B Berkhout

Structure and Function of the Human Immunodeficiency Virus Leader RNA

Publisher Summary This chapter deals with the structure and function of the leader transcript of human immunodeficiency virus (HIV-1) and HIV-2. Most of the RNA signals encoded by the untranslated leader RNA have specific nucleotide sequences critical for recognition and function, but there is accumulating evidence that their structural context can also be important. There is an intense effort to analyze the secondary structure of retroviral leader RNAs using a variety of methods, including biochemical analysis, free-energy minimization, sequence comparison, and mutant analysis. The chapter focuses primarily on the relationships between the structure of specific leader RNA motifs and their function in the retroviral life cycle. Among retroviruses, the lentiviruses have the most complex genome structure and expression strategy. The primate lentiviruses include the human and simian immunodeficiency viruses (SIV). Understanding the three-dimensional structure formed by the HIV leader RNA molecule, both free and in the ribonucleoprotein complex of virion, is crucial for analyzing its specific recognition by proteins and its interaction by other RNA molecules during virus replication. The number of HIV–SIV sequences now known is sufficiently large; hence, comparative analysis can be used effectively to deduce some of the basic design principles underlying HIV RNA structure. By understanding the molecular basis of the interactions that govern critical steps in the retroviral replication cycle, it may be possible to develop methods to intervene therapeutically in the process.

Optimization of the Tet-On system for regulated gene expression through viral evolution

The ability to control (trans)gene expression is important both for basic biological research and applications such as gene therapy. In vivo use of the inducible tetracycline (Tc)-regulated gene expression system (Tet-On system) is limited by its low sensitivity for the effector doxycycline (dox). We used viral evolution to optimize this Escherichia coli-derived regulatory system for its function in mammalian cells. The components of the Tet-On system (the transcriptional activator rtTA and its tetO DNA binding site) were incorporated into the human immunodeficiency virus (HIV)-1 genome to control viral replication. Prolonged culturing of this HIV-rtTA virus resulted in virus variants that acquired mutations in the rtTA gene. Some of these mutations enhance the transcriptional activity and dox-sensitivity of the rtTA protein. This improvement was observed with different tetO-containing promoters and was independent of the episomal or chromosomal status of the target gene. Combination of these beneficial mutations resulted in greatly improved rtTA variants that are seven-fold more active and 100-fold more dox-sensitive than the original Tet-On system. Furthermore, some of the new Tet-On systems are responsive to Tc and minocycline. Importantly, these rtTA variants show no activity in the absence of dox. The optimized rtTA variants are particularly useful for in vivo applications that require a more sensitive or more active Tet-On system.

Evaluation of safety and efficacy of RNAi against HIV-1 in the human immune system (Rag-2(-/-)gammac(-/-)) mouse model

RNA interference (RNAi) gene therapy against HIV-1 by stable expression of antiviral short hairpin RNAs (shRNAs) can potently inhibit viral replication in T cells. Recently, a mouse model with a human immune system (HIS) was developed that can be productively infected with HIV-1. In this in vivo model, in which Rag-2−/−γc−/− mice are engrafted with human CD34+CD38− hematopoietic precursor cells, we evaluated an anti-HIV RNAi gene therapy. Human hematopoietic stem cells were transduced with a lentiviral vector expressing an shRNA against the HIV-1 nef gene (shNef) or the control vector. We observed normal development of the different cell subsets of the immune system. However, although initial transduction efficiencies were similar for both vectors, a reduced percentage of transduced human immune cells was observed for the shNef vector after establishment of the HIS in vivo. Further studies are required to fully evaluate the safety implications. When we infected the mature human CD4+ T cells from the HIS mouse ex vivo with HIV-1, potent inhibition of viral replication was scored in shNef-expressing cells, confirming efficacy. When challenged with an shNef-resistant HIV-1 variant, equal replication was scored in control and shNef-expressing cells, confirming sequence-specificity of the RNAi therapy. We thus demonstrated that an antiviral RNAi-based gene therapy on blood stem cells leads to HIV-1-resistant T cells in vivo, an important proof of concept in the clinical development of RNAi against HIV-1.

Inhibition of human immunodeficiency virus type 1 by RNA interference using long-hairpin RNA

Inhibition of virus replication by means of RNA interference has been reported for several important human pathogens, including human immunodeficiency virus type 1 (HIV-1). RNA interference against these pathogens has been accomplished by introduction of virus-specific synthetic small interfering RNAs (siRNAs) or DNA constructs encoding short-hairpin RNAs (shRNAs). Their use as therapeutic antiviral against HIV-1 is limited, because of the emergence of viral escape mutants. In order to solve this durability problem, we tested DNA constructs encoding virus-specific long-hairpin RNAs (lhRNAs) for their ability to inhibit HIV-1 production. Expression of lhRNAs in mammalian cells may result in the synthesis of many siRNAs targeting different viral sequences, thus providing more potent inhibition and reducing the chance of viral escape. The lhRNA constructs were compared with in vitro diced double-stranded RNA and a DNA construct encoding an effective nef-specific shRNA for their ability to inhibit HIV-1 production in cells. Our results show that DNA constructs encoding virus-specific lhRNAs are capable of inhibiting HIV-1 production in a sequence-specific manner, without inducing the class I interferon genes.

Increased virus replication in mammalian cells by blocking intracellular innate defense responses

The mammalian innate immune system senses viral infection by recognizing viral signatures and activates potent antiviral responses. Besides the interferon (IFN) response, there is accumulating evidence that RNA silencing or RNA interference (RNAi) serves as an antiviral mechanism in mammalian cells. Mammalian viruses encode IFN antagonists to counteract the IFN response in infected cells. A number of IFN antagonists are also capable of blocking RNAi in infected cells and therefore serve as RNA-silencing suppressors. Virus replication in infected cells is restricted by these innate antiviral mechanisms, which may kick in earlier than the viral antagonistic or suppressor protein can accumulate. The yield of virus vaccines and viral gene delivery vectors produced in mammalian producer cells may therefore be suboptimal. To investigate whether blocking of the innate antiviral responses in mammalian cells leads to increased viral vector production, we expressed a number of immunity suppressors derived from plant and mammalian viruses in human cells. We measured that the yield of infectious human immunodeficiency virus-1 particles produced in these cells was increased 5- to 10-fold. In addition, the production of lentiviral and adenoviral vector particles was increased 5- to 10-fold, whereas Sindbis virus particle production was increased approximately 100-fold. These results can be employed for improving the production of viral gene transfer vectors and viral vaccine strains.

Autoregulatory lentiviral vectors allow multiple cycles of doxycycline-inducible gene expression in human hematopoietic cells in vivo

The efficient control of gene expression in vivo from lentiviral vectors remains technically challenging. To analyze inducible gene expression in a human setting, we generated ‘human immune system’ (HIS) mice by transplanting newborn BALB/c Rag2−/−IL-2Rγc−/− immunodeficient mice with human hematopoietic stem cells transduced with a doxycycline-inducible lentiviral vector. We compared several methods of doxycycline delivery to mice, and could accurately measure doxycycline in vivo using a new sensitive detection assay. Two different lentiviral vector designs with constitutive (TRECMV-V14) or autoregulatory (TREAuto-V14) expression of an optimized reverse tetracycline transactivator were used to transduce human hematopoietic stem cells. After transplantation into immunodeficient mice, we analyzed the expression of the green fluorescent protein (GFP) reporter gene in the human hematopoiesis-derived cells that develop and accumulate in the generated HIS mice. We show efficient inducible GFP expression in adult HIS mice containing TREAuto-V14-transduced human cells, whereas GFP expression is poor with the TRECMV-V14 vector. Multiple cycles of doxycycline exposure in the TREAuto-V14 group result in repeated cycles of GFP expression with no loss of intensity. These findings are of major interest for gene therapy and basic research settings that require inducible gene expression.

Novel vaccine and gene therapy approaches against HIV-AIDS

The design of novel anti-HIV vaccination and gene therapy strategies will be presented, including pilot experiments in animal models. Our gene therapy approach uses the RNA interference (RNAi) machinery to make human cells resistant to HIV-1. The problem of viral escape and the control of viral escape by means of a combination-RNAi therapy will be discussed. A humanized mouse model has been set up as pre-clinical test system to address the safety and efficacy of lentiviral vector-delivered RNAi cassettes. Our vaccination approach deals with the novel concept of a conditional-live virus that can be turned on and off at will. Live-attenuated virus confers the most potent protection against wild-type virus challenge in the SIV/macaque vaccination model. However, such a vaccine is not pursued because of safety reasons as the vaccine virus persists and may evolve into a pathogenic variant. We therefore designed an SIVmac239Δnef variant that is dependent on doxycycline for replication by replacing the natural Tat/TAR transcription mechanism by the Tet-system for inducible gene expression. Replication of this virus can be switched off after vaccination, which will prevent evolution. The first rhesus macaque test yielded a marked vaccine effect. This new conditional-live virus will be a useful tool to identify the correlates of protection by this vaccine strategy.

P11-10. Modulation of intestinal T cells following infection of macaques with live attenuated and conditionally replication-competent SIV

Background Live-attenuated SIV can induce superinfection resistance; however, the mechanism of this effect is not understood and may have implications for HIV vaccine development. To further investigate the role of virus replication in conferring protection we have analysed T cell phenotype and responses in gut tissue following infection of macaques with either SIVmac239Δnef or a doxycycline-dependent replication variant of SIVmac239 Δnef designated SIVrtTA.

P03-08. Protection against SIVmac239 challenge in a rhesus macaque model conferred by a doxycycline dependent attenuated SIVmac239

Background In the SIV/macaque model of HIV in man, live attenuated virus confers the most potent protection against wild-type virus challenge. We have developed a novel live attenuated (nef deleted) vaccine SIVmac239 (SIVrtTA) that is dependent on doxycycline for replication in vivo. Withdrawal of doxycycline prevents replication of SIVrtTA. We used this virus to vaccinate rhesus macaques to investigate the role of virus replication and persistence in protection against SIVmac239 challenge.