bluuebunny/crossref_metadata_2025_split huggingface dataset:
Fitness of a Turnip Crinkle Virus Satellite RNA Correlates with a Sequence-Nonspecific Hairpin and Flanking Sequences That Enhance Replication and Repress the Accumulation of Virions
Authors: Sun Xiaoping , Simon Anne E. 
Published: June 2003

ABSTRACT

satC, a satellite RNA associated with
Turnip crinkle virus
(TCV), enhances the ability of the virus to colonize plants by interfering with stable virion accumulation (F. Zhang and A. E. Simon, unpublished data). Previous results suggested that the motif1-hairpin (M1H), a replication enhancer on minus strands, forms a plus-strand hairpin flanked by CA-rich sequence that may be involved in enhancing systemic infection (G. Zhang and A. E. Simon, J. Mol. Biol. 326:35-48, 2003). In this study, sequence and structural requirements of the M1H were further assayed by replacing the 28-base M1H with 10 random bases and then subjecting the pool of satellite RNA to functional selection in plants. Unlike previous results with 28-base replacement sequences (G. Zhang and A. E. Simon, J. Mol. Biol. 326:35-48, 2003), only a few of the 10-base SELEX (systematic evolution of ligands by exponential enrichment) assay winners contained short motifs in their minus-sense orientation that were similar to TCV replication elements. However, all second- and third-round winning replacement sequences folded into hairpins flanked by CA-rich sequence predicted to be more stable on plus strands than minus strands. Plus strands of several of the most fit satellite RNAs contained insertions of CA-rich sequence at the base of their hairpins whose presence correlated with enhanced replication and reduced detection of virions. Deletion of the M1H resulted in no detectable virions despite very low satellite accumulation. These results support the hypothesis that a sequence-nonspecific plus-strand hairpin brings together flanking CA-rich sequences in the M1H region that confers fitness to satC by reducing the accumulation of stable virions.
Transmissible Gastroenteritis Coronavirus Packaging Signal Is Located at the 5′ End of the Virus Genome
Authors: Escors David , Izeta Ander , Capiscol Carmen , Enjuanes Luis 
Published: June 2003

ABSTRACT

To locate the transmissible gastroenteritis coronavirus (TGEV) packaging signal, the incorporation of TGEV subgenomic mRNAs (sgmRNAs) into virions was first addressed. TGEV virions were purified by three different techniques, including an immunopurification using an M protein-specific monoclonal antibody. Detection of sgmRNAs in virions by specific reverse transcription-PCRs (RT-PCRs) was related to the purity of virus preparations. Interestingly, virus mRNAs were detected in partially purified virus but not in virus immunopurified using stringent conditions. Analyses by quantitative RT-PCR confirmed that virus mRNAs were not present in highly purified preparations. Lack of sgmRNA encapsidation was probably due to the absence of a packaging signal (Ψ) within these mRNAs. This information plus that from the encapsidation of a collection of TGEV-derived minigenomes suggested that Ψ is located at the 5′ end of the genome. To confirm that this was the case, a set of minigenomes was expressed that included an expression cassette for an mRNA including the β-glucuronidase gene (GUS) plus variable sequence fragments from the 5′ end of the virus genome potentially including Ψ. Insertion of the first 649 nucleotides (nt) of the TGEV genome led to the specific encapsidation of the mRNA, indicating that a Ψ was located within this region which was absent from all of the other virus mRNAs. The presence of this packaging signal was further confirmed by showing the expression and rescue of the mRNA including the first 649 nt of the TGEV genome under control of the cytomegalovirus promoter in TGEV-infected cells. This mRNA was successfully amplified and encapsidated, indicating that the first 649 nt of TGEV genome also contained the 5′
cis
-acting replication signals. The encapsidation efficiency of this mRNA was about 30-fold higher than the genome encapsidation efficiency, as estimated by quantitative RT-PCR. In contrast, viral mRNAs presented significantly lower encapsidation efficiencies (about 100-fold) than those of the virus genome, strongly suggesting that TGEV mRNAs in fact lacked an alternative TGEV Ψ.
Sendai Virus Targets Inflammatory Responses, as Well as the Interferon-Induced Antiviral State, in a Multifaceted Manner
Authors: Strähle Laura , Garcin Dominique , Le Mercier Philippe , Schlaak Joerg F. , Kolakofsky Daniel 
Published: June 2003

ABSTRACTWe have used cDNA arrays to compare the activation of various cellular genes in response to infection with Sendai viruses (SeV) that contain specific mutations. Three groups of cellular genes activated by mutant SeV infection, but not by wild-type SeV, were identified in this way. While some of these genes are well known interferon (IFN)-stimulated genes, others, such as those for interleukin-6 (IL-6) and IL-8, are not directly induced by IFN. The gene for beta IFN (IFN-β), which is critical for initiating an antiviral response, was also specifically activated in mutant SeV infections. The SeV-induced activation of IFN-β was found to depend on IFN regulatory factor 3, and the activation of all three cellular genes was independent of IFN signaling. Mutations that disrupt four distinct elements in the SeV genome (the leader RNA, two regions of the C protein, and the V protein) all lead to enhanced levels of IFN-β mRNA, and at least three of these viral genes also appear to be involved in preventing activation of IL-8. Our results suggest that SeV targets the inflammatory and adaptive immune responses as well as the IFN-induced intracellular antiviral state by using a multifaceted approach.
Frequent Recovery and Broad Genotype 2 Diversity Characterize Hepatitis C Virus Infection in Ghana, West Africa
Authors: Candotti Daniel , Temple Jillian , Sarkodie Francis , Allain Jean-Pierre 
Published: June 2003

ABSTRACTHepatitis C virus (HCV) infection is thought to mostly become chronic and rarely resolves. HCV infection was serologically screened in 4,984 samples from Ghanaian blood donors, and 1.3% prevalence was found. At least 53% of confirmed anti-HCV carriers had no detectable viral RNA and were considered to have cleared the virus and recovered from the infection. Confirmation was authenticated by the presence of antibodies specific to at least two viral antigens, mostly NS3 and E2. Reactivity to HCV core antigens was lower in Ghanaian than United Kingdom blood donors. The minority of chronically infected donors carried a viral load significantly lower than an unselected comparative group of United Kingdom blood donors (2.5 × 105versus 2.9 × 106IU/ml;P= 0.004). HCV genotype 2 was largely predominant (87%). Sequence clustering was similarly broad in the E1/E2 and NS5 regions. The phylogenetic diversity and the incapacity to distinguish subtypes within genotype 2 in our and others' West African strains suggested that West Africa may be the origin of HCV genotype 2. The genetic diversity extended to the identification of strains clearly separated from known subtypes of genotype 2 and genotype 1. One strain appears to be part of a new HCV genotype. HCV infection in Ghana is characterized by a high rate of recovery and the predominance of broadly divergent genotype 2 strains.
Putative Phosphatidylinositol 3-Kinase (PI3K) Binding Motifs in Ovine Betaretrovirus Env Proteins Are Not Essential for Rodent Fibroblast Transformation and PI3K/Akt Activation
Authors: Liu Shan-Lu , Lerman Michael I. , Miller A. Dusty 
Published: June 2003

ABSTRACT
Jaagsiekte sheep retrovirus (JSRV) and enzootic nasal tumor virus (ENTV) are simple betaretroviruses that cause epithelial cell tumors in the lower and upper airways of sheep and goats. The envelope (Env) glycoproteins of both viruses can transform rodent and chicken fibroblasts, indicating that they play an essential role in oncogenesis. Previous studies found that a YXXM motif in the Env cytoplasmic tail, a putative docking site for phosphatidylinositol 3-kinase (PI3K) after tyrosine phosphorylation, was necessary for rodent cell transformation but was not required for transformation of DF-1 chicken fibroblasts. Here we show that JSRV and ENTV Env proteins with tyrosine or methionine mutations in the YXXM motif can still transform rodent fibroblasts, albeit with reduced efficiency. Akt was activated in cells transformed by JSRV or ENTV Env proteins and in cells transformed by the proteins with tyrosine mutations. Furthermore, the PI3K-specific inhibitor LY294002 could inhibit Akt activation and cell transformation in all cases, indicating that Akt activation and transformation is PI3K dependent. However, we could not detect tyrosine phosphorylation of JSRV or ENTV Env proteins or an interaction between the Env proteins and PI3K in the transformed cells. We found no evidence for mitogen-activated protein kinase activation in cells that were transformed by the JSRV or ENTV Env proteins. We conclude that ovine betaretrovirus Env proteins transform the rodent fibroblasts by indirectly activating the PI3K/Akt pathway.
Mutual Interference of Adenovirus Infection and
myc
Expression
Authors: Löhr Kristina , Hartmann Oliver , Schäfer Helmut , Dobbelstein Matthias 
Published: June 2003

ABSTRACT

During infection with adenovirus, massive changes in the transcription of virus genes are observed, suggesting that the expression of cellular genes may also be modulated. To characterize the levels of cellular RNA species in infected cells, cDNA arrays were screened 24 h after infection of HeLa cells with wild-type adenovirus type 5, strain dl309. Despite complete transduction of the cells, fewer than 20 cellular genes (out of 4,600 analyzed and 1,200 found detectable and expressed above background) were altered more than threefold in their corresponding RNA levels compared to mock-infected cells. In particular, the expression of the
myc
oncogene was reduced at the mRNA level. This reduction was dependent on the replication of virus DNA and partially dependent on the presence of the adenovirus gene products E1B-55 kDa and E4orf6, but not E4orf3. On the other hand, MYC protein had an increased half-life in infected cells, resulting in roughly constant steady-state protein levels. The adenovirus E1A gene product is necessary and sufficient to stabilize MYC. Overexpressed MYC inhibited adenovirus replication and the proper formation of the virus replication centers. We conclude that adenovirus infection leads to the stabilization of MYC, perhaps as a side effect of E1A activities. On the other hand,
myc
mRNA levels are negatively regulated during adenovirus infection, and this may avoid the detrimental effect of excessive MYC on adenovirus replication.
The Ebola Virus VP35 Protein Inhibits Activation of Interferon Regulatory Factor 3
Authors: Basler Christopher F. , Mikulasova Andrea , Martinez-Sobrido Luis , Paragas Jason , Mühlberger Elke , Bray Mike , Klenk Hans-Dieter , Palese Peter , García-Sastre Adolfo 
Published: June 2003

ABSTRACT
The Ebola virus VP35 protein was previously found to act as an interferon (IFN) antagonist which could complement growth of influenza delNS1 virus, a mutant influenza virus lacking the influenza virus IFN antagonist protein, NS1. The Ebola virus VP35 could also prevent the virus- or double-stranded RNA-mediated transcriptional activation of both the beta IFN (IFN-β) promoter and the IFN-stimulated ISG54 promoter (C. Basler et al., Proc. Natl. Acad. Sci. USA 97:12289-12294, 2000). We now show that VP35 inhibits virus infection-induced transcriptional activation of IFN regulatory factor 3 (IRF-3)-responsive mammalian promoters and that VP35 does not block signaling from the IFN-α/β receptor. The ability of VP35 to inhibit this virus-induced transcription correlates with its ability to block activation of IRF-3, a cellular transcription factor of central importance in initiating the host cell IFN response. We demonstrate that VP35 blocks the Sendai virus-induced activation of two promoters which can be directly activated by IRF-3, namely, the ISG54 promoter and the ISG56 promoter. Further, expression of VP35 prevents the IRF-3-dependent activation of the IFN-α4 promoter in response to viral infection. The inhibition of IRF-3 appears to occur through an inhibition of IRF-3 phosphorylation. VP35 blocks virus-induced IRF-3 phosphorylation and subsequent IRF-3 dimerization and nuclear translocation. Consistent with these observations, Ebola virus infection of Vero cells activated neither transcription from the ISG54 promoter nor nuclear accumulation of IRF-3. These data suggest that in Ebola virus-infected cells, VP35 inhibits the induction of antiviral genes, including the IFN-β gene, by blocking IRF-3 activation.
Delivery of Adeno-Associated Virus Vectors to the Fetal Retina: Impact of Viral Capsid Proteins on Retinal Neuronal Progenitor Transduction
Authors: Surace Enrico M. , Auricchio Alberto , Reich Samuel J. , Rex Tonia , Glover Ernest , Pineles Stacey , Tang Waixing , O'Connor Erin , Lyubarsky Arkady , Savchenko Andrey , Pugh Edward N. , Maguire Albert M. , Wilson James M. , Bennett Jean 
Published: June 2003

ABSTRACT
The development of fetal ocular gene transfer may be useful as a therapeutic tool for the prevention of retinal genetic disorders with congenital or early clinical manifestations. In this study we explored the neural progenitor transduction patterns of adeno-associated virus (AAV) vectors following delivery to the developing retina. Recombinant vectors with the same genome carrying the enhanced green fluorescent protein (EGFP) transgene packaged in capsids of differing serotypes (serotypes 1, 2, and 5, termed AAV2/1, AAV2/2, and AAV2/5, respectively) were created. Delivery of the AAV vectors during early retinal development resulted in efficient and stable transduction of retinal progenitors. Vector surface proteins and the developmental status of the retina profoundly affected viral tropism and transgene distribution. The procedure is not detrimental to retinal development and function and therefore provides a safe delivery vehicle for potential therapeutic applications and a means of assessing the mechanisms of retina development and disease.
The Viral σ1 Protein and Glycoconjugates Containing α2-3-Linked Sialic Acid Are Involved in Type 1 Reovirus Adherence to M Cell Apical Surfaces
Authors: Helander Anna , Silvey Katherine J. , Mantis Nicholas J. , Hutchings Amy B. , Chandran Kartik , Lucas William T. , Nibert Max L. , Neutra Marian R. 
Published: June 2003

ABSTRACT

Type 1 reoviruses invade the intestinal mucosa of mice by adhering selectively to M cells in the follicle-associated epithelium and then exploiting M cell transport activity. The purpose of this study was to identify the apical cell membrane component and viral protein that mediate the M cell adherence of these viruses. Virions and infectious subviral particles of reovirus type 1 Lang (T1L) adhered to rabbit M cells in Peyer's patch mucosal explants and to tissue sections in an overlay assay. Viral adherence was abolished by pretreatment of sections with periodate and in the presence of excess sialic acid or lectins MAL-I and MAL-II (which recognize complex oligosaccharides containing sialic acid linked α2-3 to galactose). The binding of T1L particles to polarized human intestinal (Caco-2
BBe
) cell monolayers was correlated with the presence of MAL-I and MAL-II binding sites, blocked by excess MAL-I and -II, and abolished by neuraminidase treatment. Other type 1 reovirus isolates exhibited MAL-II-sensitive binding to rabbit M cells and polarized Caco-2
BBe
cells, but type 2 or type 3 isolates including type 3 Dearing (T3D) did not. In assays using T1L-T3D reassortants and recoated viral cores containing T1L, T3D, or no σ1 protein, MAL-II-sensitive binding to rabbit M cells and polarized Caco-2
BBe
cells was consistently associated with the T1L σ1. MAL-II-recognized oligosaccharide epitopes are not restricted to M cells in vivo, but MAL-II immobilized on virus-sized microparticles bound only to the follicle-associated epithelium and M cells. The results suggest that selective binding of type 1 reoviruses to M cells in vivo involves interaction of the type 1 σ1 protein with glycoconjugates containing α2-3-linked sialic acid that are accessible to viral particles only on M cell apical surfaces.
Kaposi's Sarcoma-Associated Herpesvirus (Human Herpesvirus 8) Infection of Human Fibroblast Cells Occurs through Endocytosis
Authors: Akula Shaw M. , Naranatt Pramod P. , Walia Neelam-Sharma , Wang Fu-Zhang , Fegley Barbara , Chandran Bala 
Published: June 2003

ABSTRACT

Kaposi's sarcoma (KS)-associated herpesvirus or human herpesvirus 8 (HHV-8) DNA and transcripts have been detected in the B cells, macrophages, keratinocytes, and endothelial and epithelial cells of KS patients. In vitro, HHV-8 infects human B, endothelial, epithelial, and fibroblast cells, as well as animal cells, and the infection is characterized by (i) absence of lytic replication by the input virus and (ii) latent infection. For its initial binding to target cells, HHV-8 uses ubiquitous heparan sulfate molecules via its envelope-associated glycoproteins gB and gpK8.1A. HHV-8 also interacts with the α3β1 integrin via its glycoprotein gB, and virus binding studies suggest that α3β1 is one of the HHV-8 entry receptors (S. M. Akula, N. P. Pramod, F. Z. Wang, and B. Chandran, Cell 108:407-419, 2002). In this study, morphological and biochemical techniques were used to examine the entry of HHV-8 into human foreskin fibroblasts (HFF). HHV-8 was detected in coated vesicles and in large, smooth-surfaced endocytic vesicles. Fusion of viral envelope with the vesicle wall was also observed. In immune electron microscopy, anti-HHV-8 gB antibodies colocalized with virus-containing endocytic vesicles. In fluorescence microscopic analyses, transferrin was colocalized with HHV-8. HHV-8 infection was significantly inhibited by preincubation of cells with chlorpromazine HCl, which blocks endocytosis via clathrin-coated pits, but not by nystatin and cholera toxin B, which blocks endocytosis via caveolae and induces the dissociation of lipid rafts, respectively. Infection was also inhibited by blocking the acidification of endosomes by NH
4
Cl and bafilomycin A. Inhibition of HHV-8 open reading frame 73 gene expression by chlorpromazine HCl, bafilomycin A, and NH
4
Cl demonstrated that the virions in the vesicles could proceed to cause an infection. Taken together, these findings suggest that for its infectious entry into HFF, HHV-8 uses clathrin-mediated endocytosis and a low-pH intracellular environment.
Subclinical Prion Disease Induced by Oral Inoculation
Authors: Thackray Alana M. , Klein Michael A. , Bujdoso Raymond 
Published: June 2003

ABSTRACT

Natural transmission of prion disease is believed to occur by peripheral infection such as oral inoculation. Following this route of inoculation, both the peripheral nervous system and the lymphoreticular system may be involved in the subsequent neuroinvasion of the central nervous system by prions, which may not necessarily result in clinical signs of terminal disease. Subclinical prion disease, characterized by the presence of infectivity and PrP
Sc
in the absence of overt clinical signs, may occur. It is not known which host factors contribute to whether infection with prions culminates in a terminal or subclinical disease state. We have investigated whether the level of host PrP
c
protein expression is a factor in the development of subclinical prion disease. When RML prion inoculum was inoculated by either the i.c. or intraperitoneal route, wild-type and
tga20
mice both succumbed to terminal prion disease. In contrast, orally inoculated
tga20
mice succumbed to terminal prion disease, whereas wild-type mice showed no clinical signs. However, wild-type mice sacrificed 375 or 525 days after oral inoculation harbored significant levels of brain PrP
Sc
and infectivity. These data show that same-species transmission of prions by the oral route in animals that express normal levels of PrP
c
can result in subclinical prion disease. This indicates that the level of host PrP
c
protein expression is a contributing factor to the regulation of development of terminal prion disease. Events that increase PrP
c
expression may predispose a prion-infected animal to the more deleterious effects of prion pathology.
Bunyamwera Virus Nonstructural Protein NSs Counteracts Interferon Regulatory Factor 3-Mediated Induction of Early Cell Death
Authors: Kohl Alain , Clayton Reginald F. , Weber Friedemann , Bridgen Anne , Randall Richard E. , Elliott Richard M. 
Published: June 2003

ABSTRACTThe genome of Bunyamwera virus (BUN; familyBunyaviridae, genusOrthobunyavirus) consists of three segments of negative-sense RNA. The smallest segment, S, encodes two proteins, the nonstructural protein NSs, which is nonessential for viral replication and transcription, and the nucleocapsid protein N. Although a precise role in the replication cycle has yet to be attributed to NSs, it has been shown that NSs inhibits the induction of alpha/beta interferon, suggesting that it plays a part in counteracting the host antiviral defense. A defense mechanism to limit viral spread is programmed cell death by apoptosis. Here we show that a recombinant BUN that does not express NSs (BUNdelNSs) induces apoptotic cell death more rapidly than wild-type virus. Screening for apoptosis pathways revealed that the proapoptotic transcription factor interferon regulatory factor 3 (IRF-3) was activated by both wild-type BUN and BUNdelNSs infection, but only wild-type BUN was able to suppress signaling downstream of IRF-3. Studies with a BUN minireplicon system showed that active replication induced an IRF-3-dependent promoter, which was suppressed by the NSs protein. In a cell line (P2.1) defective in double-stranded RNA signaling due to low levels of IRF-3, induction of apoptosis was similar for wild-type BUN and BUNdelNSs. These data suggest that the BUN NSs protein can delay cell death in the early stages of BUN infection by inhibiting IRF-3-mediated apoptosis.
Hydrogen Bonding at a Conserved Threonine in Lentivirus Capsid Is Required for Virus Replication
Authors: Rue Sarah M. , Roos Jason W. , Amzel L. Mario , Clements Janice E. , Barber Sheila A. 
Published: June 2003

ABSTRACT

The N terminus of the capsid protein (CA) undergoes a considerable conformational change when the human immunodeficiency virus (HIV) protease cleaves it free from the Pr55
Gag
polyprotein. This rearrangement is thought to facilitate the establishment of specific CA-CA interactions that are required for the formation of the mature viral core. Substitution of amino acids that are critical for this refolding of the N terminus is generally detrimental to virus replication and mature virion core morphology. Here, we identify a conserved threonine in simian immunodeficiency virus (SIV) CA, T(47)
CA
, that is requisite for viral replication. Replacement of T(47)
CA
in the infectious viral clone SIVmac239 with amino acids with different hydrogen-bonding capabilities and analysis of the effects of these substitutions at key steps in the viral life cycle demonstrate that hydrogen bonding at this position is important for virus infectivity and virion release. In the HIV-based homology model of the mature SIV CA N terminus presented in this study, T(47)
CA
forms several hydrogen bonds with a proximal aspartate, D(50)
CA
. This model, coupled with strong phenotypic similarities between viral substitution mutants of each of these two residues in all of the virological assays described herein, indicates that hydrogen bonding between T(47)
CA
and D(50)
CA
is likely required for viral replication. As hydrogen bonding between these two residues is present in HIV CA as well, this interaction presents a potential target for antiviral drug design.