bluuebunny/crossref_metadata_2025_split huggingface dataset:
Lentivirus-Mediated RNA Interference of DC-SIGN Expression Inhibits Human Immunodeficiency Virus Transmission from Dendritic Cells to T Cells
Authors: Arrighi Jean-François , Pion Marjorie , Wiznerowicz Maciej , Geijtenbeek Teunis B. , Garcia Eduardo , Abraham Shahnaz , Leuba Florence , Dutoit Valérie , Ducrey-Rundquist Odile , van Kooyk Yvette , Trono Didier , Piguet Vincent 
Published: September 2004

ABSTRACT

In the early events of human immunodeficiency virus type 1 (HIV-1) infection, immature dendritic cells (DCs) expressing the DC-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) receptor capture small amounts of HIV-1 on mucosal surfaces and spread viral infection to CD4
+
T cells in lymph nodes (22, 34, 45). RNA interference has emerged as a powerful tool to gain insight into gene function. For this purpose, lentiviral vectors that express short hairpin RNA (shRNA) for the delivery of small interfering RNA (siRNA) into mammalian cells represent a powerful tool to achieve stable gene silencing. In order to interfere with DC-SIGN function, we developed shRNA-expressing lentiviral vectors capable of conditionally suppressing DC-SIGN expression. Selectivity of inhibition of human DC-SIGN and L-SIGN and chimpanzee and rhesus macaque DC-SIGN was obtained by using distinct siRNAs. Suppression of DC-SIGN expression inhibited the attachment of the gp120 envelope glycoprotein of HIV-1 to DC-SIGN transfectants, as well as transfer of HIV-1 to target cells in
trans
. Furthermore, shRNA-expressing lentiviral vectors were capable of efficiently suppressing DC-SIGN expression in primary human DCs. DC-SIGN-negative DCs were unable to enhance transfer of HIV-1 infectivity to T cells in
trans
, demonstrating an essential role for the DC-SIGN receptor in transferring infectious viral particles from DCs to T cells. The present system should have broad applications for studying the function of DC-SIGN in the pathogenesis of HIV as well as other pathogens also recognized by this receptor.
Nuclear Respiratory Factor 1 Plays an Essential Role in Transcriptional Initiation from the Hepatitis B Virus X Gene Promoter
Authors: Tokusumi Yumiko , Zhou Sharleen , Takada Shinako 
Published: September 2004

ABSTRACT

The X gene of hepatitis B virus (HBV) is one of the major factors in HBV-induced hepatocarcinogenesis and is essential for the establishment of productive HBV replication in vivo. Recent studies have shown that the X gene product targets mitochondria and induces calcium flux, thereby activating Ca
+
-dependent signal transduction pathways. However, regulatory mechanisms of X gene expression have remained unclear. Previous studies had localized a minimal promoter activity to a 21-bp GC-rich sequence located 130 bp upstream of the X protein coding region and showed that there was a cellular protein bound to this DNA. Interestingly, the 21-bp sequence identified as an X gene minimal promoter does not contain any previously identified core promoter elements, such as a TATA box. To better understand the mechanisms of transcriptional initiation of the X gene, we set out to biochemically purify the binding protein(s) for the 21-bp DNA. We report here the identification of the X gene minimal promoter-binding activity as nuclear respiratory factor 1 (NRF1), a previously known transcription factor that activates the majority of nucleus-encoded mitochondrial genes and various housekeeping genes. Primer extension analyses of the X mRNAs show that mutations at the binding site specifically inactivate transcription from this promoter and that a dominant-negative NRF1 mutant and short interfering RNAs inhibit transcription from this promoter. Therefore, NRF1 specifically binds the 21-bp minimal promoter and positively contributes to transcription of the X gene. Simultaneous activation of the X gene and mitochondrial genes by NRF1 may allow the X protein to target mitochondria most efficiently.
Identification of an hnRNP A1-Dependent Splicing Silencer in the Human Papillomavirus Type 16 L1 Coding Region That Prevents Premature Expression of the Late L1 Gene
Authors: Zhao Xiaomin , Rush Margaret , Schwartz Stefan 
Published: September 2004

ABSTRACT

We have previously identified
cis
-acting RNA sequences in the human papillomavirus type 16 (HPV-16) L1 coding region which inhibit expression of L1 from eukaryotic expression plasmids. Here we have determined the function of one of these RNA elements, and we provide evidence that this RNA element is a splicing silencer which suppresses the use of the 3′ splice site located immediately upstream of the L1 AUG. We also show that this splice site is inefficiently utilized as a result of a suboptimal polypyrimidine tract. Introduction of point mutations in the L1 coding region that altered the RNA sequence without affecting the L1 protein sequence resulted in the inactivation of the splicing silencer and induced splicing to the L1 3′ splice site. These mutations also prevented the interaction of the RNA silencer with a 35-kDa cellular protein identified here as hnRNP A1. The splicing silencer in L1 inhibits splicing in vitro, and splicing can be restored by the addition of RNAs containing an hnRNP A1 binding site to the reaction, demonstrating that hnRNP A1 inhibits splicing of the late HPV-16 mRNAs through the splicing silencer sequence. While we show that one role of the splicing silencer is to determine the ratio between partially spliced L2/L1 mRNAs and spliced L1 mRNAs, we also demonstrate that it inhibits splicing from the major 5′ splice site in the early region to the L1 3′ splice site, thereby playing an essential role in preventing late gene expression at an early stage of the viral life cycle. We speculate that the activity of the splicing silencer and possibly the concentration of hnRNP A1 in the HPV-16-infected cell determines the ability of the virus to establish a persistent infection which remains undetected by the host immune surveillance.
Splice Junction Map of Simian Parvovirus Transcripts
Authors: Vashisht Kapil , Faaberg Kay S. , Aber Amanda L. , Brown Kevin E. , O’Sullivan M. Gerard 
Published: September 2004

ABSTRACT

The transcription map of simian parvovirus (SPV), an
Erythrovirus
similar to
Parvovirus
B19, was investigated. RNA was extracted from tissues of experimentally infected cynomolgus macaques and subjected to reverse transcription-PCR with SPV-specific primers. The PCR products were cloned and sequenced to identify splice junctions. A total of 14 distinct sequences were identified as putative partial transcripts. Of these, 13 were spliced; a single unspliced transcript putatively encoded NS1. Sequence analysis revealed that spliced partial transcripts may encode portions of open reading frames for the major capsid proteins VP1 and VP2 and smaller, unknown proteins. These unspliced and spliced transcripts and putative proteins encoded by SPV were similar to those of B19. Initial splice junctions at nucleotides 279 and 333 were analogous to those at nucleotides 406 and 441, respectively, in B19. Seven of the 10 splices identified had typical GT/AG donor/acceptor junctions. The splice sites were confirmed by Northern blotting and autoradiography. In contrast to B19, which has a maximum of two splices per transcript, up to three splices were observed in SPV transcripts. A spliced transcript putatively encoding a truncated version of NS1, as seen with minute virus of mice and adeno-associated virus 2, was also observed. The findings indicate that that the splicing pattern of transcripts of SPV and B19 is similar, but SPV also has coding strategies in common with other parvoviruses.
Enhanced Expression of Cell Cycle Regulatory Genes in Virus-Specific Memory CD8
+
T Cells
Authors: Latner Donald R. , Kaech Susan M. , Ahmed Rafi 
Published: September 2004

ABSTRACT

Unlike naive CD8
+
T cells, antigen-experienced memory CD8
+
T cells persist over time due to their unique ability to homeostatically proliferate. It was hypothesized that memory cells might differentially regulate the expression of genes that control the cell cycle to facilitate homeostatic proliferation. To test this, the expression levels of 96 different cell cycle regulatory genes were compared between transgenic naive and memory CD8
+
T cells that specifically recognize the GP33-41 epitope of lymphocytic choriomeningitis virus (LCMV). It was discovered that relative to naive cells, memory cells overexpress several important genes that control the transition between G
1
and S phase. Some of these genes include those encoding cyclins D3, D2, B1, C, and H, cyclin-dependent kinases (cdk's) 4 and 6, the cdk inhibitors p16, p15, and p18, and other genes involved in protein degradation and DNA replication. Importantly, these differences were observed both in total populations of LCMV-specific naive and memory CD8
+
cells and in LCMV-specific CD8
+
T-cell populations that were in the G
1
phase of the cell cycle only. In addition, the expression differences between naive and memory cells were exaggerated following antigenic stimulation. The fact that memory cells are precharged with several of the major factors that are necessary for the G
1
- to-S-phase transition suggests they may require a lower threshold of stimulation to enter the cell cycle.
Identification of Proteins in Human Cytomegalovirus (HCMV) Particles: the HCMV Proteome
Authors: Varnum Susan M. , Streblow Daniel N. , Monroe Matthew E. , Smith Patricia , Auberry Kenneth J. , Paša-Tolić Ljiljana , Wang Dai , Camp David G. , Rodland Karin , Wiley Steven , Britt William , Shenk Thomas , Smith Richard D. , Nelson Jay A. 
Published: September 2004

ABSTRACT
Human
cytomegalovirus (HCMV), a member of the herpesvirus family, is a large
complex enveloped virus composed of both viral and cellular gene
products. While the sequence of the HCMV genome has been known for over
a decade, the full set of viral and cellular proteins that compose the
HCMV virion are unknown. To approach this problem we have utilized
gel-free two-dimensional capillary liquid chromatography-tandem mass
spectrometry (MS/MS) and Fourier transform ion cyclotron resonance MS
to identify and determine the relative abundances of viral and cellular
proteins in purified HCMV AD169 virions and dense bodies. Analysis of
the proteins from purified HCMV virion preparations has indicated that
the particle contains significantly more viral proteins than previously
known. In this study, we identified 71 HCMV-encoded proteins that
included 12 proteins encoded by known viral open reading frames (ORFs)
previously not associated with virions and 12 proteins from novel viral
ORFs. Analysis of the relative abundance of HCMV proteins indicated
that the predominant virion protein was the pp65 tegument protein and
that gM rather than gB was the most abundant
glycoprotein. We have also identified over
70 host cellular proteins in HCMV virions, which include cellular
structural proteins, enzymes, and chaperones. In addition, analysis of
HCMV dense bodies indicated that these viral particles are composed of
29 viral proteins with a reduced quantity of cellular proteins in
comparison to HCMV virions. This study provides the first comprehensive
quantitative analysis of the viral and cellular proteins that compose
infectious particles of a large complex
virus.
Maturation and Trafficking Markers on Rotavirus-Specific B Cells during Acute Infection and Convalescence in Children
Authors: Jaimes María C. , Rojas Olga L. , Kunkel Eric J. , Lazarus Nicole H. , Soler Dulce , Butcher Eugene C. , Bass Dorsey , Angel Juana , Franco Manuel A. , Greenberg Harry B. 
Published: September 2004

ABSTRACTWe have previously studied B cells, from people and mice, that express rotavirus-specific surface immunoglobulin (RV-sIg) by flow cytometry with recombinant virus-like particles that contain green fluorescent protein. In the present study we characterized circulating B cells with RV-sIg in children with acute and convalescent infection. During acute infection, circulating RV-sIgD−B cells are predominantly large, CD38high, CD27high, CD138+/−, CCR6−, α4β7+, CCR9+, CCR10+, cutaneous lymphocyte antigen-negative (CLA−), L-selectinint/−, and sIgM+, sIgG−, sIgA+/−lymphocytes. This phenotype likely corresponds to gut-targeted plasma cells and plasmablasts. During convalescence the phenotype switches to small and large lymphocytes, CD38int/−, CD27int/−, CCR6+, α4β7+/−, CCR9+/−and CCR10−, most likely representing RV-specific memory B cells with both gut and systemic trafficking profiles. Of note, during acute RV infection both total and RV-specific murine IgM and IgA antibody-secreting cells migrate efficiently to CCL28 (the CCR10 ligand) and to a lesser extent to CCL25 (the CCR9 ligand). Our results show that CCR10 and CCR9 can be expressed on IgM as well as IgA antibody-secreting cells in response to acute intestinal infection, likely helping target these cells to the gut. However, these intestinal infection-induced plasmablasts lack the CLA homing receptor for skin, consistent with mechanisms of differential CCR10 participation in skin T versus intestinal plasma cell homing. Interestingly, RV memory cells generally lack CCR9 and CCR10 and instead express CCR6, which may enable recruitment to diverse epithelial sites of inflammation.
Herpes Simplex Virus Type 1 ICP0 Protein Mediates Activation of Adeno-Associated Virus Type 2
rep
Gene Expression from a Latent Integrated Form
Authors: Geoffroy Marie-Claude , Epstein Alberto L. , Toublanc Estelle , Moullier Philippe , Salvetti Anna 
Published: September 2004

ABSTRACT

Adeno-associated virus type 2 (AAV-2) is a human parvovirus that requires the presence of a helper virus, such as the herpes simplex virus type 1 (HSV-1) to accomplish a complete productive cycle. In the absence of helper virus, AAV-2 can establish a latent infection that is characterized by the absence of expression of viral genes. So far, four HSV-1 early genes, UL5/8/52 (helicase primase complex) and UL29 (single-stranded DNA-binding protein), were defined as sufficient for AAV replication when cells were transfected with a plasmid carrying the wild-type AAV-2 genome. However, none of these viral products was shown to behave as a transcriptional factor able to activate AAV gene expression. Our study provides the first evidence that the immediate-early HSV-1 protein ICP0 can promote
rep
gene expression in cells latently infected with wild-type AAV-2. This ICP0-mediated effect occurs at the transcriptional level and involves the ubiquitin-proteasome pathway. Furthermore, using deletion mutants, we demonstrate that the localization of ICP0 to ND10 and their disruption is not required for the activation of the
rep
promoter, whereas binding of ICP0 to the ubiquitin-specific protease HAUSP makes a significant contribution to this effect.
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

ABSTRACT
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

ABSTRACTPleconaril 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

ABSTRACTThe 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

ABSTRACT
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

ABSTRACT

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.