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Volume 84,
Issue 9,
2003
Volume 84, Issue 9, 2003
- Animal
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- DNA viruses
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Herpes simplex virus type 1 exhibits a tropism for basal entry in polarized epithelial cells
More LessHerpes simplex virus type 1 (HSV-1) enters its host via epithelia and spreads to neuronal cells where latency is established. Hence, the in vivo route of infection relies on penetration and subsequent passage of HSV-1 through highly polarized cells. Infection studies were performed in both polarized MDCKII cells and primary human keratinocytes to gain insight into the pathway of virus entry into individual epithelial cells. Early viral gene expression was barely detectable in confluent MDCKII cells, even at high m.o.i. However, after wounding the cell layer, infected cells were observed next to the wound, where basolateral membranes were accessible. In subconfluent monolayers, MDCKII cells are organized in islets. After infection, viral capsids and early viral gene expression were detectable in peripheral cells of islets, supporting virus penetration via basolateral membranes. Further infection studies were performed in human keratinocytes, which represent the primary target cells for HSV-1 infection in vivo. In primary keratinocytes grown as monolayer cultures and wounded prior to infection, HSV-1 infection led to early viral gene expression predominantly in cells next to the wound. When stratifying cultures of primary human keratinocytes were infected, early viral gene expression was localized to peripheral cells of basal keratinocytes. Finally, infection of epithelial tissue such as human foreskin epithelia demonstrated HSV-1 entry exclusively via basal cell layers. Staining of the potential coreceptor nectin-1/HveC revealed no correlation of receptor localization and virus entry sites in keratinocytes. These results provide first evidence for a virus entry mechanism that relies on the accessibility to basal surfaces of epithelial tissue and to basolateral membranes, both in MDCKII and primary keratinocytes.
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Implication of the product of the bovine herpesvirus type 1 UL25 gene in capsid assembly
More LessThe UL25 ORF of bovine herpesvirus type 1 (BHV-1) was demonstrated recently to represent a gene encoding a 63 kDa viral protein. To investigate the role of this gene in virus replication, a BHV-1 UL25 deletion mutant was constructed. Although the UL25 mutant synthesizes late viral proteins and viral DNA, it fails to produce virus progeny in cells that do not express the UL25 gene, demonstrating that the UL25 protein is essential for the replicative cycle of BHV-1. Moreover, Southern blotting analyses of HindIII-digested DNA from infected non-complementing cells probed with the leftward terminal fragment of the BHV-1 linear genome revealed that the cleavage of the viral DNA produced is not impaired. However, the packaging of this cleaved DNA is compromised severely, since only few full C capsids were observed in infected non-complementing cells by transmission electron microscopy.
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Induction of RANTES/CCL5 by herpes simplex virus is regulated by nuclear factor κB and interferon regulatory factor 3
More LessChemokines regulate migration of leukocytes to sites of infection. In this work, we have shown that the chemokine RANTES/CCL5 is produced after herpes simplex virus (HSV) infection of macrophages and fibroblasts and provide data on the underlying molecular mechanism. Reporter gene assays showed HSV-induced RANTES production to be regulated at the transcriptional level. Expression of RANTES was blocked by N-tosyl-l-phenylalanine, an inhibitor of the nuclear factor κB (NF-κB) pathway and also in cell lines stably expressing a dominant-negative mutant of IκB kinase β. Cell lines stably expressing a dominant-negative mutant of interferon regulatory factor 3 (IRF-3) also produced dramatically decreased levels of RANTES after infection compared with the control cell line. In contrast, overexpression of dominant-negative p38 and also treatment with SB203580, an inhibitor of p38, did not significantly affect expression of RANTES. Taken together, these data suggest that HSV induces transcriptional activation of the RANTES gene through the transcription factors NF-κB and IRF-3.
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Interferon (IFN)-α/β, interleukin (IL)-12 and IL-18 coordinately induce production of IFN-γ during infection with herpes simplex virus type 2
More LessInterferons (IFNs) are important mediators of antiviral activity. In this study we have investigated how production of IFN-γ is induced during herpes simplex virus type 2 (HSV-2) infection in murine peritoneal cells (PCs). We found that HSV-2 infection of thioglycolate-activated PCs from BALB/c mice rapidly led to expression of type 1 IFNs (IFN-α/β) and interleukin (IL)-12, which was followed by production of IFN-γ. IL-12 alone induced the most expression of IFN-γ, which was augmented by cotreatment with IFN-α or IL-18, or combinations of IFN-α and IL-18. Moreover, neutralization of any of these cytokines in vitro strongly reduced the production of IFN-γ, and neutralization of all three cytokines totally prevented HSV-2-induced IFN-γ expression. Our data suggest that IFN-γ production is induced during HSV-2 infection through the coordinated action of IFN-α/β, IL-12 and IL-18.
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Herpes simplex virus type 1 tegument protein VP22 interacts with TAF-I proteins and inhibits nucleosome assembly but not regulation of histone acetylation by INHAT
More LessAffinity chromatography was used to identify cellular proteins that interact with the herpes simplex virus (HSV) tegument protein VP22. Among a small set of proteins that bind specifically to VP22, we identified TAF-I (template-activating factor I), a chromatin remodelling protein and close homologue of the histone chaperone protein NAP-1. TAF-I has been shown previously to promote more ordered transfer of histones to naked DNA through a direct interaction with histones. TAF-I, as a subunit of the INHAT (inhibitor of acetyltransferases) protein complex, also binds to histones and masks them from being substrates for the acetyltransferases p300 and PCAF. Using in vitro assays for TAF-I activity in chromatin assembly, we show that VP22 inhibits nucleosome deposition on DNA by binding to TAF-I. We also observed that VP22 binds non-specifically to DNA, an activity that is abolished by TAF-I. However, the presence of VP22 does not affect the property of INHAT in inhibiting the histone acetyltransferase activity of p300 or PCAF in vitro. We speculate that this interaction could be relevant to HSV DNA organization early in infection, for example, by interfering with nucleosomal deposition on the genome. Consistent with this possibility was the observation that overexpression of TAF-I in transfected cells interferes with the progression of HSV-1 infection.
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The most abundantly transcribed human cytomegalovirus gene (β2.7) is non-essential for growth in vitro
More LessThe most abundantly transcribed HCMV gene (β2.7) encodes a 2·7 kb polyadenylated RNA. Although the laboratory-adapted HCMV strains AD169 and Towne possess two copies of the β2.7 gene within an expanded b sequence element, the low passage strain Toledo and all clinical isolates analysed contain only a single copy located within the UL region. A β2.7 deletion mutant constructed based on a strain Toledo background was shown to replicate with kinetics comparable to those of the parental virus; the β2.7 gene is therefore not essential for virus replication in vitro. Sequencing the β2.7 gene from HCMV clinical isolates and the Toledo strain reveals that although the overall gene sequence is highly conserved (>99 %), the RL4 frame originally assigned in strain AD169 was disrupted in each of these viruses. Consequently, the β2.7 transcript does not encode any obvious translation product and thus may not function as an mRNA.
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The rat cytomegalovirus R78 G protein-coupled receptor gene is required for production of infectious virus in the spleen
The rat cytomegalovirus (RCMV) R33 and R78 genes are conserved within members of the subfamily Betaherpesvirinae and encode proteins (pR33 and pR78, respectively) that show sequence similarity with G protein-coupled receptors. Previously, the biological relevance of these genes was demonstrated by the finding that R33- and R78-deleted RCMV strains are severely attenuated in vivo. In addition, R78-deleted strains were found to replicate less efficiently in cell culture. To monitor of the expression of R33- and R78-encoded proteins, recombinant RCMV strains, designated RCMV33G and RCMV78G, were generated. These recombinants expressed enhanced green fluorescent protein (EGFP)-tagged versions of pR33 and pR78 instead of native pR33 and pR78, respectively. Here it is reported that, although RCMV33G replicates as efficiently as wt virus in rat embryo fibroblast cultures, strain RCMV78G produces virus titres that are 3- to 4-fold lower than wt RCMV in the culture medium. This result indicates that the pR78-EGFP protein, as expressed by RCMV78G, does not completely functionally replace its native counterpart (pR78) in vitro. Interestingly, in infected rats, infectious RCMV33G was produced in significantly lower amounts than infectious wt RCMV, as well as RCMV78G, in the salivary glands. Conversely, although RCMV33G replicated to similar levels as wt virus in the spleen, both RCMV78G and an R78 knock-out strain (RCMVΔR78a) replicated poorly in this organ. Together, these data indicate that R78 is crucial for the production of infectious RCMV in the spleen of infected rats.
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Phylogenetic analysis and possible function of bro-like genes, a multigene family widespread among large double-stranded DNA viruses of invertebrates and bacteria
More LessBaculovirus repeated open reading frame (bro) genes and their relatives constitute a multigene family, typically with multiple copies per genome, known to occur among certain insect dsDNA viruses and bacteriophages. Little is known about the evolutionary history and function of the proteins encoded by these genes. Here we have shown that bro and bro-like (bro-l) genes occur among viruses of two additional invertebrate viral families, Ascoviridae and Iridoviridae, and in prokaryotic class II transposons. Analysis of over 100 sequences showed that the N-terminal region, consisting of two subdomains, is the most conserved region and contains a DNA-binding motif that has been characterized previously. Phylogenetic analysis indicated that these proteins are distributed among eight groups, Groups 1–7 consisting of invertebrate virus proteins and Group 8 of proteins in bacteriophages and bacterial transposons. No bro genes were identified in databases of invertebrate or vertebrate genomes, vertebrate viruses and transposons, nor in prokaryotic genomes, except in prophages or transposons of the latter. The phylogenetic relationship between bro genes suggests that they have resulted from recombination of viral genomes that allowed the duplication and loss of genes, but also the acquisition of genes by horizontal transfer over evolutionary time. In addition, the maintenance and diversity of bro-l genes in different types of invertebrate dsDNA viruses, but not in vertebrate viruses, suggests that these proteins play an important role in invertebrate virus biology. Experiments with the unique orf2 bro gene of Autographa californica multicapsid nucleopolyhedrovirus showed that it is not required for replication, but may enhance replication during the occlusion phase of reproduction.
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A phage-displayed peptide can inhibit infection by white spot syndrome virus of shrimp
More LessWhite spot disease, caused by white spot syndrome virus (WSSV), results in devastating losses to the shrimp farming industry around the world, and no effective treatments have been found. Control focuses on exclusion of the virus from culture ponds but, once introduced, spread is often rapid and uncontrollable. The purpose of this study was to select a phage-displayed peptide that might be able to prevent WSSV infection. A 10-mer phage display peptide library (titre 7·2×107) was constructed and screened against immobilized WSSV. Selected peptides were assessed for specificity and efficiency of inhibition of virus infection. Of four peptides that specifically bound to WSSV one, designated 2E6, had a high specificity and blocked virus infection, with the possible critical motif for virus inhibition being VAVNNSY. The results suggest that peptide 2E6 has potential for exploitation as an antiviral peptide drug.
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- Plant
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Further variability within the genus Crinivirus, as revealed by determination of the complete RNA genome sequence of Cucurbit yellow stunting disorder virus
The complete nucleotide (nt) sequences of genomic RNAs 1 and 2 of Cucurbit yellow stunting disorder virus (CYSDV) were determined for the Spanish isolate CYSDV-AlLM. RNA1 is 9123 nt long and contains at least five open reading frames (ORFs). Computer-assisted analyses identified papain-like protease, methyltransferase, RNA helicase and RNA-dependent RNA polymerase domains in the first two ORFs of RNA1. This is the first study on the sequences of RNA1 from CYSDV. RNA2 is 7976 nt long and contains the hallmark gene array of the family Closteroviridae, characterized by ORFs encoding a heat shock protein 70 homologue, a 59 kDa protein, the major coat protein and a divergent copy of the coat protein. This genome organization resembles that of Sweet potato chlorotic stunt virus (SPCSV), Cucumber yellows virus (CuYV) and Lettuce infectious yellows virus (LIYV), the other three criniviruses sequenced completely to date. However, several differences were observed. The most striking novel features of CYSDV compared to SPCSV, CuYV and LIYV are a unique gene arrangement in the 3′-terminal region of RNA1, the identification in this region of an ORF potentially encoding a protein which has no homologues in any databases, and the prediction of an unusually long 5′ non-coding region in RNA2. Additionally, the CYSDV genome resembles that of SPCSV in having very similar 3′ regions in RNAs 1 and 2, although for CYSDV similarity in primary structures did not result in predictions of equivalent secondary structures. Overall, these data reinforce the view that the genus Crinivirus contains considerable genetic variation. Additionally, several subgenomic RNAs (sgRNAs) were detected in CYSDV-infected plants, suggesting that generation of sgRNAs is a strategy used by CYSDV for the expression of internal ORFs.
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Molecular resolution of a complex of potyviruses infecting solanaceous crops at the centre of origin in Peru
More LessPeru is a centre of origin and domestication of the potato, pepper and tomato (family Solanaceae). Many potyviruses (genus Potyvirus) that infect these crops were described 20–30 years ago. However, definitive classification of these viruses as distinct species remains unresolved for several reasons, including their close serological relationships, similar symptomatology in test plants and lack of genomic sequence data. Using samples collected from Peru, we have determined the complete genomic sequence of two strains of Peru tomato virus (PTV) as well as near-complete sequences for two additional PTV strains. We also obtained partial sequences of four strains of Potato virus V (PVV). Comparisons with genomic sequences of Wild potato mosaic virus (WPMV), Potato virus Y (PVY), Pepper mottle virus (PepMoV), Potato virus A (PVA) and other potyviruses established that all these viruses constitute different taxa (species). Phylogenetic comparisons indicated that PTV, PVV and WPMV are the most closely related species which, together with PepMoV, PVY, Pepper yellow mosaic virus and Pepper severe mosaic virus, constitute a group that is distinguishable from other potyviruses. Therefore, the members of this group may share a common ancestor. PVA does not belong to this group. PVV and PTV were also closely related serologically. However, PTV did not cross-protect against PVV and WPMV in tobacco plants or complement systemic infection of PVV and WPMV in pepper plants. Two biologically and phylogenetically distinguishable strain groups were identified within PTV and PVV. In future studies, the sequence data and virus-specific primers and probes for PTV, PVV and WPMV described in this study will enable accurate indexing of plants with respect to either single or mixed infection with these viruses.
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A single silent substitution in the genome of Apple stem grooving virus causes symptom attenuation
More LessAmong randomly mutagenized clones derived from an infectious cDNA copy of genomic RNA of Apple stem grooving virus (ASGV), we previously identified a clone, pRM21, whose in vitro transcript (ASGV-RM21) does not induce any symptoms characteristic of the original (wild-type) cDNA clone (ASGV-wt) in several host plants. Interestingly, ASGV-RM21 has only a single, translationally silent nucleotide substitution, U to C, at nucleotide 4646 of the viral genome within open reading frame (ORF) 1. Here, we characterize and verify this unprecedented silent-mutation-induced attenuation of symptoms in infected plants. Northern and Western blot analyses showed that less ASGV-RM21 accumulates in host plants than ASGV-wt. In addition, two more silent substitutions, U to A and U to G, constructed by site-directed mutagenesis at the same nucleotide (4646), also induced attenuated symptoms. This is the first report that a single silent substitution attenuates virus-infection symptoms and implicates a novel determinant of disease symptom severity.
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Mapping of viral RNA sequences required for assembly of peanut clump virus particles
More LessRNA sequences required for assembly into rod-shaped virions of RNA-1 and RNA-2 of Peanut clump virus (PCV) were mapped by testing the ability of different RNA-1 and -2 deletion mutants to be encapsidated in vivo in an RNase-resistant form. Encapsidation of RNA-1 was found to require a sequence domain in the 5′-proximal part of the P15 gene, the 3′-proximal gene of RNA-1. On the other hand, the subgenomic RNA which encodes P15 was not encapsidated, suggesting that other features of RNA-1 are important as well. Two sequences which could drive encapsidation of RNA-2 deletion mutants were located. One was in the 5′-proximal coat protein gene and the other in the P14 gene near the RNA 3′ terminus. There were no obvious sequence homologies between the different assembly initiation sequences.
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- Other Agents
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A novel generation of heparan sulfate mimetics for the treatment of prion diseases
The accumulation of PrPres, the protease-resistant abnormal form of the host-encoded cellular prion protein, PrPC, plays a central role in transmissible spongiform encephalopathies. Human contamination by bovine spongiform encephalopathy (BSE) has propelled many scientific teams on a highway for anti-prion drug development. This study reports that heparan sulfate mimetics (HMs), developed originally for their effect on tissue regeneration, abolish prion propagation in scrapie-infected GT1 cells. PrPres does not reappear for up to 50 days post-treatment. When tested in vivo, one of these compounds, HM2602, hampered PrPres accumulation in scrapie- and BSE-infected mice and prolonged significantly the survival time of 263K scrapie-infected hamsters. Interestingly, HM2602 is an apparently less toxic and more potent inhibitor of PrPres accumulation than dextran sulfate 500, a molecule known to exhibit anti-prion properties in vivo. Kinetics of PrPres disappearance in vitro and unaffected PrPC levels during treatment suggest that HMs are able to block the conversion of PrPC into PrPres. It is speculated that HMs act as competitors of endogenous heparan sulfates known to act as co-receptors for the prion protein. Since these molecules are particularly amenable to drug design, their anti-prion potential could be developed further and optimized for the treatment of prion diseases.
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Inducible cytokine gene expression in the brain in the ME7/CV mouse model of scrapie is highly restricted, is at a strikingly low level relative to the degree of gliosis and occurs only late in disease
More LessThe temporal course of cerebral cytokine gene expression was investigated in the ME7/CV murine scrapie model to determine any association with neuropathological events. Analysis by RNase protection assay (RPA) demonstrated no transcripts for ILs 2, 3, 4, 5, 6, 7, 10, 12p40 and 13, granulocyte macrophage colony-stimulating factor, IFN-γ or lymphotoxin-α at any time during the course of this disease. Transcripts for transforming growth factor-β1 were constitutively expressed in both control and scrapie-infected brain and were elevated at terminal disease. RPA and quantitative real-time RT-PCR detected low levels of transcripts for IL-1α, IL-1β and TNFα in scrapie-infected brain but only IL-1β was elevated consistently in all mice studied. Although glial cell activation within the hippocampus was evident from 100 days post-infection (p.i.), elevated IL-1β transcripts (and immunoreactivity) were evident from 180 days p.i., around the time of hippocampal pyramidal neuron loss, and increased steadily thereafter to reach a 3·5-fold increase at terminal disease. Even at their maximum, levels of these transcripts were disproportionately low relative to the degree of glial cell activation. It is concluded that cytokine gene expression in the ME7 scrapie-infected mouse brain, relative to the degree of reactive gliosis, is highly restricted, temporally late and disproportionately low.
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Volumes and issues
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