Digoxin suppresses HIV-1 replication by altering viral RNA processing.

To develop new approaches to control HIV-1 replication, we examined the capacity of recently described small molecular modulators of RNA splicing for their effects on viral RNA metabolism. Of the drugs tested, digoxin was found to induce a dramatic inhibition of HIV-1 structural protein synthesis, a response due, in part, to reduced accumulation of the corresponding viral mRNAs. In addition, digoxin altered viral RNA splice site use, resulting in loss of the essential viral factor Rev. Digoxin induced changes in activity of the CLK family of SR protein kinases and modification of several SR proteins, including SRp20 and Tra2ß, which could account for the effects observed. Consistent with this hypothesis, overexpression of SRp20 elicited changes in HIV-1 RNA processing similar to those observed with digoxin. Importantly, digoxin was also highly active against clinical strains of HIV-1 in vitro, validating this novel approach to treatment of this infection.

System-level identification of transcriptional circuits underlying mammalian circadian clocks.

Mammalian circadian clocks consist of complexly integrated regulatory loops, making it difficult to elucidate them without both the accurate measurement of system dynamics and the comprehensive identification of network circuits. Toward a system-level understanding of this transcriptional circuitry, we identified clock-controlled elements on 16 clock and clock-controlled genes in a comprehensive surveillance of evolutionarily conserved cis elements and measurement of their transcriptional dynamics. Here we report the roles of E/E' boxes, DBP/E4BP4 binding elements and RevErbA/ROR binding elements in nine, seven and six genes, respectively. Our results indicate that circadian transcriptional circuits are governed by two design principles: regulation of E/E' boxes and RevErbA/ROR binding elements follows a repressor-precedes-activator pattern, resulting in delayed transcriptional activity, whereas regulation of DBP/E4BP4 binding elements follows a repressor-antiphasic-to-activator mechanism, which generates high-amplitude transcriptional activity. Our analysis further suggests that regulation of E/E' boxes is a topological vulnerability in mammalian circadian clocks, a concept that has been functionally verified using in vitro phenotype assay systems.

The HIV-1 Rev response element: an RNA scaffold that directs the cooperative assembly of a homo-oligomeric ribonucleoprotein complex.

The HIV-1 Rev response element (RRE) is a ~350 nucleotide, highly structured, cis-acting RNA element essential for viral replication. It is located in the env coding region of the viral genome and is extremely well conserved across different HIV-1 isolates. It is present on all partially spliced and unspliced viral mRNA transcripts, and serves as an RNA framework onto which multiple molecules of the viral protein Rev assemble. The Rev-RRE oligomeric complex mediates the export of these messages from the nucleus to the cytoplasm, where they are translated to produce essential viral proteins and/or packaged as genomes for new virions.

Organization patterns of the AGFG genes: an evolutionary study.

A number of proteins which are needed for the building of new immunodeficiency virus type 1 virions can only be translated from unspliced virus-derived pre-mRNAs. These unspliced mRNAs are shuttled through the nuclear pores reaching the cytosol when bound to the viral protein Rev. However, as a cellular co-factor Rev requires a Rev-binding protein of the AGFG family (nucleoporin-related Arf-GAP domain and FG repeats-containing proteins). In this article we address the evolution of the AGFGs by analyzing the first section of the coding mRNAs. This contains a "core module" which can be traced from Drosophilae to fish, amphibia, birds, and mammals, including man. In the subfamily of AGFG1 molecules the estimated conservation from Drosophilae to primates is 67% (with limited gaps). In some Drosophilae the core module is preceded by a long stretch of more than 300 coding nucleotides, but this additional module is absent in other Drosophilae and in all AGFG1s of other species. The AGFG2 molecules emerged later in evolution, possibly deriving from a duplication of AGFG1s. AGFG2s, present in mammals only, exhibit an additional module of about 50 coding nucleotides ahead of the core module, which is significantly less conserved (54%, with more remarkable gaps). This additional module does not seem to have homologies with the additional module of Drosophilae nor with the precoding section of AGFG1s. Interestingly, in birds a highly re-edited form of the AGFG1 core module (Gallus gallus, Galliformes) coexists with a typical form of the AGFG1 core module (Taeniopygia guttata, Passeriformes).

Prevalence of transmitted antiretroviral resistance and distribution of HIV-1 subtypes among patients with recent infection in Catalonia (Spain) between 2003 and 2005.

OBJECTIVES: The objectives of this study were to assess the prevalence of transmitted HIV-1 drug resistances (TDR) and HIV-1 subtypes in recently infected patients in Catalonia between 2003 and 2005 and to describe the characteristics of these patients according to the presence or absence of TDR and HIV-1 subtype. METHODS: After application of the Serological Testing Algorithm for Recent HIV Seroconversion (STARHS), residual aliquots of serum samples from recently infected antiretroviral-naïve individuals were genotyped. FASTA sequences were analyzed using the HIVDB Program. The World Health Organization 2009 List of Mutations for Surveillance of Transmitted HIV-1 Drug Resistant HIV Strains was used to estimate the prevalence of TDR. RESULTS: Of 182 recently infected patients, 14 (7.7%) presented TDR. Seven (3.8%) had genotypic evidence of TDR against non-nucleoside reverse transcriptase inhibitors, 6 (3.3%) against nucleoside reverse transcriptase inhibitors, 3 (1.6%) against protease inhibitors (PIs), and only 2 individuals (1.1%) presented TDR against more than one class of drugs. Thirty-five (19.2%) patients were infected with a non-B HIV-1 subtype. CONCLUSION: This is the first study to estimate the prevalence of TDR in recently infected patients in Catalonia. The results are similar to those of studies performed in other Spanish regions. Correct monitoring of these parameters requires systematic epidemiologic surveillance of transmitted resistance.

Human papillomavirus infection and its association with HIV.

Human papillomavirus (HPV) can infect oral mucosa, causing asymptomatic infection or warty lesions. Several case-control studies have confirmed HPV as an independent risk factor for squamous cell carcinoma. HPV-related cancers seem to have better prognoses and different risk factors than do HPV-negative ones. HIV-infected patients are known to be at increased risk for persistent genital and anal high-risk HPV infections and intraepithelial neoplasm. Since the era of highly active antiretroviral therapy, the prevalence and persistence of warty lesions in oral mucosa have increased. Oral squamous cell carcinoma was recently added in the case definitions for common HIV-related oral mucosa lesions. The increased risk of HPV infection in HIV patients has been associated with impaired immune response to HPV, highly active antiretroviral therapy, aging of the HIV-infected patients, and direct interaction between the 2 viruses. HPV32 seems to be much more prevalent in asymptomatic HPV infections and warts among those infected with HIV than among those in the general population. Regarding HIV genes, there is evidence of an interaction between HPV and tat, rev, and vpr. HIV might play a role in HPV-associated pathogenesis by exhorting oncogenic stimuli via tat and rev or visa versa.

Halogenated analogs of 1'-acetoxychavicol acetate, Rev-export inhibitor from Alpinia galanga, designed from mechanism of action.

In the course of search for the robust analogs of 1'-acetoxychavicol acetate (ACA, 1), the Rev-export inhibitor from the medicinal plant Alpinia galanga, we clarified formation of the quinone methide intermediate ii to be essential for exerting the inhibitory activity of 1. Based on this mechanism of action, the rational design from the MO calculation of the conclusive activation energy to ii resulted in the four halogenated analogs with more potent activity than ACA (1). In particular, the difluoroanalog 20d exhibited approximately four-fold potent activity as compared with 1.

Eukaryotic initiation factor 5A is a cellular target of the human immunodeficiency virus type 1 Rev activation domain mediating trans-activation.

Expression of human immunodeficiency virus type 1 (HIV-1) structural proteins requires the presence of the viral trans-activator protein Rev. Rev is localized in the nucleus and binds specifically to the Rev response element (RRE) sequence in viral RNA. Furthermore, the interaction of the Rev activation domain with a cellular cofactor is essential for Rev function in vivo. Using cross-linking experiments and Biospecific Interaction Analysis (BIA) we identify eukaryotic initiation factor 5A (eIF-5A) as a cellular factor binding specifically to the HIV-1 Rev activation domain. Indirect immunofluorescence studies demonstrate that a significant fraction of eIF-5A localizes to the nucleus. We also provide evidence that Rev transactivation is functionally mediated by eIF-5A in Xenopus oocytes. Furthermore, we are able to block Rev function in mammalian cells by antisense inhibition of eIF-5A gene expression. Thus, regulation of HIV-1 gene expression by Rev involves the targeting of RRE-containing RNA to components of the cellular translation initiation complex.

Inhibition of Rev-mediated HIV-1 expression by an RNA binding protein encoded by the interferon-inducible 9-27 gene.

Interferon inhibits expression of human immunodeficiency virus type-1 (HIV-1) through unknown mechanisms. A gene inducible by interferon-alpha (IFN-alpha) and interferon-gamma (IFN-gamma) was isolated by screening of a human complementary DNA library for proteins binding to the Rev-responsive element (RRE) of HIV-1. The product of this gene, RBP9-27, was shown to bind RNA in vitro and to inhibit HIV-1 expression after transfection into human cells. RBP9-27 primarily inhibited Rev-dependent posttranscriptional steps of viral gene expression. Thus, RBP9-27 is a cellular factor that antagonizes Rev function. These results suggest an interferon-induced antiviral mechanism operating through the induction of RNA binding proteins such as RBP9-27. Elucidation of RBP9-27 function may lead to a better understanding of the mechanism of interferon action during HIV-1 infection.

Secondary structure is the major determinant for interaction of HIV rev protein with RNA.

A region in the human immunodeficiency virus (HIV) env message, with the potential to form a complex secondary structure (designated RRE), interacts with the rev protein (Rev). This interaction is believed to mediate export of HIV structural messenger RNAs from the nucleus to the cytoplasm. In this report the regions essential for Rev interaction with the RRE are further characterized and the functional significance of Rev-RRE interaction in vivo is examined. A single hairpin loop structure within the RRE was found to be a primary determinant for Rev binding in vitro and Rev response in vivo. Maintenance of secondary structure, rather than primary nucleotide sequence alone, appeared to be necessary for Rev-RNA interaction, which distinguishes it from the mechanism for cis-acting elements in DNA. Limited changes within the 200 nucleotides, which preserved the proper RRE conformational structure, were well tolerated for Rev binding and function. Thus, variation among the RRE elements present in the diverse HIV isolates would have little, if any, effect on Rev responsiveness.

Inducible packaging cells for large-scale production of lentiviral vectors in serum-free suspension culture.

We have developed new packaging cell lines (293SF-PacLV) that can produce lentiviral vectors (LVs) in serum-free suspension cultures. A cell line derived from 293SF cells, expressing the repressor (CymR) of the cumate switch and the reverse transactivator (rtTA2(S)-M2) of the tetracycline (Tet) switch, was established first. We next generated clones stably expressing the Gag/Pol and Rev genes of human immunodeficiency virus-1, and the glycoprotein of vesicular stomatitis virus (VSV-G). Expression of Rev and VSV-G was tightly regulated by the cumate and Tet switches. Our best packaging cells produced up to 2.6 x 10(7) transducing units (TU)/ml after transfection with the transfer vector. Up to 3.4 x 10(7) TU/ml were obtained using stable producers generated by transducing the packaging cells with conditional-SIN-LV. The 293SF-PacLV was stable, as shown by the fact that some producers maintained high-level LV production for 18 weeks without selective pressure. The utility of the 293SF-PacLV for scaling up production in serum-free medium was demonstrated in suspension cultures and in a 3.5-L bioreactor. In shake flasks, the best packaging cells produced between 3.0 and 8.0 x 10(6) TU/ml/day for 3 days, and the best producer cells, between 1.0 and 3.4 x 10(7) TU/ml/day for 5 days. In the bioreactor, 2.8 liters containing 2.0 x 10(6) TU/ml was obtained after 3 days of batch culture following the transfection of packaging cells. In summary, the 293SF-PacLV possesses all the attributes necessary to become a valuable tool for scaling up LV production for preclinical and clinical applications.

RNA recognition by the human immunodeficiency virus Tat and Rev proteins.

The human immunodeficiency virus (HIV-1) regulatory proteins, Tat and Rev, are important potential targets for the development of new drug therapies against HIV infection. Both proteins are highly specific RNA-binding proteins that recognize cis-acting regulatory elements in the viral mRNAs. These interactions are fascinating paradigms of a new principle of RNA recognition in which the protein makes contact with functional groups displayed in a distorted major groove of an RNA duplex.

The HIV-1 Rev protein: prototype of a novel class of eukaryotic post-transcriptional regulators.

Complex retroviruses, including Human Immunodeficiency Virus Type 1 (HIV-1), are characterized by the ordered temporal expression of the various viral gene products in infected cells. This effect is mediated by a novel class of RNA-sequence-specific regulatory proteins typified by the Rev trans-activator of HIV-1. Evidence suggests that Rev regulates HIV-1 gene expression by intervening in the normal pathway of eukaryotic mRNA processing and transport.

Genetic analysis and gene expression of human immunodeficiency virus.

Ten years after the initial description of acquired immune deficiency syndrome, its causative agent, the human immunodeficiency virus, remains the subject of intense scientific interest. Recent research has focused on the detailed analysis of the molecular principles governing gene expression and virion formation and on the cause of immune system dysfunction. Within the past year, considerable progress has been made regarding both the role of the regulatory proteins and the mechanism by which they function, and the determinants of cell tropism and of virion formation.

Production of human immunodeficiency virus type 1 (HIV-1) pseudoviruses using linear HIV-1 envelope expression cassettes.

HIV-1 pseudoviruses constitute an important tool in HIV-1 vaccine and entry inhibitor research. Single-cycle pseudoviruses carrying functional envelopes are generated by co-transfecting HEK293T cells with pNL4-3.LucR(-)E(-) and Env expression plasmids. However, cloning of Env genes is time consuming and single Env clones are not representative of the diversity of HIV-1 in a patient's blood sample. A new method to construct Env expression cassettes is proposed which can be used for the rapid generation of heterogeneous HIV-1 pseudoviruses without a cloning step. The linear Env expression cassettes are constructed by ligating PCR amplified Env genes between a 5' CMV promoter and 3' SV40 polyadenylation element. The resulting cassettes generate pseudoviruses carrying heterogeneous Env variants of a primary HIV-1 isolate derived from viral RNA or proviral DNA. The influence of cis-acting sequences upstream of the Env gene on infectivity was compared between pseudoviruses generated from plasmids and linear expression cassettes. The results suggest that the presence of these upstream sequences tends to result in higher infectivity of pseudoviruses when present in heterogeneous Env expression cassettes, but they do not enhance infectivity of pseudoviruses generated with homogeneous Env expression constructs. Using linear expression cassettes allows for the rapid production of heterogeneous patient-derived functional Env genes.

CRISPR/Cas9 system targeting regulatory genes of HIV-1 inhibits viral replication in infected T-cell cultures.

The CRISPR/Cas9 system provides a novel and promising tool for editing the HIV-1 proviral genome. We designed RNA-guided CRISPR/Cas9 targeting the HIV-1 regulatory genes tat and rev with guide RNAs (gRNA) selected from each gene based on CRISPR specificity and sequence conservation across six major HIV-1 subtypes. Each gRNA was cloned into lentiCRISPRv2 before co-transfection to create a lentiviral vector and transduction into target cells. CRISPR/Cas9 transduction into 293 T and HeLa cells stably expressing Tat and Rev proteins successfully abolished the expression of each protein relative to that in non-transduced and gRNA-absent vector-transduced cells. Tat functional assays showed significantly reduced HIV-1 promoter-driven luciferase expression after tat-CRISPR transduction, while Rev functional assays revealed abolished gp120 expression after rev-CRISPR transduction. The target gene was mutated at the Cas9 cleavage site with high frequency and various indel mutations. Conversely, no mutations were detected at off-target sites and Cas9 expression had no effect on cell viability. CRISPR/Cas9 was further tested in persistently and latently HIV-1-infected T-cell lines, in which p24 levels were significantly suppressed even after cytokine reactivation, and multiplexing all six gRNAs further increased efficiency. Thus, the CRISPR/Cas9 system targeting HIV-1 regulatory genes may serve as a favorable means to achieve functional cures.

Potent knock down of HIV-1 replication by targeting HIV-1 Tat/Rev RNA sequences synergistically with catalytic RNA and DNA.

OBJECTIVE: Ribozymes (Rzs) and DNA-enzymes (Dzs) possess the ability to prevent gene expression by cleaving target RNA in a catalytic and sequence-specific manner. Although Rzs or Dzs have been used earlier for HIV-1 gene suppression, the present study explored the possibility of using catalytic RNA and DNA simultaneously in a synergistic manner with the hope that this novel approach will allow more potent inhibition for a longer duration. METHODS: In order to achieve long-term inhibition of HIV-1 replication, a novel non-GUX hammerhead Rz was designed by standard recombinant DNA technology and cloned it under the powerful CMV promoter containing expression vector. A 10-23 catalytic motif containing Dz that was targeted against the conserved second exon of HIV-1 Tat/Rev region was also assembled. RESULTS: Both Rz and Dz possessed sequence-specific cleavage activities individually and simultaneously cleaved target RNA in a synergistic manner under the same in vitro cleavage conditions. These catalytic molecules inhibited HIV-1 replication in macrophages individually and exhibited potent inhibitory effects when used in combination. CONCLUSIONS: The combination strategy described here can be widely used against any target RNA to achieve more effective gene inhibition that exploits the simultaneous sequence-specific cleavage potentials of catalytic RNA and DNA.

hnRNP E1 and E2 have distinct roles in modulating HIV-1 gene expression.

Pre-mRNA processing, including 5' end capping, splicing, and 3' end cleavage/polyadenylation, are events coordinated by transcription that can influence the subsequent export and translation of mRNAs. Coordination of RNA processing is crucial in retroviruses such as HIV-1, where inefficient splicing and the export of intron-containing RNAs are required for expression of the full complement of viral proteins. RNA processing can be affected by both viral and cellular proteins, and in this study we demonstrate that a member of the hnRNP E family of proteins can modulate HIV-1 RNA metabolism and expression. We show that hnRNP E1/E2 are able to interact with the ESS3a element of the bipartite ESS in tat/rev exon 3 of HIV-1 and that modulation of hnRNP E1 expression alters HIV-1 structural protein synthesis. Overexpression of hnRNP E1 leads to a reduction in Rev, achieved in part through a decrease in rev mRNA levels. However, the reduction in Rev levels cannot fully account for the effect of hnRNP E1, suggesting that hmRNP E1 might also act to suppress viral RNA translation. Deletion mutagenesis determined that the C-terminal end of hnRNP E1 was required for the reduction in Rev expression and that replacing this portion of hnRNP E1 with that of hnRNP E2, despite the high degree of conservation, could not rescue the loss of function.

Control of human immunodeficiency virus replication by the tat, rev, nef and protease genes.

Immediately after infection, human immunodeficiency virus directs the synthesis of three regulatory proteins tat, rev and nef that together allow the synthesis of the structural proteins of the virus after a delay of several hours. Viral mRNA production is controlled by the tat gene, which appears to stimulate elongation by RNA polymerase II, and the rev gene, which allows the accumulation of unspliced or partially spliced mRNAs in the cytoplasm. The nef gene is dispensible for virus growth but may limit virus spread by downregulating the levels of cellular surface proteins such as the CD4 receptor. Virus maturation also depends critically on the protease gene which allows the orderly rearrangement of the viral core structures in newly budded virions as well as the vpu and vif genes which allow efficient production of mature envelope glycoprotein.

High frequency of defective vpu compared with tat and rev genes in brain from patients with HIV type 1-associated dementia.

Human immunodeficiency virus type 1 (HIV) infection of the central nervous system frequently causes HIV-associated dementia (HAD) and other neurological disorders. The role of HIV regulatory and accessory proteins in the pathogenesis of these disorders is unclear. Here we analyzed sequences of tat, rev, and vpu genes in 55 subgenomic clones previously shown to encode functional env genes from brain and lymphoid tissues of four AIDS patients with HAD. Phylogenetic analysis showed distinct compartmentalization of tat, rev, and vpu genes in brain versus lymphoid tissues. Nine of 19 vpu sequences from brain of two patients had premature stop codons at positions between amino acids 2 and 30, compared with 0 of 8 from lymphoid tissues. Tat sequences from brain (n = 8 of 8) but not lymphoid (n = 0 of 6) tissue from one patient had a 35 amino acid truncation at the C-terminus. Rev sequences from the brain of one patient (n = 6 of 8) had a 5 amino acid truncation. These results demonstrate a high frequency of defective vpu compared with tat and rev genes in brain from HAD patients, and identify sequence variants of these regulatory/accessory genes that may influence the pathogenesis of HIV-associated neurological disease.