bluuebunny/crossref_metadata_2025_split huggingface dataset:
Human Immunodeficiency Virus Type 1 Infection of Human Brain-Derived Progenitor Cells
Authors: Lawrence Diane M. P. , Durham Linda C. , Schwartz Lynnae , Seth Pankaj , Maric Dragan , Major Eugene O. 
Published: June 2004

ABSTRACTAlthough cells of monocytic lineage are the primary source of human immunodeficiency virus type 1 (HIV-1) in the brain, other cell types in the central nervous system, including astrocytes, can harbor a latent or persistent HIV-1 infection. In the present study, we examined whether immature, multipotential human brain-derived progenitor cells (nestin positive) are also permissive for infection. When exposed to IIIB and NL4-3 strains of HIV-1, progenitor cells and progenitor-derived astrocytes became infected, with peak p24 levels of 100 to 500 pg/ml at 3 to 6 days postinfection. After 10 days, virus production was undetectable but could be stimulated by the addition of tumor necrosis factor alpha (TNF-α). To bypass limitations to receptor entry, we compared the fate of infection in these cell populations by transfection with the infectious HIV-1 clone, pNL4-3. Again, transfected progenitors and astrocytes produced virus for 7 days but diminished to low levels beyond 8 days posttransfection. During the nonproductive phase, TNF-α stimulated virus production from progenitors as late as 5 weeks posttransfection. Astrocytes produced 5- to 20-fold more infectious virus (27 ng of p24/106cells) than progenitors at the peak of 3 days posttransfection. Differentiation of infected progenitors toward an astrocyte phenotype increased virus production to levels consistent with infected astrocytes, suggesting a phenotypic difference in viral replication. Using this cell culture system of multipotential human brain-derived progenitor cells, we provide evidence that progenitor cells may be a reservoir for HIV-1 in the brains of AIDS patients.
Sensitivity of NS3 Serine Proteases from Hepatitis C Virus Genotypes 2 and 3 to the Inhibitor BILN 2061
Authors: Thibeault Diane , Bousquet Christiane , Gingras Rock , Lagacé Lisette , Maurice Roger , White Peter W. , Lamarre Daniel 
Published: June 2004

ABSTRACTHepatitis C virus (HCV) displays a high degree of genetic variability. Six genotypes and more than 50 subtypes have been identified to date. In this report, kinetic profiles were determined for NS3 proteases of genotypes 1a, 1b, 2ac, 2b, and 3a, revealing no major differences in activity. In vitro sensitivity studies with BILN 2061 showed a decrease in affinity for proteases of genotypes 2 and 3 (Ki, 80 to 90 nM) compared to genotype 1 enzymes (Ki, 1.5 nM). To understand the reduced sensitivity of genotypes 2 and 3 to BILN 2061, active-site residues in the proximity of the inhibitor binding site were replaced in the genotype-1b enzyme with the corresponding genotype-2b or -3a residues. The replacement of five residues at positions 78, 79, 80, 122, and 132 accounted for most of the reduced sensitivity of genotype 2b, while replacement of residue 168 alone could account for the reduced sensitivity of genotype 3a. BILN 2061 remains a potent inhibitor of these non-genotype-1 NS3-NS4A proteins, withKivalues below 100 nM. This in vitro potency, in conjunction with the good pharmacokinetic data reported for humans, suggests that there is potential for BILN 2061 as an antiviral agent for individuals infected with non-genotype-1 HCV.
Elevated Expression of GM3 in Receptor-Bearing Targets Confers Resistance to Human Immunodeficiency Virus Type 1 Fusion
Authors: Rawat Satinder S. , Gallo Stephen A. , Eaton Julie , Martin Thomas D. , Ablan Sherimay , KewalRamani Vineet N. , Wang Ji Ming , Blumenthal Robert , Puri Anu 
Published: June 2004

ABSTRACT

GM3, a major ganglioside of T lymphocytes, promotes human immunodeficiency virus type 1 (HIV-1) entry via interactions with HIV-1 receptors and the viral envelope glycoprotein (Env). Increased GM3 levels in T lymphocytes and the appearance of anti-GM3 antibodies in AIDS patients have been reported earlier. In this study, we investigated the effect of GM3 regulation on HIV-1 entry by utilizing a mouse cell line (B16F10), which expresses exceptionally high levels of GM3. Strikingly, B16 cells bearing CD4, CXCR4, and/or CCR5 were highly resistant to CD4-dependent HIV-1 Env-mediated membrane fusion. In contrast, these targets supported membrane fusion mediated by CD4-requiring HIV-2, SIV, and CD4-independent HIV-1 Envs. Coreceptor function was not impaired by GM3 overexpression as indicated by Ca
2+
fluxes mediated by the CXCR4 ligand SDF-1α and the CCR5 ligand MIP-1β. Reduction in GM3 levels of B16 target cells resulted in a significant recovery of CD4-dependent HIV-1 Env-mediated fusion. We propose that GM3 in the plasma membrane blocks HIV-1 Env-mediated fusion by interfering with the lateral association of HIV-1 receptors. Our findings offer a novel mechanism of interplay between membrane lipids and receptors by which host cells may escape viral infections.
The N-Terminal 85 Amino Acids of the
Barley Stripe Mosaic Virus
γb Pathogenesis Protein Contain Three Zinc-Binding Motifs
Authors: Bragg Jennifer N. , Lawrence Diane M. , Jackson Andrew O. 
Published: June 2004

ABSTRACT

Barley stripe mosaic virus
RNAγ encodes γb, a cysteine-rich protein that affects pathogenesis. Nine of the eleven cysteines are concentrated in two clusters, designated C1 (residues 1 to 23) and C2 (residues 60 to 85), that are arranged in zinc finger-like motifs. A basic motif (BM) rich in lysine and arginine (residues 19 to 47) resides between the C1 and C2 clusters. We have demonstrated that γb binds zinc and that the C1, BM, and C2 motifs have independent zinc-binding activities. To evaluate the requirements for binding, mutations were introduced into each region. Cysteine residues at positions 7, 9, 10, 19, and 23 in the C1 motif were replaced with serines. In the BM, asparagines were substituted for lysines at positions 26 and 35, glutamine for arginine at position 25, and glycines for arginines at positions 33 and 36. The C2 mutations included cysteine replacements with serines at positions 60, 64, 71, and 81, and a histidine-to-leucine change at position 85. These mutations destroyed zinc-binding activity in each of the isolated motifs. γb derivatives containing mutations in only two of the motifs retained the ability to bind zinc, whereas a γb derivative containing mutations inactivating all three motifs destroyed the ability to bind zinc. Plants inoculated with transcripts containing combinations of the C1, BM, and C2 mutations elicited a “null” phenotype in barley characteristic of γb deletion mutants and also delayed the appearance and reduced the size of local lesions in
Chenopodium amaranticolor
. These results show that zinc binding of each of the motifs is critical for the biological activity of γb.
Human Immunodeficiency Virus Type 1-Specific Immune Responses in Primates upon Sequential Immunization with Adenoviral Vaccine Carriers of Human and Simian Serotypes
Authors: Reyes-Sandoval A. , Fitzgerald J. C. , Grant R. , Roy S. , Xiang Z. Q. , Li Y. , Gao G. P. , Wilson J. M. , Ertl H. C. J. 
Published: June 2004

ABSTRACT

Two triple immunization vaccine regimens with adenoviral vectors with E1 deleted expressing Gag of human immunodeficiency virus type 1 were tested for induction of T- and B-cell-mediated-immune responses in mice and in nonhuman primates. The vaccine carriers were derived from distinct serotypes of human and simian adenoviruses that fail to elicit cross-neutralizing antibodies expected to dampen the effect of booster immunizations. Both triple immunization regimens induced unprecedented frequencies of gamma interferon-producing CD8
+
T cells to Gag in mice and monkeys that remained remarkably stable over time. In addition, monkeys developed Gag-specific interleukin-2-secreting T cells, presumably belonging to the CD4
+
T-cell subset, and antibodies to both Gag and the adenoviral vaccine carriers.
Insertion of Green Fluorescent Protein into Nonstructural Protein 5A Allows Direct Visualization of Functional Hepatitis C Virus Replication Complexes
Authors: Moradpour Darius , Evans Matthew J. , Gosert Rainer , Yuan Zhenghong , Blum Hubert E. , Goff Stephen P. , Lindenbach Brett D. , Rice Charles M. 
Published: June 2004

ABSTRACT

Hepatitis C virus (HCV) replicates its genome in a membrane-associated replication complex, composed of viral proteins, replicating RNA and altered cellular membranes. We describe here HCV replicons that allow the direct visualization of functional HCV replication complexes. Viable replicons selected from a library of Tn
7
-mediated random insertions in the coding sequence of nonstructural protein 5A (NS5A) allowed the identification of two sites near the NS5A C terminus that tolerated insertion of heterologous sequences. Replicons encoding green fluorescent protein (GFP) at these locations were only moderately impaired for HCV RNA replication. Expression of the NS5A-GFP fusion protein could be demonstrated by immunoblot, indicating that the GFP was retained during RNA replication and did not interfere with HCV polyprotein processing. More importantly, expression levels were robust enough to allow direct visualization of the fusion protein by fluorescence microscopy. NS5A-GFP appeared as brightly fluorescing dot-like structures in the cytoplasm. By confocal laser scanning microscopy, NS5A-GFP colocalized with other HCV nonstructural proteins and nascent viral RNA, indicating that the dot-like structures, identified as membranous webs by electron microscopy, represent functional HCV replication complexes. These findings reveal an unexpected flexibility of the C-terminal domain of NS5A and provide tools for studying the formation and turnover of HCV replication complexes in living cells.
Antiviral Drug Discovery Strategy Using Combinatorial Libraries of Structurally Constrained Peptides
Authors: Real Eléonore , Rain Jean-Christophe , Battaglia Véronique , Jallet Corinne , Perrin Pierre , Tordo Noël , Chrisment Peggy , D'Alayer Jacques , Legrain Pierre , Jacob Yves 
Published: June 2004

ABSTRACT
We have developed a new strategy for antiviral peptide discovery by using lyssaviruses (rabies virus and rabies-related viruses) as models. Based on the mimicry of natural bioactive peptides, two genetically encoded combinatorial peptide libraries composed of intrinsically constrained peptides (coactamers) were designed. Proteomic knowledge concerning the functional network of interactions in the lyssavirus transcription-replication complex highlights the phosphoprotein (P) as a prime target for inhibitors of viral replication. We present an integrated, sequential drug discovery process for selection of peptides with antiviral activity directed against the P. Our approach combines (i) an exhaustive two-hybrid selection of peptides binding two phylogenetically divergent lyssavirus P's, (ii) a functional analysis of protein interaction inhibition in a viral reverse genetic assay, coupled with a physical analysis of viral nucleoprotein-P complex by protein chip mass spectrometry, and (iii) an assay for inhibition of lyssavirus infection in mammalian cells. The validity of this strategy was demonstrated by the identification of four peptides exhibiting an efficient antiviral activity. Our work highlights the importance of P as a target in anti-rabies virus drug discovery. Furthermore, the screening strategy and the coactamer libraries presented in this report could be considered, respectively, a general target validation strategy and a potential source of biologically active peptides which could also help to design pharmacologically active peptide-mimicking molecules. The strategy described here is easily applicable to other pathogens.
Exchanging the Yellow Fever Virus Envelope Proteins with Modoc Virus prM and E Proteins Results in a Chimeric Virus That Is Neuroinvasive in SCID Mice
Authors: Charlier Nathalie , Molenkamp Richard , Leyssen Pieter , Paeshuyse Jan , Drosten Christian , Panning Marcus , De Clercq Erik , Bredenbeek Peter J. , Neyts Johan 
Published: June 2004

ABSTRACTA chimeric flavivirus infectious cDNA was constructed by exchanging the premembrane (prM) and envelope (E) genes of the yellow fever virus vaccine strain 17D (YF17D) with the corresponding genes of Modoc virus (MOD). This latter virus belongs to the cluster of the “not-known vector” flaviviruses and is, unlike YF17D, neuroinvasive in SCID mice. Replication of in vitro-transcribed RNA from this chimeric flavivirus was shown by [3H]uridine labeling and RNA analysis. Expression of the MOD prM and E proteins was monitored by radioimmunoprecipitation and revealed that the MOD proteins were correctly and efficiently produced from the chimeric precursor protein. The MOD E protein was shown to be N-linked glycosylated, whereas prM, as predicted from the genome sequence, did not contain N-linked carbohydrates. In Vero cells, the chimeric virus replicated with a similar efficiency as the parental viruses, although it formed smaller plaques than YF17D and MOD. In SCID mice that had been infected intraperitoneally with the chimeric virus, the viral load increased steadily until death. The MOD/YF virus, like MOD from which it had acquired the prM and E structural proteins, but unlike YF, proved neuroinvasive in SCID mice. Animals developed neurological symptoms about 15 days after inoculation and died shortly thereafter. The distribution of MOD/YF RNA in the brain of infected mice was similar to that observed in MOD-infected mice. The observations provide compelling evidence that the determinants of neuroinvasiveness of flaviviruses are entirely located in the envelope proteins prM and E.
Structure of D-63 from Sulfolobus Spindle-Shaped Virus 1: Surface Properties of the Dimeric Four-Helix Bundle Suggest an Adaptor Protein Function
Authors: Kraft Paul , Kümmel Daniel , Oeckinghaus Andrea , Gauss George H. , Wiedenheft Blake , Young Mark , Lawrence C. Martin 
Published: June 2004

ABSTRACT
Sulfolobus spindle-shaped virus 1 (SSV1) and its fusellovirus homologues can be found in many acidic (pH ≤ 4.0) hot springs (≥70°C) around the world. SSV1 contains a 15.5-kb double-stranded DNA genome that encodes 34 proteins with greater than 50 amino acids. A site-specific integrase and a DnaA-like protein have been previously identified by sequence homology, and three structural proteins have been isolated from purified virus and identified by N-terminal sequencing (VP1, VP2, and VP3). The functions of the remaining 29 proteins are currently unknown. To assign functions to these proteins, we have initiated biochemical and structural studies on the SSV1 proteome. Here we report the structure of SSV1 D-63. The structure reveals a helix-turn-helix motif that dimerizes to form an antiparallel four-helix bundle. Mapping residues conserved among three fusellovirus isolates onto the structure shows that one face of the rod-shaped molecule is highly conserved. This conserved surface spans the dimer axis and thus exhibits 2-fold symmetry. Two smaller conserved patches, also related by 2-fold symmetry, are found on the opposite face of the molecule. All of these conserved surfaces are devoid of clefts or pockets typically used to bind small molecules, suggesting that D-63 may function as an adaptor protein in macromolecular assembly.
Characterization of the Amino Acid Residues of Sendai Virus C Protein That Are Critically Involved in Its Interferon Antagonism and RNA Synthesis Down-Regulation
Authors: Kato Atsushi , Cortese-Grogan Case , Moyer Sue A. , Sugahara Fumihiro , Sakaguchi Takemasa , Kubota Toru , Otsuki Noriyuki , Kohase Masayoshi , Tashiro Masato , Nagai Yoshiyuki 
Published: June 2004

ABSTRACT

Sendai virus (SeV) encodes two accessory proteins, V and C, in the alternative reading frames in the P gene that are accessed transcriptionally (V) or translationally (C). The C protein is expressed as a nested set of four C-coterminal proteins, C′, C, Y1, and Y2, that use different initiation codons. Using HeLa cell lines constitutively expressing the various C proteins, we previously found that the smallest (the 175-residue Y2) of the four C proteins was fully capable of counteracting the antiviral action of interferons (IFNs) and inhibiting viral RNA synthesis and that the C-terminal half of 106 residues was sufficient for both of these inhibitory functions (A. Kato et al., J. Virol.
75:
3802-3810, 2001, and A. Kato et al., J. Virol.
76:
7114-7124, 2002). Here, we further generated HeLa cell lines expressing the mutated C (Cm) proteins with charged amino acids substituted for alanine residues at either positions 77 and 80; 114 and 115; 139 and 142; 151, 153, and 154; 156; or 173, 175, and 176. We found that only the mutations at positions 151, 153, and 154 abolished IFN antagonism. All the Cm proteins lost the ability to bind with STAT1 under our assay conditions, regardless of their ability to inhibit IFN signaling. On the other hand, the Cm proteins that altered the tyrosine phosphorylation and dephosphorylation of STAT1 and STAT2 always retained IFN antagonism. Thus, the abnormality of phosphorylation or dephosphorylation appeared to be a cause of the IFN antagonism by SeV C. Regarding viral RNA synthesis inhibition, all mutants but the mutant with replacements at positions 114 and 115 greatly reduced the inhibitory activity, indicating that anti-RNA synthesis by the C protein is governed by amino acids scattered across its C-terminal half. Thus, amino acid sequence requirements differ greatly between IFN antagonism and RNA synthesis inhibition. In addition, we confirmed that another SeV accessory protein, V, does not antagonize IFN.
Short Interfering RNA Accumulation Correlates with Host Recovery in DNA Virus-Infected Hosts, and Gene Silencing Targets Specific Viral Sequences
Authors: Chellappan Padmanabhan , Vanitharani Ramachandran , Fauquet Claude M. 
Published: June 2004

ABSTRACT

Viruses are both inducers and targets of posttranscriptional gene silencing (PTGS), a natural defense mechanism in plants. Here we report molecular evidence of the ability of single-stranded DNA (ssDNA) viruses to induce PTGS in infected plants irrespective of the severity of or recovery from the symptoms. Our results reveal that five distinct species of cassava-infecting geminiviruses were capable of triggering PTGS by producing two classes of virus-specific short interfering RNAs (siRNAs) of 21 to 26 nucleotides in two plant hosts, tobacco (
Nicotiana benthamiana
) and cassava (
Manihot esculenta
, Crantz). However, the efficacy of virus-induced PTGS varied depending on the intrinsic features of the virus and its interaction with the plant host. We found that symptom recovery over time in plants infected with the isolates of
African cassava mosaic virus
(ACMV-[CM]) or
Sri Lankan cassava mosaic virus
was associated with a much higher level of virus-derived siRNA accumulation compared to plants infected with viruses that do not show symptom recovery. Furthermore, we determined that the C terminus of
AC1
that overlaps with the N terminus of
AC2
early viral genes involved in virus replication were the primary targets for ACMV-[CM]-induced PTGS, whereas the C terminus of BC1 was targeted for the
East African cassava mosaic Cameroon virus
. In addition, our results reveal the possibility for double-stranded RNA formation during transcription in ssDNA viruses, which explains in part how these viruses can trigger PTGS in plants.
Gene Profiling of Cottontail Rabbit Papillomavirus-Induced Carcinomas Identifies Upregulated Genes Directly Involved in Stroma Invasion as Shown by Small Interfering RNA-Mediated Gene Silencing
Authors: Huber Evamaria , Vlasny Daniela , Jeckel Sonja , Stubenrauch Frank , Iftner Thomas 
Published: June 2004

ABSTRACT
To investigate changes in cellular gene expression associated with malignant progression, we identified differentially expressed genes in a cottontail rabbit papillomavirus (CRPV) squamous carcinoma model employing New Zealand White rabbits. The technique of suppression subtractive cDNA hybridization was applied to pairs of mRNA isolates from CRPV-induced benign papillomas and carcinomas, with each pair derived from the same individual rabbit. The differential expression of 23 subtracted cDNAs was further confirmed by quantitative reverse transcription-PCR (RT-PCR) with additional biopsies. Eight papilloma-carcinoma pairs examined showed a constant upregulation of the transcripts for the multifunctional adaptor protein 14-3-3 ζ and the Y-box binding transcription factor YB-1, whereas transcripts for m-type calpain 2 and NB thymosin β, which are involved in cell motility and tissue invasion, as well as casein kinase 1 α, chaperonin, and annexin I, were found to be upregulated in the majority of the cases. RNA-RNA in situ hybridization and laser capture microdissection in combination with quantitative RT-PCR analysis verified the deregulated expression of the transcripts in the tumor cells. In contrast, CRPV E7 transcript levels remained rather constant indicating no requirement for a further upregulation of E7 expression following tumor induction. Small interfering RNA-mediated interference with expression of genes encoding YB-1, m-type calpain 2, or NB thymosin β in a CRPV-positive cell line established from New Zealand White rabbit keratinocytes resulted in decreased cell invasion in matrigel chamber assays.
Heterologous Envelope Immunogens Contribute to AIDS Vaccine Protection in Rhesus Monkeys
Authors: Letvin Norman L. , Huang Yue , Chakrabarti Bimal K. , Xu Ling , Seaman Michael S. , Beaudry Kristin , Korioth-Schmitz Birgit , Yu Faye , Rohne Daniela , Martin Kristi L. , Miura Ayako , Kong Wing-Pui , Yang Zhi-Yong , Gelman Rebecca S. , Golubeva Olga G. , Montefiori David C. , Mascola John R. , Nabel Gary J. 
Published: June 2004

ABSTRACT

Because a strategy to elicit broadly neutralizing anti-human immunodeficiency virus type 1 (HIV-1) antibodies has not yet been found, the role of an Env immunogen in HIV-1 vaccine candidates remains undefined. We sought to determine whether an HIV-1 Env immunogen genetically disparate from the Env of the challenge virus can contribute to protective immunity. We vaccinated Indian-origin rhesus monkeys with Gag-Pol-Nef immunogens, alone or in combination with Env immunogens that were either matched or mismatched with the challenge virus. These animals were then challenged with a pathogenic simian-human immunodeficiency virus. The vaccine regimen included a plasmid DNA prime and replication-defective adenoviral vector boost. Vaccine regimens that included the matched or mismatched Env immunogens conferred better protection against CD4
+
T-lymphocyte loss than that seen with comparable regimens that did not include Env immunogens. This increment in protective immunity was associated with anamnestic Env-specific cellular immunity that developed in the early days following viral challenge. These data suggest that T-lymphocyte immunity to Env can broaden the protective cellular immune response to HIV despite significant sequence diversity of the strains of the Env immunogens and can contribute to immune protection in this AIDS vaccine model.