bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Cytoplasmic Tail of Infectious Bronchitis Virus E Protein Directs Golgi Targeting
Authors: Corse Emily , Machamer Carolyn E. 
Published: October 2010

ABSTRACT
We have previously shown that the E protein of the coronavirus infectious bronchitis virus (IBV) is localized to the Golgi complex when expressed exogenously from cDNA. Here, we report that neither the transmembrane domain nor the short lumenal domain of IBV E is required for Golgi targeting. However, an N-terminal truncation containing only the cytoplasmic domain (CTE) was efficiently localized to the Golgi complex, and this domain could retain a reporter protein in the Golgi. Thus, the cytoplasmic tail of the E protein is necessary and sufficient for Golgi targeting. The IBV E protein is palmitoylated on one or two cysteine residues adjacent to its transmembrane domain, but palmitoylation was not required for proper Golgi targeting. Using C-terminal truncations, we determined that the IBV E Golgi targeting information is present between tail amino acids 13 and 63. Upon treatment with brefeldin A, both the E and CTE proteins redistributed to punctate structures that colocalized with the Golgi matrix proteins GM130 and p115 instead of being localized to the endoplasmic reticulum like Golgi glycosylation enzymes. This suggests that IBV E is associated with the Golgi matrix through interactions of its cytoplasmic tail and may have interesting implications for coronavirus assembly in early Golgi compartments.
Potent Immunosuppressive Activities of Cytomegalovirus- Encoded Interleukin-10
Authors: Spencer Juliet V. , Lockridge Kristen M. , Barry Peter A. , Lin Gaofeng , Tsang Monica , Penfold Mark E. T. , Schall Thomas J. 
Published: October 2010

ABSTRACTCytomegalovirus (CMV) has highly evolved mechanisms for avoiding detection by the host immune system. Recently, in the genomes of human and primate CMV, a novel gene comprising segments of noncontiguous open reading frames was identified and found to have limited predicted homology to endogenous cellular interleukin-10 (IL-10). Here we investigate the biological activities of the CMV IL-10-like gene product and show it to possess potent immunosuppressive properties. Both purified bacterium-derived recombinant CMV IL-10 and CMV IL-10 expressed in supernatants of human cells were found to inhibit proliferation of mitogen-stimulated peripheral blood mononuclear cells (PBMCs), with specific activity comparable to that of recombinant human IL-10. In addition, CMV IL-10 expressed from human cells inhibited cytokine synthesis, as treatment of stimulated PBMCs and monocytes with CMV IL-10 led to a marked decrease in production of proinflammatory cytokines. Finally, CMV IL-10 was observed to decrease cell surface expression of both major histocompatibility complex (MHC) class I and class II molecules, while conversely increasing expression of the nonclassical MHC allele HLA-G. These results demonstrate for the first time that CMV has a biologically active IL-10 homolog that may contribute to immune evasion during virus infection.
Influenza A Virus M2Ion Channel Activity Is Essential for Efficient Replication in Tissue Culture
Authors: Takeda Makoto , Pekosz Andrew , Shuck Kevin , Pinto Lawrence H. , Lamb Robert A. 
Published: October 2010

ABSTRACTThe amantadine-sensitive ion channel activity of influenza A virus M2protein was discovered through understanding the two steps in the virus life cycle that are inhibited by the antiviral drug amantadine: virus uncoating in endosomes and M2protein-mediated equilibration of the intralumenal pH of thetransGolgi network. Recently it was reported that influenza virus can undergo multiple cycles of replication without M2ion channel activity (T. Watanabe, S. Watanabe, H. Ito, H. Kida, and Y. Kawaoka, J. Virol. 75:5656–5662, 2001). An M2protein containing a deletion in the transmembrane (TM) domain (M2-del29–31) has no detectable ion channel activity, yet a mutant virus was obtained containing this deletion. Watanabe and colleagues reported that the M2-del29–31virus replicated as efficiently as wild-type (wt) virus. We have investigated the effect of amantadine on the growth of four influenza viruses: A/WSN/33; N31S-M2WSN, a mutant in which an asparagine residue at position 31 in the M2TM domain was replaced with a serine residue; MUd/WSN, which possesses seven RNA segments from WSN plus the RNA segment 7 derived from A/Udorn/72; and A/Udorn/72. N31S-M2WSN was amantadine sensitive, whereas A/WSN/33 was amantadine resistant, indicating that the M2residue N31is the sole determinant of resistance of A/WSN/33 to amantadine. The growth of influenza viruses inhibited by amantadine was compared to the growth of an M2-del29–31virus. We found that the M2-del29–31virus was debilitated in growth to an extent similar to that of influenza virus grown in the presence of amantadine. Furthermore, in a test of biological fitness, it was found that wt virus almost completely outgrew M2-del29–31virus in 4 days after cocultivation of a 100:1 ratio of M2-del29–31virus to wt virus, respectively. We conclude that the M2ion channel protein, which is conserved in all known strains of influenza virus, evolved its function because it contributes to the efficient replication of the virus in a single cycle.
The g5R (D250) Gene of African Swine Fever Virus Encodes a Nudix Hydrolase That Preferentially Degrades Diphosphoinositol Polyphosphates
Authors: Cartwright Jared L. , Safrany Stephen T. , Dixon Linda K. , Darzynkiewicz Edward , Stepinski Janusz , Burke Richard , McLennan Alexander G. 
Published: October 2010

ABSTRACT

The African swine fever virus (ASFV) g5R gene encodes a protein containing a Nudix hydrolase motif which in terms of sequence appears most closely related to the mammalian diadenosine tetraphosphate (Ap
4
A) hydrolases. However, purified recombinant g5R protein (g5Rp) showed a much wider range of nucleotide substrate specificity compared to eukaryotic Ap
4
A hydrolases, having highest activity with GTP, followed by adenosine 5′-pentaphosphate (p
5
A) and dGTP. Diadenosine and diguanosine nucleotides were substrates, but the enzyme showed no activity with cap analogues such as 7mGp
3
A. In common with eukaryotic diadenosine hexaphosphate (Ap
6
A) hydrolases, which prefer higher-order polyphosphates as substrates, g5Rp also hydrolyzes the diphosphoinositol polyphosphates PP-InsP
5
and [PP]
2
-InsP
4
. A comparison of the kinetics of substrate utilization showed that the
k
cat
/
K

m

ratio for PP-InsP
5
is 60-fold higher than that for GTP, which allows classification of g5R as a novel diphosphoinositol polyphosphate phosphohydrolase (DIPP). Unlike mammalian DIPP, g5Rp appeared to preferentially remove the 5-β-phosphate from both PP-InsP
5
and [PP]
2
-InsP
4
. ASFV infection led to a reduction in the levels of PP-InsP
5
, ATP and GTP by ca. 50% at late times postinfection. The measured intracellular concentrations of these compounds were comparable to the respective
K

m

values of g5Rp, suggesting that one or all of these may be substrates for g5Rp during ASFV infection. Transfection of ASFV-infected Vero cells with a plasmid encoding epitope-tagged g5Rp suggested localization of this protein in the rough endoplasmic reticulum. These results suggest a possible role for g5Rp in regulating a stage of viral morphogenesis involving diphosphoinositol polyphosphate-mediated membrane trafficking.
Oncoretrovirus and Lentivirus Vectors Pseudotyped with Lymphocytic Choriomeningitis Virus Glycoprotein: Generation, Concentration, and Broad Host Range
Authors: Beyer Winfried R. , Westphal Manfred , Ostertag Wolfram , von Laer Dorothee 
Published: October 2010

ABSTRACT
Lymphocytic choriomeningitis virus (LCMV) is a noncytopathic arenavirus shown to infect a broad range of different cell types. Here, we combined the beneficial characteristics of the LCMV glycoprotein (LCMV-GP) and those of retroviral vectors to generate a new, safe, and efficient gene transfer system. These LCMV-GP pseudotypes were systematically compared with vectors containing the widely used amphotropic murine leukemia virus envelope (A-MLVenv) or the vesicular stomatitis virus G protein (VSV-G). Production of LCMV-GP-pseudotyped oncoretroviral and lentiviral vectors by transient transfection resulted in vector titers similar to those with A-MLVenv or VSV-G. In contrast to A-MLVenv particles, LCMV-GP pseudotypes could be efficiently concentrated by ultracentrifugation without loss of vector titer. Unlike the cell-toxic VSV-G, a stable retroviral packaging cell line constitutively expressing LCMV-GP could be established. Vectors pseudotyped with LCMV-GP efficiently transduced many cell lines from different species and tissues relevant for gene therapy. Transduction of human glioma cells was studied in detail. These cells are a major target for cancer gene therapy and were transduced more efficiently with LCMV-GP-pseudotyped vectors than with the generally used A-MLVenv particles. The high stability, low toxicity, and broad host range make LCMV-GP-pseudotyped vectors attractive for gene transfer applications. The recombinant LCMV-GP-pseudotyped vectors will also allow functional characterization of naturally occurring and recombinant LCMV-GP variants.
Adaptation of Wild-Type Measles Virus to Tissue Culture
Authors: Waku Kouomou Diane , Wild T. Fabian 
Published: October 2010

ABSTRACT
Measles has a host range restricted to humans and monkeys in captivity. Fresh measles virus (MV) isolates replicate readily in several human and simian B-cell lines but need a period of adaptation to other types of cells. The identification of CD46 and CD150 (SLAM) as cellular receptors for MV has helped to clarify certain aspects of the immunobiology of MV infections. We have examined the properties of an MV wild-type strain grown in the epithelial cell line Vero. After adaptation, this virus expressed high levels of both the viral glycoproteins (hemagglutinin and fusion protein) but did not induce fusion (syncytia). No changes in the amino acid sequence were found in either of the viral glycoproteins. Using several approaches, the Vero-adapted virus could not be shown to interact with CD46 either in the initiation or during the course of infection. The presence of human SLAM expressed in the Vero cells rapidly gave rise to fusion and lower yields of infectious virus.
Sequence and Genetic Arrangement of the USRegion of the Monkey B Virus (Cercopithecine Herpesvirus 1) Genome and Comparison with the USRegions of Other Primate Herpesviruses
Authors: Ohsawa Kazutaka , Black Darla H. , Sato Hiroshi , Eberle R. 
Published: October 2010

ABSTRACTThe sequence of the unique short (US) region of monkey B virus (BV) was determined. The 13 genes identified are arranged in the same order and orientation as in herpes simplex virus (HSV). These results demonstrate that the BV USregion is entirely colinear with that of HSV type 1 (HSV-1), HSV-2, and simian agent 8 virus.
Kissing Interaction between 3′ Noncoding and Coding Sequences Is Essential for Porcine Arterivirus RNA Replication
Authors: Verheije M. H. , Olsthoorn R. C. L. , Kroese M. V. , Rottier P. J. M. , Meulenberg J. J. M. 
Published: October 2010

ABSTRACT

We used an infectious cDNA clone of
Porcine reproductive and respiratory syndrome virus
(PRRSV) to investigate the presence of essential replication elements in the region of the genome encoding the structural proteins. Deletion analysis showed that a stretch of 34 nucleotides (14653 to 14686) within ORF7, which encodes the nucleocapsid protein, is essential for RNA replication. Strand-specific reverse transcription-PCR analysis of viral RNA isolated from transfected BHK-21 cells revealed that this region is required for negative-strand genomic RNA synthesis. The 34-nucleotide stretch is highly conserved among PRRSV isolates and folds into a putative hairpin. A 7-base sequence within the loop of this structure was suggested to base-pair with a sequence present in the loop of a hairpin located in the 3′ noncoding region, resulting in a kissing interaction. Mutational analyses confirmed that this kissing interaction is required for RNA replication.
Second-Site Mutation Outside of the U
S
10-12 Domain of Δγ
1
34.5 Herpes Simplex Virus 1 Recombinant Blocks the Shutoff of Protein Synthesis Induced by Activated Protein Kinase R and Partially Restores Neurovirulence
Authors: Cassady Kevin A. , Gross Martin , Gillespie G. Yancey , Roizman Bernard 
Published: October 2010

ABSTRACT

Earlier studies have shown that herpes simplex virus type 1 (HSV-1) activated protein kinase R (PKR) but that the product of the product of the γ
1
34.5 gene binds and redirects the host phosphatase 1 to dephosphorylate the α subunit of eukaryotic translation initiation factor 2 (eIF-2α). In consequence, the γ
1
34.5 gene product averts the threatened shutoff of protein synthesis caused by activated PKR. Serial passages of Δγ
1
34.5 mutants in human cells led to isolation of two classes of second-site, compensatory mutants. The first, reported earlier, resulted from the juxtaposition of the α promoter of the U
S
12 gene to the coding sequence of the U
S
11 gene. The mutant blocks the phosphorylation of eIF-2α but does not restore the virulence phenotype of the wild-type virus. We report another class of second-site, compensatory mutants that do not map to the U
S
10-12 domain of the HSV-1 genome. All mutants in this series exhibit sustained late protein synthesis, higher yields in human cells, and reduced phosphorylation of PKR that appears to be phosphatase dependent. Specific dephosphorylation of eIF-2α was not demonstrable. At least one mutant in this series exhibited a partial restoration of the virulence phenotype characteristic of the wild-type virus phenotype. The results suggest that the second-site mutations reflect activation of fossilized functions designed to block the interferon response pathways in cells infected with the progenitor of present HSV.
Small DNA Hairpin Negatively Regulates In Situ Priming during Duck Hepatitis B Virus Reverse Transcription
Authors: Habig Jeffrey W. , Loeb Daniel D. 
Published: October 2010

ABSTRACT

There are two mutually exclusive pathways for plus-strand DNA synthesis in hepadnavirus reverse transcription. The predominant pathway gives rise to relaxed circular DNA, while the other pathway yields duplex linear DNA. Both pathways use the same RNA primer, which is capped and 18 or 19 nucleotides in length. At the completion of minus-strand DNA synthesis, the final RNase H cleavage generates the plus-strand primer. To make relaxed circular DNA, primer translocation must occur, resulting in the transfer of the primer generated at DR1 to the acceptor site (DR2) near the opposite end of the minus-strand DNA. A small fraction of viruses instead make duplex linear DNA after initiating plus-strand DNA synthesis from DR1, a process called in situ priming. We are interested in understanding the mechanism of discrimination between these two pathways. Some variants of duck hepatitis B virus exhibit high levels of in situ priming due to
cis-
acting mutations. The mechanism by which these mutations act has been obscure. Sequence inspection predicted formation of a small DNA hairpin in the region overlapping these mutations. We have shown that substitutions disrupting base pairing potential in this hairpin led to increased levels of in situ priming. The introduction of compensatory changes to restore base pairing potential led to reduced levels of in situ priming. Thus, formation of the small DNA hairpin overlapping the 5′ end of DR1 in the minus strand contributes to the regulation of primer translocation, at least, through inhibition of in situ priming by making the 3′ end of the minus-strand DNA a poor template for initiation.
Phosphorylation of the Epstein-Barr Virus (EBV) DNA Polymerase Processivity Factor EA-D by the EBV-Encoded Protein Kinase and Effects of the
l
-Riboside Benzimidazole 1263W94
Authors: Gershburg Edward , Pagano Joseph S. 
Published: October 2010

ABSTRACT

A member of the family of
l
-riboside benzimidazole compounds, 1263W94, was shown recently to inhibit replication of Epstein-Barr virus (EBV) (V. L. Zacny, E. Gershburg, M. G. Davis, K. K. Biron, and J. S. Pagano, J. Virol. 73:7271–7277, 1999). In the present report the effect of 1263W94 on the phosphorylation pattern of the EBV DNA polymerase processivity factor, EA-D, during viral reactivation in latently EBV-infected Akata cells is analyzed. This pattern specifically changes with progression of cytolytic infection. In the presence of 1263W94 the appearance of the hyperphosphorylated form of EA-D is mainly affected. Next, coexpression of the cloned EBV-encoded protein kinase (EBV PK), BGLF4, with EA-D demonstrated the ability of EBV PK to phosphorylate EA-D to its hyperphosphorylated form in transient assays. However, the phosphorylation of EA-D was not directly inhibited by 1263W94 in these coexpression assays. The results indicate that the EBV PK appears to be responsible for the hyperphosphorylation of EA-D, imply that the phosphorylation status of EA-D is important for viral replication, and suggest that 1263W94 acts at a level other than direct inhibition of EA-D phosphorylation by EBV PK.
Analysis of the C-Terminal Membrane Anchor Domains of Hepatitis C Virus Glycoproteins E1 and E2: toward a Topological Model
Authors: Charloteaux Benoit , Lins Laurence , Moereels Henri , Brasseur Robert 
Published: October 2010

ABSTRACT
The hepatitis C virus (HCV) glycoproteins E1 and E2 should be anchored in the viral membrane by their C-terminal domains. During synthesis, they are translocated to the endoplasmic reticulum (ER) lumen where they remain. The 31 C-terminal residues of the E1 protein and the 29 C-terminal residues of the E2 protein are implicated in the ER retention. Moreover, the E1 and E2 C termini are implicated in E1-E2 heterodimerization. We studied the E1 and E2 C-terminal sequences of 25 HCV strains in silico using molecular modeling techniques. We conclude that both C-terminal domains should adopt a similar and peculiar configuration: one amphipathic α-helix followed by a pair of transmembrane β-strands. Several three-dimensional (3-D) models were generated. After energy minimization, their ability to interact with membranes was studied using the molecular hydrophobicity potentials calculation and the IMPALA procedure. The latter simulates interactions with a membrane by a Monte Carlo minimization of energy. These methods suggest that the β-hairpins could anchor the glycoproteins in the ER membrane at least transiently. Anchoring could be stabilized by the adsorption of the nearby amphipathic α-helices at the membrane surface. The 3-D models correlate with experimental results which indicate that the E1-E2 transmembrane domains are involved in the heterodimerization and have ER retention properties.
Silencing of Autocrine Motility Factor Induces Mesenchymal-to-Epithelial Transition and Suppression of Osteosarcoma Pulmonary Metastasis
Authors: Niinaka Yasufumi , Harada Kiyoshi , Fujimuro Masahiro , Oda Masamitsu , Haga Arayo , Hosoki Misa , Uzawa Narikazu , Arai Naoya , Yamaguchi Satoshi , Yamashiro Masashi , Raz Avraham 
Published: October 2010

Abstract
Phosphoglucose isomerase (PGI) is a multifunctional enzyme that functions in glucose metabolism as a glycolytic enzyme catalyzing an interconversion between glucose and fructose inside the cell, while it acts as cytokine outside the cell, with properties that include autocrine motility factor (AMF)–regulating tumor cell motility. Overexpression of AMF/PGI induces epithelial-to-mesenchymal transition with enhanced malignancy. Recent studies have revealed that silencing of AMF/PGI resulted in mesenchymal-to-epithelial transition (MET) of human lung fibrosarcoma cells and breast cancer cells with reduced malignancy. Here, we constructed a hammerhead ribozyme specific against GUC triplet at the position G390 in the human, mouse, and rat AMF/PGI mRNA sequence. Mesenchymal human osteosarcoma MG-63, HS-Os-1, and murine LM8 cells were stably transfected with the ribozyme specific for AMF/PGI. The stable transfectant cells showed effective downregulation of AMF/PGI expression and subsequent abrogation of AMF/PGI secretion, which resulted in morphologic change with reduced growth, motility, and invasion. Silencing of AMF/PGI induced MET, in which upregulation of E-cadherin and cytokeratins, as well as downregulation of vimentin, were noted. The MET guided by AMF/PGI gene silencing induced osteosarcoma MG-63 to terminally differentiate into mature osteoblasts. Furthermore, MET completely suppressed the tumor growth and pulmonary metastasis of LM8 cells in nude mice. Thus, acquisition of malignancy might be completed in part by upregulation of AMF/PGI, and waiver of malignancy might also be controlled by downregulation of AMF/PGI. Cancer Res; 70(22); 9483–93. ©2010 AACR.
mTOR Complex Component Rictor Interacts with PKCζ and Regulates Cancer Cell Metastasis
Authors: Zhang Fei , Zhang Xiaofang , Li Menghui , Chen Peng , Zhang Bin , Guo Hua , Cao Wenfeng , Wei Xiying , Cao Xuchen , Hao Xishan , Zhang Ning 
Published: October 2010

Abstract
Epidermal growth factor (EGF) mediates breast cancer cell chemotaxis and metastasis through mechanisms that involve the growth-regulatory mammalian target of rapamycin (mTOR) complex mTORC2, but the mechanisms involved remain obscure. Here, we report that the rapamycin-insensitive mTORC2 component protein Rictor is a critical mediator of metastasis in breast cancer cells. In patients with ductal carcinoma, Rictor expression was associated with increased lymph node metastasis. EGF induced translocation and colocalization of Rictor with protein kinase Cζ (PKCζ), a pivotal molecule in chemotaxis signaling. Further, Rictor coimmunoprecipitated with PKCζ in the absence of the mTORC2 complex. Small interfering RNA–mediated knockdown of Rictor inhibited EGF-induced PKCζ phosphorylation and translocation along with phosphorylation of the key F-actin binding protein cofilin. In parallel, Rictor knockdown reduced cellular chemotactic capacity and ablated pulmonary metastasis in a xenograft mouse model of breast cancer. Our findings identify Rictor as an important mediator of chemotaxis and metastasis in breast cancer cells. Cancer Res; 70(22); 9360–70. ©2010 AACR.
BRAF Inactivation Drives Aneuploidy by Deregulating CRAF
Authors: Kamata Tamihiro , Hussain Jahan , Giblett Susan , Hayward Robert , Marais Richard , Pritchard Catrin 
Published: October 2010

Abstract
Aspartate-594 is the third most common BRAF residue mutated in human cancer. Mutants of this residue are kinase inactive, and the mechanism(s) by which they contribute to cancer has remained perplexing. Using a conditional knock-in mouse model, we show that the D594ABraf mutant does not drive tumor development per se but is able to induce aneuploidy in murine splenocytes and mouse embryonic fibroblasts and contributes to immortalization through the propagation of aneuploid cells. D594ABraf lacks kinase activity but induces the related gene product Craf as well as the mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK pathway. Here, we show that the aneuploid phenotype is dependent on Craf. Treatment with the MEK inhibitor U0126 did not attenuate the emergence of aneuploidy but prevented the growth of aneuploid cells. These results provide a previously unidentified link between Craf and chromosomal stability, with important implications for our understanding of the development of cancers with driver mutations that hyperactivate Craf. Cancer Res; 70(21); 8475–86. ©2010 AACR.