bluuebunny/crossref_metadata_2025_split huggingface dataset:
Interactions between Brain Endothelial Cells and Human T-Cell Leukemia Virus Type 1-Infected Lymphocytes: Mechanisms of Viral Entry into the Central Nervous System
Authors: Romero Ignacio A. , Prevost Marie-Christine , Perret Emmanuelle , Adamson Peter , Greenwood John , Couraud Pierre-Olivier , Ozden Simona 
Published: July 2002

ABSTRACTHuman T-cell leukemia virus type 1 (HTLV-1) is associated with a variety of clinical manifestations, including tropical spastic paraparesis or HTLV-1-associated myelopathy (TSP/HAM). Viral detection in the central nervous system (CNS) of TSP/HAM patients demonstrates the ability of HTLV-1 to cross the blood-brain barrier (BBB). To investigate viral entry into the CNS, rat brain capillary endothelial cells were exposed to human lymphocytes chronically infected by HTLV-1 (MT2), to lymphocytes isolated from a seropositive patient, or to a control lymphoblastoid cell line (CEM). An enhanced adhesion to and migration through brain endothelial cells in vitro was observed with HTLV-1-infected lymphocytes. HTLV-1-infected lymphocytes also induced a twofold increase in the paracellular permeability of the endothelial monolayer. These effects were associated with an increased production of tumor necrosis factor alpha by HTLV-1-infected lymphocytes in the presence of brain endothelial cells. Ultrastructural analysis showed that contact between endothelial cells and HTLV-1-infected lymphocytes resulted in a massive and rapid budding of virions from lymphocytes, followed by their internalization into vesicles by brain endothelial cells and apparent release onto the basolateral side, suggesting that viral particles may cross the BBB using the transcytotic pathway. Our study also demonstrates that cell-cell fusion occurs between HTLV-1-infected lymphocytes and brain endothelial cells, with the latter being susceptible to transient HTLV-1 infection. These aspects may help us to understand the pathogenic mechanisms associated with neurological diseases induced by HTLV-1 infection.
Characterization of a Macaque Recombinant Monoclonal Antibody That Binds to a CD4-Induced Epitope and Neutralizes Simian Immunodeficiency Virus
Authors: Glamann Joakim , Hirsch Vanessa M. 
Published: July 2002

ABSTRACTA potent neutralizing Fab fragment from a long-term survivor of simian immunodeficiency virus (SIVsm) infection was used to construct a recombinant macaque immunoglobulin G1κ (IgG1κ) molecule, designated IgG1-201. A Chinese hamster ovary cell line expressing IgG1-201 was derived by stable transfection and optimized for antibody secretion by methotrexate selection and dihydrofolate reductase gene amplification. IgG1-201 effectively neutralized the homologous, molecularly cloned SIVsmH4 virus but had no activity against the heterologous SIVmac251/BK28 virus. The previously characterized, neutralization-resistant SIVsmE543-3 virus was also not neutralized by IgG1-201. Binding to SIVsmH4 gp120 was enhanced in the presence of recombinant soluble CD4, suggesting that IgG1-201 bound a CD4-induced epitope. IgG1-201 immunoprecipitated the SIVsmH4 but not the SIVsmE543-3 envelope despite a close relationship between these two clones. Immunoprecipitation of a panel of SIVsmH4/SIVsmE543-3 chimeric viruses tentatively assigned the neutralization epitope to the third constant domain, immediately C terminal to the V3 loop. These findings suggest the presence of at least one CD4-induced neutralization epitope on SIV, as is the case with human immunodeficiency virus type 1.
DNA Vaccines for Influenza Virus: Differential Effects of Maternal Antibody on Immune Responses to Hemagglutinin and Nucleoprotein
Authors: Pertmer Tamera M. , Oran Alp E. , Moser Janice M. , Madorin Catherine A. , Robinson Harriet L. 
Published: July 2002

ABSTRACTMaternal antibody is the major form of protection from disease in early life when the neonatal immune system is still immature; however, the presence of maternal antibody also interferes with active immunization, placing infants at risk for severe bacterial and viral infection. We tested the ability of intramuscular and gene gun immunization with DNA expressing influenza virus hemagglutinin (HA) and nucleoprotein (NP) to raise protective humoral and cellular responses in the presence or absence of maternal antibody. Neonatal mice born to influenza virus-immune mothers raised full antibody responses to NP but failed to generate antibody responses to HA. In contrast, the presence of maternal antibody did not affect the generation of long-lived CD8+T-cell responses to both HA and NP. Thus, maternal antibody did not affect cell-mediated responses but did affect humoral responses, with the ability to limit the antibody response correlating with whether the DNA-expressed immunogen was localized in the plasma membrane or within the cell.
Association of Human Immunodeficiency Virus Type 1 Vif with RNA and Its Role in Reverse Transcription
Authors: Dettenhofer Markus , Cen Shan , Carlson Bradley A. , Kleiman Lawrence , Yu Xiao-Fang 
Published: July 2002

ABSTRACT

The
vif
gene of human immunodeficiency virus type 1 (HIV-1) is essential for viral replication, although the functional target of Vif remains elusive. HIV-1
vif
mutant virions derived from nonpermissive H9 cells displayed no significant differences in the amount, ratio, or integrity of their protein composition relative to an isogenic wild-type virion. The amounts of the virion-associated viral genomic RNA and tRNA
3
Lys
were additionally present at normal levels in
vif
mutant virions. We demonstrate that Vif associates with RNA in vitro as well as with viral genomic RNA in virus-infected cells. A functionally conserved lentivirus Vif motif was found in the double-stranded RNA binding domain of
Xenopus laevis
, Xlrbpa. The natural intravirion reverse transcriptase products were markedly reduced in
vif
mutant virions. Moreover, purified
vif
mutant genomic RNA-primer tRNA complexes displayed severe defects in the initiation of reverse transcription with recombinant reverse transcriptase. These data point to a novel role for Vif in the regulation of efficient reverse transcription through modulation of the virion nucleic acid components.
Human T-Lymphotropic Virus Type 1 p30
II
Functions as a Transcription Factor and Differentially Modulates CREB-Responsive Promoters
Authors: Zhang Weiqing , Nisbet John W. , Bartoe Joshua T. , Ding Wei , Lairmore Michael D. 
Published: July 2002

ABSTRACT

Human T-lymphotropic virus type 1 (HTLV-1), a complex retrovirus, causes adult T-cell lymphoma/leukemia and is linked to a variety of immune-mediated disorders. The roles of proteins encoded in the pX open reading frame (ORF) II gene region in HTLV-1 replication or in mediating virus-associated diseases remain to be defined. A nucleus-localizing 30-kDa protein, p30
II
, encoded within pX ORF II has limited homology with the POU family of transcription factors. Recently, we reported that selected mutations in pX ORF II diminish the ability of HTLV-1 to maintain high viral loads in infected rabbits. Herein we have tested the transcriptional ability of p30
II
in mammalian cells by using yeast Gal4 fusion protein vectors and transfection of luciferase reporter genes driven by CREB-responsive promoters. p30
II
as a Gal4 DNA-binding domain (DBD) fusion protein transactivates Gal4-driven luciferase reporter gene activity up to 25-fold in 293 and HeLa-tat cells. We confirmed nuclear localization of p30
II
and demonstrate dose-dependent binding of p30
II
-Gal4(DBD) to Gal4 DNA-binding sites. The transcriptional activity of p30
II
-Gal4(DBD) was independent of TATA box flanking sequences, as shown by using two different Gal4 reporter systems. Studies of selected p30
II
mutants indicated that domains that mediate transcription are restricted to a central core region of the protein between amino acids 62 and 220. Transfection of a p30
II
-expressing plasmid repressed cellular CRE-driven reporter gene activity, with or without Tax expression. In contrast, p30
II
at lower concentrations enhanced HTLV-1 long terminal repeat-driven reporter gene activity independent of Tax expression. These data are the first to demonstrate a transcriptional function for p30
II
and suggest a mechanism by which this nuclear protein may influence HTLV-1 replication or cellular gene expression in vivo.
Glycoprotein E of Varicella-Zoster Virus Enhances Cell-Cell Contact in Polarized Epithelial Cells
Authors: Mo Chengjun , Schneeberger Eveline E. , Arvin Ann M. 
Published: July 2002

ABSTRACT

Varicella-zoster virus (VZV) infection involves the cell-cell spread of virions, but how viral proteins interact with the host cell membranes that comprise intercellular junctions is not known. Madin-Darby canine kidney (MDCK) cells were constructed to express the glycoproteins gE, gI, or gE/gI constitutively and were used to examine the effects of these VZV glycoproteins in polarized epithelial cells. At low cell density, VZV gE induced partial tight junction (TJ) formation under low-calcium conditions, whether expressed alone or with gI. Although most VZV gE was intracellular, gE was also shown to colocalize with the TJ protein ZO-1 with or without concomitant expression of gI. Freeze fracture electron microscopy revealed normal TJ strand morphology in gE-expressing MDCK cells. Functionally, the expression of gE was associated with a marked acceleration in the establishment of maximum transepithelial electrical resistance (TER) in MDCK-gE cells; MDCK-gI and MDCK-gE/gI cells exhibited a similar pattern of early TER compared to MDCK cells, although peak resistances were lower than those of gE alone. VZV gE expression altered F-actin organization and lipid distribution, but coexpression of gI modulated these effects. Two regions of the gE ectodomain, amino acids (aa) 278 to 355 and aa 467 to 498, although lacking Ca
2+
binding motifs, exhibit similarities with corresponding regions of the cell adhesion molecules, E-cadherin and desmocollin. These observations suggest that VZV gE and gE/gI may contribute to viral pathogenesis by facilitating epithelial cell-cell contacts.
Translation Is Not Required To Generate Virion Precursor RNA in Human Immunodeficiency Virus Type 1-Infected T Cells
Authors: Butsch Melinda , Boris-Lawrie Kathleen 
Published: July 2002

ABSTRACT

The retroviral primary transcription product is a multifunctional RNA that is utilized as pre-mRNA, mRNA, and genomic RNA. The relationship between human immunodeficiency virus type 1 (HIV-1) unspliced transcripts used as mRNA for viral protein synthesis and as virion precursor RNA (vpRNA) for encapsidation remains an important question. We developed a biochemical assay to evaluate the hypothesis that prior utilization as mRNA template for protein synthesis is necessary to generate vpRNA. HIV-1-infected T cells were treated with translation inhibitors under conditions that maintain virus production. Immunoprecipitation of newly synthesized HIV-1 Gag protein revealed that de novo translation is not necessary to sustain assembly, release, or processing of Gag structural protein. Both newly synthesized protein and steady-state Gag are competent for assembly, and the extracellular accumulation of Gag is proportional to the intracellular abundance of Gag. As early as 2 h after transcription, newly synthesized RNA is detectable in cell-free virions and encapsidation is sustained upon inhibition of host cell translation. Results of both [
3
H]uridine incorporation assays and HIV-1-specific RNase protection assays (RPAs) indicate that translation inhibition reduces the absolute amounts of both cytoplasmic and virion-associated RNA. Evaluation of encapsidation efficiency by RPA revealed that the cytoplasmic availability of vpRNA is increased, indicating that HIV-1 unspliced mRNA can be rerouted to function as vpRNA. Our data contrast with results from the HIV-2 and murine leukemia virus systems and indicate that HIV-1 unspliced RNA constitutes a single functional pool that can function interchangeably as mRNA and as vpRNA.
Sendai Virus Blocks Alpha Interferon Signaling to Signal Transducers and Activators of Transcription
Authors: Komatsu Takayuki , Takeuchi Kenji , Yokoo Junko , Tanaka Yukie , Gotoh Bin 
Published: July 2002

ABSTRACTWe demonstrate here that Sendai virus (SeV) blocks alpha interferon (IFN-α) signaling to signal transducers and activators of transcription (STATs) in HeLa cells. IFN-α-stimulated tyrosine phosphorylation of STATs and subsequent formation of the IFN-stimulated gene factor 3 transcription complex were inhibited in SeV-infected cells, resulting in inefficient induction of IFN-stimulated gene products. None of the components of the signaling pathway—type I IFN receptor subunits Jak1, Tyk2, Stat1, Stat2, and p48—was degraded. Moreover, tyrosine phosphorylation of Jak1 in response to IFN-α was unaffected at the early phase of infection, suggesting that oligomerization of the receptor subunits proceeded normally. In contrast to Jak1, IFN-α-stimulated tyrosine phosphorylation of Tyk2 was partially inhibited. Therefore, this partial inhibition of activation of Tyk2 probably contributes to the subsequent failure in the activation of STATs.
Induction of Host Gene Expression following Infection of Chicken Embryo Fibroblasts with Oncogenic Marek's Disease Virus
Authors: Morgan Robin W. , Sofer Luc , Anderson Amy S. , Bernberg Erin L. , Cui Jing , Burnside Joan 
Published: July 2002

ABSTRACTMicroarrays containing 1,126 nonredundant cDNAs selected from a chicken activated T-cell expressed sequence tag database (http://chickest.udel.edu) were used to examine changes in host cell gene expression that accompany infection of chicken embryo fibroblasts (CEF) with Marek's disease virus (MDV). Host genes that were reproducibly induced by infection of CEF with the oncogenic RB1B strain of MDV included macrophage inflammatory protein, interferon response factor 1, interferon-inducible protein, quiescence-specific protein, thymic shared antigen 1, major histocompatibility complex (MHC) class I, MHC class II, β2-microglobulin, clusterin, interleukin-13 receptor alpha chain, ovotransferrin, a serine/threonine kinase, and avian leukosis virus subgroup J glycoprotein.
Rat Cytomegalovirus Major Immediate-Early Enhancer Switching Results in Altered Growth Characteristics
Authors: Sandford Gordon R. , Brock Lorine E. , Voigt Sebastian , Forester Craig M. , Burns William H. 
Published: July 2002

ABSTRACT
It has been hypothesized that the major immediate-early (MIE) enhancer of cytomegalovirus (CMV) is important in determining virus tropism and latency because of its essential role in initiating the cascade of early gene expression necessary for virus replication. Although rat CMV (RCMV) and murine CMV (MCMV) exhibit extreme species specificity in vivo, they differ in their ability to replicate in tissue culture. MCMV can replicate in a rat embryo fibroblast (REF) cell line while RCMV does not grow in murine fibroblasts. The tropism is not due to a block in virus entry into the cell. We have constructed a recombinant RCMV in which the RCMV MIE enhancer has been replaced with that of MCMV. Growth of the recombinant virus in tissue culture remains restricted to rat cells, suggesting that other viral and/or host factors are more important in determining in vitro tropism. Unlike findings using recombinant MCMV in which the human CMV (HCMV) MIE enhancer substitutes for the native one (A. Angulo, M. Messerle, U. H. Koszinowski, and P. Ghazal, J. Virol. 72:8502–8509, 1998), infection with our recombinant virus at a low multiplicity of infection resulted in a substantial decrease in virus replication. This occurred despite comparable or increased MIE transcription from the recombinant virus. In vivo experiments showed that the recombinant virus replicates normally in the spleen during acute infection. Notably, the recombinant virus appears to be deficient in spreading to the salivary gland, suggesting a role for the MIE enhancer in tropism for certain tissues involved in virus dissemination. Four months after infection, recombinant virus with the foreign MIE enhancer was reactivated from spleen explants.
Essential Role of Cyclization Sequences in Flavivirus RNA Replication
Authors: Khromykh Alexander A. , Meka Hedije , Guyatt Kimberley J. , Westaway Edwin G. 
Published: July 2002

ABSTRACT

A possible role in RNA replication for interactions between conserved complementary (cyclization) sequences in the 5′- and 3′-terminal regions of
Flavivirus
RNA was previously suggested but never tested in vivo. Using the M-fold program for RNA secondary-structure predictions, we examined for the first time the base-pairing interactions between the covalently linked 5′ genomic region (first ∼160 nucleotides) and the 3′ untranslated region (last ∼115 nucleotides) for a range of mosquito-borne
Flavivirus
species. Base-pairing occurred as predicted for the previously proposed conserved cyclization sequences. In order to obtain experimental evidence of the predicted interactions, the putative cyclization sequences (5′ or 3′) in the replicon RNA of the mosquito-borne Kunjin virus were mutated either separately, to destroy base-pairing, or simultaneously, to restore the complementarity. None of the RNAs with separate mutations in only the 5′ or only the 3′ cyclization sequences was able to replicate after transfection into BHK cells, while replicon RNA with simultaneous compensatory mutations in both cyclization sequences was replication competent. This was detected by immunofluorescence for expression of the major nonstructural protein NS3 and by Northern blot analysis for amplification and accumulation of replicon RNA. We then used the M-fold program to analyze RNA secondary structure of the covalently linked 5′- and 3′-terminal regions of three tick-borne virus species and identified a previously undescribed additional pair of conserved complementary sequences in locations similar to those of the mosquito-borne species. They base-paired with Δ
G
values of approximately −20 kcal, equivalent or greater in stability than those calculated for the originally proposed cyclization sequences. The results show that the base-pairing between 5′ and 3′ complementary sequences, rather than the nucleotide sequence per se, is essential for the replication of mosquito-borne Kunjin virus RNA and that more than one pair of cyclization sequences might be involved in the replication of the tick-borne
Flavivirus
species.
Functional Analysis of Recombinant Respiratory Syncytial Virus Deletion Mutants Lacking the Small Hydrophobic and/or Attachment Glycoprotein Gene
Authors: Techaarpornkul Sunee , Barretto Naina , Peeples Mark E. 
Published: July 2002

ABSTRACT
Respiratory syncytial virus (RSV) produces three envelope glycoproteins, the attachment glycoprotein (G), the fusion (F) protein, and the small hydrophobic (SH) protein. It had been assumed, by analogy with other paramyxoviruses, that the G and F proteins would be required for the first two steps of viral entry, attachment and fusion. However, following repeated passage in cell culture, a viable mutant RSV that lacked both the G and SH genes was isolated (R. A. Karron, D. A. Buonagurio, A. F. Georgiu, S. S. Whitehead, J. E. Adamus, M. L. Clements-Mann, D. O. Harris, V. B. Randolph, S. A. Udem, B. R. Murphy, and M. S. Sidhu, Proc. Natl. Acad. Sci. USA 94:13961–13966, 1997). To explore the roles of the G, F, and SH proteins in virion assembly, function, and cytopathology, we have modified the full-length RSV cDNA and used it to rescue infectious RSV lacking the G and/or SH genes. The three resulting viruses and the parental virus all contain the green fluorescent protein (GFP) gene that serves to identify infected cells. We have used purified, radiolabeled virions to examine virus production and function, in conjunction with GFP to quantify infected cells. We found that the G protein enhances virion binding to target cells but plays no role in penetration after attachment. The G protein also enhances cell-to-cell fusion, presumably via cell-to-cell binding, and enhances virion assembly or release. The presence or absence of the G protein in virions has no obvious effect on the content of F protein or host cell proteins in the virion. In growth curve experiments, the viruses lacking the G protein produced viral titers that were at least 10-fold lower than titers of viruses containing the G protein. This reduction is due in large part to the less efficient release of virions and the lower infectivity of the released virions. In the absence of the G protein, virus expressing both the F and SH proteins displayed somewhat smaller plaques, lower fusion activity, and slower viral entry than the virus expressing the F protein alone, suggesting that the SH protein has a negative effect on virus fusion in cell culture.
Terminal Nucleotidyl Transferase Activity of Recombinant
Flaviviridae
RNA-Dependent RNA Polymerases: Implication for Viral RNA Synthesis
Authors: Ranjith-Kumar C. T. , Gajewski J. , Gutshall L. , Maley D. , Sarisky R. T. , Kao C. Cheng 
Published: July 2002

ABSTRACT
Recombinant hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) was reported to possess terminal transferase (TNTase) activity, the ability to add nontemplated nucleotides to the 3′ end of viral RNAs. However, this TNTase was later purported to be a cellular enzyme copurifying with the HCV RdRp. In this report, we present evidence that TNTase activity is an inherent function of HCV and bovine viral diarrhea virus RdRps highly purified from both prokaryotic and eukaryotic cells. A change of the highly conserved GDD catalytic motif in the HCV RdRp to GAA abolished both RNA synthesis and TNTase activity. Furthermore, the nucleotides added via this TNTase activity are strongly influenced by the sequence near the 3′ terminus of the viral template RNA, perhaps accounting for the previous discrepant observations between RdRp preparations. Last, the RdRp TNTase activity was shown to restore the ability to direct initiation of RNA synthesis in vitro on an initiation-defective RNA substrate, thereby implicating this activity in maintaining the integrity of the viral genome termini.
Interaction with the Epstein-Barr Virus Helicase Targets Zta to DNA Replication Compartments
Authors: Liao Gangling , Wu Frederick Y. , Hayward S. Diane 
Published: July 2002

ABSTRACT
Zta has a dual role in the Epstein-Barr virus (EBV) lytic cycle, acting as a key regulator of EBV lytic gene expression and also being essential for lytic viral DNA replication. Zta's replication function is mediated in part through interactions with the core viral replication proteins. We now show interaction between Zta and the helicase (BBLF4) and map the binding region to within amino acids (aa) 22 to 86 of the Zta activation domain. In immunofluorescence assays, green fluorescent protein (GFP)-tagged BBLF4 localized to the cytoplasm of transfected cells. Cotransfection of Zta resulted in translocation of BBLF4-GFP into the nucleus indicating interaction between these two proteins. However, Zta with a deletion of aa 24 to 86 was unable to mediate nuclear translocation of BBLF4-GFP. Results obtained with Zta variants carrying deletions across the aa 24 to 86 region indicated more than one contact site for BBLF4 within this domain, and this was reinforced by the behavior of the four-point mutant Zta (m22/26,74/75), which was severely impaired for BBLF4 interaction. Binding of BBLF4 to Zta was confirmed using GST affinity assays. In both cotransfection-replication assays and replication assays performed in EBV-positive P3HR1 cells, the Zta (m22/26,74/75) mutant was replication defective. In Zta-transfected D98-HR1 cells, replication compartments could be detected by immunofluorescence staining using anti-BMRF1 monoclonal antibody. Cells transfected with Zta variants that were defective for helicase binding still formed replication compartments, but Zta was excluded from these compartments. These experiments reveal a role for the Zta-helicase interaction in targeting Zta to sites of viral DNA replication.