- Volume 85, Issue 6, 2004
Volume 85, Issue 6, 2004
- Animal
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- DNA viruses
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Enhanced oncogenicity of Asian-American human papillomavirus 16 is associated with impaired E2 repression of E6/E7 oncogene transcription
Asian-American (AA) variants of human papillomavirus 16 (HPV-16) are linked to a high incidence of cervical cancer in Mexico, with some evidence strongly suggesting that they are more oncogenic than European (E) variants, including their association with younger women and their higher associated risk of cervical cancer. Differences in the regulation of viral E6/E7 oncogene transcription by the E2 protein may be involved in the higher oncogenicity of AA variants. In E variants, E6/E7 oncogene transcription is repressed by the E2 protein and is frequently up-regulated by the destruction of the E2 gene during viral integration. In contrast, the E2 gene is retained in full in most AA-positive carcinomas, raising the possibility of alternative mechanisms for increasing viral oncogene transcription. The authors investigated whether the higher oncogenicity of AA variants is linked to differences in E6/E7 oncogene transcription and the mechanism of E2 deactivation. E6/E7 and E1/E2 transcripts were explored by RT-PCR in 53 HPV-16-positive cervical carcinomas, 39 retaining (20 European and 19 AA) and 14 having lost (12 European and 2 AA) the E1/E2 genes, and transcription repression activity of the AA E2 genes was tested in four cell lines that constitutively express the β-galactosidase reporter or E6/E7 genes driven by the viral long control region. E6/E7 oncogene transcripts were found in all carcinomas, but only those positive for AA variants with E1/E2 genes had complete E2 transcripts. E2 transcripts were down-regulated by splicing in E-positive carcinomas retaining E1/E2. AA E2 genes were impaired for repression of E6/E7 oncogene transcription in vivo. These results suggest that E6/E7 oncogene expression starts earlier in AA than E variant infections, since E variants need E2 to be destroyed or down-regulated.
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TT virus-derived apoptosis-inducing protein induces apoptosis preferentially in hepatocellular carcinoma-derived cells
TT virus (TTV) is widespread among the global population. Its pathogenic nature is still unclear but TTV seems to be more prevalent in cases of hepatitis than in healthy individuals. TTV harbours similarities to chicken anaemia virus (CAV). Here, the apoptotic potential of a putative TTV-derived 105 aa protein and of the main apoptosis-inducing agent of CAV, Apoptin, is compared. As the putative protein induced apoptosis in various human hepatocellular carcinoma (HCC) cell lines, it was named TTV-derived apoptosis-inducing protein (TAIP). The apoptotic activity of TAIP in HCC lines was comparable with that of Apoptin. Conversely, unlike Apoptin, TAIP induced only low-level apoptosis in several non-HCC human cancer cell lines. The data suggest that TAIP acts in a different way to Apoptin as it is selective to a certain degree for HCC lines. This activity of TAIP, coupled with the heterogeneity of TTV isolates, may help to explain the variable reports of TTV pathogenicity.
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- Plant Viruses
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Characterization of regulatory elements within the coat protein (CP) coding region of Tobacco mosaic virus affecting subgenomic transcription and green fluorescent protein expression from the CP subgenomic RNA promoter
More LessA replicon based on Tobacco mosaic virus that was engineered to express the open reading frame (ORF) of the green fluorescent protein (GFP) gene in place of the native coat protein (CP) gene from a minimal CP subgenomic (sg) RNA promoter was found to accumulate very low levels of GFP. Regulatory regions within the CP ORF were identified that, when presented as untranslated regions flanking the GFP ORF, enhanced or inhibited sg transcription and GFP expression. Full GFP expression from the CP sgRNA promoter required more than the first 20 nt of the CP ORF but not beyond the first 56 nt. Further analysis indicated the presence of an enhancer element between nt +25 and +55 with respect to the CP translation start site. The inclusion of this enhancer sequence upstream of the GFP ORF led to elevated sg transcription and to a 50-fold increase in GFP accumulation in comparison with a minimal CP promoter in which the entire CP ORF was displaced by the GFP ORF. Inclusion of the 3′-terminal 22 nt had a minor positive effect on GFP accumulation, but the addition of extended untranslated sequences from the 3′ terminus of the CP ORF downstream of the GFP ORF was basically found to inhibit sg transcription. Secondary structure analysis programs predicted the CP sgRNA promoter to reside within two stable stem–loop structures, which are followed by an enhancer region.
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Evidence for contribution of an internal ribosome entry site to intercellular transport of a tobamovirus
More LessPreviously, it has been shown that tobacco mosaic virus (TMV) U1 and crucifer-infecting TMV contain a 75 nt internal ribosome entry site (IRES) upstream of movement protein (MP) gene (IRESU1 MP,75 and IRESCR MP,75, respectively). A movement-deficient TMV mutant, KK6, has been constructed previously [ Lehto, K., Grantham, G. L. & Dawson, W. O. (1990). Virology 174, 145–157 ] by insertion of the second coat protein subgenomic promoter (CP SGP-2) upstream of the MP gene, in addition to the natural CP SGP-1. Here, the authors compare the efficiency of movement function expression by KK6 and a derivative, K86, obtained by insertion of IRESCR MP,75 between the CP SGP-2 and MP genes resulting in restoration of IRESCR MP,75 function in the 5′-untranslated sequence of the I2 subgenomic RNA of K86. The data indicate that the efficiency of K86 movement was largely restored by this insertion, which was apparently due to the translation-enhancing ability of IRESCR MP,75.
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Tomato yellow leaf curl Sardinia virus can overcome transgene-mediated RNA silencing of two essential viral genes
To evaluate RNA silencing for the control of geminivirus infection, two classes of post-transcriptionally silenced (PTS) plants were tested using Tomato yellow leaf curl Sardinia virus (TYLCSV) Rep-210-transgenic plants, a sense×antisense hybrid and two multicopy sense lines. In both classes, PTS plants accumulated low or undetectable amounts of Rep-210 protein and mRNA but high amounts of Rep-210 small interfering RNAs. PTS plants were susceptible to TYLCSV when challenged by agroinoculation or using high viruliferous whitefly (Bemisia tabaci) pressure, although some plants were resistant at low whitefly pressure. Delayed infections were also observed, indicating that TYLCSV could overcome transgene silencing of rep and of the nested C4 gene. TYLCSV infection boosted transgene silencing but this did not lead to recovery. The data suggest that if the virus reaches a threshold level of expression/replication in the initially infected cells then virus spreading can no longer be prevented.
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The C terminus of the movement protein of Brome mosaic virus controls the requirement for coat protein in cell-to-cell movement and plays a role in long-distance movement
More LessThe 3a movement protein (MP) plays a central role in the movement of Brome mosaic virus (BMV). To identify the functional regions in BMV MP, 24 alanine-scanning (AS) MP mutants of BMV were constructed. Infectivity of the AS mutants in the host plant Chenopodium quinoa showed that the central region of BMV MP is important for viral movement and both termini of BMV MP have effects on the development of systemic symptoms. A green-fluorescent-protein-expressing RNA3-based BMV vector containing a 2A sequence from Foot-and-mouth disease virus was also constructed. Using this vector, two AS mutants that showed more efficient cell-to-cell movement than wild-type BMV were identified. The MPs of these two AS mutants, which have mutations at their C termini, mediated cell-to-cell movement independently of coat protein (CP), unlike wild-type BMV MP. Furthermore, a BMV mutant with a truncation in the C-terminal 42 amino acids of MP was also able to move from cell to cell without CP, but did not move systemically, even in the presence of CP. These results and an encapsidation analysis suggest that the C terminus of BMV MP is involved in the requirement for CP in cell-to-cell movement and plays a role in long-distance movement. Furthermore, the ability to spread locally and form virions is not sufficient for the long-distance movement of BMV. The roles of MP and CP in BMV movement are discussed.
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Significance of the 3′-terminal region in minus-strand RNA synthesis of Hibiscus chlorotic ringspot virus
More LessRNA-dependent RNA polymerase (RdRp) was solubilized from crude extracts of Hibiscus cannabinus infected by Hibiscus chlorotic ringspot virus (HCRSV), a member of the Carmoviridae. After treatment of the extracts with micrococcal nuclease to remove the endogenous templates, the full-length genomic RNA and the two subgenomic RNAs were efficiently synthesized by the partially purified RdRp complex in vitro. When the full-length RNAs of Potato virus X, Tobacco mosaic virus, Odontoglossum ringspot virus and Cucumber mosaic virus were used as templates, no detectable RNA was synthesized. Synthesis of HCRSV minus-strand RNA was shown to initiate opposite the 3′-terminal two C residues at the 3′ end in vitro and in vivo. The CCC-3′ terminal nucleotide sequence was optimal and nucleotide variations from CCC-3′ diminished minus-strand synthesis. In addition, two putative stem–loops (SLs) located within the 3′-terminal 87 nt of HCRSV plus-strand RNA were also essential for minus-strand RNA synthesis. Deletion or disruption of the structure of these two SLs severely reduced or abolished RNA synthesis. HCRSV RNA in which the two SLs were replaced with the SLs of Turnip crinkle virus could replicate in kenaf protoplasts, indicating that functionally conserved structure, rather than nucleotide sequence, plays an important role in the minus-strand synthesis of HCRSV. Taken together, the specific sequence CCC at the 3′ terminus and the two SLs structures located in the 3′UTR are essential for efficient minus-strand synthesis of HCRSV.
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- Other Agents
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Standards for the assay of Creutzfeldt–Jakob disease specimens
Assays for the agent of Creutzfeldt–Jakob disease (CJD) include measurement of infectivity in different animal systems, such as wild-type or transgenic mice, and detection of PrPSc by different methods and formats. The various assays could be best calibrated against each other by use of uniform readily available materials, and samples of four human brains, two from sporadic CJD patients, one from a variant CJD patient and one from a non-CJD patient, have been prepared as 10 % homogenates dispensed in 2000 vials each for this purpose. Results of in vitro methods, particularly immunoblot assays, were compared in the first collaborative study described here. While dilution end-points varied, the minimum detectable volume was surprisingly uniform for most assays and differences in technical procedure, other than the sample volume tested, had no detectable systematic effect. The two specimens from sporadic CJD cases contained both type 1 and type 2 prion proteins in approximately equal proportions. The materials have been given the status of reference reagents by the World Health Organization and are available for further study and assessment of other in vitro or in vivo assay procedures.
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Amyloid imaging probes are useful for detection of prion plaques and treatment of transmissible spongiform encephalopathies
Diagnostic imaging probes have been developed to monitor cerebral amyloid lesions in patients with neurodegenerative disorders. A thioflavin derivative, 2-[4′-(methylamino)phenyl] benzothiazole (BTA-1) and a Congo red derivative, (trans, trans),-1-bromo-2,5-bis-(3-hydroxycarbonyl-4-hydroxy)styrylbenzene (BSB) are representative chemicals of these probes. In this report, the two chemicals were studied in transmissible spongiform encephalopathies (TSE). Both BTA-1 and BSB selectively bound to compact plaques of prion protein (PrP), not only in the brain specimens of certain types of human TSE, but also in the brains of TSE-infected mice when the probes were injected intravenously. The chemicals bound to plaques in the brains were stable and could be detected for more than 42 h post-injection. In addition, the chemicals inhibited abnormal PrP formation in a cellular model of TSE with IC50 values of 4 nM for BTA-1 and 1·4 μM for BSB. In an experimental mouse model, the intravenous injection of 1 mg BSB prolonged the incubation period by 14 %. This efficacy was only observed against the RML strain and not the other strains examined. These observations suggest that these chemicals bind directly to PrP aggregates and inhibit new formation of abnormal PrP in a strain-dependent manner. Both BTA-1 and BSB can be expected to be lead chemicals not only for imaging probes but also for therapeutic drugs for TSEs caused by certain strains.
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Cationic phosphorus-containing dendrimers reduce prion replication both in cell culture and in mice infected with scrapie
Over the last 30 years, many drugs have been tested both in cell culture and in vivo for their ability to prevent the generation of prions and the development of transmissible spongiform encephalopathies. Among the compounds tested, dendrimers are defined by their branched and repeating molecular structure. The anti-prion activity of new cationic phosphorus-containing dendrimers (P-dendrimers) with tertiary amine end-groups was tested. These molecules had a strong anti-prion activity, decreasing both PrPSc and infectivity in scrapie-infected cells at non-cytotoxic doses. They can bind PrP and decrease the amount of pre-existing PrPSc from several prion strains, including the BSE strain. More importantly, when tested in a murine scrapie model, the dendrimers were able to decrease PrPSc accumulation in the spleen by more than 80 %. These molecules have a high bio-availability and therefore exhibit relevant potential for prion therapeutics for at least post-exposure prophylaxis.
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Volumes and issues
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Volume 105 (2024)
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Volume 73 (1992 - 2024)
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Volume 2 (1968)
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Volume 1 (1967)