- Volume 90, Issue 5, 2009
Volume 90, Issue 5, 2009
- Animal
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- DNA viruses
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A truncated T antigen expressed from an alternatively spliced BK virus early mRNA
More LessThe early region of BK virus (BKV) is known to encode two well-characterized tumour (T) antigens, large T antigen (TAg) and small T antigen (tAg). In this study, we provide evidence of a third early BKV mRNA that codes for an additional early region product with an apparent molecular mass of 17–20 kDa. This truncated form of TAg (truncTAg) is expressed from an alternatively spliced mRNA that is derived from the excision of a second intron from the mRNA encoding TAg. The first 133 aa of truncTAg are identical to those of TAg but the additional splice results in translation from a different reading frame, adding three new amino acids before reaching a stop codon. TruncTAg is expressed in both BKV-transformed and lytically infected cells and it is found to be primarily localized to the nucleus. The function of BKV truncTAg is likely to be relevant to transformation, similar to the additional T antigens of simian virus 40, JC virus and mouse polyomavirus.
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Prospero-related homeobox protein (Prox1) inhibits hepatitis B virus replication through repressing multiple cis regulatory elements
Hepatitis B virus (HBV) gene transcription is controlled by viral promoters and enhancers, the activities of which are regulated by a number of cellular factors as well as virally encoded proteins. Negative regulation of HBV cis-element activities by cellular factors has been reported less widely than their activation. In this study, we report that nuclear factor Prospero-related homeobox protein (Prox1) represses HBV antigen expression and genome replication in cultured hepatocytes. By using reporter-gene analysis, three of the four HBV promoters, namely the enhancer II/core promoter (ENII/Cp), preS1 promoter (Sp1) and enhancer I/X promoter, were identified as targets for Prox1-mediated repression. Mechanistic analysis then revealed that, for ENII/Cp, Prox1 serves as a corepressor of liver receptor homologue 1 (LRH-1) and downregulates LRH-1-mediated activation of ENII/Cp, whereas for Sp1, Prox1 partially represses Sp1 activity by interacting directly with hepatocyte nuclear factor 1. Identification of Prox1 as an HBV repressor will help in the understanding of detailed interactions between viral cis elements and host cellular factors and may also form the basis for new anti-HBV intervention therapeutics.
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Novel anellovirus discovered from a mortality event of captive California sea lions
More LessA viral metagenomic study was performed to investigate potential viral pathogens associated with a mortality event of three captive California sea lions (Zalophus californianus). This study identified a novel California sea lion anellovirus (ZcAV), with 35 % amino acid identity in the ORF1 region to feline anelloviruses. The double-stranded replicative form of ZcAV was detected in lung tissue, suggesting that ZcAV replicates in sea lion lungs. Specific PCR revealed the presence of ZcAV in the lung tissue of all three sea lions involved in the mortality event, but not in three other sea lions from the same zoo. In addition, ZcAV was detected at low frequency (11 %) in the lungs of wild sea lions. The higher prevalence of ZcAV and presence of the double-stranded replicative form in the lungs of sea lions from the mortality event suggest that ZcAV was associated with the death of these animals.
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Analysis of promoter activity of selected Cotesia plutellae bracovirus genes
In a previous study, we cloned 27 discrete genome segments of Cotesia plutellae bracovirus (CpBV) and provided the complete nucleotide sequences and annotation. Seven putative coding regions were predicted from one of the largest segments, CpBV-S30. The activity of promoters associated with six predicted ORFs from this segment were investigated using both transient and baculovirus expression assays with enhanced green fluorescent protein as a reporter gene. CpBV promoters showed activity earlier than the polyhedrin promoter and the activity of some of these promoters was superior to that of the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ie-1 promoter in the baculovirus expression assays. The promoter of ORF3004 showed the highest level of activity in insect cells, exhibiting 24 % of the activity obtained with the AcMNPV polyhedrin promoter in Sf9 cells. In Spodoptera exigua larvae, the ORF3006 promoter showed the highest activity, with about 35 % of the activity measured with the polyhedrin promoter. In addition, analysis of the ORF3006 promoter revealed that the region between −382 and −422 from the translation start point was critical for activity of this promoter. These results suggest that the CpBV-S30 promoters characterized here could be useful tools in a variety of biotechnological applications, such as gene expression analyses and insecticide development.
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Analysis of transcripts from predicted open reading frames of Musca domestica salivary gland hypertrophy virus
More LessThe Musca domestica salivary gland hypertrophy virus (MdSGHV) is a large dsDNA virus that infects and sterilizes adult houseflies. The transcriptome of this newly described virus was analysed by rapid amplification of cDNA 3′-ends (3′-RACE) and RT-PCR. Direct sequencing of 3′-RACE products revealed 78 poly(A) transcripts containing 95 of the 108 putative ORFs. An additional six ORFs not amplified by 3′-RACE were detected by RT-PCR. Only seven of the 108 putative ORFs were not amplified by either 3′-RACE or RT-PCR. A series of 5′-RACE reactions were conducted on selected ORFs that were identified by 3′-RACE to be transcribed in tandem (tandem transcripts). In the majority of cases, the downstream ORFs were detected as single transcripts as well as components of the tandem transcripts, whereas the upstream ORFs were found only in tandem transcripts. The only exception was the upstream ORF MdSGHV084, which was differentially transcribed as a single transcript at 1 and 2 days post-infection (days p.i.) and as a tandem transcript (MdSGHV084/085) at 2 days p.i. Transcriptome analysis of MdSGHV detected splicing in the 3′ untranslated region (3′-UTR) and extensive heterogeneity in the polyadenylation signals and cleavage sites. In addition, 23 overlapping antisense transcripts were found. In conclusion, sequencing the 3′-RACE products without cloning served as an alternative approach to detect both 3′-UTRs and transcript variants of this large DNA virus.
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- Plant
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A multipartite single-stranded negative-sense RNA virus is the putative agent of fig mosaic disease
Several dsRNA bands (approx. 0.6–7 kbp in size) were recovered from tissues of mosaic-diseased fig seedlings which contained the enveloped round structures known as double membrane bodies (DMBs). blast analysis of a 4353 and a 1120 nt sequence from the two largest RNA segments showed homology with the polymerase and the putative glycoprotein precursor genes of negative-sense single-stranded RNA viruses of the family Bunyaviridae. Negative- and positive-sense riboprobes designed from both RNA segments hybridized to two bands of approximately 7 and 2.3 kbp in Northern blots of dsRNAs. Thus, these segments were identified as putative RNA-1 and RNA-2 of a novel virus for which the name fig mosaic virus (FMV) is proposed. Identity levels of predicted amino acids of the protein encoded by FMV RNA-1 with those of species of the family Bunyaviridae and European mountain ash ringspot-associated virus (EMERaV) were 28 and 54 %, respectively. RNA-2 showed 38 % identity at the amino acid level only with EMARaV. RNA-1 segment contained five conserved motifs (A–E) and an endonucleolytic centre of comparable genes of L RNA of bunyaviruses and EMARaV RNA-1. In a phylogenetic tree constructed with RdRp sequences, EMARaV grouped with FMV in a clade distinct from those of all bunyavirus genera. The consistent association of DMBs with mosaic symptoms and the results of molecular investigations strongly indicate that DMBs are particles of FMV, the aetiological agent of fig mosaic disease.
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- Other Agents
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Ovine PRNP untranslated region and promoter haplotype diversity
The diversity and possible contribution of non-coding regions of the prion protein (PrP) gene (PRNP) to transmissible spongiform encephalopathy susceptibility and PrP regulation are not fully known. This study defined ten ovine PRNP promoters and five untranslated region (UTR) haplotypes found in atypical and classical scrapie cases and healthy control sheep. A greater diversity of promoter and UTR haplotypes was observed in conjunction with the ARQ PrP allele (seven promoter and four UTR haplotypes), while it was observed that the other alleles were linked with a limited number of haplotypes, such as ARR, found to be linked to only two promoter and one UTR haplotypes. In silico analysis identified potential transcription factor binding sites that differed in the promoter haplotype variants. Furthermore, a 5′ UTR internal ribosome entry site motif was identified in exon 2 and highlights a possible role for this exon in regulating PrP expression at the translational level.
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New inhibitors of prion replication that target the amyloid precursor
At present, there is no effective therapy for any of the neurodegenerative amyloidoses, despite renewed efforts to identify compounds active against the various implicated pathogenetic molecules. We have screened a library of 2960 natural and synthetic compounds in two cell lines chronically infected with mouse prions, and have identified eight new inhibitors of prion replication in vitro. They belong to two distinct chemical families that have not previously been recognised as effective in the field of transmissible spongiform encephalopathies: seven are 3-aminosteroids and one is a derivative of erythromycin A with an oxime functionality. Our results suggest that these aminosteroids inhibit prion replication by triggering a common target, possibly implicated in the regulatory pathways of cellular prion protein metabolism. Furthermore, using a quantitative approach for the study of protein stability, it was shown that the erythromycin A derivative altered prion protein stability by direct interaction. Such direct targeting of this amyloid precursor might provide new clues for the understanding of prion diseases and, more importantly, help to define new molecules that are active against prion diseases.
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Volumes and issues
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Volume 105 (2024)
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Volume 1 (1967)