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In order to evaluate the role of persisting virus replication during occult phase immunisation in the live attenuated SIV vaccine model, a novel SIVmac239Δnef variant (SIVrtTA) genetically engineered to replicate in the presence of doxycycline was evaluated for its ability to protect against wild-type SIVmac239. Indian rhesus macaques were vaccinated either with SIVrtTA or with SIVmac239Δnef. Doxycycline was withdrawn from 4 of 8 SIVrtTA vaccinates before challenge with wild-type virus. Unvaccinated challenge controls exhibited ~107 peak plasma viral RNA copies/ml persisting beyond the acute phase. Six vaccinates, four SIVmac239Δnef and two SIVrtTA vaccinates exhibited complete protection, defined by lack of wild-type viraemia post-challenge and virus-specific PCR analysis of tissues recovered post-mortem, whereas six SIVrtTA vaccinates were protected from high levels of viraemia. Critically, the complete protection in two SIVrtTA vaccinates was associated with enhanced SIVrtTA replication in the immediate post-acute vaccination period but was independent of doxycycline status at the time of challenge. Mutations were identified in the LTR promoter region and rtTA gene that do not affect doxycycline-control but were associated with enhanced post-acute phase replication in protected vaccinates. High frequencies of total circulating CD8+T effector memory cells and a higher total frequency of SIV-specific CD8+ mono and polyfunctional T cells on the day of wild-type challenge were associated with complete protection but these parameters were not predictive of outcome when assessed 130 days after challenge. Moreover, challenge virus-specific Nef CD8+ polyfunctional T cell responses and antigen were detected in tissues post mortem in completely-protected macaques indicating post-challenge control of infection. Within the parameters of the study design, on-going occult-phase replication may not be absolutely required for protective immunity. [ABSTRACT FROM AUTHOR]

Conditionally replicating human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) variants that can be switched on and off at will are attractive tools for HIV and SIV research. We constructed HIV and SIV variants in which the natural transcription control mechanism was replaced by the doxycycline (dox)-inducible Tet-On gene expression mechanism. These HIV-rtTA and SIV-rtTA variants are fully replication-competent, but replication is critically dependent on dox administration. We here describe how the dox-dependent virus variants may improve the safety of live-attenuated virus vaccines and how they can be used to study the immune responses that correlate with vaccine-induced protection. Furthermore, we review how these variants were initially designed and subsequently optimized by spontaneous viral evolution. These efforts yielded efficiently replicating and tightly dox-controlled HIV-rtTA and SIV-rtTA variants that replicate in a variety of cell and tissue culture systems, and in human immune system (HIS) mice and macaques, respectively. These viruses can be used as a tool in HIV and SIV biology studies and in vaccine research. We review how HIV-rtTA and SIV-rtTA were used to study the role of the viral TAR and Tat elements in virus replication. [ABSTRACT FROM AUTHOR]

The 5' leader of the HIV-1 RNA genome encodes signals that control various steps in the replication cycle, including the dimerization initiation signal (DIS) that triggers RNA dimerization. The DIS folds a hairpin structure with a palindromic sequence in the loop that allows RNA dimerization via intermolecular kissing loop (KL) base pairing. The KL dimer can be stabilized by including the DIS stem nucleotides in the intermolecular base pairing, forming an extended dimer (ED). The role of the ED RNA dimer in HIV-1 replication has hardly been addressed because of technical challenges. We analyzed a set of leader mutants with a stabilized DIS hairpin for in vitro RNA dimerization and virus replication in T cells. In agreement with previous observations, DIS hairpin stability modulated KL and ED dimerization. An unexpected previous finding was that mutation of three nucleotides immediately upstream of the DIS hairpin significantly reduced in vitro ED formation. In this study, we tested such mutants in vivo for the importance of the ED in HIV-1 biology. Mutants with a stabilized DIS hairpin replicated less efficiently than WT HIV-1. This defect was most severe when the upstream sequence motif was altered. Virus evolution experiments with the defective mutants yielded fast replicating HIV-1 variants with second site mutations that (partially) restored the WT hairpin stability. Characterization of the mutant and revertant RNA molecules and the corresponding viruses confirmed the correlation between in vitro ED RNA dimer formation and efficient virus replication, thus indicating that the ED structure is important for HIV-1 replication. [ABSTRACT FROM AUTHOR]

The viral integrase (IN) is an essential protein for HIV-1 replication. IN inserts the viral dsDNA into the host chromosome, thereby aided by the cellular co-factor LEDGF/p75. Recently a new class of integrase inhibitors was described: allosteric IN inhibitors (ALLINIs). Although designed to interfere with the IN-LEDGF/p75 interaction to block HIV DNA integration during the early phase of HIV-1 replication, the major impact was surprisingly found on the process of virus maturation during the late phase, causing a reverse transcription defect upon infection of target cells. Virus particles produced in the presence of an ALLINI are misformed with the ribonucleoprotein located outside the virus core. Virus assembly and maturation are highly orchestrated and regulated processes in which several viral proteins and RNA molecules closely interact. It is therefore of interest to study whether ALLINIs have unpredicted pleiotropic effects on these RNA-related processes. We confirm that the ALLINI BI-D inhibits virus replication and that the produced virus is non-infectious. Furthermore, we show that the wild-type level of HIV-1 genomic RNA is packaged in virions and these genomes are in a dimeric state. The tRNAlys3 primer for reverse transcription was properly placed on this genomic RNA and could be extended ex vivo. In addition, the packaged reverse transcriptase enzyme was fully active when extracted from virions. As the RNA and enzyme components for reverse transcription are properly present in virions produced in the presence of BI-D, the inhibition of reverse transcription is likely to reflect the mislocalization of the components in the aberrant virus particle. [ABSTRACT FROM AUTHOR]

The 5' untranslated leader region of the human immunodeficiency virus type 1 (HIV-1) RNA genome is a strongly conserved sequence that encodes several regulatory motifs important for viral replication. Most of these motifs are exposed as hairpin structures, including the dimerization initiation signal (DIS), the major splice donor site (SD) and the packaging signal (Ψ), which are connected by short single-stranded regions. Mutational analysis revealed many functions of these hairpins, but only few studies have focused on the single-stranded purine-rich sequences. Using the in vivo SELEX approach we probed the sequence space in these regions that is compatible with efficient HIV-1 replication and we analyzed the impact on the RNA secondary structure of the leader RNA. Our results show a strong sequence requirement for the DIS hairpin flanking regions. We postulate that these sequences are important for the binding of specific protein factors that support leader RNA mediated functions. The sequence between the SD and Ψ hairpins seems to have a less prominent role, despite the strong conservation of the stretch of 5 A residues in natural isolates. We hypothesize that this may reflect the subtle evolutionary pressure on HIV-1 to acquire an A-rich RNA genome. In silico analyses indicate that sequences are avoided in all 3 single-stranded domains that affect the local or overall leader RNA folding. [ABSTRACT FROM AUTHOR]

Background: Two hairpin structures that are present at both the 5' and 3' end of the HIV-1 RNA genome have important functions in the viral life cycle. The TAR hairpin binds the viral Tat protein and is essential for Tat-mediated activation of transcription. The adjacent polyA hairpin encompasses the polyadenylation signal AAUAAA and is important for the regulation of polyadenylation. Specifically, this RNA structure represses polyadenylation at the 5' side, and enhancer elements on the 3' side overcome this suppression. We recently described that the replication of an HIV-1 variant that does not need TAR for transcription was severely impaired by destabilization of the TAR hairpin, even though a complete TAR deletion was acceptable. Results: In this study, we show that the TAR-destabilizing mutations result in reduced 3' polyadenylation of the viral transcripts due to an extension of the adjacent polyA hairpin. Thus, although the TAR hairpin is not directly involved in polyadenylation, mutations in TAR can affect this process. Conclusion: The stability of the HIV-1 TAR hairpin structure is important for the proper folding of the viral RNA transcripts. This study illustrates how mutations that are designed to study the function of a specific RNA structure can change the structural presentation of other RNA domains and thus affect viral replication in an indirect way. [ABSTRACT FROM AUTHOR]

Background: We have previously constructed a doxycycline (dox)-dependent HIV-1 variant by incorporating the Tet-On gene regulatory system into the viral genome. Replication of this HIVrtTA virus is driven by the dox-inducible transactivator protein rtTA, and can be switched on and off at will. We proposed this conditional-live virus as a novel vaccine approach against HIV-1. Upon vaccination, replication of HIV-rtTA can be temporarily activated by transient dox administration and controlled to the extent needed for optimal induction of the immune system. However, subsequent dox-withdrawal may impose a selection for virus variants with reduced dox-dependence. Results: We simulated this on/off switching of virus replication in multiple, independent cultures and could indeed select for HIV-rtTA variants that replicated without dox. Nearly all evolved variants had acquired a typical amino acid substitution at position 56 in the rtTA protein. We developed a novel rtTA variant that blocks this undesired evolutionary route and thus prevents HIV-rtTA from losing dox-control. Conclusion: The loss of dox-control observed upon evolution of the dox-dependent HIV-variant was effectively blocked by modification of the Tet-On regulatory system. [ABSTRACT FROM AUTHOR]

Live attenuated human immunodeficiency virus type 1 (HIV-1) vaccines are considered unsafe because faster-replicating pathogenic virus variants may evolve after vaccination. We previously presented a conditional-live HIV-1 variant of which replication can be switched off as an alternative vaccination strategy. To improve the safety of such a vaccine, we constructed a new HIV-1 variant that depends not only on doxycycline for gene expression but also on the T20 peptide for cell entry. Replication of this virus can be limited to the level required to induce the immune system by transient administration of doxycycline and T20. Subsequent withdrawal of these inducers efficiently blocks viral replication and evolution. [ABSTRACT FROM AUTHOR]

Short-term assays have suggested that RNA interference (RNAi) may be a powerful new method for intracellular immunization against human immunodeficiency virus type 1 (HIV-1) infection. However, RNAi has not yet been shown to protect cells against HIV-1 in long-term virus replication assays. We stably introduced vectors expressing small interfering RNAs (siRNAs) directed against the HIV-1 genome into human T cells by retroviral transduction. We report here that an siRNA directed against the viral Nef gene (siRNA-Nef) confers resistance to HIV-1 replication. This block in replication is not absolute, and HIV-1 escape variants that were no longer inhibited by siRNA-Nef appeared after several weeks of culture. These RNAi-resistant viruses contained nucleotide substitutions or deletions in the Nef gene that modified or deleted the siRNA-Nef target sequence. These results demonstrate that efficient inhibition of HIV-1 replication through RNAi is possible in stably transduced cells. Therefore, RNAi could become a realistic gene therapy approach with which to overcome the devastating effect of HIV-1 on the immune system. However, as is known for antiviral drug therapy against HIV-1, antiviral approaches involving RNAi should be used in a combined fashion to prevent the emergence of resistant viruses. [ABSTRACT FROM AUTHOR]

Novel therapeutic strategies aiming at the permanent inactivation of the HIV-1 reservoir in infected individuals are currently being explored, including approaches based on CRISPR-Cas gene editing. Extinction of all infectious HIV provirus in infected T-cell cultures was previously achieved when cells were transduced with lentiviral vectors for the stable expression of CRISPR-Cas9 or Cas12a systems targeting HIV DNA. Because lentiviral transduction and long-term CRISPR-Cas activity are less suitable for in vivo application of this antiviral strategy, we investigated whether HIV can also be completely inactivated by transient CRISPR-Cas activity. Latently infected SupT1 T-cells were repeatedly transfected with different Cas9 and Cas12a mRNA/protein sources in combination with dual gRNAs/crRNAs targeting highly conserved viral sequences. Upon repeated Cas9 protein treatment, viral replication could no longer be reactivated. We demonstrate that this was due to complete mutational inactivation of the proviral DNA, mostly through mutations at the target sites, but also through excision or inversion of the viral DNA fragment between the two target sites. These results demonstrate that repeated transient CRISPR-Cas treatment of a latently infected T-cell culture can lead to the permanent inactivation of HIV replication, indicating that transient CRISPR-Cas delivery methods can be considered for in vivo application. [ABSTRACT FROM AUTHOR]

In the quest for an effective vaccine against human immunodeficiency virus (HIV), live attenuated virus vaccines have proven to be very effective in the experimental model system of simian immunodeficiency virus (SIV) in macaques. However, live attenuated HIV vaccines are considered unsafe for use in humans because the attenuated virus may accumulate genetic changes during persistence and evolve to a pathogenic variant. As an alternative approach, we earlier presented a conditionally live HIV-1 variant that replicates exclusively in the presence of doxycycline (DOX). Replication of this vaccine strain can be limited to the time that is needed to provide full protection through transient DOX administration. Since the effectiveness and safety of such a conditionally live AIDS vaccine should be tested in macaques, we constructed a similar DOX-dependent SIVmac239 variant in which the Tat-TAR (trans-acting responsive) transcription control mechanism was functionally replaced by the DOX-inducible Tet-On regulatory mechanism. Moreover, this virus can be used as a tool in SIV biology studies and vaccine research because both the level and duration of replication can be controlled by DOX administration. Unexpectedly, the new SIV variant required a wild-type Tat protein for replication, although gene expression was fully controlled by the incorporated Tet-On system. This result suggests that Tat has a second function in SIV replication in addition to its role in the activation of transcription. [ABSTRACT FROM AUTHOR]

The human immunodeficiency virus type 1 (HIV-1) RNA genome contains a terminal repeat (R) region that encodes the transacting responsive (TAR) hairpin, which is essential for Tat-mediated activation of gene expression. TAR has also been implicated in several other processes during viral replication, including translation, dimerization, packaging, and reverse transcription. However, most studies in which replication of TAR-mutated viruses was analyzed were complicated by the dominant negative effect of the mutations on transcription. We therefore used an HIV-1 variant that does not require TAR for transcription to reinvestigate the role of TAR in HIV-1 replication. We demonstrate that this virus can replicate efficiently upon complete deletion of TAR. Furthermore, evolution of a TAR-deleted variant in long-term cultures indicates that HIV-1 requires a stable stem-loop structure at the start of the viral transcripts in which the 5′-terminal nucleotides are base paired. This prerequisite for efficient replication can be fulfilled by the TAR hairpin but also by unrelated stem-loop structures. We therefore conclude that TAR has no essential function in HIV-1 replication other than to accommodate Tat-mediated activation of transcription. [ABSTRACT FROM AUTHOR]

The untranslated leader of the HIV-1 RNA genome is highly structured and contains multiple replication signals. We probed in detail the sequence requirements of a small single-stranded domain using a combination of in silico, in vitro and in vivo virus experiments. Although ‘structure-neutral’ mutations can be designed by RNA prediction algorithms, experimental follow-up studies nearly always demonstrate local or regional RNA structure changes. Our results indicate that the wild-type HIV-1 RNA sequence has been selected from total sequence space as a unique solution to present critical replication signals in the context of a complex leader structure with small intervening single-stranded segments. [ABSTRACT FROM AUTHOR]

Productive infection and successful replication of human immunodeficiency virus 1 (HIV-1) requires the balanced expression of all viral genes. This is achieved by a combination of alternative splicing events and regulated nuclear export of viral RNA. Because viral splicing is incomplete and intron-containing RNAs must be exported from the nucleus where they are normally retained, it must be ensured that the unspliced HIV-1 RNA is actively exported from the nucleus and protected from degradation by processes such as nonsense-mediated decay. Here we report the identification of a novel 178-nt-long exon located in the gag-pol gene of HIV-1 and its inclusion in at least two different mRNA species. Although efficiently spliced in vitro, this exon appears to be tightly repressed and infrequently used in vivo. The splicing is activated or repressed in vitro by the splicing factors ASF/SF2 and heterogeneous nuclear ribonucleoprotein A1, respectively, suggesting that splicing is controlled by these factors. Interestingly, mutations in the 5′-splice site resulted in a dramatic reduction in the steady-state level of HIV-1 RNA, and this effect was partially reversed by expression of U1 small nuclear RNA harboring the compensatory mutation. This implies that U1 small nuclear RNA binding to optimal but non-functional splice sites might have a role in protecting unspliced HIV-1 mRNA from degradation. [ABSTRACT FROM AUTHOR]

Conditionally replicating HIV-1 variants that can be switched on and off at will are attractive tools for HIV research. We previously developed a genetically modified HIV-1 variant that replicates exclusively when doxycycline (dox) is administered. The nef gene in this HIV-rtTA variant was replaced with the gene encoding the dox-dependent rtTA transcriptional activator. Because loss of Nef expression compromises virus replication in primary cells and precludes studies on Nef function, we tested different approaches to restore Nef production in HIV-rtTA. Strategies that involved translation via an EMCV or synthetic internal ribosome entry site (IRES) failed because these elements were incompatible with efficient virus replication. Fusion protein approaches with the FMDV 2A peptide and human ubiquitin were successful and resulted in genetically-stable Nef-expressing HIV-rtTA strains that replicate more efficiently in primary T-cells and human immune system (HIS) mice than Nef-deficient variants, thus confirming the positive effect of Nef on in vivo virus replication. [ABSTRACT FROM AUTHOR]

The 5′ leader region of the HIV-1 RNA contains the major 5′ splice site (ss) that is used in the production of all spliced viral RNAs. This splice-donor (SD) region can fold a stem-loop structure. We demonstrate that whereas stabilization of this SD hairpin reduces splicing efficiency, destabilization increases splicing. Both stabilization and destabilization reduce viral fitness. These results demonstrate that the stability of the SD hairpin can modulate the level of splicing, most likely by controlling the accessibility of the 5′ss for the splicing machinery. The natural stability of the SD hairpin restricts splicing and this stability seems to be fine-tuned to reach the optimal balance between unspliced and spliced RNAs for efficient virus replication. The 5′ss region of different HIV-1 isolates and the related SIVmac239 can fold a similar structure. This evolutionary conservation supports the importance of this structure in viral replication. [ABSTRACT FROM AUTHOR]

Human pathogenic viruses can be targeted by therapeutic strategies based on RNA interference. Whereas the administration of synthetic short interfering RNAs (siRNAs) may transiently inhibit viral replication, long-term inhibition may be achieved through stable intracellular expression of siRNAs or short hairpin RNAs (shRNAs). Both approaches face serious problems with delivery to the right cells in an infected individual. We explored the potential of a replicating HIV-based vector to deliver an antiviral shRNA cassette into HIV-1-susceptible target cells to block chronic HIV-1 infection. The vector is based on a doxycycline (dox)-dependent HIV-1 variant that we previously proposed as a conditional-live HIV-1 vaccine. With dox, this virus spreads efficiently to all HIV-susceptible cells. Subsequent dox withdrawal generates cells with a transcriptionally silent integrated provirus, but with an active shRNA expression cassette. Because the shRNA targets viral sequences that are removed from the vector construct, there is no self-targeting, yet there is specific shutdown of HIV-1 replication.Molecular Therapy (2006) 14, 268–275; doi: 10.1016/j.ymthe.2006.03.018 [ABSTRACT FROM AUTHOR]