Моделирование геомеханического состояния повторно подрабатываемых массивов горных пород

The geomechanical model is proposed and the technology of numerical simulation is developed. Carried out computer simulation of the geomechanical state of the being undermined repeatedly rock massifs of the 3rd potash level of the Starobin deposit taking into account its structural and strength features, as well as the technological schemes of the primary mining. The regularities of the stress-strain zones formation in the undermined rock mass containing mined out mine workings and inter-panel pillars were determined. It is shown that the stability of the workings located in the undermined areas significantly depends on the time passed since the primary mining and on the location of the workings in the massif relative to the location of the primary mining operations. It is determined that the most dangerous for repeated mining are the areas of generalized shear, since the processes of rock mass movement and failure are most likely to be active in these areas. In the areas of generalized compression, the processes of compaction of caved rock take place. As a consequence, after a considerable period of time, the state of the rock massif in these areas can be treated as approximating to the natural state, without additional structural failures. In such areas, the effective mechanical characteristics of the rock massifs are practically restored. Therefore, the greatest stability of mine workings will be achieved when they are placed in the area of generalized compression stress state in the zone of caved, compacted rocks of the mined out roadways and faces. Предложена геомеханическая модель, разработана технология численного моделирования и выполнено компьютерное моделирование геомеханического состояния повторно подрабатываемого массива горных пород Третьего калийного горизонта Старобинского месторождения с учетом его структурных и прочностных особенностей, а также технологических схем первичной отработки. Установлены закономерности формирования зон напряженно-деформированного состояния в подработанном массиве, вмещающем погашенные очистные выработки и межпанельные целики. Показано, что устойчивость выработок, расположенных в областях повторной подработки, существенным образом зависит от времени, прошедшего со времени первичной подработки, и от места расположения выработки в массиве относительно места проведения первичных горных работ. Установлено, что наиболее опасными для повторного размещения выработок являются зоны обобщенного сдвига, так как в них наиболее вероятна активизация процессов сдвижения и разрушения породных масс. В областях обобщенного сжатия происходят процессы уплотнения обрушенных породных масс. Вследствие этого по истечении значительного периода времени состояние массива горных пород в данных зонах можно рассматривать как приближенное к естественному, без дополнительных структурных нарушений. В таких областях эффективные механические характеристики массивов горных пород практически восстанавливаются. Поэтому наибольшая устойчивость выработок будет достигнута при размещении их в области напряженного состояния обобщенного сжатия в зоне обрушенных, уплотненных пород отработанных очистных ходов и лав

Probing the HIV-1 Genomic RNA Trafficking Pathway and Dimerization by Genetic Recombination and Single Virion Analyses

Once transcribed, the nascent full-length RNA of HIV-1 must travel to the appropriate host cell sites to be translated or to find a partner RNA for copackaging to form newly generated viruses. In this report, we sought to delineate the location where HIV-1 RNA initiates dimerization and the influence of the RNA transport pathway used by the virus on downstream events essential to viral replication. Using a cell-fusion-dependent recombination assay, we demonstrate that the two RNAs destined for copackaging into the same virion select each other mostly within the cytoplasm. Moreover, by manipulating the RNA export element in the viral genome, we show that the export pathway taken is important for the ability of RNA molecules derived from two viruses to interact and be copackaged. These results further illustrate that at the point of dimerization the two main cellular export pathways are partially distinct. Lastly, by providing Gag in trans, we have demonstrated that Gag is able to package RNA from either export pathway, irrespective of the transport pathway used by the gag mRNA. These findings provide unique insights into the process of RNA export in general, and more specifically, of HIV-1 genomic RNA trafficking

Conserved determinants of lentiviral genome dimerization

Abstract Background Retroviruses selectively package two copies of their unspliced genomes by what appears to be a dimerization-dependent RNA packaging mechanism. Dimerization of human immunodeficiency virus Type-1 (HIV-1) genomes is initiated by “kissing” interactions between GC-rich palindromic loop residues of a conserved hairpin (DIS), and is indirectly promoted by long-range base pairing between residues overlapping the gag start codon (AUG) and an upstream Unique 5′ element (U5). The DIS and U5:AUG structures are phylogenetically conserved among divergent retroviruses, suggesting conserved functions. However, some studies suggest that the DIS of HIV-2 does not participate in dimerization, and that U5:AUG pairing inhibits, rather than promotes, genome dimerization. We prepared RNAs corresponding to native and mutant forms of the 5′ leaders of HIV-1 (NL4-3 strain), HIV-2 (ROD strain), and two divergent strains of simian immunodeficiency virus (SIV; cpz-TAN1 and -US strains), and probed for potential roles of the DIS and U5:AUG base pairing on intrinsic and NC-dependent dimerization by mutagenesis, gel electrophoresis, and NMR spectroscopy. Results Dimeric forms of the native HIV-2 and SIV leaders were only detectable using running buffers that contained Mg2+, indicating that these dimers are more labile than that of the HIV-1 leader. Mutations designed to promote U5:AUG base pairing promoted dimerization of the HIV-2 and SIV RNAs, whereas mutations that prevented U5:AUG pairing inhibited dimerization. Chimeric HIV-2 and SIV leader RNAs containing the dimer-promoting loop of HIV-1 (DIS) exhibited HIV-1 leader-like dimerization properties, whereas an HIV-1NL4-3 mutant containing the SIVcpzTAN1 DIS loop behaved like the SIVcpzTAN1 leader. The cognate NC proteins exhibited varying abilities to promote dimerization of the retroviral leader RNAs, but none were able to convert labile dimers to non-labile dimers. Conclusions The finding that U5:AUG formation promotes dimerization of the full-length HIV-1, HIV-2, SIVcpzUS, and SIVcpzTAN1 5′ leaders suggests that these retroviruses utilize a common RNA structural switch mechanism to modulate function. Differences in native and NC-dependent dimerization propensity and lability are due to variations in the compositions of the DIS loop residues rather than other sequences within the leader RNAs. Although NC is a well-known RNA chaperone, its role in dimerization has the hallmarks of a classical riboswitch.http://deepblue.lib.umich.edu/bitstream/2027.42/113284/1/12977_2015_Article_209.pd

Mechanisms of Human Immunodeficiency Virus Type 2 RNA Packaging: Efficient trans Packaging and Selection of RNA Copackaging Partners▿

Human immunodeficiency virus type 2 (HIV-2) has been reported to have a distinct RNA packaging mechanism, referred to as cis packaging, in which Gag proteins package the RNA from which they were translated. We examined the progeny generated from dually infected cell lines that contain two HIV-2 proviruses, one with a wild-type gag/gag-pol and the other with a mutant gag that cannot express functional Gag/Gag-Pol. Viral titers and RNA analyses revealed that mutant viral RNAs can be packaged at efficiencies comparable to that of viral RNA from which wild-type Gag/Gag-Pol is translated. These results do not support the cis-packaging hypothesis but instead indicate that trans packaging is the major mechanism of HIV-2 RNA packaging. To further characterize the mechanisms of HIV-2 RNA packaging, we visualized HIV-2 RNA in individual particles by using fluorescent protein-tagged RNA-binding proteins that specifically recognize stem-loop motifs in the viral genomes, an assay termed single virion analysis. These studies revealed that >90% of the HIV-2 particles contained viral RNAs and that RNAs derived from different viruses were copackaged frequently. Furthermore, the frequencies of heterozygous particles in the viral population could be altered by changing a 6-nucleotide palindromic sequence at the 5′-untranslated region of the HIV-2 genome. This finding indicates that selection of copackaging RNA partners occurs prior to encapsidation and that HIV-2 Gag proteins primarily package one dimeric RNA rather than two monomeric RNAs. Additionally, single virion analyses demonstrated a similar RNA distribution in viral particles regardless of whether both viruses had a functional gag or one of the viruses had a nonfunctional gag, providing further support for the trans-packaging hypothesis. Together, these results revealed mechanisms of HIV-2 RNA packaging that are, contrary to previous studies, in many respects surprisingly similar to those of HIV-1

High efficiency of HIV-1 genomic RNA packaging and heterozygote formation revealed by single virion analysis

A long-standing question in retrovirus biology is how RNA genomes are distributed among virions. In the studies presented in this report, we addressed this issue by directly examining HIV-1 RNAs in virions using a modified HIV-1 genome that contained recognition sites for BglG, an antitermination protein in the Escherichia coli bgl operon, which was coexpressed with a fragment of BglG RNA binding protein fused to a fluorescent protein. Our results demonstrate that the majority of virions (>90%) contain viral RNAs. We also coexpressed HIV-1 genomes containing binding sites for BglG or the bacteriophage MS2 coat protein along with 2 fluorescent protein-tagged RNA binding proteins. This method allows simultaneously labeling and discrimination of 2 different RNAs at single-RNA-detection sensitivity. Using this strategy, we obtained physical evidence that virions contain RNAs derived from different parental viruses (heterozygous virion) at ratios expected from a random distribution, and we found that this ratio can be altered by changing the dimerization sequences. Our studies of heterozygous virions also support a generally accepted but unproven assumption that most particles contain 1 dimer. This study provides answers to long-standing questions in HIV-1 biology and illustrates the power and sensitivity of the 2-RNA labeling method, which can also be adapted to analyze various issues of RNA biogenesis including the detection of different RNAs in live cell imaging