bluuebunny/crossref_metadata_2025_split huggingface dataset:
Role of a Mutation in Human Cytomegalovirus Gene UL104 in Resistance to Benzimidazole Ribonucleosides
Authors: Komazin Gloria , Townsend Leroy B. , Drach John C. 
Published: December 2003

ABSTRACT

The benzimidazole
d
-ribonucleosides TCRB and BDCRB are potent and selective inhibitors of human cytomegalovirus (HCMV) replication. Two HCMV strains resistant to these compounds were selected and had resistance mutations in genes UL89 and UL56. Proteins encoded by these two genes are the two subunits of the HCMV “terminase” and are necessary for cleavage and packaging of viral genomic DNA, a process inhibited by TCRB and BDCRB. We now report that both strains also have a previously unidentified mutation in UL104, the HCMV portal protein. This mutation, which results in L21F substitution, was introduced into the genome of wild-type HCMV by utilizing a recently cloned genome of HCMV as a bacterial artificial chromosome. The virus with this mutation alone was not resistant to BDCRB, suggesting that this site is not involved in binding benzimidazole nucleosides. As in previous proposals for mutations in UL104 of murine cytomegalovirus and HCMV strains resistant to BAY 38-4766, we hypothesize that this mutation could compensate for conformational changes in mutant UL89 and UL56 proteins, since the HCMV terminase is likely to interact with the portal protein during cleavage and packaging of genomic DNA.
Late Domain-Dependent Inhibition of Equine Infectious Anemia Virus Budding
Authors: Shehu-Xhilaga Miranda , Ablan Sherimay , Demirov Dimiter G. , Chen Chaoping , Montelaro Ronald C. , Freed Eric O. 
Published: December 2003

ABSTRACT
The Gag proteins of a number of different retroviruses contain late or L domains that promote the release of virions from the plasma membrane. Three types of L domains have been identified to date: Pro-Thr-Ala-Pro (PTAP), Pro-Pro-X-Tyr, and Tyr-Pro-Asp-Leu. It has previously been demonstrated that overexpression of the N-terminal, E2-like domain of the endosomal sorting factor TSG101 (TSG-5′) inhibits human immunodeficiency virus type 1 (HIV-1) release but does not affect the release of the PPPY-containing retrovirus murine leukemia virus (MLV), whereas overexpression of the C-terminal portion of TSG101 (TSG-3′) potently disrupts both HIV-1 and MLV budding. In addition, it has been reported that, while the release of a number of retroviruses is disrupted by proteasome inhibitors, equine infectious anemia virus (EIAV) budding is not affected by these agents. In this study, we tested the ability of TSG-5′, TSG-3′, and full-length TSG101 (TSG-F) overexpression, a dominant negative form of the AAA ATPase Vps4, and proteasome inhibitors to disrupt the budding of EIAV particles bearing each of the three types of L domain. The results indicate that (i) inhibition by TSG-5′ correlates with dependence on PTAP; (ii) the release of wild-type EIAV (EIAV/WT) is insensitive to TSG-3′, whereas this C-terminal TSG101 fragment potently impairs the budding of EIAV when it is rendered PTAP or PPPY dependent; (iii) budding of all EIAV clones is blocked by dominant negative Vps4; and (iv) EIAV/WT release is not impaired by proteasome inhibitors, while EIAV/PTAP and EIAV/PPPY release is strongly disrupted by these compounds. These findings highlight intriguing similarities and differences in host factor utilization by retroviral L domains and suggest that the insensitivity of EIAV to proteasome inhibitors is conferred by the L domain itself and not by determinants in Gag outside the L domain.
Efficient Intracellular Assembly of Papillomaviral Vectors
Authors: Buck Christopher B. , Pastrana Diana V. , Lowy Douglas R. , Schiller John T. 
Published: December 2003

ABSTRACTAlthough the papillomavirus structural proteins, L1 and L2, can spontaneously coassemble to form virus-like particles, currently available methods for production of L1/L2 particles capable of transducing reporter plasmids into mammalian cells are technically demanding and relatively low-yield. In this report, we describe a simple 293 cell transfection method for efficient intracellular production of papillomaviral-based gene transfer vectors carrying reporter plasmids. Using bovine papillomavirus type 1 (BPV1) and human papillomavirus type 16 as model papillomaviruses, we have developed a system for producing papillomaviral vector stocks with titers of several billion transducing units per milliliter. Production of these vectors requires both L1 and L2, and transduction can be prevented by papillomavirus-neutralizing antibodies. The stocks can be purified by an iodixanol (OptiPrep) gradient centrifugation procedure that is substantially more effective than standard cesium chloride gradient purification. Although earlier data had suggested a potential role for the viral early protein E2, we found that E2 protein expression did not enhance the intracellular production of BPV1 vectors. It was also possible to encapsidate reporter plasmids devoid of BPV1 DNA sequences. BPV1 vector production efficiency was significantly influenced by the size of the target plasmid being packaged. Use of 6-kb target plasmids resulted in BPV1 vector yields that were higher than those with target plasmids closer to the native 7.9-kb size of papillomavirus genomes. The results suggest that the intracellular assembly of papillomavirus structural proteins around heterologous reporter plasmids is surprisingly promiscuous and may be driven primarily by a size discrimination mechanism.
Human Immunodeficiency Virus Type 1 Tat Regulates Endothelial Cell Actin Cytoskeletal Dynamics through PAK1 Activation and Oxidant Production
Authors: Wu Ru Feng , Gu Ying , Xu You Cheng , Mitola Stefania , Bussolino Federico , Terada Lance S. 
Published: December 2003

ABSTRACT

Human immunodeficiency virus type 1 Tat exerts prominent angiogenic effects which may lead to a variety of vasculopathic conditions in AIDS patients. Because endothelial cells undergo prominent cytoskeletal rearrangement during angiogenesis, we investigated the specific effects of Tat on the endothelial cell actin cytoskeleton. Glutathione
S
-transferase (GST)-Tat, at a level of 200 ng/ml (equivalent to 52 ng of Tat/ml), caused stress fiber disassembly, peripheral retraction, and ruffle formation in human umbilical vein endothelial cells (HUVEC) and human lung microvascular endothelial cells. At 600 ng of GST-Tat/ml (157 ng of Tat/ml), actin structures were lost, and severe cytoskeletal collapse occurred. In contrast, GST-Tat harboring mutations within either the cysteine-rich or basic domains exerted minimal effects on the endothelial cytoskeleton. HUVEC expressing a DsRed-Tat fusion protein displayed similar actin rearrangements, followed by actin collapse, whereas neighboring nontransfected cells retained normal actin structures. Because active mutants of p21-activated kinase 1 (PAK1) induce identical changes in actin dynamics, we hypothesized that Tat exerts its cytoskeletal effects through PAK1. GST-Tat activated PAK1 within 5 min, and adenovirus delivery of a kinase-dead PAK1 [PAK1(K298A)] completely prevented cytoskeletal collapse induced by GST-Tat or DsRed-Tat and also blocked downstream activation of c-Jun N-terminal kinase. Further, GST-Tat increased phosphorylation of the NADPH oxidase subunit p47

phox

and caused its rapid redistribution to membrane ruffles. PAK1(K298A) blocked p47

phox

phosphorylation, and interference with NADPH oxidase function through superoxide scavenging or through expression of a transdominant inhibitor, p67(V204A), prevented GST-Tat-induced alterations in the actin cytoskeleton. We conclude that Tat induces actin cytoskeletal rearrangements through PAK1 and downstream activation of the endothelial NADPH oxidase.
NS-3 Protein of the
Junonia coenia
Densovirus Is Essential for Viral DNA Replication in an Ld 652 Cell Line and
Spodoptera littoralis
Larvae
Authors: Abd-Alla Adly , Jousset Françoise-Xavière , Li Yi , Fédière Gilles , Cousserans François , Bergoin Max 
Published: December 2003

ABSTRACT

The genome of
Junonia coenia
densovirus (JcDNV) shares with members of the genus
Densovirus
the property of possessing structural (VP) and nonstructural (NS) genes in opposite orientations. The three NS genes located in the 5′ half on one strand encode three NS proteins assumed to be involved in viral DNA replication: NS-1 and NS-2, which are common to all DNVs, and a 28-kDa polypeptide, NS-3, with a unique sequence. Whereas the essential role played by JcDNV NS-1 in viral DNA replication has been clearly established (C. Ding, M. Urabe, M. Bergoin, and R. M. Kotin, J. Virol.
76:
338-345, 2002), nothing is known of the biological function(s) of NS-3. To investigate this function, we designed constructs derived from pBRJ, a plasmid encompassing an infectious sequence of JcDNV DNA (M. Jourdan, F. X. Jousset, M. Gervais, S. Skory, M. Bergoin, and B. Dumas, Virology
179:
403-409, 1990), with partial or complete deletion of NS-3 sequence or with the ATG initiation codon mutated by site-directed mutagenesis. Transfection of these constructs to sensitive Ld 652 cells or
Spodoptera littoralis
larvae prevented the accomplishment of a productive cycle. We clearly established that the blocking of the replicative cycle in the absence of NS-3 expression occurred at the level of viral DNA replication. Replication of viral DNA could be restored by cotransfecting Ld 652 cells with a plasmid expressing JcDNV-NS-3 protein in
trans
. Time course analysis showed that NS-3 is produced early (6 h posttransfection) in the replicative cycle, and its production parallels that of replicative-form viral DNA. Finally, we present evidence that NS-1 and NS-2 proteins are synthesized at apparently the same levels whether or not NS-3 is expressed.
Functional Analysis of the Human Papillomavirus Type 16 E1
∧
E4 Protein Provides a Mechanism for In Vivo and In Vitro Keratin Filament Reorganization
Authors: Wang Qian , Griffin Heather , Southern Shirley , Jackson Deborah , Martin Ana , McIntosh Pauline , Davy Clare , Masterson Phillip J. , Walker Philip A. , Laskey Peter , Omary M. Bishr , Doorbar John 
Published: December 2003

ABSTRACT

High-risk human papillomaviruses, such as human papillomavirus type 16 (HPV16), are the primary cause of cervical cancer. The HPV16 E1
∧
E4 protein associates with keratin intermediate filaments and causes network collapse when expressed in epithelial cells in vitro. Here, we show that keratin association and network reorganization also occur in vivo in low-grade cervical neoplasia caused by HPV16. The 16E1
∧
E4 protein binds to keratins directly and interacts strongly with keratin 18, a member of the type I intermediate-filament family. By contrast, 16E1
∧
E4 bound only weakly to keratin 8, a type II intermediate-filament protein, and showed no detectable affinity for the type III protein, vimentin. The N-terminal 16 amino acids of the 16E1
∧
E4 protein, which contains the YPLLXLL motif that is conserved among supergroup A viruses, were sufficient to target green fluorescent protein to the keratin network. When expressed in the SiHa cervical epithelial cell line, the full-length 16E1
∧
E4 protein caused an almost total inhibition of keratin dynamics, despite the phosphorylation of keratin 18 at serine 33, which normally leads to 14-3-3-mediated keratin solubilization. Mutant 16E1
∧
E4 proteins which lack the LLKLL motif, or which have lost amino acids from their C termini, and which were compromised in the ability to associate with keratins did not disturb normal keratin dynamics. 16E1
∧
E4 was found to exist as dimers and hexamers, whereas a C-terminal deletion mutant (16E1
∧
E4Δ87-92) existed as monomers and formed multimeric structures only poorly. Considered together, our results suggest that by associating with keratins through its N terminus, and by associating with itself through its C terminus, 16E1
∧
E4 may act as a keratin cross-linker and prevent the movement of keratins between the soluble and insoluble compartments. The increase in avidity associated with multimeric binding may contribute to the ability of 16E1
∧
E4 to sequester its cellular targets in the cytoplasm.
Nipah Virus: Vaccination and Passive Protection Studies in a Hamster Model
Authors: Guillaume V. , Contamin H. , Loth P. , Georges-Courbot M.-C. , Lefeuvre A. , Marianneau P. , Chua K. B. , Lam S. K. , Buckland R. , Deubel V. , Wild T. F. 
Published: December 2003

ABSTRACT
Nipah virus, a member of the paramyxovirus family, was first isolated and identified in 1999 when the virus crossed the species barrier from fruit bats to pigs and then infected humans, inducing an encephalitis with up to 40% mortality. At present there is no prophylaxis for Nipah virus. We investigated the possibility of vaccination and passive transfer of antibodies as interventions against this disease. We show that both of the Nipah virus glycoproteins (G and F) when expressed as vaccinia virus recombinants induced an immune response in hamsters which protected against a lethal challenge by Nipah virus. Similarly, passive transfer of antibody induced by either of the glycoproteins protected the animals. In both the active and passive immunization studies, however, the challenge virus was capable of hyperimmunizing the vaccinated animals, suggesting that although the virus replicates under these conditions, the immune system can eventually control the infection.
Evidence for Frequent Recombination within SpeciesHuman Enterovirus BBased on Complete Genomic Sequences of All Thirty-Seven Serotypes
Authors: Oberste M. Steven , Maher Kaija , Pallansch Mark A. 
Published: December 2003

ABSTRACTThe speciesHuman enterovirus B(HEV-B) in the familyPicornaviridaeconsists of coxsackievirus A9; coxsackieviruses B1 to B6; echoviruses 1 to 7, 9, 11 to 21, 24 to 27, and 29 to 33; and enteroviruses 69 and 73. We have determined complete genome sequences for the remaining 22 HEV-B serotypes whose sequences were not represented in public databases and analyzed these in conjunction with previously available complete sequences in GenBank. Members of HEV-B were monophyletic relative to all other human enterovirus species in all regions of the genome except in the 5′-nontranslated region (NTR), where they are known to cluster with members of HEV-A. Within HEV-B, phylogenies constructed from the structural (P1) and nonstructural regions of the genome (P2 and P3) are incongruent, suggesting that recombination had occurred. Similarity plots and bootscanning analysis across the complete genome identified multiple sites at which the phylogeny of a given strain's sequence shifted, indicating potential recombination points. These points are distributed in the 5′-NTR and throughout P2 and P3, but no sites with >80% bootstrap support were identified within the capsid. Individual sequence comparisons and phylogenetic analyses suggest that members of HEV-B have recombined with one another on multiple occasions, resulting in a complex mosaic of sequences derived from multiple parental viruses in the nonstructural regions of the genome. We conclude that RNA recombination is a common mechanism for enterovirus evolution and that recombination within the nonstructural regions of the genome (P2 and P3) has been observed only among members of the same species.
Differential Effects of Actin Cytoskeleton Dynamics on Equine Infectious Anemia Virus Particle Production
Authors: Chen Chaoping , Weisz Ora A. , Stolz Donna B. , Watkins Simon C. , Montelaro Ronald C. 
Published: December 2003

ABSTRACTRetrovirus assembly and budding involve a highly dynamic and concerted interaction of viral and cellular proteins. Previous studies have shown that retroviral Gag proteins interact with actin filaments, but the significance of these interactions remains to be defined. Using equine infectious anemia virus (EIAV), we now demonstrate differential effects of cellular actin dynamics at distinct stages of retrovirus assembly and budding. First, virion production was reduced when EIAV-infected cells were treated with phallacidin, a cell-permeable reagent that stabilizes actin filaments by slowing down their depolymerization. Confocal microscopy confirmed that the inhibition of EIAV production correlated temporally over several days with the incorporation dynamics of phallacidin into the actin cytoskeleton. Although the overall structure of the actin cytoskeleton and expression of viral protein appeared to be unaffected, phallacidin treatment dramatically reduced the amount of full-length Gag protein associated with the actin cytoskeleton. These data suggest that an association of full-length Gag proteins with de novo actin filaments might contribute to Gag assembly and budding. On the other hand, virion production was enhanced when EIAV-infected cells were incubated briefly (2 h) with the actin-depolymerizing drugs cytochalasin D and latrunculin B. Interestingly, the enhanced virion production induced by cytochalasin D required a functional late (L) domain, either the EIAV YPDL L-domain or the proline-rich L domains derived from human immunodeficiency virus type 1 or Rous sarcoma virus, respectively. Thus, depolymerization of actin filaments may be a common function mediated by retrovirus L domains during late stages of viral budding. Taken together, these observations indicate that dynamic actin polymerization and depolymerization may be associated with different stages of viral production.
A Reovirus of the Fungus
Cryphonectria parasitica
That Is Infectious as Particles and Related to the
Coltivirus
Genus of Animal Pathogens
Authors: Hillman Bradley I. , Supyani S. , Kondo Hideki , Suzuki Nobuhiro 
Published: December 2003

ABSTRACT

RNA viruses of filamentous fungi fall into two broad categories, those that contain double-stranded RNA (dsRNA) genomes in rigid particles and those that are more closely related to positive-sense, single-stranded RNA viruses with dsRNA replicative intermediates found within lipid vesicles. Effective infectivity systems have been described for the latter, using RNA transcripts, but not for the former. We report the characterization of a reovirus from
Cryphonectria parasitica
, the filamentous fungus that causes chestnut blight disease. The virus substantially reduces the virulence of the fungus and results in dramatically altered colony morphology, as well as changes in other associated fungal traits, relative to the virus-free isogenic strain. Virus particles from infected mycelium contained 11 segments of dsRNA and showed characteristics typical of the family
Reoviridae
. Sequences of the largest three segments revealed that the virus is closely related to the
Coltivirus
genus of animal pathogens, which includes the human pathogen
Colorado tick fever virus
. The introduction of purified virus particles into protoplasts from virus-free isolates of the fungus resulted in a newly infected mycelium with the same morphology and virus composition as the original virus-infected isolate. This represents the completion of Koch's postulates for a true dsRNA virus from a filamentous fungus and the description of a definitive fungal member of the family
Reoviridae
.
Genome Structure and Thymic Expression of an Endogenous Retrovirus in Zebrafish
Authors: Shen Ching-Hung , Steiner Lisa A. 
Published: December 2003

ABSTRACT

In a search for previously unknown genes that are required for lymphocyte development in zebrafish, a retroviral sequence was identified in a subtracted thymus cDNA library and in genomic DNA libraries. The provirus is 11.2 kb and contains intact open reading frames for the
gag
,
pol
, and
env
genes, as well as nearly identical flanking long terminal repeat sequences. As determined by in situ hybridization, the thymus appears to be a major tissue for retroviral expression in both larval and adult fish. Several viral transcripts were found by Northern blotting in the adult thymus. The provirus was found at the same genomic locus in sperm from four fish, suggesting that it is an endogenous retrovirus. Phylogenetic analysis indicates that it is closest to, yet distinct from, the cluster of murine leukemia virus-related retroviruses, suggesting that this virus represents a new group of retroviruses.
Secretory Immunoglobulin A Antibodies against the σ1 Outer Capsid Protein of Reovirus Type 1 Lang Prevent Infection of Mouse Peyer's Patches
Authors: Hutchings Amy B. , Helander Anna , Silvey Katherine J. , Chandran Kartik , Lucas William T. , Nibert Max L. , Neutra Marian R. 
Published: December 2003

ABSTRACTReovirus type 1 Lang (T1L) adheres to M cells in the follicle-associated epithelium of mouse intestine and exploits the transport activity of M cells to enter and infect the Peyer's patch mucosa. Adult mice that have previously cleared a reovirus T1L infection have virus-specific immunoglobulin G (IgG) in serum and IgA in secretions and are protected against reinfection. Our aim in this study was to determine whether secretory IgA is sufficient for protection of Peyer's patches against oral reovirus challenge and, if so, against which reovirus antigen(s) the IgA may be directed. Monoclonal antibodies (MAbs) of the IgA isotype, directed against the σ1 protein of reovirus T1L, the viral adhesin, were produced and tested along with other, existing IgA and IgG MAbs against reovirus T1L outer capsid proteins. Anti-σ1 IgA and IgG MAbs neutralized reovirus T1L in L cell plaque reduction assays and inhibited T1L adherence to L cells and Caco-2BBeintestinal epithelial cells in vitro, but MAbs against other proteins did not. Passive oral administration of anti-σ1 IgA and IgG MAbs prevented Peyer's patch infection in adult mice, but other MAbs did not. When anti-σ1 IgA and IgG MAbs were produced in mice from hybridoma backpack tumors, however, the IgA prevented Peyer's patch infection, but the IgG did not. The results provide evidence that neutralizing IgA antibodies specific for the σ1 protein are protective in vitro and in vivo and that the presence of these antibodies in intestinal secretions is sufficient for protection against entry of reovirus T1L into Peyer's patches.
Unintentional and undetermined firearm related deaths: a preventable death analysis for three safety devices
Authors: Vernick J S , O’Brien M , Hepburn L M , Johnson S B , Webster D W , Hargarten S W 
Published: December 2003

Objective:To determine the proportion of unintentional and undetermined firearm related deaths preventable by three safety devices: personalization devices, loaded chamber indicators (LCIs), and magazine safeties. A personalized gun will operate only for an authorized user, a LCI indicates when the gun contains ammunition, and a magazine safety prevents the gun from firing when the ammunition magazine is removed.Design:Information about all unintentional and undetermined firearm deaths from 1991–98 was obtained from the Office of the Chief Medical Examiner for Maryland, and from the Wisconsin Firearm Injury Reporting System for Milwaukee. Data regarding the victim, shooter, weapon, and circumstances were abstracted. Coding rules to classify each death as preventable, possibly preventable, or not preventable by each of the three safety devices were also applied.Results:There were a total of 117 firearm related deaths in our sample, 95 (81%) involving handguns. Forty three deaths (37%) were classified as preventable by a personalized gun, 23 (20%) by a LCI, and five (4%) by a magazine safety. Overall, 52 deaths (44%) were preventable by at least one safety device. Deaths involving children 0–17 (relative risk (RR) 3.3, 95% confidence interval (CI) 2.1 to 5.1) and handguns (RR 8.1, 95% CI 1.2 to 53.5) were more likely to be preventable. Projecting the findings to the entire United States, an estimated 442 deaths might have been prevented in 2000 had all guns been equipped with these safety devices.Conclusion:Incorporating safety devices into firearms is an important injury intervention, with the potential to save hundreds of lives each year.