Authors: Haglund Karl , Leiner Ingrid , Kerksiek Kristen , Buonocore Linda , Pamer Eric , Rose John K.
Published: July 2002

We investigated long-term memory and recall cellular immune responses to human immunodeficiency virus type 1 (HIV-1) Env and Gag proteins elicited by recombinant vesicular stomatitis viruses (VSVs) expressing Env and Gag. More than 7 months after a single vaccination with VSV-Env, ∼6% of CD8+splenocytes stained with major histocompatibility complex class I tetramers containing the Env p18-I10 immunodominant peptide and showed a memory phenotype (CD44Hi). The level of tetramer-positive cells in memory was about 14% of the peak primary response. Recall responses elicited in these mice 5 days after boosting with a heterologous recombinant vaccinia virus expressing HIV-1 Env showed that 40 to 45% of CD8+splenocytes were tetramer positive and activated (CD62LLo), and these cells produced gamma interferon after stimulation with Env peptide, indicating that they were functional. Five months after the boost, the long-term memory cell population (tetramer positive, CD44Hi) constituted 30% of the CD8+splenocytes. Recall responses to HIV-1 Gag were examined in mice primed with VSV recombinants expressing HIV-1 Gag protein and boosted with a vaccinia virus recombinant expressing Gag. Using this protocol, we found that ∼40% of CD8+splenocytes were activated (CD62LLo) and specific for a Gag immunodominant peptide (tetramer positive). The high-level Gag recall response elicited by the vaccinia virus-Gag was greater than that obtained by boosting with a VSV-Gag vector with a different VSV glycoprotein. The corresponding levels of CD44Himemory cells were also higher long after boosting with vaccinia virus-Gag than after boosting with a glycoprotein exchange VSV-Gag. Our results show that VSV vectors elicit high-level memory CTL responses and that these can be amplified as much as six- to sevenfold using a heterologous boosting vector.
Enhanced Presentation of Major Histocompatibility Complex Class I-Restricted Human Immunodeficiency Virus Type 1 (HIV-1) Gag-Specific Epitopes after DNA Immunization with Vectors Coding for Vesicular Stomatitis Virus Glycoprotein- Pseudotyped HIV-1 Gag Particles
Authors: Marsac D. , Loirat D. , Petit C. , Schwartz O. , Michel M.-L.
Published: July 2002

In vivo priming of cytotoxic T lymphocytes (CTL) by DNA injection predominantly occurs by antigen transfer from DNA-transfected cells to antigen-presenting cells. A rational strategy for increasing DNA vaccine potency would be to use a delivery system that facilitates antigen uptake by antigen-presenting cells. Exogenous antigen presentation through the major histocompatibility complex (MHC) class I-restricted pathway of some viral antigens is increased after adequate virus-receptor interaction and the fusion of viral and cellular membranes. We used DNA-based immunization with plasmids coding for human immunodeficiency virus type 1 (HIV-1) Gag particles pseudotyped with vesicular stomatitis virus glycoprotein (VSV-G) to generate Gag-specific CTL responses. The presence of the VSV-G-encoding plasmid not only increased the number of mice displaying anti-Gag-specific cytotoxic response but also increased the efficiency of specific lysis. In vitro analysis of processing confirmed that exogenous presentation of Gag epitopes occurred much more efficiently when Gag particles were pseudotyped with the VSV-G envelope. We show that the VSV-G-pseudotyped Gag particles not only entered the MHC class II processing pathway but also entered the MHC class I processing pathway. In contrast, naked Gag particles entered the MHC class II processing pathway only. Thus, the combined use of DNA-based immunization and nonreplicating pseudotyped virus to deliver HIV-1 antigen to the immune system in vivo could be considered in HIV-1 vaccine design.
Virus-Mediated Transduction of Murine Retina with Adeno-Associated Virus: Effects of Viral Capsid and Genome Size
Authors: Yang Grace S. , Schmidt Michael , Yan Ziying , Lindbloom Jonathan D. , Harding Thomas C. , Donahue Brian A. , Engelhardt John F. , Kotin Robert , Davidson Beverly L.
Published: July 2002

Gene therapy vectors based on adeno-associated viruses (AAVs) show promise for the treatment of retinal degenerative diseases. In prior work, subretinal injections of AAV2, AAV5, and AAV2 pseudotyped with AAV5 capsids (AAV2/5) showed variable retinal pigmented epithelium (RPE) and photoreceptor cell transduction, while AAV2/1 predominantly transduced the RPE. To more thoroughly compare the efficiencies of gene transfer of AAV2, AAV3, AAV5, and AAV6, we quantified, using stereological methods, the kinetics and efficiency of AAV transduction to mouse photoreceptor cells. We observed persistent photoreceptor and RPE transduction by AAV5 and AAV2 up to 31 weeks and found that AAV5 transduced a greater volume than AAV2. AAV5 containing full-length or half-length genomes and AAV2/5 transduced comparable numbers of photoreceptor cells with similar rates of onset of expression. Compared to AAV2, AAV5 transduced significantly greater numbers of photoreceptor cells at 5 and 15 weeks after surgery (greater than 1,000 times and up to 400 times more, respectively). Also, there were 30 times more genome copies in eyes injected with AAV2/5 than in eyes injected with AAV2. Comparing AAVs with half-length genomes, AAV5 transduced only four times more photoreceptor cells than AAV2 at 5 weeks and nearly equivalent numbers at 15 weeks. The enhancement of transduction was seen at the DNA level, with 50 times more viral genome copies in retinas injected with AAV having short genomes than in retinas injected with AAV containing full-length ones. Subretinal injection of AAV2/6 showed only RPE transduction at 5 and 15 weeks, while AAV2/3 did not transduce retinal cells. We conclude that varying genome length and AAV capsids may allow for improved expression and/or gene transfer to specific cell types in the retina.
Determination of the Structure of a Decay Accelerating Factor-Binding Clinical Isolate of Echovirus 11 Allows Mapping of Mutants with Altered Receptor Requirements for Infection
Authors: Stuart Amanda D. , McKee Thomas A. , Williams Pamela A. , Harley Chris , Shen Shuo , Stuart David I. , Brown T. David K. , Lea Susan M.
Published: July 2002

We have used X-ray crystallography to determine the structure of a decay accelerating factor (DAF)-binding, clinic-derived isolate of echovirus 11 (EV11-207). The structures of the capsid proteins closely resemble those of capsid proteins of other picornaviruses. The structure allows us to interpret a series of amino acid changes produced by passaging EV11-207 in different cell lines as highlighting the locations of multiple receptor-binding sites on the virion surface. We suggest that a DAF-binding site is located at the fivefold axes of the virion, while the binding site for a distinct but as yet unidentified receptor is located within the canyon surrounding the virion fivefold axes.
Priming of Human Papillomavirus Type 11-Specific Humoral and Cellular Immune Responses in College-Aged Women with a Virus-Like Particle Vaccine
Authors: Emeny Rebecca T. , Wheeler Cosette M. , Jansen Kathrin U. , Hunt William C. , Fu Tong-Ming , Smith Judith F. , MacMullen Stefani , Esser Mark T. , Paliard Xavier
Published: July 2002

In this study, we evaluated the potency of a human papillomavirus (HPV) virus-like particle (VLP)-based vaccine at generating HPV type 11 (HPV-11)-specific cellular and humoral immune responses in seronegative women. The vaccine was administered by intramuscular immunizations at months 0, 2, and 6. A fourth immunization was administered to approximately half of the women at month 12. All vaccine recipients had positive HPV-11 VLP-specific lymphoproliferative responses at month 3 following the second immunization (geometric mean lymphoproliferative stimulation index [SI] = 28.4; 95% confidence interval [CI] = 16.9 to 48.0) and HPV-11 VLP-specific antibody titers following the first immunization at month 1 (geometric mean antibody titer = 53.9 milli-Merck units/ml, 95% CI, 34.8 to 83.7). In contrast, lymphoproliferative and antibody titer responses were never detected in the participants who received placebo. Relatively homogeneous lymphoproliferative responses were observed in all vaccinated women. The mean lymphoproliferative SI of the vaccinated group over the first 12 months of the study was 7.6-fold greater than that of the placebo group following the initial immunization. The cellular immune responses generated by VLP immunization were both Th1 and Th2, since peripheral blood mononuclear cells from vaccinees, but not placebo recipients, secreted interleukin 2 (IL-2), IL-5, and gamma interferon (IFN-γ) in response to in vitro stimulation with HPV-11 VLP. The proliferation-based SI was moderately correlated with IFN-γ production and significantly correlated with IL-2 production after the third immunization (
P
= 0.078 and 0.002, respectively). The robust lymphoproliferative responses were specific for HPV-11, since SIs generated against bovine papillomavirus and HPV-16 VLPs were not generally observed and when detected were similar pre- and postimmunization.
Analysis of Natural Variants of the Human Immunodeficiency Virus Type 1
gag-pol
Frameshift Stem-Loop Structure
Authors: Telenti Amalio , Martinez Raquel , Munoz Miguel , Bleiber Gabriela , Greub Gilbert , Sanglard Dominique , Peters Solange
Published: July 2002

Human immunodeficiency virus type 1 uses ribosomal frameshifting for translation of the Gag-Pol polyprotein. Frameshift activities are thought to be tightly regulated. Analysis of
gag p1
sequences from 270 plasma virions identified in 64% of the samples the occurrence of polymorphism that could lead to changes in thermodynamic stability of the stem-loop. Expression in
Saccharomyces cerevisiae
of p1-β-galactosidase fusion proteins from 10 representative natural stem-loop variants and three laboratory mutant constructs (predicted the thermodynamic stability [Δ
G
°] ranging from −23.0 to −4.3 kcal/mol) identified a reduction in frameshift activity of 13 to 67% compared with constructs with the wild-type stem-loop (Δ
G
°, −23.5 kcal/mol). Viruses carrying stem-loops associated with greater than 60% reductions in frameshift activity presented profound defects in viral replication. In contrast, viruses with stem-loop structures associated with 16 to 42% reductions in frameshift efficiency displayed no significant viral replication deficit.
Rapid Progression to Simian AIDS Can Be Accompanied by Selection of CD4-Independent gp120 Variants with Impaired Ability To Bind CD4
Authors: Ryzhova Elena , Whitbeck J. Charles , Canziani Gabriela , Westmoreland Susan V. , Cohen Gary H. , Eisenberg Roselyn J. , Lackner Andrew , González-Scarano Francisco
Published: July 2002

Aspartate 368 on human immunodeficiency virus type 1 (HIV-1) gp120 forms multiple contacts with CD4; in mutagenesis studies, its replacement by asparagine and corresponding changes in simian immunodeficiency virus SIVmac (D385N) reduced binding with CD4. Nevertheless, simian immunodeficiency virus envelopes with D385N were prevalent in several studies. Extending these observations, we also found D385N to be dominant among
env
clones from two rhesus macaques that progressed rapidly to simian AIDS. These envelopes showed a CD4-independent phenotype as well as reduced affinity to CD4. Moreover, an adjacent change, G383R, which was frequently coselected with D385N, further decreased binding. An optical biosensor study demonstrated that the SIVmac239 gp120 bound to CD4 with kinetics similar to those of HIV-1. However, the gp120s with D385N and G383R showed a 40-fold reduction in affinity, with a drastic increase in dissociation rate, indicating an inherently unstable complex. This finding showed that rapid progression to simian AIDS may be accompanied by the selection of CD4-independent gp120 variants with impaired CD4 binding ability.
Bovine Leukemia Virus SU Protein Interacts with Zinc, and Mutations within Two Interacting Regions Differently Affect Viral Fusion and Infectivity In Vivo
Authors: Gatot Jean-Stéphane , Callebaut Isabelle , Van Lint Carine , Demonté Dominique , Kerkhofs Pierre , Portetelle Daniel , Burny Arsène , Willems Luc , Kettmann Richard
Published: July 2002

Bovine leukemia virus (BLV) and human T-cell lymphotropic virus type 1 (HTLV-1) belong to the genus of deltaretroviruses. Their entry into the host cell is supposed to be mediated by interactions of the extracellular (SU) envelope glycoproteins with cellular receptors. To gain insight into the mechanisms governing this process, we investigated the ability of SU proteins to interact with specific ligands. In particular, by affinity chromatography, we have shown that BLV SU protein specifically interacted with zinc ions. To identify the protein domains involved in binding, 16 peptides distributed along the sequence were tested. Two of them appeared to be able to interact with zinc. To unravel the role of these SU regions in the biology of the virus, mutations were introduced into theenvgene of a BLV molecular clone in order to modify residues potentially interacting with zinc. The fusogenic capacity of envelope mutated within the first zinc-binding region (104 to 123) was completely abolished. Furthermore, the integrity of this domain was also required for in vivo infectivity. In contrast, mutations within the second zinc-binding region (218 to 237) did not hamper the fusogenic capacity; indeed, the syncytia were even larger. In sheep, mutations in region 218 to 237 did not alter infectivity or viral spread. Finally, we demonstrated that the envelope of the related HTLV-1 was also able to bind zinc. Interestingly, zinc ions were found to be associated with the receptor-binding domain (RBD) of Friend murine leukemia virus (Fr-MLV) SU glycoprotein, further supporting their relevance in SU structure. Based on the sequence similarities shared with the Fr-MLV RBD, whose three-dimensional structure has been experimentally determined, we located the BLV zinc-binding peptide 104-123 on the opposite side of the potential receptor-binding surface. This observation supports the hypothesis that zinc ions could mediate interactions of the SU RBD either with the C-terminal part of SU, thereby contributing to the SU structural integrity, or with a partner(s) different from the receptor.
Multiple Transmembrane Amino Acid Requirements Suggest a Highly Specific Interaction between the Bovine Papillomavirus E5 Oncoprotein and the Platelet-Derived Growth Factor Beta Receptor
Authors: Nappi Valerie M. , Petti Lisa M.
Published: July 2002

The bovine papillomavirus E5 protein activates the cellular platelet-derived growth factor β receptor (PDGFβR) tyrosine kinase in a ligand-independent manner. Evidence suggests that the small transmembrane E5 protein homodimerizes and physically interacts with the transmembrane domain of the PDGFβR, thereby inducing constitutive dimerization and activation of this receptor. Amino acids in the receptor previously found to be required for the PDGFβR-E5 interaction are a transmembrane Thr513 and a juxtamembrane Lys499. Here, we sought to determine if these are the only two receptor amino acids required for an interaction with the E5 protein. Substitution of large portions of the PDGFβR transmembrane domain indicated that additional amino acids in both the amino and carboxyl halves of the receptor transmembrane domain are required for a productive interaction with the E5 protein. Indeed, individual amino acid substitutions in the receptor transmembrane domain identified roles for the extracellular proximal transmembrane residues in the interaction. These data suggest that multiple amino acids within the transmembrane domain of the PDGFβR are required for a stable interaction with the E5 protein. These may be involved in direct protein-protein contacts or may support the proper transmembrane alpha-helical conformation for optimal positioning of the primary amino acid requirements.
A Novel Herpes Simplex Virus Type 1 Transcript (AL-RNA) Antisense to the 5′ End of the Latency-Associated Transcript Produces a Protein in Infected Rabbits
Authors: Perng Guey-Chuen , Maguen Barak , Jin Ling , Mott Kevin R. , Kurylo John , BenMohamed Lbachir , Yukht Ada , Osorio Nelson , Nesburn Anthony B. , Henderson Gail , Inman Melissa , Jones Clinton , Wechsler Steven L.
Published: July 2002

Following primary ocular infection, herpes simplex virus type 1 (HSV-1) establishes a lifelong latent infection in sensory neurons of the trigeminal ganglia. Latency-associated transcript (LAT), the only known viral gene abundantly transcribed during HSV-1 neuronal latency, is required for high levels of reactivation. Recently we showed that three different mutants that do not alter the LAT promoter but contain deletions within the 5′ end of the primary LAT transcript affect viral virulence (G. C. Perng et al., J. Virol. 75:9018-9028, 2001). In contrast, in LAT-null mutants viral virulence appears unaltered (T. M. Block et al., Virology 192:618-630, 1993; D. C. Bloom et al., J. Virol. 68:1283-1292, 1994; J. M. Hill et al., Virology 174:117-125, 1990; G. C. Perng et al., J. Virol. 68:8045-8055, 1994; F. Sedarati, K. M. Izumi, E. K. Wagner, and J. G. Stevens, J. Virol. 63:4455-4458, 1989). We therefore hypothesized that the 5′ end of LAT and/or an as yet unidentified gene that overlaps part of this region is involved in viral virulence. We report here on the discovery and initial characterization of a novel HSV-1 RNA consistent with such a putative gene. The novel RNA was antisense to the 5′ end of LAT and was designated AL-RNA (anti-LAT sense RNA). The AL-RNA overlapped the core LAT promoter and the first 158 nucleotides of the 5′ end of the primary LAT transcript. AL-RNA was detected in extracts from neuron-like cells (PC-12) infected with wild-type HSV-1 but not in cells infected with a mutant with the AL region deleted. The deletions in each of the above three mutants with altered virulence encompass the 5′ end of the AL-RNA, and these mutants cannot transcribe AL. This supports the hypothesis that the AL gene may play a role in viral virulence. Based on comparison to the corresponding genomic sequence, the AL-RNA did not appear to be spliced. The AL-RNA was polyadenylated and contained an open reading frame capable of encoding a protein 56 amino acids in length with a predicted molecular mass of 6.8 kDa. Sera from three of three rabbits infected with wild-type HSV-1 but not sera from any of three rabbits infected with a mutant with the AL-RNA region deleted recognized the
Escherichia coli
recombinantly expressed AL open reading frame on Western blots. In addition, four of six rabbits infected with wild-type virus developed enzyme-linked immunosorbent assay titers against one or more AL synthetic peptides. These results suggest that an AL protein is produced in vivo.
The Lytic Cycle of Epstein-Barr Virus Is Associated with Decreased Expression of Cell Surface Major Histocompatibility Complex Class I and Class II Molecules
Authors: Keating Sinéad , Prince Stuart , Jones Matthew , Rowe Martin
Published: July 2002

Human herpesviruses utilize an impressive range of strategies to evade the immune system during their lytic replicative cycle, including reducing the expression of cell surface major histocompatibility complex (MHC) and immunostimulatory molecules required for recognition and lysis by virus-specific cytotoxic T cells. Study of possible immune evasion strategies by Epstein-Barr virus (EBV) in lytically infected cells has been hampered by the lack of an appropriate permissive culture model. Using two-color immunofluorescence staining of cell surface antigens and EBV-encoded lytic cycle antigens, we examined EBV-transformed B-cell lines in which a small subpopulation of cells had spontaneously entered the lytic cycle. Cells in the lytic cycle showed a four- to fivefold decrease in cell surface expression of MHC class I molecules relative to that in latently infected cells. Expression of MHC class II molecules, CD40, and CD54 was reduced by 40 to 50% on cells in the lytic cycle, while no decrease was observed in cell surface expression of CD19, CD80, and CD86. Downregulation of MHC class I expression was found to be an early-lytic-cycle event, since it was observed when progress through late lytic cycle was blocked by treatment with acyclovir. The immediate-early transactivator of the EBV lytic cycle, BZLF1, did not directly affect expression of MHC class I molecules. However, BZLF1 completely inhibited the upregulation of MHC class I expression mediated by the EBV cell-transforming protein, LMP1. This novel function of BZLF1 elucidates the paradox of how MHC class I expression can be downregulated when LMP1, which upregulates MHC class I expression in latent infection, remains expressed in the lytic cycle.
Physical Interaction between Envelope Glycoproteins E and M of Pseudorabies Virus and the Major Tegument Protein UL49
Authors: Fuchs Walter , Klupp Barbara G. , Granzow Harald , Hengartner Christoph , Brack Alexandra , Mundt Alice , Enquist Lynn W. , Mettenleiter Thomas C.
Published: July 2002

Envelope glycoprotein M (gM) and the complex formed by glycoproteins E (gE) and I (gI) are involved in the secondary envelopment of pseudorabies virus (PrV) particles in the cytoplasm of infected cells. In the absence of the gE-gI complex and gM, envelopment is blocked and capsids surrounded by tegument proteins accumulate in the cytoplasm (A. R. Brack, J. Dijkstra, H. Granzow, B. G. Klupp, and T. C. Mettenleiter, J. Virol.
73:
5364-5372, 1999). Here we demonstrate by yeast two-hybrid analyses that the cytoplasmic domains of gE and gM specifically interact with the C-terminal part of the UL49 gene product of PrV, which represents a major tegument protein and which is homologous to VP22 of herpes simplex virus type 1. However, deletion of the UL49 gene from PrV had only minor effects on viral replication, and ultrastructural analyses of infected cells confirmed that virus maturation and egress, including secondary envelopment in the cytoplasm, were not detectably affected by the absence of UL49. Moreover, the UL49 gene product was shown to be dispensable for virion localization of gE and gM, and mutants lacking either gE or gM incorporated the UL49 protein efficiently into virus particles. In contrast, a PrV mutant with deletions of gE-gI and gM failed to incorporate the UL49 protein despite apparently unaltered intracytoplasmic UL49 expression. In summary, we describe specific interactions between herpesvirus envelope and tegument proteins which may play a role in secondary envelopment during herpesvirus virion maturation.
Mammalian Reovirus Nonstructural Protein μNS Forms Large Inclusions and Colocalizes with Reovirus Microtubule-Associated Protein μ2 in Transfected Cells
Authors: Broering Teresa J. , Parker John S. L. , Joyce Patricia L. , Kim Jonghwa , Nibert Max L.
Published: July 2002

Cells infected with mammalian orthoreoviruses contain large cytoplasmic phase-dense inclusions believed to be the sites of viral replication and assembly, but the morphogenesis, structure, and specific functions of these “viral factories” are poorly understood. Using immunofluorescence microscopy, we found that reovirus nonstructural protein μNS expressed in transfected cells forms inclusions that resemble the globular viral factories formed in cells infected with reovirus strain type 3 Dearing from our laboratory (T3D
N
). In the transfected cells, the formation of μNS large globular perinuclear inclusions was dependent on the microtubule network, as demonstrated by the appearance of many smaller μNS globular inclusions dispersed throughout the cytoplasm after treatment with the microtubule-depolymerizing drug nocodazole. Coexpression of μNS and reovirus protein μ2 from a different strain, type 1 Lang (T1L), which forms filamentous viral factories, altered the distributions of both proteins. In cotransfected cells, the two proteins colocalized in thick filamentous structures. After nocodazole treatment, many small dispersed globular inclusions containing μNS and μ2 were seen, demonstrating that the microtubule network is required for the formation of the filamentous structures. When coexpressed, the μ2 protein from T3D
N
also colocalized with μNS, but in globular inclusions rather than filamentous structures. The morphology difference between the globular inclusions containing μNS and μ2 protein from T3D
N
and the filamentous structures containing μNS and μ2 protein from T1L in cotransfected cells mimicked the morphology difference between globular and filamentous factories in reovirus-infected cells, which is determined by the μ2-encoding M1 genome segment. We found that the first 40 amino acids of μNS are required for colocalization with μ2 but not for inclusion formation. Similarly, a fusion of μNS amino acids 1 to 41 to green fluorescent protein was sufficient for colocalization with the μ2 protein from T1L but not for inclusion formation. These observations suggest a functional difference between μNS and μNSC, a smaller form of the protein that is present in infected cells and that is missing amino acids from the amino terminus of μNS. The capacity of μNS to form inclusions and to colocalize with μ2 in transfected cells suggests a key role for μNS in forming viral factories in reovirus-infected cells.
Nef Enhances Human Immunodeficiency Virus Type 1 Infectivity and Replication Independently of Viral Coreceptor Tropism
Authors: Papkalla Armin , Münch Jan , Otto Claas , Kirchhoff Frank
Published: July 2002

We investigated the infectivities and replicative capacities of a large panel of variants of the molecular human immunodeficiency virus type 1 (HIV-1) NL4-3 clone that differ exclusively in the V3 region of the viral envelope glycoprotein and thenefgene. Our results demonstrate that Nef enhances virion infectivity and HIV-1 replication independently of the viral coreceptor tropism.
Glycoprotein I of Varicella-Zoster Virus Is Required for Viral Replication in Skin and T Cells
Authors: Moffat Jennifer , Ito Hideki , Sommer Marvin , Taylor Shannon , Arvin Ann M.
Published: July 2002

Varicella-zoster virus (VZV) glycoprotein I (gI) is dispensable in cell culture; the SCIDhu model of VZV pathogenesis was used to determine whether gI is necessary in vivo. The parental and repaired viruses grew in human skin and thymus/liver implants, but the gI deletion mutant was not infectious. Thus, gI is essential for VZV infectivity in skin and T cells.
Reactivity of [1]Benzothieno[3,2-b][1]benzothiophene - Electrophilic and Metallation Reactions
Authors: Košata Bedřich , Kozmík Václav , Svoboda Jiří
Published: July 2002

Electrophilic nitration, formylation and acetylation of the title heterocycle 1 were studied. Monosubstitution proceeds preferentially in position 2 and to a lesser extent in position 4. Lowering the reaction temperature substantially increases regioselectivity of nitration and acetylation. Substitution reactions of the 2-substituted derivatives of 1 led to the corresponding 2,7-disubstituted derivatives as major products. Formation of other regioisomers was also observed. Metallation of 1 with butyllithium preferably proceeds in position 1; subsequent reaction with N,N-dimethylformamide, carbon dioxide or iodine gives rise to corresponding 1-substituted derivatives. Long-chain 2,7-disubstitued derivatives exhibit liquid-crystalline properties.
The Reaction of Cp*ReH6, Cp* = C5Me5, with Monoborane to Yield a Novel Rhenaborane. Synthesis and Characterization of arachno-Cp*ReH3B3H8
Authors: Ghosh Sundargopal , Beatty Alicia M. , Fehlner Thomas P.
Published: July 2002

The rhenaborane Cp*ReH3B3H8, which is generated by the reaction of Cp*ReH6 with BH3·THF, has been characterized spectroscopically in solution and by a single-crystal X-ray diffraction study in the solid state as a hydrogen-rich arachno-metallatetraborane.
Overtime work, insufficient sleep, and risk of non-fatal acute myocardial infarction in Japanese men
Authors: Liu Y , Tanaka H
Published: July 2002

Objectives:To examine the relation between working hours and hours of sleep and the risk of acute myocardial infarction (AMI), with special reference to the joint effect of these two factors.

Method:Case-control study in Japan. Cases were 260 men aged 40–79 admitted to hospitals with AMI during 1996–8. Controls were 445 men free from AMI matched for age and residence who were recruited from the resident registers. Odds ratios of AMI relative to mean weekly working hours and daily hours of sleep in the past year or in the recent past were calculated.

Results:Weekly working hours were related to progressively increased odds ratios of AMI in the past year as well as in the past month, with a twofold increased risk for overtime work (weekly working hours ≥61) compared with working hours ≤40. Short time sleep (daily hours of sleep ≤5) and frequent lack of sleep (2 or more days/week with <5 hours of sleep) were also associated with a two to threefold increased risk. Frequent lack of sleep and few days off in the recent past showed greater odds ratios than those in the past year.

Conclusions:Overtime work and insufficient sleep may be related to increased risk of AMI.
Comparison of two different approaches for the analysis of data from a prospective cohort study: an application to work related risk factors for low back pain
Authors: Hoogendoorn W E , Bongers P M , de Vet H C W , Twisk J W R , van Mechelen W , Bouter L M
Published: July 2002

Aims:To compare the results of a traditional approach using standard regression for the analysis of data from a prospective cohort study with the results of generalised estimating equations (GEE) analysis.

Methods:The research was part of a three year prospective cohort study on work related risk factors for low back pain. The study population consisted of a cohort of 1192 workers with no low back pain at baseline. Information on work related physical and psychosocial factors and the occurrence of low back pain was obtained by means of questionnaires at baseline and at the three annual follow up measurements. In a traditional standard logistic regression model, physical and psychosocial risk factors at baseline were related to the cumulative incidence of low back pain during the three year follow up period. In a GEE logistic model, repeated measurements of the physical and psychosocial risk factors were related to low back pain reported at one measurement point later.

Results:The traditional standard regression model showed a significant effect of flexion and/or rotation of the upper part of the body (OR = 1.8; 95% CI: 1.2 to 3.0), but not of moving heavy loads (OR = 1.4; 95% CI: 0.7 to 3.1). The GEE model showed a significant effect of both flexion and/or rotation of the upper part of the body (OR = 2.2; 95% CI: 1.5 to 3.3) and moving heavy loads (OR = 1.5; 95% CI: 1.0 to 2.4). No significant associations with low back pain were found for the psychosocial work characteristics with either method, but the GEE model showed weaker odds ratios for these variables than the traditional standard regression model.

Conclusions:Results show that there are differences between the two analytical approaches in both the magnitude and the precision of the observed odds ratios.
The prevalence of occupational dermatitis in the UK printing industry
Authors: Livesley E J , Rushton L , English J S , Williams H C
Published: July 2002

Aims:To quantify occupational ill health resulting from dermatitis in the UK printing industry and to explore links with particular processes and activities.

Methods:Approximately 2600 members of the Graphical, Paper and Media Union living in Nottinghamshire were sent a self completion questionnaire. A sample of respondents, both those who reported current skin problems and those who did not, were invited for a short dermatological examination.

Results:The overall response rate was 62%. A total of 1189 respondents were directly involved in the printing industry and categorised according to work in pre-press (25%), printing (46%), or finishing (42%) processes. A total of 490 respondents (41%) self reported having a skin complaint at some time. Prevalence was highest in males (43%) and those working in printing (49%), in particular those who cleaned rollers and cylinders or who came into contact with substances containing isocyanates on a daily basis. The most commonly affected areas reported were the fingers and webs between the fingers. Twenty six per cent of the 490 reported a current problem on the hand. Reported symptoms included itching (61%), rash (58%), and dry skin (56%). Although certain printing industry substances were thought by respondents to aggravate their condition, constant washing and friction was most often cited. Reported use of protective equipment and cleansing products was generally high, particularly by printers. Clinical examination confirmed the high self reported prevalence and also identified a substantial proportion of mild cases which were not reported. The overall prevalence of occupationally related skin complaints is estimated to be 40%.

Conclusions:A much higher prevalence of dermatitis has been identified than from routine surveillance schemes. The use of good quality records from unions with high membership facilitated access to workers across a range of company sites and printing processes. Validation of self reported symptoms through clinical examination was shown to be essential. The importance of non-chemical causes of dermatitis was highlighted. The findings point towards the need for the development of effective and acceptable risk reduction strategies, in particular to reduce water contact and friction.
A cullin gene is induced in tomato roots forming arbuscular mycorrhizae
Authors: Tahiri-Alaoui Abdessamad , Lingua Guido , Avrova Anna , Sampò Simonetta , Fusconi Anna , Antoniw John , Berta Graziella
Published: July 2002

We have isolated a cDNA clone, Le-MI-13 (Lycopersicon esculentum mycorrhizal induced) by differential screening of a cDNA library prepared from mRNA extracted from tomato roots colonized by the arbuscular mycorrhizal (AM) fungus Glomus mosseae. The Le-MI-13 clone encodes a polypeptide that shows a high degree of amino acid sequence similarity with members of the recently identified multigene family, the cullins. Northern blot analyses demonstrated that the Le-MI-13 transcript accumulated in tomato roots forming arbuscular mycorrhizal symbiosis. Only very little Le-MI-13 transcript was detected in control roots. Tomato roots infected by the pathogenic fungus Phytophthora nicotianae var. parasitica did not accumulate Le-MI-13 transcript, indicating that upregulation of the Le-MI-13 gene is specific to roots forming arbuscular mycorrhizal symbiosis. Indirect evidence suggesting that a Le-MI-13-mediated cell-cycle-like control might operate in AM-colonized cells came from flow cytometry and static micro fluorimetry analysis. There was a strong correlation between nuclear polyploidization and AM colonization.Key words: tomato, arbuscular mycorrhizae, Phytophthora, cullins, polyploidy.
Ontogenetic niche shifts and their implications for recruitment in three clonalVacciniumshrubs:Vaccinium myrtillus,Vaccinium vitis-idaea, andVaccinium oxycoccos
Authors: Eriksson Ove
Published: July 2002

Ontogenetic niche shifts, i.e., changes in niche breadth and (or) position during the life of individuals, have received rather limited attention in plant ecology. An experiment was designed to assess ontogenetic niche shifts in three clonal Vaccinium shrubs in order to examine their recruitment behavior. Seeds were sown, and juveniles transplanted, at sites along a gradient of changing occurrence of the three species. Recruitment was seed limited, varied among sites, and was correlated with juvenile survivorship. No niche shift occurred during seed to seedling stages for any of the plants. For Vaccinium myrtillus L., recruitment was not related to adult occurrence, whereas for Vaccinium oxycoccos L., recruitment was highest at the site where adults dominated. However, for both of these species, recruitment also occurred outside the range of adult occurrence. Vaccinium vitis-idaea L. exhibited a negative relationship between adult occurrence and recruitment and between adult occurrence and juvenile survivorship, indicating an ontogenetic niche shift. For V. myrtillus the results suggested an uncoupling of niche requirements of juvenile and adult stages, whereas for V. oxycoccos the results suggested a niche contraction towards the requirements for adults. Thus, the form of ontogenetic niche shifts differs among the species. These differences in niche shifts contribute to understanding dispersal and recruitment behavior of the species.Key words: clonal plants, dispersal, niche shifts, recruitment.