bluuebunny/crossref_metadata_2025 huggingface dataset:
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

A 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

Maternal 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

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

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

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

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

We 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

Microarrays 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

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

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

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

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

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.
Replication of the Wild Type and a Natural Hepatitis B Virus Nucleocapsid Promoter Variant Is Differentially Regulated by Nuclear Hormone Receptors in Cell Culture
Authors: Tang Hong , Raney Anneke K. , McLachlan Alan
Published: July 2002

A natural hepatitis B virus (HBV) variant associated with seroconversion from HBeAg to anti-HBe antibody contains two nucleotide substitutions (A1764T and G1766A) in the proximal nuclear hormone receptor binding site in the nucleocapsid promoter. These nucleotide substitutions prevent the binding of the retinoid X receptor α (RXRα)–peroxisome proliferator-activated receptor α (PPARα) heterodimer without greatly altering the efficiency of binding of hepatocyte nuclear factor 4 (HNF4) to this recognition sequence. In addition, these nucleotide substitutions create a new binding site for HNF1. Analysis of HBV transcription and replication in nonhepatoma cells indicates that RXRα-PPARα heterodimers support higher levels of pregenomic RNA transcription from the wild-type than from the variant nucleocapsid promoter, producing higher levels of wild-type than of variant replication intermediates. In contrast, HNF4 supports higher levels of pregenomic RNA transcription from the variant than from the wild-type nucleocapsid promoter, producing higher levels of variant than of wild-type replication intermediates. HNF1 can support variant virus replication at a low level but is unable to support replication of the wild-type HBV genome. These observations indicate that the replication of wild-type and variant viruses can be differentially regulated by the liver-specific transcription factors that bind to the proximal nuclear hormone receptor binding site of the nucleocapsid promoter. Differential regulation of viral replication may be important in the selection of specific viral variants as a result of an antiviral immune response.
A Cucumber Mosaic Virus (CMV) RNA 1 Transgene Mediates Suppression of the Homologous Viral RNA 1 Constitutively and Prevents CMV Entry into the Phloem
Authors: Canto Tomas , Palukaitis Peter
Published: July 2002

Resistance to
Cucumber mosaic virus
(CMV) in tobacco lines transformed with CMV RNA 1 is characterized by reduced virus accumulation in the inoculated leaf, with specific suppression of accumulation of the homologous viral RNA 1, and by the absence of systemic infection. We show that the suppression of viral RNA 1 occurs in protoplasts from resistant transgenic plants and therefore is not due to a host response activated by the cell-to-cell spread of virus. In contrast, suppression of
Tobacco rattle virus
vectors carrying CMV RNA 1 sequences did not occur in protoplasts from resistant plants. Furthermore, steady-state levels of transgene mRNA 1 were higher in resistant than in susceptible lines. Thus, the data indicate that sequence homology is not sufficient to induce suppression. Grafting experiments using transgenic resistant or susceptible rootstocks and scions demonstrated that the resistance mechanism exhibited an additional barrier to phloem entry, preventing CMV from moving a long distance in resistant plants. On the other hand, virus from susceptible rootstocks could systemically infect grafted resistant scions via the phloem. Analysis of viral RNA accumulation in the infected scions showed that the mechanism that suppresses the accumulation of viral RNA 1 at the single-cell level was overcome. The data indicate that this transgene-mediated systemic resistance probably is not based on a posttranscriptional gene-silencing mechanism.
Human T-Lymphotropic Virus Type 1 p30
II
Regulates Gene Transcription by Binding CREB Binding Protein/p300
Authors: Zhang Weiqing , Nisbet John W. , Albrecht Bjorn , Ding Wei , Kashanchi Fatah , Bartoe Joshua T. , Lairmore Michael D.
Published: July 2002

The highly conserved coadapters CREB binding protein (CBP) and p300 form complexes with CREB as well as other DNA binding transcription factors to modulate chromatin remodeling and thus transcription. Human T-lymphotropic virus type 1 (HTLV-1) transcription is controlled, in part, by the CREB/ATF family of transcription factors which bind promoter sequences and function as complexes with the viral oncogenic protein Tax. We have reported that the nuclear localizing protein p30
II
of HTLV-1 functions as a transcription factor, differentially modulates CREB-responsive promoters, and is critical for maintenance of proviral loads in rabbits. In this study, we tested whether p30
II
directly interacts with CBP/p300 to modulate gene transcription. Gal4(BD)-p30
II
-mediated transactivation was enhanced following exogenous expression of p300 and was competitively repressed by the p300 binding protein, adenovirus E1A, and E1ACR2 (mutated for retinoblastoma binding but retaining p300 binding). In contrast, E1ACR1 (mutated for p300 binding) failed to alter Gal4(BD)-p30
II
-mediated transactivation. In addition, Gal4(BD)-p30
II
-mediated transactivation was competitively inhibited by the cotransfection of CMV-p30
II
-HA and CMV-Tax but could be rescued by exogenous p300. Importantly, we demonstrate that p30
II
colocalizes with p300 in cell nuclei and directly binds to CBP/p300 in cells. Deletion mutants of CBP/p300 were used to localize the site critical for binding p30
II
to a highly conserved KIX region. DNA binding assays confirmed the interference of p30
II
with the assembly of CREB-Tax-p300/CBP multiprotein complexes on 21-bp repeat oligonucleotides in vitro. Collectively, our results demonstrate that CBP/p300 is a cellular protein target for HTLV-1 p30
II
and mediates its transcriptional effects in vivo.
Role of bcl-2 in Epstein-Barr Virus-Induced Malignant Conversion of Burkitt's Lymphoma Cell Line Akata
Authors: Komano Jun , Takada Kenzo
Published: July 2002

We have demonstrated that Epstein-Barr virus (EBV) confers enhanced growth capability in soft agarose, tumorigenesis in the SCID mouse, and resistance to apoptosis in the Burkitt's lymphoma cell line Akata. Subsequently, we have shown that EBV-encoded small RNAs (EBERs) are responsible for these phenotypes. We constantly observed the upregulation of bcl-2 oncoprotein expression upon EBV infection and expression of EBERs. To test whether these phenotypes were due to the upregulation of bcl-2 expression, we introduced bcl-2 into EBV-negative Akata cells at various levels encompassing the range at which EBV-positive cells expressed it. As cells expressed bcl-2 at higher levels, they became more capable of growing in soft agarose and became resistant to apoptosis. However, clones expressing bcl-2 at a higher level than EBV-positive Akata cells were negative in the tumorigenesis assay in the SCID mouse. On the other hand, introduction of bax into EBV-positive Akata cells reduced the resistance to apoptosis; however, it failed to reduce the growth capability in soft agarose. These data indicate that EBV targets not only bcl-2, but also an unknown pathway(s) to enhance the oncogenic potential of Akata cells.
Human Papillomavirus Infection Requires Cell Surface Heparan Sulfate
Authors: Giroglou Tzenan , Florin Luise , Schäfer Frank , Streeck Rolf E. , Sapp Martin
Published: July 2002

Using pseudoinfection of cell lines, we demonstrate that cell surface heparan sulfate is required for infection by human papillomavirus type 16 (HPV-16) and HPV-33 pseudovirions. Pseudoinfection was inhibited by heparin but not dermatan or chondroitin sulfate, reduced by reducing the level of surface sulfation, and abolished by heparinase treatment. Carboxy-terminally deleted HPV-33 virus-like particles still bound efficiently to heparin. The kinetics of postattachment neutralization by antiserum or heparin indicated that pseudovirions were shifted on the cell surface from a heparin-sensitive into a heparin-resistant mode of binding, possibly involving a secondary receptor. Alpha-6 integrin is not a receptor for HPV-33 pseudoinfection.
Antibody-Mediated Neutralization of Primary Human Immunodeficiency Virus Type 1 Isolates: Investigation of the Mechanism of Inhibition
Authors: Spenlehauer Catherine , Kirn André , Aubertin Anne-Marie , Moog Christiane
Published: July 2002

Human immunodeficiency virus type 1 (HIV-1) neutralization occurs when specific antibodies, mainly those directed against the envelope glycoproteins, inhibit infection, most frequently by preventing the entry of the virus into target cells. However, the precise mechanisms of neutralization remain unclear. Previous studies, mostly with cell lines, have produced conflicting results involving either the inhibition of virus attachment or interference with postbinding events. In this study, we investigated the mechanisms of neutralization by immune sera and compared the inhibition of peripheral blood mononuclear cells (PBMC) infection by HIV-1 primary isolates (PI) with the inhibition of T-cell line infection by T-cell line-adapted (TCLA) strains. We followed the kinetics of neutralization to determine at which step of the viral cycle the antibodies act. We found that neutralization of the TCLA strain HIV-1MN/MT-4 required an interaction between antibodies and cell-free virions before the addition of MT-4 cells, whereas PI were neutralized even after adsorption onto PBMC. In addition, the dose-dependent inhibition of HIV-1MNbinding to MT-4 cells was strongly correlated with serum-induced neutralization. In contrast, neutralizing sera did not reduce the adhesion of PI to PBMC. Postbinding inhibition was also detected for HIV-1MNproduced by and infecting PBMC, demonstrating that the mechanism of neutralization depends on the target cell used in the assay. Finally, we considered whether the different mechanisms of neutralization may reflect the recognition of qualitatively different epitopes on the surface of PI and HIV-1MNor whether they reflect differences in virus attachment to PBMC and MT-4 cells.
Use of Helper-Free Replication-Defective Simian Immunodeficiency Virus-Based Vectors To Study Macrophage and T Tropism: Evidence for Distinct Levels of Restriction in Primary Macrophages and a T-Cell Line
Authors: Kim Steve S. , You Xue Juan , Harmon Mary-Elizabeth , Overbaugh Julie , Fan Hung
Published: July 2002

Cell tropism of human and simian immunodeficiency viruses (HIV and SIV, respectively) is governed in part by interactions between the viral envelope protein and the cellular receptors. However, there is evidence that envelope-host cell interactions also affect postentry steps in viral replication. We used a helper-free replication-defective SIV macaque (SIVmac)-based retroviral vector carrying the enhanced jellyfish green fluorescent protein inserted into thenefregion (V1EGFP) to examine SIV tropism in a single cycle of infection. Vector stocks containing envelope proteins from three different SIVmac clones, namely, SIVmac239 (T-lymphocyte tropic [T-tropic]), SIVmac316 (macrophage tropic [M-tropic]), and SIVmac1A11 (dualtropic), were tested. SIVmac239 replicates efficiently in many human T-cell lines, but it does not efficiently infect primary rhesus macrophages. Conversely, SIVmac316 efficiently infects primary macrophages, but it does not replicate in Molt4-Clone8 (M4C8) T cells. SIVmac1A11 replicates efficiently in both cell types. When primary macrophages were infected with V1EGFP pseudotyped by SIVmac316 or SIVmac1A11 envelopes, the infection was substantially (ca. 200- to 300-fold) more efficient than for the SIVmac239 pseudotype. Thus, in primary macrophages, a major component of M versus T tropism involves relatively early events in the infection cycle. Quantitative PCR studies indicated that synthesis and transport of vector DNA into the nucleus were similar for macrophages infected with the clone 239 and 316 pseudotypes, suggesting that the restriction for SIVmac239 infection is after reverse transcription and nuclear import of viral DNA. When the same vector pseudotypes were used to infect M4C8 cells, they all showed approximately equivalent infectivities, even though replication-competent SIVmac316 does not continue to replicate in these cells. Therefore, in M4C8 cells, restriction involves a late step in the infection cycle (after proviral integration and expression). Thus, depending on the cell type infected, envelope-dependent cell interactions that govern SIV M and T tropism may involve different steps in infection.
Epstein-Barr Virus Immediate-Early Protein BZLF1 Is SUMO-1 Modified and Disrupts Promyelocytic Leukemia Bodies
Authors: Adamson Amy L. , Kenney Shannon
Published: July 2002

Although the immediate-early proteins of both herpes simplex virus (HSV) and cytomegalovirus (CMV) are known to modify promyelocytic leukemia (PML) (ND10) bodies in the nucleus of the host cell, it has been unclear whether lytic infection with gamma herpesviruses induces a similar effect. The PML protein is induced by interferon, involved in major histocompatibility complex class I presentation, and necessary for certain types of apoptosis. Therefore, it is likely that PML bodies function in an antiviral capacity. SUMO-1 modification of PML is known to be required for the formation of PML bodies. To examine whether Epstein-Barr virus (EBV) lytic replication interferes with PML bodies, we expressed the EBV immediate-early genes BZLF1 (Z) and BRLF1 (R) in EBV-positive cell lines and examined PML localization. Both Z and R expression resulted in PML dispersion in EBV-positive cells. Z but not R expression is sufficient to disrupt PML bodies in EBV-negative cell lines. We show that dispersion of PML bodies by Z requires a portion of the transcriptional activation domain of Z but not the DNA-binding function. As was previously reported for the HSV-1 ICP0 and CMV IE1 proteins, Z reduces the amount of SUMO-1-modified PML. We also found that Z itself is SUMO-1 modified (through amino acid 12) and that Z competes with PML for limiting amounts of SUMO-1. These results suggest that disruption of PML bodies is important for efficient lytic replication of EBV. Furthermore, Z may potentially alter the function of a variety of cellular proteins by inhibiting SUMO-1 modification.
Simian Immunodeficiency Virus (SIV) Infection of a Rhesus Macaque Induces SIV-Specific CD8+T Cells with a Defect in Effector Function That Is Reversible on Extended Interleukin-2 Incubation
Authors: Xiong Yelin , Luscher Mark A. , Altman John D. , Hulsey Michael , Robinson Harriet L. , Ostrowski Mario , Barber Brian H. , MacDonald Kelly S.
Published: July 2002

A vigorous expansion of antigen-specific CD8+T cells lacking apparent effector function was observed in a rhesus macaque acutely infected with the simian immunodeficiency virus (SIV) strain SIVmac239. Antigen-specific CD8+T cells were identified using antigenic-peptide class I major histocompatibility complex tetramers. As many as 8.3% of CD8+cells recognized the Mamu-A*01-associated SIV epitope Gag181–189(CTPYDINQM); however, these cells demonstrated no effector function when presented with peptide-incubated targets, as measured by intracellular cytokine staining for gamma interferon (IFN-γ), interleukin-2 (IL-2) production, or direct cellular lysis. Similar results were observed with three other SIV peptide antigens. Nonresponsiveness did not correlate with apoptosis of the CD8+cells, nor were cells from this macaque impaired in their ability to present peptide antigens. Associated with the nonresponsive state was a lack of IL-2 production and decreased IL-2 receptor expression. Exogenous IL-2 treatment for 1 week in the absence of antigenic stimulation restored antigen-specific responses and the quantitative correlation between tetramer recognition and antigen-responsive IFN-γ secretion. This case report suggests a regulatory mechanism that may impede the effector function of antigen-specific T cells during acute infection with SIV or human immunodeficiency virus in some cases. This mechanism may participate in the failure of the immune system to limit infection.
Tissue-Specific, Tumor-Selective, Replication-Competent Adenovirus Vector for Cancer Gene Therapy
Authors: Doronin Konstantin , Kuppuswamy Mohan , Toth Karoly , Tollefson Ann E. , Krajcsi Peter , Krougliak Valeri , Wold William S. M.
Published: July 2002

We have previously described two replication-competent adenovirus vectors, named KD1 and KD3, for potential use in cancer gene therapy. KD1 and KD3 have two small deletions in theE1Agene that restrict efficient replication of these vectors to human cancer cell lines. These vectors also have increased capacity to lyse cells and spread from cell to cell because they overexpress the adenovirus death protein, an adenovirus protein required for efficient cell lysis and release of adenovirus from the cell. We now describe a new vector, named KD1-SPB, which is the KD1 vector with the E4 promoter replaced by the promoter for surfactant protein B (SPB). SPB promoter activity is restricted in the adult to type II alveolar epithelial cells and bronchial epithelial cells. Because KD1-SPB has the E1A mutations, it should replicate within and destroy only alveolar and bronchial cancer cells. We show that KD1-SPB replicates, lyses cells, and spreads from cell to cell as well as does KD1 in H441 cells, a human cancer cell line where the SPB promoter is active. KD1-SPB replicates, lyses cells, and spreads only poorly in Hep3B liver cancer cells. Replication was determined by expression of the E4ORF3 protein, viral DNA accumulation, fiber synthesis, and virus yield. Cell lysis and vector spread were measured by lactate dehydrogenase release and a “vector spread” assay. In addition to Hep3B cells, KD1-SPB also did not express E4ORF3 in HT29.14S (colon), HeLa (cervix), KB (nasopharynx), or LNCaP (prostate) cancer cell lines, in which the SPB promoter is not expected to be active. Following injection into H441 or Hep3B tumors growing in nude mice, KD1-SPB caused a three- to fourfold suppression of growth of H441 tumors, similar to that seen with KD1. KD1-SPB had only a minimal effect on the growth of Hep3B tumors, whereas KD1 again caused a three- to fourfold suppression. These results establish that the adenovirus E4 promoter can be replaced by a tissue-specific promoter in a replication-competent vector. The vector has three engineered safety features: the tissue-specific promoter, the mutations in E1A that preclude efficient replication in nondividing cells, and a deletion of the E3 genes which shield the virus from attack by the immune system. KD1-SPB may have use in treating human lung cancers in which the SPB promoter is active.
Herpesvirus Saimiri Open Reading Frame 50 (Rta) Protein Reactivates the Lytic Replication Cycle in a Persistently Infected A549 Cell Line
Authors: Goodwin Delyth J. , Walters Matthew S. , Smith Peter G. , Thurau Mathias , Fickenscher Helmut , Whitehouse Adrian
Published: July 2002

Herpesviruses occur in two distinct forms of infection, lytic replication and latent persistence. In this study, we investigated the molecular mechanisms that govern the latent-lytic switch in the prototype gamma-2 herpesvirus, herpesvirus saimiri (HVS). We utilized a persistently HVS-infected A549 cell line, in which HVS DNA is stably maintained as nonintegrated circular episomes, to assess the role of the open reading frame 50 (ORF 50) (Rta) proteins in the latent-lytic switch. Northern blot analysis and virus recovery assays determined that the ORF 50a gene product, when expressed under the control of a constitutively active promoter, was sufficient to reactivate the entire lytic replication cycle, producing infectious virus particles. Furthermore, although the ORF 50 proteins of HVS strains A11 and C488 are structurally divergent, they were both capable of inducing the lytic replication cycle in this model of HVS latency.
In Vitro Reconstitution of Functional Hepadnavirus Reverse Transcriptase with Cellular Chaperone Proteins
Authors: Hu Jianming , Toft David , Anselmo Dana , Wang Xingtai
Published: July 2002

Initiation of reverse transcription in hepadnaviruses (hepatitis B viruses) depends on the specific binding of an RNA signal (the packaging signal, ε) on the pregenomic RNA template by the viral reverse transcriptase (RT) and is primed by the RT itself (protein priming). We have previously shown that the RT-ε interaction and protein priming require the cellular heat shock protein, Hsp90. However, additional host factors required for these reactions remained to be identified. We now report that five cellular chaperone proteins, all known cofactors of Hsp90, were sufficient to reconstitute a duck hepatitis B virus RT active in ε binding and protein priming in vitro. Four proteins, Hsp90, Hsp70, Hsp40, and Hop, were required for reconstitution of RT activity, and the fifth protein, p23, further enhanced the kinetics of reconstitution. RT activation by the chaperone proteins is a dynamic process dependent on ATP hydrolysis and the Hsp90 ATPase activity. Thus, our results have defined a minimal complement of host factors necessary and sufficient for RT activation. Furthermore, this defined in vitro reconstitution system has now paved the way for future biochemical and structural studies to elucidate the mechanisms of RT activation and chaperone functions.
Terminally Repeated Sequences on a Herpesvirus Genome Are Deleted following Circularization but Are Reconstituted by Duplication during Cleavage and Packaging of Concatemeric DNA
Authors: Nixon Daniel E. , McVoy Michael A.
Published: July 2002

The mechanisms underlying cleavage of herpesvirus genomes from replicative concatemers are unknown. Evidence from herpes simplex virus type 1 suggests that cleavage occurs by a nonduplicative process; however, additional evidence suggests that terminal repeats may also be duplicated during the cleavage process. This issue has been difficult to resolve due to the variable numbers of reiterated terminal repeats that the herpes simplex virus type 1 genome can contain. Guinea pig cytomegalovirus is a herpesvirus with a simple terminal repeat arrangement that defines two genome types. Type II genomes have a single copy of a 1-kb terminal repeat at both their left and right termini, whereas type I genomes have only one copy at their left termini and lack the repeat at their right termini. In a previous study, we constructed a recombinant guinea pig cytomegalovirus in which certain
cis
elements were disrupted such that only type II genomes were produced. Here we show that double repeats that are formed by circularization of infecting genomes are rapidly converted to single repeats, such that the junctions between genomes within replicative concatemers formed late in infection almost exclusively contain single copies of the terminal repeat. Therefore, for the recombinant virus, each cleavage event begins with a single repeat within a concatemer yet produces two repeats, one at each of the resulting termini, demonstrating that terminal repeat duplication occurs in conjunction with cleavage. For wild-type guinea pig cytomegalovirus, the formation of type I genomes further suggests that cleavage can also occur by a nonduplicative process and that duplicative and nonduplicative cleavage can occur concurrently. Other herpesviruses having terminal repeats, such as the herpes simplex viruses and human cytomegalovirus, may also utilize repeat duplication and deletion; however, the biological importance of these events remains unknown.
High-Level Primary CD8+T-Cell Response to Human Immunodeficiency Virus Type 1 Gag and Env Generated by Vaccination with Recombinant Vesicular Stomatitis Viruses
Authors: Haglund Karl , Leiner Ingrid , Kerksiek Kristen , Buonocore Linda , Pamer Eric , Rose John K.
Published: July 2002

We investigated the primary cellular immune responses to human immunodeficiency virus type 1 (HIV-1) Env and Gag proteins elicited by recombinant vesicular stomatitis viruses (rVSVs). The primary response to Env peaked 5 to 7 days after intraperitoneal vaccination, at which time 40% of CD8+cells were Env tetramer positive and activated (CD62LLo). These freshly isolated cells actively lysed target cells pulsed with the p18-I10 peptide and secreted gamma interferon and tumor necrosis factor alpha after stimulation with the Env p18-I10 peptide. The primary response to Env elicited by rVSVs was sixfold higher than that elicited by recombinant vaccinia viruses (rVVs) at 5 days postvaccination. An intranasal route of vaccination with VSV-Env also elicited a strong primary response to Env. The primary immune response to Gag elicited by rVSV peaked 7 days after vaccination, at which time 3% of CD8+cells were Gag tetramer positive and CD62LLoand functional by intracellular cytokine staining. This response was eightfold higher than that elicited by rVV expressing Gag. VSV-GagEnv, which expresses both Gag and Env from a single recombinant, also induced strong cytotoxic T-lymphocyte (CTL) responses to both Env and Gag. Our quantitative analyses illustrate the potency of the VSV vector system in CTL induction.
Characterization and Complete Nucleotide Sequence of an Unusual Reptilian Retrovirus Recovered from the Order Crocodylia
Authors: Martin Joanne , Kabat Peter , Herniou Elisabeth , Tristem Michael
Published: July 2002

A novel group of retroviruses found within the order Crocodylia are described. Phylogenetic analyses demonstrate that they are probably the most divergent members of the
Retroviridae
described to date; even the most conserved regions of Pol show an average of only 23% amino acid identity when compared to other retroviruses.
Sesquiterpenic Lactones of Some Species of Genus Vernonia SCHREB.
Authors: Buděšínský Miloš , Perez Souto Nestor , Holub Miroslav
Published: July 2002

Four species of the genus Vernonia, endemic in Cuba, have been investigated on the presence of sesquiterpenic lactones. In addition to two derivatives of lupeol, scopoletin, genkwanin and velutin, total 15 sesquiterpenic lactones have been isolated, four of which are described for the first time.