HIV-1 LTR C/EBP binding site sequence configurations preferentially encountered in brain lead to enhanced C/EBP factor binding and increased LTR-specific activity.

Recent studies have shown that two CAAT/enhancer binding protein (C/EBP) sites are critically important for efficient human immunodeficiency virus (HIV) type 1 (HIV-1) replication within cells of the monocyte/macrophage lineage, a primary cell type infected by HIV-1 and a potentially important vehicle for transport of virus to the central nervous system (CNS). Given the relevance of HIV-1 LTR sequence variation with respect to HIV-1 replication within monocyte populations and the important role that monocyte tropism likely plays in HIV-1 infection of the brain, C/EBP site sequence variation was examined within peripheral blood- and brain-derived LTR populations. Brain-derived LTRs commonly possessed a C/EBP site I configuration (6G, comprised of a thymidine to guanosine substitution with respect to the clade B consensus sequence at position 6 of C/EBP site I) that leads to enhanced binding of C/EBP proteins over that observed with the HIV-1 clade B consensus sequence at this site. In contrast, the 6GC/EBP site I configuration appeared infrequently within sequenced peripheral blood-derived LTRs. In addition, C/EBP site II was even more highly conserved in brain-derived HIV-1 LTR populations than site I. This was not the case with peripheral blood-derived LTR C/EBP site II sequences. The high degree of C/EBP site II conservation in brain-derived LTRs was likely important in LTR regulation since the clade B consensus sequence conserved at C/EBP site II recruited high amounts of C/EBP family members. Transient transfection analyses indicated that conservation of the strong C/EBP site II in brain-derived LTRs was likely due to important interactions with Tat. Overall, brain-derived HIV-1 LTRs preferentially contained two highly reactive C/EBP binding sites, which may suggest that these sites play important roles in LTR-directed transcription during invasion and maintenance of HIV-1 in the central nervous system.

Interaction between CCAAT/enhancer binding protein and cyclic AMP response element binding protein 1 regulates human immunodeficiency virus type 1 transcription in cells of the monocyte/macrophage lineage.

Recent observations have shown two CCAAT/enhancer binding protein (C/EBP) binding sites to be critically important for efficient human immunodeficiency virus type 1 (HIV-1) replication within cells of the monocyte/macrophage lineage, a cell type likely involved in transport of the virus to the brain. Additionally, sequence variation at C/EBP site I, which lies immediately upstream of the distal nuclear factor kappa B site and immediately downstream of a binding site for activating transcription factor (ATF)/cyclic AMP response element binding protein (CREB), has been shown to affect HIV-1 long terminal repeat (LTR) activity. Given that C/EBP proteins have been shown to interact with many other transcription factors including members of the ATF/CREB family, we proceeded to determine whether an adjacent ATF/CREB binding site could affect C/EBP protein binding to C/EBP site I. Electrophoretic mobility shift analyses indicated that selected ATF/CREB site variants assisted in the recruitment of C/EBP proteins to an adjacent, naturally occurring, low-affinity C/EBP site. This biophysical interaction appears to occur via at least two mechanisms. First, low amounts of CREB-1 and C/EBP appear to heterodimerize and bind to a site consisting of a half site from both the ATF/CREB and C/EBP binding sites. In addition, CREB-1 homodimers bind to the ATF/CREB site and recruit C/EBP dimers to their cognate weak binding sites. This interaction is reciprocal, since C/EBP dimer binding to a strong C/EBP site leads to enhanced CREB-1 recruitment to ATF/CREB sites that are weakly bound by CREB. Sequence variation at both C/EBP and ATF/CREB sites affects the molecular interactions involved in mediating both of these mechanisms. Most importantly, sequence variation at the ATF/CREB binding site affected basal LTR activity as well as LTR function following interleukin-6 stimulation, a treatment that leads to increases in C/EBP activation. Thus, HIV-1 LTR ATF/CREB binding site sequence variation may modulate cellular signaling at the viral promoter through the C/EBP pathway.

HIV-1-associated central nervous system dysfunction.

Despite more than 15 years of extensive investigative efforts, a complete understanding of the neurological consequences of HIV-1 CNS infection remains elusive. Although the resources of numerous investigators have been focused on studies of HIV-1-associated CNS disease, the complex nature of the disease processes that underlie the clinical, pathological, and cellular manifestations of HIV-1 CNS infection have required a larger volume of studies than was initially envisioned. Several major areas remain as the focus of current research efforts. One of the more pressing issues facing researchers and clinicians alike is the search for correlates to the development of HIV-1-associated CNS neuropathology and the onset of HIVD. Although numerous parameters have been studied, none have been shown to be absolute predictors or markers of HIV-1-related CNS dysfunction. The identification of solid correlates of HIVD is an important goal that would permit clinical identification of individuals at risk for developing potentially crippling, life-threatening CNS abnormalities and would facilitate early treatment of nascent neurological problems. A more complete comprehension of the cellular foundations of CNS dysfunction and HIVD is also a fundamental part of strategies designed to treat or prevent HIV-1-associated CNS disease. Future investigations will strive to expand the body of knowledge concerning the complex interactions between infected and uninfected neuroglial cells and the roles of numerous cytokines, chemokines, and other soluble agents that are deregulated during HIV-1 CNS infection. In particular, a thorough understanding of the mechanisms of neurotoxicity may facilitate the development of new therapies that alleviate or eliminate the clinical consequences of CNS infection. Finally, investigators will continue to study HIVD within the context of single and combination drug therapies used in the treatment of HIV-1 infection and AIDS. As newer and more effective systemic treatments for HIV-1 infection and AIDS are introduced, the effects of these treatments on the onset, incidence, and severity of HIVD will also require intensive study. The impact of drug therapies on the ability of the CNS to act as an HIV-1 reservoir will also need to be addressed. Introduction of each new drug or drug combination will necessitate studies of drug penetration into the CNS and efficacy against the development of CNS abnormalities. Furthermore, as more effective treatments prolong the lifespan of individuals infected with HIV-1, the impact of extended survival on the occurrence and severity of HIVD will also require further investigations. The quest for answers to these and other questions will be complicated by the diversity of experimental systems used to study different aspects of HIV-1 CNS infection and HIVD. Each system has its own unique strengths and weaknesses. Clinical observations provide a continuous spectrum of symptomatic findings but reveal little about the underlying mechanisms of disease. In vivo imaging techniques, such as CT and MRI, also provide a continuum of observations, but the images are limited in their resolution. Neuropathological examinations of postmortem HIV-1-infected brains offer gross, cellular, and molecular views (including phenotypic and genotypic analyses of CNS viral isolates) of the diseased brain, but only provide a snapshot of the end-stage neurologic dysfunction. Studies that rely on animal surrogates for HIV-1, including SIV, simian-HIV (SHIV), feline immunodeficiency virus (FIV), visna virus, and HIV-1 SCID-hu models, permit experimental protocols that cannot be carried out in humans, but are limited by the fidelity with which each virus and animal model emulates the conditions and events observed in the human host. Finally, in vitro techniques, which include the use of primary cells and cell lines, adult or fetal human cell cultures, and BBB barrier model systems, are also convenient means by which aspe

Analysis of the HIV-1 LTR NF-kappaB-proximal Sp site III: evidence for cell type-specific gene regulation and viral replication.

It has been widely demonstrated that the human immunodeficiency virus type 1 (HIV-1) envelope, specifically the V3 loop of the gp120 spike, evolves to facilitate adaptation to different cellular populations within an infected host. Less energy has been directed at determining whether the viral promoter, designated the long terminal repeat (LTR), also exhibits this adaptive quality. Because of the unique nature of the cell populations infected during the course of HIV-1 infection, one might expect the opportunity for such adaptation to exist. This would permit select viral species to take advantage of the different array of conditions and factors influencing transcription within a given cell type. To investigate this hypothesis, the function of natural variants of the NF-kappaB-proximal Sp element (Sp site III) was examined in human cell line models of the two major cell types infected during the natural course of HIV-1 infection, T cells and monocytes. Utilizing the HIV-1 LAI molecular clone, which naturally contains a high-affinity Sp site III, substitution of low-affinity Sp sites in place of the natural site III element markedly decreased viral replication in Jurkat T cells. However, these substitutions had relatively small effects on viral replication in U-937 monocytic cells. Transient transfections of HIV-1 LAI-based LTR-luciferase constructs into these cell lines suggest that the large reduction in viral replication in Jurkat T cells, caused by low-affinity Sp site III variants, may result from reduced basal as well as Vpr- and Tat-activated LTR activities in Jurkat T cells compared to those in U-937 monocytic cells. When the function of Sp site III was examined in the context of HIV-1 YU-2-based LTR-luciferase constructs, substitution of a high-affinity element in place of the natural low-affinity element resulted in increased basal YU-2 LTR activity in Jurkat T cells and reduced activity in U-937 monocytic cells. These observations suggest that recruitment of Sp family members to Sp site III is of greater importance to the function of the viral promoter in the Jurkat T cell line as compared to the U-937 monocytic cell line. These observations also suggest that other regions of the LTR may compensate for Sp recruitment defects in specific cell populations.

Sodium dodecyl sulfate and C31G as microbicidal alternatives to nonoxynol 9: comparative sensitivity of primary human vaginal keratinocytes.

A broad-spectrum vaginal microbicide must be effective against a variety of sexually transmitted disease pathogens and be minimally toxic to the cell types found within the vaginal epithelium, including vaginal keratinocytes. We assessed the sensitivity of primary human vaginal keratinocytes to potential topical vaginal microbicides nonoxynol-9 (N-9), C31G, and sodium dodecyl sulfate (SDS). Direct immunofluorescence and fluorescence-activated cell sorting analyses demonstrated that primary vaginal keratinocytes expressed epithelial cell-specific keratin proteins. Experiments that compared vaginal keratinocyte sensitivity to each agent during a continuous, 48-h exposure demonstrated that primary vaginal keratinocytes were almost five times more sensitive to N-9 than to either C31G or SDS. To evaluate the effect of multiple microbicide exposures on cell viability, primary vaginal keratinocytes were exposed to N-9, C31G, or SDS three times during a 78-h period. In these experiments, cells were considerably more sensitive to C31G than to N-9 or SDS at lower concentrations within the range tested. When agent concentrations were chosen to result in an endpoint of 25% viability after three daily exposures, each exposure decreased cell viability at the same constant rate. When time-dependent sensitivity during a continuous 48-h exposure was examined, exposure to C31G for 18 h resulted in losses in cell viability not caused by either N-9 or SDS until at least 24 to 48 h. Cumulatively, these results reveal important variations in time- and concentration-dependent sensitivity to N-9, C31G, or SDS within populations of primary human vaginal keratinocytes cultured in vitro. These investigations represent initial steps toward both in vitro modeling of the vaginal microenvironment and studies of factors that impact the in vivo efficacy of vaginal topical microbicides.

Molecular circuitry regulating herpes simplex virus type 1 latency in neurons.

Herpes simplex virus (HSV) type 1 (HSV-1) enters nerve endings during a primary infection, is transported to sensory ganglia, and establishes latency within nuclei of a subpopulation of neurons. The latent state is characterized by absence of detectable HSV-1 antigen, minimal transcription of productive cycle genes, and high expression of 1.5- and 2-kb viral transcripts, termed the major latency-associated transcripts (LATs), within nuclei of a subpopulation of infected neurons. Transcription within the HSV-1 LAT genomic locus has been reported to both facilitate the establishment of latency in additional neurons and to increase the frequency of spontaneous and induced viral reactivation in animal model systems. More recent evidence suggests some possible mechanisms that may explain the relationship between LATs and both reactivation and establishment of latency. This review summarizes general aspects of latency, but focuses on the structure, expression, and function of LATs, and the interaction between host transcriptional regulators and viral gene expression that may impact latency and reactivation. A model that incorporates evidence from a number of experimental studies is proposed that summarizes the involvement of the LAT locus in the biology of HSV-1 latency.

Human T cell lymphotropic virus type I genomic expression and impact on intracellular signaling pathways during neurodegenerative disease and leukemia.

HTLV-I has been identified as the etiologic agent of neoplasia within the human peripheral blood T lymphocyte population, and a progressive neurologic disorder based primarily within the central nervous system. We have examined the role of HTLV-I in these two distinctly different clinical syndromes by examining the life cycle of the virus, with emphasis on the regulation of viral gene expression within relevant target cell populations. In particular, we have examined the impact of specific viral gene products, particularly Tax, on cellular metabolic function. Tax is a highly promiscuous and pleiotropic viral oncoprotein, and is the most important factor contributing to the initial stages of viral-mediated transformation of T cells after HTLV-I infection. Tax, which weakly binds to Tax response element 1 (TRE-1) in the viral long terminal repeat (LTR), can dramatically trans-activate viral gene expression by interacting with cellular transcription factors, such as activated transcription factors and cyclic AMP response element binding proteins (ATF/CREB), CREB binding protein (CBP/p300), and factors involved with the basic transcription apparatus. At the same time, Tax alters cellular gene expression by directly or indirectly interacting with a variety of cellular transcription factors, cell cycle control elements, and cellular signal transduction molecules ultimately resulting in dysregulated cell proliferation. The mechanisms associated with HTLV-I infection, leading to tropical spastic paraparesis (TSP) are not as clearly resolved. Possible explanations of viral-induced neurologic disease range from central nervous system (CNS) damage caused by direct viral invasion of the CNS to bystander CNS damage caused by the immune response to HTLV-I infection. It is interesting to note that it is very rare for an HTLV-I infected individual to develop both adult T cell leukemia (ATL) and TSP in his/her life time, suggesting that the mechanisms governing development of these two diseases are mutually exclusive.

Inactivation of human immunodeficiency virus type 1 by nonoxynol-9, C31G, or an alkyl sulfate, sodium dodecyl sulfate.

A highly desirable approach to prevention of human immunodeficiency virus type 1 (HIV-1) transmission during sexual intercourse is the development of nontoxic, topical, broad spectrum microbicides effective against transmission of cell-associated and cell-free virus. Toward this end, the HIV-1 inactivation potential of surface active agents C31G and an alkyl sulfate, sodium dodecyl sulfate (SDS) was assessed. Because of its extensive use as a microbicidal agent, nonoxynol-9 (N-9) was used as a reference against which C31G and SDS were compared. Viral inactivation was measured using HIV-1 LTR-beta-galactosidase indicator cells (expressing CD4 or CD4/CCR5) derived from HeLa cells, a cell line of human cervical adenocarcinoma origin. In experiments which examined inactivation of cell-free HIV-1, C31G was generally more effective than N-9. Viral inactivation by SDS occurred at twice the concentration necessary to achieve similar levels of inactivation using either N-9 or C31G. Using HeLa and HeLa-derived cells in cytotoxicity studies, it was demonstrated that SDS is as much as 11 and five times less cytotoxic than N-9 or C31G, respectively, during 48 h of continuous exposure. SDS (unlike C31G and N-9) can inactivate non-enveloped viruses such as human papillomavirus (HPV) [Howett, M.K., Neely, E.B., Christensen, N.D., Wigdahl, B., Krebs, F.C., Malamud, D., Patrick, S.D., Pickel, M.D., Welsh, P.A., Reed, C.A., Ward, M.G., Budgeon, L.R., Kreider, J.W., 1999. A broad-spectrum microbicide with virucidal activity against sexually transmitted viruses. Antimicrob. Agents Chemother. 43(2), 314-321]. Since addition of SDS to C31G or N-9 may make the resulting microbicidal mixtures broadly effective against both enveloped and non-enveloped viruses, several surface active agent combinations were evaluated for their abilities to inactivate HIV-1. Addition of SDS to either C31G or N-9 resulted in mixtures that were only slightly less effective than equivalent concentrations of C31G or N-9 alone. To investigate inactivation of cell-associated infectivity, HIV-1 IIIB-infected SupT1 cells were treated with N-9, C31G, or SDS. Inactivation of cell-associated infectivity required higher microbicide concentrations than were needed for inactivation of cell-free virus. However, the relative activities of N-9, C31G, or SDS were similar to those seen in assays of inactivation using cell-free virus. These studies suggest that C31G and SDS may be attractive candidates for human trials as topical microbicides effective against HIV-1 transmission since both function at concentrations that provide effective viral inactivation with low levels of cytotoxicity. SDS microbicides (used alone or with other microbicides) may provide the added advantage of protection from HPV infection.

A broad-spectrum microbicide with virucidal activity against sexually transmitted viruses.

Sodium dodecyl sulfate (SDS), an alkyl sulfate surfactant derived from an organic alcohol, possesses surfactant properties but also denatures and unfolds both monomeric and subunit proteins. In preliminary experiments, we demonstrated that SDS is a potent inactivator of herpes simplex virus type 2 and human immunodeficiency virus type 1 at concentrations comparable to those used for the surfactant nonoxynol-9. We hypothesized that SDS might be capable of denaturing the capsid proteins of nonenveloped viruses. In this report, we demonstrate inactivation of rabbit, bovine, and human papillomaviruses after brief treatment with dilute solutions of SDS. Effective concentrations were nontoxic to rabbit skin and to split-thickness grafts of human foreskin epithelium. This is the first report of a microbicidal surfactant that will inactivate papillomaviruses. We propose that SDS is now a candidate microbicide for formulation and testing with humans.

ATF/CREB elements in the herpes simplex virus type 1 latency-associated transcript promoter interact with members of the ATF/CREB and AP-1 transcription factor families.

The herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) promoter 1 (LP1) is an inducible and cell type-specific promoter involved in regulating the production of an 8.3-kb primary LAT transcript during acute and latent infection of peripheral sensory neurons and during subsequent virus reactivation. A number of cis-acting regulatory elements have been identified in LP1, including two cyclic-AMP (cAMP) response element (CRE)-like sequences, designated CRE-1 and CRE-2. CRE-1 has previously been shown to confer cAMP responsiveness to LP1 and to regulate reactivation of HSV-1 from latency in vivo. A role for CRE-2 in modulating inducible activity is not yet as clear; however, it has been shown to support basal expression in neuronal cells in vitro. Electrophoretic mobility shift (EMS) analyses demonstrate that the LP1 CRE-like elements interact with distinct subsets of neuronal ATF/CREB and Jun/Fos proteins including CREB-1, CREB-2, ATF-1, and JunD. The factor-binding properties of each LP1 CRE element distinguish them from each other and from a highly related canonical CRE binding site and the TPA response element (TRE). LP1 CRE-1 shares binding characteristics of both a canonical CRE and a TRE. LP1 CRE-2 is more unusual in that it shares more features of a canonical CRE site than a TRE with two notable exceptions: it does not bind CREB-1 very well and it binds CREB-2 better than the canonical CRE. Interestingly, a substantial proportion of the C1300 neuroblastoma factors that bind to CRE-1 and CRE-2 have been shown to be immunologically related to JunD, suggesting that the AP-1 family of transcription factors may be important in regulating CRE-dependent LP1 transcriptional activity. In addition, we have demonstrated the two HSV-1 LP1 CRE sites to be unique with respect to their ability to bind neuronal AP1-related factors that are regulated by cAMP. These studies suggest that both factor binding and activation of bound factors may be involved in cAMP regulation of HSV-1 LP1 through the CRE elements, and indicate the necessity of investigating the expression and posttranslational modification of a variety of ATF/CREB and AP-1 factors during latency and reactivation.

Sp1 and related factors fail to interact with the NF-kappaB-proximal G/C box in the LTR of a replication competent, brain-derived strain of HIV-1 (YU-2).

The HIV-1 LTR promoter proximal G/C box array has been demonstrated to function by interacting with the Sp1 transcription factor family whose members can act as either activators or repressors of transcription. In this regard, we have examined the interaction of the HIV-1 Sp binding sites with nuclear factors that are present in cell types that support HIV-1 replication, including those of lymphocytic, monocytic, and astrocytic origin. As determined by electrophoretic mobility shift (EMS) competition analyses using oligonucleotides containing the sequences of each of the Sp1 sites of HIV-1 strain LAI, the NF-kappaB-proximal Sp site (site III) displayed the highest binding activity compared to sites I and II with regard to Sp1 and related factors present in lymphocytic (Jurkat) and astrocytic (U-373 MG) nuclear extracts. Sp1 and two Sp3 isoforms were detected as the primary cellular constituents of DNA-protein complexes formed with the NF-kappaB-proximal site. Only modest differences in Sp1:Sp3 binding ratios were observed when this site was reacted with either astrocytic or lymphocytic nuclear extract. However, when nuclear extracts derived from two monocytic cell types that differ in the degree of differentiation were reacted with the HIV-1 LAI Sp site III, a large difference in Sp1 and Sp3 binding was observed. To determine if naturally occurring and replication-competent strains of HIV-1 contain base pair alterations within the Sp elements that affect the ability of the site to interact with Sp1 and related factors, a series of Sp site III variants were constructed and examined by EMS analyses. One of these sites, obtained from the published sequence of the YU-2 strain (a brain-derived macrophage tropic strain of HIV-1), displayed almost no Sp1 or Sp3 binding activity as a result of a single base pair alteration in Sp site III. This base-pair alteration, when placed in the context of an HIV-1 LAI LTR-luciferase construct, resulted in a 40-50% reduction in LTR activity in transiently transfected Jurkat and U-373 MG cells. Overall, these results suggest that specific G/C box sequence alterations present in the brain-derived HIV-1 variant YU-2, or possibly other brain-derived variants, may exhibit altered replication properties as a result of the low affinity of the NF-kappaB-proximal G/C box for members of the Sp transcription factor family.

Human immunodeficiency virus type 1 long terminal repeat quasispecies differ in basal transcription and nuclear factor recruitment in human glial cells and lymphocytes.

The generation of genomic diversity during the course of infection has the potential to affect all aspects of HIV-1 replication, including expression of the proviral genome. To gain a better understanding of the impact of long terminal repeat (LTR) sequence diversity on LTR-directed gene expression in cells of the central nervous system (CNS) and immune system, we amplified and cloned LTRs from proviral DNA in HIV-1-infected peripheral blood. Sequence analysis of nineteen LTRs cloned from 2 adult and 3 pediatric patients revealed an average of 33 nucleotide changes (with respect to the sequence of the LAI LTR) within the 455-bp U3 region. Transient expression analyses in cells of neuroglial and lymphocytic origin demonstrated that some of these LTRs had activities which varied significantly from the LAI LTR in U-373 MG cells (an astrocytoma cell line) as well as in Jurkat cells (a CD4-positive lymphocyte cell line). While LTRs which demonstrated the highest activities in U-373 MG cells also yielded high activities in Jurkat cells, the LTRs were generally more active in Jurkat cells when compared to the LAI LTR. Differences in LTR sequence also resulted in differences in transcription factor recruitment to cis-acting sites within the U3 region of the LTR, as demonstrated by electrophoretic mobility shift assays. In particular, naturally occurring sequence variation impacted transcription factor binding to an activating transcription factor/cAMP response element binding (ATF/CREB) binding site (located between the LEF-1 and distal NF-kappaB transcription factor binding sites) that we identified in previous studies of the HIV-1 LTR. These findings suggest that LTR sequence changes can significantly affect basal LTR function and transcription factor recruitment, which may, in turn, alter the course of viral replication in cells of CNS and immune system origin.

Sp family members preferentially interact with the promoter proximal repeat within the HTLV-I enhancer.

Human T cell lymphotropic virus type I (HTLV-I) encodes the transactivator protein, Tax, which facilitates viral transcription from three 21 bp repeated elements within the U3 region of the long terminal repeat (LTR). Examination of the basal factors interacting with the 21 bp repeat elements through electrophoretic mobility shift (EMS) analyses has demonstrated the formation of DNA-protein complexes common to each of the 21 bp repeats (C1-C3) as well as three DNA-protein complexes specific to the promoter proximal (pp) repeat (U1 (U1A/U1B) and U2; 1-4). These studies have indicated that the individual repeats are not identical with respect to the cellular factors with which they interact. EMS analyses utilizing a series of mutated pp repeat elements demonstrate that the nucleotide sequence requirements for U1 (U1A/U1B) and U2 formation are separable from those required for C1-C3 formation. Competition EMS analyses utilizing Sp1 and CREB binding site oligonucleotides demonstrate that Sp family members are critical components of U1 (U1A/U1B) and U2 and that ATF/CREB family members are critical components of C1-C3. EMS supershift analyses have demonstrated that Sp1 is involved in U1A formation while Sp3 is involved in U1B and U2 formation. EMS analyses performed with nuclear extracts from Tax-expressing Jurkat cells and HTLV-I-transformed peripheral blood mononuclear cells demonstrate that Tax prevents the formation of U1 (U1A/U1B) and U2 DNA-protein complexes. Therefore, Tax appears to inhibit the interaction of Sp family members with the pp repeat. Based on these observations, it is possible that the interaction of Sp and ATF/CREB family members with the pp repeat during basal and Tax-mediated transcription may play a critical role in viral gene expression during the initial stages of virus infection or during activation of a latent infection.

AP-1 derived from mature monocytes and astrocytes preferentially interacts with the HTLV-I promoter central 21 bp repeat.

Characterization of the cellular transcription factors interacting with the human T cell lymphotropic virus type I (HTLV-I) long terminal repeat (LTR) is essential to dissecting the mechanisms involved in viral transcription that may be pertinent to the oncogenic and neuropathogenic processes associated with HTLV-I infection in both the immune and nervous systems. Electrophoretic mobility shift (EMS) analyses utilizing oligonucleotides homologous to each of the 21 bp repeat elements reacted with nuclear extracts derived from cell lines of lymphocytic, monocytic, neuronal, and glial cell origin have demonstrated differential binding of cellular factors to the three 21 bp repeats (1-4). ATF/CREB and Sp family members interacted with the 21 bp repeats to form DNA-protein complexes common to all cell types examined. However, a unique DNA-protein complex was detected when the promoter central 21 bp repeat was reacted with nuclear extracts derived from either the U-373 MG glioblastoma cell line or the THP-1 mature monocytic cell line. Based on nucleotide sequence requirements and immunoreactivity, we demonstrate that this DNA-protein complex is comprised of the AP-1 components, Fos and Jun.

Upstream stimulatory factor family binds to the herpes simplex virus type 1 latency-associated transcript promoter.

The herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) promoter 1 (LP1) is the only viral promoter that exhibits detectable transcriptional activity during a latent HSV infection. The LAT promoter-binding factor (LPBF) regulatory sequence (nucleotides -65 to -72 relative to the transcriptional start site of the 8.3-kb primary transcript) closely resembles the core recognition sequence required for binding members of the upstream stimulatory factor (USF)/major late transcription factor (MLTF) family. In this analysis, we demonstrate that oligonucleotides containing either the LPBF recognition sequence or the USF/MLTF recognition sequences from previously described promoters bind cellular factors which exhibit very similar mobilities in electrophoretic mobility shift (EMS) analyses. We also observe a high degree of similarity in competition profiles obtained in competition EMS analyses utilizing oligonucleotides containing recognition sequences for either LPBF or USF/MLTF. Furthermore, antibody supershift EMS analyses have demonstrated that the factors binding the LPBF or USF/MLTF recognition sites in these oligonucleotides are antigenically related, if not identical, and that greater than 90% of the LPBF-binding activity is antigenically related to USF. In addition, we demonstrate that both forms of in vitro translated USF proteins (43 and 44 kDa) bind to the LPBF recognition sequence within HSV-1 LP1. Taken together, these data indicate that USF is capable of binding to the HSV-1 LPBF recognition sequence and that USF is a major LPBF-binding activity in cells of neuronal and nonneuronal lineage. These data further support the hypothesis that USF may indeed play a significant role in the transcriptional activity of HSV-1 LP1.

Characterization of a glial cell-specific DNA-protein complex formed with the human T cell lymphotropic virus type I (HTLV-I) enhancer.

Human T cell lymphotropic virus type I (HTLV-I) encodes the trans-activator, Tax, which facilitates viral transcription from three 21 bp repeated elements within the U3 region of the long terminal repeat (LTR). Electrophoretic mobility shift (EMS) analyses utilizing double-stranded (ds) oligonucleotides homologous to each of the 21 bp repeats and nuclear extracts derived from selected cell lines of lymphocytic, neuronal, and glial origin have demonstrated the differential binding of cellular factors to each of the three 21 bp repeats. Specifically, both a glial cell-specific DNA-protein complex (designated GCS) and 21 bp repeat-specific DNA-protein complexes (designated U1 and U2) were detected. The formation of the GCS DNA-protein complex may involve activating transcription factor (ATF)/cAMP-response element (CRE) binding protein (CREB) family member(s) while the formation of the U1 and U2 DNA-protein complexes may involve an Sp1-related factor. In addition, three ATF-CREB-related DNA-protein complexes common to each individual 21 bp repeat (designated C1-C3) were also detected. However, we demonstrated that the abundance of the C1 and C2 DNA-protein complexes detected with the individual 21 bp repeats and glial cell nuclear extract was relatively low compared to that obtained with lymphocyte, monocyte, or neuronal nuclear extracts. We also have demonstrated that the ATF-CREB factors participating formation of the GCS DNA-protein complex are distinct from those participating in formation of the C1-C3 DNA-protein complexes. Based on nucleotide sequence requirements and immunoreactivity, we suggest that the GCS DNA-protein complex may contain a novel glial cell type specific ATF-CREB-related factor(s). Furthermore, we demonstrate that the CRE modulator (CREM) protein in conjunction with the ATF/CREB factor, CREBP1, interact with each of the three 21 bp repeats to form the C3 DNA-protein complex. However, the abundance of the C3 DNA-protein complex formed utilizing the promoter proximal repeat is dramatically lower compared to either of the other two 21 bp repeat elements. Based on these observations, we suggest that the differential binding of cellular factors to each of the three 21 bp repeat elements may play a role in basal as well as Tax-mediated LTR-directed transcription within cell populations of either immune or nervous system origin.

Identification of human T-cell lymphotropic virus type I 21-base-pair repeat-specific and glial cell-specific DNA-protein complexes.

The human T-cell lymphotropic virus type I (HTLV-I)-encoded protein, Tax, is capable of trans-activating HTLV-I transcription by interacting with specific sequences in the HTLV-I long terminal repeat (LTR) which comprise an inducible enhancer containing three imperfect tandem repeats of a 21-bp sequence. There is no evidence that purified Tax can bind to DNA in the absence of cellular factors, suggesting that Tax most likely regulates transcription via interaction with cellular factors. Since HTLV-I is a documented agent of adult T-cell leukemia and tropical spastic paraparesis, disorders of the immune and nervous systems, respectively, characterization of cellular factors of lymphoid and neuroglial origin which interact with the 21-bp repeat elements is essential to understanding of the mechanisms involved in basal and Tax-mediated transcription in cells of immune and nervous system origin. Utilizing electrophoretic mobility shift (EMS) analyses, we have detected both 21-bp repeat-specific and glial cell-specific DNA-protein complexes. Several 21-bp repeat-specific DNA-protein complexes were detected when nuclear extracts derived from cells of lymphoid (Jurkat, SupT1, and H9), neuronal (IMR-32 and SK-N-MC), and glial (U-373 MG, Hs683, and U-118) origin were used in reactions with each of the three 21-bp repeat elements. In addition, a glial cell-specific DNA-protein complex was detected when nuclear extracts derived from U-373 MG, Hs683, and U-118 glial cell lines reacted with the promoter-distal and central 21-bp repeat elements. Furthermore, EMS analyses performed with nuclear extracts derived from lymphocytic and glial cell origin and a 223-bp fragment of the HTLV-I long terminal repeat encompassing the three 21-bp repeat elements (designated Tax-responsive elements 1 and 2, TRE-1/-2) have also resulted in the detection of glial cell type-specific DNA-protein complexes. Competition EMS analyses with oligonucleotides containing transcription factor binding site sequences indicate the involvement of a cyclic AMP response element binding protein in the formation of DNA-protein complexes which form with all three 21-bp repeat elements and the glial cell-specific DNA-protein complex as well as the involvement of Sp1 or an Sp1-related factor in the formation of the 21-bp repeat III-specific DNA-protein complexes.

Identification of HTLV-I 21 bp repeat-specific DNA-protein complexes.

The human T cell lymphotropic virus type 1 (HTLV-I)-encoded protein, Tax, is capable of transcriptionally trans-activating HTLV-I by interacting with specific sequences in the HTLV-I long terminal repeat (LTR) which comprise an inducible enhancer containing three imperfect tandem repeats of a 21 bp sequence. Evidence suggests that Tax is incapable of directly interacting with DNA; therefore, Tax most likely regulates transcription via interaction with cellular factors. In addition to a role in Tax-mediated trans-activation, cellular factors are also critical elements in basal HTLV-I LTR-directed transcription. Therefore, characterization of cellular factors which interact with the 21 bp repeat elements is essential to understanding the molecular mechanisms involved in both basal and Tax-mediated transcription from the HTLV-I LTR. Utilizing electrophoretic mobility shift (EMS) analyses, we have detected 21 bp repeat-specific DNA-protein complexes when nuclear extracts derived from cells of lymphoid (Jurkat, SupT1, and H9) and monocytoid (U937) origin were reacted with each of the three 21 bp repeat elements. Furthermore, results from EMS competition analyses utilizing unlabeled 21 bp repeats as competitor DNAs have indicated a difference in the ability of each unlabeled 21 bp repeat to compete for the specific DNA-protein complexes formed between the nuclear extracts and radiolabeled 21 bp repeats. In each case, the most effective competitor was the homologous, unlabeled 21 bp repeat element. These results demonstrate that there are 21 bp repeat-specific DNA-protein complexes and suggest functional differences among the three 21 bp repeat elements.