Wild-type Long Terminal Repeat Research Articles

Mechanism of Reduction in Titers From Lentivirus Vectors Carrying Large Inserts in the 3′LTR

Self-inactivating (SIN) lentiviruses flanked by the 1.2-kb chicken hypersensitive site-4 (cHS4) insulator element provide consistent, improved expression of transgenes, but have significantly lower titers. The mechanism by which this occurs is unknown. Lengthening the lentiviral (LV) vector transgene cassette by an additional 1.2 kb by an internal cassette caused no further reduction in titers. However, when cHS4 sequences or inert DNA spacers of increasing size were placed in the 3'-long terminal repeat (LTR), infectious titers decreased proportional to the length of the insert. The stage of vector life cycle affected by vectors carrying the large cHS4 3'LTR insert was compared to a control vector: there was no increase in read-through transcription with insertion of the 1.2-kb cHS4 in the 3'LTR. Equal amount of full-length viral mRNA was produced in packaging cells and viral assembly/packaging was unaffected, resulting in comparable amounts of intact vector particles produced by either vectors. However, LV vectors carrying cHS4 in the 3'LTR were inefficiently processed following target-cell entry, with reduced reverse transcription and integration efficiency, and hence lower transduction titers. Therefore, vectors with large insertions in the 3'LTR are transcribed and packaged efficiently, but the LTR insert hinders viral-RNA (vRNA) processing and transduction of target cells. These studies have important implications in design of integrating vectors.

Bovine immunodeficiency virus: identification of a long terminal repeat sequence with enhanced promoter activity

We previously identified a new bovine immunodeficiency virus (BIV) trans-activator factor of transcription (Tat236) that was derived from a variant of BIV. Here, we report a new BIV long terminal repeat (LTR) sequence (LTRn) that was obtained by PCR from the DNA of cells infected with the BIV variant mentioned above. Sequence analysis indicated that the LTRn U3 region harbors three nucleic acid mutations at residue positions -194, -135 and -114 when compared to the original (wild-type) LTR sequence. Reporter gene assays indicated that LTRn promotes basal and Tat-mediated transactivation activity to levels significantly higher than those obtained with the wild-type LTR. Restoration experiments to the wild-type genotype indicated that both the -135 and -114 nucleic acid substitutions were responsible for the enhanced promoter activity of BIV LTRn.

Transgene expression in the mouse cerebellar Purkinje cells with a minimal level of integration using long terminal repeat–modified lentiviral vectors

Lentiviral vectors (LVs), which preferentially target nondividing cells, such as neurons, are promising tools for gene therapy. However, these vectors are still unsuitable as they result in insertional mutagenesis. It is therefore essential to prevent insertional mutagenesis if these vectors are to be adopted for safe next generation clinical applications. In order to establish safe genetic therapy with LVs, we focused on the integrase recognition sequence (att) in the long terminal repeat (LTR), which is localized at the edge of the preintegrated viral DNA. We generated LTR-modified LVs (LMLVs), by altering the conserved sequences located just before the cleavage site; this alteration prevented the integration of viral DNA into the host genome. In this study, the LMLVs significantly decreased the LV-mediated transgene expression in HeLa cells compared to the control, i.e., wild-type LTR LVs; this supposedly occurred because integration was prevented. In addition, LMLVs exhibited gene expression in vivo when they were injected into the mouse cerebellum. Moreover, quantitative Alu element-mediated polymerase chain reaction (Alu-PCR), which detects integrated viral DNA, revealed that rate of LMLV-suppressed integration was approximately 1/500-fold compared to that in the case of the wild-type LTR LV. These data suggest that LMLVs efficiently prevent integration as well as exhibit LV-mediated gene expression in mouse cerebellar Purkinje cells in vivo.

Correction of Laminin-5 Deficiency in Human Epidermal Stem Cells by Transcriptionally Targeted Lentiviral Vectors

Deficiency of the basement membrane component laminin-5 (LAM5) causes junctional epidermolysis bullosa (JEB), a severe and often fatal skin adhesion defect. Autologous transplantation of epidermal stem cells genetically corrected with a Moloney leukemia virus (MLV)-derived retroviral vector reconstitutes LAM5 synthesis, and corrects the adhesion defect in JEB patients. However, MLV-derived vectors have genotoxic characteristics, and are unable to reproduce the physiological, basal layer-restricted expression of LAM5 chains. We have developed an alternative gene transfer strategy based on self-inactivating (SIN) or long terminal repeat (LTR)-modified lentiviral vectors, in which transgene expression is under the control of different combinations of promoter-enhancer elements derived from the keratin-14 (K14) gene. Analysis in human keratinocyte cultures and in fully differentiated skin regenerated onto immunodeficient mice showed that gene expression directed by K14 enhancers is tissue-specific and restricted to the basal layer of the epidermis. Transcriptionally targeted lentiviral vectors efficiently transduced clonogenic stem/progenitor cells derived from a skin biopsy of a JEB patient, restored normal synthesis of LAM5 in cultured keratinocytes, and reconstituted normal adhesion properties in human skin equivalents transplanted onto immunodeficient mice. These vectors are therefore an effective, and potentially more safe, alternative to MLV-based retroviral vectors in gene therapy of JEB.Molecular Therapy (2008) 16 12, 1977-1985 doi:10.1038/mt.2008.204.

Long terminal repeat sequences from virulent and attenuated equine infectious anemia virus demonstrate distinct promoter activities

In the early 1970s, the Chinese Equine Infectious Anemia Virus (EIAV) vaccine, EIAV DLA, was developed through successive passages of a wild-type virulent virus (EIAV L) in donkeys in vivo and then in donkey macrophages in vitro. EIAV attenuation and cell tropism adaptation are associated with changes in both envelope and long terminal repeat (LTR). However, specific LTR changes during Chinese EIAV attenuation have not been demonstrated. In this study, we compared LTR sequences from both virulent and attenuated EIAV strains and documented the diversities of LTR sequence from in vivo and in vitro infections. We found that EIAV LTRs of virulent strains were homologous, while EIAV vaccine have variable LTRs. Interestingly, experimental inoculation of EIAV DLA into a horse resulted in a restriction of the LTR variation. Furthermore, LTRs from EIAV DLA showed higher Tat transactivated activity than LTRs from virulent strains. By using chimeric clones of wild-type LTR and vaccine LTR, the main difference of activity was mapped to the changes of R region, rather than U3 region.

A Moloney Murine Leukemia Virus Driven by the Jaagsiekte Sheep Retrovirus Enhancers Shows Enhanced Specificity for Infectivity in Lung Epithelial Cells

Jaagsiekte sheep retrovirus (JSRV) is the etiologic agent of ovine pulmonary adenocarcinoma (OPA), a transmissible lung cancer in sheep. One of the unique features of this virus is that in infected animals, the only tissues that show expression of the virus are the tumor cells in the lung. We previously showed that the JSRV long terminal repeat (LTR) is preferentially active in murine lung epithelial cell lines (MLE-15 and mtCC1-2). To further explore the tissue specificity, we inserted the JSRV enhancer sequences from the U3 region of the LTR into a Moloney murine leukemia virus (M-MuLV) LTR lacking its own enhancer sequences, to give the chimeric LTR DeltaMo + JS. Transient transfection assays indicated that the DeltaMo + JS LTR is > 5-fold more active in lung epithelial cell lines than in non-lung lines, compared to the wild-type M-MuLV LTR. This was due to preferential activity of the JSRV enhancers in lung epithelial cells. Moreover, M-MuLV driven by the DeltaMo + JS LTR was > 3 logs more infectious in MLE-15 cells compared to non-lung cell lines. This chimeric virus may facilitate investigations of the tissue-specificity of JSRV.

Involvement of Ethylene in Stress-Induced Expression of the TLC1.1 Retrotransposon from Lycopersicon chilense Dun.

The TLC1 family is one of the four families of long terminal repeat (LTR) retrotransposons identified in the genome of Lycopersicon chilense. Here, we show that this family of retroelements is transcriptionally active and its expression is induced in response to diverse stress conditions such as wounding, protoplast preparation, and high salt concentrations. Several stress-associated signaling molecules, including ethylene, methyl jasmonate, salicylic acid, and 2,4-dichlorophenoxyacetic acid, are capable of inducing TLC1 family expression in vivo. A representative of this family, named TLC1.1, was isolated from a genomic library from L. chilense. Transient expression assays in leaf protoplasts and stably transformed tobacco (Nicotiana tabacum) plants demonstrate that the U3 domain of the 5'-LTR region of this element can drive stress-induced transcriptional activation of the beta-glucuronidase reporter gene. Two 57-bp tandem repeated sequences are found in this region, including an 8-bp motif, ATTTCAAA, previously identified as an ethylene-responsive element box in the promoter region of ethylene-induced genes. Expression analysis of wild-type LTR and single and double ethylene-responsive element box mutants fused to the beta-glucuronidase gene shows that these elements are required for ethylene-responsive gene expression in protoplasts and transgenic plants. We suggest that ethylene-dependent signaling is the main signaling pathway involved in the regulation of the expression of the TLC1.1 element from L. chilense.

9. Mobilization of Sendai Virus Vectors (SeVV) by Phylogenetic Related Human Pathogens: Implications for Future Vector Generations

In an attempt to make HIV-1 based vectors safer for use in the research and clinical setting, a significant modification to the HIV-1 genome has been the deletion of the TATA box and enhancer elements from the U3 region of the long terminal repeat (LTR). Vectors containing this 400bp U3 deletion are thought to have no LTR-directed transcription, and are thus called self-inactivating (SIN) lentivectors. As with wild-type LTR vectors, any transcripts initiated at the U3-R junction in the 5' LTR of SIN lentivectors contain the packaging signal found in the psi region and 5' portion of gag. Transcripts containing the packaging signal have the potential to be encapsidated and mobilized by wild-type HIV-1. Using four distinct approaches, we show that SIN lentivectors continue to have promoter activity near the 5' LTR which is responsible for the production of full-length vector genome transcripts. To verify that transcripts derived from the LTR in SIN lentivectors are competent for encapsidation and integration, we transduced a lentiviral packaging cell line with a standard SIN lentivector and then observed production of viable vector particles containing full-length SIN lentivector genomes. We also demonstrate that a Herpes Simplex Virus thymidine kinase gene placed downstream of the SIN LTR, but upstream of the vector's internal promoter, is produced in quantities high enough to render cells containing this vector partially succeptible to gancyclovir-induced cell death. Additionally, we show that SIN lentivectors containing no internal promoter express an eGFP transgene. Using RT-PCR we demonstrate that transcripts initiated at the 5' LTR in SIN lentivectors can be readily detected, and using this assay, we conclude that the SIN LTR retains approximately 15% of the transcriptional activity of the wild-type LTR (in the absence of Tat). We have also identified sequences in the SIN lentivector which are responsible for transcriptional activation at the 5' LTR. With a promoter assay using different segments of the LTR and leader region of the SIN lentivector, we have determined that the residual promoter activity does not depend on any LTR sequence, including the HIV-1 start-site initiator (Inr) element. Rather, the promoter activity is contained entirely within the leader region, and although this element is downstream of the transcription initiation site, it is capable of initiating transcription from the 5' end of R in the LTR. Mutation of leader region binding sites for the transcriptional activators DBF1 and SP1 reduces transcription from the SIN LTR by up to 80%; however, a significant decrease in vector titer results from this mutation, perhaps owing to disruption of the folding of psi, which overlaps the SP1 binding site. In conclusion, knowledge of the potential for mobilization of SIN lentivectors by HIV-1 will be important for the design of future gene therapy trials using such vectors. It is also possible that low level expression from the SIN LTR may, in some instances, be adequate for the production of small amounts of transgene in a non-cell-type specific manner, which may be relevant to studies using SIN lentivectors to introduce cell-specific expression cassettes.

Engineered long terminal repeats of retroviral vectors enhance transgene expression in hepatocytes in vitro and in vivo

To analyze the important elements for retroviral expression in hepatocytes, cis-acting elements in the U3 region of the long terminal repeat (LTR) of the polycythemic strain of spleen focus-forming virus (SFFVp) were analyzed in a hepatocellular carcinoma cell line. Two cis-acting elements located within the upstream region of the direct repeat, which positively regulated retroviral expression, were identified. Transcription factors NFAT5 and Sp1, which are ubiquitously expressed in a variety of tissues, bound to these elements. To increase specificity without lowering the potency of retroviral expression in hepatocytes, these elements were replaced by a sequence derived from the hepatitis B virus enhancer II region. Novel vectors, SF-Hep3 and SF-Hep5 (SFFVp-based vector for hepatocytes 3 and 5), were developed with these engineered LTRs. The engineered LTRs of these vectors enhanced the retroviral expression only in hepatocellular carcinoma cell lines in vitro. These vectors also increased transgene expression 4- to 9-fold or 3.5- to 5-fold in comparison with a Moloney murine leukemia virus-based vector or a vector containing the wild-type LTR of SFFVp, respectively, in murine hepatocytes in vivo.

A truncated form of Nef selected during pathogenic reversion of simian immunodeficiency virus SIVmac239Deltanef increases viral replication.

The live, attenuated vaccine simian immunodeficiency virus SIVmac239Deltanef efficiently protects rhesus macaques against infection with wild-type SIVmac but occasionally causes CD4(+) T-cell depletion and progression to simian AIDS (SAIDS). Virus recovered from a vaccinated macaque (Rh1490) that progressed to SAIDS had acquired an additional deletion in the nef gene, resulting in a frameshift that restored the original nef open reading frame (R. I. Connor, D. C. Montefiori, J. M. Binley, J. P. Moore, S. Bonhoeffer, A. Gettie, E. A. Fenamore, K. E. Sheridan, D. D. Ho, P. J. Dailey, and P. A. Marx, J. Virol. 72:7501-7509, 1998). Intravenous inoculation of the Rh1490 viral isolate into four naive rhesus macaques induced CD4(+) T-cell depletion and disease in three out of four animals within 2 years, indicating a restoration of virulence. A DNA fragment encompassing the truncated nef gene amplified from the Rh1490 isolate was inserted into the genetic backbone of SIVmac239. The resulting clone, SIVmac239-Delta2nef, expressed a Nef protein of approximately 23 kDa, while the original SIVmac239Deltanef clone expressed a shorter protein of 8 kDa. The revertant form of Nef did not cause downregulation of CD4, CD3, or major histocompatibility complex class I. The infectivity of SIVmac239-Delta2nef was similar to that of SIVmac239Deltanef in single-cycle assays using indicator cell lines. In contrast, SIVmac239-Delta2nef replicated more efficiently than SIVmac239Deltanef in peripheral blood mononuclear cell (PBMC) cultures infected under unstimulated conditions. The p27 Gag antigen levels in SIVmac239-Delta2nef-infected cultures were still lower than those obtained with wild-type SIVmac239, consistent with a partial recovery of Nef function. The transcriptional activity of long terminal repeat (LTR)-luciferase constructs containing the nef deletions did not differ markedly from that of wild-type LTR. Introduction of a premature stop codon within Nef-Delta2 abolished the replicative advantage in PBMCs, demonstrating that the Nef-Delta2 protein, rather than the structure of the U3 region of the LTR, was responsible for the increase in viral replication. Taken together, these results show that SIV with a deletion in the nef gene can revert to virulence and that expression of a form of nef with multiple deletions may contribute to this process by increasing viral replication.

Efficient human immunodeficiency virus replication requires a fine-tuned level of transcription.

Transcription represents a crucial step in the life cycle of human immunodeficiency virus (HIV) and is highly regulated. Here we show that the strength of the viral long terminal repeat (LTR) promoter is optimized for efficient replication. Artificially increasing the rate of LTR-driven transcription was strongly detrimental for viral fitness, and HIV was able to regain replication capacity by selecting for variants with a weaker LTR. Strikingly, the strength of the evolved promoter was equivalent to that of the wild-type LTR.

Lentiviral vectors approach the clinic but fall back: National Institutes of Health Recombinant DNA Advisory Committee review of a first clinical protocol for use of a lentiviral vector.

Lentiviral vectors approach the clinic but fall back: National Institutes of Health Recombinant DNA Advisory Committee review of a first clinical protocol for use of a lentiviral vector.

Attenuated and wild-type HIV-1 infections and long terminal repeat-mediated gene expression from plasmids delivered by gene gun to human skin ex vivo and macaques in vivo.

Gene expression from HIV-based gene therapy vectors or live-attenuated HIV-1 vaccines requires RNA transcription supported by the HIV-1 promoter, the long terminal repeat (LTR). Delivery of live-attenuated HIV-1 vaccines as plasmid DNA would overcome problems associated with production of attenuated HIV-1 strains. We investigated the expression of reporter plasmids and proviral HIV-1 constructs driven by either the HIV-1 LTR or LTRs with deletions in the U3 enhancer regions. LTR-driven plasmids were inoculated by gene gun into both human epidermis ex vivo and macaques in vivo. The HIV-1 LTR drove reporter gene expression in human and macaque skin, although with 15- to 20-fold less efficiency compared to the immediate-early cytomegalovirus promoter. A deleted LTR derived from a naturally attenuated HIV-1 strain infecting a member of the well-characterized Sydney Blood Bank Cohort of long-term nonprogressors was 5-fold less efficient in expression of the reporter gene compared to wild-type LTR. Delivery of proviral wild-type HIV-1 DNA constructs to human skin resulted in recovery of HIV-1 from cells emigrating from the epidermis, providing an ex vivo model of the infectivity of proviral HIV-1 DNA. However, delivery of proviral HIV-1 DNA containing deletions in either the LTR, Nef, or the secondary viral transcription activator,Vpr, significantly reduced HIV-1 replication in this model. The early coexpression of Tat from a second plasmid did not restore replication. Thus, although attenuated lentiviral vaccines might be deliverable as proviral DNA constructs in primate subjects, significant improvements are needed to enhance the efficiency of this method.

Modulation of Moloney Leukemia Virus Long Terminal Repeat Transcriptional Activity by the Murine CD4 Silencer in Retroviral Vectors

We investigated whether CD4 gene regulatory sequences might be useful for developing transcriptionally targeted Moloney murine leukemia virus (Mo-MLV)-based retroviral vectors for gene expression specifically in CD4+ cells. We could modulate Mo-MLV long terminal repeat (LTR) activity by inserting a 438-bp-long fragment containing the murine CD4 silencer in the LTR of the vector; both β-galactosidase and green fluorescent protein reporter gene activities were strongly down-regulated in both murine and human CD8+ cells, but not in CD4+ lymphoid cell lines and freshly isolated lymphocytes transduced with this vector, compared with the findings using a control vector carrying wild-type LTRs. Titration experiments on NIH-3T3 cells revealed that inclusion of the CD4 silencer in the LTRs did not reduce the titer of the vectors. These findings indicate that a cellular silencer can be successfully included in retroviral vectors, where it maintains its transcription-regulatory function, thus suggesting a novel approach to transcriptional targeting.

CDP is a repressor of mouse mammary tumor virus expression in the mammary gland.

Mouse mammary tumor virus (MMTV) transcription is highest in the lactating mammary gland but is detectable in a variety of other tissues. Previous results have shown that MMTV expression is suppressed in lymphoid and other tissues through the binding of the homeodomain-containing repressor special AT-rich binding protein 1 to a negative regulatory element (NRE) in the MMTV long terminal repeat (LTR). Another homeoprotein repressor, CCAAT displacement protein (CDP), also binds to the MMTV NRE, but a role for CDP in MMTV transcriptional suppression has not yet been demonstrated. In this paper, we show that the level of CDP decreases during development of the mammary gland and that this decline in CDP level correlates with the known increase in MMTV expression observed during mammary gland differentiation. Moreover, CDP overexpression was able to suppress MMTV LTR-reporter gene activity up to 20-fold in transient-transfection assays of mouse mammary cells. To determine if this effect was due to direct binding of CDP to the promoter-proximal NRE, we performed DNase I protection assays to map two CDP-binding sites from +835 to +845 and +920 to +931 relative to the first base of the LTR. Mutations engineered into each of these sites decreased CDP binding to the proximal NRE, whereas a combination of these mutations further reduced binding. Subsequently, each of these mutations was introduced into the full-length MMTV LTR upstream of the luciferase reporter gene. Analysis of stable transfectants of LTR constructs showed that CDP binding site mutations in the proximal NRE elevated reporter gene expression two- to sixfold compared to wild-type LTR constructs. Thus, MMTV expression increases during mammary gland development, in part due to decreased CDP levels and CDP binding to the LTR. Together, these experiments provide the first evidence that CDP acts as a repressor of MMTV transcription in the mammary gland.

Retrovirus DNA Termini Bound by Integrase Communicate in Trans for Full-Site Integration in Vitro

Integration of linear retrovirus DNA involves the concerted insertion of the viral termini (full-site integration) into the host chromosome. We investigated the interactions that occur between long terminal repeat (LTR) termini bound by avian retrovirus integrase (IN) for full-site integration in vitro. Wild-type (wt) or mutant LTR donors that possess gain-of-function (“G”) or loss-of-function (“L”) for full-site integration activity were used. G LTR termini are characterized as having significantly higher strand transfer activity than the wt and the L LTR termini. L LTR mutations are classified as partially or extremely defective for strand transfer activity. The L mutations were further classified by their ability to either permit or block the assembly of G or wt LTR termini into nucleoprotein complexes capable of full-site strand transfer. We demonstrated that avian myeloblastosis virus IN bound to G LTR termini increased the incorporation of partially defective L LTR termini into nucleoprotein complexes that were capable of full-site integration. The observed full-site integration activity of these assembled nucleoprotein complexes appeared to be influenced by each individual IN–LTR complex in trans. In contrast, extremely defective L LTR termini exhibited the ability to effectively block the assembly of wt LTR termini into nucleoprotein complexes capable of full-site strand transfer. Data from nonspecific DNA competition experiments suggested that IN had an apparent higher affinity for G LTR donor termini than for partially defective L LTR donor termini as measured by full-site integration activity. However, assembled nucleoprotein complexes containing either two G or two L LTR donors were stable, having a similar half-life of ∼2 h on ice. The results suggest that LTR termini bound by IN exhibit an allosteric effect to modulate full-site integration in vitro. Similar regulatory controls also appear to exist in vivo between the wt U3 and wt U5 LTR termini in retroviruses as well as purified retrovirus preintegration complexes that promoted full-site integration in vitro.

Stimulation of Tat-associated kinase-independent transcriptional elongation from the human immunodeficiency virus type-1 long terminal repeat by a cellular enhancer.

The human immunodeficiency virus type-1 (HIV-1) long terminal repeat (LTR) initiates transcription efficiently but produces only short transcripts in the absence of the trans-activator protein, Tat. To determine whether a cellular enhancer could provide the signals required to recruit an elongation-competent polymerase to the HIV-1 LTR, the B cell-specific immunoglobulin heavy chain gene enhancer (IgHE) was inserted upstream of the LTR. The enhancer increased transcription in the absence of Tat between 6- and 7-fold in transfected B cells, but the full-length transcripts remained at basal levels in HeLa cells, where the enhancer is inactive. RNase-protection studies showed that initiation levels in the presence and absence of the enhancer were constant, but the enhancer significantly increased the elongation capacity of the polymerases. Tat-stimulated elongation is strongly inhibited by the nucleoside analogue 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), which inhibits the Tat-associated kinase, TAK (CDK9). However, polymerases initiating transcription from LTRs carrying the enhancer were able to efficiently elongate in the presence of DRB. Specific repression of TAK by expression in trans of the CDK9 kinase also inhibited Tat-stimulated elongation but did not inhibit enhancer-dependent transcription significantly. Thus, the activation of polymerase processivity by the IgHE involves a unique mechanism which is independent of TAK.

Evolution of the human immunodeficiency virus type 1 long terminal repeat promoter by conversion of an NF-kappaB enhancer element into a GABP binding site.

Human immunodeficiency virus type 1 (HIV-1) transcription is regulated by the viral Tat protein and cellular factors, of which the concentration and activity may depend on the cell type. Viral long terminal repeat (LTR) promoter sequences are therefore optimized to suit the specific nuclear environment of the target host cell. In long-term cultures of a Tat-defective, poorly replicating HIV-1 mutant, we selected for a faster-replicating virus with a 1-nucleotide deletion in the upstream copy of two highly conserved NF-kappaB binding sites. The variant enhancer sequence demonstrated a severe loss of NF-kappaB binding in protein binding assays. Interestingly, we observed a new binding activity that is specific for the variant NF-kappaB sequence and is present in the nuclear extract of unstimulated cells that lack NF-kappaB. These results suggest that inactivation of the NF-kappaB site coincides with binding of another transcription factor. Fine mapping of the sequence requirements for binding of this factor revealed a core sequence similar to that of Ets binding sites, and supershift assays with antibodies demonstrated the involvement of the GABP transcription factor. Transient transfection experiments with LTR-chloramphenicol acetyltransferase constructs indicated that the variant LTR promoter is specifically inhibited by GABP in the absence of Tat, but this promoter was dramatically more responsive to Tat than the wild-type LTR. Introduction of this GABP site into the LAI virus yielded a specific gain of fitness in SupT1 cells, which contain little NF-kappaB protein. These results suggest that GABP potentiates Tat-mediated activation of LTR transcription and viral replication in some cell types. Conversion of an NF-kappaB into a GABP binding site is likely to have occurred also during the worldwide spread of HIV-1, as we noticed the same LTR modification in subtype E isolates from Thailand. This typical LTR promoter configuration may provide these viruses with unique biological properties.

The matrix attachment region-binding protein SATB1 participates in negative regulation of tissue-specific gene expression.

The nuclear matrix has been implicated in several cellular processes, including DNA replication, transcription, and RNA processing. In particular, transcriptional regulation is believed to be accomplished by binding of chromatin loops to the nuclear matrix and by the concentration of specific transcription factors near these matrix attachment regions (MARs). A number of MAR-binding proteins have been identified, but few have been directly linked to tissue-specific transcription. Recently, we have identified two cellular protein complexes (NBP and UBP) that bind to a region of the mouse mammary tumor virus (MMTV) long terminal repeat (LTR) previously shown to contain at least two negative regulatory elements (NREs) termed the promoter-proximal and promoter-distal NREs. These NREs are absent from MMTV strains that cause T-cell lymphomas instead of mammary carcinomas. We show here that NBP binds to a 22-bp sequence containing an imperfect inverted repeat in the promoter-proximal NRE. Previous data showed that a mutation (p924) within the inverted repeat elevated basal transcription from the MMTV promoter and destabilized the binding of NBP, but not UBP, to the proximal NRE. By using conventional and affinity methods to purify NBP from rat thymic nuclear extracts, we obtained a single major protein of 115 kDa that was identified by protease digestion and partial sequencing analysis as the nuclear matrix-binding protein special AT-rich sequence-binding protein 1 (SATB1). Antibody ablation, distamycin inhibition of binding, renaturation and competition experiments, and tissue distribution data all confirmed that the NBP complex contained SATB1. Similar types of experiments were used to show that the UBP complex contained the homeodomain protein Cux/CDP that binds the MAR of the intronic heavy-chain immunoglobulin enhancer. By using the p924 mutation within the MMTV LTR upstream of the chloramphenicol acetyltransferase gene, we generated two strains of transgenic mice that had a dramatic elevation of reporter gene expression in lymphoid tissues compared with reporter gene expression in mice expressing wild-type LTR constructs. Thus, the 924 mutation in the SATB1-binding site dramatically elevated MMTV transcription in lymphoid tissues. These results and the ability of the proximal NRE in the MMTV LTR to bind to the nuclear matrix clearly demonstrate the role of MAR-binding proteins in tissue-specific gene regulation and in MMTV-induced oncogenesis.

TAR and Sp1-independent transactivation of HIV long terminal repeat by the Tat protein in the presence of human cytomegalovirus IE1/IE2.

The HIV Tat protein is a transcriptional transactivator of the HIV-1 long terminal repeat (LTR) promoter element. Its activity depends on its direct interaction with the trans-activation response (TAR) element, although TAR-independent activation by Tat has been demonstrated in different cells. Herpesviruses in general and human cytomegalovirus (HCMV) in particular are often isolated from HIV-1-infected patients and could play a role in the activation of latent HIV and in a subsequent increase in HIV replication. HCMV immediate early gene products (IE1 and IE2) are nuclear phosphoproteins that play a pivotal role in HCMV replication and have been shown to transregulate both viral and cellular gene expression. It has repeatedly been shown that HCMV IE1/IE2 can independently transactivate HIV-1 LTR. The aim of this study was to investigate IE1/IE2 transactivation of HIV-1 LTR in a CD4+ T-cell line in the absence and presence of HIV-1 Tat to establish whether IE1/IE2 can synergize with Tat. HIV-1 LTR transactivation by HCMV IE1/IE2 in the presence and absence of HIV-1 Tat was determined by transient transfection experiments of J-Jhan lymphoblastoid cells with a series of different expression vectors. We found a strong synergistic transactivation between HIV Tat and the IE1-IE2 complex on HIV LTR activity using vectors driven either by wild-type LTR or by the nuclear factor NF-kappa(B) response element-mutated HIV LTR. IE1/IE2 synergism with HIV Tat was also observed in Sp1 binding site-mutated for TAR-deleted LTR, which cannot be activated by Tat alone. This cooperation is abolished when the region in IE2 that binds the TATA box binding protein is deleted. The results obtained indicate that Sp1-binding and TAR sequences are not strictly required for Tat responsiveness when Tat is directed to the HIV promoter by HCMV IE1-IE2. This synergistic effect is mediated by the IE2 and TATA-binding region, and could play a major role in HIV activation when cells are infected by both viruses, a feature often observed in AIDS patients.