bluuebunny/crossref_metadata_2025_split huggingface dataset:
Correlation between Point Mutations in NS2 and the Viability and Cytopathogenicity of Bovine Viral Diarrhea Virus Strain Oregon Analyzed with an Infectious cDNA Clone
Authors: Kümmerer Beate M. , Meyers Gregor 
Published: April 2009

ABSTRACTCytopathogenicity ofBovine viral diarrhea virus(BVDV) is correlated with expression of the nonstructural protein NS3, which can be generated by processing of a fusion protein termed NS2-3. For the cytopathogenic (cp) BVDV strain Oregon, NS2-3 processing is based on a set of point mutations within NS2. To analyze the correlation between NS2-3 cleavage and cytopathogenicity, a full-length cDNA clone composed of cDNA from BVDV Oregon and the utmost 5′- and 3′-terminal sequences of a published infectious BVDV clone was established. After transfection of RNA transcribed from this cDNA clone, infectious virus with similar growth characteristics to wild-type BVDV Oregon could be recovered that also exhibited a cytopathic effect. Based on this cDNA construct and published cp and noncp infectious clones, chimeric full-length cDNA clones were constructed. Analysis of the recovered viruses demonstrated that the presence of the NS2 gene of BVDV Oregon in a chimeric construct is sufficient for NS2-3 processing and a cp phenotype. Since previous studies had revealed that the amino acid serine at position 1555 of BVDV Oregon plays an important role in efficient NS2-3 cleavage, mutants of BVDV Oregon with different amino acids at this position were constructed. Some of these mutants showed NS2-3 cleavage efficiencies in the range of the wild-type sequence and allowed the recovery of viruses that behaved similarly to wild-type virus with regard to growth characteristics and cytopathogenicity. In contrast, other mutants with considerably reduced NS2-3 cleavage efficiencies propagated much more slowly and reverted to viruses expressing polyproteins with sequences allowing efficient NS2-3 cleavage. These viruses apparently induced cytopathic effects only after reversion.
Upregulation of the Genes Encoding Lysosomal Hydrolases, a Perforin-Like Protein, and Peroxidases in the Brains of Mice Affected with an Experimental Prion Disease
Authors: Kopacek Juraj , Sakaguchi Suehiro , Shigematsu Kazuto , Nishida Noriyuki , Atarashi Ryuichirou , Nakaoke Ryota , Moriuchi Ryozo , Niwa Masami , Katamine Shigeru 
Published: April 2009

ABSTRACT

In an attempt to identify the molecules involved in the pathogenesis of prion diseases, we performed cDNA subtraction on the brain tissues of mice affected with an experimental prion disease and the unaffected control. The genes identified as being upregulated in the prion-affected brain tissue included those encoding a series of lysosomal hydrolases (lysozyme M and both isoforms of β-
N
-acetylhexosaminidase), a perforin-like protein (macrophage proliferation-specific gene-1 [MPS-1]), and an oxygen radical scavenger (peroxiredoxin). Dramatic increases in the expression level occurred at between 12 and 16 weeks after intracerebral inoculation of the prion, coinciding with the onset of spongiform degeneration. The proteinase K-resistant prion protein (PrP
Sc
) became detectable by immunoblotting well before 12 weeks, suggesting a causal relationship between this and the gene activation. Immunohistochemistry paired with in situ hybridization on sections of the affected brain tissue revealed that expression of the peroxiredoxin gene was detectable only in astrocytes and was noted throughout the affected brain tissue. On the other hand, the genes for the lysosomal hydrolases and MPS-1 were overexpressed exclusively by microglia, which colocalized with the spongiform morphological changes. A crucial role for microglia in the spongiform degeneration by their production of neurotoxic substances, and possibly via the aberrant activation of the lysosomal system, would have to be considered.
Adenovirus Type 37 Uses Sialic Acid as a Cellular Receptor
Authors: Arnberg Niklas , Edlund Karin , Kidd Alistair H. , Wadell Göran 
Published: April 2009

ABSTRACTTwo cellular receptors for adenovirus, coxsackievirus-adenovirus receptor (CAR) and major histocompatibility complex class I (MHC-I) α2, have recently been identified. In the absence of CAR, MHC-I α2 has been suggested to serve as a cellular attachment protein for subgenus C adenoviruses, while members from all subgenera except subgenus B have been shown to interact with CAR. We have found that adenovirus type 37 (Ad37) attachment to CAR-expressing CHO cells was no better than that to CHO cells lacking CAR expression, suggesting that CAR is not used by Ad37 during attachment. Instead, we have identified sialic acid as a third adenovirus receptor moiety. First, Ad37 attachment to both CAR-expresing CHO cells and MHC-I α2-expressing Daudi cells was sensitive to neuraminidase treatment, which eliminates sialic acid on the cell surface. Second, Ad37 attachment to sialic acid-expressing Pro-5 cells was more than 10-fold stronger than that to the Pro-5 subline Lec2, which is deficient in sialic acid expression. Third, neuraminidase treatment of A549 cells caused a 60% decrease in Ad37 replication in a fluorescent-focus assay. Moreover, the receptor sialoconjugate is most probably a glycoprotein rather than a ganglioside, since Ad37 attachment to sialic acid-expressing Pro-5 cells was sensitive to protease treatment. Ad37 attachment to Pro-5 cells occurs via α(2→3)-linked sialic acid saccharides rather than α(2→6)-linked ones, since (i) α(2→3)-specific but not α(2→6)-specific lectins blocked Ad37 attachment to Pro-5 cells and (ii) pretreatment of Pro-5 cells with α(2→3)-specific neuraminidase resulted in decreased Ad37 binding. Taken together, these results suggest that, unlike Ad5, Ad37 makes use of α(2→3)-linked sialic acid saccharides on glycoproteins for entry instead of using CAR or MHC-I α2.
Development of Human T-Cell Leukemia Virus Type 1-Transformed Tumors in Rats following Suppression of T-Cell Immunity by CD80 and CD86 Blockade
Authors: Hanabuchi Shino , Ohashi Takashi , Koya Yoshihiro , Kato Hirotomo , Takemura Fumiyo , Hirokawa Katsuiku , Yoshiki Takashi , Yagita Hideo , Okumura Ko , Kannagi Mari 
Published: April 2009

ABSTRACTHost immunity influences clinical manifestations of human T-cell leukemia virus type 1 (HTLV-1) infection. In this study, we demonstrated that HTLV-1-transformed tumors could develop in immunocompetent rats by blocking a costimulatory signal for T-cell immune responses. Four-week-old WKA/HKm rats were treated with monoclonal antibodies (MAbs) to CD80 and CD86 and subcutaneously inoculated with syngeneic HTLV-1-infected TARS-1 cells. During MAb treatment for 14 days, TARS-1 inoculation resulted in the development of solid tumors at the site of inoculation, which metastasized to the lungs. In contrast, rats not treated with MAbs promptly rejected tumor cells. Splenic T cells from MAb-treated rats indicated impairment of proliferative and cytotoxic T-lymphocyte responses against TARS-1 in vitro compared to untreated rats. However, tumors grown in MAb-treated rats regressed following withdrawal of MAb therapy. Recovery of TARS-1-specific T-cell immune responses was associated with tumor regression in these rats. Our results suggest that HTLV-1-specific cell-mediated immunity plays a critical role in immunosurveillance against HTLV-1-transformed tumor development in vivo.
Secondary Structure Analysis of a Minimal Avian Leukosis-Sarcoma Virus Packaging Signal
Authors: Banks Jennifer D. , Linial Maxine L. 
Published: April 2009

ABSTRACT

We previously identified a 160-nucleotide packaging signal, MΨ, from the 5′ end of the
Rous sarcoma virus
genome. In this study, we determine the secondary structure of MΨ by using phylogenetic analysis with computer modeling and heterologous packaging assays of point mutants. The results of the in vivo studies are in good agreement with the computer model. Additionally, the packaging studies indicate several structures which are important for efficient packaging, including a single-stranded bulge containing the initiation codon for the short open reading frame, uORF3, as well as adjacent stem structures. Finally, we show that the L3 stem-loop at the 3′ end of MΨ is dispensable for packaging, thus identifying an 82-nucleotide minimal packaging signal, μΨ, composed of the O3 stem-loop.
Brain Infection by Neuroinvasive but Avirulent Murine Oncornaviruses
Authors: Ašković Srdjan , McAtee Frank J. , Favara Cynthia , Portis John L. 
Published: April 2009

ABSTRACT

The chimeric murine oncornavirus FrCas
E
causes a rapidly progressive noninflammatory spongiform encephalomyelopathy after neonatal inoculation. The virus was constructed by the introduction of
pol-env
sequences from the wild mouse virus CasBrE into the genome of a neuroinvasive but nonneurovirulent strain of Friend murine leukemia virus (FMuLV), FB29. Although the brain infection by FrCas
E
as well as that by other neurovirulent murine retroviruses has been described in detail, little attention has been paid to the neuroinvasive but nonneurovirulent viruses. The purpose of the present study was to compare brain infection by FrCas
E
with that by FB29 and another nonneurovirulent virus, F43, which contains
pol-env
sequences from FMuLV 57. Both FB29 and F43 infected the same spectrum of cell types in the brain as that infected by FrCas
E
, including endothelial cells, microglia, and populations of neurons which divide postnatally. Viral burdens achieved by the two nonneurovirulent viruses in the brain were actually higher than that of FrCas
E
. The widespread infection of microglia by the two nonneurovirulent viruses is notable because it is infection of these cells by FrCas
E
which is thought to be a critical determinant of its neuropathogenicity. These results indicate that although the sequence of the envelope gene determines neurovirulence, this effect appears to operate through a mechanism which does not influence either viral tropism or viral burden in the brain. Although all three viruses exhibited similar tropism for granule neurons in the cerebellar cortex, there was a striking difference in the distribution of envelope proteins in those cells in vivo. The FrCas
E
envelope protein accumulated in terminal axons, whereas those of FB29 and F43 remained predominantly in the cell bodies. These observations suggest that differences in the intracellular sorting of these proteins may exist and that these differences appear to correlate with neurovirulence.
The UL25 Protein of Pseudorabies Virus Associates with Capsids and Localizes to the Nucleus and to Microtubules
Authors: Kaelin Karin , Dezélée Sybille , Masse Marie Jo , Bras Françoise , Flamand Anne 
Published: April 2009

ABSTRACT
The UL25 gene of pseudorabies virus (PrV) can encode a protein of about 57 kDa which is well conserved among herpesviruses. The UL25 protein of herpes simplex virus type 1 is a capsid constituent involved in virus penetration and capsid maturation. To identify and characterize the UL25 gene product of PrV, polyclonal mouse anti-UL25 antibodies were raised to a bacterially expressed fusion protein. In immunoblotting and immunoprecipitation assays of PrV-infected cell lysates, these anti-UL25 antisera specifically recognized a protein of the expected size with late expression kinetics. This 57-kDa product was also present in purified virions and was found to be associated with all types of capsids. Synthesis of a protein migrating at the same size point was directed from the eukaryotic expression plasmid pCG-UL25. To determine the subcellular localization of UL25, immunofluorescence studies with anti-UL25 antisera were performed on Nonidet P-40-extracted COS-7 cells infected with PrV or transfected with pCG-UL25. In PrV-infected cells, newly synthesized UL25 is directed mainly to distinct nuclear compartments, whereas UL25 expressed in the absence of other viral proteins is distributed more uniformly in the nucleus and colocalizes also with microtubules. To study the fate of UL25 at very early stages of infection, immunofluorescence experiments were performed on invading PrV particles in the presence or absence of drugs that specifically depolymerize components of the cytoskeleton. We found that the incoming nucleocapsids colocalize with microtubules during their transport to the nucleus and that UL25 remains associated with nucleocapsids during this transport.
cisExpression of the F12 Human Immunodeficiency Virus (HIV) Nef Allele Transforms the Highly Productive NL4-3 HIV Type 1 to a Replication-Defective Strain: Involvement of both Env gp41 and CD4 Intracytoplasmic Tails
Authors: Olivetta Eleonora , Pugliese Katherina , Bona Roberta , D'Aloja Paola , Ferrantelli Flavia , Santarcangelo Anna Claudia , Mattia Gianfranco , Verani Paola , Federico Maurizio 
Published: April 2009

ABSTRACTF12 human immunodeficiency virus type 1 (HIV-1)nefis a naturally occurringnefmutant cloned from the provirus of a nonproductive, nondefective, and interfering HIV-1 variant (F12-HIV). We have already shown that cells stably transfected with a vector expressing the F12-HIVnefallele do not downregulate CD4 receptors and, more peculiarly, become resistant to the replication of wild type (wt) HIV. In order to investigate the mechanism of action of such an HIV inhibition, the F12-HIVnefgene was expressed in the context of the NL4-3 HIV-1 infectious molecular clone by replacing the wtnefgene (NL4-3/chi). Through this experimental approach we established the following. First, NL4-3/chi andnef-defective (Δnef) NL4-3 viral particles behave very similarly in terms of viral entry and HIV protein production during the first replicative cycle. Second, no viral particles were produced from cells infected with NL4-3/chi virions, whatever the multiplicity of infection used. The viral inhibition apparently occurs at level of viral assembling and/or release. Third, this block could not be relieved by in-transexpression of wtnef. Finally, NL4-3/chi reverts to a producer HIV strain when F12-HIV Nef is deprived of its myristoyl residue. Through a CD4 downregulation competition assay, we demonstrated that F12-HIV Nef protein potently inhibits the CD4 downregulation induced by wt Nef. Moreover, we observed a redistribution of CD4 receptors at the cell margin induced by F12-HIV Nef. These observations strongly suggest that F12-HIV Nef maintains the ability to interact with the intracytoplasmic tail of the CD4 receptor molecule. Remarkably, we distinguished the intracytoplasmic tails of Env gp41 and CD4 as, respectively, viral and cellular targets of the F12-HIV Nef-induced viral retention. For the first time, the inhibition of the viral life cycle by means of in-cisexpression of a Nef mutant is here reported. Delineation of the F12-HIV Nef mechanism of action may offer additional approaches to interference with the propagation of HIV infection.
Extended Analysis of the In Vitro Tropism of Porcine Endogenous Retrovirus
Authors: Wilson Carolyn A. , Wong Susan , VanBrocklin Matthew , Federspiel Mark J. 
Published: April 2009

ABSTRACTWe previously reported that mitogenic activation of porcine peripheral blood mononuclear cells resulted in production of porcine endogenous retrovirus(es) (PERV[s]) capable of productively infecting human cells (C. Wilson et al., J. Virol. 72:3082–3087, 1998). We now extend that analysis to show that additional passage of isolated virus, named here PERV-NIH, through a human cell line yielded a viral population with a higher titer of infectious virus on human cells than the initial isolate. We show that in a single additional passage on a human cell line, the increase in infectivity for human cells is accounted for by selection against variants carrying pig-tropic envelope sequences (PERV-C) as well as by enrichment for replication-competent genomes. Sequence analysis of the envelope cDNA present in virions demonstrated that the envelope sequence of PERV-NIH is related to but distinct from previously reported PERV envelopes. The in vitro host range of PERV was studied in human primary cells and cell lines, as well as in cell lines from nonhuman primate and other species. This analysis reveals three patterns of susceptibility to infection among these host cells: (i) cells are resistant to infection in our assay; (ii) cells are infected by virus, as viral RNA is detected in the supernatant by reverse transcription-PCR, but the cells are not permissive to productive replication and spread; and (iii) cells are permissive to low-level productive replication. Certain cell lines were permissive for efficient productive infection and spread. These results may prove useful in designing appropriate animal models to assess the in vivo infectivity properties of PERV.
Structural Fingerprinting: Subgrouping of Comoviruses by Structural Studies of Red Clover Mottle Virus to 2.4-Å Resolution and Comparisons with Other Comoviruses
Authors: Lin Tianwei , Clark Anthony J. , Chen Zhongguo , Shanks Michael , Dai Jin-Bi , Li Ying , Schmidt Tim , Oxelfelt Per , Lomonossoff George P. , Johnson John E. 
Published: April 2009

ABSTRACT

Red clover mottle virus
(RCMV) is a member of the comoviruses, a group of picornavirus-like plant viruses. The X-ray structure of RCMV strain S has been determined and refined to 2.4 Å. The overall structure of RCMV is similar to that of two other comoviruses,
Cowpea mosaic virus
(CPMV) and
Bean pod mottle virus
(BPMV). The sequence of the coat proteins of RCMV strain O were modeled into the capsid structure of strain S without causing any distortion, confirming the close resemblance between the two strains. By comparing the RCMV structure with that of other comoviruses, a structural fingerprint at the N terminus of the small subunit was identified which allowed subgrouping of comoviruses into CPMV-like and BPMV-like viruses.
Human Cytomegalovirus Infects Caco-2 Intestinal Epithelial Cells Basolaterally Regardless of the Differentiation State
Authors: Esclatine Audrey , Lemullois Michel , Servin Alain L. , Quero Anne-Marie , Geniteau-Legendre Monique 
Published: April 2009

ABSTRACT
Human cytomegalovirus (CMV) causes severe disease in immunosuppressed patients and notably infects the gastrointestinal tract. To understand the interaction of CMV with intestinal epithelial cells, which are highly susceptible to CMV infection in vivo, we used the intestinal epithelial cell line Caco-2 and demonstrated that CMV enters predominantly through the basolateral surface of polarized Caco-2 cells. As shown by expression of all three classes of CMV proteins and by visualization of nucleocapsids by transmission electron microscopy, both poorly and fully differentiated Caco-2 cells were permissive to CMV replication. However, infection failed to produce infectious particles in Caco-2 cells, irrespective of the state of differentiation.
Avian Reticuloendotheliosis Virus Strain A and Spleen Necrosis Virus Do Not Infect Human Cells
Authors: Gautier R. , Jiang A. , Rousseau V. , Dornburg R. , Jaffredo T. 
Published: April 2009

ABSTRACT

Spleen necrosis virus
(SNV) and
Reticuloendotheliosis virus
strain A (REV-A) belong to the family of reticuloendotheliosis viruses and are 90% sequence related. SNV-derived retroviral vectors produced by the REV-A-based D17.2G packaging cell line were shown to infect human cells (H.-M. Koo, A. M. C. Brown, Y. Ron, and J. P. Dougherty, J. Virol. 65:4769–4776, 1991), while similar vectors produced by another SNV-based packaging cell line, DSH134G, are not infectious in human cells (reviewed by R. Dornburg, Gene Ther. 2:301–310, 1995). Here we describe a careful reevaluation of the infectivity of vectors produced from the most commonly used REV-A- or SNV-based packaging cells obtained from various sources with, among them, one batch of D17.2G packaging cells obtained from the American Type Culture Collection. None of these packaging cells produced vectors able to infect human cells. Thus, contrary to previously published data, we conclude that REV-based vectors are not infectious in human cells.
Incorporation of Adeno-Associated Virus in a Calcium Phosphate Coprecipitate Improves Gene Transfer to Airway Epithelia In Vitro and In Vivo
Authors: Walters Robert W. , Duan Dongsheng , Engelhardt John F. , Welsh Michael J. 
Published: April 2009

ABSTRACT

Adeno-associated virus (AAV) is inefficient at infecting differentiated airway epithelia because of a lack of receptors at the apical surface. We hypothesized that incorporation of AAV in a calcium phosphate coprecipitate would circumvent this barrier. Interestingly, coprecipitation of AAV type 2 improved gene transfer to differentiated human airway epithelia in vitro and to the mouse lung in vivo. These results suggest that delivery of AAV as a CaP
i
coprecipitate may significantly enhance its utility for gene transfer to the airway epithelia in vivo.
A Heterologous, High-Affinity RNA Ligand for Human Immunodeficiency Virus Gag Protein Has RNA Packaging Activity
Authors: Clever Jared L. , Taplitz Randy A. , Lochrie Michael A. , Polisky Barry , Parslow Tristram G. 
Published: April 2009

ABSTRACT

Retroviral RNA encapsidation depends on the specific binding of Gag proteins to packaging (ψ) signals in genomic RNA. We investigated whether an in vitro-selected, high-affinity RNA ligand for the nucleocapsid (NC) portion of the Gag protein from human immunodeficiency virus type 1 (HIV-1) could mediate packaging into HIV-1 virions. We find that this ligand can functionally substitute for one of the Gag-binding elements (termed SL3) in the HIV-1 ψ locus to support packaging and viral infectivity in
cis
. By contrast, this ligand, which fails to dimerize spontaneously in vitro, is unable to replace a different ψ element (termed SL1) which is required for both Gag binding and dimerization of the HIV-1 genome. A single point mutation within the ligand that eliminates high-affinity in vitro Gag binding also abolishes its packaging activity at the SL3 position. These results demonstrate that specific binding of Gag or NC protein is a critical determinant of genomic RNA packaging.
MxA GTPase Blocks Reporter Gene Expression of Reconstituted Thogoto Virus Ribonucleoprotein Complexes
Authors: Weber Friedemann , Haller Otto , Kochs Georg 
Published: April 2009

ABSTRACT

Human MxA protein accumulates in the cytoplasm of interferon-treated cells and inhibits the multiplication of several RNA viruses, including
Thogoto virus
(THOV), a tick-borne orthomyxovirus that transcribes and replicates its genome in the cell nucleus. The antiviral mechanism of MxA was investigated by using two alternative minireplicon systems in which recombinant viral ribonucleoprotein complexes (vRNPs) of THOV were reconstituted from cloned cDNAs. A chloramphenicol acetyltransferase reporter minigenome RNA was expressed either by T7 RNA polymerase in the cytoplasm of transfected cells or, alternatively, by RNA polymerase I in the nucleus. The inhibitory effect of MxA was studied in both cellular compartments by coexpressing wild-type MxA or TMxA, an artificial nuclear form of MxA. Our results indicate that both MxA proteins recognize the assembled vRNP rather than the newly synthesized unassembled components. The present findings are consistent with previous data which indicated that cytoplasmic MxA prevents transport of vRNPs into the nucleus, whereas nuclear MxA directly inhibits the viral polymerase activity in the nucleus.