Sequences in the 5' and 3' R elements of human immunodeficiency virus type 1 critical for efficient reverse transcription.
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The genome of human immunodeficiency virus type 1 (HIV-1) contains two direct repeats (R) of 97 nucleotides at each end. These elements are of critical importance during the first-strand transfer of reverse transcription, during which the minus-strand strong-stop DNA (-sssDNA) is transferred from the 5' end to the 3' end of the genomic RNA. This transfer is critical for the synthesis of the full-length minus-strand cDNA. These repeats also contain a variety of other functional domains involved in many aspects of the viral life cycle. In this study, we have introduced a series of mutations into the 5', the 3', or both R sequences designed to avoid these other functional domains. Using a single-round infectivity assay, we determined the ability of these mutants to undergo the various steps of reverse transcription utilizing a semiquantitative PCR analysis. We find that mutations within the first 10 nucleotides of either the 5' or the 3' R sequence resulted in virions that were markedly defective for reverse transcription in infected cells. These mutations potentially introduce mismatches between the full-length -sssDNA and 3' acceptor R. Even mutations that would create relatively small mismatches, as little as 3 bp, resulted in inefficient reverse transcription. In contrast, virions containing identically mutated R elements were not defective for reverse transcription or infectivity. Using an endogenous reverse transcription assay with disrupted virus, we show that virions harboring the 5' or the 3' R mutations were not intrinsically defective for DNA synthesis. Similarly sized mismatches slightly further downstream in either the 5', the 3', or both R sequences were not detrimental to continued reverse transcription in infected cells. These data are consistent with the idea that certain mismatches within 10 nucleotides downstream of the U3-R junction in HIV-1 cause defects in the stability of the cDNA before or during the first-strand transfer of reverse transcription leading to the rapid disappearance of the -sssDNA in infected cells. These data also suggest that the great majority of first-strand transfers in HIV-1 occur after the copying of virtually the entire 5' R. (+info)
Natural genetic exchanges between vaccine and wild poliovirus strains in humans.
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In a previous study of poliovirus vaccine-derived strains isolated from patients with vaccine-associated paralytic poliomyelitis (VAPP) (9, 11), we reported that a high proportion (over 50%) of viruses had a recombinant genome. Most were intertypic vaccine/vaccine recombinants. However, some had restriction fragment length polymorphism (RFLP) profiles different from those of poliovirus vaccine strains. We demonstrate here that five such recombinants, of 88 VAPP strains examined, carried sequences of wild (nonvaccine) origin. To identify the parental wild donor of these sequences, we used RFLP profiles and nucleotide sequencing to look for similarity in the 3D polymerase-coding region of 61 wild, cocirculating poliovirus isolates (43 type 1, 16 type 2, and 2 type 3 isolates). In only one case was the donor identified, and it was a wild type 1 poliovirus. For the other four vaccine/wild recombinants, the wild parent could not be identified. The possibility that the wild sequences were of a non-poliovirus-enterovirus origin could not be excluded. Another vaccine/wild recombinant, isolated in Belarus from a VAPP case, indicated that the poliovirus vaccine/wild recombination is not an isolated phenomenon. We also found wild polioviruses (2 of 15) carrying vaccine-derived sequences in the 3' moiety of their genome. All these results suggest that genetic exchanges with wild poliovirus and perhaps with nonpoliovirus enteroviruses, are also a natural means of evolution for poliovirus vaccine strains. (+info)
Avian nephritis virus (ANV) as a new member of the family Astroviridae and construction of infectious ANV cDNA.
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The complete RNA genome of the avian nephritis virus (ANV) associated with acute nephritis in chickens has been molecularly cloned and sequenced. Excluding the poly(A) tail, the genome comprises 6,927 nucleotides and contains three sequential open reading frames (ORFs). The first ORF (ORF 1a) contains a sequence encoding a serine protease motif, and the second ORF (ORF 1b) has a sequence encoding an RNA-dependent RNA polymerase. ORF 1a may be linked to the second ORF by a ribosomal frameshifting mechanism. The third ORF (ORF 2) may encode the virion structural proteins as a polyprotein precursor. Two RNAs, probably genonic and subgenonic RNA (7.5 and 3.0 kb), were detected in the cytoplasm of ANV-infected cells. ANV and human astroviruses have the same genonic organization, and both are characterized by the presence of two RNA bands. The amino acid homologies of the products of ORF 1a, 1b, and 2 were 20.3, 41.9, and 25.8% to products of the corresponding ORFs of human astrovirus serotype 1 (A/88 Newcastle strain). We have constructed a genonic-length cDNA clone of ANV to test whether the in vitro transcript is infectious. When a chicken kidney cell culture was transfected with RNA transcribed in vitro and the cDNA clone, infectious virus was produced with cytopathic effects in the absence of trypsin. These observations suggested that the ANV (G-4260 strain) is a new genus of the family Astroviridae. (+info)
The valine anticodon and valylatability of Peanut clump virus RNAs are not essential but provide a modest competitive advantage in plants.
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The role of valine aminoacylation of the two genomic RNAs of Peanut clump virus (PCV) was studied by comparing the amplification in vivo of RNAs with GAC, GDeltaC, or CCA anticodons in the tRNA-like structure (TLS) present at the 3' end of each viral RNA. The PCV RNA1 TLS of isolate PCV2 possesses a GAC anticodon and is capable of highly efficient valylation, whereas the RNA2 TLS has a GDeltaC anticodon that does not support valylation. The presence in RNA1 of GDeltaC or CCA anticodons that conferred nonvalylatability resulted in about 2- to 4-fold and a 14- to 24-fold reduction, respectively, in RNA accumulations in tobacco BY-2 protoplasts inoculated with the RNA1 variants together with wild-type RNA2(GDeltaC). No differences in RNA levels were observed among protoplasts inoculated with the three variant RNA2s in the presence of wild-type RNA1(GAC). All combinations of valylatable and nonvalylatable RNAs 1 and 2 were similarly infectious in Nicotiana benthamiana plants, and viral RNAs accumulated to similar levels; all input TLS sequences were present unchanged in apical leaves. In direct competition experiments in N. benthamiana plants, however, both RNA1 and RNA2 with GAC valylatable anticodons outcompeted the nonvalylatable variants. We conclude that valylation provides a small but significant replicational advantage to both PCV RNAs. Sequence analysis of the TLS from RNA2 of a second PCV isolate, PO2A, revealed the presence of an intact GAC valine anticodon, suggesting that the differential valylation of the genomic RNAs of isolate PCV2 is not a general characteristic of PCV. (+info)
Porcine CTLA4-Ig lacks a MYPPPY motif, binds inefficiently to human B7 and specifically suppresses human CD4+ T cell responses costimulated by pig but not human B7.
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The CTLA4 receptor (CD152) on activated T lymphocytes binds B7 molecules (CD80 and CD86) on APC and delivers a signal that inhibits T cell proliferation. Several regions involved in binding to B7 are known, but the relative importance of these is not clear. We have cloned porcine CTLA4 (pCTLA4). Although highly homologous to human CTLA4 (hCTLA4), the predicted protein sequence contains a leucine for methionine substitution at position 97 in the MYPPPY sequence. A fusion protein constructed from the extracellular regions of pCTLA4 and the constant regions of human IgG1 (pCTLA4-Ig) bound porcine CD86 with equivalent affinity to that of hCTLA4-Ig. However, pCTLA4-Ig bound poorly to human CD80 and CD86 expressed on transfectants and EBV-transformed human B cells. In functional assays with MHC class II-expressing porcine endothelial cells and human B cells, pCTLA4-Ig blocked human CD4+ T cell responses to pig but not human cells, whereas control hCTLA4-Ig inhibited responses to both. Comparison between mouse, human, and porcine CTLA4-Ig suggests that the selective binding of pCTLA4-Ig to porcine CD86 molecules is due to the L for M substitution at position 97. Our results indicate that pCTLA4-Ig may be a useful reagent to define the precise nature of the interaction between B7 and CTLA4. By failing to inhibit the delivery of costimulatory signals provided by human B7, it may also prove to be a relatively specific inhibitor of the direct human T cell response to immunogenic pig tissue. (+info)
Phylogenetically enhanced statistical tools for RNA structure prediction.
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MOTIVATION: Methods that predict the structure of molecules by looking for statistical correlation have been quite effective. Unfortunately, these methods often disregard phylogenetic information in the sequences they analyze. Here, we present a number of statistics for RNA molecular-structure prediction. Besides common pair-wise comparisons, we consider a few reasonable statistics for base-triple predictions, and present an elaborate analysis of these methods. All these statistics incorporate phylogenetic relationships of the sequences in the analysis to varying degrees, and the different nature of these tests gives a wide choice of statistical tools for RNA structure prediction. RESULTS: Starting from statistics that incorporate phylogenetic information only as independent sequence evolution models for each position of a multiple alignment, and extending this idea to a joint evolution model of two positions, we enhance the usual purely statistical methods (e.g. methods based on the Mutual Information statistic) with the use of phylogenetic information available in the sequences. In particular, we present a joint model based on the HKY evolution model, and consequently a X(2) test of independence for two positions. A significant part of this work is devoted to some mathematical analysis of these methods. We tested these statistics on regions of 16S and 23S rRNA, and tRNA. (+info)
Atypical Chryseobacterium meningosepticum and meningitis and sepsis in newborns and the immunocompromised, Taiwan.
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From 1996 to 1999, 17 culture-documented systemic infections due to novel, atypical strains of Chryseobacterium meningosepticum occurred in two newborns and 15 immunocompromised patients in a medical center in Taiwan. All clinical isolates, which were initially misidentified as Aeromonas salmonicida by an automated bacterial identification system, were resistant to a number of antimicrobial agents. The isolates were characterized as atypical strains of C. meningosepticum by complete biochemical investigation, 16S rRNA gene sequence analysis, cellular fatty acid analysis, and random amplified polymorphic DNA fingerprinting (RAPD). This is the first report of a cluster of atypically variant strains of C. meningosepticum, which may be an emerging pathogen in newborns and the immunocompromised. (+info)
A pathogenic point mutation reduces stability of mitochondrial mutant tRNA(Ile).
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Point mutations in mitochondrial tRNA genes are responsible for individual subgroups of mitochondrial encephalomyopathies. We have recently reported that point mutations in the tRNA(Leu)(UUR) and tRNA(Lys) genes cause a defect in the normal modification at the first nucleotide of the anticodon. As part of a systematic analysis of pathogenic mutant mitochondrial tRNAs, we purified tRNA(Ile) with a point mutation at nucleotide 4269 to determine its nucleotide sequence, including modified nucleotides. We found that, instead of causing a defect in the post-transcriptional modification, a pathogenic point mutation in the mitochondrial tRNA(Ile) reduced the stability of the mutant tRNA molecule, resulting in a low steady-state level of aminoacyl-tRNA. The reduced stability was confirmed by examining the life-span of the mutant tRNA(Ile) both in vitro and in vivo, as well as by monitoring its melting profile. Our finding indicates that the mutant tRNA(Ile) itself is intrinsically unstable. (+info)