bluuebunny/crossref_metadata_2025_split huggingface dataset:
A G Protein-Coupled Receptor Encoded by Rhesus Rhadinovirus Is Similar to ORF74 of Kaposi's Sarcoma-Associated Herpesvirus
Authors: Estep Ryan D. , Axthelm Michael K. , Wong Scott W. 
Published: January 2003

ABSTRACTRhesus rhadinovirus (RRV) is a gamma-2 herpesvirus and is the rhesus macaque homologue of human herpesvirus 8 (HHV-8), also known as Kaposi's sarcoma-associated herpesvirus. DNA sequence analysis of RRV indicates that it shares numerous open reading frames (ORFs) with HHV-8, including one (ORF74) encoding a seven-transmembrane-spanning G protein-coupled receptor (GPCR) with similarity to cellular chemokine receptors. Examination of the predicted amino acid sequence of RRV ORF74 reveals that it encodes a seven-transmembrane-spanning GPCR sharing 40.8% amino acid sequence identity with HHV-8 ORF74 and 24.1% amino acid sequence identity with rhesus macaque CXCR2. In addition, immunofluorescence studies indicate that an epitope-tagged version of RRV ORF74 is expressed on the surfaces of transfected cells, suggesting that this protein is in fact a membrane receptor. In in vitro cell culture assays, RRV ORF74 possesses transforming potential, as NIH 3T3 clones stably expressing the receptor demonstrate an increased ability to grow in soft agarose and to induce tumor formation in nude mice. Further analysis of RRV ORF74 indicates that expression of the receptor in NIH 3T3 cells causes an increased secretion of vascular endothelial growth factor and activation of the ERK1/2 (p44/42) mitogen-activated protein kinase signaling pathway. The results of these studies suggest that RRV ORF74 encodes a GPCR with properties similar to those of its homologue in HHV-8 and that this gene may play a role in RRV-associated pathogenesis.
Rotavirus Nonstructural Protein NSP5 Interacts with Major Core Protein VP2
Authors: Berois Mabel , Sapin Catherine , Erk Inge , Poncet Didier , Cohen Jean 
Published: January 2003

ABSTRACTRotavirus is a nonenveloped virus with a three-layered capsid. The inner layer, made of VP2, encloses the genomic RNA and two minor proteins, VP1 and VP3, with which it forms the viral core. Core assembly is coupled with RNA viral replication and takes place in definite cellular structures termed viroplasms. Replication and encapsidation mechanisms are still not fully understood, and little information is available about the intermolecular interactions that may exist among the viroplasmic proteins. NSP2 and NSP5 are two nonstructural viroplasmic proteins that have been shown to interact with each other. They have also been found to be associated with precore replication intermediates that are precursors of the viral core. In this study, we show that NSP5 interacts with VP2 in infected cells. This interaction was demonstrated with recombinant proteins expressed from baculovirus recombinants or in bacterial systems. NSP5-VP2 interaction also affects the stability of VP6 bound to VP2 assemblies. The data presented showed evidence, for the first time, of an interaction between VP2 and a nonstructural rotavirus protein. Published data and the interaction demonstrated here suggest a possible role for NSP5 as an adapter between NSP2 and the replication complex VP2-VP1-VP3 in core assembly and RNA encapsidation, modulating the role of NSP2 as a molecular motor involved in the packaging of viral mRNA.
Phosphorylation of Rubella Virus Capsid Regulates Its RNA Binding Activity and Virus Replication
Authors: Law Lok Man J. , Everitt Jason C. , Beatch Martin D. , Holmes Charles F. B. , Hobman Tom C. 
Published: January 2003

ABSTRACT

Rubella virus
is an enveloped positive-strand RNA virus of the family
Togaviridae
. Virions are composed of three structural proteins: a capsid and two membrane-spanning glycoproteins, E2 and E1. During virus assembly, the capsid interacts with genomic RNA to form nucleocapsids. In the present study, we have investigated the role of capsid phosphorylation in virus replication. We have identified a single serine residue within the RNA binding region that is required for normal phosphorylation of this protein. The importance of capsid phosphorylation in virus replication was demonstrated by the fact that recombinant viruses encoding hypophosphorylated capsids replicated at much lower titers and were less cytopathic than wild-type virus. Nonphosphorylated mutant capsid proteins exhibited higher affinities for viral RNA than wild-type phosphorylated capsids. Capsid protein isolated from wild-type strain virions bound viral RNA more efficiently than cell-associated capsid. However, the RNA-binding activity of cell-associated capsids increased dramatically after treatment with phosphatase, suggesting that the capsid is dephosphorylated during virus assembly. In vitro assays indicate that the capsid may be a substrate for protein phosphatase 1A. As capsid is heavily phosphorylated under conditions where virus assembly does not occur, we propose that phosphorylation serves to negatively regulate binding of viral genomic RNA. This may delay the initiation of nucleocapsid assembly until sufficient amounts of virus glycoproteins accumulate at the budding site and/or prevent nonspecific binding to cellular RNA when levels of genomic RNA are low. It follows that at a late stage in replication, the capsid may undergo dephosphorylation before nucleocapsid assembly occurs.
Biochemical and Functional Characterization of the Ebola Virus VP24 Protein: Implications for a Role in Virus Assembly and Budding
Authors: Han Ziying , Boshra Hani , Sunyer J. Oriol , Zwiers Susan H. , Paragas Jason , Harty Ronald N. 
Published: January 2003

ABSTRACT
The VP24 protein of Ebola virus is believed to be a secondary matrix protein and minor component of virions. In contrast, the VP40 protein of Ebola virus is the primary matrix protein and the most abundant virion component. The structure and function of VP40 have been well characterized; however, virtually nothing is known regarding the structure and function of VP24. Wild-type and mutant forms of VP24 were expressed in mammalian cells to gain a better understanding of the biochemical and functional nature of this viral protein. Results from these experiments demonstrated that (i) VP24 localizes to the plasma membrane and perinuclear region in both transfected and Ebola virus-infected cells, (ii) VP24 associates strongly with lipid membranes, (iii) VP24 does not contain N-linked sugars when expressed alone in mammalian cells, (iv) VP24 can oligomerize when expressed alone in mammalian cells, (v) progressive deletions at the N terminus of VP24 resulted in a decrease in oligomer formation and a concomitant increase in the formation of high-molecular-weight aggregates, and (vi) VP24 was present in trypsin-resistant virus like particles released into the media covering VP24-transfected cells. These data indicate that VP24 possesses structural features commonly associated with viral matrix proteins and that VP24 may have a role in virus assembly and budding.
Phosphorylated Serine Residues and an Arginine-Rich Domain of the Moloney Murine Leukemia Virus p12 Protein Are Required for Early Events of Viral Infection
Authors: Yueh Andrew , Goff Stephen P. 
Published: January 2003

ABSTRACT

Mutational analyses of the p12 Gag phosphoprotein of Moloney murine leukemia virus have demonstrated its participation in both virus assembly and the early stages of infection. The molecular mechanisms by which p12 functions in these events are still poorly understood. We performed studies to examine the significance of p12 phosphorylation in the viral life cycle. Alanine substitutions were introduced at the potential phosphorylation sites in p12, and the resulting mutants were tested for replication. Mutant viruses with changes at S61 and S78 were severely impaired, whereas the other mutant viruses were viable. S61 was shown to be required for normal levels of phosphorylation of p12 in vivo. These defective mutant viruses showed no apparent alteration to Gag protein processing or reduction in the yield of virions after transient transfection, but the mutants failed to form circular viral DNAs in acutely infected cells. Sequence analysis of revertant clones derived from S(61,65)A mutant virus revealed two classes: one group with a single mutation at a residue adjacent to S61 and another group with mutations introducing new positive charges surrounding S61. In vivo [
32
P]orthophosphate labeling indicated that the rescue of the S(61,65)A mutant virus did not result in a significant increase in the phosphorylation level of p12. Alanine substitutions of an arginine-rich stretch near S61 (at R-66, -68, -70, and -71) resulted in the same phenotype as the S(61,65)A mutant virus. The restored function of S(61,65)A mutant virus by second or third site mutations may result from a structural change or the addition of positively charged residues in the arginine-rich region.
Enhancer-Like Activity of a Brome Mosaic Virus RNA Promoter
Authors: Ranjith-Kumar C. T. , Zhang Xin , Kao C. Cheng 
Published: January 2003

ABSTRACT
As with transcription from DNA templates, RNA synthesis from viral RNA templates must initiate accurately. RNA sequences named specificity and initiation determinants allow recognition of and coordinated interaction with the viral replication enzyme. Using enriched replicase from brome mosaic virus (BMV)-infected plants and variants of the promoter template for minus-strand and subgenomic RNA initiation, we found that a specificity determinant for minus-strand initiation could function at variable distances and positions from the 3′ initiation site in a manner similar to enhancers of transcription from DNA templates. This determinant's addition could convert a cellular tRNA into a template for RNA synthesis by the BMV replicase in vitro. Furthermore, the same specificity element could direct internal initiation, which occurred at a highly preferred site in a manner distinct from initiation at the 3′ terminus of the template. These results document two distinct modes of initiation site recognition by a viral RNA replicase.
Peripheral Blood Cytotoxic γδ T Lymphocytes from Patients with Human Immunodeficiency Virus Type 1 Infection and AIDS Lyse Uninfected CD4+T Cells, and Their Cytocidal Potential Correlates with Viral Load
Authors: Sindhu Sardar T. A. K. , Ahmad Rasheed , Morisset Richard , Ahmad Ali , Menezes José 
Published: January 2003

ABSTRACTProgression of human immunodeficiency virus type 1 (HIV-1) infection in humans is marked by declining CD4+-T-cell counts and increasing virus load (VL). Cytotoxic T lymphocytes (CTL) play an important role in the lysis of HIV-infected cells, especially during the early phase of asymptomatic infection. CTL responses in the later phase of disease progression may not be as effective since progressors with lower CD4+-T-cell counts have consistently higher VL despite having elevated CTL counts. We hypothesized that, apart from antiviral effects, some CTL might also contribute to AIDS pathogenesis by depleting CD4+T cells and that this CTL activity may correlate with the VL in AIDS patients. Therefore, a cross-sectional study of 31 HIV-1-infected patients at various clinical stages was carried out. Purified CTL from these donors as well as HIV-seronegative controls were used as effectors against different human cell targets by using standard51Cr release cytolytic assays. A direct correlation between VL and CTL-mediated, major histocompatibility complex (MHC)-unrestricted lysis of primary CD4+-T-cell, CEM.NKR, and K562 targets was observed. CD4+-T-cell counts and duration of infection also correlated with MHC-unrestricted cytolytic activity. Our data clearly show that γδ CTL are abnormally expanded in the peripheral blood of HIV-infected patients and that the Vδ1 subset of γδ T cells is the main effector population responsible for this type of cytolysis. The present data suggest that γδ CTL can contribute to the depletion of bystander CD4+T cells in HIV-infected patients as a parallel mechanism to HIV-associated immunopathogenesis and hence expedite AIDS progression.
Amino Acid Changes within Conserved Region III of the Herpes Simplex Virus and Human Cytomegalovirus DNA Polymerases Confer Resistance to 4-Oxo-Dihydroquinolines, a Novel Class of Herpesvirus Antiviral Agents
Authors: Thomsen Darrell R. , Oien Nancee L. , Hopkins Todd A. , Knechtel Mary L. , Brideau Roger J. , Wathen Michael W. , Homa Fred L. 
Published: January 2003

ABSTRACT
The 4-oxo-dihydroquinolines (PNU-182171 and PNU-183792) are nonnucleoside inhibitors of herpesvirus polymerases (R. J. Brideau et al., Antiviral Res. 54:19-28, 2002; N. L. Oien et al., Antimicrob. Agents Chemother. 46:724-730, 2002). In cell culture these compounds inhibit herpes simplex virus type 1 (HSV-1), HSV-2, human cytomegalovirus (HCMV), varicella-zoster virus (VZV), and human herpesvirus 8 (HHV-8) replication. HSV-1 and HSV-2 mutants resistant to these drugs were isolated and the resistance mutation was mapped to the DNA polymerase gene. Drug resistance correlated with a point mutation in conserved domain III that resulted in a V823A change in the HSV-1 or the equivalent amino acid in the HSV-2 DNA polymerase. Resistance of HCMV was also found to correlate with amino acid changes in conserved domain III (V823A+V824L). V823 is conserved in the DNA polymerases of six (HSV-1, HSV-2, HCMV, VZV, Epstein-Barr virus, and HHV-8) of the eight human herpesviruses; the HHV-6 and HHV-7 polymerases contain an alanine at this amino acid. In vitro polymerase assays demonstrated that HSV-1, HSV-2, HCMV, VZV, and HHV-8 polymerases were inhibited by PNU-183792, whereas the HHV-6 polymerase was not. Changing this amino acid from valine to alanine in the HSV-1, HCMV, and HHV-8 polymerases alters the polymerase activity so that it is less sensitive to drug inhibition. In contrast, changing the equivalent amino acid in the HHV-6 polymerase from alanine to valine alters polymerase activity so that PNU-183792 inhibits this enzyme. The HSV-1, HSV-2, and HCMV drug-resistant mutants were not altered in their susceptibilities to nucleoside analogs; in fact, some of the mutants were hypersensitive to several of the drugs. These results support a mechanism where PNU-183792 inhibits herpesviruses by interacting with a binding determinant on the viral DNA polymerase that is less important for the binding of nucleoside analogs and deoxynucleoside triphosphates.
Activation of Natural Killer (NK) T Cells during Murine Cytomegalovirus Infection Enhances the Antiviral Response Mediated by NK Cells
Authors: van Dommelen Serani L. H. , Tabarias Hyacinth A. , Smyth Mark J. , Degli-Esposti Mariapia A. 
Published: January 2003

ABSTRACTNK1.1+T (NKT) cells are efficient regulators of early host responses which have been shown to play a role in tumor surveillance. The relevance of NKT cells in immune surveillance of viral infections, however, is not well understood. In this study, we investigated the functional relevance of NKT cells in controlling herpesvirus infections by using challenge with murine cytomegalovirus (MCMV) as the study model. This model has proven to be one of the best systems for evaluating the role of NK cells during virus infection. Using gene-targeted mice and α-galactosylceramide (α-GalCer) as an exogenous stimulator of NKT cells, we have analyzed the role of these cells in the immune surveillance of MCMV infection. Our studies in NKT-cell-deficient, T-cell receptor Jα281 gene-targeted mice have established that classical NKT cells do not play a critical role in the early clearance of MCMV infection. Importantly, however, activation of NKT cells by α-GalCer resulted in reduced viral replication in visceral organs. Depletion studies, coupled with analysis of gene-targeted mice lacking perforin and gamma interferon (IFN-γ), have revealed that the antiviral effects of α-GalCer involve NK cells and have clearly demonstrated that the antiviral activity of α-GalCer, unlike the antitumor one, is critically dependent on both perforin and IFN-γ.
Conditional Replication of Duck Hepatitis B Virus in Hepatoma Cells
Authors: Guo Ju-Tao , Pryce Melissa , Wang Xingtai , Barrasa M. Inmaculada , Hu Jianming , Seeger Christoph 
Published: January 2003

ABSTRACT
To facilitate investigations of replication and host cell interactions in the hepadnavirus system, we have developed cell lines permitting the conditional replication of duck hepatitis B virus (DHBV). With the help of this system, we devised conditions for core particle isolation that preserve replicase activity, which was not found in previous preparations. Investigations of the stability of viral DNA intermediates indicated that both encapsidated DNA and covalently closed circular DNA (cccDNA) were turned over independently of cell division. Moreover, we showed that alpha interferon reduced the accumulation of RNA-containing viral particles. The availability of a synchronized replication system will permit the biochemical analysis of individual steps of the viral replication cycle, including the mechanism and regulation of cccDNA formation.
Identification and Characterization of an Adeno-Associated Virus Integration Site in CV-1 Cells from the African Green Monkey
Authors: Amiss Terry J. , McCarty Doug M. , Skulimowski Anna , Samulski R. Jude 
Published: January 2003

ABSTRACT
Adeno-associated virus (AAV) is a classification given to a group of nonpathogenic, single-stranded DNA viruses known to reside latently in primates. During latency in humans, AAV type 2 (AAV2) preferentially integrates at a site on chromosome 19q13.3ter by targeting a sequence composed of an AAV Rep binding element (RBE), a spacer, and a nicking site. Here, we report the DNA sequence of an African green monkey AAV integration site isolated from CV-1 cells. Overall, it has 98% homology to the analogous human site, including identical spacer and nicking sequences. However, the simian RBE is expanded, having five perfect directly repeated GAGC tetramers. We carried out a number of in vitro and in vivo assays to determine the effect of this expanded RBE sequence on the Rep-RBE interaction and AAV targeted integration. Using electromobility shift assays it was demonstrated that AAV4 Rep68 bound the expanded RBE with a sixfold-greater affinity than the human RBE. To determine the basis for the affinity increase, DNase I protection and methylation interference (MI) assays were performed. Comparison of footprints on both the human and simian RBEs revealed nearly identical protection; however, MI analysis suggested greater interaction with the guanine nucleotides of the expanded RBE, thus providing a biochemical basis for the increased binding activity. In vivo, integration targeted to the simian RBE was demonstrated by PCR analysis of latently infected Cos-7 cells. Interestingly, the frequency of site-specific integration was twofold greater in Cos-7 cells than in HeLa cells. Overall, these experiments establish that the simian RBE, identified in CV-1 cells, functions analogously to the human RBE and provide further evidence for a developing model that proposes individual roles for the RBE and the spacer and nicking site elements.
Evidence for Mixed Membrane Topology of the Newcastle Disease Virus Fusion Protein
Authors: McGinnes Lori W. , Reitter Julie N. , Gravel Kathy , Morrison Trudy G. 
Published: January 2003

ABSTRACT

The synthesis of the Newcastle disease virus (NDV) fusion (F) protein in a cell-free protein-synthesizing system containing membranes was characterized. The membrane-associated products were in at least two different topological forms with respect to the membranes. The properties of one form were consistent with the expected membrane insertion as a classical type 1 glycoprotein. This form of the protein was fully glycosylated, and sequences amino terminal to the transmembrane domain were protected from protease digestion by the membranes. The second form of membrane-associated F protein was partially glycosylated and partially protected from protease digestion by the membranes. Protease digestion resulted in a 23-kDa protease-protected polypeptide derived from F
2
sequences and sequences from the amino-terminal end of the F
1
domain. Furthermore, a 10-kDa polypeptide derived from the cytoplasmic domain (CT) was also protected from protease digestion by the membranes. Protease resistance of the 23- and 10-kDa polypeptides suggested that this second form of F protein inserted in membranes in a polytopic conformation with both the amino-terminal end and the carboxyl-terminal end translocated across membranes. To determine if this second form of the fusion protein could be found in cells expressing the F protein, two different approaches were taken. A polypeptide with the size of the partially translocated F protein was detected by Western analysis of proteins in total-cell extracts of NDV strain B1 (avirulent)-infected Cos-7 cells. Using antibodies raised against a peptide with sequences from the cytoplasmic domain, CT sequences were detected on surfaces of F protein-expressing Cos-7 cells by immunofluorescence and by flow cytometry. This antibody also inhibited the fusion of red blood cells to cells expressing F and HN proteins. These results suggest that NDV F protein made both in a cell-free system and in Cos-7 cells may exist in two topological forms with respect to membranes and that the second form of the protein may be involved in cell-cell fusion.
New Hepatitis B Virus of Cranes That Has an Unexpected Broad Host Range
Authors: Prassolov Alexej , Hohenberg Heinz , Kalinina Tatyana , Schneider Carola , Cova Lucyna , Krone Oliver , Frölich Kai , Will Hans , Sirma Hüseyin 
Published: January 2003

ABSTRACTAll hepadnaviruses known so far have a very limited host range, restricted to their natural hosts and a few closely related species. This is thought to be due mainly to sequence divergence in the large envelope protein and species-specific differences in host components essential for virus propagation. Here we report an infection of cranes with a novel hepadnavirus, designated CHBV, that has an unexpectedly broad host range and is only distantly evolutionarily related to avihepadnaviruses of related hosts. Direct DNA sequencing of amplified CHBV DNA as well a sequencing of cloned viral genomes revealed that CHBV is most closely related to, although distinct from, Ross' goose hepatitis B virus (RGHBV) and slightly less closely related to duck hepatitis B virus (DHBV). Phylogenetically, cranes are very distant from geese and ducks and are most closely related to herons and storks. Naturally occurring hepadnaviruses in the last two species are highly divergent in sequence from RGHBV and DHBV and do not infect ducks or do so only marginally. In contrast, CHBV from crane sera and recombinant CHBV produced from LMH cells infected primary duck hepatocytes almost as efficiently as DHBV did. This is the first report of a rather broad host range of an avihepadnavirus. Our data imply either usage of similar or identical entry pathways and receptors by DHBV and CHBV, unusual host and virus adaptation mechanisms, or divergent evolution of the host genomes and cellular components required for virus propagation.