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PubMed2005

Integrase of Mason-Pfizer monkey virus.

The gene encoding an integrase of Mason-Pfizer monkey virus (M-PMV) is located at the 3'-end of the pol open reading frame. The M-PMV integrase has not been previously isolated and characterized. We have now cloned, expressed, isolated, and characterized M-PMV integrase and compared its activities and primary structure with those of HIV-1 and other retroviral integrases. M-PMV integrase prefers untranslated 3'-region-derived long-terminal repeat sequences in both the 3'-processing and the strand transfer activity assays. While the 3'-processing reaction catalyzed by M-PMV integrase was significantly increased in the presence of Mn(2+) and Co(2+) and was readily detectable in the presence of Mg(2+) and Ni(2+) cations, the strand transfer activity was strictly dependent only on Mn(2+). M-PMV integrase displays more relaxed substrate specificity than HIV-1 integrase, catalyzing the cleavage and the strand transfer of M-PMV and HIV-1 long-terminal repeat-derived substrates with similar efficiency. The structure-based sequence alignment of M-PMV, HIV-1, SIV, and ASV integrases predicted critical amino acids and motifs of M-PMV integrase for metal binding, interaction with nucleic acids, dimerization, protein structure maintenance and function, as well as for binding of human immunodeficiency virus type 1 and Rous avian sarcoma virus integrase inhibitors 5-CI-TEP, DHPTPB and Y-3.

Amino Acid Sequence↗
PubMed1984

Replication of adenovirus mini-chromosomes.

We have isolated adenovirus origins of DNA replication from both the right and left ends of the genome, which are functional on linear autonomously replicating mini-chromosomes. The mini-chromosomes contain two cloned inverted adenovirus termini and require non-defective adenovirus as a helper. Replicated molecules are covalently attached to protein, and DNA synthesis is initiated at the correct nucleotide even when the origins are not located at molecular ends. The activity of embedded origins leads to the generation of linear mini-chromosomes from circular or linear molecules. These observations therefore suggest that sequences within the adenovirus origin of replication position the protein priming event at the adenovirus terminus. Experiments investigating the regeneration of deleted viral inverted terminal repeat sequences show a sequence-independent requirement for inverted sequences in this process. This result strongly suggests that repair results from the formation of a panhandle structure by a displaced single strand. On the basis of these observations we propose a model for the generation of adenovirus mini-chromosomes from larger molecules.

Adenoviridae↗
PubMed1983

In vivo incorporation of Drosophila H2a histone into mammalian chromatin.

Hybrid prokaryotic/eukaryotic expression vectors have been used to introduce Drosophila histone genes into CV-1 African green monkey tissue culture cells. Transfection of CV-1 cells with Drosophila genes under the control of insect DNA promoter sequences results in low level expression of histone genes. On the other hand, when the Drosophila H2a gene is juxtaposed downstream from the long terminal repeat sequence of Rous sarcoma virus (RSV) expression of the insect gene is considerably more efficient; both 3' polyadenylated insect histone messenger RNA and putative Drosophila H2a histone protein can be readily detected in the transduced cells. Using this RSV/H2a vector, we have been able to demonstrate the presence of Drosophila H2a histone in monomer nucleosome preparations isolated from transfected CV-1 cells. These results suggest the feasibility of 'remodeling' cellular chromatin in vivo in precisely defined ways. The techniques described may be generally applicable to other genes coding for chromosomal proteins.

Animals↗
PubMed1996

The noncoding and surface envelope coding sequences of myeloblastosis-associated virus are respectively responsible for nephroblastoma development and renal hyperplasia.

The use of chimeric viruses allowed us to establish that myeloblastosis-associated virus long terminal repeat sequences are necessary and sufficient for induction of nephroblastoma in chickens and that the blastemal hyperplasia induced by env SU is not a prerequisite for tumor development but rather constitutes a predisposing stage.

Animals↗
PubMed2002

A sensitive, quantitative assay for human immunodeficiency virus type 1 integration.

Quantitative methods to measure human immunodeficiency virus type 1 (HIV-1) integration promise to be important tools in dissecting the mechanisms whereby latent reservoirs of provirus are established, most notably in the resting T cells of patients receiving antiretroviral therapy. Here we describe a fluorescence-monitored, nested PCR assay that is able to quantify the relatively rare integration events that occur within these cells. Following DNA extraction, a nonkinetic preamplification step is performed with primers that bind genomic Alu elements and HIV-1 gag sequences, under conditions where primers, deoxynucleoside triphosphates, and enzyme are not limiting. This is followed by a kinetic PCR that quantitates HIV-1 long terminal repeat sequences. A T-cell-based integration standard which reflects the randomness of HIV-1 integration is also described. The assay is 10 to 100 times more sensitive than previously reported quantitative Alu PCR-based integration assays. It is specific for integration events, since no proviruses are detected in cells infected either in the presence of an integrase inhibitor or with an integrase-deficient virus. This method promises to provide important new insights into the processes underlying the accumulation and persistence of latent HIV-1 reservoirs and may eventually be useful clinically in monitoring the eradication of latent virus by novel therapies.

CD4-Positive T-Lymphocytes↗
PubMed2002

Analysis of the disease potential of a recombinant retrovirus containing Friend murine leukemia virus sequences and a unique long terminal repeat from feline leukemia virus.

We have molecularly cloned a feline leukemia virus (FeLV) (clone 33) from a domestic cat with acute myeloid leukemia (AML). The long terminal repeat (LTR) of this virus, like the LTRs present in FeLV proviruses from other cats with AML, contains an unusual structure in its U3 region upstream of the enhancer (URE) consisting of three tandem direct repeats of 47 bp. To test the disease potential and specificity of this unique FeLV LTR, we replaced the U3 region of the LTR of the erythroleukemia-inducing Friend murine leukemia virus (F-MuLV) with that of FeLV clone 33. When the resulting virus, F33V, was injected into newborn mice, almost all of the mice eventually developed hematopoietic malignancies, with a significant percentage being in the myeloid lineage. This is in contrast to mice injected with an F-MuLV recombinant containing the U3 region of another FeLV that lacks repetitive URE sequences, none of which developed myeloid malignancies. Examination of tumor proviruses from F33V-infected mice failed to detect any changes in FeLV U3 sequences other than that in the URE. Like F-MuLV-infected mice, those infected with the F-MuLV/FeLV recombinants were able to generate and replicate mink cell focus-inducing viruses. Our studies are consistent with the idea that the presence of repetitive sequences upstream of the enhancer in the LTR of FeLV may favor the activation of this promoter in myeloid cells and contribute to the development of malignancies in this hematopoietic lineage.

Animals↗
PubMed1984

Co-transfection of normal NIH/3T3 DNA and retroval LTR sequences: a novel strategy for the detection of potential c-onc genes.

Morphologically transformed, tumorigenic cell lines were obtained after co-transfecting normal NIH/3T3 DNA and cloned 3'-long terminal repeat sequences of Moloney leukemia virus (Mo-LTR) onto NIH/3T3 recipient cells. In four such cell lines the malignant phenotype was found to be associated with single and specific Mo-LTR integration sites that were retained after serial passages through NIH/3T3 and rat 208F cells, indicating that Mo-LTR sequences are linked to the activated oncogenes. In one of these clones the activated transforming gene was identified as c-raf, the cellular homologue of a recently described retroviral oncogene. This finding not only demonstrates that the mouse c-raf gene can be activated to exhibit an oncogenic potential but also that the approach chosen in this study is suitable for the detection of potential c-onc genes. In contrast to this clone, the activated transforming genes in other cell lines appear to be different from 19 previously isolated v-onc and c-onc genes. These results demonstrate the potential of the established transformation system for the detection and isolation of previously unidentified c-onc genes.

Animals↗
PubMed1983

Complete nucleotide sequence of the gene for the specific glycoprotein (gp55) of Friend spleen focus-forming virus.

The complete nucleotide sequence of the gene for the specific glycoprotein (gp55) of the polycythemic strain of Friend spleen focus-forming virus (SFFV) was derived from the cloned SFFV DNA intermediate. The gp55 gene is present within 1.4 kilobases of the 5' side of the 3'long terminal repeat sequence. The open reading frame predicts the primary translation product has a total of 409 amino acids with a Mr of 44,752. Comparisons of the deduced amino acid sequence of gp55 with those of the envelope (env) gene products of murine leukemia viruses (MuLVs) revealed that gp55 is composed of three distinct regions. The amino-terminal 80% of the molecule has a high degree of sequence homology with the amino-terminal portion of the gp70 of the Moloney mink cell focus-forming virus (BALB/Mo-MCFV). This portion of the BALB/Mo-MCFV gp70 is known to be coded for by the acquired xenotropic env-like sequence. The sequence of the following 66 amino acids of gp55 is highly homologous to that of the middle portion of the p15E of Moloney MuLV (Mo-MuLV). The sequence of the Carboxyl-terminal 12 amino acids is specific to gp55 and a comparison of the nucleotide sequence showed that this specific amino acid sequence is due to the presence of seven extra nucleotides compared with the sequence of the Mo-MuLV.

Amino Acid Sequence↗
PubMed1988

Somatic and germ-line reverse mutation rates of the retrovirus-induced dilute coat-color mutation of DBA mice.

At present, the dilute (dv) coat-color mutation of DBA mice provides the only simple means for measuring the relative somatic and germ-line reverse mutation rates of retrovirus-induced mutations in mammals. The dv mutation was generated by the spontaneous integration of an ecotropic murine leukemia virus into noncoding sequences of the dilute locus. Reversion of the dv mutation occurs by provirus excision and is mediated by homologous recombination events involving the viral long terminal repeat sequences. Although numerous independent germ-line d+ revertants have been identified, somatic d+ revertants have not been reported previously. During the past 5 years, we have screened more than one million mice homozygous for the dv mutation to determine whether we could identify somatic d+ revertants. This survey has resulted in the identification of a somatic d+ revertant and has provided a data base from which we can estimate the relative somatic and germ-line excision frequencies of retroviruses in mice and speculate about the nature of homologous recombination events producing d+ revertant alleles.

Animals↗
PubMed1999

Nup124p is a nuclear pore factor of Schizosaccharomyces pombe that is important for nuclear import and activity of retrotransposon Tf1.

The long terminal repeat (LTR)-containing retrotransposon Tf1 propagates within the fission yeast Schizosaccharomyces pombe as the result of several mechanisms that are typical of both retrotransposons and retroviruses. To identify host factors that contribute to the transposition process, we mutagenized cultures of S. pombe and screened them for strains that were unable to support Tf1 transposition. One such strain contained a mutation in a gene we named nup124. The product of this gene contains 11 FXFG repeats and is a component of the nuclear pore complex. In addition to the reduced levels of Tf1 transposition, the nup124-1 allele caused a significant reduction in the nuclear localization of Tf1 Gag. Surprisingly, the mutation in nup124-1 did not cause any reduction in the growth rate, the nuclear localization of specific nuclear localization signal-containing proteins, or the cytoplasmic localization of poly(A) mRNA. A two-hybrid analysis and an in vitro precipitation assay both identified an interaction between Tf1 Gag and the N terminus of Nup124p. These results provide evidence for an unusual mechanism of nuclear import that relies on a direct interaction between a nuclear pore factor and Tf1 Gag.

Alleles↗
PubMed2003

HIV-1 p55Gag encoded in the lysosome-associated membrane protein-1 as a DNA plasmid vaccine chimera is highly expressed, traffics to the major histocompatibility class II compartment, and elicits enhanced immune responses.

Several genetic vaccines encoding antigen chimeras containing the lysosome-associated membrane protein (LAMP) translocon, transmembrane, and cytoplasmic domain sequences have elicited strong mouse antigen-specific immune responses. The increased immune response is attributed to trafficking of the antigen chimera to the major histocompatibility class II (MHC II) compartment where LAMP is colocalized with MHC II. In this report, we describe a new form of an HIV-1 p55gag DNA vaccine, with the gag sequence incorporated into the complete LAMP cDNA sequence. Gag encoded with the translocon, transmembrane and cytoplasmic lysosomal membrane targeting sequences of LAMP, without the luminal domain, was poorly expressed, did not traffic to lysosomes or MHC II compartments of transfected cells, and elicited a limited immune response from DNA immunized mice. In contrast, addition of the LAMP luminal domain sequence to the construct resulted in a high level of expression of the LAMP/Gag protein chimera in transfected cells that was further increased by including the inverted terminal repeat sequences of the adeno-associated virus to the plasmid vector. This LAMP/Gag chimera with the complete LAMP protein colocalized with endogenous MHC II of transfected cells and elicited strong cellular and humoral immune responses of immunized mice as compared with the response to DNA-encoding native Gag, with a 10-fold increase in CD4+ responses, a 4- to 5-fold increase in CD8+ T-cell responses, and antibody titers of >100,000. These results reveal novel roles of the LAMP luminal domain as a determinant of Gag protein expression, lysosomal trafficking, and possibly of the immune response to Gag.

3T3 Cells↗
PubMed1996

Nucleotide sequence of the Kaposi sarcoma-associated herpesvirus (HHV8).

The genome of the Kaposi sarcoma-associated herpesvirus (KSHV or HHV8) was mapped with cosmid and phage genomic libraries from the BC-1 cell line. Its nucleotide sequence was determined except for a 3-kb region at the right end of the genome that was refractory to cloning. The BC-1 KSHV genome consists of a 140.5-kb-long unique coding region flanked by multiple G + C-rich 801-bp terminal repeat sequences. A genomic duplication that apparently arose in the parental tumor is present in this cell culture-derived strain. At least 81 ORFs, including 66 with homology to herpesvirus saimiri ORFs, and 5 internal repeat regions are present in the long unique region. The virus encodes homologs to complement-binding proteins, three cytokines (two macrophage inflammatory proteins and interleukin 6), dihydrofolate reductase, bcl-2, interferon regulatory factors, interleukin 8 receptor, neural cell adhesion molecule-like adhesin, and a D-type cyclin, as well as viral structural and metabolic proteins. Terminal repeat analysis of virus DNA from a KS lesion suggests a monoclonal expansion of KSHV in the KS tumor.

Base Sequence↗
PubMed1997

Yeast telomeric sequences function as chromosomal anchorage points in vivo.

Site-specific recombination in Saccharomyces cerevisiae was used to generate non-replicative DNA rings containing yeast telomeric sequences. In topoisomerase mutants expressing Escherichia coli topoisomerase I, the rings adopted a novel DNA topology consistent with the ability of yeast telomeric DNA to block or retard the axial rotation of DNA. DNA fragments bearing portions of the terminal repeat sequence C1-3 A/TG1-3 were both necessary and sufficient to create a barrier to DNA rotation. Synthetic oligonucleotide sequences containing Rap1p binding sites, a well represented motif in naturally occurring C1-3A arrays, also conferred immobilization; mutant Rap1p binding sites and telomeric sequences from other organisms were not sufficient. DNA anchoring was diminished by addition of competing telomeric sequences, implicating a role for an as yet unidentified limiting trans-acting factor. Though Rap1p is a likely protein constituent of the DNA anchor, deletion of the non-essential C-terminal domain did not affect the topology of telomeric DNA rings. Similarly, disruption of SIR2, SIR3 and SIR4, genes which influence a variety of telomere functions in yeast, also had no effect. We propose that telomeric DNA supports the formation of a SIR-independent macromolecular protein-DNA assembly that hinders the motion of DNA because of its linkage to an insoluble nuclear structure. Potential roles for DNA anchoring in telomere biology are discussed.

Binding Sites↗
PubMed1985

Nucleotide sequence analysis of a variant human T-cell leukemia virus (HTLV-Ib) provirus with a deletion in pX-I.

A variant of human T-cell leukemia virus subgroup I (HTLV-I), designated HTLV-Ib, has been isolated from a transformed T-lymphocytic cell line established from a Zairian patient with adult T-cell lymphoma. A recombinant phage clone of the variant provirus, denoted lambda MC-1, hybridizes under high stringency to HTLV-I DNA probes, but 17 of 43 restriction enzyme sites differ from those of HTLV-I, 10 of them clustering within 1.5 kilobases in the env-pX region. Since this variant virus retains its capacity to transform T-cells in vitro, and since a pX product is suspected to be important in transformation, we have determined the nucleotide sequence of the entire pX region of this virus for comparison to the prototype HTLV-I. In addition, the region between the gag and pol genes, parts of the pol and env genes, and a portion of the U3 region of the long terminal repeat sequence were also analyzed. We noted 141 single-base-pair changes among 3,897 base pairs, which were relatively well distributed over those portions of the provirus that were examined. In addition, an 11-base-pair deletion was found which included the potential initiator ATG codon of the first open reading frame of pX (pX-I). The next potential initiator codon predicted by the sequence is followed by 10 codons and then a termination codon. An identical deletion was also demonstrated in the only provirus present in another cell line established from the same patient on a different occasion after transformation in vitro of normal human umbilical cord blood cells. These results indicate that pX-I is not required for transformation.

Amino Acid Sequence↗
PubMed1987

Complete nucleotide sequences of functional clones of the AIDS virus.

To examine the mechanism of lymphocytotoxicity induced by human T-lymphotropic virus type III/lymphadenopathy associated virus (HTLV-III/LAV), an in vitro model has been developed. Introduction of an HTLV-III/LAV proviral clone, HXB2, into normal lymphocytes results in the production of virions and cell death. The complete nucleotide sequence of the proviral form of HXB2 has now been determined. Its structure is quite similar to that previously determined for HTLV-III/LAV clones whose biological capacities had not previously been demonstrated. The biological function of two additional clones of HTLV-III/LAV, BH10 and HXB3, are reported. Clone BH10 which lacks the 5' long terminal repeat sequences (LTR) and a portion of the 3' LTR is reconstituted by substituting the corresponding sequences of HXB2 and is shown to be capable of generating infectious cytopathic virions. Clone HXB3, which has been partially sequenced, is also found to be capable of producing lymphocytopathic virus. Clone HXB3 differs from HXB2 in its lack of a termination codon in 3' orf, demonstrating that 3' orf plays no major role in virus replication or cytopathic activity. These data provide the necessary background to allow the identification of viral determinants of replication, cytopathic activity, and antigenicity using these functional proviral clones.

Base Sequence↗
PubMed1992

Interest in and limits to the utilization of reporter genes for the analysis of transcriptional regulation of nitrate reductase.

Reporter gene techniques and mutant analysis were used to identify the molecular basis of the regulation of the expression of nitrate reductase (NR) by nitrate and nitrate-, or ammonium-derived metabolites (N-metabolites), in the true diploïd species Nicotiana plumbaginifolia and in the amphidiploïd species Nicotiana tabacum. The N. plumbaginifolia mutant E23 results from the insertion of a Tnt1-like retrotransposon (Tnp2) in the first exon of the single-copy nia gene, which encodes nitrate reductase. One of the resulting transcripts ends in the 5' LTR (long terminal repeat) sequence of this retrotransposon, and another one in the 3' LTR. Nitrate and N-metabolites modulate the expression of these truncated transcripts, indicating that intron splicing and termination processes are not essential to these regulatory events. A GUS reporter sequence was transcriptionally linked to the promoter of the nia-1 gene of N. tabacum. This fusion was functional in transient expression assays done with protoplasts derived from mesophyll cells of N. tabacum. However none of the regulatory mechanisms known to affect steady-state levels of the nia-1 transcript were operative under these experimental conditions. Transgenic plants carrying either this fusion or translational fusions of GUS linked to the promoter of either the nia-1 or nia-2 gene of N. tabacum were obtained by Agrobacterium-mediated transfer. A low proportion of the transgenic plants (22 out of 105 independent transformants) expressed GUS activity although at a low level. Only 4 plants exhibited a detectable level of GUS mRNA. The concentration of this mRNA increased significantly in an NR-deficient background, indicating regulation by N-metabolites. Only 2 plants, however, showed regulation (induction) by nitrate. Attempts to use aux2 or nptII reporter sequences linked to either the nia-1 or nia-2 promoter as marker genes for the selection of regulatory mutants of the nitrate assimilation pathway were unsuccessful because of our inability to isolate transgenic plants in which these reporter genes were properly regulated by nitrate. The implications of these results are discussed.

Circadian Rhythm↗
PubMed1999

Integrating adenovirus-adeno-associated virus hybrid vectors devoid of all viral genes.

Recently, we demonstrated that inverted repeat sequences inserted into first-generation adenovirus (Ad) vector genomes mediate precise genomic rearrangements resulting in vector genomes devoid of all viral genes that are efficiently packaged into functional Ad capsids. As a specific application of this finding, we generated adenovirus-adeno-associated virus (AAV) hybrid vectors, first-generation Ad vectors containing AAV inverted terminal repeat sequences (ITRs) flanking a reporter gene cassette inserted into the E1 region. We hypothesized that the AAV ITRs present within the hybrid vector genome could mediate the formation of rearranged vector genomes (DeltaAd.AAV) and stimulate transgene integration. We demonstrate here that DeltaAd.AAV vectors are efficiently generated as by-products of first-generation adenovirus-AAV vector amplification. DeltaAd.AAV genomes contain only the transgene flanked by AAV ITRs, Ad packaging signals, and Ad ITRs. DeltaAd.AAV vectors can be produced at a high titer and purity. In vitro transduction properties of these deleted hybrid vectors were evaluated in direct comparison with first-generation Ad and recombinant AAV vectors (rAAVs). The DeltaAd.AAV hybrid vector stably transduced cultured cells with efficiencies comparable to rAAV. Since cells transduced with DeltaAd.AAV did not express cytotoxic viral proteins, hybrid viruses could be applied at very high multiplicities of infection to increase transduction rates. Southern analysis and pulsed-field gel electrophoresis suggested that DeltaAd.AAV integrated randomly as head-to-tail tandems into the host cell genome. The presence of two intact AAV ITRs was crucial for the production of hybrid vectors and for transgene integration. DeltaAd.AAV vectors, which are straightforward in their production, represent a promising tool for stable gene transfer in vitro and in vivo.

Adenoviridae↗
PubMed2003

Endogenous retroviruses and MS: using ERVs as disease markers.

Multiple sclerosis (MS) has an unknown cause, but its epidemiology suggests an interplay between environmental factors, possibly including viruses, and genetic components. Endogenous retroviruses (ERVs) are elements of the human genome that potentially may act as either genetic markers for polymorphisms related to MS, or markers of environmental/endogenous stress. Activation of the ERVs HERV-H/RGH, HERV-W and ERV-9 was reported when specific cell types (mainly B cells) from MS patients were cultivated in vitro. Viral RNA from these ERVs has been detected by reverse-transcriptase polymerase chain reaction in sera/plasma and brain tissues from MS patients, although not exclusively from these patients. ERVs play unknown roles in MS: their activation may represent an inflammatory cytokine-mediated epiphenomenon; alternatively, preliminary evidence suggests that specific ERVs may act as auto-, super- or neoantigens with the potential to enhance inflammatory responses or induce autoimmune reactions. ERVs that occur in few copies in the human genome (e.g. ERV-3 and human endogenous retrovirus, HRES-1) may show polymorphic patterns in MS. Studies show that the sequences encoding the envelope protein of ERV-3 are polymorphic to a degree where it becomes impossible to link them with MS. In contrast, the HRES-1 long terminal repeat sequence has a polymorphic pattern with haplotypes characteristic of MS. Haplotypes from non-MS control groups were identical between different topographic areas, but haplotypes from MS patients were different, depending on the population.

Biomarkers↗