bluuebunny/crossref_metadata_2025_split huggingface dataset:
Role of the Proline-Rich Motif of Bovine Leukemia Virus Transmembrane Protein gp30 in Viral Load and Pathogenicity in Sheep
Authors: Reichert M. , Winnicka A. , Willems L. , Kettmann R. , Cantor G. H. 
Published: July 2002

ABSTRACT

The cytoplasmic tail of bovine leukemia virus (BLV) transmembrane protein gp30 has multiple amino acid motifs that mimic those present in signaling proteins associated with B-cell and T-cell receptors. The proline-rich motif of gp30, PX
2
PX
4–5
P, is analogous to the recognition site of Src homology 3 (SH3) domains of signaling molecules. Using site-directed mutagenesis of an infectious molecular clone of BLV, point mutations were introduced which changed three of the prolines of the motif to alanines. The influence of these mutations on the pathogenicity of BLV was studied in sheep which received either (i) plasmid DNA with provirus containing proline-to-alanine mutations (pppBLV), (ii) plasmid DNA with wild-type provirus (wtBLV), or (iii) transfection reagent alone. Although all of the BLV-injected animals seroconverted at approximately the same time, viral loads at later time points were high in five of five of the wtBLV group and two of five of the pppBLV group but low in three of five of the pppBLV group, as determined by semiquantitative PCR. Viral expression was lower in the pppBLV-transfected sheep, as measured by p24 antigen enzyme-linked immunosorbent assay in cultured cells, and serologic titers were lower. Thirty-one months after transfection, four of four wtBLV-transfected sheep had died of leukemia and lymphoma, and all five of the pppBLV-transfected sheep were clinically healthy and had normal peripheral blood lymphocyte counts. These data indicate that the proline-rich motif of gp30 is not required for viral infectivity but is important for high viral load in vivo, suggesting that SH3-mediated gp30 interactions are critical for viral pathogenesis following infection. Absence of interactions with the proline-rich motif may prevent or delay tumorigenesis in sheep.
Functional Analysis of PA Binding by Influenza A Virus PB1: Effects on Polymerase Activity and Viral Infectivity
Authors: Perez Daniel R. , Donis Ruben O. 
Published: July 2002

ABSTRACT
Influenza A virus expresses three viral polymerase (P) subunits—PB1, PB2, and PA—all of which are essential for RNA and viral replication. The functions of P proteins in transcription and replication have been partially elucidated, yet some of these functions seem to be dependent on the formation of a heterotrimer for optimal viral RNA transcription and replication. Although it is conceivable that heterotrimer subunit interactions may allow a more efficient catalysis, direct evidence of their essentiality for viral replication is lacking. Biochemical studies addressing the molecular anatomy of the P complexes have revealed direct interactions between PB1 and PB2 as well as between PB1 and PA. Previous studies have shown that the N-terminal 48 amino acids of PB1, termed domain α, contain the residues required for binding PA. We report here the refined mapping of the amino acid sequences within this small region of PB1 that are indispensable for binding PA by deletion mutagenesis of PB1 in a two-hybrid assay. Subsequently, we used site-directed mutagenesis to identify the critical amino acid residues of PB1 for interaction with PA in vivo. The first 12 amino acids of PB1 were found to constitute the core of the interaction interface, thus narrowing the previous boundaries of domain α. The role of the minimal PB1 domain α in influenza virus gene expression and genome replication was subsequently analyzed by evaluating the activity of a set of PB1 mutants in a model reporter minigenome system. A strong correlation was observed between a functional PA binding site on PB1 and P activity. Influenza viruses bearing mutant PB1 genes were recovered using a plasmid-based influenza virus reverse genetics system. Interestingly, mutations that rendered PB1 unable to bind PA were either nonviable or severely growth impaired. These data are consistent with an essential role for the N terminus of PB1 in binding PA, P activity, and virus growth.
Proteolytic Processing of the P2/Nucleocapsid Cleavage Site Is Critical for Human Immunodeficiency Virus Type 1 RNA Dimer Maturation
Authors: Shehu-Xhilaga M. , Kraeusslich H. G. , Pettit S. , Swanstrom R. , Lee J. Y. , Marshall J. A. , Crowe S. M. , Mak J. 
Published: July 2002

ABSTRACT
Differences in virion RNA dimer stability between mature and protease-defective (immature) forms of human immunodeficiency virus type 1 (HIV-1) suggest that maturation of the viral RNA dimer is regulated by the proteolytic processing of the HIV-1 Gag and Gag-Pol precursor proteins. However, the proteolytic processing of these proteins occurs in several steps denoted primary, secondary, and tertiary cleavage events and, to date, the processing step associated with formation of stable HIV-1 RNA dimers has not been identified. We show here that a mutation in the primary cleavage site (p2/nucleocapsid [NC]) hinders formation of stable virion RNA dimers, while dimer stability is unaffected by mutations in the secondary (matrix/capsid [CA], p1/p6) or a tertiary cleavage site (CA/p2). By introducing mutations in a shared cleavage site of either Gag or Gag-Pol, we also show that the cleavage of the p2/NC site in Gag is more important for dimer formation and stability than p2/NC cleavage in Gag-Pol. Electron microscopy analysis of viral particles shows that mutations in the primary cleavage site in Gag but not in Gag-Pol inhibit viral particle maturation. We conclude that virion RNA dimer maturation is dependent on proteolytic processing of the primary cleavage site and is associated with virion core formation.
A Single Amino Acid Substitution in the ICP27 Protein of Herpes Simplex Virus Type 1 Is Responsible for Its Resistance to Leptomycin B
Authors: Murata Takayuki , Goshima Fumi , Koshizuka Tetsuo , Takakuwa Hiroki , Nishiyama Yukihiro 
Published: July 2002

ABSTRACT
Leptomycin B (LMB) is a specific inhibitor of Crm1-dependent nuclear export of proteins. The replication of herpes simplex virus (HSV) is normally highly sensitive to LMB; a resistant HSV variant, however, was isolated by serial passages of the virus. Analysis of marker transfer and viral DNA sequences revealed that a single amino acid substitution within the ICP27 gene is responsible for conferring this resistance.
Identification of Three Functions of the Adenovirus E4orf6 Protein That Mediate p53 Degradation by the E4orf6-E1B55K Complex
Authors: Querido Emmanuelle , Morisson Megan R. , Chu-Pham-Dang Huan , Thirlwell Sarah W.-L. , Boivin Dominique , Branton Philip E. 
Published: July 2002

ABSTRACT
Complexes containing adenovirus E4orf6 and E1B55K proteins play critical roles in productive infection. Both proteins interact directly with the cellular tumor suppressor p53, and in combination they promote its rapid degradation. To examine the mechanism of this process, degradation of exogenously expressed p53 was analyzed in p53-null human cells infected with adenovirus vectors encoding E4orf6 and/or E1B55K. Coexpression of E4orf6 and E1B55K greatly reduced both the level and the half-life of wild-type p53. No effect was observed with the p53-related p73 proteins, which did not appear to interact with E4orf6 or E1B55K. Mutant forms of p53 were not degraded if they could not efficiently bind E1B55K, suggesting that direct interaction between p53 and E1B55K may be required. Degradation of p53 was independent of both MDM2 and p19ARF, regulators of p53 stability in mammalian cells, but required an extended region of E4orf6 from residues 44 to 274, which appeared to possess three separate biological functions. First, residues 39 to 107 were necessary to interact with E1B55K. Second, an overlapping region from about residues 44 to 218 corresponded to the ability of E4orf6 to form complexes with cellular proteins of 19 and 14 kDa. Third, the nuclear retention signal/amphipathic arginine-rich α-helical region from residues 239 to 253 was required. Interestingly, neither the E4orf6 nuclear localization signal nor the nuclear export signal was essential. These results suggested that if nuclear-cytoplasmic shuttling is involved in this process, it must involve another export signal. Degradation was significantly blocked by the 26S proteasome inhibitor MG132, but unlike the HPV E6 protein, E4orf6 and E1B55K were unable to induce p53 degradation in vitro in reticulocyte lysates. Thus, this study implies that the E4orf6-E1B55K complex may direct p53 for degradation by a novel mechanism.
Transcriptional Activation of the Interleukin-2 Promoter by Hepatitis C Virus Core Protein
Authors: Bergqvist Anders , Rice Charles M. 
Published: July 2002

ABSTRACTMost patients infected with hepatitis C virus (HCV) become chronic carriers. Viruses that efficiently establish persistent infections must have effective ways of evading host defenses. In the case of HCV, little is known about how chronic infections are established or maintained. Besides hepatocytes, several reports suggest that HCV can infect T and B lymphocytes. Since T cells are essential for viral clearance, direct or indirect effects of HCV on T-cell function could influence the outcome of infection. Given that T-cell growth and differentiation require the cytokine interleukin 2 (IL-2), we asked whether HCV might modulate synthesis of IL-2. Portions of the HCV polyprotein were expressed in Jurkat cells under a variety of conditions. We found that the highly conserved HCV core protein, in combination with other stimuli, was able to dramatically activate transcription from the IL-2 promoter. The carboxy-terminal hydrophobic portion of the core protein was required for this activity. Activation was dependent on nuclear factor of activated T cells (NFAT), occurred in cells deficient in the tyrosine kinase p56lck, and could be blocked by addition of cyclosporin A and by depletion of calcium. These results suggest that the HCV core protein can activate transcription of the IL-2 promoter through the NFAT pathway. This novel activity may have consequences for T-cell development and establishment of persistent infections.
JC Virus Multiplication in Human Hematopoietic Progenitor Cells Requires the NF-1 Class D Transcription Factor
Authors: Monaco Maria Chiara G. , Sabath Bruce F. , Durham Linda C. , Major Eugene O. 
Published: July 2002

ABSTRACTJCV, a small DNA virus of the polyomavirus family, has been shown to infect glial cells of the central nervous system, hematopoietic progenitor cells, and immune system lymphocytes. A family of DNA binding proteins called nuclear factor-1 (NF-1) has been linked with site-coding specific transcription of cellular and viral genes and replication of some viruses, including JC virus (JCV). It is unclear which NF-1 gene product must be expressed by cells to promote JCV multiplication. Previously, it was shown that elevated levels of NF-1 class D mRNA were expressed by human brain cells that are highly susceptible to JCV infection but not by JCV nonpermissive HeLa cells. Recently, we reported that CD34+precursor cells of the KG-1 line, when treated with the phorbol ester phorbol 12-myristate 13-acetate (PMA), differentiated to cells with macrophage-like characteristics and lost susceptibility to JCV infection. These studies have now been extended by asking whether loss of JCV susceptibility by PMA-treated KG-1 cells is linked with alterations in levels of NF-1 class D expression. Using reverse transcription-PCR, we have found that PMA-treated KG-1 cells express mRNA that codes for all four classes of NF-1 proteins, although different levels of RNA expression were observed in the hematopoietic cells differentiated into macrophages. Northern hybridization confirms that the expression of NF-1 class D gene is lower in JCV nonpermissive PMA-treated KG-1 cells compared with non-PMA-treated cells. Further, using gel mobility shift assays, we were able to show the induction of specific NF-1–DNA complexes in KG-1 cells undergoing PMA treatment. The binding increases in direct relation to the duration of PMA treatment. These results suggest that the binding pattern of NF-1 class members may change in hematopoietic precursor cells, such as KG-1, as they undergo differentiation to macrophage-like cells. Transfection of PMA-treated KG-1 cells with an NF-1 class D expression vector restored the susceptibility of these cells to JCV infection, while the transfection of PMA-treated KG-1 cells with NF-1 class A, B, and C vectors was not able to restore JCV susceptibility. These data collectively suggest that selective expression of NF-1 class D has a regulatory role in JCV multiplication.
Inhibition of Human Immunodeficiency Virus Type 1 gp120 Presentation to CD4 T Cells by Antibodies Specific for the CD4 Binding Domain of gp120
Authors: Hioe Catarina E. , Tuen Michael , Chien Peter C. , Jones Gareth , Ratto-Kim Silvia , Norris Philip J. , Moretto Walter J. , Nixon Douglas F. , Gorny Miroslaw K. , Zolla-Pazner Susan 
Published: July 2002

ABSTRACTHuman immunodeficiency virus (HIV)-specific CD4 T-cell responses, particularly to the envelope glycoproteins of the virus, are weak or absent in most HIV-infected patients. Although these poor responses can be attributed simply to the destruction of the specific CD4 T cells by the virus, other factors also appear to contribute to the suppression of these virus-specific responses. We previously showed that human monoclonal antibodies (MAbs) specific for the CD4 binding domain of gp120 (gp120CD4BD), when complexed with gp120, inhibited the proliferative responses of gp120-specific CD4 T-cells. MAbs to other gp120 epitopes did not exhibit this activity. The present study investigated the inhibitory mechanisms of the anti-gp120CD4BDMAbs. The anti-gp120CD4BDMAbs complexed with gp120 suppressed gamma interferon production as well as proliferation of gp120-specific CD4 T cells. Notably, the T-cell responses to gp120 were inhibited only when the MAbs were added to antigen-presenting cells (APCs) during antigen pulse; the addition of the MAbs after pulsing caused no inhibition. However, the anti-gp120CD4BDMAbs by themselves, or as MAb/gp120 complexes, did not affect the presentation of gp120-derived peptides by the APCs to T cells. These MAb/gp120 complexes also did not inhibit the ability of APCs to process and present unrelated antigens. To test whether the suppressive effect of anti-gp120CD4BDantibodies is caused by the antibodies' ability to block gp120-CD4 interaction, APCs were treated during antigen pulse with anti-CD4 MAbs. These treated APCs remained capable of presenting gp120 to the T cells. These results suggest that anti-gp120CD4BDAbs inhibit gp120 presentation by altering the uptake and/or processing of gp120 by the APCs but their inhibitory activity is not due to blocking of gp120 attachment to CD4 on the surface of APCs.
Papillomavirus Type 16 Oncogenes Downregulate Expression of Interferon-Responsive Genes and Upregulate Proliferation-Associated and NF-κB-Responsive Genes in Cervical Keratinocytes
Authors: Nees Matthias , Geoghegan Joel M. , Hyman Tehila , Frank Stephan , Miller Lance , Woodworth Craig D. 
Published: July 2002

ABSTRACTInfection with high-risk human papillomaviruses (HPV) is a major risk factor for development of cervical cancer. Expression of the HPV E6 and E7 oncoproteins increases in differentiating keratinocytes, resulting in inactivation of the p53 and retinoblastoma proteins, two important transcriptional regulators. We used cDNA microarrays to examine global alterations in gene expression in differentiating cervical keratinocytes after infection with retroviruses encoding HPV type 16 (HPV-16) E6 and E7. Expression of 80 cellular genes (approximately 4% of the genes on the array) was altered reproducibly by E6 and/or E7. Cluster analysis classified these genes into three functional groups: (i) interferon (IFN)-responsive genes, (ii) genes stimulated by NF-κB, and (iii) genes regulated in cell cycle progression and DNA synthesis. HPV-16 E6 or a dominant negative p53 protein downregulated multiple IFN-responsive genes. E6 decreased expression of IFN-α and -β, downregulated nuclear STAT-1 protein, and decreased binding of STAT-1 to the IFN-stimulated response element. E7 alone was less effective; however, coexpression of E6 and E7 downregulated IFN-responsive genes more efficiently than E6. The HPV-16 E6 protein also stimulated expression of multiple genes known to be inducible by NF-κB and AP-1. E6 enhanced expression of functional components of the NF-κB signal pathway, including p50, NIK, and TRAF-interacting protein, and increased binding to NF-κB and AP-1 DNA consensus binding sites. Secretion of interleukin-8, RANTES, macrophage inflammatory protein 1α, and 10-κDa IFN-γ-inducible protein were increased in differentiating keratinocytes by E6. Thus, high-level expression of the HPV-16 E6 protein in differentiating keratinocytes directly alters expression of genes that influence host resistance to infection and immune function.
Human Papillomavirus Type 16 E6-Induced Degradation of E6TP1 Correlates with Its Ability To Immortalize Human Mammary Epithelial Cells
Authors: Gao Qingshen , Singh Latika , Kumar Ajay , Srinivasan Seetha , Wazer David E. , Band Vimla 
Published: July 2002

ABSTRACT
Recent analyses have identified a number of binding partners for E6, including E6AP, ERC55, paxillin, hDlg, p300, interferon regulatory factor 3, hMCM7, Bak, and E6TP1. Notably, association with E6 targets p53, E6TP1, myc, hMCM7, and Bak for degradation. However, the relative importance of the various E6 targets in cellular transformation remains unclear. E6 alone can dominantly immortalize normal human mammary epithelial cells (MECs), permitting an assessment of the importance of various E6 targets in cellular transformation. Studies in this system indicate that E6-induced degradation of p53 and E6 binding to ERC55 or hDlg do not correlate with efficient immortalization. Here, we have examined the role of E6TP1, a Rap GTPase-activating protein, in E6-induced immortalization of MECs. We tested a large set of human papillomavirus type 16 E6 mutants for their ability to bind and target E6TP1 for degradation in vitro and in vivo. We observed a strict correlation between the ability of E6 protein to target E6TP1 for degradation and its ability to immortalize MECs. Recent studies have identified telomerase as a target of E6 protein. Previous analyses of E6 mutants have revealed this trait to closely correlate with MEC immortalization. We examined our entire panel of E6 mutants for rapid induction of telomerase activity and found in general a strong correlation with immortalizing ability. The tight correlation between E6TP1 degradation and MEC immortalization strongly supports a critical role of functional inactivation of E6TP1 in E6-induced cellular immortalization.
Recombinant Wild-Type and Edmonston Strain Measles Viruses Bearing Heterologous H Proteins: Role of H Protein in Cell Fusion and Host Cell Specificity
Authors: Takeuchi Kaoru , Takeda Makoto , Miyajima Naoko , Kobune Fumio , Tanabayashi Kiyoshi , Tashiro Masato 
Published: July 2002

ABSTRACT
Wild-type measles virus (MV) isolated from B95a cells has a restricted host cell specificity and hardly replicates in Vero cells, whereas the laboratory strain Edmonston (Ed) replicates in a variety of cell types including Vero cells. To investigate the role of H protein in the differential MV host cell specificity and cell fusion activity, H proteins of wild-type MV (IC-B) and Ed were coexpressed with the F protein in Vero cells. Cell-cell fusion occurred in Vero cells when Ed H protein, but not IC-B H protein, was expressed. To analyze the role of H protein in the context of viral infection, a recombinant IC-B virus bearing Ed H protein (IC/Ed-H) and a recombinant Ed virus bearing IC-B H protein (Ed/IC-H) were generated from cloned cDNAs. IC/Ed-H replicated efficiently in Vero cells and induced small syncytia in Vero cells, indicating that Ed H protein conferred replication ability in Vero cells on IC/Ed-H. On the other hand, Ed/IC-H also replicated well in Vero cells and induced small syncytia, although parental Ed induced large syncytia in Vero cells. These results indicated that an MV protein(s) other than H protein was likely involved in determining cell fusion and host cell specificity of MV in the case of our recombinants. SLAM (CDw150), a recently identified cellular receptor for wild-type MV, was not expressed in Vero cells, and a monoclonal antibody against CD46, a cellular receptor for Ed, did not block replication or syncytium formation of Ed/IC-H in Vero cells. It is therefore suggested that Ed/IC-H entered Vero cells through another cellular receptor.
Expression of Human Immunodeficiency Virus Type 1 gp120 from Herpes Simplex Virus Type 1-Derived Amplicons Results in Potent, Specific, and Durable Cellular and Humoral Immune Responses
Authors: Hocknell Peter K. , Wiley Rebecca D. , Wang Xiuqing , Evans Thomas G. , Bowers William J. , Hanke Tomas , Federoff Howard J. , Dewhurst Stephen 
Published: July 2002

ABSTRACTHerpes simplex virus type 1 (HSV-1) infects a wide range of cells, including dendritic cells. Consequently, HSV-1 vectors may be capable of eliciting strong immune responses to vectored antigens. To test this hypothesis, an HSV-1 amplicon plasmid encoding human immunodeficiency virus type 1 gp120 was constructed, and murine immune responses to helper virus-free amplicon preparations derived from this construct were evaluated. Initial studies revealed that a single intramuscular (i.m.) injection of 106infectious units (i.u.) of HSV:gp120 amplicon particles (HSV:gp120) elicited Env-specific cellular and humoral immune responses. A potent, CD8+-T-cell-mediated response to an H-2Dd-restricted peptide from gp120 (RGPGRAFVTI) was measured by a gamma interferon ELISPOT and was confirmed by standard cytotoxic-T-lymphocyte assays. Immunoglobulin G enzyme-linked immunosorbent assay analysis showed the induction of a strong, Env-specific antibody response. An i.m. or an intradermal administration of HSV:gp120 at the tail base elicited a more potent cellular immune response than did an intraperitoneal (i.p.) inoculation, although an i.p. introduction generated a stronger humoral response. The immune response to HSV:gp120 was durable, with robust cellular and humoral responses persisting at 171 days after a single 106-i.u. inoculation. The immune response to HSV:gp120 was also found to be dose dependent: as few as 104i.u. elicited a strong T-cell response. Finally, HSV:gp120 elicited significant Env-specific cellular immune responses even in animals that had been previously infected with wild-type HSV-1. Taken together, these data strongly support the use of helper-free HSV-1 amplicon particles as vaccine delivery vectors.