bluuebunny/crossref_metadata_2025_split huggingface dataset:
Alphavirus RNA Genome Repair and Evolution: Molecular Characterization of Infectious Sindbis Virus Isolates Lacking a Known Conserved Motif at the 3′ End of the Genome
Authors: George Jyothi , Raju Ramaswamy 
Published: July 2002

ABSTRACT
The 3′ nontranslated region of the genomes of Sindbis virus (SIN) and other alphaviruses carries several repeat sequence elements (RSEs) as well as a 19-nucleotide (nt) conserved sequence element (3′CSE). The 3′CSE and the adjoining poly(A) tail of the SIN genome are thought to act as viral promoters for negative-sense RNA synthesis and genome replication. Eight different SIN isolates that carry altered 3′CSEs were studied in detail to evaluate the role of the 3′CSE in genome replication. The salient findings of this study as it applies to SIN infection of BHK cells are as follows: i) the classical 19-nt 3′CSE of the SIN genome is not essential for genome replication, long-term stability, or packaging; ii) compensatory amino acid or nucleotide changes within the SIN genomes are not required to counteract base changes in the 3′ terminal motifs of the SIN genome; iii) the 5′ 1-kb regions of all SIN genomes, regardless of the differences in 3′ terminal motifs, do not undergo any base changes even after 18 passages; iv) although extensive addition of AU-rich motifs occurs in the SIN genomes carrying defective 3′CSE, these are not essential for genome viability or function; and v) the newly added AU-rich motifs are composed predominantly of RSEs. These findings are consistent with the idea that the 3′ terminal AU-rich motifs of the SIN genomes do not bind directly to the viral polymerase and that cellular proteins with broad AU-rich binding specificity may mediate this interaction. In addition to the classical 3′CSE, other RNA motifs located elsewhere in the SIN genome must play a major role in template selection by the SIN RNA polymerase.
Persistence and Tissue Distribution of DNA in Normal and Immunodeficient Mice Inoculated with Polyomavirus VP1 Pseudocapsid Complexes or Polyomavirus
Authors: Heidari S. , Krauzewicz N. , Kalantari M. , Vlastos A. , Griffin B. E. , Dalianis T. 
Published: July 2002

ABSTRACT
Introduction of DNA into normal and immunodeficient mice, alone or in complex with VP1 pseudocapsids, has been compared to DNA transfer by viral infection. Similar to natural infection and in contrast to plasmid alone, VP1 pseudocapsids efficiently introduced DNA, which remained for months in normal mice and possibly longer in B- and T-cell-deficient mice.
Humoral Immunity to Adeno-Associated Virus Type 2 Vectors following Administration to Murine and Nonhuman Primate Muscle
Authors: Chirmule Narendra , Xiao Weidong , Truneh Alemseged , Schnell Michael A. , Hughes Joseph V. , Zoltick Philip , Wilson James M. 
Published: July 2002

ABSTRACTAdeno-associated virus (AAV) is being developed as a vector capable of conferring long-term gene expression, which is useful in the treatment of chronic diseases. In most therapeutic applications, it is necessary to readminister the vector. This study characterizes the humoral immune response to AAV capsid proteins following intramuscular injection and its impact on vector readministration. Studies of mice and rhesus monkeys demonstrated the formation of neutralizing antibodies to AAV capsid proteins that persisted for over 1 year and then diminished, but this did not prevent the efficacy of vector readministration. More-detailed studies strongly suggested that the B-cell response was T cell dependent. This was further evaluated with a blocking antibody to human CD4, primatized for clinical trials, in a biologically compatible mouse in which the endogenous murine CD4 gene was functionally replaced with the human counterpart. Transient pharmacologic inhibition of CD4 T cells with CD4 antibody prevented an antivector response long after the effects of the CD4 antibody diminished; readministration of vector without diminution of gene expression was possible. Our studies suggest that truly durable transgene expression (i.e., prolonged genetic engraftment together with vector readministration) is possible with AAV in skeletal muscle, although it will be necessary to transiently inhibit CD4 T-cell function to avoid the activation of memory B cells.
Rinderpest Viruses Lacking the C and V Proteins Show Specific Defects in Growth and Transcription of Viral RNAs
Authors: Baron Michael D. , Barrett Thomas 
Published: July 2002

ABSTRACT

Rinderpest virus
is a morbillivirus and the causative agent of an important disease of cattle and wild bovids. The P genes of all morbilliviruses give rise to two proteins in addition to the P protein itself: use of an alternate start translation site, in a second open reading frame, gives rise to the C protein, while cotranscriptional insertion of an extra base gives rise to the V protein, a fusion of the amino-terminal half of P to a short, highly conserved, cysteine-rich zinc binding domain. Little is known about the function of either of these two proteins in the rinderpest virus life cycle. We have constructed recombinant rinderpest viruses in which the expression of these proteins has been suppressed, individually and together, and studied the replication of these viruses in tissue culture. We show that the absence of the V protein has little effect on the replication rate of the virus but does lead to an increase in synthesis of genome and antigenome RNAs and a change in cytopathic effect to a more syncytium-forming phenotype. Virus that does not express the C protein, on the other hand, is clearly defective in growth in all cell lines tested, and this defect appears to be related to a decreased transcription of mRNA from viral genes. The phenotypes of both individual mutant virus types are both expressed in the double mutant expressing neither V nor C.
Activation of Lymphocyte Signaling by the R1 Protein of Rhesus Monkey Rhadinovirus
Authors: Damania Blossom , DeMaria Maryann , Jung Jae U. , Desrosiers Ronald C. 
Published: July 2002

ABSTRACTRhesus monkey rhadinovirus(RRV) is a gamma-2 herpesvirus that exhibits a considerable degree of similarity to the humanKaposi's sarcoma-associated herpesvirus(KSHV). The R1 protein of RRV is distantly related to the K1 protein of KSHV, and R1, like K1, can contribute to cell growth transformation. In this study we analyzed the ability of the cytoplasmic tail of R1 to function as a signal transducer. The cytoplasmic domain of the R1 protein contains several tyrosine residues whose phosphorylation is induced in cells expressing Syk kinase. Expression of a CD8 chimera protein containing the extracellular and transmembrane domains of CD8 fused to the cytoplasmic domain of R1 mobilized intracellular calcium and induced cellular tyrosine phosphorylation in B cells upon stimulation with anti-CD8 antibody. None of the CD8-R1 cytoplasmic deletion mutants tested were able to mobilize intracellular calcium or to induce tyrosine phosphorylation to a significant extent upon addition of anti-CD8 antibody. Expression of wild-type R1 protein activated nuclear factor of activated T lymphocytes (NFAT) eightfold in B cells in the absence of antibody stimulation; expression of the CD8-R1C chimera strongly induced NFAT activity (60-fold) but only upon the addition of anti-CD8 antibody. We conclude that the cytoplasmic domain of R1 is capable of transducing signals that elicit B-lymphocyte activation events. The signal-inducing properties of R1 appear to be similar to those of K1 but differ in that the required sequences are distributed over a much longer stretch of the cytoplasmic domain (>150 amino acids). In addition, the induction of calcium mobilization was considerably longer in duration and stronger with R1 than with K1.
Mengovirus and Encephalomyocarditis Virus Poly(C) Tract Lengths Can Affect Virus Growth in Murine Cell Culture
Authors: Martin Lee R. , Neal Zane C. , McBride M. Scott , Palmenberg Ann C. 
Published: July 2002

ABSTRACT

Many virulent aphthoviruses and cardioviruses have long homopolymeric poly(C) tracts in the 5′ untranslated regions of their RNA genomes. A panel of genetically engineered mengo-type cardioviruses has been described which contain a variety of different poly(C) tract lengths. Studies of these viruses have shown the poly(C) tract to be dispensable for growth in HeLa cells, although the relative murine virulence of the viruses correlates directly and positively with tract length. Compared with wild-type mengovirus strain M, mutants with shortened poly(C) tracts grow poorly in mice and protectively immunize rather than kill recipient animals. In the present study, several murine cell populations were tested to determine whether, unlike HeLa cells, they allowed a differential amplification of viruses with long or short poly(C) tracts. Replication and cytopathic studies with four hematopoietically derived cell lines (CH2B, RAW 264.7, A20.J, and P815) and two murine fibroblast cell lines [L929 and L(Y)] demonstrated that several of these cell types indeed allowed differential virus replication as a function of viral poly(C) tract length. Among the most discerning of these cells, RAW 264.7 macrophages supported vigorous lytic growth of a long-tract virus, vMwt (C
44
UC
10
), but supported only substantially diminished and virtually nonlytic growth of vMC
24
(C
13
UC
10
) and vMC
0
short-tract viruses. The viral growth differences evident in all cell lines were apparent early and continuously during every cycle of virus amplification. The data suggest that poly(C) tract-dependent attenuation of mengovirus may be due in part to a viral replication defect manifest in similar hematopoietic-type cells shortly after murine infection. The characterized cultures should provide excellent tools for molecular study of poly(C) tract-mediated virulence.
An Adeno-Associated Virus (AAV) Initiator Protein, Rep78, Catalyzes the Cleavage and Ligation of Single-Stranded AAV
ori
DNA
Authors: Smith Richard H. , Kotin Robert M. 
Published: July 2002

ABSTRACT

The Rep78 protein of adeno-associated virus (AAV) contains amino acid sequence motifs common to rolling-circle replication (RCR) initiator proteins. In this report, we describe RCR initiator-like activities of Rep78. We demonstrate that a maltose-binding protein (MBP)–Rep78 fusion protein can catalyze the cleavage and ligation of single-stranded DNA substrates derived from the AAV origin of replication. Rep-mediated single-stranded DNA cleavage was strictly dependent on the presence of certain divalent cations (e.g., Mn
2+
or Mg
2+
) but did not require the presence of a nucleoside triphosphate cofactor. Electrophoretic mobility shift assays demonstrated that binding of single-stranded DNA by MBP-Rep78 was influenced by the length of the substrate as well as the presence of potential single-stranded
cis
-acting sequence elements. Site-directed mutagenesis was used to examine the role of specific tyrosine residues within a conserved RCR motif (motif 3) of Rep78. Replacement of Tyr-156 with phenylalanine abolished the ability of MBP-Rep78 to mediate the cleavage and ligation of single-stranded DNA substrates but not the ability to stably bind single-stranded DNA. The cleaving-joining activity of Rep78 is consistent with the mechanism of replicative intermediate dimer resolution proposed for the autonomous parvoviruses and may have implications for targeted integration of recombinant AAV vectors.
Induction of Potent Human Immunodeficiency Virus Type 1-Specific T-Cell-Restricted Immunity by Genetically Modified Dendritic Cells
Authors: Lisziewicz Julianna , Gabrilovich Dmitry I. , Varga Georg , Xu Jianqing , Greenberg Philip D. , Arya Suresh K. , Bosch Marnix , Behr Jean-Paul , Lori Franco 
Published: July 2002

ABSTRACTA novel technology combining replication- and integration-defective human immunodeficiency virus type 1 (HIV-1) vectors with genetically modified dendritic cells was developed in order to induce T-cell immunity. We introduced the vector into dendritic cells as a plasmid DNA using polyethylenimine as the gene delivery system, thereby circumventing the problem of obtaining viral vector expression in the absence of integration. Genetically modified dendritic cells (GMDC) presented viral epitopes efficiently, secreted interleukin 12, and primed both CD4+and CD8+HIV-specific T cells capable of producing gamma interferon and exerting potent HIV-1-specific cytotoxicity in vitro. In nonhuman primates, subcutaneously injected GMDC migrated into the draining lymph node at an unprecedentedly high rate and expressed the plasmid DNA. The animals presented a vigorous HIV-specific effector cytotoxic-T-lymphocyte (CTL) response as early as 3 weeks after a single immunization, which later developed into a memory CTL response. Interestingly, antibodies did not accompany these CTL responses, indicating that GMDC can induce a pure Th1 type of immune response. Successful induction of a broad and long-lasting HIV-specific cellular immunity is expected to control virus replication in infected individuals.
Ex Vivo Stimulation and Expansion of both CD4+and CD8+T Cells from Peripheral Blood Mononuclear Cells of Human Cytomegalovirus-Seropositive Blood Donors by Using a Soluble Recombinant Chimeric Protein, IE1-pp65
Authors: Vaz-Santiago Jocelyn , Lulé Jacqueline , Rohrlich Pierre , Jacquier Céline , Gibert Nicolas , Le Roy Emmanuelle , Betbeder Didier , Davignon Jean-Luc , Davrinche Christian 
Published: July 2002

ABSTRACTThe transfer of anti-human cytomegalovirus (HCMV) effector T cells to allogeneic bone marrow recipients results in protection from HCMV disease associated with transplantation, suggesting the direct control of CMV replication by T cells. IE1 and pp65 proteins, both targets of CD4+and CD8+T cells, are considered the best candidates for immunotherapy and vaccine design against HCMV. In this report, we describe the purification of a 165-kDa chimeric protein, IE1-pp65, and its use for in vitro stimulation and expansion of anti-HCMV CD4+and CD8+T cells from peripheral blood mononuclear cells (PBMC) of HCMV-seropositive donors. We demonstrate that an important proportion of anti-HCMV CD4+T cells was directed against IE1-pp65 in HCMV-seropositive donors and that the protein induced activation of HLA-DR3-restricted anti-IE1 CD4+T-cell clones, as assessed by gamma interferon (IFN-γ) secretion and cytotoxicity. Moreover, soluble IE1-pp65 stimulated and expanded anti-pp65 CD8+T cells from PBMC of HLA-A2, HLA-B35, and HLA-B7 HCMV-seropositive blood donors, as demonstrated by cytotoxicity, intracellular IFN-γ labeling, and quantitation of peptide-specific CD8+cells using an HLA-A2–peptide tetramer and staining of intracellular IFN-γ. These results suggest that soluble IE1-pp65 may provide an alternative to infectious viruses used in current adoptive strategies of immunotherapy.
Human T-Cell Leukemia Virus Type 1 Receptor Expression among Syncytium-Resistant Cell Lines Revealed by a Novel Surface Glycoprotein-Immunoadhesin
Authors: Jassal Sushma R. , Pöhler Richard G. , Brighty David W. 
Published: July 2002

ABSTRACTThe envelope glycoproteins of human T-cell leukemia virus type 1 (HTLV-1) perform functions that are crucial for virus entry into cells. The surface glycoprotein (SU) is responsible for viral recognition of, and binding to, target cells through its interaction with an unknown cell surface receptor. To facilitate molecular analysis of the receptor-binding properties of SU and to characterize the cellular receptor employed by HTLV-1, we have expressed a recombinant SU fused to the Fc domain of human immunoglobulin G. Here, we demonstrate that this novel SU-immunoadhesin retains both the biochemical properties of Fc and the receptor-binding specificity of the HTLV-1 SU. We use this SU-immunoadhesin to demonstrate, by direct cell surface binding assays, that the receptor used by HTLV-1 has been conserved through vertebrate evolution. Moreover, using murine-human somatic cell hybrids we provide data that do not support the previously assigned location for the HTLV-1 receptor on human chromosome 17. Most importantly, we show that many cell lines that are resistant to HTLV-1 envelope-mediated infection and syncytium formation express functional receptors that are recognized by the HTLV-1 SU. Based on our results, we suggest that for some HTLV-1-resistant cell lines the block to viral entry occurs at a late post-receptor-binding step of the entry process. Our findings will be of value in developing new strategies to identify the cellular receptor used by HTLV-1.
Transient Disruption of Intercellular Junctions Enables Baculovirus Entry into Nondividing Hepatocytes
Authors: Bilello John P. , Delaney William E. , Boyce Frederick M. , Isom Harriet C. 
Published: July 2002

ABSTRACT
Baculovirus infection has extended the capabilities for transfection of exogenous genes into a variety of mammalian cell types. Because rat hepatocytes plated on collagen-coated dishes and maintained in dimethyl sulfoxide (DMSO)-supplemented chemically defined medium are an excellent model system for studying liver function in vitro, we investigated the ability of baculoviruses to infect and deliver exogenous genes to cells in this culture system. Efficient delivery to hepatocytes in short-term culture becomes restricted to peripheral cells, or “edge” cells, as the hepatocytes acquire intercellular junctions and form islands with time in culture. This barrier to baculovirus entry can be overcome, and the percentage of internal cells within the hepatocyte islands that are infected with the baculovirus can be increased more than 100-fold, when cells are subjected to transient calcium depletion before and during infection. These findings suggest that at least in some cell types, such as hepatocytes, baculovirus entry may require contact with the basolateral surface. We conclude from this study that recombinant baculovirus infection following transient depletion of extracellular calcium results in delivery of exogenous genes to at least 75% of hepatocytes in long-term DMSO culture, thereby making it possible for the first time to carry out gain-of-function and loss-of-function studies in this cell system.
Inhibition of Major Histocompatibility Complex Class II-Dependent Antigen Presentation by Neutralization of Gamma Interferon Leads to Breakdown of Resistance against Measles Virus-Induced Encephalitis
Authors: Weidinger Gerald , Henning Golo , ter Meulen Volker , Niewiesk Stefan 
Published: July 2002

ABSTRACTBALB/c mice are resistant to measles virus (MV)-induced encephalitis due to their strong MV-specific CD4+T-cell response. Resistance is broken by neutralization of gamma interferon with monoclonal antibodies, indicating an important role for this pleiotropic cytokine. Here, we demonstrate that mouse gamma interferon has no direct antiviral effect in vitro and in vivo. The breakdown of resistance is due neither to a switch in the T-helper response nor to an impaired migration of CD4+T cells. Neutralization of gamma interferon interferes with the major histocompatibility complex class II-dependent antigen presentation and subsequent proliferation of CD4+T cells in vitro and in vivo. In consequence, the reduction in numbers of CD4+T cells below a protective threshold leads to susceptibility to MV-induced encephalitis.
Kaposi's Sarcoma-Associated Herpesvirus K-bZIP Protein Is Phosphorylated by Cyclin-Dependent Kinases
Authors: Polson Andrew G. , Huang Lan , Lukac David M. , Blethrow Justin D. , Morgan David O. , Burlingame Alma L. , Ganem Don 
Published: July 2002

ABSTRACT
The K8 locus in Kaposi's sarcoma-associated herpesvirus (KSHV) is syntenic with the Epstein-Barr virus (EBV) BZLF (Z) locus and expresses three alternatively spliced transcripts. The fully spliced transcript encodes K-bZIP, the KSHV homologue of the EBV immediate-early transcriptional transactivator Z. Here we show that despite the presence of alternatively spliced transcripts, the protein from the fully spliced RNA, K-bZIP, is the principal product detectable in KSHV-infected B cells. The protein is detected only in lytically infected cells and is localized to the nucleus. We further characterized K-bZIP by determining its phosphorylation status. Phosphoamino acid analysis revealed phosphorylation on serine and threonine. Analysis of the sites of K-bZIP phosphorylation by tandem mass spectrometry revealed that K-bZIP was phosphorylated on Thr 111 and Ser 167. These phosphorylation sites are contained within cyclin-dependent kinase (CDK) recognition sites with the consensus sequence (S/T)PXR, suggesting that K-bZIP could be phosphorylated by CDKs. We tested this hypothesis using an in vitro kinase reaction performed in whole-cell extracts that resemble in vivo conditions more closely than standard in vitro kinase reactions. We found that the three CDK-cyclin complexes we tested phosphorylated K-bZIP but not the control ORF 73 protein, which contains four (S/T)PXR sites. Ectopic expression of K-bZIP cannot reactivate KSHV from latency, and single and double mutants of K-bZIP in which alanines replaced the phosphorylated serine and/or threonine also failed to induce lytic replication. These studies indicate that K-bZIP is a substrate for CDKs and should inform further functional analyses of the protein.
Restoration of Anti-Human Immunodeficiency Virus Type 1 (HIV-1) Responses in CD8+T Cells from Late-Stage Patients on Prolonged Antiretroviral Therapy by Stimulation In Vitro with HIV-1 Protein-Loaded Dendritic Cells
Authors: Fan Zheng , Huang Xiao-Li , Borowski Luann , Mellors John W. , Rinaldo Charles R. 
Published: July 2002

ABSTRACTWe demonstrate that dendritic cells loaded in vitro with human immunodeficiency virus type 1 (HIV-1) protein-liposome complexes activate HLA class I-restricted anti-HIV-1 cytotoxic T-lymphocyte and gamma interferon (IFN-γ) responses in autologous CD8+T cells from late-stage HIV-1-infected patients on prolonged combination drug therapy. Interleukin-12 enhanced this effect through an interleukin-2- and IFN-γ-mediated pathway. This suggests that dendritic cells from HIV-1-infected persons can be engineered to evoke stronger anti-HIV-1 CD8+T-cell reactivity as a strategy to augment antiretroviral therapy.