Structural Mimicry Drives HIV-1 Rev-Mediated HERV-K Expression.

Expression of the Human Endogenous Retrovirus Type K (HERV-K), the youngest and most active HERV, has been associated with various cancers and neurodegenerative diseases. As in all retroviruses, a fraction of HERV-K transcripts is exported from the nucleus in unspliced or incompletely spliced forms to serve as templates for translation of viral proteins. In a fraction of HERV-K loci (Type 2 proviruses), nuclear export of the unspliced HERV-K mRNA appears to be mediated by a cis-acting signal on the mRNA, the RcRE, and the protein Rec-these are analogous to the RRE-Rev system in HIV-1. Interestingly, the HIV-1 Rev protein is able to mediate the nuclear export of the HERV-K RcRE, contributing to elevated HERV-K expression in HIV-infected patients. We aimed to understand the structural basis for HIV Rev-HERV-K RcRE recognition. We examined the conformation of the RcRE RNA in solution using small-angle X-ray scattering (SAXS) and atomic force microscopy (AFM). We found that the 433-nt long RcRE can assume folded or extended conformations as observed by AFM. SAXS analysis of a truncated RcRE variant revealed an "A"-shaped topological structure similar to the one previously reported for the HIV-1 RRE. The effect of the overall topology was examined using several deletion variants. SAXS and biochemical analyses demonstrated that the "A" shape is necessary for efficient Rev-RcRE complex formation in vitro and nuclear export activity in cell culture. The findings provide insight into the mechanism of HERV-K expression and a structural explanation for HIV-1 Rev-mediated expression of HERV-K in HIV-infected patients. IMPORTANCE: Expression of the human endogenous retrovirus type K (HERV-K) has been associated with various cancers and autoimmune diseases. Nuclear export of both HIV-1 and HERV-K mRNAs is dependent on the interaction between a small viral protein (Rev in HIV-1 and Rec in HERV-K) and a region on the mRNA (RRE in HIV-1 and RcRE in HERV-K). HIV-1 Rev is able to mediate the nuclear export of RcRE-containing HERV-K mRNAs, which contributes to elevated production of HERV-K proteins in HIV-infected patients. We report the solution conformation of the RcRE RNA-the first three-dimensional topological structure for a HERV molecule-and find that the RcRE resembles the HIV-1 nuclear export signal, RRE. The finding reveals the structural basis for the increased HERV-K expression observed in HIV-infected patients. Elevated HERV expression, mediated by HIV infection or other stressors, can have various HERV-related biological consequences. The findings provide structural insight for regulation of HERV-K expression.

Distinct Contributions of Different Domains within the HIV-1 Gag Polyprotein to Specific and Nonspecific Interactions with RNA.

Viral genomic RNA is packaged into virions with high specificity and selectivity. However, in vitro the Gag specificity towards viral RNA is obscured when measured in buffers containing physiological salt. Interestingly, when the binding is challenged by increased salt concentration, the addition of competing RNAs, or introducing mutations to Gag protein, the specificity towards viral RNA becomes detectable. The objective of this work was to examine the contributions of the individual HIV-1 Gag polyprotein domains to nonspecific and specific RNA binding and stability of the initial protein-RNA complexes. Using a panel of Gag proteins with mutations disabling different Gag-Gag or Gag-RNA interfaces, we investigated the distinct contributions of individual domains which distinguish the binding to viral and nonviral RNA by measuring the binding of the proteins to RNAs. We measured the binding affinity in near-physiological salt concentration, and then challenged the binding by increasing the ionic strength to suppress the electrostatic interactions and reveal the contribution of specific Gag-RNA and Gag-Gag interactions. Surprisingly, we observed that Gag dimerization and the highly basic region in the matrix domain contribute significantly to the specificity of viral RNA binding.

SELECTION OF SUITABLE SAMPLING TOOL FOR RELIABLE SOIL SAMPLING NOT ONLY AFTER RADIOLOGICAL ACCIDENT.

The objective of this research was to develop a sampling protocol for contaminated soils after a large radiological accident. One of the criteria for good sampling method is reproducibility and accuracy of large number of samples collected in short time. Members of the chemical laboratories of the Fire Rescue Service of the Czech Republic (FRS CR), which are included in Radiation Monitoring Network, tested four tools in different soil types. More than 100 samples were collected. Root auger and square soil sampler seem to be the best for reliable soil sampling. Modifications of the root auger decreased of relative error of sampling to 7.5%. The root auger is recommended as a first-choice sampling tool based on: (1) ease, (2) operator safety, (3) collection of a representative sample (4) applicability to most soil types and (5) enabling collection of depth-discrete samples.

REFERENCE SURFACE FOR IN SITU GAMMA SPECTROMETRY.

Chemical laboratories of the Fire Rescue Service of the Czech Republic are part of the radiation monitoring network and participate in the radiation situation monitoring in the Czech Republic. Measurements in situ are crucial for monitoring the radiation situation in emergencies associated with the deposition of radioactive substances on a large area. Those data can be used for estimating a possible dose obtained either by staying in a contaminated area or by consumption of food produced in the area. For correct setting of device parameters (e.g. efficiency calibration), standard samples should be measured regularly. Unlike in laboratory, verification in field conditions is difficult. Therefore, a search for suitable reference areas containing a higher amount of 137Cs homogeneously dispersed after the fall of a radioactive cloud passing through our territory following the Chernobyl accident was conducted. Small airports in the East Bohemia regions were identified as suitable candidates.

Micromotors as "Motherships": A Concept for the Transport, Delivery, and Enzymatic Release of Molecular Cargo via Nanoparticles.

Nano/micromotors based on biodegradable and biocompatible polymers represent a progressively developing group of self-propelled artificial devices capable of delivering biologically active compounds to target sites. The majority of these machines are micron sized, and biologically active compounds are simply attached to their surface. Micron-sized devices cannot enter cells, but they provide rapid velocity, which scales down with the size of the device; nanosized devices can enter cells, but their velocity is negligible. An advanced hierarchical design of the micro/nanodevices is an important tool in the development of functional biocompatible transport systems and their implementation in real in vivo applications. In this work, we demonstrate a "mothership" concept, whereby self-propelled microrobots transport smaller cargo-carrying nanorobots that are released by enzymatic degradation.

Efficient support of virus-like particle assembly by the HIV-1 packaging signal.

The principal structural component of a retrovirus particle is the Gag protein. Retroviral genomic RNAs contain a 'packaging signal' ('Ψ') and are packaged in virus particles with very high selectivity. However, if no genomic RNA is present, Gag assembles into particles containing cellular mRNA molecules. The mechanism by which genomic RNA is normally selected during virus assembly is not understood. We previously reported (Comas-Garcia et al., 2017) that at physiological ionic strength, recombinant HIV-1 Gag binds with similar affinities to RNAs with or without Ψ, and proposed that genomic RNA is selectively packaged because binding to Ψ initiates particle assembly more efficiently than other RNAs. We now present data directly supporting this hypothesis. We also show that one or more short stretches of unpaired G residues are important elements of Ψ; Ψ may not be localized to a single structural element, but is probably distributed over >100 bases.

Membrane Interactions of the Mason-Pfizer Monkey Virus Matrix Protein and Its Budding Deficient Mutants.

Matrix proteins (MAs) play a key role in the transport of retroviral proteins inside infected cells and in the interaction with cellular membranes. In most retroviruses, retroviral MAs are N-terminally myristoylated. This modification serves as a membrane targeting signal and also as an anchor for membrane interaction. The aim of this work was to characterize the interactions anchoring retroviral MA at the plasma membrane of infected cell. To address this issue, we compared the structures and membrane affinity of the Mason-Pfizer monkey virus (M-PMV) wild-type MA with its two budding deficient double mutants, that is, T41I/T78I and Y28F/Y67F. The structures of the mutants were determined using solution NMR spectroscopy, and their interactions with water-soluble phospholipids were studied. Water-soluble phospholipids are widely used models for studying membrane interactions by solution NMR spectroscopy. However, this approach might lead to artificial results due to unnatural hydrophobic interactions. Therefore, we used a new approach based on the measurement of the loss of the 1H NMR signal intensity of the protein sample induced by the addition of the liposomes containing phospholipids with naturally long fatty acids. HIV-1 MA was used as a positive control because its ability to interact with liposomes has already been described. We found that in contrast to HIV-1, the M-PMV MA interacted with the liposomes differently and much weaker. In our invivo experiments, the M-PMV MA did not co-localize with lipid rafts. Therefore, we concluded that M-PMV might adopt a different membrane binding mechanism than HIV-1.

Cost-effective method for the preparation of uniformly labeled myristoylated proteins for NMR measurements.

Nuclear magnetic resonance (NMR) is a powerful technique for solving protein structures or studying their interactions. However, it requires molecules labeled with NMR sensitive isotopes like carbon (13)C and nitrogen (15)N. The recombinant expression of labeled proteins is simple to perform but requires quite expensive chemicals. When there is a need for special labeled chemicals, like uniformly (13)C-labeled myristic acid, the price significantly rises. Here we describe a cost-effective method for the recombinant expression of uniformly labeled myristoylated proteins in Escherichia coli demonstrated on the production of Mason-Pfizer monkey virus matrix protein. We used the ability of E. coli to naturally synthetize myristic acid. When grown in isotopically labeled medium the myristic acid will be labelled as well. Bacteria were co-transfected with plasmid carrying gene for yeast N-myristoyltransferase which ensures myristoylation of expressed protein. This process provided 1.8mg of the myristoylated, doubly labeled ((13)C/(15)N)M-PMV matrix protein from 1L of (15)N/(13)C labeled M9 medium. The price represents approximately 50% cost reduction of conventional method using commercially available [U-(13)C]myristic acid.

Interaction of Mason-Pfizer monkey virus matrix protein with plasma membrane.

Budding is the final step of the late phase of retroviral life cycle. It begins with the interaction of Gag precursor with plasma membrane (PM) through its N-terminal domain, the matrix protein (MA). However, single genera of Retroviridae family differ in the way how they interact with PM. While in case of Lentiviruses (e.g., human immunodeficiency virus) the structural polyprotein precursor Gag interacts with cellular membrane prior to the assembly, Betaretroviruses [Mason-Pfizer monkey virus (M-PMV)] first assemble their virus-like particles (VLPs) in the pericentriolar region of the infected cell and therefore, already assembled particles interact with the membrane. Although both these types of retroviruses use similar mechanism of the interaction of Gag with the membrane, the difference in the site of assembly leads to some differences in the mechanism of the interaction. Here we describe the interaction of M-PMV MA with PM with emphasis on the structural aspects of the interaction with single phospholipids.