CBP80/20-dependent translation initiation factor (CTIF) inhibits HIV-1 Gag synthesis by targeting the function of the viral protein Rev.

Translation initiation of the human immunodeficiency virus type-1 (HIV-1) full-length RNA has been shown to occur through cap-dependent and IRES-driven mechanisms. Previous studies suggested that the nuclear cap-binding complex (CBC) rather than eIF4E drives cap-dependent translation of the full-length RNA and we have recently reported that the CBC subunit CBP80 supports the function of the viral protein Rev during nuclear export and translation of this viral transcript. Ribosome recruitment during CBC-dependent translation of cellular mRNAs relies on the activity CBP80/20 translation initiation factor (CTIF), which bridges CBP80 and the 40S ribosomal subunit through interactions with eIF3g. Here, we report that CTIF inhibits HIV-1 and HIV-2 Gag synthesis from the full-length RNA. Our results indicate that CTIF associates with HIV-1 Rev through its N-terminal domain and is recruited onto the full-length RNA ribonucleoprotein complex in order to interfere with Gag synthesis. We also demonstrate that CTIF induces the cytoplasmic accumulation of Rev impeding the association of the viral protein with CBP80. We finally show that Rev interferes with the association of CTIF with CBP80 indicating that CTIF and Rev compete for the CBC subunit.

HIV-1 Rev multimerization: mechanism and insights.

To export intron-containing viral mRNAs that encode the structural components of new viral particles from the nucleus to the cytoplasm, HIV-1 uses the cellular CRM1 export pathway that is exploited by the viral Rev protein. Rev multimerizes on the Rev response element (RRE) present in the intron-containing RNA species to bridge these to the cellular export factor CRM1. As a result, this Rev-RRE complex is exported to the cytoplasm. This review provides a systematic overview of different aspects of the crucial function of Rev multimerization, such as co-operative Rev-Rev and Rev-RNA interactions, the biological function of Rev multimerization, the relevance of Rev multimerization in the absence of RRE and its potential as a therapeutic target.

Human immunodeficiency virus type 1 (HIV-1) transactivator of transcription through its intact core and cysteine-rich domains inhibits Wnt/ß-catenin signaling in astrocytes: relevance to HIV neuropathogenesis.

Wnt/ß-catenin is a neuroprotective pathway regulating cell fate commitment in the CNS and many vital functions of neurons and glia. Its dysregulation is linked to a number of neurodegenerative diseases. Wnt/ß-catenin is also a repressor of HIV transcription in multiple cell types, including astrocytes, which are dysregulated in HIV-associated neurocognitive disorder. Given that HIV proteins can overcome host restriction factors and that perturbations of Wnt/ß-catenin signaling can compromise astrocyte function, we evaluated the impact of HIV transactivator of transcription (Tat) on Wnt/ß-catenin signaling in astrocytes. HIV clade B Tat, in primary progenitor-derived astrocytes and U87MG cells, inhibited Wnt/ß-catenin signaling as demonstrated by its inhibition of active ß-catenin, TOPflash reporter activity, and Axin-2 (a downstream target of Wnt/ß-catenin signaling). Point mutations in either the core region (K41A) or the cysteine-rich region (C30G) of Tat abrogated its ability to inhibit ß-catenin signaling. Clade C Tat, which lacks the dicysteine motif, did not alter ß-catenin signaling, confirming that the dicysteine motif is critical for Tat inhibition of ß-catenin signaling. Tat coprecipitated with TCF-4 (a transcription factor that partners with ß-catenin), suggesting a physical interaction between these two proteins. Furthermore, knockdown of ß-catenin or TCF-4 enhanced docking of Tat at the TAR region of the HIV long terminal repeat. These findings highlight a bidirectional interference between Tat and Wnt/ß-catenin that negatively impacts their cognate target genes. The consequences of this interaction include alleviation of Wnt/ß-catenin-mediated suppression of HIV and possible astrocyte dysregulation contributing to HIV neuropathogenesis.

The HIV-1 Rev protein enhances encapsidation of unspliced and spliced, RRE-containing lentiviral vector RNA.

BACKGROUND: During the RNA encapsidation process of human immunodeficiency virus (HIV) viral genomic, unspliced RNA (gRNA) is preferentially incorporated into assembling virions. However, a certain amount of spliced viral transcripts can also be detected in viral particles. Recently, we observed that nuclear export of HIV and lentiviral vector gRNA by Rev is required for efficient encapsidation. Since singly-spliced HIV transcripts also contain the Rev-response element (RRE), we investigated if the encapsidation efficiency of RRE-containing spliced HIV-vector transcripts is also increased by the viral Rev protein. FINDINGS: Starting with a lentiviral vector imitating the splicing pattern of HIV, we constructed vectors that express an unspliced transcript either identical in sequence to the singly-spliced or the fully-spliced RNA of the parental construct. After transfection of the different lentiviral vectors cytoplasmic and virion-associated RNA levels and vector titers were determined in the presence and absence of Rev. Rev enhanced the infectious titer of vectors containing an RRE 6 to 37-fold. Furthermore, Rev strongly increased encapsidation efficiencies of all RRE-containing transcripts up to 200-fold. However, a good correlation between encapsidation efficiency and lentiviral vector titer could only be observed for the gRNA. The infectious titer of the vector encoding the fully-spliced RNA without RRE as well as the encapsidation efficiency of all transcripts lacking the RRE was not influenced by Rev. Interestingly, the splicing process itself did not seem to interfere with packaging, since the encapsidation efficiencies of the same RNA expressed either by splicing or as an unspliced transcript did not differ significantly. CONCLUSIONS: Rev-mediated nuclear export enhances the encapsidation efficiency of RRE-containing lentiviral vector RNAs independently of whether they have been spliced or not.

Production of HIV particles is regulated by altering sub-cellular localization and dynamics of Rev induced by double-strand RNA binding protein.

Human immunodeficiency virus (HIV)-1 encoded Rev is essential for export from the nucleus to the cytoplasm, of unspliced and singly spliced transcripts coding for structural and nonstructural viral proteins. This process is spatially and temporally coordinated resulting from the interactions between cellular and viral proteins. Here we examined the effects of the sub-cellular localization and dynamics of Rev on the efficiency of nucleocytoplasmic transport of HIV-1 Gag transcripts and virus particle production. Using confocal microscopy and fluorescence recovery after bleaching (FRAP), we report that NF90ctv, a cellular protein involved in Rev function, alters both the sub-cellular localization and dynamics of Rev in vivo, which drastically affects the accumulation of the viral protein p24. The CRM1-dependent nuclear export of Gag mRNA linked to the Rev Response Element (RRE) is dependent on specific domains of the NF90ctv protein. Taken together, our results demonstrate that the appropriate intracellular localization and dynamics of Rev could regulate Gag assembly and HIV-1 replication.

Structural basis for cooperative RNA binding and export complex assembly by HIV Rev.

HIV replication requires nuclear export of unspliced viral RNAs to translate structural proteins and package genomic RNA. Export is mediated by cooperative binding of the Rev protein to the Rev response element (RRE) RNA, to form a highly specific oligomeric ribonucleoprotein (RNP) that binds to the Crm1 host export factor. To understand how protein oligomerization generates cooperativity and specificity for RRE binding, we solved the crystal structure of a Rev dimer at 2.5-Å resolution. The dimer arrangement organizes arginine-rich helices at the ends of a V-shaped assembly to bind adjacent RNA sites and structurally couple dimerization and RNA recognition. A second protein-protein interface arranges higher-order Rev oligomers to act as an adaptor to the host export machinery, with viral RNA bound to one face and Crm1 to another, the oligomers thereby using small, interconnected modules to physically arrange the RNP for efficient export.

The human immunodeficiency virus type 1 Rev protein: ménage à trois during the early phase of the lentiviral replication cycle.

The Rev protein of human immunodeficiency viruses (HIV) has long been recognized to be essential for the late phase of the virus replication cycle, due to its strong enhancement of expression of viral structural proteins. Surprisingly, a number of recent papers have demonstrated that Rev can also interfere with integration of the reverse-transcribed cDNA into the host-cell genome. This seems to be due to Rev's binding to integrase and LEDGF/p75, an important cellular cofactor of HIV-1 integration. As Rev is presumably expressed at sufficiently high levels only after the encoding genome has already integrated, the main function of Rev during the early phase might be to reduce genotoxicity due to excessive integration events after superinfection of the same cell by subsequent viruses. Other potential consequences for HIV-1 replication and evolution after co-infection of the same cell with two viruses are discussed.

Preintegration HIV-1 inhibition by a combination lentiviral vector containing a chimeric TRIM5 alpha protein, a CCR5 shRNA, and a TAR decoy.

Human immunodeficiency virus (HIV) gene therapy offers a promising alternative approach to current antiretroviral treatments to inhibit HIV-1 infection. Various stages of the HIV life cycle including pre-entry, preintegration, and postintegration can be targeted by gene therapy to block viral infection and replication. By combining multiple highly potent anti-HIV transgenes in a single gene therapy vector, HIV-1 resistance can be achieved in transduced cells while prohibiting the generation of escape mutants. Here, we describe a combination lentiviral vector that encodes three highly effective anti-HIV genes functioning at separate stages of the viral life cycle including a CCR5 short hairpin RNA (shRNA) (pre-entry), a human/rhesus macaque chimeric TRIM5 alpha (postentry/preintegration), and a transactivation response element (TAR) decoy (postintegration). The major focus on designing this anti-HIV vector was to block productive infection of HIV-1 and to inhibit any formation of provirus that would maintain the viral reservoir. Upon viral challenge, potent preintegration inhibition of HIV-1 infection was achieved in combination vector-transduced cells in both cultured and primary CD34(+) hematopoietic progenitor cell (HPC)-derived macrophages. The generation of escape mutants was also blocked as evaluated by long-term culture of challenged cells. The ability of this combination anti-HIV lentiviral vector to prevent HIV-1 infection, in vitro, warrants further evaluation of its in vivo efficacy.

HIV-1 Rev protein assembles on viral RNA one molecule at a time.

Oligomerization of the HIV-1 protein Rev on the Rev Response Element (RRE) regulates nuclear export of genomic viral RNA and partially spliced viral mRNAs encoding for structural proteins. Single-molecule fluorescence spectroscopy has been used to dissect the multistep assembly pathway of this essential ribonucleoprotein, revealing dynamic intermediates and the mechanism of assembly. Assembly is initiated by binding of Rev to a high-affinity site in stem-loop IIB of the RRE and proceeds rapidly by addition of single Rev monomers, facilitated by cooperative Rev-Rev interactions on the RRE. Dwell-time analysis of fluorescence trajectories recorded during individual Rev-RRE assembly reactions has revealed the microscopic rate constants for several of the Rev monomer binding and dissociation steps. The high-affinity binding of multiple Rev monomers to the RRE is achieved on a much faster timescale than reported in previous bulk kinetic studies of Rev-RRE association, indicating that oligomerization is an early step in complex assembly.

TAT-PAX6 protein transduction in neural progenitor cells: a novel approach for generation of dopaminergic neurones in vitro.

Neural stem cells (NSCs) have the potential to be used for the treatment of Parkinson's disease (PD), as they can be expanded, manipulated and differentiated in vitro to generate dopaminergic neurones which are suitable for transplantation. Since NSCs have a tendency to follow an astrocytic lineage after differentiation in vitro, researchers are investigating ways to induce a neuronal phenotype in these cells. In this study, the human immunodeficiency virus 1 (HIV-1) transactivator of transcription (TAT) protein transduction domain (PTD) system was used in an attempt to promote neuronal differentiation in rodent NSCs. A fusion protein that incorporated both the TAT PTD and the Pax6 protein (a determinant of neurogenesis) was created and added to the differentiation phase of embryonic day (E) 12 rat ventral mesencephalic (VM) neurosphere cultures. Subsequently, application of dopaminergic growth factors (GFs) was used in an attempt to induce the newly-generated neuronal progenitors to adopt a dopaminergic phenotype. In addition, a technique involving the differentiation of intact neurospheres (instead of the differentiation of neurosphere-derived dissociated cells) was investigated for its ability to promote dopaminergic neurogenesis. Immunocytochemical analysis of the differentiated neurosphere cultures indicated that both of these techniques had a significant effect on the emergence of dopaminergic neurones. Moreover, upon combination of these techniques, a further increase in dopaminergic neuronal generation was observed. Based on the findings of the present study, it is clear that NSCs are greatly influenced by their environment and that optimised in vitro conditions can support the potential of these cells to differentiate into dopaminergic neurones.

Oral malignancies associated with HIV.

Advances in the management of HIV infection have resulted in significant changes in survival and in the prevalence and incidence of oral diseases found in persons infected with HIV (as discussed in other articles in this series). HIV is associated with an increased risk of malignant disease that is related to immunosuppression and the activity of the HIV transactivator of transcription protein, coviral infection and exposure to carcinogens. The presence of oral malignancies varies with the route of the transmission of HIV and varies geographically, based on behaviour, viral cofactors, HIV therapy and genetic variation. Oral health care providers can identify these lesions early.