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At least 811 records · Page 45Linked to original sources
PubMed2004

Anti-termination by SIV Tat requires flexibility of the nascent TAR structure.

Substitution of the SIVmac239 promoter/enhancer by the strong EF1alpha promoter results in a severe replication defect due to a failure to respond to Tat. Revertant viruses with minimal promoter sequences (two Sp1 sites and a TATA box) were obtained that had fully restored their replicative potential. Comparison of the different LTRs indicated that structural alterations in the TAR stem due to a 31bp exon of the EF1alpha promoter rather than the mere presence of transcription factor binding sites within U3 were responsible for the attenuation. Structural models based on genuine RNA sequences combined with a refined algorithm to calculate the probability of the looping-mediated interaction between protein complexes bound to nucleic acid polymers indicated that the local concentration of TAR-bound Tat close to the RNA polymerase II complex was reduced more than 100-fold for the mutant as compared to SIVmac239. These results show that HIV/SIV replication requires only a minimal set of cis-acting elements in the promoter and suggest a hitherto unrecognised requirement of flexibility for the nascent TAR structure to allow anti-termination by Tat.

Amino Acid Sequence↗
PubMed2006

A novel deletion in the LTR region of a Greek small ruminant lentivirus may be associated with low pathogenicity.

Greek small ruminant lentivirus (SRLV) strains remain relatively uncharacterized at the molecular level, despite the fact that lentiviral diseases of small ruminants are known to be widespread in the country. In the present study, we investigated the sequence diversity of the LTR region in Greek SRLV strains from sheep with and without disease symptoms, since sequence differences within this genomic area have been shown to lead to SRLVs with distinct replication rates. The AP-4 and AML (vis) motifs and the TATA-box were highly conserved among Greek strains, whereas the two AP-1 sites exhibited some substitutions. Pairwise comparisons with reference strains revealed that Greek LTR sequences were closer to the ovine strains (25.7% average divergence) rather than the caprine strain CAEV (59.1% average divergence). The most striking difference observed between the two groups of animals was a 13-14 nucleotide deletion in the strains obtained from the asymptomatic sheep. The deletion was located within the R region of LTR, which was also found to be much less homologous (39.6% average divergence) than the U3 and U5. Taken together, our data suggest that the R region of LTR may be involved in virus transcriptional activation. Furthermore, a specific deletion within this region may, at least in part, be associated with low pathogenicity of some SRLV strains.

Animals↗
PubMed2002

Evidence of recombination between 3' and 5' LTRs in macaques inoculated with SIV DNA.

Proviral SIV DNA inoculation of macaques is an efficient method to initiate wild-type and attenuated SIV infections. However, we found that macaques inoculated with SIV DNA engineered to contain a single 105-bp deletion in the 3' nef/LTR overlap region had SIV sequences subsequently isolated that had partially or fully repaired the deletion with wild-type sequence. Animals inoculated with SIV DNA containing identical deletions in both the 5' and 3' LTRs did not repair the deletion. Recombination events occurred early, most likely by homologous recombination with sequences from the wild-type 5' LTR. This sequence analysis is the first demonstration of homologous recombination in vivo following administration of a single SIV strain.

Amino Acid Sequence↗
PubMed2001

An ancient retrovirus-like element contains hot spots for SINE insertion.

Vertebrate retrotransposons have been used extensively for phylogenetic analyses and studies of molecular evolution. Information can be obtained from specific inserts either by comparing sequence differences that have accumulated over time in orthologous copies of that insert or by determining the presence or absence of that specific element at a particular site. The presence of specific copies has been deemed to be an essentially homoplasy-free phylogenetic character because the probability of multiple independent insertions into any one site has been believed to be nil. Mys elements are a type of LTR-containing retrotransposon present in Sigmodontine rodents. In this study we have shown that one particular insert, mys-9, is an extremely old insert present in multiple species of the genus Peromyscus. We have found that different copies of this insert show a surprising range of sizes, due primarily to a continuing series of SINE (short interspersed element) insertions into this locus. We have identified two hot spots for SINE insertion within mys-9 and at each hot spot have found that two independent SINE insertions have occurred at identical sites. These results have major repercussions for phylogenetic analyses based on SINE insertions, indicating the need for caution when one concludes that the existence of a SINE at a specific locus in multiple individuals is indicative of common ancestry. Although independent insertions at the same locus may be rare, SINE insertions are not homoplasy-free phylogenetic markers.

Alleles↗
PubMed1999

Forced recombination of psi-modified murine leukaemia virus-based vectors with murine leukaemia-like and VL30 murine endogenous retroviruses.

Co-encapsidation of retroviral RNAs into virus particles allows for the generation of recombinant proviruses through events of template switching during reverse transcription. By use of a forced recombination system based on recombinational rescue of replication- defective primer binding site-impaired Akv-MLV-derived vectors, we here examine putative genetic interactions between vector RNAs and copackaged endogenous retroviral RNAs of the murine leukaemia virus (MLV) and VL30 retroelement families. We show (i) that MLV recombination is not blocked by nonhomology within the 5' untranslated region harbouring the supposed RNA dimer-forming cis -elements and (ii) that copackaged retroviral RNAs can recombine despite pronounced sequence dissimilarity at the cross-over site(s) and within parts of the genome involved in RNA dimerization, encapsidation and strand transferring during reverse transcription. We note that recombination-based rescue of primer binding site knock-out retroviral vectors may constitute a sensitive assay to register putative genetic interactions involving endogenous retroviral RNAs present in cells of various species.

5' Untranslated Regions↗
PubMed2002

Studies on intracellular processing of the capsid protein of human astrovirus serotype 1 in infected cells.

Astroviruses are non-enveloped, positive-strand RNA viruses. Their structural (capsid) protein is processed extracellularly into several smaller fragments which are found on the mature viral particle. In addition, intracellular cleavage of the capsid protein has been proposed. However, analysis of capsid protein processing has been hampered by the lack of antibodies to regions near the N and C termini of the protein. Here we describe the construction of two infectious mutants of human astrovirus serotype 1 (HAstV-1), in which amino acids (aa) 11-30 or aa 783-787, respectively, of the 787 aa capsid protein were replaced by tag sequences. Processing of the tagged capsid proteins in infected Caco-2 cells was analysed by immunoprecipitation with specific reagents directed against the tags or against native internal regions of the capsid protein. No intracellular processing of the capsid protein in infected cells could be detected, while assembled viral particles were readily observed within cells.

Amino Acid Sequence↗
PubMed2001

Human chromosome 19 and related regions in mouse: conservative and lineage-specific evolution.

To illuminate the function and evolutionary history of both genomes, we sequenced mouse DNA related to human chromosome 19. Comparative sequence alignments yielded confirmatory evidence for hypothetical genes and identified exons, regulatory elements, and candidate genes that were missed by other predictive methods. Chromosome-wide comparisons revealed a difference between single-copy HSA19 genes, which are overwhelmingly conserved in mouse, and genes residing in tandem familial clusters, which differ extensively in number, coding capacity, and organization between the two species. Finally, we sequenced breakpoints of all 15 evolutionary rearrangements, providing a view of the forces that drive chromosome evolution in mammals.

Animals↗
PubMed2001

Use of intron-disrupted polyadenylation sites to enhance expression and safety of retroviral vectors.

Normal mRNA polyadenylation signals are composed of an AAUAAA motif and G/U box spaced 20 to 30 bp apart. If this spacing is increased further, then polyadenylation is disrupted. Previously it has been demonstrated that insertion of an intron will similarly disrupt this signal even though such introns are removed during a nuclear splicing reaction (X. Liu and J. Mertz, Nucleic Acids Res. 21:5256-5263, 1993). This observation has led to the suggestion that polyadenylation site selection is undertaken prior to intron excision. We now present results that both support and extend these observations and in doing so create a novel class of retroviral expression vector with improved qualities. We found that when an intron-disrupted polyadenylation signal is inserted within a retroviral expression vector, such a signal, although reformed in the producer cell, remains benign until transduction, where it is then preferentially used. Thus, we demonstrate that upon transduction these vectors now produce a majority of shortened subgenomic species and as a consequence have a reduced tendency for subsequent mobilization from transduced cells. In addition, we demonstrate that the use of this internal signal leads to enhanced expression from such vectors and that this is achieved without any loss in titer. Therefore, split polyadenylation signals confer enhanced performance and improved safety upon retroviral expression vectors into which they are inserted. Such split signals may prove useful for the future optimization of retroviral vectors in gene therapy.

Base Sequence↗
PubMed2001

Mapping full-length porcine endogenous retroviruses in a large white pig.

Xenotransplantation may bridge the widening gap between the shortage of donor organs and the increasing number of patients waiting for transplantation. However, a major safety issue is the potential cross-species transmission of porcine endogenous retroviruses (PERV). This problem could be resolved if it is possible to produce pigs that do not contain replication-competent copies of this virus. In order to determine the feasibility of this, we have determined the number of potentially replication-competent full-length PERV proviruses and obtained data on their integration sites within the porcine genome. We have screened genomic DNA libraries from a Large White pig for potentially intact proviruses. We identified six unique PERV B proviruses that were apparently intact in all three genes, while the majority of isolated proviruses were defective in one or more genes. No intact PERV A proviruses were found in this pig, despite the identification of multiple defective A proviruses. Genotyping of 30 unrelated pigs for these unique proviruses showed a heterogeneous distribution. Two proviruses were uncommon, present in 7 of 30 and 3 of 30 pigs, while three were each present in 24 of 30 pigs, and one was present in 30 of 30 animals examined. Our data indicate that few PERV proviruses in Large White pigs are capable of productive infection and suggest that many could be removed by selective breeding. Further studies are required to determine if all potentially functional proviruses could be removed by breeding or whether gene knockout techniques will be required to remove the residuum.

Amino Acid Sequence↗
PubMed2003

Antigenic subclasses of polytropic murine leukemia virus (MLV) isolates reflect three distinct groups of endogenous polytropic MLV-related sequences in NFS/N mice.

Polytropic murine leukemia viruses (MLVs) are generated by recombination of ecotropic MLVs with members of a family of endogenous proviruses in mice. Previous studies have indicated that polytropic MLV isolates comprise two mutually exclusive antigenic subclasses, each of which is reactive with one of two monoclonal antibodies termed MAb 516 and Hy 7. A major determinant of the epitopes distinguishing the subclasses mapped to a single amino acid difference in the SU protein. Furthermore, distinctly different populations of the polytropic MLV subclasses are generated upon inoculation of different ecotropic MLVs. Here we have characterized the majority of endogenous polytropic MLV-related proviruses of NFS/N mice. Most of the proviruses contain intact sequences encoding the receptor-binding region of the SU protein and could be distinguished by sequence heterogeneity within that region. We found that the endogenous proviruses comprise two major groups that encode the major determinant for Hy 7 or MAb 516 reactivity. The Hy 7-reactive proviruses correspond to previously identified polytropic proviruses, while the 516-reactive proviruses comprise the modified polytropic proviruses as well as a third group of polytropic MLV-related proviruses that exhibit distinct structural features. Phylogenetic analyses indicate that the latter proviruses reflect features of phylogenetic intermediates linking xenotropic MLVs to the polytropic and modified polytropic proviruses. These studies elucidate the relationships of the antigenic subclasses of polytropic MLVs to their endogenous counterparts, identify a new group of endogenous proviruses, and identify distinguishing characteristics of the proviruses that should facilitate a more precise description of their expression in mice and their participation in recombination to generate recombinant viruses.

Animals↗
PubMed2004

sigma marY1, the LTR of the gypsy-type retroelement marY1 from the basidiomycete tricholoma matsutake, allows multicopy DNA integration in Lentinula edodes.

sigma marY1 is the LTR of the retroelement marY1 from the homobasidiomycete Tricholoma matsutake. Upon integration through transformation, pLC1-hph carrying a sigma marY1 derivative, sigma* marY1, conferred the hygromycin-resistant phenotype stronger than the vector without sigma* marY1 on Lentinula edodes. Based on the densitometric analysis after Southern hybridization, a copy number of the former construct integrated in the genome is much higher than that of the latter. We conclude that sigma marY1 allows multicopy DNA integration and will be useful in the genetic research on this fungal group.

Agaricales↗
PubMed2000

Phylogenetic analysis of retroposon family as exemplified on human chromosome 13: further evidence for recent proliferation.

The retroposon SINE-R.C2 was first identified as a human-specific insertion in the complement C2 gene. In our previous study, SINE-R type retroposons, derived from the endogenous retrovirus HERV-K family, have been found to be hominoid specific. In this report on human chromosome 13, we identified eighteen new SINE-R retroposons resembling those we have previously reported on the sex chromosomes and on chromosomes 7 and 17. Phylogenetic analysis using the neighbor-joining method revealed that four SINE-R retroposons (13-16, 21, 23, 25) on chromosome 13 were closely related to the human-specific retroposon SINE-R.C2, with a high degree of sequence homology (95-97%). Such elements differ from the HERV-K10. LTR sequence from which they are derived in being deleted for the promoter region. Therefore while the evidence adds to the case that some classes of SINE-R element have continued to proliferate in hominid and hominoid evolution and may, as in the case of Fukuyama type muscular dystrophy, be a cause of insertional mutagenesis, they are less likely than the HERV-K10 LTR to have a positive effect on host gene activity.

Chromosomes, Human, Pair 13↗
PubMed2006

Distinctive repertoire of contingency genes conferring mutation- based phase variation and combinatorial expression of surface lipoproteins in Mycoplasma capricolum subsp. capricolum of the Mycoplasma mycoides phylogenetic cluster.

The generation of surface variation among many divergent species of Mollicutes (mycoplasmas) occurs through stochastic expression patterns of diverse lipoprotein genes. The size and wide distribution of such variable gene sets in minimal (approximately 0.6- to 1.4-Mb) mycoplasmal genomes suggest their key role in the adaptation and survival of these wall-less monoderms. Diversity through variable genes is less clearly established among phylogenetically similar mycoplasmas, such as the Mycoplasma mycoides cluster of ruminant pathogens, which vary widely in host range and pathobiology. Using (i) genome sequences from two members of this clade, Mycoplasma capricolum subsp. capricolum and M. mycoides subsp. mycoides small colony biotype (SC), (ii) antibodies to specific peptide determinants of predicted M. capricolum subsp. capricolum gene products, and (iii) analysis of the membrane-associated proteome of M. capricolum subsp. capricolum, a novel set of six genes (vmcA to vmcF) expressing distinct Vmc (variable M. capricolum subsp. capricolum) lipoproteins is demonstrated. These occur at two separate loci in the M. capricolum subsp. capricolum genome, which shares striking overall similarity and gene synteny with the M. mycoides subsp. mycoides SC genome. Collectively, Vmc expression is noncoordinate and combinatorial, subject to a single-unit insertion/deletion in a 5' flanking dinucleotide repeat that governs expression of each vmc gene. All vmc genes share modular regions affecting expression and membrane translocation. In contrast, vmcA to vmcD genes at one locus express surface proteins with highly structured size-variable repeating domains, whereas vmcE to vmcF genes express products with short repeats devoid of predicted structure. These genes confer a distinctive, dynamic surface architecture that may represent adaptive differences within this important group of pathogens as well as exploitable diagnostic targets.

5' Flanking Region↗
PubMed2000

Cloning and characterization of Vine-1, a LTR-retrotransposon-like element in Vitis vinifera L., and other Vitis species.

We report the organization of a grapevine chimeric gene Adhr-Vine-1, composed by an Adhr gene, into which a retroelement, Vine-1, was inserted. Sequence analysis revealed that Adhr is a member of the Adh multigene family, but does not correspond to any other grapevine Adh described to date. Vine-1, albeit defective, is the most complete LTR (long terminal repeat)-retrotransposon-like element described in Vitis vinifera L. It is 2392 bp long, with two almost identical LTRs (287 bp) in the same orientation, and flanked by direct repeats of a 5 bp host DNA. This element presents other features, characteristic of retroviruses and retrotransposons including inverted repeats, a primer binding site, and a polypurine tract. It has a single open reading frame (ORF) of 581 amino acids, potentially encoding for a gag protein and parts of the protease and integrase proteins. Vine-1 is most likely related to the copia-like type family, but with no significant similarity to any previously described plant retrotransposon or inserted element, nor to any eukaryotic element described to date. Vine-1 element has been found in Adhr at the same location in different V. vinifera cultivars, but not in some other analyzed Vitis species. These data suggest that Vine-1 insertion in Adhr is specific to V. vinifera, and has occurred after the Adh isogene separation, but prior to cultivar development. Sequences related to Vine-1 were revealed in multiple copies in the V. vinifera genome and, to a lesser extent, in other analyzed Vitis species. The polymorphism observed prompts us to question the role played by transposition in the evolution of the Vitis genus.

Alcohol Dehydrogenase↗
PubMed1995

Defective accessory genes in a human immunodeficiency virus type 1-infected long-term survivor lacking recoverable virus.

We have been studying a patient who acquired human immunodeficiency virus (HIV) infection via a blood transfusion 13 years ago. She has remained asymptomatic since that time. The blood donor and two other recipients have all died of AIDS. Although this patient has shown persistently strong seroreactivity to HIV type 1 (HIV-1) antigens by Western blot (immunoblot), she has been continually HIV culture negative in results from multiple laboratories over the last 6 years and has a very low viral burden. Her CD4+ T-cell count has fluctuated around a mean of 399 cells per microliters, with little change in lymphocyte subset percentages. Strong cellular immune responses to HIV-1 epitopes by this patient have been demonstrated. We now report the results of an intensive molecular genetic analysis of the HIV-1 proviral quasispecies from this patient sampled over 5 years. Long terminal repeat region sequences supported the argument for normal basal and Tat-mediated promoter activities. Sequential sequencing of the nef gene revealed a low frequency (8.3%) of defective genes and a striking lack of sequence evolution. Functional analysis of predominant nef genes by both a cell surface CD4 downregulation and a viral infectivity complementation assay showed wild-type function. In contrast, sequential analysis of an amplicon containing the vif, vpr, vpu, tat1, and rev1 genes revealed the presence of inactivating mutations in 64% of the clones. These data suggest that this patient, initially infected with a virulent swarm of HIV-1, is presently infected with a more-attenuated viral quasispecies as a result of effective host immunity.

Acquired Immunodeficiency Syndrome↗
PubMed2003

Relative age of proviral porcine endogenous retrovirus sequences in Sus scrofa based on the molecular clock hypothesis.

Porcine endogenous retroviruses (PERV) are discussed as putative infectious agents in xenotransplantation. PERV classes A, B, and C harbor different envelope proteins. Two different types of long terminal repeat (LTR) structures exist, of which both are present only in PERV-A. One type of LTR contains a distinct repeat structure in U3, while the other is repeatless, conferring a lower level of transcriptional activity. Since the different LTR structures are distributed unequally among the proviruses and, apparently, PERV is the only virus harboring two different LTR structures, we were interested in determining which LTR is the ancestor. Replication-competent viruses can still be found today, suggesting an evolutionary recent origin. Our studies revealed that the age of PERV is at most 7.6 x 10(6) years, whereas the repeatless LTR type evolved approximately 3.4 x 10(6) years ago, being the phylogenetically younger structure. The age determined for PERV correlates with the time of separation between pigs (Suidae, Sus scrofa) and their closest relatives, American-born peccaries (Tayassuidae, Pecari tajacu), 7.4 x 10(6) years ago.

Animals↗
PubMed2006

Identification and characterization of the telomerase reverse transcriptase of Bombyx mori (silkworm) and Tribolium castaneum (flour beetle).

Chromosomal ends of most eukaryotes are composed of simple telomeric repeats. Arthropod telomeres are generally constituted by TTAGG pentanucleotide repeats; however, some insect species including Drosophila melanogaster do not have telomeric repeats. In contrast, the domestic silkworm Bombyx mori contains TTAGG-type telomeric repeats, but the telomerase activity has not been detected in all investigated tissues. To search for a cause of unusual telomere structure in insects, we here identified telomerase reverse transcriptase (TERT) subunit from the domestic silkworm B. mori and the flour beetle Tribolium castaneum. This is the first report of telomerase genes from arthropods. The domestic silkworm TERT gene (BmoTERT) and the flour beetle TERT gene (TcasTERT) both did not have the N-terminal GQ motif. Comparison between cDNA and genomic DNA of BmoTERT revealed that it includes no introns. BmoTERT contains five ATG codons in its 5'UTR, which could reduce the translation of BmoTERT proteins. Also, Northern hybridization indicated that BmoTERT is transcribed at a very low level. These unique features of BmoTERT possibly explain the undetectable Bombyx telomerase activity.

5' Untranslated Regions↗