bluuebunny/crossref_metadata_2025_split huggingface dataset:
Treatment with Anti-LFA-1 Delays the CD8+Cytotoxic-T-Lymphocyte Response and Viral Clearance in Mice with Primary Respiratory Syncytial Virus Infection
Authors: Rutigliano John A. , Johnson Teresa R. , Hollinger Tonya N. , Fischer Julie E. , Aung Sandra , Graham Barney S. 
Published: February 2004

ABSTRACTCytotoxic T lymphocytes (CTLs) play an important role in the immune response against respiratory syncytial virus (RSV) infection. The cell surface molecule lymphocyte function-associated antigen 1 (LFA-1) is an important contributor to CTL activation, CTL-mediated direct cell lysis, and lymphocyte migration. In an attempt to determine the role of LFA-1 during RSV infection, we treated BALB/c mice with monoclonal antibodies to LFA-1 at days −1, +1, and +4 relative to primary RSV infection. Anti-LFA-1 treatment during primary RSV infection led to reduced illness and delayed clearance of virus-infected cells. CTLs from RSV-infected mice that were treated with anti-LFA-1 exhibited diminished cytolytic activity and reduced gamma interferon production. In addition, studies with BrdU (5-bromo-2′-deoxyuridine)- and CFSE [5-(and 6)-carboxyfluorescein diacetate succinimidyl ester]-labeled lymphocytes showed that anti-LFA-1 treatment led to delayed proliferation during RSV infection. These results indicate that LFA-1 plays a critical role in the initiation of the immune response to RSV infection by facilitating CTL activation. These results may prove useful in the development of new therapies to combat RSV infection or other inflammatory diseases.
Simultaneous Deletion of Pseudorabies Virus Tegument Protein UL11 and Glycoprotein M Severely Impairs Secondary Envelopment
Authors: Kopp Martina , Granzow Harald , Fuchs Walter , Klupp Barbara , Mettenleiter Thomas C. 
Published: February 2004

ABSTRACT
The pseudorabies virus (PrV) proteins UL11, glycoprotein E (gE), and gM are involved in secondary envelopment of tegumented nucleocapsids in the cytoplasm. To assess the relative contributions of these proteins to the envelopment process, virus mutants with deletions of either UL11, gM, or gE as well as two newly constructed mutant viruses with simultaneous deletions of UL11 and gE or of UL11 and gM were analyzed in cell culture for their growth phenotype. We show here that simultaneous deletion of UL11 and gE reduced plaque size in an additive manner over the reduction observed by deletion of only UL11 or gE. However, one-step growth was not further impaired beyond the level of the UL11 deletion mutant. Moreover, in electron microscopic analyses PrV-ΔUL11/gE exhibited a phenotype similar to that of the UL11 mutant virus. In contrast, plaque formation was virtually abolished by the simultaneous absence of UL11 and gM, and one-step growth was significantly reduced. Electron microscopy showed the presence of huge intracytoplasmic inclusions in PrV-ΔUL11/gM-infected cells, with a size reaching 3 μm and containing nucleocapsids embedded in tegument. We hypothesize that UL11 and gM are involved in different steps during secondary envelopment and that simultaneous deletion of both interrupts both processes, resulting in the observed drastic impairment of secondary envelopment.
Impaired Lymphoid Chemokine-Mediated Migration due to a Block on the Chemokine Receptor Switch in Human Cytomegalovirus-Infected Dendritic Cells
Authors: Moutaftsi Magdalena , Brennan Paul , Spector Stephen A. , Tabi Zsuzsanna 
Published: February 2004

ABSTRACTDendritic cell (DC) migration from the site of infection to the site of T-cell priming is a crucial event in the generation of antiviral T-cell responses. Here we present to our knowledge the first functional evidence that human cytomegalovirus (HCMV) blocks the migration of infected monocyte-derived DCs toward lymphoid chemokines CCL19 and CCL21. DC migration is blocked by viral impairment of the chemokine receptor switch at the level of the expression of CCR7 molecules. The inhibition occurs with immediate-early-early kinetics, and viral interference with NF-κB signaling is likely to be at least partially responsible for the lack of CCR7 expression. DCs which migrate from the infected cultures are HCMV antigen negative, and consequently they do not stimulate HCMV-specific CD8+T cells, while CD4+-T-cell activation is not impaired. Although CD8+T cells can also be activated by alternative antigen presentation mechanisms, the spatial segregation of naive T cells and infected DCs seems a potent mechanism of delaying the generation of primary CD8+-T-cell responses and aiding early viral spread.
Identification and Characterization of Viral Structural Proteins of Infectious Salmon Anemia Virus
Authors: Falk Knut , Aspehaug Vidar , Vlasak Reinhard , Endresen Curt 
Published: February 2004

ABSTRACT

Infectious salmon anemia virus
(ISAV) is an unclassified
Orthomyxovirus
that has been shown to contain a segmented genome with eight single-stranded RNA species coding for 10 viral proteins. Four major structural proteins were characterized in the present study: two glycosylated proteins with estimated molecular masses of 42 and 50 kDa, one 66-kDa phosphoprotein, and one 22-kDa protein. Examination of lysed virions revealed the two glycoproteins and the 22-kDa protein in the soluble fraction, while the 66-kDa phosphoprotein and a minor part of the 22-kDa protein were found in the pelleted fraction. Immunofluorescence staining of infected cells demonstrated that the 22-kDa protein was a late protein accumulating in the nucleus. We conclude that the 66-kDa protein is the nucleoprotein, the 22-kDa protein is the matrix protein, and the 42- and 50-kDa proteins are the surface proteins. Radioimmunoprecipitation analysis of the 42-kDa glycoprotein, which was previously shown to represent the ISAV hemagglutinin, indicated that this protein exists at least as dimers. Further, by labeling of purified ISAV with [1,3-
3
H]diisopropyl fluorophosphate, it was also demonstrated that the viral esterase is located with the hemagglutinin. This finding was confirmed by demonstration of acetylesterase activity in affinity-purified hemagglutinin preparations. Finally, the active-site serine residue could be tentatively identified at position 32 within the amino acid sequence of the hemagglutinin of ISAV strain Glesvaer/2/90. It is proposed that the ISAV vp66 protein be termed nucleoprotein, the gp42 protein be termed HE protein, and the vp22 protein be termed matrix protein.
cis
and
trans
Requirements for Rolling Circle Replication of a Satellite RNA
Authors: Song Sang Ik , Miller W. Allen 
Published: February 2004

ABSTRACT

Satellite RNAs usurp the replication machinery of their helper viruses, even though they bear little or no sequence similarity to the helper virus RNA. In
Cereal yellow dwarf polerovirus
serotype RPV (CYDV-RPV), the 322-nucleotide satellite RNA (satRPV RNA) accumulates to high levels in the presence of the CYDV-RPV helper virus. Rolling circle replication generates multimeric satRPV RNAs that self-cleave via a double-hammerhead ribozyme structure. Alternative folding inhibits formation of a hammerhead in monomeric satRPV RNA. Here we determine helper virus requirements and the effects of mutations and deletions in satRPV RNA on its replication in oat cells. Using in vivo selection of a satRPV RNA pool randomized at specific bases, we found that disruption of the base pairing necessary to form the non-self-cleaving conformation reduced satRPV RNA accumulation. Unlike other satellite RNAs, both the plus and minus strands proved to be equally infectious. Accordingly, very similar essential replication structures were identified in each strand. A different region is required only for encapsidation. The CYDV-RPV RNA-dependent RNA polymerase (open reading frames 1 and 2), when expressed from the nonhelper
Barley yellow dwarf luteovirus,
was capable of replicating satRPV RNA. Thus, the helper virus's polymerase is the sole determinant of the ability of a virus to replicate a rolling circle satellite RNA. We present a framework for functional domains in satRPV RNA with three types of function: (i) conformational control elements comprising an RNA switch, (ii) self-functional elements (hammerhead ribozymes), and (iii)
cis
-acting elements that interact with viral proteins.
Characterization of a Neuraminidase-Deficient Influenza A Virus as a Potential Gene Delivery Vector and a Live Vaccine
Authors: Shinya Kyoko , Fujii Yutaka , Ito Hiroshi , Ito Toshihiro , Kawaoka Yoshihiro 
Published: February 2004

ABSTRACT

We recently identified a packaging signal in the neuraminidase (NA) viral RNA (vRNA) segment of an influenza A virus, allowing us to produce a mutant virus [GFP(NA)-Flu] that lacks most of the NA open reading frame but contains instead the gene encoding green fluorescent protein (GFP). To exploit the expanding knowledge of vRNA packaging signals to establish influenza virus vectors for the expression of foreign genes, we studied the replicative properties of this virus in cell culture and mice. Compared to wild-type virus, GFP(NA)-Flu was highly attenuated in normal cultured cells but was able to grow to a titer of >10
6
PFU/ml in a mutant cell line expressing reduced levels of sialic acid on the cell surface. GFP expression from this virus was stable even after five passages in the latter cells. In intranasally infected mice, GFP was detected in the epithelial cells of nasal mucosa, bronchioles, and alveoli for up to 4 days postinfection. We attribute the attenuated growth of GFP(NA)-Flu to virion aggregation at the surface of bronchiolar epithelia. In studies to test the potential of this mutant as a live attenuated influenza vaccine, all mice vaccinated with ≥10
5
PFU of GFP(NA)-Flu survived when challenged with lethal doses of the parent virus. These results suggest that influenza virus could be a useful vector for expressing foreign genes and that a sialidase-deficient virus may offer an alternative to the live influenza vaccines recently approved for human use.
Extracellular Nef Protein Targets CD4+T Cells for Apoptosis by Interacting with CXCR4 Surface Receptors
Authors: James Cleve O. , Huang Ming-Bo , Khan Mafuz , Garcia-Barrio Minerva , Powell Michael D. , Bond Vincent C. 
Published: February 2004

ABSTRACTThe effects of soluble Nef protein on CD4+T cells were examined. CD4+-T-cell cultures exposed to soluble Nef were analyzed for apoptosis by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling and hallmarks of apoptosis including cytoplasmic shrinkage, nuclear fragmentation, DNA laddering, and caspase activation. We observed dose- and time-dependent inductions of apoptosis. DNA laddering and activated caspase 3 were also evident. Cells treated with Nef/protein kinase inhibitor complexes were protected from Nef-induced apoptosis, suggesting possible roles for protein kinases in the apoptosis pathway. Similarly, cells treated with Nef/anti-Nef antibody complexes were protected from Nef-induced apoptosis. The cellular receptor responsible for Nef-induced apoptosis was identified through antibody- and ligand-blocking experiments as a receptor commonly involved in viral entry. CXCR4 antibodies, as well as the endogenous ligand SDF-1α, were effective in blocking Nef-induced apoptosis, while CCR5 and CD4 antibodies were ineffective. Moreover, a CXCR4-deficient cell line, MDA-MB-468, which was resistant to Nef-induced apoptosis, became sensitive upon transfection with a CXCR4-expressing vector. This study suggests that extracellular Nef protein could contribute to the decline of CD4 counts prior to and during the onset of AIDS in patients with human immunodeficiency virus type 1 infections.
Crystal Structures of a Multidrug-Resistant Human Immunodeficiency Virus Type 1 Protease Reveal an Expanded Active-Site Cavity
Authors: Logsdon Bradley C. , Vickrey John F. , Martin Philip , Proteasa Gheorghe , Koepke Jay I. , Terlecky Stanley R. , Wawrzak Zdzislaw , Winters Mark A. , Merigan Thomas C. , Kovari Ladislau C. 
Published: February 2004

ABSTRACTThe goal of this study was to use X-ray crystallography to investigate the structural basis of resistance to human immunodeficiency virus type 1 (HIV-1) protease inhibitors. We overexpressed, purified, and crystallized a multidrug-resistant (MDR) HIV-1 protease enzyme derived from a patient failing on several protease inhibitor-containing regimens. This HIV-1 variant contained codon mutations at positions 10, 36, 46, 54, 63, 71, 82, 84, and 90 that confer drug resistance to protease inhibitors. The 1.8-angstrom (Å) crystal structure of this MDR patient isolate reveals an expanded active-site cavity. The active-site expansion includes position 82 and 84 mutations due to the alterations in the amino acid side chains from longer to shorter (e.g., V82A and I84V). The MDR isolate 769 protease “flaps” stay open wider, and the difference in the flap tip distances in the MDR 769 variant is 12 Å. The MDR 769 protease crystal complexes with lopinavir and DMP450 reveal completely different binding modes. The network of interactions between the ligands and the MDR 769 protease is completely different from that seen with the wild-type protease-ligand complexes. The water molecule-forming hydrogen bonds bridging between the two flaps and either the substrate or the peptide-based inhibitor are lacking in the MDR 769 clinical isolate. The S1, S1′, S3, and S3′ pockets show expansion and conformational change. Surface plasmon resonance measurements with the MDR 769 protease indicate higherkoffrates, resulting in a change of binding affinity. Surface plasmon resonance measurements providekonandkoffdata (Kd=koff/kon) to measure binding of the multidrug-resistant protease to various ligands. This MDR 769 protease represents a new antiviral target, presenting the possibility of designing novel inhibitors with activity against the open and expanded protease forms.
Vaccinia Virus Infection during Murine Pregnancy: a New Pathogenesis Model for Vaccinia Fetalis
Authors: Benning Nicola , Hassett Daniel E. 
Published: February 2004

ABSTRACTVaccinia fetalis, the vertical transfer of vaccinia virus from mother to fetus, is a relatively rare but often fatal complication of primary vaccinia virus vaccination during pregnancy. To date there has been no attempt to develop an animal model to study the pathogenesis of this acute viral infection in vivo. Here we report that infection of gestating BALB/c mice by either intravenous or intraperitoneal routes with the Western Reserve strain of vaccinia virus results in the rapid colonization of the placenta and vertical transfer of virus to the developing fetus. Systemic maternal infections during gestation lead to the death of all offspring prior to or very shortly after birth. Using in situ hybridization for vaccinia virus mRNA to identify infected cells, we show that the virus initially colonizes cells lining maternal lacunae within the trophospongium layer of the placenta. The study of this model will significantly enhance our understanding of the pathogenesis of fetal vaccinia virus infections and aid in the development of effective treatments designed to reduce the risk of vaccinia virus-associated complications during pregnancy.
Repeated Low-Dose Mucosal Simian Immunodeficiency Virus SIVmac239 Challenge Results in the Same Viral and Immunological Kinetics as High-Dose Challenge: a Model for the Evaluation of Vaccine Efficacy in Nonhuman Primates
Authors: McDermott Adrian B. , Mitchen Jacque , Piaskowski Shari , De Souza Ivna , Yant Levi J. , Stephany Jason , Furlott Jessica , Watkins David I. 
Published: February 2004

ABSTRACT

Simian immunodeficiency virus (SIV) challenge of rhesus macaques provides a relevant model for the assessment of human immunodeficiency virus (HIV) vaccine strategies. To ensure that all macaques become infected, the vaccinees and controls are exposed to large doses of pathogenic SIV. These nonphysiological high-dose challenges may adversely affect vaccine evaluation by overwhelming potentially efficacious vaccine responses. To determine whether a more physiologically relevant low-dose challenge can initiate infection and cause disease in Indian rhesus macaques, we used a repeated low-dose challenge strategy designed to reduce the viral inoculum to more physiologically relevant doses. In an attempt to more closely mimic challenge with HIV, we administered repeated mucosal challenges with 30, 300, and 3,000 50% tissue culture infective doses (TCID
50
) of pathogenic SIVmac239 to six animals in three groups. Infection was assessed by sensitive quantitative reverse transcription-PCR and was achieved following a mean of 8, 5.5, and 1 challenge(s) in the 30, 300, and 3,000 TCID
50
groups, respectively. Mortality, humoral immune responses, and peak plasma viral kinetics were similar in five of six animals, regardless of challenge dose. Interestingly, macaques challenged with lower doses of SIVmac239 developed broad T-cell immune responses as assessed by ELISPOT assay. This low-dose repeated challenge may be a valuable tool in the evaluation of potential vaccine regimes and offers a more physiologically relevant regimen for pathogenic SIVmac239 challenge experiments.
Cell-to-Cell, but Not Long-Distance, Spread of RNA Silencing That Is Induced in Individual Epidermal Cells
Authors: Ryabov Eugene V. , van Wezel Rene , Walsh John , Hong Yiguo 
Published: February 2004

ABSTRACT

A
Turnip crinkle virus
(TCV)-based system was devised to discriminate cell-to-cell and systemic long-distance spread of RNA silencing in plants. Modified TCV-GFPΔCP, constructed by replacing the coat protein (CP) gene with the green fluorescent protein (GFP) gene, replicated in single epidermal cells but failed to move from cell to cell in
Nicotiana benthamiana
. Mechanical inoculation of TCV-GFPΔCP induced effective RNA silencing in single epidermal cells which spread from cell to cell to form silenced foci on inoculated leaves, but no long-distance systemic spread of RNA silencing occurred. Agroinfiltration of TCV-GFPΔCP was, however, able to induce both local and systemic RNA silencing. TCV coinfection arrested TCV-GFPΔCP-mediated local induction of RNA silencing. Possible mechanisms involved in cell-to-cell and long-distance spread of RNA silencing are discussed.
Inhibition of Different Lassa Virus Strains by Alpha and Gamma Interferons and Comparison with a Less Pathogenic Arenavirus
Authors: Asper Marcel , Sternsdorf Thomas , Hass Meike , Drosten Christian , Rhode Antje , Schmitz Herbert , Günther Stephan 
Published: February 2004

ABSTRACT
The high pathogenicity of Lassa virus is assumed to involve resistance to the effects of interferon (IFN). We have analyzed the effects of alpha IFN (IFN-α), IFN-γ, and tumor necrosis factor alpha (TNF-α) on replication of Lassa virus compared to the related, but less pathogenic, lymphocytic choriomeningitis virus (LCMV). Three low-passage Lassa virus strains (AV, NL, and CSF), isolated from humans with mild to fulminant Lassa fever, were tested. Lassa virus replication was inhibited by IFN-α and IFN-γ, but not TNF-α, in Huh7 and Vero cells. The degree of IFN sensitivity of a Lassa virus isolate did not correlate with disease severity in human patients. Furthermore, cytokine effects observed for Lassa virus and LCMV (strains CH-5692, Armstrong, and WE) were similar. To address the mechanisms involved in the IFN effect, we used cell lines in which overexpression of IFN-stimulated proteins promyelocytic leukemia protein (PML) and Sp100 could be induced. Both proteins reside in PML bodies, a cellular target of the LCMV and Lassa virus Z proteins. Overexpression of PML or Sp100 did not affect replication of either virus. This, together with the previous finding that PML knockout facilitates LCMV replication in vitro and in vivo (M. Djavani, J. Rodas, I. S. Lukashevich, D. Horejsh, P. P. Pandolfi, K. L. Borden, and M. S. Salvato, J. Virol. 75:6204-6208, 2001; W. V. Bonilla, D. D. Pinschewer, P. Klenerman, V. Rousson, M. Gaboli, P. P. Pandolfi, R. M. Zinkernagel, M. S. Salvato, and H. Hengartner, J. Virol. 76:3810-3818, 2002), describes PML as a mediator within the antiviral pathway rather than as a direct effector protein. In conclusion, the high pathogenicity of Lassa virus compared to LCMV is probably not due to increased resistance to the effects of IFN-α or IFN-γ. Both cytokines inhibit replication which is relevant for the design of antiviral strategies against Lassa fever with the aim of enhancing the IFN response.
A Mutation in the Latency-Related Gene of Bovine Herpesvirus 1 Inhibits Protein Expression from Open Reading Frame 2 and an Adjacent Reading Frame during Productive Infection
Authors: Jiang Yunquan , Inman Melissa , Zhang Yange , Posadas Nuria Alemañ , Jones Clinton 
Published: February 2004

ABSTRACT
The latency-related (LR) gene of bovine herpesvirus 1 (BHV-1) is abundantly expressed during latency. A mutant BHV-1 strain that contains three stop codons at the 5′ terminus of the LR gene (LR mutant) does not reactivate from latency. This study demonstrates that the LR mutant does not express open reading frame 2 or an adjacent reading frame that lacks an initiating ATG (reading frame C). Since the LR mutant and wild-type BHV-1 express similar levels of LR RNA, we conclude that LR protein expression plays an important role in regulating the latency reactivation cycle in cattle.
Human Cytomegalovirus Stimulates Cellular IKK2 Activity and Requires the Enzyme for Productive Replication
Authors: Caposio Patrizia , Dreano Michel , Garotta Gianni , Gribaudo Giorgio , Landolfo Santo 
Published: February 2004

ABSTRACT
Human cytomegalovirus (HCMV) exploits the host transcription factor NF-κB to enhance its own replication, dissemination, and reactivation from latency. Here we report that HCMV infection activates the upstream IκB kinase (IKK) complex and that its catalytic IKK2 subunit is required for HCMV-induced NF-κB activation, as well as the replication of different HCMV strains. These results indicate that IKK2 is essential for HCMV replication and emphasize the feasibility of blocking NF-κB activation as a way of inhibiting infection.
Mathematics and Time
Authors:   Fraser
Published: February 2004

AbstractThe structure of mathematics, as revealed by the exploration of axiomatic systems, bears striking similarities to the structure of nature, as revealed by the hierarchical theory of time. It is assumed that this isomorphism is not accidental but reflects the evolutionary development of the human capacity of handling numbers. This assumption permits a conjecture. Namely, if mathematics is found to possess certain systematic uncertainties, than nature must also possess corresponding qualities which may be identified. The paper proposes that the theme of the conference, "time and uncertainty," be understood in this broad context. I would like to demonstrate the existence of certain striking similarities between the structure and properties of mathematics on the one hand and, on the other hand, the structures and processes of nature at large, as revealed by the hierarchical theory of time. Then, using these correspondences, I propose a framework that promises to provide a unified perspective for the rich program we have ahead.