bluuebunny/crossref_metadata_2025_split huggingface dataset:
J Domain-Independent Regulation of the Rb Family by Polyomavirus Large T Antigen
Authors: Sheng Qing , Love Tara M. , Schaffhausen Brian 
Published: July 2002

ABSTRACT

The ability of polyomavirus large T antigen (LT) to promote cell cycling, to immortalize primary cells, and to block differentiation has been linked to its effects on tumor suppressors of the retinoblastoma susceptibility (Rb) gene family. Our previous studies have shown that LT requires an intact N-terminal DnaJ domain, in addition to an Rb binding site, for activation of simple E2F-containing promoters and stimulation of cell cycle progression. Here we show that some LT effects dependent on interaction with the Rb family are largely DnaJ independent. In differentiating C2C12 myoblasts, overexpression of LT caused apoptosis. Although this activity of LT completely depended on Rb binding, LTs with mutations in the J domain remained able to kill. Comparisons of Rb
−
and J
−
LTs revealed additional differences. Wild-type but not Rb
−
LT activated the cyclin A promoter under serum starvation conditions. Genetic analysis of the promoter linked the Rb requirement to an E2F site in the promoter. LTs with mutations in the J domain were still able to activate the promoter. Finally, J mutant LTs caused changes in phosphorylation of both pRb and p130. In the case of p130, Thr-986 was shown to be a site that is regulated by J mutant LT. Taken together, these observations reveal that LT regulation of Rb function can be separated into both DnaJ-dependent and DnaJ-independent pathways.
Incorporation of Tumor Vasculature Targeting Motifs into Moloney Murine Leukemia Virus Env Escort Proteins Enhances Retrovirus Binding and Transduction of Human Endothelial Cells
Authors: Liu Liqiong , Liu Ling , Anderson W. French , Beart Robert W. , Gordon Erlinda M. , Hall Frederick L. 
Published: July 2002

ABSTRACT

Adhesion receptors expressed on the surfaces of tumor-activated endothelial cells provide an advantageous locus for targeting gene therapy vectors to angiogenic tissues and/or tumor vasculature. In this study, we engineered a series of Asn-Gly-Arg (NGR)-containing congeners of the presumptive cell binding motif contained within the ninth type III repeat of fibronectin and displayed these tumor vasculature targeting motifs (TVTMs) within the context of Moloney murine leukemia envelope “escort” proteins. Comparative studies of envelope incorporation into viral particles and evaluation of the cell binding properties of the targeted vectors revealed critical structural features, thus identifying a subset of optimal TVTMs. Utilizing a modified ELISA to evaluate viral binding to target cells, we observed a significant down-regulation of TVTM-virion binding to human endothelial cells following sustained (48-h) exposure to VEGF. Normalized for equivalent titers (10
6
CFU/ml), as assayed on NIH 3T3 cells, vectors displaying TVTM escort proteins significantly enhanced the transduction efficiency from 12.2 to 37.4% in human KSY-1 endothelial cell cultures (
P
< 0.001) and from 0.4 to 4.1% in human umbilical vein endothelial cell (HUVEC) cultures (
P
< 0.001). In summary, these studies utilized an engineering approach to identify a subset of TVTMs that are stably incorporated as envelope “escort” proteins into retroviral vectors and that, by functioning to improve the binding efficiency and transduction of both HUVEC and KSY1 endothelial cells, may have therapeutic potential for targeting gene delivery to the tumor-associated vasculature.
Activation of Divergent Neuronal Cell Death Pathways in Different Target Cell Populations during Neuroadapted Sindbis Virus Infection of Mice
Authors: Havert Michael B. , Schofield Brian , Griffin Diane E. , Irani David N. 
Published: July 2002

ABSTRACTInfection of adult mice with neuroadapted Sindbis virus (NSV) results in a severe encephalomyelitis accompanied by prominent hindlimb paralysis. We find that the onset of paralysis parallels morphologic changes in motor neuron cell bodies in the lumbar spinal cord and in motor neuron axons in ventral nerve roots, many of which are eventually lost over time. However, unlike NSV-induced neuronal cell death found in the brain of infected animals, the loss of motor neurons does not appear to be apoptotic, as judged by morphologic and biochemical criteria. This may be explained in part by the lack of detectable caspase-3 expression in these cells.
Major Histocompatibility Complex Class I Gene Controls the Generation of Gamma Interferon-Producing CD4+and CD8+T Cells Important for Recovery from Friend Retrovirus-Induced Leukemia
Authors: Peterson Karin E. , Iwashiro Michihiro , Hasenkrug Kim J. , Chesebro Bruce 
Published: July 2002

ABSTRACTRecovery from leukemia induced by Friend virus complex (FV) requires strong CD4+helper, CD8+cytotoxic T-lymphocyte, and B-cell responses. The development of these immune responses is dependent on the major histocompatibility complex (MHC) (H-2) genotype of the mouse. InH-2b/bmice, which spontaneously recover from FV-induced erythroleukemia, neutralization of gamma interferon (IFN-γ) in vivo inhibited recovery, which indicated that IFN-γ was a necessary component of the immune response to FV. Furthermore, inH-2b/bmice, high numbers of IFN-γ-producing cells were detected after FV infection, whereas inH-2a/bmice, which have a low-recovery phenotype, only low numbers of IFN-γ-producing cells were detected. Similarly,H-2bm14/bmice, which cannot recover from FV infection due to a point mutation in one allele of theH-2Dbgene, also had low numbers of IFN-γ-producing T cells. Surprisingly, this effect was observed for both CD8+and CD4+T cells. These findings reveal a novel influence of MHC class I genes on CD4+T-cell responses to viral infection. Furthermore, the influence of MHC class I genotype on the generation of both IFN-γ-producing CD4+and CD8+T cells helps explain the major impact of theH-2Dgene on recovery from FV disease.
Structure-Function Studies of the Self-Assembly Domain of the Human Immunodeficiency Virus Type 1 Transmembrane Protein gp41
Authors: Weng Yongkai , Yang Zhongning , Weiss Carol D. 
Published: July 2002

ABSTRACT
The coiled-coil region of the human immunodeficiency virus type 1 transmembrane protein (gp41) makes up the interior core of the six-helix bundle structure of the gp41 self-assembly domain. We extended our previous study of this domain (Y. Weng and C. D. Weiss, J. Virol. 72:9676–9682, 1998) by analyzing 23 additional mutants at positions that lie at the interface of the interior core and outer helices. We found nine new functional mutants. For most mutants, the activity could be explained by the ability of the modeled mutants to stabilize the six-helix bundle structure. The present study provides insights into the envelope glycoprotein fusion mechanism and information for rational drug and vaccine design.
Probing the Structure of Rotavirus NSP4: a Short Sequence at the Extreme C Terminus Mediates Binding to the Inner Capsid Particle
Authors: O'Brien Judith A. , Taylor John A. , Bellamy A. R. 
Published: July 2002

ABSTRACT

The rotavirus nonstructural glycoprotein NSP4 functions as the receptor for the inner capsid particle (ICP) which buds into the lumen of the endoplasmic reticulum during virus maturation. The structure of the cytoplasmic domain of NSP4 from rotavirus strain SA11 has been investigated by using limited proteolysis and mass spectrometry. Digestion with trypsin and V8 protease reveals a C-terminal protease-sensitive region that is 28 amino acids long. The minimal sequence requirements for receptor function have been defined by constructing fusions with glutathione
S
-transferase and assessing their ability to bind ICPs. These experiments demonstrate that 17 to 20 amino acids from the extreme C terminus are necessary and sufficient for ICP binding and that this binding is cooperative. These observations are consistent with a model for the structure of the NSP4 cytoplasmic region in which four flexible regions of 28 amino acids are presented by a protease-resistant coiled-coil tetramerization domain, with only the last ∼20 amino acids of each peptide interacting with the surface binding sites on the ICP.
Gamma Interferon Is Not Required for Mucosal Cytotoxic T-Lymphocyte Responses or Heterosubtypic Immunity to Influenza A Virus Infection in Mice
Authors: Nguyen Huan H. , van Ginkel Frederik W. , Vu Huong L. , Novak Miroslav J. , McGhee Jerry R. , Mestecky Jiri 
Published: July 2002

ABSTRACTHeterosubtypic immunity (HSI) is defined as cross-protection against influenza virus of a different serotype than the virus initially encountered and is thought to be mediated by influenza virus-specific cytotoxic T lymphocytes (CTL). Since gamma interferon (IFN-γ) stimulates cytotoxic cells, including antigen-specific CTL which may control virus replication by secretion of antiviral cytokines such as tumor necrosis factor alpha and IFN-γ, we have investigated the mechanism of HSI by analyzing the role of IFN-γ for HSI in IFN-γ gene-deleted (IFN-γ−/−) mice. It has been reported that IFN-γ is not required for recovery from primary infection with influenza virus but is important for HSI. Here, we conclusively show that IFN-γ is not required for induction of secondary influenza virus-specific CTL responses in mediastinal lymph nodes and HSI to lethal influenza A virus infection. Although T helper 2 (Th2)-type cytokines were upregulated in the lungs of IFN-γ−/−mice after virus challenge, either Th1- or Th2-biased responses could provide heterosubtypic protection. Furthermore, titers of serum-neutralizing and cross-reactive antibodies to conserved nucleoprotein in IFN-γ−/−mice did not differ significantly from those in immunocompetent mice. These results indicate that lack of IFN-γ does not impair cross-reactive virus-specific immune responses and HSI to lethal infection with influenza virus. Our findings provide new insight for the mechanisms of HSI and should be valuable in the development of protective mucosal vaccines against variant virus strains, such as influenza and human immunodeficiency virus.
Rubella Virus Capsid Associates with Host Cell Protein p32 and Localizes to Mitochondria
Authors: Beatch Martin D. , Hobman Tom C. 
Published: July 2002

ABSTRACT
Togavirus nucleocapsids have a characteristic icosahedral structure and are composed of multiple copies of a capsid protein complexed with genomic RNA. The assembly of rubella virus nucleocapsids is unique among togaviruses in that the process occurs late in virus assembly and in association with intracellular membranes. The goal of this study was to identify host cell proteins which may be involved in regulating rubella virus nucleocapsid assembly through their interactions with the capsid protein. Capsid was used as bait to screen a CV1 cDNA library using the yeast two-hybrid system. One protein that interacted strongly with capsid was p32, a cellular protein which is known to interact with other viral proteins. The interaction between capsid and p32 was confirmed using a number of different in vitro and in vivo methods, and the site of interaction between these two proteins was shown to be at the mitochondria. Interestingly, overexpression of the rubella virus structural proteins resulted in clustering of the mitochondria in the perinuclear region. The p32-binding site in capsid is a potentially phosphorylated region that overlaps the viral RNA-binding domain of capsid. Our results are consistent with the possibility that the interaction of p32 with capsid plays a role in the regulation of nucleocapsid assembly and/or virus-host interactions.
Application of the Intracellular Gamma Interferon Assay To Recalculate the Potency of CD8+T-Cell Responses to Herpes Simplex Virus
Authors: Kumaraguru Udayasankar , Rouse Barry T. 
Published: July 2002

ABSTRACTEnumeration and characterization of herpes simplex virus (HSV)-specific CD8+T lymphocytes are tedious and indirect. We quantitated antigen-specific CD8+T cells during acute and secondary stages of HSV infection using intracellular gamma interferon production upon stimulation with virus or immunodominant peptide. Results show a substantial increase in the number of CD8+T cells which was otherwise underestimated with the conventional limiting dilution analysis.
Role of NH
2
- and COOH-Terminal Domains of the P Protein of Human Parainfluenza Virus Type 3 in Transcription and Replication
Authors: De Bishnu P. , Hoffman Michael A. , Choudhary Suresh , Huntley Clayton C. , Banerjee Amiya K. 
Published: July 2002

ABSTRACT
The phosphoproteins (P proteins) of paramyxoviruses play a central role in transcription and replication of the viruses by forming the RNA polymerase complex L-P and encapsidation complex (N-P) with nucleocapsid protein (N) and binding to N protein-encapsidated genome RNA template (N-RNA template). We have analyzed the human parainfluenza virus type 3 (HPIV3) P protein and deletion mutants thereof in an in vitro transcription and in vivo replication system. The in vitro system utilizes purified N-RNA template and cell extract containing L and P proteins coexpressed via plasmids using a recombinant vaccinia virus expression system. The in vivo system takes advantage of minigenome replication, which measures luciferase reporter gene expression from HPIV3 minigenomes by viral proteins in a recombinant vaccinia virus expression system. These studies revealed that the C-terminal 20-amino-acid region of P is absolutely required for transcription in vitro and luciferase expression in vivo, suggesting its critical role in viral RNA synthesis. The N-terminal 40-amino-acid region, on the other hand, is essential for luciferase expression but dispensable for transcription in vitro. Consistent with these findings, the C-terminal domain is required for binding of P protein to the N-RNA template involved in both transcription and replication, whereas the N-terminal domain is required for the formation of soluble N-P complex involved in encapsidation of nascent RNA chains during replication. Coimmunoprecipitation analysis showed that the P protein forms a stable homooligomer (perhaps a trimer) that is present in L-P and N-P complexes in the higher oligomeric forms (at least a pentamer). Interestingly, coexpression of a large excess of N- or C-terminally deleted P with wild-type P had no effect on minigenome replication in vivo, notwithstanding the formation of heterooligomeric complexes. These data indicate that P protein with a deleted terminal domain can function normally within the P heterooligomeric complex to carry out transcription and replication in vivo.
Interleukin-12 (IL-12) Enhancement of the Cellular Immune Response against Human Immunodeficiency Virus Type 1 Env Antigen in a DNA Prime/Vaccinia Virus Boost Vaccine Regimen Is Time and Dose Dependent: Suppressive Effects of IL-12 Boost Are Mediated by Nitric Oxide
Authors: Gherardi M. Magdalena , Ramírez Juan C. , Esteban Mariano 
Published: July 2002

ABSTRACTWe previously demonstrated that codelivery of interleukin-12 (IL-12) with the human immunodeficiency virus type 1 (HIV-1) Env antigen from a recombinant vaccinia virus (rVV) can enhance the specific anti-Env cell-mediated immune (CMI) response. In the present study, we have investigated the effects of IL-12 in mice when it is expressed in a DNA prime/VV boost vaccine regimen. The delivery of IL-12 and Env product during priming with a DNA vector, followed by a booster with VV expressing the Env gene (rVVenv), was found to trigger the optimal CMI response compared with other immunization schedules studied. Significantly, if IL-12 is also delivered as a booster from the viral vector, an impairment of the effects of IL-12 was observed involving nitric oxide (NO), since it was overcome by specific inhibitors of inducible NO synthase. NO caused transient immunosuppression rather than impairment of viral replication. Moreover, at certain viral doses, coadministration of the NO inhibitor during the booster resulted in IL-12-mediated enhancement of the specific CD8+T-cell response. In addition, the dose of the IL-12-encoding plasmid (pIL-12) and the route of administration of both vectors were relevant factors for optimal CMI responses. Maximal numbers of Env-specific CD8+gamma interferon-secreting cells were obtained when 50 μg of pIL-12 was administered intramuscularly at priming, followed by an intravenous rVVenv boost. Our results demonstrate, in a murine model, critical parameters affecting the success of vaccination schedules based on a combination of DNA and VV vectors in conjunction with immunomodulators.
DNA Vaccines Encoding Viral Glycoproteins Induce Nonspecific Immunity and Mx Protein Synthesis in Fish
Authors: Kim Carol H. , Johnson Marc C. , Drennan John D. , Simon Benjamin E. , Thomann Estela , Leong Jo-Ann C. 
Published: July 2002

ABSTRACTProtective immunity by vaccination with plasmid DNA encoding a viral glycoprotein (G) has long been assumed to result from the induction of a specific immune response. We report here that the initial protection may be due to the induction of alpha/beta interferon, with long-term protection due to a specific response to the encoded viral G. DNA vaccines encoding the Gs of three serologically unrelated fish rhabdoviruses were used to vaccinate rainbow trout against a lethal challenge with infectious hematopoietic necrosis virus (IHNV). All three vaccines, each encoding the G gene of either IHNV (IHNV-G), snakehead rhabdovirus (SHRV) (SHRV-G), or spring viremia of carp virus (SVCV) (SVCV-G), elicited protective immunity against IHNV. Vaccinated fish were challenged at 30 or 70 days postvaccination with lethal doses of IHNV. At 30 days postvaccination, only 5% of fish that had received any of the G vaccines died, whereas more than 50% of the control fish succumbed to virus challenge. When fish were vaccinated and challenged at 70 days postvaccination, only 12% of the IHNV-G-vaccinated fish died compared to 68% for the SHRV-G- and 76% for the SVCV-G-vaccinated fish. Assays for trout Mx protein, an indicator of alpha/beta interferon induction, showed that only fish vaccinated with a G-containing plasmid produced high levels of Mx protein in the kidneys and liver. Interestingly, at day 7 after virus challenge, all of the fish vaccinated with the IHNV-G plasmid were negative for Mx, but the SHRV-G- and SVCV-G-vaccinated fish still showed detectable levels of Mx. These results suggest that DNA vaccines in fish induce an early, nonspecific antiviral protection mediated by an alpha/beta interferon and, later, a specific immune response.
Use of a Transient Assay for Studying the Genetic Determinants of
Fv1
Restriction
Authors: Bock Michael , Bishop Kate N. , Towers Greg , Stoye Jonathan P. 
Published: July 2002

ABSTRACT

To probe the genetic determinants controlling the interaction between the retroviral restriction gene
Fv1
and its murine leukemia virus target, we set out to develop rapid, transient assays for
Fv1
function. Cells were transfected or transduced with
Fv1
expression plasmids which can produce green fluorescent protein via an internal ribosome entry site positioned between the
Fv1
and green fluorescent protein coding sequences.
Fv1
function was then assessed by comparing virus replication in green fluorescent protein-positive and -negative cells, using retroviral vectors encoding a second fluorescent marker, yellow fluorescent protein, or β-galactosidase. Using this assay, we could show that
Fv1
specificities were not as absolute as previously thought, since the

Fv1
b

allele was capable of interacting with “nonrestricted” B- and NB-tropic viruses and by shuffling the n- and b-alleles of
Fv1
, it was possible to generate a Fv1 molecule capable of restricting N-, B-, and NB-tropic viruses equally efficiently. Further, we could show that the presence of nonrestricting Fv1 in the same cell as restrictive Fv1 abrogates restriction, implying competition for binding to the retroviral target.
Analysis of the Pore Structure of the Influenza A Virus M
2
Ion Channel by the Substituted-Cysteine Accessibility Method
Authors: Shuck Kevin , Lamb Robert A. , Pinto Lawrence H. 
Published: July 2002

ABSTRACT

The M
2
ion channel of influenza A virus is a small integral membrane protein whose active form is a homotetramer with each polypeptide chain containing 96-amino-acid residues. To identify residues of the transmembrane (TM) domain that line the presumed central ion-conducting pore, a set of mutants was generated in which each residue of the TM domain (residues 25 to 44) was replaced by cysteine. The accessibility of the cysteine mutants to modification by the sulfhydryl-specific reagents methane thiosulfonate ethylammonium (MTSEA) and MTS tetraethylammonium (MTSET) was tested. Extracellular application of MTSEA evoked decreases in the conductances measured from two mutants, M
2
-A30C and M
2
-G34C. The changes observed were not reversible on washout, indicative of a covalent modification. Inhibition by MTSEA, or by the larger reagent MTSET, was not detected for residues closer to the extracellular end of the channel than Ala-30, indicating the pore may be wider near the extracellular opening. To investigate the accessibility of the cysteine mutants to reagents applied intracellularly, oocytes were microinjected directly with reagents during recordings. The conductance of the M
2
-W41C mutant was decreased by intracellular injection of a concentrated MTSET solution. However, intracellular application of MTSET caused no change in the conductance of the M
2
-G34C mutant, a result in contrast to that obtained when the reagent was applied extracellularly. These data suggest that a constriction in the pore exists between residues 34 and 41 which prevents passage of the MTS reagent. These findings are consistent with the proposed role for His-37 as the selectivity filter. Taken together, these data confirm our earlier model that Ala-30, Gly-34, His-37, and Trp-41 line the channel pore (L. H. Pinto, G. R. Dieckmann, C. S. Gandhi, C. G. Papworth, J. Braman, M. A. Shaughnessy, J. D. Lear, R. A. Lamb, and W. F. DeGrado, Proc. Natl. Acad. Sci. USA 94:11301–11306, 1997).
Poxvirus-Induced Immunostimulating Effects on Porcine Leukocytes
Authors: Fachinger Vicky , Schlapp Tobias , Strube Walter , Schmeer Norbert , Saalmüller Armin 
Published: July 2002

ABSTRACTThe prophylactic application of inactivated parapox ovis viruses (Baypamun; Bayer AG, Leverkusen, Germany) has been shown to reduce efficiently the outbreak of stress-mediated diseases in different species. However, little is known about the basic mechanism behind this observed stimulatory property. We therefore tested eight inactivated poxvirus strains belonging to three different genera (Orthopoxvirus,Avipoxvirus, andParapoxvirus) for their capacity to activate cells of the porcine innate and specific immune systems in vitro. The results indicated that poxviruses failed to induce increased phagocytosis, oxidative burst, or natural killer cell activity in swine. In contrast, enhanced release of interleukin-2, alpha interferon, and gamma interferon, as well as strong proliferation, could be measured. Flow cytometric analyses and cell sorting experiments identified T-helper cells as the main target responding to inactivated poxviruses: the activated cells had a CD4highCD25+major histocompatibility complex type II-positive phenotype and were the major source of secreted cytokines. Together, the results demonstrated that all tested poxviruses possessed immunostimulating capacity. These in vitro poxvirus-induced effects may be responsible at least in part for the in vivo immunostimulating capacity of inactivated poxviruses.