Katarzyna Pachulska-Wieczorek

The Apical Loop of the HIV-1 TAR RNA Hairpin Is Stabilized by a Cross-loop Base Pair

The TAR hairpin of the HIV-1 RNA genome is indispensable for trans-activation of the viral promoter and virus replication. The TAR structure has been studied extensively, but most attention has been directed at the three-nucleotide bulge that constitutes the binding site of the viral Tat protein. In contrast, the conformational properties of the apical loop have remained elusive. We performed biochemical studies and molecular dynamics simulations, which indicate that the TAR loop is structured and stabilized by a cross-loop base pair between residues C30 and G34. Mutational disruption of the cross-loop base pair results in reduced Tat response of the LTR promoter, which can be rescued by compensatory mutations that restore the base pair. Thus, Tat-mediated transcriptional activation depends on the structure of the TAR apical loop. The C30-G34 cross-loop base pair classes TAR in a growing family of hairpins with a structured loop that was recently identified in ribosomal RNA, tRNA, and several viral and cellular mRNAs.

The in vitro loose dimer structure and rearrangements of the HIV-2 leader RNA

RNA dimerization is an essential step in the retroviral life cycle. Dimerization and encapsidation signals, closely linked in HIV-2, are located in the leader RNA region. The SL1 motif and nucleocapsid protein are considered important for both processes. In this study, we show the structure of the HIV-2 leader RNA (+1–560) captured as a loose dimer. Potential structural rearrangements within the leader RNA were studied. In the loose dimer form, the HIV-2 leader RNA strand exists in vitro as a single global fold. Two kissing loop interfaces within the loose dimer were identified: SL1/SL1 and TAR/TAR. Evidence for these findings is provided by RNA probing using SHAPE, chemical reagents, enzymes, non-denaturing PAGE mobility assays, antisense oligonucleotides hybridization and analysis of an RNA mutant. Both TAR and SL1 as isolated domains are bound by recombinant NCp8 protein with high affinity, contrary to the hairpins downstream of SL1. Foot-printing of the SL1/NCp8 complex indicates that the major binding site maps to the SL1 upper stem. Taken together, these data suggest a model in which TAR hairpin III, the segment of SL1 proximal to the loop and the PAL palindromic sequence play specific roles in the initiation of dimerization.

Ty1 retrovirus-like element Gag contains overlapping restriction factor and nucleic acid chaperone functions

Ty1 Gag comprises the capsid of virus-like particles and provides nucleic acid chaperone (NAC) functions during retrotransposition in budding yeast. A subgenomic Ty1 mRNA encodes a truncated Gag protein (p22) that is cleaved by Ty1 protease to form p18. p22/p18 strongly inhibits transposition and can be considered an element-encoded restriction factor. Here, we show that only p22 and its short derivatives restrict Ty1 mobility whereas other regions of GAG inhibit mobility weakly if at all. Mutational analyses suggest that p22/p18 is synthesized from either of two closely spaced AUG codons. Interestingly, AUG1p18 and AUG2p18 proteins display different properties, even though both contain a region crucial for RNA binding and NAC activity. AUG1p18 shows highly reduced NAC activity but specific binding to Ty1 RNA, whereas AUG2p18 shows the converse behavior. p22/p18 affects RNA encapsidation and a mutant derivative defective for RNA binding inhibits the RNA chaperone activity of the C-terminal region (CTR) of Gag-p45. Moreover, affinity pulldowns show that p18 and the CTR interact. These results support the idea that one aspect of Ty1 restriction involves inhibition of Gag-p45 NAC functions by p22/p18-Gag interactions.

New, extended hairpin form of the TAR-2 RNA domain points to the structural polymorphism at the 5′ end of the HIV-2 leader RNA

The HIV-2 TAR RNA domain (TAR-2) plays a key role in the trans-activation of HIV-2 transcription as it is the target for the Tat-2 protein and several cell factors. Here, we show that the TAR-2 domain exists in vitro in two global, alternative forms: a new, extended hairpin form with two conformers and the already proposed branched hairpins form. This points strongly to the structural polymorphism of the 5′ end of the HIV-2 leader RNA. The evidence comes from the non-denaturing PAGE mobility assay, 2D structure prediction, enzymatic and Pb2+- or Mg2+-induced RNA cleavages. Existence of the TAR-2 extended form was further proved by the examination of engineered TAR-2 mutants stabilized either in the branched or extended structure. The TAR-2 extended form predominates with an increasing magnesium concentration. Gel retardation assays reveal that both TAR-2 wt and its mutant, unable to form branched structure, bind Tat-2 protein with comparable, high affinity, while RNA hairpins I and II, derived from TAR-2 branched structure model, show much less protein binding. We propose that an internal loop region of the TAR-2 extended hairpin form is a potential Tat-2 binding site.

A self-encoded capsid derivative restricts Ty1 retrotransposition in Saccharomyces

Retrotransposons and retroviral insertions have molded the genomes of many eukaryotes. Since retroelements transpose via an RNA intermediate, the additive nature of the replication cycle can result in massive increases in copy number if left unchecked. Host organisms have countered with several defense systems, including domestication of retroelement genes that now act as restriction factors to minimize propagation. We discovered a novel truncated form of the Saccharomyces Ty1 retrotransposon capsid protein, dubbed p22 that inhibits virus-like particle (VLP) assembly and function. The p22 restriction factor expands the repertoire of defense proteins targeting the capsid and highlights a novel host–parasite strategy. Instead of inhibiting all transposition by domesticating the restriction gene as a distinct locus, Ty1 and budding yeast may have coevolved a relationship that allows high levels of transposition when Ty1 copy numbers are low and progressively less transposition as copy numbers rise. Here, we offer a perspective on p22 restriction, including its mode of expression, effect on VLP functions, interactions with its target, properties as a nucleic acid chaperone, similarities to other restriction factors, and future directions.

The matrix domain contributes to the nucleic acid chaperone activity of HIV-2 Gag.

BackgroundThe Gag polyprotein is a multifunctional regulator of retroviral replication and major structural component of immature virions. The nucleic acid chaperone (NAC) activity is considered necessary to retroviral Gag functions, but so far, NAC activity has only been confirmed for HIV-1 and RSV Gag polyproteins. The nucleocapsid (NC) domain of Gag is proposed to be crucial for interactions with nucleic acids and NAC activity. The major function of matrix (MA) domain is targeting and binding of Gag to the plasma membrane but MA can also interact with RNA and influence NAC activity of Gag. Here, we characterize RNA binding properties and NAC activity of HIV-2 MA and Gag, lacking p6 domain (GagΔp6) and discuss potential contribution of NC and MA domains to HIV-2 GagΔp6 functions and interactions with RNA.ResultsWe found that HIV-2 GagΔp6 is a robust nucleic acid chaperone. HIV-2 MA protein promotes nucleic acids aggregation and tRNALys3 annealing in vitro. The NAC activity of HIV-2 NC is affected by salt which is in contrast to HIV-2 GagΔp6 and MA. At a physiological NaCl concentration the tRNALys3 annealing activity of HIV-2 GagΔp6 or MA is higher than HIV-2 NC. The HIV-2 NC and GagΔp6 show strong binding to the packaging signal (Ψ) of HIV-2 RNA and preference for the purine-rich sequences, while MA protein binds mainly to G residues without favouring Ψ RNA. Moreover, HIV-2 GagΔp6 and NC promote HIV-2 RNA dimerization while our data do not support MA domain participation in this process in vitro.ConclusionsWe present that contrary to HIV-1 MA, HIV-2 MA displays NAC activity and we propose that MA domain may enhance the activity of HIV-2 GagΔp6. The role of the MA domain in the NAC activity of Gag may differ significantly between HIV-1 and HIV-2. The HIV-2 NC and MA interactions with RNA are not equivalent. Even though both NC and MA can facilitate tRNALys3 annealing, MA does not participate in RNA dimerization in vitro. Our data on HIV-2 indicate that the role of the MA domain in the NAC activity of Gag differs not only between, but also within, retroviral genera.

Similarities and differences in the nucleic acid chaperone activity of HIV-2 and HIV-1 nucleocapsid proteins in vitro

BackgroundThe nucleocapsid domain of Gag and mature nucleocapsid protein (NC) act as nucleic acid chaperones and facilitate folding of nucleic acids at critical steps of retroviral replication cycle. The basic N-terminus of HIV-1 NC protein was shown most important for the chaperone activity. The HIV-2 NC (NCp8) and HIV-1 NC (NCp7) proteins possess two highly conserved zinc fingers, flanked by basic residues. However, the NCp8 N-terminal domain is significantly shorter and contains less positively charged residues. This study characterizes previously unknown, nucleic acid chaperone activity of the HIV-2 NC protein.ResultsWe have comparatively investigated the in vitro nucleic acid chaperone properties of the HIV-2 and HIV-1 NC proteins. Using substrates derived from the HIV-1 and HIV-2 genomes, we determined the ability of both proteins to chaperone nucleic acid aggregation, annealing and strand exchange in duplex structures. Both NC proteins displayed comparable, high annealing activity of HIV-1 TAR DNA and its complementary nucleic acid. Interesting differences between the two NC proteins were discovered when longer HIV substrates, particularly those derived from the HIV-2 genome, were used in chaperone assays. In contrast to NCp7, NCp8 weakly facilitates annealing of HIV-2 TAR RNA to its complementary TAR (−) DNA. NCp8 is also unable to efficiently stimulate tRNALys3 annealing to its respective HIV-2 PBS motif. Using truncated NCp8 peptide, we demonstrated that despite the fact that the N-terminus of NCp8 differs from that of NCp7, this domain is essential for NCp8 activity.ConclusionOur data demonstrate that the HIV-2 NC protein displays reduced nucleic acid chaperone activity compared to that of HIV-1 NC. We found that NCp8 activity is limited by substrate length and stability to a greater degree than that of NCp7. This is especially interesting in light of the fact that the HIV-2 5′UTR is more structured than that of HIV-1. The reduced chaperone activity observed with NCp8 may influence the efficiency of reverse transcription and other key steps of the HIV-2 replication cycle.

Determinants of Genomic RNA Encapsidation in the Saccharomyces cerevisiae Long Terminal Repeat Retrotransposons Ty1 and Ty3

Long-terminal repeat (LTR) retrotransposons are transposable genetic elements that replicate intracellularly, and can be considered progenitors of retroviruses. Ty1 and Ty3 are the most extensively characterized LTR retrotransposons whose RNA genomes provide the template for both protein translation and genomic RNA that is packaged into virus-like particles (VLPs) and reverse transcribed. Genomic RNAs are not divided into separate pools of translated and packaged RNAs, therefore their trafficking and packaging into VLPs requires an equilibrium between competing events. In this review, we focus on Ty1 and Ty3 genomic RNA trafficking and packaging as essential steps of retrotransposon propagation. We summarize the existing knowledge on genomic RNA sequences and structures essential to these processes, the role of Gag proteins in repression of genomic RNA translation, delivery to VLP assembly sites, and encapsidation.

RNAComposer and RNA 3D structure prediction for nanotechnology

RNAs adopt specific, stable tertiary architectures to perform their activities. Knowledge of RNA tertiary structure is fundamental to understand RNA functions beginning with transcription and ending with turnover. Contrary to advanced RNA secondary structure prediction algorithms, which allow good accuracy when experimental data are integrated into the prediction, tertiary structure prediction of large RNAs still remains a significant challenge. However, the field of RNA tertiary structure prediction is rapidly developing and new computational methods based on different strategies are emerging. RNAComposer is a user-friendly and freely available server for 3D structure prediction of RNA up to 500 nucleotide residues. RNAComposer employs fully automated fragment assembly based on RNA secondary structure specified by the user. Importantly, this method allows incorporation of distance restraints derived from the experimental data to strengthen the 3D predictions. The potential and limitations of RNAComposer are discussed and an application to RNA design for nanotechnology is presented.

A normal genetic variation modulates synaptic MMP?9 protein levels and the severity of schizophrenia symptoms

Matrix metalloproteinase 9 (MMP‐9) has recently emerged as a molecule that contributes to pathological synaptic plasticity in schizophrenia, but explanation of the underlying mechanisms has been missing. In the present study, we performed a phenotype‐based genetic association study (PGAS) in > 1,000 schizophrenia patients from the Göttingen Research Association for Schizophrenia (GRAS) data collection and found an association between the MMP‐9 rs20544 C/T single‐nucleotide polymorphism (SNP) located in the 3′untranslated region (UTR) and the severity of a chronic delusional syndrome. In cultured neurons, the rs20544 SNP influenced synaptic MMP‐9 activity and the morphology of dendritic spines. We demonstrated that Fragile X mental retardation protein (FMRP) bound the MMP‐9 3′UTR. We also found dramatic changes in RNA structure folding and alterations in the affinity of FMRP for MMP‐9 RNA, depending on the SNP variant. Finally, we observed greater sensitivity to psychosis‐related locomotor hyperactivity in Mmp‐9 heterozygous mice. We propose a novel mechanism that involves MMP‐9‐dependent changes in dendritic spine morphology and the pathophysiology of schizophrenia, providing the first mechanistic insights into the way in which the single base change in the MMP‐9 gene (rs20544) influences gene function and results in phenotypic changes observed in schizophrenia patients.