bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Reovirus S4 Gene 3′ Nontranslated Region Contains a Translational Operator Sequence
Authors: Mochow-Grundy Michelle , Dermody Terence S. 
Published: July 2002

ABSTRACT
Reovirus mRNAs are efficiently translated within host cells despite the absence of 3′ polyadenylated tails. The 3′ nontranslated regions (3′NTRs) of reovirus mRNAs contain sequences that exhibit a high degree of gene-segment-specific conservation. To determine whether the 3′NTRs of reovirus mRNAs serve to facilitate efficient translation of viral transcripts, we used T7 RNA polymerase to express constructs engineered with full-length S4 gene cDNA or truncation mutants lacking sequences in the 3′NTR. Full-length and truncated s4 mRNAs were translated using rabbit reticulocyte lysates, and translation product ς3 was quantitated by phosphorimager analysis. In comparison to full-length s4 mRNA, translation of the s4 mRNA lacking the 3′NTR resulted in a 20 to 50% decrease in ς3 produced. Addition to translation reactions of an RNA oligonucleotide corresponding to the S4 3′NTR significantly enhanced translation of full-length s4 mRNA but had no effect on s4 mRNA lacking 3′NTR sequences. Translation of s4 mRNAs with smaller deletions within the 3′NTR identified a discrete region capable of translational enhancement and a second region capable of translational repression. Differences in translational efficiency of full-length and deletion-mutant mRNAs were independent of RNA stability. Protein complexes in reticulocyte lysates that specifically interact with the S4 3′NTR were identified by RNA mobility shift assays. RNA oligonucleotides lacking either enhancer or repressor sequences did not efficiently compete the binding of these complexes to full-length 3′NTR. These results indicate that the reovirus S4 gene 3′NTR contains a translational operator sequence that serves to regulate translational efficiency of the s4 mRNA. Moreover, these findings suggest that cellular proteins interact with reovirus 3′NTR sequences to regulate translation of the nonpolyadenylated reovirus mRNAs.
Gag-Pol Supplied in
trans
Is Efficiently Packaged and Supports Viral Function in Human Immunodeficiency Virus Type 1
Authors: Hill M. K. , Hooker C. W. , Harrich D. , Crowe S. M. , Mak J. 
Published: July 2002

ABSTRACT
The intracellular trafficking and subsequent incorporation of Gag-Pol into human immunodeficiency virus type 1 (HIV-1) remains poorly defined. Gag-Pol is encoded by the same mRNA as Gag and is generated by ribosomal frameshifting. The multimerization of Gag and Gag-Pol is an essential step in the formation of infectious viral particles. In this study, we examined whether the interaction between Gag and Gag-Pol is initiated during protein translation in order to facilitate the trafficking and subsequent packaging of Gag-Pol into the virion. A conditional cotransfection system was developed in which virion formation required the coexpression of two HIV-1-based plasmids, one that produces both Gag and Gag-Pol and one that only produces Gag-Pol. The Gag-Pol proteins were either immunotagged with a His epitope or functionally tagged with a mutation (K65R) in reverse transcriptase that is associated with drug resistance. Gag-Pol packaging was assessed to determine whether the Gag-Pol incorporated into the virion was preferentially packaged from the plasmid that expressed both Gag and Gag-Pol or whether it could be packaged from either plasmid. Our data show that translation of Gag and Gag-Pol from the same mRNA is not critical for virion packaging of the Gag-Pol polyprotein or for viral function.
Construction, Safety, and Immunogenicity in Nonhuman Primates of a Chimeric Yellow Fever-Dengue Virus Tetravalent Vaccine
Authors: Guirakhoo F. , Arroyo J. , Pugachev K. V. , Miller C. , Zhang Z.-X. , Weltzin R. , Georgakopoulos K. , Catalan J. , Ocran S. , Soike K. , Ratterree M. , Monath T. P. 
Published: July 2002

ABSTRACTWe previously reported construction of a chimeric yellow fever-dengue type 2 virus (YF/DEN2) and determined its safety and protective efficacy in rhesus monkeys (F. Guirakhoo et al., J. Virol. 74:5477–5485, 2000). In this paper, we describe construction of three additional YF/DEN chimeras using premembrane (prM) and envelope (E) genes of wild-type (WT) clinical isolates: DEN1 (strain PUO359, isolated in 1980 in Thailand), DEN3 (strain PaH881/88, isolated in 1988 in Thailand), and DEN4 (strain 1228, isolated in 1978 in Indonesia). These chimeric viruses (YF/DEN1, YF/DEN3, and YF/DEN4) replicated to ∼7.5 log10PFU/ml in Vero cells, were not neurovirulent in 3- to 4-week-old ICR mice inoculated by the intracerebral route, and were immunogenic in monkeys. All rhesus monkeys inoculated subcutaneously with one dose of these chimeric viruses (as monovalent or tetravalent formulation) developed viremia with magnitudes similar to that of the YF 17D vaccine strain (YF-VAX) but significantly lower than those of their parent WT viruses. Eight of nine monkeys inoculated with monovalent YF/DEN1 -3, or -4 vaccine and six of six monkeys inoculated with tetravalent YF/DEN1-4 vaccine seroconverted after a single dose. When monkeys were boosted with a tetravalent YF/DEN1-4 dose 6 months later, four of nine monkeys in the monovalent YF/DEN groups developed low levels of viremia, whereas no viremia was detected in any animals previously inoculated with either YF/DEN1-4 vaccine or WT DEN virus. An anamnestic response was observed in all monkeys after the second dose. No statistically significant difference in levels of neutralizing antibodies was observed between YF virus-immune and nonimmune monkeys which received the tetravalent YF/DEN1-4 vaccine or between tetravalent YF/DEN1-4-immune and nonimmune monkeys which received the YF-VAX. However, preimmune monkeys developed either no detectable viremia or a level of viremia lower than that in nonimmune controls. This is the first recombinant tetravalent dengue vaccine successfully evaluated in nonhuman primates.
Serum and Mucosal Immune Responses to an Inactivated Influenza Virus Vaccine Induced by Epidermal Powder Immunization
Authors: Chen Dexiang , Periwal Sangeeta B. , Larrivee Katherine , Zuleger Cindy , Erickson Cherie A. , Endres Ryan L. , Payne Lendon G. 
Published: July 2002

ABSTRACTBoth circulating and mucosal antibodies are considered important for protection against infection by influenza virus in humans and animals. However, current inactivated vaccines administered by intramuscular injection using a syringe and needle elicit primarily circulating antibodies. In this study, we report that epidermal powder immunization (EPI) via a unique powder delivery system elicits both serum and mucosal antibodies to an inactivated influenza virus vaccine. Serum antibody responses to influenza vaccine following EPI were enhanced by codelivery of cholera toxin (CT), a synthetic oligodeoxynucleotide containing immunostimulatory CpG motifs (CpG DNA), or the combination of these two adjuvants. In addition, secretory immunoglobulin A (sIgA) antibodies were detected in the saliva and mucosal lavages of the small intestine, trachea, and vaginal tract, although the titers were much lower than the IgG titers. The local origin of the sIgA antibodies was further shown by measuring antibodies released from cultured tracheal and small intestinal fragments and by detecting antigen-specific IgA-secreting cells in the lamina propria using ELISPOT assays. EPI with a single dose of influenza vaccine containing CT or CT and CpG DNA conferred complete protection against lethal challenges with an influenza virus isolated 30 years ago, whereas a prime and boost immunizations were required for protection in the absence of an adjuvant. The ability to elicit augmented circulating antibody and mucosal antibody responses makes EPI a promising alternative to needle injection for administering vaccines against influenza and other diseases.
In Vitro Activation of Feline Immunodeficiency Virus in Ramified Microglial Cells from Asymptomatically Infected Cats
Authors: Hein Andreas , Martin Jean-Pierre , Dörries Rüdiger 
Published: July 2002

ABSTRACTIntravenous infection of cats with feline immunodeficiency virus was used as a model system to study activation of virus replication in brain-resident microglial cells in vitro. Virus release by ramified microglial cells isolated from subclinically infected animals was detectable in cell-free tissue culture supernatant only by reverse transcription and nested PCR ofgag-specific RNA sequences and not by virion-associated reverse transcriptase activity. In contrast, cocultivation of in vivo-infected microglial cells with mitogen-activated peripheral blood mononuclear cells (PBMC) regularly allows detection of high virus yields in cell-free tissue culture fluid. Besides uptake and multiplication of microglia-derived virus in PBMC, release of virus from microglia is stimulated by cell contact with PBMC. The data suggest that T lymphocytes patrolling the central nervous system could reactivate the semilatent state of lentiviruses in microglial cells in the course of clinically silent central nervous system infection.
An Epstein-Barr Virus Isolated from a Lymphoblastoid Cell Line Has a 16-Kilobase-Pair Deletion Which Includes gp350 and the Epstein-Barr Virus Nuclear Antigen 3A
Authors: Lee Wonkeun , Hwang Yoon-Ha , Lee Suk-Kyeong , Subramanian Chitra , Robertson Erle S. 
Published: July 2002

ABSTRACT

Epstein-Barr virus
(EBV) is associated with human cancers, including nasopharyngeal carcinoma, Burkitt's lymphoma, gastric carcinoma and, somewhat controversially, breast carcinoma. EBV infects and efficiently transforms human primary B lymphocytes in vitro. A number of EBV-encoded genes are critical for EBV-mediated transformation of human B lymphocytes. In this study we show that an EBV-infected lymphoblastoid cell line obtained from the spontaneous outgrowth of B cells from a leukemia patient contains a deletion, which involves a region of approximately 16 kbp. This deletion encodes major EBV genes involved in both infection and transformation of human primary B lymphocytes and includes the glycoprotein gp350, the entire open reading frame of EBNA3A, and the amino-terminal region of EBNA3B. A fusion protein created by this deletion, which lies between the BMRF1 early antigen and the EBNA3B latent antigen, is truncated immediately downstream of the junction 21 amino acids into the region of the EBNA3B sequence, which is out of frame with respect to the EBNA3B protein sequence, and indicates that EBNA3B is not expressed. The fusion is from EBV coordinate 80299 within the BMRF1 sequence to coordinate 90998 in the EBNA3B sequence. Additionally, we have shown that there is no detectable induction in viral replication observed when SNU-265 is treated with phorbol esters, and no transformants were detected when supernatant is used to infect primary B lymphocytes after 8 weeks in culture. Therefore, we have identified an EBV genome with a major deletion in critical genes involved in mediating EBV infection and the transformation of human primary B lymphocytes that is incompetent for replication of this naturally occurring EBV isolate.
Caspase Activation by Adenovirus E4orf4 Protein Is Cell Line Specific and Is Mediated by the Death Receptor Pathway
Authors: Livne Adi , Shtrichman Ronit , Kleinberger Tamar 
Published: July 2002

ABSTRACTAdenovirus E4orf4 protein has been shown to induce transformed cell-specific, protein phosphatase 2A-dependent, and p53-independent apoptosis. It has been further reported that the E4orf4 apoptotic pathway is caspase-independent in CHO cells. Here, we show that E4orf4 induces caspase activation in the human cell lines H1299 and 293T. Caspase activation is required for apoptosis in 293T cells, but not in H1299 cells. Dominant negative mutants of caspase-8 and the death receptor adapter protein FADD/MORT1 inhibit E4orf4-induced apoptosis in 293T cells, suggesting that E4orf4 activates the death receptor pathway. Cytochromecis released into the cytosol in E4orf4-expressing cells, but caspase-9 is not required for induction of apoptosis. Furthermore, E4orf4 induces accumulation of reactive oxygen species (ROS) in a caspase-8- and FADD/MORT1-dependent manner, and inhibition of ROS generation by 4,5-dihydroxy-1,3-benzene-disulfonic acid (Tiron) inhibits E4orf4-induced apoptosis. Thus, our results demonstrate that E4orf4 engages the death receptor pathway to generate at least part of the molecular events required for E4orf4-induced apoptosis.
Clustered Charge-to-Alanine Mutagenesis of the Vaccinia Virus A20 Gene: Temperature-Sensitive Mutants Have a DNA-Minus Phenotype and Are Defective in the Production of Processive DNA Polymerase Activity
Authors: Punjabi Almira , Boyle Kathleen , DeMasi Joseph , Grubisha Olivera , Unger Beth , Khanna Marilyn , Traktman Paula 
Published: July 2002

ABSTRACT

Although the vaccinia virus DNA polymerase is inherently distributive, a highly processive form of the enzyme exists within the cytoplasm of infected cells (W. F. McDonald, N. Klemperer, and P. Traktman, Virology 234:168–175, 1997). In the accompanying report we outline the purification of the 49-kDa A20 protein as a stoichiometric component of the processive polymerase complex (N. Klemperer, W. McDonald, K. Boyle, B. Unger, and P. Traktman, J. Virol. 75:12298–12307, 2001). To complement this biochemical analysis, we undertook a genetic approach to the analysis of the structure and function of the A20 protein. Here we report the application of clustered charge-to-alanine mutagenesis of the A20 gene. Eight mutant viruses containing altered A20 alleles were isolated using this approach; two of these,
ts
A20-6 and
ts
A20-ER5, have tight temperature-sensitive phenotypes. At the nonpermissive temperature, neither virus forms macroscopic plaques and the yield of infectious virus is <1% of that obtained at the permissive temperature. Both viruses show a profound defect in the accumulation of viral DNA at the nonpermissive temperature, although both the A20 protein and DNA polymerase accumulate to wild-type levels. Cytoplasmic extracts prepared from cells infected with the
ts
A20 viruses show a defect in processive polymerase activity; they are unable to direct the formation of RFII product using a singly primed M13 template. In sum, these data indicate that the A20 protein plays an essential role in the viral life cycle and that viruses with A20 lesions exhibit a DNA
−
phenotype that is correlated with a loss in processive polymerase activity as assayed in vitro. The vaccinia virus A20 protein can, therefore, be considered a new member of the family of proteins (E9, B1, D4, and D5) with essential roles in vaccinia virus DNA replication.
NSP4 Enterotoxin of Rotavirus Induces Paracellular Leakage in Polarized Epithelial Cells
Authors: Tafazoli Farideh , Zeng Carl Q. , Estes Mary K. , Magnusson Karl-Erik , Svensson Lennart 
Published: July 2002

ABSTRACT
The nonstructural NSP4 protein of rotavirus has been described as the first viral enterotoxin. In this study we have examined the effect of NSP4 on polarized epithelial cells (MDCK-1) grown on permeable filters. Apical but not basolateral administration of NSP4 was found to cause a reduction in the transepithelial electrical resistance, redistribution of filamentous actin, and an increase in paracellular passage of fluorescein isothiocyanate-dextran. Significant effects on transepithelial electrical resistance were noted after a 20- to 30-h incubation with 1 nmol of NSP4. Most surprisingly, the epithelium recovered its original integrity and electrical resistance upon removal of NSP4. Preincubation of nonconfluent MDCK-1 cells with NSP4 prevented not only development of a permeability barrier but also lateral targeting of the tight-junction-associated Zonula Occludens-1 (ZO-1) protein. Taken together, these data indicate new and specific effects of NSP4 on tight-junction biogenesis and show a novel effect of NSP4 on polarized epithelia.
L1 Interaction Domains of Papillomavirus L2 Necessary for Viral Genome Encapsidation
Authors: Okun Martin M. , Day Patricia M. , Greenstone Heather L. , Booy Frank P. , Lowy Douglas R. , Schiller John T. , Roden Richard B. S. 
Published: July 2002

ABSTRACT
BPHE-1 cells, which harbor 50 to 200 viral episomes, encapsidate viral genome and generate infectious bovine papillomavirus type 1 (BPV1) upon coexpression of capsid proteins L1 and L2 of BPV1, but not coexpression of BPV1 L1 and human papillomavirus type 16 (HPV16) L2. BPV1 L2 bound in vitro via its C-terminal 85 residues to purified L1 capsomers, but not with intact L1 virus-like particles in vitro. However, when the efficiency of BPV1 L1 coimmunoprecipitation with a series of BPV1 L2 deletion mutants was examined in vivo, the results suggested that residues 129 to 246 and 384 to 460 contain independent L1 interaction domains. An L2 mutant lacking the C-terminal L1 interaction domain was impaired for encapsidation of the viral genome. Coexpression of BPV1 L1 and a chimeric L2 protein composed of HPV16 L2 residues 1 to 98 fused to BPV1 L2 residues 99 to 469 generated infectious virions. However, inefficient encapsidation was seen when L1 was coexpressed with either BPV1 L2 with residues 91 to 246 deleted or with BPV1 L2 with residues 1 to 225 replaced with HPV16 L2. Impaired genome encapsidation did not correlate closely with impairment of the L2 proteins either to localize to promyelocytic leukemia oncogenic domains (PODs) or to induce localization of L1 or E2 to PODs. We conclude that the L1-binding domain located near the C terminus of L2 may bind L1 prior to completion of capsid assembly, and that both L1-binding domains of L2 are required for efficient encapsidation of the viral genome.
Calcineurin-Dependent Mitochondrial Disturbances in Calcium-Induced Apoptosis of Human Immunodeficiency Virus gp160-Expressing CD4+Cells
Authors: Sasaki Masafumi , Miyazaki Kozo , Koga Yasuhiro , Kimura Genki , Nomoto Kikuo , Yoshida Hiroki 
Published: July 2002

ABSTRACTIn CD4+UE160 cells with inducible expression of gp160, mechanisms of apoptosis induced by human immunodeficiency virus (HIV) Env protein were analyzed. Induction of gp160 caused intracellular calcium increase followed by the release of cytochromecfrom mitochondria, which was inhibited by calcineurin inhibitors. Association of BAD with Bcl-xL was observed, and a portion of BAD was dephosphorylated after induction of gp160. These data suggested that calcineurin plays a role in the HIV Env-induced apoptosis in a mitochondrion-dependent way.
Substitution in the Murine Nectin1 Receptor of a Single Conserved Amino Acid at a Position Distal from the Herpes Simplex Virus gD Binding Site Confers High-Affinity Binding to gD
Authors: Menotti Laura , Casadio Rita , Bertucci Carlo , Lopez Marc , Campadelli-Fiume Gabriella 
Published: July 2002

ABSTRACT
By analogy with its human nectin1 counterpart, murine nectin1 serves as a cellular receptor for the entry of herpes simplex virus (HSV) into murine cells. HSV entry mediated by either receptor is dependent on the viral glycoprotein D (gD). Whereas human nectin1 binds gD at high affinity and in a saturable manner, murine nectin1 binds gD in a barely detectable fashion, depending on the sensitivity of the assay. The immunoglobulin type V domain of murine nectin differs from its human counterpart in 11 amino acids. To identify the key residues responsible for the high-affinity binding of gD to human nectin1, we replaced each of the 11 divergent amino acids with the human counterparts singly or in groups in an incremental manner. Replacement in murine nectin1 of six amino acids that lie within the gD binding region of human nectin1 (previously mapped to residues 64 to 94, likely the CC′C″ surface) increased the gD binding activity to a limited extent. In contrast, the single P138L substitution, which lies distal from the gD binding site, markedly increased gD binding. This substitution, when coupled with downstream substitutions, exerted the greatest effect. Three-dimensional modeling of the nectin1 V domain suggested that P138 in murine nectin1 might decrease the stability of the V domain by reducing the size of β-strand G. The results support the notion that the overall structure of V nectin1 plays a pivotal role in its ability to bind HSV gD.
Vector Infection Determinants of Venezuelan Equine Encephalitis Virus Reside within the E2 Envelope Glycoprotein
Authors: Brault Aaron C. , Powers Ann M. , Weaver Scott C. 
Published: July 2002

ABSTRACT

Epizootic subtype IAB and IC Venezuelan equine encephalitis viruses (VEEV) readily infect the epizootic mosquito vector
Aedes taeniorhynchus
. The inability of enzootic subtype IE viruses to infect this mosquito species provides a model system for the identification of natural viral determinants of vector infectivity. To map mosquito infection determinants, reciprocal chimeric viruses generated from epizootic subtype IAB and enzootic IE VEEV were tested for mosquito infectivity. Chimeras containing the IAB epizootic structural gene region and, more specifically, the IAB PE2 envelope glycoprotein E2 precursor gene demonstrated an efficient infection phenotype. Introduction of the PE2 gene from an enzootic subtype ID virus into an epizootic IAB or IC genetic backbone resulted in lower infection rates than those of the epizootic parent. The finding that the E2 envelope glycoprotein, the site of epitopes that define the enzootic and epizootic subtypes, also encodes mosquito infection determinants suggests that selection for efficient infection of epizootic mosquito vectors may mediate VEE emergence.
Parvovirus Initiator Protein NS1 and RPA Coordinate Replication Fork Progression in a Reconstituted DNA Replication System
Authors: Christensen Jesper , Tattersall Peter 
Published: July 2002

ABSTRACT

We show here that the DNA helicase activity of the parvoviral initiator protein NS1 is highly directional, binding to the single strand at a recessed 5′ end and displacing the other strand while progressing in a 3′-to-5′ direction on the bound strand. NS1 and a cellular site-specific DNA binding factor, PIF, also known as glucocorticoid modulating element binding protein, bind to the left-end minimal replication origin of minute virus of mice, forming a ternary complex. In this complex, NS1 is activated to nick one DNA strand, becoming covalently attached to the 5′ end of the nick in the process and providing a 3′ OH for priming DNA synthesis. In this situation, the helicase activity of NS1 did not displace the nicked strand, but the origin duplex was distorted by the NS1-PIF complex, as assayed by its sensitivity to KMnO
4
oxidation, and a stretch of about 14 nucleotides on both strands of the nicked origin underwent limited unwinding. Addition of
Escherichia coli
single-stranded DNA binding protein (SSB) did not lead to further unwinding. However, addition of recombinant human single-stranded DNA binding protein (RPA) to the initiation reaction catalyzed extensive unwinding of the nicked origin, suggesting that RPA may be required to form a functional replication fork. Accordingly, the unwinding mediated by NS1 and RPA promoted processive leading-strand synthesis catalyzed by recombinant human DNA polymerase δ, PCNA, and RFC, using the minimal left-end origin cloned in a plasmid as a template. The requirement for RPA, rather than SSB, in the unwinding reaction indicated that specific NS1-RPA protein interactions were formed. NS1 was tested by enzyme-linked immunosorbent assay for binding to two- or three-subunit RPA complexes expressed from recombinant baculoviruses. NS1 efficiently bound each of the baculovirus-expressed complexes, indicating that the small subunit of RPA is not involved in specific NS1 binding. No NS1 interactions were observed with
E. coli
SSB or other proteins included as controls.