[Show abstract][Hide abstract] ABSTRACT: The 5' leader region of the HIV-1 RNA genome contains the major 5' splice site (ss) that is used in the production of the many spliced viral RNAs. This splice-donor (SD) region can fold into a stable stem-loop structure and the thermodynamic stability of this RNA hairpin influences splicing efficiency. In addition, splicing may be modulated by binding of splicing-regulatory proteins, in particular SR proteins SF2/ASF (SRSF1), SC35 (SRSF2), SRp40 (SRSF5) and SRp55 (SRSF6), to sequence elements in the SD region. The role of RNA structure and SR protein binding in splicing control was previously studied by functional analysis of mutant SD sequences. The interpretation of these studies was complicated by the fact that most mutations simultaneously affect both structure and sequence elements. We therefore tried to disentangle the contribution of these two variables by designing more precise SD region mutants with a single effect on either the sequence or the structure. The current analysis indicates that HIV-1 splicing at the major 5'ss is modulated by both the stability of the local RNA structure and the binding of splicing regulatory proteins.
Journal of General Virology 03/2015; DOI:10.1099/vir.0.000122 · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This review summarizes recent findings concerning the ever-growing HIV-1 RNA population.
The retrovirus HIV-1 has an RNA genome that is converted into DNA and is integrated into the genome of the infected host cell. Transcription from the long terminal repeat-encoded promoter results in the production of a full-length genomic RNA and multiple spliced mRNAs. Recent experiments, mainly based on next-generation sequencing, provided evidence for several additional HIV-encoded RNAs, including antisense RNAs and virus-encoded microRNAs.
We will survey recent findings related to HIV-1 RNA biosynthesis, especially regulatory mechanisms that control initiation of transcription, capping and polyadenylation. We zoom in on the diversity of HIV-1 derived RNA transcripts, their mode of synthesis and proposed functions in the infected cell. Special attention is paid to the viral transacting responsive RNA hairpin motif that has been suggested to encode microRNAs.
[Show abstract][Hide abstract] ABSTRACT: 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 wild-type (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.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: A r t i c l e s Current methods in genetic diagnostics and research are limited in their ability to uncover all possible genetic variation in genes of inter-est 1 . Clinical genetic tests, for example, often focus only on exons and therefore miss variants in the noncoding regulatory sequences (pro-moters and enhancers) of genes that can also disrupt gene function 2 . In addition, structural variants—such as copy number variants, trans-locations, insertions and inversions—are difficult to detect, particu-larly those that are balanced (i.e., inversions and translocations that are not accompanied by a loss or gain of sequences). Also, balanced structural variants can cause disease through the disruption of genes, creation of gene fusions or position effects—i.e., when a gene is placed under the control of different regulatory DNA elements 3,4 . Reliable detection of structural variants is hampered by the hypothesis-driven nature of current methods for targeted re-sequencing, in which the sequences to be analyzed are determined by the set of probes used in hybridization-based capture methods 5 or the primers in polymerase or ligase-based re-sequencing approaches 6 . Unknown sequences, such as those introduced by chromosomal rearrangements, are difficult to capture and re-sequence with these methods 7,8 . In addition, none of the existing targeted sequencing methods allow haplotyping, the allelic phasing of genetic variation. This information is useful, for example, when a person carries multiple recessive genetic variants that may coexist on one allele (giving carrier status) or be divided over both alleles (giving disease status). In chromosome-conformation capture (3C) 9 and related methods such as 3C on chip or combined with sequencing 10,11 (4C, which ena-bles searching of the genome for sequences contacting a site of inter-est), chromatin is crosslinked, fragmented and re-ligated to identify, on the basis of their ligation efficiency, genomic loci that are in close spatial proximity in the nucleus. Although these methods are used to detect genome folding inside cells, the resulting contact profiles typically show high enrichment of sequences directly neighboring targeted sequencing by proximity ligation for comprehensive variant detection and local haplotyping
[Show abstract][Hide abstract] ABSTRACT: 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.
PLoS ONE 07/2014; 9(7):e103552. DOI:10.1371/journal.pone.0103552 · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Eukaryotic cells and several DNA viruses encode miRNAs to regulate the expression of specific target genes. It has been controversial whether RNA viruses can encode such miRNAs as miRNA excision may lead to cleavage of the viral RNA genome. We will focus on the retrovirus family, HIV-1 in particular, and discuss the production of virus-encoded miRNAs and their putative function in the viral replication cycle. An intricate scenario of multi-layer virus-host interactions becomes apparent with small RNAs as the regulatory molecules.
[Show abstract][Hide abstract] ABSTRACT: Eukaryotic cells and several DNA viruses encode miRNAs to regulate the expression of specific target genes. It has been controversial whether RNA viruses can encode such miRNAs as miRNA excision may lead to cleavage of the viral RNA genome. We will focus on the retrovirus family, HIV-1 in particular, and discuss the production of virus-encoded miRNAs and their putative function in the viral replication cycle. An intricate scenario of multi-layer virus–host interactions becomes apparent with small RNAs as the regulatory molecules.
Current Opinion in Virology 01/2014; 7:47–54. · 6.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Journal of Virology 12/2013; 88(4). DOI:10.1128/JVI.02942-13 · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background
Live attenuated SIV induces potent protection against superinfection with virulent virus; however the mechanism of this vaccine effect is poorly understood. Such knowledge is important for the development of clinically acceptable vaccine modalities against HIV.
Using a novel, doxycycline dependent, replication-competent live-attenuated SIVmac239Δnef (SIV-rtTAΔnef), we show that under replication-permissive conditions SIV-rtTAΔnef is fully viable. Twelve rhesus macaques were infected with a peak plasma vRNA on average two log10 lower than in 6 macaques infected with unconditionally replication-competent SIVΔnef. Consistent with the attenuated phenotype of the viruses the majority of animals displayed low or undetectable levels of viraemia by 42-84 days after infection. Next, comparison of circulating T cells before and after chronic infection with parental SIVΔnef revealed a profound global polarisation toward CD28-CCR7- T-effector memory 2 (TEM2) cells within CD95+CD4+ and CD95+CD8+ populations. Critically, a similar effect was seen in the CD95+ CD4+ population and to somewhat lesser extent in the CD95+ CD8+ population of SIV-rtTAΔnef chronically infected macaques that were maintained on doxycycline, but was not seen in animals from which doxycycline had been withdrawn. The proportions of gut-homing T-central memory (TCM) and TEM defined by the expression of α4β7 and CD95 and differential expression of CD28 were increased in CD4 and CD8 cells under replication competent conditions and gut-homing CD4 TCM were also significantly increased under non-permissive conditions. TEM2 polarisation was seen in the small intestines of animals under replication permissive conditions but the effect was less pronounced than in the circulation. Intracellular cytokine staining of circulating SIV-specific T cells for IL-2, IFN-γ, TNF-α and IL-17 showed that the extent of polyfunctionality in CD4 and CD8 T cells was associated with replication permissivity; however, signature patterns of cytokine combinations were not distinguishable between groups of macaques.
Taken together our results show that the global T memory cell compartment is profoundly skewed towards a mature effector phenotype by attenuated SIV. Results with the replication-conditional mutant suggest that maintenance of this effect, that may be important in vaccine design, might require persistence of replicating virus.
[Show abstract][Hide abstract] ABSTRACT: The mRNAs encoding the Rev and Env proteins of simian immunodeficiency virus (SIV) are unique because upstream translation start codons are present that may modulate the expression of these viral proteins. We previously reported the regulatory effect of a small upstream open reading frame (ORF) on Rev and Env translation. Here we study this mechanism in further detail by modulating the strength of the translation signals upstream of the open reading frames in subgenomic reporters. Furthermore, the effects of these mutations on SIV gene expression and viral replication are analyzed. An intricate regulatory mechanism is disclosed that allows the virus to express a balanced amount of these two proteins.
[Show abstract][Hide abstract] ABSTRACT: The mRNAs encoding the Rev and Env proteins of simian immunodeficiency virus (SIV) are unique because upstream translation start codons are present that may modulate the expression of these viral proteins. This is true for the regular mRNAs, but we also report novel mRNA splicing variants that encode up to five upstream AUG (uAUG) codons. Their influence on Rev and Env translation was measured by mutational inactivation in reporter constructs and in the SIVmac239 strain. An intricate regulatory mechanism was disclosed that allows the virus to express a balanced amount of these two proteins. This insight also allows the design of vector constructs that efficiently express these proteins.
Journal of Virology 09/2012; 86(22):12362-71. DOI:10.1128/JVI.01532-12 · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The TAR hairpin is present at both the 5' and 3' end of the HIV-1 RNA genome. The 5' element binds the viral Tat protein and is essential for Tat-mediated activation of transcription. We recently observed that complete TAR deletion is allowed in the context of an HIV-1 variant that does not depend on this Tat-TAR axis for transcription. Mutations that open the 5' stem-loop structure did however affect the leader RNA conformation and resulted in a severe replication defect. In this study, we set out to analyze which step of the HIV-1 replication cycle is affected by this conformational change of the leader RNA.
We demonstrate that opening the 5' TAR structure through a deletion in either side of the stem region caused aberrant dimerization and reduced packaging of the unspliced viral RNA genome. In contrast, truncation of the TAR hairpin through deletions in both sides of the stem did not affect RNA dimer formation and packaging.
These results demonstrate that, although the TAR hairpin is not essential for RNA dimerization and packaging, mutations in TAR can significantly affect these processes through misfolding of the relevant RNA signals.
[Show abstract][Hide abstract] ABSTRACT: Tat has a pivotal role in human and simian immunodeficiency virus (HIV and SIV) replication because it stimulates transcription by binding to the trans-activator response (TAR) element. In addition, several other Tat functions have been proposed. Most studies have focused on HIV-1 Tat and much less is known about SIV Tat. An SIVmac239 variant was constructed previously in which the Tat-TAR transcription mechanism is functionally replaced by the doxycycline-inducible Tet-On gene expression mechanism (SIV-rtTA). In this study, SIV-rtTA variants were used to analyse the functions of SIV Tat. It was shown that Tat-minus SIV-rtTA variants replicated efficiently in PM1 T-cells, ruling out an additional essential Tat function. Nevertheless, replication was suboptimal in other cells, and evolutionary pressure to repair Tat expression was documented. It was demonstrated that SIV-rtTA required Tat for optimal gene expression, despite the absence of the Tat-TAR axis. This Tat effect was lost upon replacement of the long terminal repeat promoter region by a non-related promoter. These results indicate that Tat can activate SIV transcription via TAR RNA and U3 DNA elements but has no other essential function in replication in cultured cells. The experiments were limited to cell lines and PBMCs, and did not exclude an accessory Tat function under specific conditions or in vivo.
Journal of General Virology 07/2012; 93(Pt 10):2279-89. DOI:10.1099/vir.0.044511-0 · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A novel genetic approach for the control of virus replication was used for the design of a conditionally replicating human immunodeficiency virus (HIV) variant, HIV-rtTA. HIV-rtTA gene expression and virus replication are strictly dependent on the presence of a non-toxic effector molecule, doxycycline (dox), and thus can be turned on and off at will in a graded and reversible manner. The in vivo replication capacity, pathogenicity and genetic stability of this HIV-rtTA variant were evaluated in a humanized mouse model of haematopoiesis that harbours lymphoid and myeloid components of the human immune system (HIS). Infection of dox-fed BALB Rag/γc HIS (BRG-HIS) mice with HIV-rtTA led to the establishment of a productive infection without CD4(+) T-cell depletion. The virus did not show any sign of escape from dox control for up to 10 weeks after the onset of infection. No reversion towards a functional Tat-transactivating responsive (TAR) RNA element axis was observed, confirming the genetic stability of the HIV-rtTA variant in vivo. These results demonstrate the proof of concept that HIV-rtTA replicates efficiently in vivo. HIV-rtTA is a promising tool for fundamental research to study virus-host interactions in vivo in a controlled fashion.
Journal of General Virology 05/2012; 93(Pt 9):2017-27. DOI:10.1099/vir.0.042796-0 · 3.53 Impact Factor