bluuebunny/crossref_metadata_2025 huggingface dataset:
Spike Protein VP4 Assembly with Maturing Rotavirus Requires a Postendoplasmic Reticulum Event in Polarized Caco-2 Cells
Authors: Delmas Olivier , Durand-Schneider Anne-Marie , Cohen Jean , Colard Odile , Trugnan Germain
Published: September 2004

Rotavirus assembly is a multistep process that requires the successive association of four major structural proteins in three concentric layers. It has been assumed until now that VP4, the most external viral protein that forms the spikes of mature virions, associates with double-layer particles within the endoplasmic reticulum (ER) in conjunction with VP7 and with the help of a nonstructural protein, NSP4. VP7 and NSP4 are two glycosylated proteins. However, we recently described a strong association of VP4 with raft-type membrane microdomains, a result that makes the ER a highly questionable site for the final assembly of rotavirus, since rafts are thought to be absent from this compartment. In this study, we used tunicamycin (TM), a drug known to block the first step of protein N glycosylation, as a tool to dissect rotavirus assembly. We show that, as expected, TM blocks viral protein glycosylation and also decreases virus infectivity. In the meantime, viral particles were blocked as enveloped particles in the ER. Interestingly, TM does not prevent the targeting of VP4 to the cell surface nor its association with raft membranes, whereas the infectivity associated with the raft fractions strongly decreased. VP4 does not colocalize with the ER marker protein disulfide-isomerase even when viral particles were blocked by TM in this compartment. These results strongly support a primary role for raft membranes in rotavirus final assembly and the fact that VP4 assembly with the rest of the particle is an extrareticular event.
Structural and Virological Studies of the Stages of Virus Replication That Are Affected by Antirhinovirus Compounds
Authors: Zhang Ying , Simpson Alan A. , Ledford Rebecca M. , Bator Carol M. , Chakravarty Sugoto , Skochko Gregory A. , Demenczuk Tina M. , Watanyar Adiba , Pevear Daniel C. , Rossmann Michael G.
Published: September 2004

Pleconaril is a broad-spectrum antirhinovirus and antienterovirus compound that binds into a hydrophobic pocket within viral protein 1, stabilizing the capsid and resulting in the inhibition of cell attachment and RNA uncoating. When crystals of human rhinovirus 16 (HRV16) and HRV14 are incubated with pleconaril, drug occupancy in the binding pocket is lower than when pleconaril is introduced during assembly prior to crystallization. This effect is far more marked in HRV16 than in HRV14 and is more marked with pleconaril than with other compounds. These observations are consistent with virus yield inhibition studies and radiolabeled drug binding studies showing that the antiviral effect of pleconaril against HRV16 is greater on the infectivity of progeny virions than the parent input viruses. These data suggest that drug integration into the binding pocket during assembly, or at some other late stage in virus replication, may contribute to the antiviral activity of capsid binding compounds.
Characterization of Nef-CXCR4 Interactions Important for Apoptosis Induction
Authors: Huang Ming-Bo , Jin Ling Ling , James Cleve O. , Khan Mahfuz , Powell Michael D. , Bond Vincent C.
Published: September 2004

The HIV-1 Nef protein was analyzed for apoptotic structural motifs that interact with the CXCR4 receptor and induce apoptosis in CD4+lymphocytes. Two apoptotic motifs were identified. One centered on Nef amino acids (aa) 50 to 60, with the overlapping 20-mer peptides retaining about 82% of the activity of the full Nef protein. The second centered on aa 170 to 180, with the overlapping 20-mer peptides retaining about 30% of the activity of the full protein. Significant apoptotic abilities were observed for 11-mer motif peptides spanning aa 50 to 60 and aa 170 to 180, with a scrambled version of the 11-mer motif peptide corresponding to aa 50 to 60 showing no apoptotic ability. Hallmarks of apoptosis, such as the formation of DNA ladders and caspase activation, that were observed with the full-length protein were equally evident upon exposure of cells to these motif peptides. A CXCR4 antibody and the endogenous ligand SDF-1α were effective in blocking Nef peptide-induced apoptosis as well as the physical binding of a fluorescently tagged Nef protein, while CCR5 antibodies were ineffective. The CXCR4-negative cell line MDA-MB-468 was resistant to the apoptotic peptides and became sensitive to the apoptotic peptides upon transfection with a CXCR4-expressing vector. A fluorescently tagged motif peptide and Nef protein displayed physical binding to CXCR4-transfected MDA-MB-468 cells, but not to CCR5-transfected cells. The removal of the apoptotic motif sequences from the full-length protein completely eliminated the ability of Nef to induce apoptosis. However, these modified Nef proteins still retained the ability to enhance viral infectivity. Thus, specific sequences in the Nef protein appear to be necessary for Nef protein-induced apoptosis as well as for physical interaction with CXCR4 receptors.
Molecular Mechanisms of Attenuation of the Sabin Strain of Poliovirus Type 3
Authors: Guest Stephen , Pilipenko Evgeny , Sharma Kamal , Chumakov Konstantin , Roos Raymond P.
Published: September 2004

Mutations critical for the central nervous system (CNS) attenuation of the Sabin vaccine strains of poliovirus (PV) are located within the viral internal ribosome entry site (IRES). We examined the interaction of the IRESs of PV type 3 (PV3) and Sabin type 3 (Sabin3) with polypyrimidine tract-binding protein (PTB) and a neural cell-specific homologue, nPTB. PTB and nPTB were found to bind to a site directly adjacent to the attenuating mutation, and binding at this site was less efficient on the Sabin3 IRES than on the PV3 IRES. Translation mediated by the PV3 and Sabin3 IRESs in neurons of the chicken embryo spinal cord demonstrated a translation deficit for the Sabin3 IRES that could be rescued by increasing PTB expression in the CNS. These data suggest that the low levels of PTB available in the CNS, coupled to a reduced binding of PTB on the Sabin3 IRES, leads to its CNS-specific attenuation. This study also demonstrates the use of the chicken embryo to easily investigate translation of RNA within a neuron in the CNS of an intact living organism.
Lytic Replication-Defective Kaposi's Sarcoma-Associated Herpesvirus: Potential Role in Infection and Malignant Transformation
Authors: Deng Jian-Hong , Zhang Yan-Jin , Wang Xin-Ping , Gao Shou-Jiang
Published: September 2004

Defective viruses often have pivotal roles in virus-induced diseases. Although Kaposi's sarcoma-associated herpesvirus (KSHV) is etiologically associated with Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL), defective KSHV has not been reported. Using differential genetic screening methods, we show that defective KSHV is present in KS tumors and PEL cell lines. To investigate the role of defective viruses in KSHV-induced pathogenesis, we isolated and characterized a lytic replication-defective KSHV, KV-1, containing an 82-kb genomic deletion of solely lytic genes. Cells harboring KV-1 escaped G
0
/G
1
apoptosis induced by spontaneous lytic replication occurred in cells infected with regular KSHV but maintained efficient latent replication. Consequently, KV-1-infected cells had phenotypes of enhanced cell proliferation and transformation potentials. Importantly, KV-1 was packaged as infectious virions by using regular KSHV as helpers, and KV-1-like variants were detected in cultures of two of five KSHV cell lines and 1 of 18 KS tumors. These results point to a potential role for defective viruses in the regulation of KSHV infection and malignant transformation.
Relationships between Infectious Titer, Capsid Protein Levels, and Reverse Transcriptase Activities of Diverse Human Immunodeficiency Virus Type 1 Isolates
Authors: Marozsan Andre J. , Fraundorf Erika , Abraha Awet , Baird Heather , Moore Dawn , Troyer Ryan , Nankja Immaculate , Arts Eric J.
Published: September 2004

Most studies on human immunodeficiency virus type 1 (HIV-1) replication kinetics or fitness must rely on a particular assay to initially standardize inocula from virus stocks. The most accurate measure of infectious HIV-1 titers involves a limiting dilution-infection assay and a calculation of the dose required for 50% infectivity of susceptible cells in tissue culture (TCID
50
). Surrogate assays are now commonly used to measure the amount of p24 capsid, the endogenous reverse transcriptase (RT) activity, or the amount of viral genomic RNA in virus particles. However, a direct comparison of these surrogate assays and actual infectious HIV-1 titers from TCID
50
assays has not been performed with even the most conserved laboratory strains, let alone the highly divergent primary HIV-1 isolates of different subtypes. This study indicates that endogenous RT activity, not p24 content or viral RNA load, is the best surrogate measure of infectious HIV-1 titer in both cell-free supernatants and viruses purified on sucrose cushions. Sequence variation between HIV-1 subtypes did not appear to affect the function or activity of the RT enzyme in this endogenous assay but did affect the detection of p24 capsid by both enzyme immunoassays and Western blots. Clear groupings of non-syncytium-inducing (NSI), CCR5-tropic (R5), and SI/CXCR4-tropic (X4) HIV-1 isolates were observed when we compared the slopes derived from correlations of RT activity with infectious titers. Finally, the replication efficiency or fitness of both the NSI/R5 and SI/X4 HIV-1 isolates was not linked to the titers of the virus stocks.
Papillomavirus-Like Particles Stimulate Murine Bone Marrow-Derived Dendritic Cells To Produce Alpha Interferon and Th1 Immune Responses via MyD88
Authors: Yang Rongcun , Murillo Francisco Martinez , Cui Hengmi , Blosser Richard , Uematsu Satoshi , Takeda Kiyoshi , Akira Shizuo , Viscidi Raphael P. , Roden Richard B. S.
Published: September 2004

Dendritic cells (DCs) link innate and adaptive immunity by sensing pathogens or vaccinogens and signaling a variety of defense responses. Since human papillomavirus type 16 L1 virus-like particles (HPV16 VLPs) induce a potent, protective immune response after vaccination, we examined their recognition by DCs. HPV16 VLPs cause phenotypic maturation of murine bone marrow-derived DCs (BMDCs), and immunization of mice with HPV16 VLP-loaded BMDCs or HPV16 VLPs alone induced T helper 1 (Th1)-biased immune responses. Analysis of transcriptional responses of murine BMDCs by microarray suggested that alpha/beta interferon (IFN-α/β) transcripts and numerous proinflammatory cytokines and chemokines are up regulated in response to HPV16 VLPs. Indeed, the induction of IFN-α, IFN-γ, and interleukin-12 (IL-12) production by BMDCs after stimulation with HPV16 VLPs was demonstrated by quantitative enzyme-linked immunosorbent assay. Many microbial products that induce proinflammatory responses are recognized via Toll-like receptor (TLR) signaling through the key adaptor protein MyD88 and activation of NF-κB, nuclear factor of activated T cells (NF-AT), and activating protein 1 (AP-1). Reporter assays indicated that HPV16 VLPs activated NF-κB-, NF-AT-, and AP-1-dependent transcription in the RAW264.7 macrophage cell line. Knockdown of MyD88 transcripts by small interfering RNA in the RAW264.7 macrophage cell line inhibited the activation of NF-κB-, NF-AT- and AP-1-dependent transcription by HPV16 VLP. Furthermore, MyD88
−/−
BMDCs failed to up regulate IL-12 and IFN-α and -γ in response to HPV16 VLPs. Finally, Th1-biased immune responses to HPV16 VLPs are dramatically impaired in MyD88 and IFN-α/β receptor-deficient mice. This implicates TLR recognition as central to immune recognition of HPV16 L1 VLPs.
Interaction of the Movement Protein NSP and the
Arabidopsis
Acetyltransferase AtNSI Is Necessary for Cabbage Leaf Curl Geminivirus Infection and Pathogenicity
Authors: Carvalho Miguel F. , Lazarowitz Sondra G.
Published: September 2004

DNA viruses can modulate the activity of cellular acetyltransferases to regulate virus gene expression and to affect cell cycle progression in order to support virus replication. A role for protein acetylation in regulating the nuclear export of the bipartite geminivirus DNA genome was recently suggested by the findings that the viral movement protein NSP, which shuttles the viral genome between the nucleus and the cytoplasm, interacts with a novel
Arabidopsis
acetyltransferase, AtNSI, and the increased expression of AtNSI enhances susceptibility to
Cabbage leaf curl virus
infection. To further investigate the interaction of NSP and AtNSI and to establish the importance of this interaction in virus infections, we used a reverse yeast two-hybrid selection and deletion analysis to identify NSP mutants that were impaired in their ability to bind AtNSI. These mutants identified a 38-amino-acid region of NSP, to which no function had so far been assigned, as being necessary for NSP-AtNSI interaction. Three NSP missense mutants were analyzed in detail and were found to be comparable to wild-type NSP in their levels of accumulation, nucleocytoplasmic shuttling, DNA binding, and cooperative interaction with the viral cell-to-cell movement protein MP. Despite this,
Cabbage leaf curl virus
that expressed each mutated NSP was defective in its ability to infect
Arabidopsis
, exhibiting lower levels of infectivity than the wild-type virus, and delayed systemic spread of the virus and attenuated disease symptoms. Our data demonstrate the importance of the interaction of NSP with AtNSI for virus infection and pathogenicity.
Clonal Selection for Transcriptionally Active Viral Oncogenes during Progression to Cancer
Authors: Van Tine Brian A. , Kappes John C. , Banerjee N. Sanjib , Knops Judith , Lai Lilin , Steenbergen Renske D. M. , Meijer Chris L. J. M. , Snijders Peter J. F. , Chatis Pamela , Broker Thomas R. , Moen Phillip T. , Chow Louise T.
Published: September 2004

Primary keratinocytes immortalized by human papillomaviruses (HPVs), along with HPV-induced cervical carcinoma cell lines, are excellent models for investigating neoplastic progression to cancer. By simultaneously visualizing viral DNA and nascent viral transcripts in interphase nuclei, we demonstrated for the first time a selection for a single dominant papillomavirus transcription center or domain (PVTD) independent of integrated viral DNA copy numbers or loci. The PVTD did not associate with several known subnuclear addresses but was almost always perinucleolar. Silent copies of the viral genome were activated by growth in the DNA methylation inhibitor 5-azacytidine. HPV-immortalized keratinocytes supertransduced with HPV oncogenes and selected for marker gene coexpression underwent crisis, and the surviving cells transcribed only the newly introduced genes. Thus, transcriptional selection in response to environmental changes is a dynamic process to achieve optimal gene expression for cell survival. This phenomenon may be critical in clonal selection during carcinogenesis. Examination of HPV-associated cancers supports this hypothesis.
Identification of Proteins Associated with Murine Cytomegalovirus Virions
Authors: Kattenhorn Lisa M. , Mills Ryan , Wagner Markus , Lomsadze Alexandre , Makeev Vsevolod , Borodovsky Mark , Ploegh Hidde L. , Kessler Benedikt M.
Published: September 2004

Proteins associated with the murine cytomegalovirus (MCMV) viral particle were identified by a combined approach of proteomic and genomic methods. Purified MCMV virions were dissociated by complete denaturation and subjected to either separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and in-gel digestion or treated directly by in-solution tryptic digestion. Peptides were separated by nanoflow liquid chromatography and analyzed by tandem mass spectrometry (LC-MS/MS). The MS/MS spectra obtained were searched against a database of MCMV open reading frames (ORFs) predicted to be protein coding by an MCMV-specific version of the gene prediction algorithm GeneMarkS. We identified 38 proteins from the capsid, tegument, glycoprotein, replication, and immunomodulatory protein families, as well as 20 genes of unknown function. Observed irregularities in coding potential suggested possible sequence errors in the 3′-proximal ends of m20 and M31. These errors were experimentally confirmed by sequencing analysis. The MS data further indicated the presence of peptides derived from the unannotated ORFs ORF
c225441-226898
(m166.5) and ORF
105932-106072
. Immunoblot experiments confirmed expression of m166.5 during viral infection.
Parainfluenza Virus Type 3 Expressing the Native or Soluble Fusion (F) Protein of Respiratory Syncytial Virus (RSV) Confers Protection from RSV Infection in African Green Monkeys
Authors: Tang Roderick S. , MacPhail Mia , Schickli Jeanne H. , Kaur Jasmine , Robinson Christopher L. , Lawlor Heather A. , Guzzetta Jeanne M. , Spaete Richard R. , Haller Aurelia A.
Published: September 2004

Respiratory syncytial virus (RSV) causes respiratory disease in young children, the elderly, and immunocompromised individuals, often resulting in hospitalization and/or death. After more than 40 years of research, a Food and Drug Administration-approved vaccine for RSV is still not available. In this study, a chimeric bovine/human (b/h) parainfluenza virus type 3 (PIV3) expressing the human PIV3 (hPIV3) fusion (F) and hemagglutinin-neuraminidase (HN) proteins from an otherwise bovine PIV3 (bPIV3) genome was employed as a vector for RSV antigen expression with the aim of generating novel RSV vaccines. b/h PIV3 vaccine candidates expressing native or soluble RSV F proteins were evaluated for efficacy and immunogenicity in a nonhuman primate model. b/h PIV3 is suited for development of pediatric vaccines since bPIV3 had already been evaluated in clinical studies in 1- and 2-month-old infants and was found to be safe, immunogenic, and nontransmissible in a day care setting (Karron et al., Pediatr. Infect. Dis. J.
15:
650-654, 1996; Lee et al., J. Infect. Dis.
184:
909-913, 2001). African green monkeys immunized with b/h PIV3 expressing either the native or soluble RSV F protein were protected from challenge with wild-type RSV and produced RSV neutralizing and RSV F-protein specific immunoglobulin G serum antibodies. The PIV3-vectored RSV vaccines evaluated here further underscore the utility of this vector system for developing safe and immunogenic pediatric respiratory virus vaccines.
Cyclin-Dependent Kinase Activity Is Required at Early Times for Accurate Processing and Accumulation of the Human Cytomegalovirus UL122-123 and UL37 Immediate-Early Transcripts and at Later Times for Virus Production
Authors: Sanchez Veronica , McElroy Anita K. , Yen Judy , Tamrakar Sama , Clark Charles L. , Schwartz Rachel A. , Spector Deborah H.
Published: September 2004

Human cytomegalovirus (HCMV) infection leads to dysregulation of multiple cell cycle-regulatory proteins. In this study, we examined the effects of inhibition of cyclin-dependent kinase (cdk) activity on viral replication. With the drug Roscovitine, a specific inhibitor of cyclin-dependent kinases 1, 2, 5, 7, and 9, we have shown that during the first 6 h of infection, cyclin-dependent kinase-dependent events occurred that included the regulated processing and accumulation of the immediate-early (IE) UL122-123 transcripts and UL36-37 transcripts. Altered processing of UL122-123 led to a loss of IE1-72 and an increase in IE2-86. The ratio of spliced to unspliced UL37 transcripts also changed. These effects did not require de novo protein synthesis or degradation of proteins by the proteasome. Addition of Roscovitine at the beginning of the infection was also associated with inhibition of expression of selected viral early gene products, viral DNA replication, and late viral gene expression. When Roscovitine was added after the first 6 h of infection, the effects on IE gene expression were no longer observed and viral replication proceeded through the late phase, but viral titers were reduced. The reduction in viral titer was observed even when Roscovitine was first added at 48 h postinfection, indicating that cyclin-dependent kinase activity is required at both IE and late times. Flavopiridol, another specific inhibitor of cyclin-dependent kinases, had similar effects on IE and early gene expression. These results underscore the importance of accurate RNA processing and reiterate the significant role of cell cycle-regulatory factors in HCMV infection.
Multiple Epitopes in the Murine Cytomegalovirus Early Gene Product M84 Are Efficiently Presented in Infected Primary Macrophages and Contribute to Strong CD8
+
-T-Lymphocyte Responses and Protection following DNA Immunization
Authors: Ye Ming , Morello Christopher S. , Spector Deborah H.
Published: September 2004

We previously demonstrated that after vaccination of BALB/c mice with DNA encoding murine cytomegalovirus (MCMV) IE1 or M84, a similar level of protection against MCMV infection was achieved. However, the percentage of antigen-specific CD8
+
T cells elicited by IE1 was higher than that by M84 as measured by intracellular cytokine staining when splenocytes were stimulated with an epitope peptide (M. Ye at al., J. Virol. 76:2100-2112, 2002). We show here that after DNA vaccination with M84, a higher percentage of M84-specific CD8
+
T cells was detected when splenocytes were stimulated with J774 cells expressing full-length M84. When the defined M84 epitope 297-305 was deleted, the mutant DNA vaccine was still protective against MCMV replication and induced strong M84-specific CD8
+
-T-cell responses. The M84 gene was subsequently subcloned into three fragments encoding overlapping protein fragments. When mice were immunized with each of the M84 subfragment DNAs, at least two additional protective CD8
+
-T-cell epitopes were detected. In contrast to strong responses after DNA vaccination, M84-specific CD8
+
-T-cell responses were poorly induced during MCMV infection. The weak M84-specific response after MCMV infection was not due to poor antigen presentation in antigen-presenting cells, since both J774 macrophages and primary peritoneal macrophages infected with MCMV in vitro were able to efficiently and constitutively present M84-specific epitopes starting at the early phase of infection. These results indicate that antigen presentation by macrophages is not sufficient for M84-specific CD8
+
-T-cell responses during MCMV infection.
Antioxidant Treatment Reduces Expansion and Contraction of Antigen-Specific CD8+T Cells during Primary but Not Secondary Viral Infection
Authors: Laniewski Nathan G. , Grayson Jason M.
Published: September 2004

During many viral infections, antigen-specific CD8+T cells undergo large-scale expansion. After viral clearance, the vast majority of effector CD8+T cells undergo apoptosis. Previous studies have implicated reactive oxygen intermediates (ROI) in lymphocyte apoptosis. The purpose of the experiments presented here was to determine the role of ROI in the expansion and contraction of CD8+T cells in vivo during a physiological response such as viral infection. Mice were infected with lymphocytic choriomeningitis virus (LCMV) and treated with Mn(III)tetrakis(4-benzoic acid)porphyrin chloride (MnTBAP), a metalloporphyrin-mimetic compound with superoxide dismutase activity, from days 0 to 8 postinfection. At the peak of CD8+-T-cell response, on day 8 postinfection, the numbers of antigen-specific cells were 10-fold lower in MnTBAP-treated mice than in control mice. From days 8 to 30, a contraction phase ensued where the numbers of antigen-specific CD8+T cells declined 25-fold in vehicle-treated mice compared to a 3.5-fold decrease in MnTBAP-treated mice. Differences in contraction appeared to be due to greater proliferation in drug-treated mice. By day 38, the numbers of antigen-specific CD8+memory T cells were equivalent for the two groups. The administration of MnTBAP during secondary viral infection had no effect on the expansion of antigen-specific CD8+secondary effector T cells. These data suggest that ROI production is critical for the massive expansion and contraction of antigen-specific CD8+T cells during primary, but not secondary, viral infection.
Cytotoxic T-Lymphocyte Antigen 4 Gene and Recovery from Hepatitis B Virus Infection
Authors: Thio Chloe L. , Mosbruger Timothy L. , Kaslow Richard A. , Karp Christopher L. , Strathdee Steffanie A. , Vlahov David , O'Brien Stephen J. , Astemborski Jacquie , Thomas David L.
Published: September 2004

Cytotoxic T-lymphocyte antigen 4 (CTLA-4) is an inhibitory T-cell receptor expressed by activated and regulatory T cells. We hypothesized that single-nucleotide polymorphisms (SNPs) in the gene encoding CTLA-4 may affect the vigor of the T-cell response to hepatitis B virus (HBV) infection, thus influencing viral persistence. To test this hypothesis, we genotyped sixCTLA4SNPs, from which all frequent haplotypes can be determined, using a large, matched panel of subjects with known HBV outcomes. Haplotypes with these SNPs were constructed for each subject using PHASE software. The haplotype distribution differed between those with viral persistence and those with clearance. Two haplotypes were associated with clearance of HBV infection, which was most likely due to associations with the SNPs −1722C (odds ratio [OR] = 0.60,P= 0.06) and +49G (OR = 0.73,P= 0.02). The wild-type haplotype, which contains an SNP leading to a decreased T-cell response (+6230A), was associated with viral persistence (OR = 1.32,P= 0.04). These data suggest thatCTLA4influences recovery from HBV infection, which is consistent with the emerging role of T regulatory cells in the pathogenesis of disease.
Rapid Human Immunodeficiency Virus Decay in Highly Active Antiretroviral Therapy (HAART)-Experienced Children after Starting Mega-HAART
Authors: Plipat Nottasorn , Ruan Ping K. , Fenton Terence , Yogev Ram
Published: September 2004

Increasing numbers of patients are treated with mega-highly active antiretroviral therapy (HAART), or multiple-combination antiretroviral therapy, in an attempt to overcome the viral resistance that has contributed to treatment failure. Studies of human immunodeficiency virus (HIV) viral dynamics are used to quantify the potency of a given regimen. While mega-HAART is expected to provide potent therapy, its potency among heavily experienced HIV-infected children who have failed previous treatment is untested. HIV dynamics studies performed in children have provided minimal information on viral dynamics during mega-HAART. The present study estimates first- and second-phase viral dynamics in six children on mega-HAART, following failure of combination therapy. The first phase of viral decay was rapid, relative to rates reported in previous pediatric studies (median δ = 0.778
d
−
1
, range = 0.583 to 1.088, half-life 1 [
t
1
1/2
] = 0.894
d
), while the second phase revealed results similar to those of previous studies (median μ = 0.026
d
−
1
, range = −0.005 to 0.206,
t
2
1/2
= 9.316d). This indicates that mega-HAART can provide potent therapy among heavily experienced pediatric patients.
Diversity of Human Immunodeficiency Virus Type 1 Subtype A and CRF03_AB Protease in Eastern Europe: Selection of the V77I Variant and Its Rapid Spread in Injecting Drug User Populations
Authors: Roudinskii Nikita I. , Sukhanova Anna L. , Kazennova Elena V. , Weber Jonathan N. , Pokrovsky Vadim V. , Mikhailovich Vladimir M. , Bobkov Aleksei F.
Published: September 2004

To characterize polymorphisms of the subtype A protease in the former Soviet Union, proviral DNA samples were obtained, with informed consent, from 119 human immunodeficiency virus type 1 (HIV-1)-positive untreated injecting drug users (IDUs) from 16 regions. All individuals studied have never been treated with antiretroviral drugs. The isolates were defined as IDU-A (
n
= 115) and CRF03_AB (
n
= 4) by using
gag/env
HMA/sequencing. The
pro
region was analyzed by using sequencing and original HIV-ProteaseChip hybridization technology. The mean of pairwise nucleotide distance between 27
pro
sequences (23 IDU-A and 4 CRF03_AB) was low (1.38 ± 0.79; range, 0.00 to 3.23). All sequences contained no primary resistance mutations. However, 13 of 23 (56.5%) subtype A isolates bore the V77I substitution known as the secondary protease mutation. V77I was associated with two synonymous substitutions in triplets 31 and 78, suggesting that all V77I-bearing viruses evolved from a single source in 1997. Hybridization analysis showed that 55 of 115 (47.8%) HIV-1 isolates contained V77I, but this variant was not found in any of 31 DNA samples taken from regions, where the HIV-1 epidemic among IDUs started earlier 1997, as well as in any of four CRF03_AB isolates. The results of analysis of 12 additional samples derived from epidemiologically linked subjects showed that in all four epidemiological clusters the genotype of the donor and the recipients was the same irrespective of the route of transmission. This finding demonstrates the transmission of the V77I mutant variant, which is spreading rapidly within the circulating viral pool in Russia and Kazakhstan. The continued molecular epidemiological and virological monitoring of HIV-1 worldwide thus remains of great importance.
Evolution of Human Immunodeficiency Virus Type 1 Coreceptor Usage during Antiretroviral Therapy: a Bayesian Approach
Authors: Kitchen Christina M. R. , Philpott Sean , Burger Harold , Weiser Barbara , Anastos Kathryn , Suchard Marc A.
Published: September 2004

There is substantial evidence for ongoing replication and evolution of human immunodeficiency virus type 1 (HIV-1), even in individuals receiving highly active antiretroviral therapy. Viral evolution in the presence of antiviral therapy needs to be considered when developing new therapeutic strategies. Phylogenetic analyses of HIV-1 sequences can be used for this purpose but may give rise to misleading results if rates of intrapatient evolution differ significantly. To improve analyses of HIV-1 evolution relevant to studies of pathogenesis and treatment, we developed a Bayesian hierarchical model that incorporates all available sequence data while simultaneously allowing the phylogenetic parameters of each patient to vary. We used this method to examine evolutionary changes in HIV-1 coreceptor usage in response to treatment. We examined patients whose viral populations exhibited a shift in coreceptor utilization in response to therapy. CXCR4 (X4) strains emerged in each patient but were suppressed following initiation of new antiretroviral regimens, so that CCR5-utilizing (R5) strains predominated. By phylogenetically reconstructing the evolutionary relationship of HIV-1 obtained longitudinally from each patient, it was possible to examine the origin of the reemergent R5 virus. Using our Bayesian hierarchical approach, we found that the reemergent R5 virus detectable after therapy was more closely related to the predecessor R5 virus than to the X4 strains. The Bayesian hierarchical approach, unlike more traditional methods, makes it possible to evaluate competing hypotheses across patients. This model is not limited to analyses of HIV-1 but can be used to elucidate evolutionary processes for other organisms as well.
Human Immunodeficiency Virus Type 2 Gag Interacts Specifically with PRP4, a Serine-Threonine Kinase, and Inhibits Phosphorylation of Splicing Factor SF2
Authors: Bennett Erin M. , Lever Andrew M. L. , Allen Jane F.
Published: September 2004

Using a yeast two-hybrid screen of a T-cell cDNA library to identify cellular proteins that bind to the human immunodeficiency virus type 2 (HIV-2) Gag polyprotein, we identified PRP4, a serine-threonine protein kinase. Specific interaction of PRP4 and HIV-2 Gag was confirmed in in vitro and in vivo assays. The interacting region of HIV-2 Gag is located in the conserved matrix and capsid domains, while both the RS (arginine-serine-rich) domain and the KS (kinase) domain of PRP4 are able to bind to HIV-2 Gag. PRP4 is not incorporated into virus particles. HIV-2 Gag is able to inhibit PRP4-mediated phosphorylation of the splicing factor SF2. This is also observed with Gag from simian immunodeficiency virus, a closely related virus, but not with Gag from human T-cell lymphotropic virus type 1. Our results provide evidence for a novel interaction between Gag and a cellular protein kinase involved in the control of constitutive splicing in two closely related retroviruses. We hypothesize that as Gag accumulates in the cell, down regulation of splicing occurs through reduced phosphorylation of SF2. At late stages of infection, this interaction may replace the function of the early viral regulatory protein Rev.
Coxsackievirus B4-Induced Cytokine Production in Pancreatic Cells Is Mediated through Toll-Like Receptor 4
Authors: Triantafilou Kathy , Triantafilou Martha
Published: September 2004

Coxsackievirus B4 (CBV4), a member of the
Picornavirus
genus, has long been implicated in the development of insulin-dependent diabetes mellitus (IDDM) caused by virus-induced pancreatic cell damage. The progressive destruction of pancreatic β cells is responsible for the development of IDDM. It has recently been suggested that CBV4 infection can induce the production of proinflammatory cytokines, and these cytokines seem to be involved in the damage to the insulin-producing cells. In this study we investigated whether toll-like receptors (TLRs) are responsible for triggering the proinflammatory cytokine production in human pancreatic cells in response to CBV4. Here we demonstrate that CBV4 triggers cytokine production through a TLR4-dependent pathway. This interaction seems to be independent of virus attachment and cell entry.
Small Molecules Blocking the Entry of Severe Acute Respiratory Syndrome Coronavirus into Host Cells
Authors: Yi Ling , Li Zhengquan , Yuan Kehu , Qu Xiuxia , Chen Jian , Wang Guangwen , Zhang Hong , Luo Hongpeng , Zhu Lili , Jiang Pengfei , Chen Lirong , Shen Yan , Luo Min , Zuo Guoying , Hu Jianhe , Duan Deliang , Nie Yuchun , Shi Xuanling , Wang Wei , Han Yang , Li Taisheng , Liu Yuqing , Ding Mingxiao , Deng Hongkui , Xu Xiaojie
Published: September 2004

Severe acute respiratory syndrome coronavirus (SARS-CoV) is the pathogen of SARS, which caused a global panic in 2003. We describe here the screening of Chinese herbal medicine-based, novel small molecules that bind avidly with the surface spike protein of SARS-CoV and thus can interfere with the entry of the virus to its host cells. We achieved this by using a two-step screening method consisting of frontal affinity chromatography-mass spectrometry coupled with a viral infection assay based on a human immunodeficiency virus (HIV)-luc/SARS pseudotyped virus. Two small molecules, tetra-
O
-galloyl-β-
d
-glucose (TGG) and luteolin, were identified, whose anti-SARS-CoV activities were confirmed by using a wild-type SARS-CoV infection system. TGG exhibits prominent anti-SARS-CoV activity with a 50% effective concentration of 4.5 μM and a selective index of 240.0. The two-step screening method described here yielded several small molecules that can be used for developing new classes of anti-SARS-CoV drugs and is potentially useful for the high-throughput screening of drugs inhibiting the entry of HIV, hepatitis C virus, and other insidious viruses into their host cells.
A Lentiviral cDNA Library Employing Lambda Recombination Used To Clone an Inhibitor of Human Immunodeficiency Virus Type 1-Induced Cell Death
Authors: Kawano Yuji , Yoshida Takeshi , Hieda Kuniko , Aoki Jun , Miyoshi Hiroyuki , Koyanagi Yoshio
Published: September 2004

Expression cloning technology of cDNAs is a suitable tool for identifying novel functional properties of genes. Here, we generated a lentiviral cDNA library-expressing system for human T cells based on a site-specific recombination system of phage lambda for transferring cDNA libraries with a minimum loss of its complexity. The library-transduced CD4
+
T cells were challenged with wild-type human immunodeficiency virus type 1 (HIV-1), and the cells that acquired resistance to HIV-1-induced cytopathic effect (CPE) were selected. From these cells, CD14 was isolated and proved to inhibit the entry of HIV-1 and the HIV-1-induced CPE. This cloning system allows rapid identification of genes encoding novel properties in human T cells and probably other mammalian cells.
Enhanced Apoptosis of Peripheral CD5-Negative B Lymphocytes from Chronically Hepatitis C Virus-Infected Patients: Reversal after Antiviral Treatment
Authors: Toubi Elias , Kessel Aharon , Peri Regina , Shmuel Zehava , Bamberger Ellen , Sabo Edmond , Zuckerman Eli
Published: September 2004

Whereas enhanced peripheral T-cell apoptosis and its association with autoimmunity have recently been reported, the apoptotic status of peripheral B cells in chronic hepatitis C virus (HCV) infection remains ambiguous. We therefore sought to investigate the sensitivity of peripheral B cells to apoptosis and to assess the possible benefits of antiviral treatment in mitigating these effects. Spontaneous apoptosis, the extent of apoptosis rescue, and NF-κB expression in peripheral B cells were studied in patients with chronic HCV infections (group 1), in sustained responders after antiviral treatment (group 2), and in healthy controls (group 3). For group 1, spontaneous B-cell apoptosis was increased (26% ± 4.6%) and apoptosis rescue was altered (39%) compared to group 3 (18% ± 5% and 50%, respectively;
P
= 0.001). In contrast, apoptosis and apoptosis rescue were similar for groups 2 and 3. Enhanced B-cell apoptosis was associated with decreased NF-κB expression and was found only in CD5-negative (CD5
neg
) B cells, whereas CD5
pos
cells were apoptosis resistant. Chronic HCV infection is associated with enhanced peripheral B-cell apoptosis and decreased apoptosis rescue. Successful antiviral treatment reverses these abnormalities to the levels seen in healthy individuals. The relative resistance of the CD5
pos
B-cell subpopulation to apoptosis may play a role in HCV-related autoimmunity and lymphoproliferation.
Targeting c-Mpl for Revival of Human Immunodeficiency Virus Type 1-Induced Hematopoietic Inhibition When CD34+Progenitor Cells Are Re-Engrafted into a Fresh Stromal Microenvironment In Vivo
Authors: Koka Prasad S. , Kitchen Christina M. R. , Reddy Srinivasa T.
Published: September 2004

The inhibition of multilineage hematopoiesis which occurs in the severe combined immunodeficiency mouse with transplanted human fetal thymus and liver tissues (SCID-hu Thy/Liv) due to human immunodeficiency virus type 1 (HIV-1) infection is also accompanied by a severe loss of c-Mpl expression on these progenitor cells. Inhibition of colony-forming activity (CFA) of the CD34+progenitor cells is partially revived to about 40% of mock-infected Thy/Liv implants, following reconstitution of the CD34+cells that were exposed to HIV-1 infection, in a new Thy/Liv stromal microenvironment of irradiated secondary SCID-hu recipients at 3 weeks post-re-engraftment. In addition, in these reconstituted animals, the proportion of c-Mpl+CD34+cells relative to c-Mpl−CD34+cells increased by about 25%, to 35% of mock-infected implants, suggesting a reacquirement of c-Mpl phenotype by the c-Mpl−CD34+cells. These results suggest a correlation between c-Mpl expression and multilineage CFA of the human CD34+progenitor cells that have experienced the effects of HIV-1 infection. Treatment of the secondary-recipient animals with the c-Mpl ligand, thrombopoietin (Tpo), further increased c-Mpl expression and CFA of re-engrafted CD34+cells previously exposed to virus in the primary implants to about 50 to 70% over that of those re-engrafted CD34+cells derived from implants of untreated animals. Blocking of c-Mpl with anti-c-Mpl monoclonal antibody in vivo by injecting the SCID-hu animals resulted in the reduction or loss of CFA. Thus, inhibition, absence, or loss of c-Mpl expression as in the c-Mpl−CD34+subset of cells is the likely cause of CFA inhibition. Further, CFA of the CD34+cells segregates with their c-Mpl expression. Therefore, c-Mpl may play a role in hematopoietic inhibition during HIV-1 infection, and control of its expression levels may aid in hematopoietic recovery and thereby reduce the incidence of cytopenias occurring in infected individuals.
Visualization by Live-Cell Microscopy of Disruption of ND10 during Herpes Simplex Virus Type 1 Infection
Authors: Everett Roger D. , Zafiropoulos Alexandros
Published: September 2004

ND10 structures are disrupted during herpes simplex virus type 1 (HSV-1) infection by viral regulatory protein ICP0. The significance of this effect remains controversial, partly because of a report that high-level expression of the major ND10 promyelocytic leukemia (PML) protein precludes ND10 disruption yet does not inhibit HSV-1 infection. Here we demonstrate dramatic reorganization of ND10 during HSV-1 infection by live-cell microscopy, even in the presence of overexpressed PML.
Thogoto Virus Lacking Interferon-Antagonistic Protein ML Is Strongly Attenuated in Newborn
Mx1
-Positive but Not
Mx1
-Negative Mice
Authors: Pichlmair Andreas , Buse Johanna , Jennings Stephanie , Haller Otto , Kochs Georg , Staeheli Peter
Published: September 2004

The Thogoto virus ML protein suppresses interferon synthesis in infected cells. Nevertheless, a virus mutant lacking ML remained highly pathogenic in standard laboratory mice. It was strongly attenuated, however, in mice carrying the interferon-responsive
Mx1
gene found in wild mice, demonstrating that enhanced interferon synthesis is protective only if appropriate antiviral effector molecules are present. Our study shows that the virulence-enhancing effects of some viral interferon antagonists may escape detection in conventional animal models.
Modulation by Morphine of Viral Set Point in Rhesus Macaques Infected with Simian Immunodeficiency Virus and Simian-Human Immunodeficiency Virus
Authors: Kumar Rakesh , Torres Cynthia , Yamamura Yasuhiro , Rodriguez Idia , Martinez Melween , Staprans Silvija , Donahoe Robert M. , Kraiselburd Edmundo , Stephens Edward B. , Kumar Anil
Published: September 2004

Six rhesus macaques were adapted to morphine dependence by injecting three doses of morphine (5 mg/kg of body weight) for a total of 20 weeks. These animals along with six control macaques were infected intravenously with mixture of simian-human immunodeficiency virus KU-1B (SHIV
KU-1B
), SHIV
89.6P
, and simian immunodeficiency virus 17E-Fr. Levels of circulating CD4
+
T cells and viral loads in the plasma and the cerebrospinal fluid were monitored in these macaques for a period of 12 weeks. Both morphine and control groups showed precipitous loss of CD4
+
T cells. However this loss was more prominent in the morphine group at week 2 (
P
= 0.04). Again both morphine and control groups showed comparable peak plasma viral load at week 2, but the viral set points were higher in the morphine group than that in the control group. Likewise, the extent of virus replication in the cerebral compartment was more pronounced in the morphine group. These results provide a definitive evidence for a positive correlation between morphine and levels of viral replication.
Efficient Replication of Severe Acute Respiratory Syndrome Coronavirus in Mouse Cells Is Limited by Murine Angiotensin-Converting Enzyme 2
Authors: Li Wenhui , Greenough Thomas C. , Moore Michael J. , Vasilieva Natalya , Somasundaran Mohan , Sullivan John L. , Farzan Michael , Choe Hyeryun
Published: September 2004

Replication of viruses in species other than their natural hosts is frequently limited by entry and postentry barriers. The coronavirus that causes severe acute respiratory syndrome (SARS-CoV) utilizes the receptor angiotensin-converting enzyme 2 (ACE2) to infect cells. Here we compare human, mouse, and rat ACE2 molecules for their ability to serve as receptors for SARS-CoV. We found that, compared to human ACE2, murine ACE2 less efficiently bound the S1 domain of SARS-CoV and supported less-efficient S protein-mediated infection. Rat ACE2 was even less efficient, at near background levels for both activities. Murine 3T3 cells expressing human ACE2 supported SARS-CoV replication, whereas replication was less than 10% as efficient in the same cells expressing murine ACE2. These data imply that a mouse transgenically expressing human ACE2 may be a useful animal model of SARS.
Expression of thyroid transcription factor 1 in primary brain tumours
Authors: Zamecnik J , Chanova M , Kodet R
Published: September 2004

Background:Thyroid transcription factor 1 (TTF-1) is expressed in a proportion of carcinomas derived from follicular thyroid cells and respiratory epithelium. Immunohistochemical detection of this protein was shown previously to be a helpful aid in tumour diagnosis, specifically in deciding whether a tumour is primary to the lung/thyroid gland or metastatic. Recently, TTF-1 expression was also observed in certain areas of postnatal brain.

Aim/Method:To investigate the expression of TTF-1 protein in a spectrum of 73 primary brain tumours including astrocytomas, glioblastomas, ependymomas, oligodendrogliomas, medulloblastomas, and gangliogliomas of different sites.

Results:All the tumours were negative for TTF-1 except for two ependymomas of the third ventricle.

Conclusions:The expression of TTF-1 in brain tumours appears to be site specific rather than associated with tumour dedifferentiation. The presented expression of TTF-1 protein in certain primary brain tumours should be taken into consideration when interpreting the immunohistochemical staining of brain tumours of uncertain primary site.
Endometrial coccidiosis
Authors: de Otazu R D , García-Nieto L , Izaguirre-Gondra E , Mayayo E , Ciani S , Nogales F F
Published: September 2004

This report describes a case of granulomatous endometritis caused by coccidiosis in an immunologically uncompromised 63 year old patient. The glandular epithelium of the endometrium contained numerous intracytoplasmic cysts, corresponding to periodic acid Schiff positive and methenamine silver negative sporoblasts. The endometrial glands revealed reactive phenomena, such as eosinophilic and squamous glandular metaplasia and intraluminal desquamation. Non-necrotising epithelioid granulomata, lacking the presence of parasites, were present in the stroma. Although not detected in the stool examination, the organisms were probablyIsospora belli. There was no evidence of other foci of the disease. Coccidiosis should be differentiated from the more commonly occurring coccidiomycosis.
The prognostic value of progesterone receptor status in meningiomas
Authors: Roser F , Nakamura M , Bellinzona M , Rosahl S K , Ostertag H , Samii M
Published: September 2004

Background:Meningiomas are known to recur frequently, and their longterm management remains controversial. Previous studies indicate that progesterone and its receptors can play a role in the recurrence of meningiomas, but the correlation between the presence of these receptors and patients’ outcome is unclear.

Aim:To conduct a retrospective analysis to investigate the prognostic relevance of progesterone receptor (PR) expression in meningiomas.

Methods:Five hundred and eighty eight meningiomas operated on over a period of 10 years were examined immunohistochemically to determine the PR status using monoclonal antibodies. Several factors including recurrence (mean follow up of 65 month), sex, tumour tissue consistency, location, vascularity, and en plaque appearance were analysed.

Results:PR status showed comparable values for men and women. World Health Organisation (WHO) grade II and III tumours had significantly fewer receptors than benign meningiomas. There was no significant correlation between PR status and recurrence rates in WHO grade I totally removed meningiomas. However, a combination of PR status and proliferation indices was shown to predict recurrence reliably.

Conclusions:Together with routine histological evaluation, PR status can help to describe the biological behaviour of meningiomas. Only a combination of clinical and biological features can describe the behaviour of meningiomas, predict their recurrence, and help to devise more effective follow up strategies.