Gene silencing: plants and viruses fight it out.
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Plants can become 'immune' to attack by viruses by degrading specific viral RNA, but some plant viruses have evolved the general capacity to suppress this resistance mechanism. (+info)
Structural basis for the specificity of the initiation of HIV-1 reverse transcription.
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Initiation of human immunodeficiency virus type 1 (HIV-1) reverse transcription requires specific recognition of the viral genome, tRNA3Lys, which acts as primer, and reverse transcriptase (RT). The specificity of this ternary complex is mediated by intricate interactions between HIV-1 RNA and tRNA3Lys, but remains poorly understood at the three-dimensional level. We used chemical probing to gain insight into the three-dimensional structure of the viral RNA-tRNA3Lys complex, and enzymatic footprinting to delineate regions interacting with RT. These and previous experimental data were used to derive a three-dimensional model of the initiation complex. The viral RNA and tRNA3Lys form a compact structure in which the two RNAs fold into distinct structural domains. The extended interactions between these molecules are not directly recognized by RT. Rather, they favor RT binding by preventing steric clashes between the nucleic acids and the polymerase and inducing a viral RNA-tRNA3Lys conformation which fits perfectly into the nucleic acid binding cleft of RT. Recognition of the 3' end of tRNA3Lys and of the first template nucleotides by RT is favored by a kink in the template strand promoted by the short junctions present in the previously established secondary structure. (+info)
A premature termination codon interferes with the nuclear function of an exon splicing enhancer in an open reading frame-dependent manner.
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Premature translation termination codon (PTC)-mediated effects on nuclear RNA processing have been shown to be associated with a number of human genetic diseases; however, how these PTCs mediate such effects in the nucleus is unclear. A PTC at nucleotide (nt) 2018 that lies adjacent to the 5' element of a bipartite exon splicing enhancer within the NS2-specific exon of minute virus of mice P4 promoter-generated pre-mRNA caused a decrease in the accumulated levels of P4-generated R2 mRNA relative to P4-generated R1 mRNA, although the total accumulated levels of P4 product remained the same. This effect was seen in nuclear RNA and was independent of RNA stability. The 5' and 3' elements of the bipartite NS2-specific exon enhancer are redundant in function, and when the 2018 PTC was combined with a deletion of the 3' enhancer element, the exon was skipped in the majority of the viral P4-generated product. Such exon skipping in response to a PTC, but not a missense mutation at nt 2018, could be suppressed by frame shift mutations in either exon of NS2 which reopened the NS2 open reading frame, as well as by improvement of the upstream intron 3' splice site. These results suggest that a PTC can interfere with the function of an exon splicing enhancer in an open reading frame-dependent manner and that the PTC is recognized in the nucleus. (+info)
High level inhibition of HIV replication with combination RNA decoys expressed from an HIV-Tat inducible vector.
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Intracellular immunization, an antiviral gene therapy approach based on the introduction of DNA into cells to stably express molecules for the inhibition of viral gene expression and replication, has been suggested for inhibition of HIV infection. Since the Tat and Rev proteins play a critical role in HIV regulation, RNA decoys and ribozymes of these sequences have potential as therapeutic molecular inhibitors. In the present study, we have generated several anti-HIV molecules; a tat-ribozyme, RRE, RWZ6 and TAR decoys and combinations of decoys, and tested them for inhibition of HIV-1 replication in vitro. We used T cell specific CD2 gene elements and regulatory the HIV inducible promoter to direct high level expression and a 3' UTR sequence for mRNA stabilization. We show that HIV replication was most strongly inhibited with the combination TAR + RRE decoy when compared with the single decoys or the tat-ribozyme. We also show that the Tat-inducible HIV promoter directs a higher level of steady-state transcription of decoys and inhibitors and that higher levels of expression directly relate to increased levels of inhibition of HIV infection. Furthermore, a stabilization of the 3' end of TAR + RRE inhibitor transcripts using a beta-globin 3' UTR sequence leads to an additional 15-fold increase in steady-state RNA levels. This cassette when used to express the best combination decoy inhibitor TAR + RRE, yields high level HIV inhibition for greater than 3 weeks. Taken together, both optimization for high level expression of molecular inhibitors and use of combinations of inhibitors suggest better therapeutic application in limiting the spread of HIV. (+info)
Reverse transcription-nested polymerase chain reaction for detecting p40 RNA of Borna disease virus, without risk of plasmid contamination.
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Several methods for the detection of Borna disease virus (BDV) RNA have been reported, one being the reverse transcription-nested polymerase chain reaction (RT-nested PCR) method. However, due to the possibility of contamination of the cloned DNA in a reaction tube, false-positive results might be obtained by RT-nested PCR. To detect only BDV RNA without anxiety of contamination, we developed an RT-nested PCR system using "mRNA selective PCR kit". Using this system, cDNA of BDV p40 in the plasmid (up to 5 x 10(7) molecules) was not amplified. BDV specific sequence was amplified from total RNA (more than 50 pg) of MDCK/BDV cells, which were persistently infected with BDV. These results indicate that this mRNA selective RT-nested PCR system can specifically amplify target RNA as distinguished from plasmid contaminated. (+info)
Enteroviral RNA replication in the myocardium of patients with left ventricular dysfunction and clinically suspected myocarditis.
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BACKGROUND: Previous studies dealing with the detection of enteroviral RNA in human endomyocardial biopsies have not differentiated between latent persistence of the enteroviral genome and active viral replication. Enteroviruses that are considered important factors for the development of myocarditis have a single-strand RNA genome of positive polarity that is transcribed by a virus-encoded RNA polymerase into a minus-strand mRNA during active viral replication. The synthesis of multiple copies of minus-strand enteroviral RNA therefore occurs only at sites of active viral replication but not in tissues with mere persistence of the viral genome. METHODS AND RESULTS: We investigated enteroviral RNA replication versus enteroviral RNA persistence in endomyocardial biopsies of 45 patients with left ventricular dysfunction and clinically suspected myocarditis. Using reverse-transcriptase polymerase chain reaction in conjunction with Southern blot hybridization, we established a highly sensitive assay to specifically detect plus-strand versus minus-strand enteroviral RNA in the biopsies. Plus-strand enteroviral RNA was detected in endomyocardial biopsies of 18 (40%) of 45 patients, whereas minus-strand RNA as an indication of active enteroviral RNA replication was detected in only 10 (56%) of these 18 plus-strand-positive patients. Enteroviral RNA was not found in biopsies of the control group (n=26). CONCLUSIONS: These data demonstrate that a significant fraction of patients with left ventricular dysfunction and clinically suspected myocarditis had active enteroviral RNA replication in their myocardium (22%). Differentiation between patients with active viral replication and latent viral persistence should be particularly important in future studies evaluating different therapeutic strategies. In addition, molecular genetic detection of enteroviral genome and differentiation between replicating versus persistent viruses is possible in a single endomyocardial biopsy. (+info)
HIV-associated nephropathy is a late, not early, manifestation of HIV-1 infection.
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BACKGROUND: Human immunodeficiency virus-associated nephropathy (HIVAN) can be the initial presentation of HIV-1 infection. As a result, many have assumed that HIVAN can occur at any point in the infection. This issue has important implications for appropriate therapy and, perhaps, for pathogenesis. Since the development of new case definitions for acquired immunodeficiency syndrome (AIDS) and better tools to assess infection, the relationship of HIVAN to the time of AIDS infection has not been addressed. In this study, we reassessed the stage of infection at the time of HIVAN diagnosis in 10 patients, and we reviewed all previously published cases applying the new case definitions to assess stage of infection. METHODS: HIVAN was confirmed by kidney biopsy in HIV seropositive patients with azotemia and/or proteinuria. CD4+ cell count and plasma HIV-1 RNA copy number were measured. We also reviewed all published cases of HIVAN to determine if AIDS-defining conditions, by current Centers for Disease Control definitions, were present in patients with biopsy-proven HIVAN. RESULTS: Twenty HIV-1 seropositive patients with proteinuria and an elevated creatinine concentration were biopsied. HIVAN was the single most common cause of renal disease. CD4+ cell count was below 200/mm3 in all patients with HIVAN, fulfilling Centers for Disease Control criteria for an AIDS-defining condition. HIV-1 plasma RNA was detectable in all patients with HIVAN. In reviewing previous reports, an AIDS-defining condition was present in virtually all patients with HIVAN. CONCLUSION: HIVAN develops late, not early, in the course of HIV-1 infection following the development of AIDS. This likely accounts for the poor prognosis noted in previous publications and has implications for pathogenesis. In addition, given the detectable viral RNA levels, highly active antiretroviral therapy is indicated in HIVAN. Highly active antiretroviral therapy may improve survival as well as alter the natural history of HIVAN. (+info)
A multistate, foodborne outbreak of hepatitis A. National Hepatitis A Investigation Team.
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BACKGROUND: We investigated a large, foodborne outbreak of hepatitis A that occurred in February and March 1997 in Michigan and then extended the investigation to determine whether it was related to sporadic cases reported in other states among persons who had consumed frozen strawberries, the food suspected of causing the outbreak. METHODS: The cases of hepatitis A were serologically confirmed. Epidemiologic studies were conducted in the two states with sufficient numbers of cases, Michigan and Maine. Hepatitis A virus RNA detected in clinical specimens was sequenced to determine the relatedness of the virus from outbreak-related cases and other cases. RESULTS: A total of 213 cases of hepatitis A were reported from 23 schools in Michigan and 29 cases from 13 schools in Maine, with the median rate of attack ranging from 0.2 to 14 percent. Hepatitis A was associated with the consumption of frozen strawberries in a case-control study (odds ratio for the disease, 8.3; 95 percent confidence interval, 2.1 to 33) and a cohort study (relative risk of infection, 7.5; 95 percent confidence interval, 1.1 to 53) in Michigan and in a case-control study in Maine (odds ratio for infection, 3.4; 95 percent confidence interval, 1.0 to 14). The genetic sequences of viruses from 126 patients in Michigan and Maine were identical to one another and to those from 5 patients in Wisconsin and 7 patients in Arizona, all of whom attended schools where frozen strawberries from the same processor had been served, and to those in 2 patients from Louisiana, both of whom had consumed commercially prepared products containing frozen strawberries from the same processor. CONCLUSIONS: We describe a large outbreak of hepatitis A in Michigan that was associated with the consumption of frozen strawberries. We found apparently sporadic cases in other states that could be linked to the same source by viral genetic analysis. (+info)