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What are all of those retrotransposons doing buzzing about in our genome? According to Kazazian, in his Perspective this week, these mobile pieces of DNA are busy reshaping our genome, making it more diverse and enabling us to survive and thrive through the vagaries of evolution. And just how do they do this?...zip to page 1152 to find out.
The transcription initiation signals for retroviruses lie within the long terminal repeat (LTR) sequences that flank the integrated provirus. This study shows that factors present in cells infected with bovine leukemia virus (BLV) mediate transcriptional trans activation of the BLV LTR. This phenomenon is similar to that reported for the human T-cell leukemia virus LTR and establishes both structural and functional criteria for inclusion of BLV and human T-cell leukemia viruses in the same family of transforming retroviruses.
In several bursal lymphoma cell lines in which c-myc transcription is regulated by avian leukosis virus (ALV) long terminal repeat (LTR) sequences, protein synthesis inhibition decreases the transcriptional activity of c-myc as well as other LTR driven viral genes. This decrease in transcription is associated with a change in the chromatin structure of c-myc, as measured by deoxyribonuclease I (DNase I) hypersensitivity, and a shift of transcription from the LTR to the normal c-myc promoter. In contrast, cycloheximide had little or no effect on the transcription of LTR driven genes in infected chicken embryo fibroblasts treated with the drug. These results suggest that a labile, cell type-specific protein may interact with the retroviral LTR and regulate transcription of genes under LTR control. Further, the results demonstrate that the increase in intracellular concentration of c-myc RNA induced by cycloheximide treatment of normal cells is the result of stabilization of this message.
The transcription initiation signals for retroviruses lie within the long terminal repeat (LTR) sequences that flank the integrated provirus. Two subtypes of human T lymphotropic virus (HTLV) are associated with different disease phenotypes. In this article it is shown that marked differences exist in the ability of LTR sequences of these subtypes to function as transcriptional elements in differentiated cell types. It is also shown that trans-acting regulatory factors present in HTLV-infected cells stimulate gene expression directed by these LTR sequences in a type-specific manner. These results have implications for understanding the diverse biological effects of HTLV infection.
Two constructs derived from the alpha-amylase gene (amyA) of Lactobacillus amylovorus were expressed in Lactobacillus plantarum, and their expression products were purified, characterized, and compared. These products correspond to the complete (AmyA) and truncated (AmyADelta) forms of alpha-amylase; AmyADelta lacks the 66-kDa carboxyl-terminal direct-repeating-unit region. AmyA and AmyADelta exhibit similar amylase activities towards a range of soluble substrates (amylose, amylopectin and alpha-cyclodextrin, and soluble starch). The specific activities of the enzymes towards soluble starch are similar, but the K(M) and V(max) values of AmyADelta were slightly higher than those of AmyA, whereas the thermal stability of AmyADelta was lower than that of AmyA. In contrast to AmyA, AmyADelta is unable to bind to beta-cyclodextrin and is only weakly active towards glycogen. More striking is the fact that AmyADelta cannot bind or hydrolyze raw starch, demonstrating that the carboxyl-terminal repeating-unit domain of AmyA is required for raw-starch binding activity.
The Escherichia coli ClpYQ (HslUV) is an ATP-dependent protease that consists of an ATPase large subunit with homology to other Clp family ATPases and a peptidase small subunit related to the proteasomal beta-subunits of eukaryotes. Six identical subunits of both ClpY and ClpQ self-assemble into an oligomeric ring, and two rings of each subunit, two ClpQ rings surrounded by single ClpY rings, form a dumbbell shape complex. The ClpYQ protease degrades the cell division inhibitor, SulA, and a positive regulator of capsule transcription, RcsA, as well as RpoH, a heat shock sigma transcription factor. Using the yeast-two hybrid system, we explored the in vivo protein-protein interactions of the individual subunits of the ClpYQ protease involved in self-oligomerization, as well as in recognition of specific substrates. Interactions were detected with ClpQ/ClpQ, ClpQ/ClpY, and ClpY/SulA. No interactions were observed in experiments with ClpY/ClpY, ClpQ/RcsA, and ClpQ/SulA. However, ClpY, lacking domain I (ClpY(Delta I)) was able to interact with itself and with intact ClpY. The C-terminal region of ClpY is important for interaction with other ClpY subunits. The previously defined PDZ-like domains at the C terminus of ClpY, including both D1 and D2, were determined to be indispensable for substrate binding. Various deletion and random point mutants of SulA were also made to verify significant interactions with ClpY. Thus, we demonstrated in vivo hetero- and homointeractions of ClpQ and ClpY molecules, as well as a direct association between ClpY and substrate SulA, thereby supporting previous in vitro biochemical findings.
The prevalence of human T-cell lymphotropic virus type 1 (HTLV-1) and HTLV-2 in blood donors in Guyana has never been estimated. We evaluated the prevalence of these viruses in blood donors by enzyme-linked immunosorbent assay and Western blotting and showed a prevalence of HTLV-1 of 1.3%; no HTLV-2 was detected. Female donors had a much higher HTLV-1 seroprevalence (3.6%) than male donors (0.7%). HTLV-1-seropositive donors tended to be slightly older than the average age for the total pool of donors. We also investigated the phylogenetic and molecular characteristics of HTLV-1 strains in Guyana and compared them with those identified in Suriname and French Guiana. Analysis of portions of the env and long terminal repeat nucleotide sequences showed that all the strains in Guyana and Suriname, like those in French Guiana, belonged to the transcontinental group of cosmopolitan subtype A. The similarities were greater between strains from Suriname and Guyana than between strains from Suriname and Guyana and those from French Guiana. Nevertheless, our results confirm that the HTLV-1 strains in all three countries have a common African origin.
Human T-cell leukemia virus type 1 (HTLV-1) Tax protein activates viral transcription from the long terminal repeats (LTR). Mechanisms through which Tax activates LTR have been established, but coactivators of this process remain to be identified and characterized. Here we show that all three members of the TORC family of transcriptional regulators are coactivators of Tax for LTR-driven expression. TORC coactivation requires CREB, but not ATF4 or other bZIP factors. Tax physically interacts with TORC1, TORC2, and TORC3 (TORC1/2/3), and the depletion of TORC1/2/3 inhibited Tax activity. TORC coactivation can be further enhanced by transcriptional coactivator p300. In addition, coactivators in the p300 family are required for full activity of Tax independently of TORC1/2/3. Thus, both TORC and p300 families of coactivators are essential for optimal activation of HTLV-1 transcription by Tax.
In this study, we demonstrate that the coactivator-associated arginine methyltransferase 1 (CARM1), which methylates histone H3 and other proteins such as p300/CBP, is positively involved in the regulation of Tax transactivation. First, transfection studies demonstrated that overexpression of CARM1 wild-type protein resulted in increased Tax transactivation of the human T-cell lymphotropic virus type 1 (HTLV-1) long terminal repeat (LTR). In contrast, transfection of a catalytically inactive CARM1 methyltransferase mutant did not enhance Tax transactivation. CARM1 facilitated Tax transactivation of the CREB-dependent cellular GEM promoter. A direct physical interaction between HTLV-1 Tax and CARM1 was demonstrated using in vitro glutathione S-transferase-Tax binding assays, in vivo coimmunoprecipitation, and confocal microscopy experiments. Finally, chromatin immunoprecipitation analysis of the activated HTLV-1 LTR promoter showed the association of CARM1 and methylated histone H3 with the template DNA. In vitro, Tax facilitates the binding of CARM1 to the transcription complex. Together, our data provide evidence that CARM1 enhances Tax transactivation of the HTLV-1 LTR through a direct interaction between CARM1 and Tax and this binding promotes methylation of histone H3 (R2, R17, and R26).
Terminal repeat (TR) elements of Kaposi's sarcoma-associated herpesvirus (KSHV), the potential origin sites of KSHV replication, have been demonstrated to play important roles in viral replication and transcription and are most likely also critical for the segregation of the KSHV genome to daughter cells. To search for the cellular proteins potentially involved in KSHV genome maintenance, we performed affinity chromatography analysis, using KSHV TR DNA as the affinity ligand. Proteomic analysis was then carried out to identify the TR-interacting proteins. We identified a total of 123 proteins from both KSHV-positive and -negative cells, among which most were identified exclusively from KSHV-positive cells. These proteins were categorized as proliferation/cell cycle regulatory proteins, proteins involved in spliceosome components, such as heterogeneous nuclear ribonuclear proteins, the DEAD/H family, the switch/sucrose nonfermenting protein family, splicing factors, RNA binding proteins, transcription regulation proteins, replication factors, modifying enzymes, and a number of proteins that could not be broadly categorized. To support the proteomic results, the presence of four candidate proteins, ATR, BRG1, NPM1 and PARP-1, in the elutions was further characterized in this study. The binding and colocalization of these proteins with the TR were verified using chromatin immunoprecipitation and immunofluorescence in situ hybridization analysis. These newly identified TR binding proteins provide a number of clues and potential links to understanding the mechanisms regulating the replication, transcription, and genome maintenance of KSHV. This study will facilitate the generation and testing of new hypotheses to further our understanding of the mechanisms involved in KSHV persistence and its associated pathogenesis.
The long terminal repeat retrotransposon Tf1 of Schizosaccharomyces pombe uses a unique mechanism of self priming to initiate reverse transcription. Instead of using a tRNA, Tf1 primes minus-strand synthesis with an 11-nucleotide RNA removed from the 5' end of its own transcript. We tested whether the self primer of Tf1 was similar to tRNA primers in being removed from the cDNA by RNase H. Our analysis of Tf1 cDNA extracted from virus-like particles revealed the surprising observation that the dominant species of cDNA retained the self primer. This suggests that integration of the cDNA relies on mechanisms other than reverse transcription to remove the primer.
Approximately 15% of the polyadenylic acid-containing cytoplasmic RNA labeled from 5 to 7 h after vaccinia virus infection formed intermolecular duplex structures characterized as double-stranded RNA by RNase resistance, density in Cs2SO4, base composition, chromatography on cellulose, and ability to inhibit reticulocyte cell-free protein synthesis. Both sucrose gradient sedimentation and electron microscopic analysis indicated that the double-stranded regions were several hundred to more than a thousand nucleotide base pairs long. The double-stranded RNA, after denaturation, hybridized to approximately 25% of the vaccinia virus genome, whereas total late RNA hybridized to 42%. The finding that the duplex RNA, after denaturation, hybridized to most HindIII restriction endonuclease fragments of vaccinia virus DNA indicated that symmetrical transcription is not confined to the terminal inverted repeat sequence or to one contiguous region of the genome. Although relatively little labeled, early, polyadenylic acid-containing RNA formed RNase-resistant hybrids upon self-annealing, the percentage increased upon addition of unlabeled late RNA, indicating that the latter contains "anti-early" sequences.
We have detected a mouse mammary tumor virus (MMTV)-specific 1.7-kilobase (kb) polyadenylated RNA in mammary glands of several mouse strains. In BALB/c mice, it is the only MMTV-specific RNA species present. C3H and GR mammary glands and tumors contain, in addition, 3.8- and 7.8-kb MMTV RNAs. Nuclease S1 analysis was performed to map 1.7-kb polyadenylated RNA. It contains predominantly long terminal repeat (LTR) sequences. The 5' end maps approximately 134 nucleotides upstream from the 3' end of the LTR. Colinearity with complete proviral DNA continues to a site about 153 nucleotides downstream from the left (5') LTR. No sequences from the middle part of proviral DNA were found. Colinearity with proviral DNA is resumed 72 nucleotides upstream from the right (3') LTR. The nucleotide sequence in this area is TTCCAGT, which is a splice acceptor consensus sequence. The anatomy of 1.7-kb RNA indicates that it may serve as a messenger for the 36,700-dalton protein encoded by the LTRs of MMTV.
Herpes simplex virus type 1 (HSV-1) and some of its immediate-early genes stimulate expression of the human immunodeficiency virus (HIV) long terminal repeat (LTR) sequences and the replication of HIV itself. To demonstrate this, the HIV LTR was linked to the indicator gene chloramphenicol acetyltransferase (CAT) and transfected into Vero cells with or without the trans-activating gene (tat) of HIV. Infection of these cells with HSV-1 strain KOS or temperature-sensitive mutant tsB21 or tsE6 resulted in a large increase in CAT activity in the absence of tat and further augmentation in the presence of tat. This stimulation was seen at both their permissive (34 degrees C) and nonpermissive (39 degrees C) temperatures, implying either that HSV-1 infection or immediate-early gene expression is all that is required. In cotransfection assays in Vero cells, cloned HSV-1 immediate-early genes ICP0 and ICP4 stimulated CAT activity in the presence of tat, while ICP27 had no effect. On the other hand, in SW480 cells, ICP4 and, to a lesser extent, ICP0 genes caused stimulation of CAT activity in the absence of tat. Deletion mutants within the HIV LTR showed that the target for HSV stimulation is distinct from the tat-responsive area and maps near the SP1 binding sites. In Hela cells, ICP0 or ICP4 stimulated the replication of a cotransfected clone of HIV, as shown by an increase in reverse transcriptase activity in the culture supernatant.
We have studied the replication of ecotropic murine leukemia viruses (MuLV) in the spleens and thymuses of mice infected with the lymphocytic leukemia-inducing virus Moloney MuLV (M-MuLV), with the erythroleukemia-inducing virus Friend MuLV (F-MuLV), or with in vitro-constructed recombinants between these viruses in which the long terminal repeat (LTR) sequences have been exchanged. At 1 week after infection both the parents and the LTR recombinants replicated predominantly in the spleens with only low levels of replication in the thymus. At 2 weeks after infection, the patterns of replication in the spleens and thymuses were strongly influenced by the type of LTR. Viruses containing the M-MuLV LTR exhibited a remarkable elevation in thymus titers which frequently exceeded the spleen titers, whereas viruses containing the F-MuLV LTR replicated predominantly in the spleen. In older preleukemic mice (5 to 8 weeks of age) the structural genes of M-MuLV or F-MuLV predominantly influenced the patterns of replication. Viruses containing the structural genes of M-MuLV replicated efficiently in both the spleen and thymus, whereas viruses containing the structural genes of F-MuLV replicated predominantly in the spleen. In leukemic mice infected with the recombinant containing F-MuLV structural genes and the M-MuLV LTR, high levels of virus replication were observed in splenic tumors but not in thymic tumors. This phenotypic difference suggested that tumors of the spleen and thymus may have originated by the independent transformation of different cell types. Quantification of polytropic MulVs in late-preleukemic mice infected with each of the ecotropic MuLVs indicated that the level of polytropic MuLV replication closely paralleled the level of replication of the ecotropic MuLVs in all instances. These studies indicated that determinants of tissue tropism are contained in both the LTR and structural gene sequences of F-MuLV and M-MuLV and that high levels of ecotropic or polytropic MuLV replication, per se, are not sufficient for leukemia induction. Our results further suggested that leukemia induction requires a high level of virus replication in the target organ only transiently during an early preleukemic stage of disease.
Long terminal repeat (LTR) sequences of murine leukemia viruses (MLVs) have been demonstrated to be mainly responsible for the pathogenic differences in these retroviruses. A region of the LTR which is downstream of the enhancer elements has been shown to contribute both to enhancer activity as well as to disease specificity of MLVs. We have identified protein-DNA complexes generated by this region of a lymphomagenic MLV (MCF13) and one which is nonpathogenic (Akv). One protein-DNA complex we have observed for this region is unique to MCF13 DNA sequences. Detection of protein involved in this unique MCF13 complex in different cell lines revealed that it was ubiquitous.
The long terminal repeat (LTR) sequences of Moloney murine leukemia virus and its closely related derivative Moloney murine sarcoma virus (Mo-MSV) are incapable of directing transcription in embryonal carcinoma (EC) stem cells. The myeloproliferative sarcoma virus, a derivative of Mo-MSV, has several point mutations in the LTR and is transcribed more efficiently to allow productive infection of F9 EC cells. One of these mutations, at -166 with respect to the transcriptional start, creates a consensus binding site for the well-characterized mammalian transcription factor Sp1. We used gel retardation assays to demonstrate that F9 EC cell extracts form several complexes with the myeloproliferative sarcoma virus sequence around -166. One of these complexes involves a murine Sp1-like protein, which has immunoreactivity, DNA binding specificity, and electrophoretic mobility equivalent to those of purified human Sp1 protein. An equivalent complex forms on the corresponding Mo-MSV sequence but with a fivefold-lower affinity. Consistent with these observations, introduction of the single point mutation at -166 into the Mo-MSV LTR, creating a consensus Sp1 binding site, increases expression in F9 EC cells sixfold.
We report the construction and characterization of several replication-competent simian immunodeficiency virus (SIV) vectors with a deletion in the viral nef gene (SIV(delta nef)) that express gamma interferon (IFN-gamma). The expression of the cytokine gene was controlled either by the simian virus 40 early promoter or by the SIV 5' long terminal repeat regulatory sequences, utilizing the nef gene splice signals. To enhance the expression of IFN-gamma, the two in-frame nef start codons were mutated without altering the Env amino acid sequence (SIV(HyIFN)). Plasmids containing full-length proviral genomes were used to obtain high-titer stocks of each recombinant virus in cell cultures. Expression of IFN-gamma by SIV(HyIFN) reached levels as high as 10(6) U/ml after 11 days in culture. The IFN-gamma gene was unstable and sustained deletions after serial passage of SIV(delta nef) vectors in CEM-X-174 cells. The degree of instability appears to depend on size and orientation of the insert and the expression of IFN-gamma. Only one virus, SIV(HyIFN), expressed detectable levels of IFN-gamma up to the sixth passage. Prospects for the use of IFN-gamma and other lymphokines to enhance the safety and efficacy of live attenuated vaccines are discussed.