Retrovirus Integrase-DNA Structure Elucidates Concerted Integration Mechanisms

Commentary on Hare, S.; Gupta, S.S.; Valkov, E.; Engelman, A.; Cherepanov, P. Retroviral intasome assembly and inhibition of DNA strand transfer. Nature 2010, 464, 232–236

Protein Kinase C: One Pathway towards the Eradication of Latent HIV-1 Reservoirs

An effective means to eradicate latent reservoirs in HIV-1-infected individuals remains elusive. Attempts to purge these reservoirs were undertaken over a decade ago without success. The subsequent lapse in further clinical attempts since may have been justified as our knowledge of the mechanisms which underpin the latent state still evolves. Although additional novel molecular antagonists of HIV-1 latency have subsequently been reported, these candidate agents have not been tested in human trials for reservoir ablation. This review provides an overview of the protein kinase C (PKC) pathway which can be modulated by small molecular agents to induce the expression of latent HIV-1 from within infected reservoir cells. Some of these agents have been tested against select cancers with seemingly tolerable side effects. As such, modulation of the PKC pathway may yet be a viable mechanism toward HIV-1 reservoir eradication

A bifunctional DNA binding region in Tn5 transposase

© 2008 The Authors. This is an open-access article distributed under the terms of the Creative Commons Attribution License 2.5. The definitive version was published in Molecular Microbiology 67 (2008): 528-540, doi:10.1111/j.1365-2958.2007.06056.x.Tn5 transposition is a complicated process that requires the formation of a highly ordered protein–DNA structure, a synaptic complex, to catalyse the movement of a sequence of DNA (transposon) into a target DNA. Much is known about the structure of the synaptic complex and the positioning of protein–DNA contacts, although many protein–DNA contacts remain largely unstudied. In particular, there is little evidence for the positioning of donor DNA and target DNA. In this communication, we describe the isolation and analysis of mutant transposases that have, for the first time, provided genetic and biochemical evidence for the stage-specific positioning of both donor and target DNAs within the synaptic complex. Furthermore, we have provided evidence that some of the amino acids that contact donor DNA also contact target DNA, and therefore suggest that these amino acids help define a bifunctional DNA binding region responsible for these two transposase–DNA binding events.This work was supported by the NIH [GM50693], the University of Wisconsin at Madison [WIS04792], and through the Evelyn Mercer Professorship in Biochemistry and Molecular Biology

Effects of prostratin on Cyclin T1/P-TEFb function and the gene expression profile in primary resting CD4(+ )T cells

BACKGROUND: The latent reservoir of human immunodeficiency virus type 1 (HIV-1) in resting CD4(+ )T cells is a major obstacle to the clearance of infection by highly active antiretroviral therapy (HAART). Recent studies have focused on searches for adjuvant therapies to activate this reservoir under conditions of HAART. Prostratin, a non tumor-promoting phorbol ester, is a candidate for such a strategy. Prostratin has been shown to reactivate latent HIV-1 and Tat-mediated transactivation may play an important role in this process. We examined resting CD4(+ )T cells from healthy donors to determine if prostratin induces Cyclin T1/P-TEFb, a cellular kinase composed of Cyclin T1 and Cyclin-dependent kinase-9 (CDK9) that mediates Tat function. We also examined effects of prostratin on Cyclin T2a, an alternative regulatory subunit for CDK9, and 7SK snRNA and the HEXIM1 protein, two factors that associate with P-TEFb and repress its kinase activity. RESULTS: Prostratin up-regulated Cyclin T1 protein expression, modestly induced CDK9 protein expression, and did not affect Cyclin T2a protein expression. Although the kinase activity of CDK9 in vitro was up-regulated by prostratin, we observed a large increase in the association of 7SK snRNA and the HEXIM1 protein with CDK9. Using HIV-1 reporter viruses with and without a functional Tat protein, we found that prostratin stimulation of HIV-1 gene expression appears to require a functional Tat protein. Microarray analyses were performed and several genes related to HIV biology, including APOBEC3B, DEFA1, and S100 calcium-binding protein genes, were found to be regulated by prostratin. CONCLUSION: Prostratin induces Cyclin T1 expression and P-TEFb function and this is likely to be involved in prostratin reactivation of latent HIV-1 proviruses. The large increase in association of 7SK and HEXIM1 with P-TEFb following prostratin treatment may reflect a requirement in CD4(+ )T cells for a precise balance between active and catalytically inactive P-TEFb. Additionally, genes regulated by prostratin were identified that have the potential to regulate HIV-1 replication both positively and negatively

Drug-Class Specific Impact of Antivirals on the Reproductive Capacity of HIV

Predictive markers linking drug efficacy to clinical outcome are a key component in the drug discovery and development process. In HIV infection, two different measures, viral load decay and phenotypic assays, are used to assess drug efficacy in vivo and in vitro. For the newly introduced class of integrase inhibitors, a huge discrepancy between these two measures of efficacy was observed. Hence, a thorough understanding of the relation between these two measures of drug efficacy is imperative for guiding future drug discovery and development activities in HIV. In this article, we developed a novel viral dynamics model, which allows for a mechanistic integration of the mode of action of all approved drugs and drugs in late clinical trials. Subsequently, we established a link between in vivo and in vitro measures of drug efficacy, and extract important determinants of drug efficacy in vivo. The analysis is based on a new quantity—the reproductive capacity—that represents in mathematical terms the in vivo analog of the read-out of a phenotypic assay. Our results suggest a drug-class specific impact of antivirals on the total amount of viral replication. Moreover, we showed that the (drug-)target half life, dominated by immune-system related clearance processes, is a key characteristic that affects both the emergence of resistance as well as the in vitro–in vivo correlation of efficacy measures in HIV treatment. We found that protease- and maturation inhibitors, due to their target half-life, decrease the total amount of viral replication and the emergence of resistance most efficiently

NFAT5 Regulates HIV-1 in Primary Monocytes via a Highly Conserved Long Terminal Repeat Site

To replicate, HIV-1 capitalizes on endogenous cellular activation pathways resulting in recruitment of key host transcription factors to its viral enhancer. RNA interference has been a powerful tool for blocking key checkpoints in HIV-1 entry into cells. Here we apply RNA interference to HIV-1 transcription in primary macrophages, a major reservoir of the virus, and specifically target the transcription factor NFAT5 (nuclear factor of activated T cells 5), which is the most evolutionarily divergent NFAT protein. By molecularly cloning and sequencing isolates from multiple viral subtypes, and performing DNase I footprinting, electrophoretic mobility shift, and promoter mutagenesis transfection assays, we demonstrate that NFAT5 functionally interacts with a specific enhancer binding site conserved in HIV-1, HIV-2, and multiple simian immunodeficiency viruses. Using small interfering RNA to ablate expression of endogenous NFAT5 protein, we show that the replication of three major HIV-1 viral subtypes (B, C, and E) is dependent upon NFAT5 in human primary differentiated macrophages. Our results define a novel host factor–viral enhancer interaction that reveals a new regulatory role for NFAT5 and defines a functional DNA motif conserved across HIV-1 subtypes and representative simian immunodeficiency viruses. Inhibition of the NFAT5–LTR interaction may thus present a novel therapeutic target to suppress HIV-1 replication and progression of AIDS

De novo assembly of Euphorbia fischeriana root transcriptome identifies prostratin pathway related genes

Background Euphorbia fischeriana is an important medicinal plant found in Northeast China. The plant roots contain many medicinal compounds including 12-deoxyphorbol-13-acetate, commonly known as prostratin that is a phorbol ester from the tigliane diterpene series. Prostratin is a protein kinase C activator and is effective in the treatment of Human Immunodeficiency Virus (HIV) by acting as a latent HIV activator. Latent HIV is currently the biggest limitation for viral eradication. The aim of this study was to sequence, assemble and annotate the E. fischeriana transcriptome to better understand the potential biochemical pathways leading to the synthesis of prostratin and other related diterpene compounds. Results In this study we conducted a high throughput RNA-seq approach to sequence the root transcriptome of E. fischeriana. We assembled 18,180 transcripts, of these the majority encoded protein-coding genes and only 17 transcripts corresponded to known RNA genes. Interestingly, we identified 5,956 protein-coding transcripts with high similarity (>=75%) to Ricinus communis, a close relative to E. fischeriana. We also evaluated the conservation of E. fischeriana genes against EST datasets from the Euphorbeacea family, which included R. communis, Hevea brasiliensis and Euphorbia esula. We identified a core set of 1,145 gene clusters conserved in all four species and 1,487 E. fischeriana paralogous genes. Furthermore, we screened E. fischeriana transcripts against an in-house reference database for genes implicated in the biosynthesis of upstream precursors to prostratin. This identified 24 and 9 candidate transcripts involved in the terpenoid and diterpenoid biosyntehsis pathways, respectively. The majority of the candidate genes in these pathways presented relatively low expression levels except for 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase (HDS) and isopentenyl diphosphate/dimethylallyl diphosphate synthase (IDS), which are required for multiple downstream pathways including synthesis of casbene, a proposed precursor to prostratin. Conclusion The resources generated in this study provide new insights into the upstream pathways to the synthesis of prostratin and will likely facilitate functional studies aiming to produce larger quantities of this compound for HIV research and/or treatment of patients

Comparison of metal-dependent catalysis by HIV-1 and ASV integrase proteins using a new and rapid, moderate throughput assay for joining activity in solution

<p>Abstract</p> <p>Background</p> <p>HIV-1 integrase (IN) is an attractive target for the development of drugs to treat AIDS, and inhibitors of this viral enzyme are already in the clinic. Nevertheless, there is a continuing need to devise new approaches to block the activity of this viral protein because of the emergence of resistant strains. To facilitate the biochemical analysis of wild-type IN and its derivatives, and to measure the potency of prospective inhibitory compounds, a rapid, moderate throughput solution assay was developed for IN-catalyzed joining of viral and target DNAs, based on the detection of a fluorescent tag.</p> <p>Results</p> <p>A detailed, step-by-step description of the new joining assay is provided. The reactions are run in solution, the products captured on streptavidin beads, and activity is measured by release of a fluorescent tag. The procedure can be scaled up for the analysis of numerous samples, and is substantially more rapid and sensitive than the standard radioactive gel methods. The new assay is validated and its utility demonstrated via a detailed comparison of the Mg⁺⁺- and Mn⁺⁺-dependent activities of the IN proteins from human immunodeficiency virus type 1 (HIV-1) and the avian sarcoma virus (ASV). The results confirm that ASV IN is considerably more active than HIV-1 IN, but with both enzymes the initial rates of joining, and the product yields, are higher in the presence of Mn⁺⁺than Mg⁺⁺. Although the pH optima for these two enzymes are similar with Mn⁺⁺, they differ significantly in the presence of Mg⁺⁺, which is likely due to differences in the molecular environment of the binding region of this physiologically relevant divalent cation. This interpretation is strengthened by the observation that a compound that can inhibit HIV-1 IN in the presence of either metal cofactors is only effective against ASV in the presence of Mn⁺⁺.</p> <p>Conclusion</p> <p>A simplified, assay for measuring the joining activity of retroviral IN in solution is described, which offers several advantages over previous methods and the standard radioactive gel analyses. Based on comparisons of signal to background ratios, the assay is 10–30 times more sensitive than gel analysis, allows more rapid and accurate biochemical analyses of IN catalytic activity, and moderate throughput screening of inhibitory compounds. The assay is validated, and its utility demonstrated in a comparison of the metal-dependent activities of HIV-1 and ASV IN proteins.</p