Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.27.5 (RNase)
17,967 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We previously identified sequences downstream of the SL4 region of HIV-1 RNA that are involved in the recognition of the 5' leader region of HIV-1 RNA by a minimal version of the HIV-1 Gag protein (mGag). These sequences increase the affinity of this interaction, promote Gag multimerization, and enhance formation of an early mGag-RNA complex. Now, we provide protein footprinting results on the +350 to +500 nucleotide region of viral RNA, based on use of different single-stranded and base-paired ribonucleases. Use of the mfold program confirmed the existence of both a stem-loop 5 (SL5), downstream of SL4, and a more complex multi-stem-loop structure (SL6). Footprinting analysis using mGag and single-stranded-specific nucleases showed almost complete protection of the single-stranded region. In contrast, results obtained with RNase V1, a double-stranded-specific nuclease, suggest that the RNA structure is changed upon protein binding, presumably because of formation of novel and longer stems. Furthermore, RNA footprinting, using viral nucleocapsid protein (NC) and RNase VI, indicate a highly double-stranded structure in several regions. These findings show that viral RNA structure is modified by interaction with proteins, and that NC may possess different chaperone activity in the context of the Gag precursor than in its mature form.
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PMID:Structural changes in the SL5 and SL6 leader sequences of HIV-1 RNA following interactions with the viral mGag protein. 2085 22

During HIV-1 assembly, Gag polypeptides multimerize to form an immature capsid and also package HIV-1 genomic RNA. Assembling Gag forms immature capsids by progressing through a stepwise pathway of assembly intermediates containing the cellular ATPase ABCE1, which facilitates capsid formation. The NC domain of Gag is required for ABCE1 binding, acting either directly or indirectly. NC is also critical for Gag multimerization and RNA binding. Previous studies of GagZip chimeric proteins in which NC was replaced with a heterologous leucine zipper that promotes protein dimerization but not RNA binding established that the RNA binding properties of NC are dispensable for capsid formation per se. Here we utilized GagZip proteins to address the question of whether the RNA binding properties of NC are required for ABCE1 binding and for the formation of ABCE1-containing capsid assembly intermediates. We found that assembly-competent HIV-1 GagZip proteins formed ABCE1-containing intermediates, while assembly-incompetent HIV-1 GagZip proteins harboring mutations in residues critical for leucine zipper dimerization did not. Thus, these data suggest that ABCE1 does not bind to NC directly or through an RNA bridge, and they support a model in which dimerization of Gag, mediated by NC or a zipper, results in exposure of an ABCE1-binding domain located elsewhere in Gag, outside NC. Additionally, we demonstrated that immature capsids formed by GagZip proteins are insensitive to RNase A, as expected. However, unexpectedly, immature HIV-1 capsids were almost as insensitive to RNase A as GagZip capsids, suggesting that RNA is not a structural element holding together immature wild-type HIV-1 capsids.
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PMID:HIV Gag-leucine zipper chimeras form ABCE1-containing intermediates and RNase-resistant immature capsids similar to those formed by wild-type HIV-1 Gag. 2154 80

We have used mild detergent to analyze the core of Moloney Murine Leukemia Virus (MoMLV) and core-like complexes in infected cells. The immature core consists of the Gag polyprotein (PrGag) and viral RNA (vRNA). It is known to be detergent-resistant, in contrast to the mature Gag core. The core matures by cleavage of PrGag into MA (matrix), p12, CA (capsid) and NC (nucleocapsid) protein. We found that mature Gag proteins were bound to the PrGag cores. The degree of binding differed widely. No (<0.1%) p12 bound, low amount of CA (3-5%), and higher amount of MA (13-20%) bound. Varying NC was bound (5-15%). NC could be released by RNase A in agreement with its binding to viral RNA. The TM (transmembrane) protein was also examined. A low amount of TM was bound to the PrGag core (approximately 5%), whereas a very high amount (65%) of the PreTM (TM with the cytoplasmic R peptide tail) bound. The binding in the PrGag core appears to occur by direct protein-protein interactions as only minute amounts of lipids including raft lipids were observed after detergent treatment.
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PMID:The retrovirus MA and PreTM proteins follow immature MLV cores. 2364 91

Mov10 and APOBEC3G (A3G) localize to cytoplasmic granules called processing bodies (P bodies), incorporate into human immunodeficiency virus type 1 (HIV-1) virions, and inhibit viral replication. The functional relevance of Mov10/A3G P-body localization to virion incorporation and antiviral activity has not been fully explored. We found that a helicase V mutant of Mov10 exhibits significantly reduced localization to P bodies but still efficiently inhibits viral infectivity via virion incorporation. Disruption of the P bodies by DDX6 knockdown also confirmed Mov10 antiviral activity without P-body localization. In addition, overexpression of SRP19, which binds to 7SL RNA, depleted A3G from P bodies but did not affect its virion incorporation. Sucrose gradient sedimentation assays revealed that the majority of Mov10, A3G, HIV-1 RNA, and Gag formed high-molecular-mass (HMM) complexes that are converted to low-molecular-mass (LMM) complexes after RNase A treatment. In contrast, the P-body markers DCP2, LSM1, eIF4e, DDX6, and AGO1 were in LMM complexes, whereas AGO2, an effector protein of the RNA-induced silencing complex that localizes to P bodies, was present in both LMM and HMM complexes. Depletion of AGO2 indicated that RNA-induced silencing function is required for Mov10's ability to reduce Gag expression upon overexpression, but not its virion incorporation or effect on virus infectivity. We conclude that the majority of Mov10 and A3G are in HMM complexes, whereas most of the P-body markers are in LMM complexes, and that virion incorporation and the antiviral activities of Mov10 and A3G do not require their localization to P bodies.
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PMID:Mov10 and APOBEC3G localization to processing bodies is not required for virion incorporation and antiviral activity. 2392 32

In most retroviruses, plasma membrane (PM) association of the Gag structural protein is a critical step in viral assembly, relying in part on interaction between the highly basic Gag MA domain and the negatively charged inner leaflet of the PM. Assembly is thought to begin with Gag dimerization followed by multimerization, resulting in a hexameric lattice. To directly address the role of multimerization in membrane binding, we fused the MA domains of Rous sarcoma virus (RSV) and HIV-1 to the chemically inducible dimerization domain FK506-binding protein (FKBP) or to the hexameric protein CcmK4 from cyanobacteria. The cellular localization of the resulting green fluorescent protein (GFP)-tagged chimeric proteins was examined by fluorescence imaging, and the association of the proteins with liposomes was quantified by flotation in sucrose gradients, following synthesis in a reticulocyte extract or as purified proteins. Four lipid compositions were tested, representative of liposomes commonly reported in flotation experiments. By themselves, GFP-tagged RSV and HIV-1 MA proteins were largely cytoplasmic, but both hexamerized proteins were highly concentrated at the PM. Dimerization led to partial PM localization for HIV-1 MA. These in vivo effects of multimerization were reproduced in vitro. In flotation analyses, the intact RSV and HIV-1 Gag proteins were more similar to multimerized MA than to monomeric MA. RNA is reported to compete with acidic liposomes for HIV-1 Gag binding, and thus we also examined the effects of RNase treatment or tRNA addition on flotation. tRNA competed with liposomes in the case of some but not all lipid compositions and ionic strengths. Taken together, our results further underpin the model that multimerization is critical for PM association of retroviral Gag proteins. In addition, they suggest that the modulation of membrane binding by RNA, as previously reported for HIV-1, may not hold for RSV.
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PMID:Effect of multimerization on membrane association of Rous sarcoma virus and HIV-1 matrix domain proteins. 2410 16

A known HIV-1-positive intravenous drug user was found to be human T cell lymphoma/leukemia virus-II (HTLV-II) DNA positive by polymerase chain reaction but seronegative in a screening ELISA. He was consistently DNA positive but took 2 years to fully seroconvert. Sequencing of the HTLV-II strain in his cultured T lymphocytes indicated that it is a prototypical type A strain with no major differences in the long terminal repeat DNA sequence, nor major amino acid differences in the Gag, Env, Tax, and Rex proteins. However, a mutation in its pol gene created a stop codon at amino acid 543 of the Pol protein, a region that encodes for the RNase function. This mutation may account for the subject's slow seroconversion.
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PMID:Delayed Seroconversion to HTLV-II Is Associated with a Stop-Codon Mutation in the pol Gene. 2789 35

HIV-1 particle assembly, which occurs at the plasma membrane (PM) of cells, is driven by the viral polyprotein Gag. Gag recognizes phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P₂], a PM-specific phospholipid, via the highly basic region (HBR) in its N-terminal matrix (MA) domain. The HBR is also known to bind to RNA. We have previously shown, using an in vitro liposome binding assay, that RNA inhibits Gag binding to membranes that lack PI(4,5)P₂ If this RNA block is removed by RNase treatment, Gag can bind nonspecifically to other negatively charged membranes. In an effort to identify the RNA species that confer this inhibition of Gag membrane binding, we have tested the impact of purified RNAs on Gag interactions with negatively charged liposomes lacking PI(4,5)P₂ We found that some tRNA species and RNAs containing stem-loop 1 of the psi region in the 5' untranslated region of the HIV-1 genome impose inhibition of Gag binding to membranes lacking PI(4,5)P₂ In contrast, a specific subset of tRNAs, as well as an RNA sequence previously selected in vitro for MA binding, failed to suppress Gag-membrane interactions. Furthermore, switching the identity of charged residues in the HBR did not diminish the susceptibility of Gag-liposome binding for each of the RNAs tested, while deletion of most of the NC domain abrogates the inhibition of membrane binding mediated by the RNAs that are inhibitory to WT Gag-liposome binding. These results support a model in which NC facilitates binding of RNA to MA and thereby promotes RNA-based inhibition of Gag-membrane binding.
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PMID:Inhibition of HIV-1 Gag-membrane interactions by specific RNAs. 2793 83

HIV-1 reverse transcriptase (RT) is translated as part of the Gag-Pol polyprotein that is proteolytically processed by HIV-1 protease (PR) to finally become a mature heterodimer, composed of a p66 and a p66-derived 51-kDa subunit, p51. Our previous work suggested that tRNA^Lys3 binding to p66/p66 introduces conformational changes in the ribonuclease (RNH) domain of RT that facilitate efficient cleavage of p66 to p51 by PR. In this study, we characterized the conformational changes in the RNH domain of p66/p66 imparted by tRNA^Lys3 using NMR. Moreover, the importance of tRNA^Lys3 in RT maturation was confirmed in cellulo by modulating the levels of Lys-tRNA synthetase, which affects recruitment of tRNA^Lys3 to the virus. We also employed nonnucleoside RT inhibitors, to modulate the p66 dimer-monomer equilibrium and monitor the resulting structural changes. Taken together, our data provide unique insights into the conformational changes in p66/p66 that drive PR cleavage.
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PMID:Conformational Changes in HIV-1 Reverse Transcriptase that Facilitate Its Maturation. 3147 Nov 29


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