Andrea L. Ferris

H3K4me3 Interactions with TAF3 Regulate Preinitiation Complex Assembly and Selective Gene Activation

Histone modifications regulate chromatin-dependent processes, yet the mechanisms by which they contribute to specific outcomes remain unclear. H3K4me3 is a prominent histone mark that is associated with active genes and promotes transcription through interactions with effector proteins that include initiation factor TFIID. We demonstrate that H3K4me3-TAF3 interactions direct global TFIID recruitment to active genes, some of which are p53 targets. Further analyses show that (1) H3K4me3 enhances p53-dependent transcription by stimulating preinitiation complex (PIC) formation; (2) H3K4me3, through TAF3 interactions, can act either independently or cooperatively with the TATA box to direct PIC formation and transcription; and (3) H3K4me3-TAF3/TFIID interactions regulate gene-selective functions of p53 in response to genotoxic stress. Our findings indicate a mechanism by which H3K4me3 directs PIC assembly for the rapid induction of specific p53 target genes.

Nature, Position, and Frequency of Mutations Made in a Single Cycle of HIV-1 Replication

ABSTRACT There is considerable HIV-1 variation in patients. The extent of the variation is due to the high rate of viral replication, the high viral load, and the errors made during viral replication. Mutations can arise from errors made either by host DNA-dependent RNA polymerase II or by HIV-1 reverse transcriptase (RT), but the relative contributions of these two enzymes to the mutation rate are unknown. In addition, mutations in RT can affect its fidelity, but the effect of mutations in RT on the nature of the mutations that arise in vivo is poorly understood. We have developed an efficient system, based on existing technology, to analyze the mutations that arise in an HIV-1 vector in a single cycle of replication. A lacZα reporter gene is used to identify viral DNAs that contain mutations which are analyzed by DNA sequencing. The forward mutation rate in this system is 1.4 × 10−5 mutations/bp/cycle, equivalent to the retroviral average. This rate is about 3-fold lower than previously reported for HIV-1 in vivo and is much lower than what has been reported for purified HIV-1 RT in vitro. Although the mutation rate was not affected by the orientation of lacZα, the sites favored for mutations (hot spots) in lacZα depended on which strand of lacZα was present in the viral RNA. The pattern of hot spots seen in lacZα in vivo did not match any of the published data obtained when purified RT was used to copy lacZα in vitro.

Replication of Phenotypically Mixed Human Immunodeficiency Virus Type 1 Virions Containing Catalytically Active and Catalytically Inactive Reverse Transcriptase

ABSTRACT The amount of excess polymerase and RNase H activity in human immunodeficiency virus type 1 virions was measured by using vectors that undergo a single round of replication. Vectors containing wild-type reverse transcriptase (RT), vectors encoding the D110E mutation to inactivate polymerase, and vectors encoding mutations D443A and E478Q to inactivate RNase H were constructed. 293 cells were cotransfected with different proportions of plasmids encoding these vectors to generate phenotypically mixed virions. The resulting viruses were used to infect human osteosarcoma cells, and the relative infectivity of the viruses was determined by measuring transduction of the murine cell surface marker CD24, which is encoded by the vectors. The results indicated that there is an excess of both polymerase and RNase H activities in virions. Viral replication was reduced to 42% of wild-type levels in virions with where half of the RT molecules were predicted to be catalytically active but dropped to 3% of wild-type levels when 25% of the RT molecules were active. However, reducing RNase H activity had a lesser effect on viral replication. As expected, based on previous work with murine leukemia virus, there was relatively inefficient virus replication when the RNase H and polymerase activities were encoded on separate vectors (D110E plus E478Q and D110E plus D443A). To determine how virus replication failed when polymerase and RNase H activities were reduced, reverse transcription intermediates were measured in vector-infected cells by using quantitative real-time PCR. The results indicated that using the D11OE mutation to reduce the amount of active polymerase reduced the number of reverse transcripts that were initiated and also reduced the amounts of products from the late stages of reverse transcription. If the E478Q mutation was used to reduce RNase H activity, the number of reverse transcripts that were initiated was reduced; there was also a strong effect on minus-strand transfer.

Lens epithelium-derived growth factor fusion proteins redirect HIV-1 DNA integration

Lens epithelium-derived growth factor (LEDGF) fusion proteins can direct HIV-1 DNA integration to novel sites in the host genome. The C terminus of LEDGF contains an integrase binding domain (IBD), and the N terminus binds chromatin. LEDGF normally directs integrations to the bodies of expressed genes. Replacing the N terminus of LEDGF with chromatin binding domains (CBDs) from other proteins changes the specificity of HIV-1 DNA integration. We chose two well-characterized CBDs: the plant homeodomain (PHD) finger from ING2 and the chromodomain from heterochromatin binding protein 1α (HP1α). The ING2 PHD finger binds H3K4me3, a histone mark that is associated with the transcriptional start sites of expressed genes. The HP1α chromodomain binds H3K9me2,3, histone marks that are widely distributed throughout the genome. A fusion protein in which the ING2 PHD finger was linked to the LEDGF IBD directed integrations near the start sites of expressed genes. A similar fusion protein in which the HP1α chromodomain was linked to the LEDGF IBD directed integrations to sites that differed from both the PHD finger fusion–directed and LEDGF-directed integration sites. The ability to redirect HIV-1 DNA integration may help solve the problems associated with the activation of oncogenes when retroviruses are used in gene therapy.

Differential Effects of Human Immunodeficiency Virus Type 1 Capsid and Cellular Factors Nucleoporin 153 and LEDGF/p75 on the Efficiency and Specificity of Viral DNA Integration

ABSTRACT Retroviruses integrate into cellular DNA nonrandomly. Lentiviruses such as human immunodeficiency virus type 1 (HIV-1) favor the bodies of active genes and gene-enriched transcriptionally active regions of chromosomes. The interaction between lentiviral integrase and the cellular protein lens epithelium-derived growth factor (LEDGF)/p75 underlies the targeting of gene bodies, whereas recent research has highlighted roles for the HIV-1 capsid (CA) protein and cellular factors implicated in viral nuclear import, including transportin 3 (TNPO3) and nucleoporin 358 (NUP358), in the targeting of gene-dense regions of chromosomes. Here, we show that CA mutations, which include the substitution of Asp for Asn74 (N74D), significantly reduce the dependency of HIV-1 on LEDGF/p75 during infection and that this difference correlates with the efficiency of viral DNA integration. The distribution of integration sites mapped by Illumina sequencing confirms that the N74D mutation reduces integration into gene-rich regions of chromosomes and gene bodies and reveals previously unrecognized roles for NUP153 (another HIV-1 cofactor implicated in viral nuclear import) and LEDGF/p75 in the targeting of the viral preintegration complex to gene-dense regions of chromatin. A role for the CA protein in determining the dependency of HIV-1 on LEDGF/p75 during infection highlights a connection between the viral capsid and chromosomal DNA integration.

HRP2 Determines the Efficiency and Specificity of HIV-1 Integration in LEDGF/p75 Knockout Cells but Does Not Contribute to the Antiviral Activity of a Potent LEDGF/p75-Binding Site Integrase Inhibitor

The binding of integrase (IN) to lens epithelium-derived growth factor (LEDGF)/p75 in large part determines the efficiency and specificity of HIV-1 integration. However, a significant residual preference for integration into active genes persists in Psip1 (the gene that encodes for LEDGF/p75) knockout (KO) cells. One other cellular protein, HRP2, harbors both the PWWP and IN-binding domains that are important for LEDGF/p75 co-factor function. To assess the role of HRP2 in HIV-1 integration, cells generated from Hdgfrp2 (the gene that encodes for HRP2) and Psip1/Hdgfrp2 KO mice were infected alongside matched control cells. HRP2 depleted cells supported normal infection, while disruption of Hdgfrp2 in Psip1 KO cells yielded additional defects in the efficiency and specificity of integration. These deficits were largely restored by ectopic expression of either LEDGF/p75 or HRP2. The double-KO cells nevertheless supported residual integration into genes, indicating that IN and/or other host factors contribute to integration specificity in the absence of LEDGF/p75 and HRP2. Psip1 KO significantly increased the potency of an allosteric inhibitor that binds the LEDGF/p75 binding site on IN, a result that was not significantly altered by Hdgfrp2 disruption. These findings help to rule out the host factor-IN interactions as the primary antiviral targets of LEDGF/p75-binding site IN inhibitors.

Immunologic and proteolytic analysis of HIV-1 reverse transcriptase structure.

HIV-1 virions contain two reverse transcriptase polypeptides that have apparent molecular weights of 66 and 51 kDa. The 51-kDa form lacks the carboxy-terminal sequences found in the 66-kDa form, and is believed to be a proteolytic digestion product. We have treated purified 66-kDa reverse transcriptase with viral and nonviral proteases. The digestion products were characterized by their ability to react with monoclonal antibodies known to recognize particular segments of the HIV-1 reverse transcriptase. The approximate location of the segments recognized by the monoclonal antibodies was determined by testing the ability of the antibodies to recognize a series of amino- and carboxy-terminal-deleted forms of HIV-1 reverse transcriptase. The segments recognized are not uniformly distributed along the primary amino acid sequence of HIV-1 reverse transcriptase. We suggest that these segments are probably on the surface of the properly folded form of reverse transcriptase. Of the tested proteases, only the viral protease was able to cleave the 66-kDa form to the 51-kDa form without producing additional cleavage products, suggesting that the viral protease cleaves the 66-kDa protein to the 51-kDa form in virions.

Reduced Genotoxicity of Avian Sarcoma Leukosis Virus Vectors in Rhesus Long-term Repopulating Cells Compared to Standard Murine Retrovirus Vectors

Insertional mutagenesis continues to be a major concern in hematopoietic stem-cell gene therapy. Nonconventional gene transfer vectors with more favorable integration features in comparison with conventional retrovirus and lentivirus vectors are being developed and optimized. In this study, we report for the first time a systematic analysis of 198 avian sarcoma leukosis virus (ASLV) insertion sites identified in rhesus long-term repopulating cells, and a comparison of ASLV insertion patterns to Moloney murine leukemia virus (MLV) (n = 396) and simian immunodeficiency virus (SIV) (n = 289) using the newly released rhesus genome databank. Despite a weak preference toward gene-coding regions, ASLV integration is nonclustered, does not favor gene-rich regions, transcription start sites, or CpG islands. There was no propensity for ASLV insertions within or near proto-oncogenes, and most importantly, no insertions close to or within the Mds1-Evi1 locus, which is in contrast to the significant over-representation of this insertion site for MLV vectors in the same transplantation model. Furthermore, ASLV long terminal repeats (LTRs) do not have detectable promoter and enhancer activity in a quantitative luciferase assay to measure neighboring gene activation. The combination of these features is unique for ASLV and suggests that optimized vectors based on this virus could be useful and safe for gene transfer to hematopoietic stem cells and progenitor cells.Insertional mutagenesis continues to be a major concern in hematopoietic stem-cell gene therapy. Nonconventional gene transfer vectors with more favorable integration features in comparison with conventional retrovirus and lentivirus vectors are being developed and optimized. In this study, we report for the first time a systematic analysis of 198 avian sarcoma leukosis virus (ASLV) insertion sites identified in rhesus long-term repopulating cells, and a comparison of ASLV insertion patterns to Moloney murine leukemia virus (MLV) (n = 396) and simian immunodeficiency virus (SIV) (n = 289) using the newly released rhesus genome databank. Despite a weak preference toward gene-coding regions, ASLV integration is nonclustered, does not favor gene-rich regions, transcription start sites, or CpG islands. There was no propensity for ASLV insertions within or near proto-oncogenes, and most importantly, no insertions close to or within the Mds1-Evi1 locus, which is in contrast to the significant over-representation of this insertion site for MLV vectors in the same transplantation model. Furthermore, ASLV long terminal repeats (LTRs) do not have detectable promoter and enhancer activity in a quantitative luciferase assay to measure neighboring gene activation. The combination of these features is unique for ASLV and suggests that optimized vectors based on this virus could be useful and safe for gene transfer to hematopoietic stem cells and progenitor cells.

A homology model of HIV-1 integrase and analysis of mutations designed to test the model.

Although there are structures of the different domains of human immunodeficiency virus type 1 (HIV-1) integrase (IN), there is no structure of the entire protein. The recently determined crystal structures of the prototype foamy virus (PFV) IN tetramer, in complexes with viral DNA, led to the generation of models of full-length HIV-1 IN. These models were generated, in part, by superimposing the structures of the domains of HIV-1 IN onto the structure of full-length PFV IN. We developed a model for HIV-1 IN-based solely on its sequence alignment with PFV IN-that differs in several ways from the previous models. Specifically, in our model, the junction between the catalytic core domain and C-terminal domain adopts a helix-loop-helix motif that is similar to the corresponding segment of PFV IN and differs from the crystal structures of these two HIV-1 IN domains. The alignment of residues in the C-terminal domain also differs from the previous models. Our model can be used to explain the phenotype of previously published HIV-1 IN mutants. We made additional mutants, and the behavior of these new mutants provides additional support for the model.

The Integrator complex controls the termination of transcription at diverse classes of gene targets.

Complexes containing INTS3 and either NABP1 or NABP2 were initially characterized in DNA damage responses, but their biochemical function remained unknown. Using affinity purifications and HIV Integration targeting-sequencing (HIT-Seq), we find that these complexes are part of the Integrator complex, which binds RNA Polymerase II and regulates specific target genes. Integrator cleaves snRNAs as part of their processing to their mature form in a mechanism that is intimately coupled with transcription termination. However, HIT-Seq reveals that Integrator also binds to the 3′ end of replication-dependent histones and promoter proximal regions of genes with polyadenylated transcripts. Depletion of Integrator subunits results in transcription termination failure, disruption of histone mRNA processing, and polyadenylation of snRNAs and histone mRNAs. Furthermore, promoter proximal binding of Integrator negatively regulates expression of genes whose transcripts are normally polyadenylated. Integrator recruitment to all three gene classes is DSIF-dependent, suggesting that Integrator functions as a termination complex at DSIF-dependent RNA Polymerase II pause sites.