Search

Enhancers are important non-coding elements, but they have traditionally been hard to characterize experimentally. The development of massively parallel assays allows the characterization of large numbers of enhancers for the first time. Here, we developed a framework using Drosophila STARR-seq to create shape-matching filters based on meta-profiles of epigenetic features. We integrated these features with supervised machine-learning algorithms to predict enhancers. We further demonstrated that our model could be transferred to predict enhancers in mammals. We comprehensively validated the predictions using a combination of in vivo and in vitro approaches, involving transgenic assays in mice and transduction-based reporter assays in human cell lines (153 enhancers in total). The results confirmed that our model can accurately predict enhancers in different species without re-parameterization. Finally, we examined the transcription factor binding patterns at predicted enhancers versus promoters. We demonstrated that these patterns enable the construction of a secondary model that effectively distinguishes enhancers and promoters.

Mice have multiple obstacles to HIV replication, including a block of unspliced and partially spliced viral mRNA nuclear export. In human, Rev binds to the Rev-response element and human (h) Crm1, facilitating nuclear export of RRE-containing viral RNAs. Murine (m) Crm1 is less functional than hCrm1 in this regard. Here we demonstrated that in biochemical experiments mCrm1 failed to interact with HIV Rev whereas hCrm1 did. In genetic experiments in human cells, we observed a modest but significant differential effect between mCrm1 and hCrm1, which was also true of other lentiviral Revs tested. Triple mutant hCrm1 P411T-M412V-F414S behaved similarly to mCrm1, whereas mCrm1 with T411P-V412M-S414F regained some activity, although contribution of additional residues to its function can not be excluded. Similar results were observed in murine cells. This suggests a differential interaction between hCrm1 and mCrm1 and many lentiviral Revs, which may partially explain the HIV replicative defect in mice.

HIV +Elite and Viremic controllers (EC/VCs) are able to control virus infection, perhaps because of host genetic determinants. We identified 16% (21 of 131) EC/VCs with CD4 +T cells with resistance specific to R5-tropic HIV, reversed after introduction of ccr5. R5 resistance was not observed in macrophages and depended upon the method of T cell activation. CD4 +T cells of these EC/VCs had lower ccr2 and ccr5 RNA levels, reduced CCR2 and CCR5 cell-surface expression, and decreased levels of secreted chemokines. T cells had no changes in chemokine receptor mRNA half-life but instead had lower levels of active transcription of ccr2 and ccr5, despite having more accessible chromatin by ATAC-seq. Other nearby genes were also down-regulated, over a region of ~500 kb on chromosome 3p21. This same R5 resistance phenotype was observed in family members of an index VC, also associated with ccr2/ccr5 down-regulation, suggesting that the phenotype is heritable.

Attachment inhibitor (AI) BMS-626529 (fostemsavir) represents a novel class of antiretrovirals which target human immunodeficiency virus type 1 (HIV-1) gp120 and block CD4-induced conformational changes required for viral entry. It is now in phase III clinical trials and is expected to be approved by the U.S. Food and Drug Administration (FDA) in the near future. Although fostemsavir is very potent against HIV in vitro and in vivo, a number of resistant mutants have already been identified. Broadly neutralizing HIV antibodies (bNAbs) can potently inhibit a wide range of HIV-1 strains by binding to viral Env and are very promising candidates for HIV-1 prevention and therapy. Since both target viral Env to block viral entry, we decided to investigate the relationship between these two inhibitors. Our data show that Env mutants resistant to BMS-626529 retained susceptibility to bNAbs. A single treatment of bNAb NIH45-46(G54W) completely inhibited the replication of these escape mutants. Remarkable synergy was observed between BMS-626529 and CD4 binding site (CD4bs)-targeting bNAbs in neutralizing HIV-1 strains at low concentrations. This synergistic effect was enhanced against virus harboring mutations conferring resistance to BMS-626529. The mechanistic basis of the observed synergy is likely enhanced inhibition of CD4 binding to the HIV-1 Env trimer by the combination of BMS-626529 and CD4bs-targeting bNAbs. This work highlights the potential for positive interplay between small- and large-molecule therapeutics against HIV entry, which may prove useful as these agents enter clinical use. IMPORTANCE As the worldwide HIV pandemic continues, there is a continued need for novel drugs and therapies. A new class of drug, the attachment inhibitors, will soon be approved for the treatment of HIV. Broadly neutralizing antibodies are also promising candidates for HIV prevention and therapy. We investigated how this drug might work with these exciting antibodies that are very potent in blocking HIV infection of cells. These antibodies worked against virus known to be resistant to the new drug. In addition, a specific type of antibody worked really well with the new drug in blocking virus infection of cells. This work has implications for both the new drug and the antibodies that are poised to be used against HIV.

There is strong evidence that overproduction of soluble fms-like tyrosine kinase-1 (sFLT1) in the placenta is a major cause of vascular dysfunction in preeclampsia through sFLT1-dependent antagonism of VEGF. However, the cause of placental sFLT1 upregulation is not known. Here we demonstrated that in women with preeclampsia, sFLT1 is upregulated in placental trophoblasts, while VEGF is upregulated in adjacent maternal decidual cells. In response to VEGF, expression of sFlt1 mRNA, but not full-length Flt1 mRNA, increased in cultured murine trophoblast stem cells. We developed a method for transgene expression specifically in mouse endometrium and found that endometrial-specific VEGF overexpression induced placental sFLT1 production and elevated sFLT1 levels in maternal serum. This led to pregnancy losses, placental vascular defects, and preeclampsia-like symptoms, including hypertension, proteinuria, and glomerular endotheliosis in the mother. Knockdown of placental sFlt1 with a trophoblast-specific transgene caused placental vascular changes that were consistent with excess VEGF activity. Moreover, sFlt1 knockdown in VEGF-overexpressing animals enhanced symptoms produced by VEGF overexpression alone. These findings indicate that sFLT1 plays an essential role in maintaining vascular integrity in the placenta by sequestering excess maternal VEGF and suggest that a local increase in VEGF can trigger placental overexpression of sFLT1, potentially contributing to the development of preeclampsia and other pregnancy complications.

Here we report on the structure-based optimization of antibody-recruiting molecules targeting HIV gp120 (ARM-H). These studies have leveraged a combination of medicinal chemistry, biochemical and cellular assay analysis, and computation. Our findings have afforded an optimized analog of ARM-H, which is ~1000 fold more potent in gp120-binding and MT-2 antiviral assays than our previously reported derivative. Furthermore, computational analysis, taken together with experimental data, provides evidence that azaindole- and indole-based attachment inhibitors bind gp120 at an accessory hydrophobic pocket beneath the CD4-binding site and can also adopt multiple unique binding modes in interacting with gp120. These results are likely to prove highly enabling in the development of novel HIV attachment inhibitors, and more broadly, they suggest novel applications for ARMs as probes of conformationally flexible systems.

Epigenetic control of human immunodeficiency virus-1 (HIV-1) genes is critical for viral integration and latency. However, epigenetic changes in the HIV-1-infected host genome have not been well characterized. Here, we report the first large-scale epigenome-wide association study of DNA methylation for HIV-1 infection. We recruited HIV-infected (n = 261) and uninfected (n = 117) patients from the Veteran Aging Cohort Study (VACS) and all samples were profiled for 485,521 CpG sites in DNA extracted from the blood. After adjusting for cell type and clinical confounders, we identified 20 epigenome-wide significant CpGs for HIV-1 infection. Importantly, 2 CpGs in the promoter of the NLR family, CARD domain containing gene 5 (NLRC5), a key regulator of major histocompatibility complex class I gene expression, showed significantly lower methylation in HIV-infected subjects than in uninfected subjects (cg07839457: t = −6.03, Pnominal = 4.96 × 10−9; cg16411857: t = −7.63, Pnominal = 3.07 × 10−13). Hypomethylation of these 2 CpGs was replicated in an independent sample (GSE67705: cg07839457: t = −4.44, Pnominal = 1.61 × 10−5; cg16411857: t = −5.90; P = 1.99 × 10−8). Methylation of these 2 CpGs in NLRC5 was negatively correlated with viral load in the 2 HIV-infected samples (cg07839457: P = 1.8 × 10−4; cg16411857: P = 0.03 in the VACS; and cg07839457: P = 0.04; cg164111857: P = 0.01 in GSE53840). Our findings demonstrate that differential DNA methylation is associated with HIV infection and suggest the involvement of a novel host gene, NLRC5, in HIV pathogenesis.

Productive replication of human immunodeficiency virus type 1 (HIV-1) occurs efficiently only in humans. The posttranscriptional stages of the HIV-1 life cycle proceed poorly in mouse cells, with a resulting defect in viral assembly and release. Previous work has shown that the presence of human chromosome 2 increases HIV-1 production in mouse cells. Recent studies have shown that human chromosome region maintenance 1 (hCRM1) stimulates Gag release from rodent cells. Here we report that expressions of hCRM1 in murine cells resulted in marked increases in the production of infectious HIV-1 and feline immunodeficiency virus (FIV). HIV-1 production was also increased by hSRp40, and a combination of hCRM1 and hSRp40 resulted in a more-than-additive effect on HIV-1 release. In contrast, the overexpression of mouse CRM1 (mCRM1) minimally affected HIV-1 and FIV production and did not antagonize hCRM1. In the presence of hCRM1 there were large increases in the amounts of released capsid, which paralleled the increases in the infectious titers. Consistent with this finding, the ratios of unspliced to spliced HIV-1 mRNAs in mouse cells expressing hCRM1 and SRp40 became similar to those of human cells. Furthermore, imaging of intron-containing FIV RNA showed that hCRM1 increased RNA export to the cytoplasm.By testing chimeras between mCRM1 and hCRM1 and comparing those sequences to feline CRM1, we mapped the functional domain to HEAT (Huntingtin, elongation factor 3, protein phosphatase 2A, and the yeast kinase TOR1) repeats 4A to 9A and a triple point mutant in repeat 9A, which showed a loss of function. Structural analysis suggested that this region of hCRM1 may serve as a binding site for viral or cellular factors to facilitate lentiviral RNA nuclear export.

Notch signaling is a key mechanism in the control of embryogenesis. However, its in vivo function during mesenchymal cell differentiation, and, specifically, in bone homeostasis, remains largely unknown. Here, we show that osteoblast-specific gain of Notch function causes severe osteosclerosis owing to increased proliferation of immature osteoblasts. Under these pathological conditions, Notch stimulates early osteoblastic proliferation by upregulating the genes encoding cyclin D, cyclin E and Sp7 (osterix). The intracellular domain of Notch1 also regulates terminal osteoblastic differentiation by directly binding Runx2 and repressing its transactivation function. In contrast, loss of all Notch signaling in osteoblasts, generated by deletion of the genes encoding presenilin-1 and presenilin-2 in bone, is associated with late-onset, age-related osteoporosis, which in turn results from increased osteoblast-dependent osteoclastic activity due to decreased osteoprotegerin mRNA expression in these cells. Together, these findings highlight the potential dimorphic effects of Notch signaling in bone homeostasis and may provide direction for novel therapeutic applications.

Gene transfer vectors encoding short hairpin RNAs (shRNAs) are useful in deciphering gene function, and are being considered for therapeutic knockdown of target genes in humans. We constructed HIV-based vectors encoding shRNA against HIV coreceptor chemokine (C-C motif) receptor 5 (CCR5). Initially we noted that vectors encoding CCR5 shRNA showed >30-fold lower viral titers than those of the empty vector. Co-transfection of expression plasmids encoding CCR5 in the producer cells yielded a tenfold increase in viral titer, thereby indicating that CCR5 mRNA, rather than HIV vector mRNA, could be the target of CCR5 shRNA. Similar increases in vector titer were observed after the H1 promoter was deleted. When Nodamura-virus B2 protein or Adenovirus VA.1 RNA (inhibitors of the Dicer-dependent siRNA pathway) were added to the producer cells, the vector titer rose almost to the level of that of the empty vector. Near identical increases in titer were observed with siRNA specifically directed against Dicer. Quantitative reverse transcriptase–polymerase chain reaction (RT-PCR) suggested that the effects were in part caused by reduction in vector RNA in the producer cells. Similar results were observed with a retroviral vector. These results suggest that retrovirally-encoded shRNAs reduce vector titer in the producer cells through a Dicer-dependent mechanism which, to a large extent, can be reversed by inhibiting that pathway. This may have important implications for large-scale production of RNA vectors encoding shRNAs.

The majority of current anti-HIV drugs target the viral reverse transcriptase or protease enzymes. However, enfuvirtide and maraviroc are drugs that have been US FDA approved recently and which function by inhibiting virus cell binding and entry which normally occurs through the interaction of the viral envelope protein with its cellular coreceptor. As HIV-1 utilizes many cellular cofactors during its replication cycle, there are a number of other protein–protein interactions that can serve as targets for anti-HIV drug development. In this review article we discuss the general method used to identify anti-HIV drugs that function through targeting protein–protein interactions. We also discuss the currently known cellular cofactors that may serve as targets in future drugs screens.

Mouse cells are non-permissive to human immunodeficiency virus type 1 (HIV) in that there is a pronounced post-integration block to viral replication. We have recently demonstrated that mouse–human somatic cell hybrids that contain human chromosome 2 increase both HIV Capsid (CA) production and infectious virus release. Here we report on the isolation of three mouse–human microcell hybrids (MCHs) that behave similarly, starting from a pool of 500 MCH clones. Release of virus was specific to HIV and cell revertants that no longer contained any human chromosome fragments did not release CA or infectious virus. Two of the three cell clones were identical as judged by PCR STS content and fluorescence in situ hybridization (FISH) and contained a single 2–12 human chromosome chimera. The third cell clone only contained human chromosome 12, as determined by PCR, FISH, and microarray analyses. There were no consistent differences in Gag protein and spliced/unspliced viral RNA levels between mouse cell lines. CMV promoter-driven, codon-optimized gag-pol had no effect on infectious HIV release from these mouse cells, despite allowing Gag targeting and increasing CA production. These permissive mouse–human MCHs and their corresponding non-permissive revertants may prove useful for mechanistic studies and also for identifying the responsible gene(s) or factor(s) involved in the production of HIV.

Despite nearly three decades of research, a safe and effective vaccine against human immunodeficiency virus type 1 (HIV-1) has yet to be achieved. More recently, the discovery of highly potent anti-gp160 broadly neutralizing antibodies (bNAbs) has garnered renewed interest in using antibody-based prophylactic and therapeutic approaches. Here, we encoded bNAbs in first-generation adenoviral (ADV) vectors, which have the distinctive features of a large coding capacity and ease of propagation. A single intramuscular injection of ADV-vectorized bNAbs in humanized mice generated high serum levels of bNAbs that provided protection against multiple repeated challenges with a high dose of HIV-1, prevented depletion of peripheral CD4(+) T cells, and reduced plasma viral loads to below detection limits. Our results suggest that ADV vectors may be a viable option for the prophylactic and perhaps therapeutic use of bNAbs in humans.

Here we describe as a potential vaccine candidate a replication-defective HIV that encodes multiple viral genes in addition to a cassette that includes both truncated cyclin T1 and an autofluorescent protein. After confirming functionality of the cyclin T1, we immunized mice intramuscularly once or twice with the replication-defective HIV vector pseudotyped with vesicular stomatitis virus (VSV) G protein (RD HIV), a plasmid encoding CMV-driven gag (gag DNA), or adenovirus gag (Ad5-gag). Capsid-specific antibody titers following RD HIV immunization were10(6)/ml and approximately equivalent to those induced by gag DNA and Ad5-gag. Antibodies against the autofluorescent protein and VSV G were also detected. After RD HIV immunization ELISpot assays demonstrated Gag-specific interferon-gamma (IFN-gamma) SFU equivalent to that of Ad5-gag and fourfold greater than that of gag DNA. HIV polymerase-specific IFN-gamma SFU values were similar, and boosting increased both antibody titers and the IFN-gamma response. Challenge using vaccinia virus (VV)-gag demonstrated significantly lower recoverable VV for RD HIV-immunized mice compared to controls. No significant differences were observed in vaccinated mice challenged with wild-type VV. This study demonstrates the efficacy of RD HIV in conferring HIV-specific immunity and protection in mice and suggests its potential use in humans as either a prophylactic or a therapeutic vaccine.

Human immunodeficiency virus type 1 (HIV) replicates only in certain primate cells. In murine cells expressing cyclin T1, a posttranscriptional block exists such that small amounts of capsid and little infectious virus are released. This block is relieved in part by fusion with human cells. Here we have tested a panel of mouse-human somatic cell hybrids for production of infectious virus. Only those containing human chromosome 2 were permissive, which correlated with capsid production. The effect was specific to HIV in that release of murine leukemia virus was minimally affected by the presence of chromosome 2. Although expression of Vpu markedly increased capsid production in the absence of chromosome 2, it did not result in a corresponding increase in infectious HIV. The presence of chromosome 2 did not have consistent effects on the amount of unspliced viral RNA, whereas the amount of cell-associated Gag p55 was increased a fewfold. These results suggest that processing of HIV Gag can be corrected by one or more genes present on human chromosome 2 to allow production of infectious HIV from murine cells.

The p53-induced serine/threonine phosphatase, protein phosphatase 1D magnesium-dependent, delta isoform (PPM1D) (or wild-type p53-induced phosphatase 1 (Wip1)), exhibits oncogenic activity in vitro and in vivo. It behaves as an oncogene in rodent fibroblast transformation assays and is amplified and overexpressed in several human tumor types. It may contribute to oncogenesis through functional inactivation of p53. Here, we show that the oncogenic function of PPM1D is associated with its phosphatase activity. While overexpressed PPM1D may be oncogenic, PPM1D null mice are resistant to spontaneous tumors over their entire lifespan. This cancer resistance may be based in part on an augmented stress response following DNA damage. PPM1D null mice treated with ionizing radiation display increased p53 protein levels and increased phosphorylation of p38 MAP kinase, p53, checkpoint kinase 1 (Chk1), and checkpoint kinase 2 (Chk2) in their tissues compared to their wild-type (WT) counterparts. Male PPM1D null mice show a modest reduction in longevity, reduced serum insulin-like growth factor 1 (IGF-1) levels, and reduced body weight compared to WT mice. The PPM1D null mouse phenotypes indicate that PPM1D has a homeostatic role in abrogating the DNA damage response and may regulate aspects of male longevity.

The 5' ends of trypanosome mRNAs consist of an identical sequence of 35 nucleotides. This "mini-exon" sequence is derived from the 5' end of a 137 nucleotide RNA (medRNA). The remainder of each mRNA is derived from a protein-coding exon that is not linked to the mini-exon. We propose that medRNA is spliced in trans to de-novo-initiated transcripts of protein-coding genes. This trans splicing model predicts that the downstream portion of medRNA will be part of a branched structure and then be released as a free product (minRNA). We demonstrate that significant levels of minRNA exist in trypanosome RNA. Furthermore, minRNA can be released from high molecular weight RNA by a HeLa cell S100 "debranching" extract. We conclude that trans splicing is the physiological process by which mature mRNA molecules are synthesized in trypanosomes.

Replication-competent lentivirus (RCL) may be generated during the production phase or subsequently after introduction of a lentiviral vector into target cells, potentially by homologous or nonhomologous recombination. Because most gene transfer of HIV-based vectors involves the use of high-titer vesicular stomatitis virus (VSV) G-pseudotyped particles, one particular concern would be the generation of an RCL of altered host range, i.e., one that has incorporated the VSV G envelope in cis configuration. We report here on the artificial generation and properties of such a virus, including its detection after biological amplification. Viral spread, beginning with a very low inoculum, takes several weeks in culture and is characterized by "autoinfection," resulting in multiple proviral copies per cell, higher levels of viral gene expression, and eventual cell death. After this initial amplification step, the RCL is easily detectable by standard p24 assay or by "marker-rescue" assay. For the latter, a 293T-based cell line that has an integrated replication-defective provirus encoding alkaline phosphatase (AP) was used and mobilization of AP-containing virus was detected by transduction of naïve cells. Replication-defective virus was not amplified nor detected, demonstrating assay specificity. These results suggest that these artificial RCLs of broad host range have slightly different biological properties compared to wild-type HIV but still spread and are readily detectable.

Human immunodeficiency virus type 1 (HIV-1) utilizes CD4 as a primary receptor for viral entry and any of several 7-transmembrane chemokine receptors, including CCR5, as a co-receptor. Previous studies have demonstrated that multiple extracellular domains (ECDs) of CCR5 contribute to co-receptor function; here we applied genetic footprinting to CCR5 to confirm and extend those investigations. In genetic footprinting, a duplex oligonucleotide is inserted into the DNA sequence of interest by use of either a bacterial transposase or retroviral integrase. Here, CCR5 mutants were analyzed in bulk for their ability to be expressed on the recipient cell surface and to mediate viral entry of R5 HIV isolates. Most of the approximately 150 CCR5 mutants were not expressed on the cell surface. Of those remaining, 8 were specifically reduced or absent after macrophage (M)-tropic HIV infection, confirming a critical role of ECDs three (extracellular loop 2 or ECL2) and possibly four (ECL3) in viral entry. Mutational and functional analyses of ECD4 (ECL3) suggest it is under severe topological constraint for CCR5 surface expression and are consistent with it contributing to co-receptor function.

Elite controllers (ECs) are a rare group of HIV seropositive individuals who are able to control viral replication without antiretroviral therapy. The mechanisms responsible for this phenotype, however, have not been fully elucidated. In this study, we examined CD4 + T cell resistance to HIV in a cohort of elite controllers and explored transcriptional signatures associated with cellular resistance. We demonstrate that a subgroup of elite controllers possess CD4 + T cells that are specifically resistant to R5-tropic HIV while remaining fully susceptible to X4-tropic and vesicular stomatitis virus G (VSV-G)-pseudotyped viruses. Transcriptome analysis revealed 17 genes that were differentially regulated in resistant elite controllers relative to healthy controls. Notably, the genes encoding macrophage inflammatory protein 1α (MIP-1α), CCL3 and CCL3L1 , were found to be upregulated. The MIP-1α, MIP-1β, and RANTES chemokines are natural ligands of CCR5 and are known to interfere with HIV replication. For three elite controllers, we observed increased production of MIP-1α and/or MIP-1β at the protein level. The supernatant from resistant EC cells contained MIP-1α and MIP-1β and was sufficient to confer R5-tropic resistance to susceptible CD4 + T cells. Additionally, this effect was reversed by using inhibitory anti-MIP antibodies. These results suggest that the T cells of these particular elite controllers may be naturally resistant to HIV infection by blocking R5-tropic viral entry. IMPORTANCE HIV is a pandemic health problem, and the majority of seropositive individuals will eventually progress to AIDS unless antiretroviral therapy (ART) is administered. However, rare patients, termed elite controllers, have a natural ability to control HIV infection in the absence of ART, but the mechanisms by which they achieve this phenotype have not been fully explored. This paper identifies one mechanism that may contribute to this natural resistance: some elite controllers have CD4 + T cells that produce high levels of MIP chemokines, which block R5-tropic HIV entry. This mechanism could potentially be exploited to achieve a therapeutic effect in other HIV-seropositive individuals.

Because of their ability to transduce nondividing cells, human immunodeficiency virus type 1 (HIV)-based vectors have great potential for the therapeutic delivery of genes to cells. We describe here a systematic study of the packaging limit of HIV-based vectors. Restriction endonuclease-generated bacterial chromosomal DNA fragments of different lengths were cloned at three different positions within a lentiviral vector. Vesicular stomatitis virus G protein (VSV G) pseudotyped lentiviral particles were prepared and the different clones were titered on mammalian cells. We observed that the restriction endonuclease site positions at the 5' and 3' ends of the genome were superior with regard to insertional capacity of foreign DNA. In all cases, viral titers decreased semi-logarithmically with increasing vector length. There appears to be no absolute packaging limit because measurable titers were obtained even when the proviral length was in excess of 18 kb. The reduction in titer appears to occur at the level of viral encapsidation, although we cannot exclude limitations in nuclear export of proviral RNA. These results suggest that HIV-based vectors may have a secondary advantage over oncoretroviral vectors because of their greater packaging limit, although the very low titers of the larger vectors will be of limited utility.

The scaffold attachment region of the human interferon beta gene (IFN-SAR) inserted into a retroviral vector improved transgene expression in human primary CD4+ and CD8+ T cells, and in primary monocytemacrophages. In T cells, expression of the Maloney murine leukemia virus (Mo-MuLV)-based retroviral vectors was high in activated cells but low in resting cells. Addition of the IFN-SAR sequence enhanced vector expression 2- to 10-fold, and the effect was particularly pronounced in resting T cells. In CD33+CD14+CD4+ monocyte-macrophages derived from transduced hematopoietic stem/progenitor cells (HSPCs) in vitro, the IFN-SAR enhanced vector expression three- to sixfold. We have used the IFN-SAR-containing vectors to express the RevM10 gene, a trans-dominant mutant of the human immunodeficiency virus type 1 (HIV-1) rev gene. Compared with a standard retroviral vector, the IFN-SAR-containing vector was significantly (p0.01) more potent at inhibiting HIV-1 replication in infected CD4+ peripheral blood lymphocytes. In monocytes, however, addition of the IFN-SAR did not significantly improve antiviral efficacy. To understand better the reason for the strong effect of the SAR on antiviral efficacy in T cells we have studied the expression of HIV, Mo-MuLV, and Mo-MuLV + SAR vectors in resting and activated cells. While the expression of all three vectors was lower in resting compared with activated cells, the kinetics of the decrease in expression were fastest for the Mo-MuLV vector, followed by the HIV vector and then the Mo-MuLV + SAR vector. Thus, higher level expression of the Mo-MuLV + SAR vector relative to wild-type HIV at all stages of T cell activation is the most likely explanation for the strong antiviral efficacy. Overall, this study demonstrates the utility of the IFN-SAR sequence for achieving high-level retroviral vector expression in lymphoid and myeloid hematopoietic cells.

Human immunodeficiency virus (HIV) type 1 vectors are highly efficient in their ability to transduce human progenitor hematopoietic stem cells (PHSC). Although mitosis was not required for transduction of these cells, transduction rates were much greater once cells had been cultured in the presence of cytokines. Transduction rates, however, rarely exceeded 70%. We demonstrate here that there is a distinct subpopulation that is more easily transduced by HIV vectors. These cells were distinguished by a disproportionate population in the S/G 2 /M phases of the cell cycle. By sorting them prior to transduction, we found that those cells in either the G 1 or S/G 2 /M fraction were more readily transduced than G 0 cells. Maintaining the cells in G 0 by omitting cytokines from the medium reduced transduction rates by up to 10-fold. Addition of cytokines to the medium immediately after transduction did not improve the transduction efficiency as measured by expression of the transgene. Analysis of replication intermediates indicated that the block to transduction of G 0 cells operated near the time of initiation of reverse transcription. These results suggest that although lentivirus vectors can transduce nondividing PHSC, transduction efficiency is severalfold greater once the cells exit G 0 and enter G 1 . Further characterization of these more transducible cells and identification of the cellular factors responsible may enhance transduction while maintaining the pluripotentiality of the PHSC.

Babesia microti is the primary causative agent of human babesiosis, an emerging pathogen that causes a malaria-like illness with possible fatal outcome in immunocompromised patients. The genome sequence of the B. microti R1 strain was reported in 2012 and revealed a distinct evolutionary path for this pathogen relative to that of other apicomplexa. Lacking from the first genome assembly and initial molecular analyses was information about the terminal ends of each chromosome, and both the exact number of chromosomes in the nuclear genome and the organization of the mitochondrial genome remained ambiguous. We have now performed various molecular analyses to characterize the nuclear and mitochondrial genomes of the B. microti R1 and Gray strains and generated high-resolution Whole Genome maps. These analyses show that the genome of B. microti consists of four nuclear chromosomes and a linear mitochondrial genome present in four different structural types. Furthermore, Whole Genome mapping allowed resolution of the chromosomal ends, identification of areas of misassembly in the R1 genome, and genomic differences between the R1 and Gray strains, which occur primarily in the telomeric regions. These studies set the stage for a better understanding of the evolution and diversity of this important human pathogen.

Induction of type I interferon is a central event of innate immunity, essential for host defense. Here we report that the transcription factor ELF4 is induced by type I interferon and upregulates interferon expression in a feed-forward loop. ELF4 deficiency leads to reduced interferon production, resulting in enhanced susceptibility to West Nile virus encephalitis in mice. After viral infection, ELF4 is recruited by STING, interacts with and is activated by the MAVS-TBK1 complex, and translocates into the nucleus to bind interferon promoters. Cooperative binding with ELF4 increases the binding affinity of interferon regulatory factors IRF3 and IRF7, which is mediated by EICE elements. Thus, in addition to identifying a regulator of innate immune signaling, we uncovered a role for EICE elements in interferon transactivation.

SummaryRNA viruses are sensed by RIG-I-like receptors (RLRs), which signal through a mitochondria-associated adaptor molecule, MAVS, resulting in systemic antiviral immune responses. Although RLR signaling is essential for limiting RNA virus replication, it must be stringently controlled to prevent damage from inflammation. We demonstrate here that among all tested UBX-domain-containing protein family members, UBXN1 exhibits the strongest inhibitory effect on RNA-virus-induced type I interferon response. UBXN1 potently inhibits RLR- and MAVS-induced, but not TLR3-, TLR4-, or DNA-virus-induced innate immune responses. Depletion of UBXN1 enhances virus-induced innate immune responses, including those resulting from RNA viruses such as vesicular stomatitis, Sendai, West Nile, and dengue virus infection, repressing viral replication. Following viral infection, UBXN1 is induced, binds to MAVS, interferes with intracellular MAVS oligomerization, and disrupts the MAVS/TRAF3/TRAF6 signalosome. These findings underscore a critical role of UBXN1 in the modulation of a major antiviral signaling pathway.

UBXN proteins likely participate in the global regulation of protein turnover, and we have shown that UBXN1 interferes with RIG-I-like receptor (RLR) signaling by interacting with MAVS and impeding its downstream effector functions. Here we demonstrate that over-expression of multiple UBXN family members decreased lentivirus and retrovirus production by several orders-of-magnitude in single cycle assays, at the level of long terminal repeat-driven transcription, and three family members, UBXN1, N9, and N11 blocked the canonical NFκB pathway by binding to Cullin1 (Cul1), inhibiting IκBα degradation. Multiple regions of UBXN1, including its UBA domain, were critical for its activity. Elimination of UBXN1 resulted in early murine embryonic lethality. shRNA-mediated knockdown of UBXN1 enhanced human immunodeficiency virus type 1 (HIV) production up to 10-fold in single cycle assays. In primary human fibroblasts, knockdown of UBXN1 caused prolonged degradation of IκBα and enhanced NFκB signaling, which was also observed after CRISPR-mediated knockout of UBXN1 in mouse embryo fibroblasts. Knockout of UBXN1 significantly up- and down-regulated hundreds of genes, notably those of several cell adhesion and immune signaling pathways. Reduction in UBXN1 gene expression in Jurkat T cells latently infected with HIV resulted in enhanced HIV gene expression, consistent with the role of UBXN1 in modulating the NFκB pathway. Based upon co-immunoprecipitation studies with host factors known to bind Cul1, models are presented as to how UBXN1 could be inhibiting Cul1 activity. The ability of UBXN1 and other family members to negatively regulate the NFκB pathway may be important for dampening the host immune response in disease processes and also re-activating quiescent HIV from latent viral reservoirs in chronically infected individuals., Author summary A family of human genes termed UBXN is thought to control many cellular processes, including protein destruction. While working with these proteins, we noticed several profoundly blocked the production of human immunodeficiency virus (HIV) and other, similar viruses. We delved into the how this occurs, and it appears that at least three of the proteins affect a central pathway in man’s immunologic response to viral and other pathogens, termed NFκB, by a mechanism not previously described. When UBXN1 protein levels were reduced, HIV synthesis was enhanced in certain cells. We also tested what happens when UBXN1 is removed or blocked in human and mouse cells, and saw consistent effects on NFκB. Removing UBXN1 from mouse cells changed the expression hundreds of genes (about 5% of all mouse genes), notably those involved in cell stickiness and also the immune response. Blocking UBXN1 in T cells harboring a silent HIV caused HIV to be resynthesized. We believe that the UBXN gene family members that negatively impact NFκB may be important for dampening immune responses and also re-animating silent HIV, important for curing or eliminating HIV in man.

We have exploited the ability of transmembrane domains to engage in highly specific protein-protein interactions to construct a new class of small proteins that inhibit HIV infection. By screening a library encoding hundreds of thousands of artificial transmembrane proteins with randomized transmembrane domains (termed “traptamers,” for transmembrane aptamers), we isolated six 44- or 45-amino-acid proteins with completely different transmembrane sequences that inhibited cell surface and total expression of the HIV coreceptor CCR5. The traptamers inhibited transduction of human T cells by HIV reporter viruses pseudotyped with R5-tropic gp120 envelope proteins but had minimal effects on reporter viruses with X4-tropic gp120. Optimization of two traptamers significantly increased their activity and resulted in greater than 95% inhibition of R5-tropic reporter virus transduction without inhibiting expression of CD4, the primary HIV receptor, or CXCR4, another HIV coreceptor. In addition, traptamers inhibited transduction mediated by a mutant R5-tropic gp120 protein resistant to maraviroc, a small-molecule CCR5 inhibitor, and they dramatically inhibited replication of an R5-tropic laboratory strain of HIV in a multicycle infection assay. Genetic experiments suggested that the active traptamers specifically interacted with the transmembrane domains of CCR5 and that some of the traptamers interacted with different portions of CCR5. Thus, we have constructed multiple proteins not found in nature that interfere with CCR5 expression and inhibit HIV infection. These proteins may be valuable tools to probe the organization of the transmembrane domains of CCR5 and their relationship to its biological activities, and they may serve as starting points to develop new strategies to inhibit HIV infection.

While hemopoietic stem cells have been thought to reside predominantly in the CD34(+) population, recent data suggests that repopulating cells may, in fact, also reside in the lin(-)/CD34(-) population. Transduction of both these populations by murine retroviral vectors is limited by quiescence of hemopoietic stem cells.We therefore sought to transduce these populations using a VSV-G pseudotyped, HIV-based, human lentiviral vector, encoding eGFP. CD34(+) cells and lin(-)/CD34(-) cells were selected from the same BM samples by immunomagnetic beads (StemSep) to deplete lineage-positive cells and by CD34 selection columns (Miltenyi) to separate CD34(+) and CD34(-) populations. We transduced target cells, with or without prestimulation with cytokines, using conventional suspension culture, or fibronectin plates, or flow-through transduction. Transduction efficiency was analyzed by flow cytometry and clonogenic assay.We found that transduction on fibronectin plates was more efficient than flow-through transduction, or suspension cultures, for both cell populations. Mean transduction rates on fibronectin plates, analyzed by flow cytometry, were 14% and 5% for CD34(+) cells and lin(-)/CD34(-) cells respectively, without prestimulation, and 31% and 20% with prestimulation. By contrast, a murine retroviral vector transduces CD34(+) cells with lower efficiency (mean 16.1% with prestimulation) and does not induce any significant transduction of the CD34(-)/lin(-) population (mean2%). When lentiviral transduction was assayed in short- and long-term clonogenic assays there was minimal transduction of CD34 cells without prestimulation, increasing to 20% with prestimulation.Lentiviral eGFP vectors can transduce hematopoietic progenitors effectively and efficiency is improved by cytokine prestimulation and the use of fibronectin. Moreover, the human viral vectors can transduce a candidate stem-cell population that is resistant to murine retroviral transduction.

We investigated the ability of western equine encephalitis virus envelope glycoproteins (WEEV GP) to pseudotype lentiviral vectors. The titers of WEEV GP-pseudotyped human immunodeficiency virus type 1 (HIV) ranged as high as 8.0 × 10 4 IU/ml on permissive cells. Sera from WEEV-infected mice specifically neutralized these pseudotypes; cell transduction was also sensitive to changes in pH. The host range of the pseudotyped particles in vitro was somewhat limited, which is atypical for most alphaviruses. HIV vectors pseudotyped by WEEV GP may be a useful tool for characterizing WEEV cell binding and entry and screening for small-molecule inhibitors.

Previous studies from this laboratory evaluated the role of p68 kinase (PKR) in the control of HIV-1 replication via retrovirus-mediated gene transfer. PKR was studied because it is a key component of the interferon (IFN)-associated innate antiviral defense pathway in mammalian cells. In this study, CD34(+) hematopoietic stem cells (HSC) were transduced with an HIV-1-based lentiviral vector encoding the PKR transgene (pHIV-PIB) and cultured under conditions that support in vitro differentiation. With high-titer pseudotyped vector stocks, the histogram suggests 100% transduction of the HSC because the cells were blasticidin resistant. Analysis of transduced cells by hybridization revealed an average proviral vector copy number of 1.8 and 2.1 copies of vector sequence per cell. Increased PKR expression and activity (phosphorylation of eukaryotic initiation factor 2alpha [eIF2alpha]) were demonstrated in PKR-transduced, differentiated HSC. There was minimal reduction in cell viability and no induction of apoptosis after transduction of PKR. HSC transduced with the pHIV-PIB lentiviral vector demonstrated normal differentiation into CD34-derived T cell progeny. Two days after HIV-1 infection, lentivirus-mediated transduction of PKR inhibited HIV-1 replication by 72% in T cell progeny compared with cells transduced with the empty vector control (pHIV-IB). By days 5 and 7 post-HIV-1 infection, the surviving PKR-transduced cells were protected from HIV-1 infection, as evidenced by a decrease in p24 antigen expression of at least two orders of magnitude. Our results demonstrate that PKR can be effectively delivered to HSC by a lentiviral vector and can protect CD34-derived T cell progeny from HIV-1 infection. These results provide support for application of the innate antiviral defense pathway in a gene therapy setting to the treatment of HIV-1 infection.

Human T-cell leukemia virus type 1 (HTLV-1) was the first human retrovirus identified and causes both adult T-cell leukemia/lymphoma and tropical spastic paraparesis/HTLV-1-associated myelopathy, among other disorders. In vitro, HTLV-1 has an extremely broad host cell tropism in that it is capable of infecting most mammalian cell types, although at the same time viral titers remain relatively low. Despite years of study, only recently has a bona fide candidate cellular receptor, glucose transporter 1 (glut-1), been identified. Although glut-1 was shown to bind specifically to the ectodomain of HTLV-1 and HTLV-2 envelope glycoproteins, which was reversible with small interfering RNA directed against glut-1, cellular susceptibility to HTLV upon expression of glut-1 was not established. Here we show that expression of glut-1 in relatively resistant MDBK cells conferred increased susceptibility to both HTLV-1- and HTLV-2-pseudotyped particles. glut-1 also markedly increased syncytium formation in MDBK cells after exposure to HTLV-1. Another assay also demonstrated HTLV-1 envelope-cell fusion in the presence of glut-1. Taken together, these results provide additional evidence that glut-1 is a receptor for HTLV.

Transcriptional targeting is a desirable property for many gene transfer applications. Because endothelial cells line most blood vessels, they are attractive candidates for the introduction of therapeutic gene products. As a proof-of-concept study, we attempted to identify a synthetic, endothelial cell-specific promoter by use of a high-throughput screen involving self-inactivating (SIN) human immunodeficiency virus type 1 (HIV-1)-based vectors. Select duplex oligodeoxynucleotides recognized by transcription factors and located 5′ of endothelial cell-specific mRNA transcripts were randomly ligated and cloned upstream of a minimal ICAM-2 promoter driving enhanced green fluorescent protein (eGFP) in a SIN HIV-1-based vector. Vesicular stomatitis virus G protein-pseudotyped particles were prepared from a library of >10 6 vector recombinants and used to transduce an endothelial cell line. The highest eGFP expressers were repeatedly sorted, and the synthetic promoters were recovered and retested by a luciferase reporter. Several promoters were active and specific to endothelial cells of varied species, with high selectivity indexes and inducibility under hypoxia-mimetic conditions. One in particular was then introduced back into a SIN HIV-1-based vector to confirm its endothelial cell activity and specificity. This study suggests that SIN vectors may be used in a high-throughput manner to identify tissue-specific promoters of high activity, with potential applications for both transcriptional targeting and gene transfer.

The development of a predictive, small animal model for human immunodeficiency virus type 1 (HIV-1) disease would greatly facilitate the analysis of many aspects of viral infection, pathogenesis and treatment. While numerous small animal models exist which emulate various aspects of HIV-1 infection and/or disease in humans, none of these models support robust HIV-1 replication within the context of an intact immune system. Despite this major limitation, these models have helped to elucidate different aspects of HIV-1 pathogenesis in humans. Moreover, recent advances regarding the underlying nature of the blocks to viral replication in non-human cells have raised the possibility that rodents may be engineered to support HIV-1 infection. This review will focus on recent attempts to develop a rodent model for HIV-1 disease, and will also describe currently available systems for studying HIV-1.

ABSTARCT: Lentiviral vectors based upon human immunodeficiency type I (HIV) are increasingly being used to transduce nondividing or terminally differentiated cells, despite the fact HIV is a known, lethal pathogen. This is because lentiviruses contain multiple gene products that allow infection of cells independent of mitosis. Typically, plasmid DNA is transiently transfected into a producer-cell line, and viral supernatant is harvested a few days later. The supernatant can be used as is to transduce targets of choice, or it can be concen trated up to 1000-fold by ultracentrifugation and then used on difficult targets, such as hematopoietic stem cells (HSC). The ease of production permits any modern molecular biology laboratory to produce reasonable amounts of repli cation-defective virus for research purposes. The entire process is illustrated schematically in Figure 1 and typically takes a week, depending upon the nature of the transgene. Fig. 1. Production of pseudotyped particles. At top are the three plasmids pHIVPV, pHIV-TV, and the VSV G expression plasmid. A tri-transfection of 293T cells is performed by the calcium-phosphate technique. After 2-3 d, the supernatant is harvested (pseudotyped particles shown), and used to transduce targets. The TV becomes integrated in the host genome (note duplication of the ΔU3); and the transgene product is transcribed (bent arrow) and elaborated in the cytoplasm (light gray circles).

Despite the tremendous success of highly active antiretroviral treatment (HAART) introduced nearly 8 years ago for the treatment of human immunodeficiency virus (HIV), innovative therapies, including gene transfer approaches, are still required for nearly half of the general patient population. A number of potential gene therapeutic targets for HIV have been identified and include both viral and cellular genes essential for viral replication. The diverse methods used to inhibit viral replication comprise RNA-based strategies such as ribozymes, RNA decoys, antisense messenger RNAs and small interfering RNA (siRNA) molecules. Other potential anti-HIV genes include dominant negative viral proteins, intracellular antibodies, intrakines and suicide genes, all of which have had a modicum of success in vitro. Cellular targets include CD4+ T cells, macrophages and their progenitors. The greatest gene transfer efficiency has been achieved using retroviral or, more recently, lentiviral vectors. A limited number of Phase I clinical trials suggest that the general method is safe. It is proposed that a national network for HIV gene therapy (similar to the AIDS Clinical Trial Groups) may be the best way to determine which approaches should proceed clinically.

Expression cloning of cDNAs is a powerful tool with which to identify genes based on their specific functional properties. Here we describe the development of a cDNA library transfer system based on the human immunodeficiency virus type-1 (HIV). This system represents an improvement over current oncoretroviral cDNA expression systems in terms of target cell range and the inclusion of a selectable marker. By use of a simple packaging system, we were able to produce high-titer vector stocks from HIV vector-based cDNA libraries and demonstrate highly efficient cDNA expression cloning in three model experiments. First, HOS TK(-) cells, which are null for thymidine kinase (TK) expression, were transduced with an HIV-based cDNA library derived from primary human foreskin fibroblasts (HFFs) and functionally selected for TK expression. In a second experiment, hypoxanthine guanine phosphoribosyltransferase-1-deficient (HPRT(-)) fibroblasts were transduced with a T cell (PM1) line-derived cDNA library and selected for HPRT expression. Both TK (frequency 1 in 5.0 x 10(4)) and HPRT (frequency 1 in 2.0 x 10(4)) cDNAs were readily isolated from these HIV-based cDNA libraries. As a third example, we demonstrated the ability of this vector system to allow functional cDNA library screens to be performed in primary, mitotically inactive cell types. Using senescent HFFs as a target cell population, we were able to isolate SV40 large T antigen cDNA-containing clones (frequency 1 in 2.5 x 10(4)) based on their ability to overcome the senescence-induced block to cell proliferation. Thus, this system can be used to clone relatively low-abundance cDNAs based upon their expression. Because of the ability of HIV-based vectors to transduce primary and nondividing cells efficiently, this vector system will further broaden the range of cell types in which expression cloning studies can be performed.

Human immunodeficiency virus type I (HIV) is the etiologic agent of acquired immunodeficiency syndrome or AIDS. Vectors based upon HIV have been in use for over a decade. Beginning in 1996, with the demonstration of improved pseudotyping using vesicular stomatitis virus (VSV) G protein along with transduction of resting mammalian cells, a series of improvements have been made in these vectors, making them both safer and more efficacious. Taking a cue from vector development of murine leukemia virus (MLV), split coding and self-inactivating HIV vectors now appear quite suitable for phase I clinical trials. In parallel, a number of pre-clinical efficacy studies in animals have demonstrated the utility of these vectors for various diseases processes, especially neurodegenerative and hematopoietic illnesses. These vectors are also appropriate for the study of other viruses (specifically of viral entry) and investigation of the HIV replicative cycle, along with straightforward transgene delivery to target cells of interest. Vectors based upon other lentiviruses have shown similar abilities and promise. Although concerns remain, particularly with regards to detection and propagation of replication-competent lentivirus, it is almost certain that these vectors will be introduced into the clinic within the next 3-5 years.

Gene transfer vectors based upon human immunodeficiency virus type 1 (HIV) are widely used in bench research applications and increasingly in clinical investigations, both to introduce novel genes but also to reduce expression of unwanted genes of the host and pathogen. At present, the vast majority of HIV-based vector supernatants are produced in 293T cells by cotransfection of up to five DNA plasmids, which is subject to variability and difficult to scale. Here we report the development of a HIV-based vector production system that utilizes helper-dependent adenovirus (HDAd). All necessary HIV vector components were inserted into one or more HDAds, which were then amplified to very high titers of ~10(13) vp/ml. These were then used to transduce 293-based cells to produce HIV-based vector supernatants, and resultant VSV G-pseudotyped lentiviral vector (LV) titers and total IU were 10- to 30-fold higher, compared to plasmid transfection. Optimization of HIV-based vector production depended upon maximizing expression of all HIV vector components from HDAd. Supernatants contained trace amounts of HDAd but were free of replication-competent lentivirus. This production method should be applicable to other retroviral vector systems. Scalable production of HIV-based vectors using this two-step procedure should facilitate their clinical advancement.

Direct isolation of human central nervous system stem cells (CNS-SC) based on cell surface markers yields a highly purified stem cell population that can extensively expand in vitro and exhibit multilineage differentiation potential both in vitro and in vivo. The CNS-SC were isolated from fetal brain tissue using the cell surface markers CD133(+), CD34(-), CD45(-), and CD24(-/lo) (CD133(+) cells). Fluorescence-activated cell sorted (FACS) CD133(+) cells continue to expand exponentially as neurospheres while retaining multipotential differentiation capacity for10 passages. CD133(-), CD34(-), and CD45(-) sorted cells (approximately 95% of total fetal brain tissue) fail to initiate neurospheres. Neurosphere cells transplanted into neonatal immunodeficient NOD-SCID mice proliferated, migrated, and differentiated in a site-specific manner. However, it has been difficult to evaluate human cell engraftment, because many of the available monoclonal antibodies against neural cells (beta-tubulin III and glial fibrillary acidic protein) are not species specific. To trace the progeny of human cells after transplantation, CD133(+)-derived neurosphere cells were transduced with lentiviral vectors containing enhanced green fluorescent protein (eGFP) expressed downstream of the phosphoglycerate kinase promoter. After transduction, GFP(+) cells were enriched by FACS, expanded, and transplanted into the lateral ventricular space of neonatal immunodeficient NOD-SCID brain. The progeny of transplanted cells were detected by either GFP fluorescence or antibody against GFP. GFP(+) cells were present in the subventricular zone-rostral migrating stream, olfactory bulb, and hippocampus as well as nonneurogenic sites, such as cerebellum, cerebral cortex, and striatum. Antibody against GFP revealed that some of the cells displayed differentiating dendrites and processes with neurons or glia cells. Thus, marking human CNS-SC with reporter genes introduced by lentiviral vectors is a useful tool with which to characterize migration and differentiation of human cells in this mouse transplantation model.

Immortalization of B cells by Epstein-Barr virus (EBV) depends on the virally encoded EBNA2 protein. Although not related by sequence, the cellular Notch protein and EBNA2 share several biochemical and functional properties, such as interaction with CBF1 and the ability to activate transcription of a number of cellular and viral genes. Whether these similarities are coincidental or exemplify EBNA2 mimicry of evolutionarily conserved cellular signaling pathways is unclear. We therefore investigated whether activated forms of Notch could substitute for EBNA2 in maintaining the immortalized phenotype of EBV-infected B cells. To address this question, we devised a transcomplementation system using EREB2.5 cells. EREB2.5 cells are immortalized by EBV expressing a conditional estrogen receptor EBNA2 fusion protein (EREBNA2), and cellular proliferation is dependent on the availability of estrogen. Withdrawal of estrogen results in inactivation of EREBNA2, leading to growth arrest and eventually to cell death. Transduction of EREB2.5 cells with a lentiviral vector expressing wild-type EBNA2 rescued EREB2.5 cells from the growth-inhibitory effects of estrogen deprivation, in contrast to transduction with the lentivirus vector alone. EREB2.5 cells were also rescued by enforced expression of human Notch1IC after estrogen starvation, but this effect was restricted to cells expressing high levels of the transcription factor. Compared to wild-type EBNA2-expressing EREB2.5 cells, the Notch-expressing cells expanded more slowly after estrogen starvation, and once established, they continued to display a lower proliferation rate. Analysis of viral and cellular gene expression from transduced EREB2.5 cells after estrogen withdrawal indicated that both wild-type EBNA2- and Notch1IC-positive cells expressed c-Myc at levels similar to those found in parental EREB2.5 cells. However, the latter cells expressed LMP-1 far less efficiently than cells transduced with the wild-type EBNA2 gene. Cells rescued by either wild-type EBNA2 or Notch1IC expressed surface CD21 and CD23 proteins, but not CD10, indicating that induction of relevant type III latency markers was maintained. The data imply that both Notch and EBNA2 activate an important subset of cellular genes associated with type III latency and B-cell growth, while EBNA2 more efficiently induces important viral genes, such as LMP-1. Thus, exploitation of conserved Notch-related signaling pathways may represent a key mechanism by which EBNA2 contributes to EBV-induced cell immortalization.

Professional antigen-presenting cells, such as dendritic cells (DCs) and macrophages, are target cells for gene therapy of infectious disease and cancer. However, transduction of DCs and macrophages has proved difficult by most currently available gene transfer methods. Several recent studies have shown that lentiviral vector systems can efficiently transduce many nondividing and differentiated cell types. In this study, we examined the gene transfer to DCs and macrophages using a lentiviral vector system. Human DCs were propagated from the adherent fraction of peripheral blood mononuclear cells (PBMCs) by culture in medium containing GM-CSF, IL-4, and TNF-alpha. Human macrophages were propagated from adherent PBMCs in medium containing GM-CSF. High titers of a replication-defective vesicular stomatitis virus glycoprotein G pseudotyped HIV-1-based vector encoding the enhanced yellow fluorescent protein were produced. In immature DCs (culture days 3 and 5), transduction efficiencies of 25 to 35% were achieved at a multiplicity of infection of 100. However, the transduction efficiency was decreased in more mature DCs (culture day 8 or later). Furthermore, monocyte-derived macrophages were also transduced by the lentiviral vector system. In addition, Alu-LTR PCR demonstrated the integration of the HIV-1 provirus into the cellular genome of the transduced DCs and macrophages. Allogeneic mixed lymphocyte reactions revealed similar antigen-presenting functions of untransduced and lentivirally transduced DCs. Thus, the results of this study demonstrate that both PBMC-derived DCs and macrophages can be transduced by lentiviral vectors.

Recent studies have opened the possibility that quiescent, G 0 /G 1 hematopoietic stem cells (HSC) can be gene transduced; lentiviruses (such as HIV type 1, HIV) encode proteins that permit transport of the viral genome into the nucleus of nondividing cells. We and others have recently demonstrated efficient transduction by using an HIV-1-based vector gene delivery system into various human cell types including human CD34 + cells or terminally differentiated neurons. Here we compare the transduction efficiency of two vectors, HIV-based and murine leukemia virus (MuLV)-based vectors, on untreated and highly purified human HSC subsets that are virtually all in G 0 /G 1 . The HIV vector, but not MuLV vector supernatants, transduced freshly isolated G 0 /G 1 HSC from mobilized peripheral blood. Single-step transduction using replication-defective HIV resulted in HSC that expressed the green fluorescent protein (GFP) transgene while retaining their stem cell phenotype; clonal outgrowths of these GFP + HSC on bone marrow stromal cells fully retained GFP expression for at least 5 weeks. MuLV-based vectors did not transduce resting HSC, as measured by transgene expression, but did so readily when the HSC were actively cycling after culture in vitro for 3 days in a cytokine cocktail. These results suggest that resting HSC may be transduced by lentiviral-based, but not MuLV, vectors and maintain their primitive phenotype, pluripotentiality, and at least in vitro , transgene expression.

Lentiviruses are potentially advantageous compared to oncoretroviruses as gene transfer agents because they can infect nondividing cells. We demonstrate here that human immunodeficiency virus type 1 (HIV-1)-based vectors were highly efficient in transducing purified human hematopoietic stem cells. Transduction rates, measured by marker gene expression or by PCR of the integrated provirus, exceeded 50%, and transduction appeared to be independent of mitosis. Derivatives of HIV-1 were constructed to optimize the vector, and a deletion of most of Vif and Vpr was required to ensure the long-term persistence of transduced cells with relatively stable expression of the marker gene product. These results extend the utility of this lentivirus vector system.

The 5' ends of trypanosome mRNAs consist of an identical sequence of 39 nucleotides which is derived from a discrete transcript of approximately 140 nucleotides (medRNA). It has been proposed that generation of chimeric mRNAs in trypanosomes occurs by the process of trans-splicing involving medRNA and an acceptor RNA. Part of the basis for this suggestion comes from the ability of HeLa cell extracts (known to contain debranching activity) to catalyze the release of the intron portion of medRNA (minRNA) implying a Y-branched intermediate in the splicing process. Here we provide direct chemical analysis that miniRNA is attached to higher mol. wt RNA molecules by a 2'-5' phosphodiester bond (i.e. as a branched structure). We also demonstrate that trypanosomes have substantial amounts of debranching activity which is similar in nature to that of HeLa cells. These results provide further evidence for trans-splicing in trypanosomes and highlights its similarity to cis-splicing in other eukaryotes.

Ovine corticotropin-releasing factor (oCRF) was administered to male rats for 3-15 days. Continuous intravenous infusion, repeated subcutaneous, and intracerebroventricular injections of oCRF resulted in a 50-100% increase in messenger ribonucleic acid (mRNA) coding for proopiomelanocortin (POMC) in the anterior pituitaries (AP) of these animals. The synthetic glucocorticoid, dexamethasone, reversed the actions of oCRF. None of the above treatments altered POMC mRNA content in the neurointermediate lobe. Bilateral adrenalectomy enhanced POMC mRNA levels 4.8-fold. To determine whether this effect was dependent on increased hypothalamic production and release of CRF, a bilateral ablation of the paraventricular nucleus (PVN), known to contain CRF neurons directly involved in ACTH secretion, was performed. 6 days after the PVN destruction a parallel 75% reduction of both stalk-median eminence CRF-like immunoreactivity and plasma ACTH had occurred. The AP content of POMC mRNA decreased by 53%, while the ACTH content increased by 56%. These observations suggest that CRF regulates the biosynthesis of POMC-derived polypeptide products in the AP, while the peptide does not seem to be directly involved in intermediate lobe POMC biosynthesis. The reduction of POMC mRNA levels following PVN destruction demonstrates that CRF and other substances located in the nucleus may cause the increased POMC gene transcription following adrenalectomy.

The influence of cortical neurotransmitters and cyclic AMP on the release of immunoreactive somatostatin (IRS) from cultured cortical cells was examined. Cells were obtained by mechanoenzymatic dispersal of telencephalons of 17-day-old rat embryos and were maintained as monolayers in minimum essential medium with 10% heat-inactivated horse serum. After the cultures had stabilized morphologically and in cellular IRS content they were subjected to rapid sequential changes of a buffered salt solution with or without test substances added. The amount of somatostatin released was measured by a specific radioimmunoassay. Acetylcholine and the GABA antagonist, picrotoxin, both stimulated IRS release. The cholinergic stimulation was predominantly muscarinic. GABA and histamine, to a lesser extent, were inhibitory and norepinephrine and serotonin produced no net change in IRS release. Both cAMP and theophylline (DMX) stimulated IRS release. These results confirm the potential of intrinsic cortical somatostatinergic neurons to respond to endogenous neurotransmitters and further establishes somatostatin as a cortical neuromodulator.