Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.27.1 (RNase)
 16,360 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The selective encapsidation of retroviral RNA requires sequences in the Gag protein, as well as a cis-acting RNA packaging signal (psi site) near the 5' end of the genomic transcript. Gag protein of human immunodeficiency virus type 1 (HIV-1) has recently been found to bind specifically to the HIV-1 psi element in vitro. Here we report studies aimed at mapping features within the genetically defined psi locus that are required for binding of HIV-1 Gag or of its processed nucleocapsid derivative. The full-length HIV-1 Gag (p55) and nucleocapsid (p15) sequences were expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli. In a gel shift assay containing excess competitor tRNA, affinity-purified GST-p15 and GST-p55 proteins bound to a 206-nucleotide psi RNA element spanning the major splice donor and gag start codons but did not bind to antisense psi transcripts. Quantitative filter-binding assays revealed that both GST-p55 and GST-p15 bound to this RNA sequence with identical affinities (apparent Kd congruent to 5 x 10(-8) M), indicating that all major determinants of psi binding affinity reside within the nucleocapsid portion of Gag. Chemical and RNase accessibility mapping, coupled with computerized sequence analysis, suggested a model for psi RNA structure comprising four independent stem-loops. Filter-binding studies revealed that RNAs corresponding to three of these hypothetical stem-loops can each function as a independent Gag binding site and that each is bound with approximately fourfold-lower apparent affinity than the full-length psi locus. Interaction of Gag with these regions is likely to play a major role in directing HIV-1 RNA encapsidation in vivo.
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PMID:RNA secondary structure and binding sites for gag gene products in the 5' packaging signal of human immunodeficiency virus type 1. 788 56

In all retrovirus systems studied, the leader region of the RNA contains a cis-acting sequence called psi that is required for packaging the viral RNA genome. Since the pol and env genes are dispensable for formation of RNA-containing particles, the gag gene product must have an RNA binding domain(s) capable of recognizing psi. To gain information about which portion(s) of Gag is required for RNA packaging in the avian sarcoma and leukemia virus system, we utilized a series of gag deletion mutants that retain the ability to assemble virus-like particles. COS cells were cotransfected with these mutant DNAs plus a tester DNA containing psi, and incorporation of RNA into particles were measured by RNase protection. The efficiency of packaging was determined by normalization of the amount of psi+ RNA to the amount of Gag protein released in virus-like particles. Specificity of packaging was determined by comparisons of psi+ and psi- RNA in particles and in cells. The results indicate that much of the MA domain, much of the p10 domain, half of the CA domain, and the entire PR domain of Gag are unnecessary for efficient packaging. In addition, none of these deleted regions is needed for specific selection of the psi RNA. Deletions within the NC domain, as expected, reduce or eliminate both the efficiency and the specificity of packaging. Among mutants that retain the ability to package, a deletion within the CA domain (which includes the major homology region) is the least efficient. We also examined particles of the well-known packaging mutant SE21Q1b. The data suggest that the random RNA packaging behavior of this mutant is not due to a specific defect but rather is the result of the cumulative effect of many point mutations throughout the gag gene.
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PMID:Efficiency and selectivity of RNA packaging by Rous sarcoma virus Gag deletion mutants. 805 73

The capsid domain (CA) of the retroviral Gag protein is a major constituent of the virion core. To examine the role of this protein in M-MuLV morphogenesis and replication, a series of substitution mutations affecting the central region of CA were introduced into an infectious proviral DNA. The altered DNAs were introduced into cells, and the resulting lines were analyzed for production of infectious virions. Only one of the replication defective mutants analyzed was blocked in virion assembly. The remaining mutant DNAs induced the formation and release of particles containing genomic RNA and polymerase protein. The reverse transcriptase associated with these mutant virions was capable of transcribing both minus strand strong stop and extended DNA products using the endogenous genomic RNA as template in vitro. Upon infection of fresh cells, however, no viral DNA synthesis could be detected either by Southern analysis or by an RNase protection assay developed specifically to detect intermediate products of reverse transcription. The results indicate that the bulk of the CA mutants are blocked before reverse transcription of the viral genome and suggest an important role for the capsid protein in an early stage of viral replication.
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PMID:Amino acid substitutions in the CA protein of Moloney murine leukemia virus that block early events in infection. 880 18

The retroviral primary transcription product is a multifunctional RNA that is utilized as pre-mRNA, mRNA, and genomic RNA. The relationship between human immunodeficiency virus type 1 (HIV-1) unspliced transcripts used as mRNA for viral protein synthesis and as virion precursor RNA (vpRNA) for encapsidation remains an important question. We developed a biochemical assay to evaluate the hypothesis that prior utilization as mRNA template for protein synthesis is necessary to generate vpRNA. HIV-1-infected T cells were treated with translation inhibitors under conditions that maintain virus production. Immunoprecipitation of newly synthesized HIV-1 Gag protein revealed that de novo translation is not necessary to sustain assembly, release, or processing of Gag structural protein. Both newly synthesized protein and steady-state Gag are competent for assembly, and the extracellular accumulation of Gag is proportional to the intracellular abundance of Gag. As early as 2 h after transcription, newly synthesized RNA is detectable in cell-free virions and encapsidation is sustained upon inhibition of host cell translation. Results of both [(3)H]uridine incorporation assays and HIV-1-specific RNase protection assays (RPAs) indicate that translation inhibition reduces the absolute amounts of both cytoplasmic and virion-associated RNA. Evaluation of encapsidation efficiency by RPA revealed that the cytoplasmic availability of vpRNA is increased, indicating that HIV-1 unspliced mRNA can be rerouted to function as vpRNA. Our data contrast with results from the HIV-2 and murine leukemia virus systems and indicate that HIV-1 unspliced RNA constitutes a single functional pool that can function interchangeably as mRNA and as vpRNA.
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PMID:Translation is not required To generate virion precursor RNA in human immunodeficiency virus type 1-infected T cells. 1109 Jan 50

The capsid (CA) sequence of human immunodeficiency virus type 1 (HIV-1) Gag protein consists of two independently folded domains named the N-terminal domain (NTD) and C-terminal domain (CTD) that are connected by a flexible linker. Most of the CTD sequence adopts rigid structure except for the last 11 amino acids (positions 354 to 364) that are disordered even in the context of the downstream SP1 and nucleocapsid (NC) sequence. Although disordered, this short peptide region plays a crucial role in HIV-1 replication. In this study, we identified three second-site mutations within Gag named A238T, G358S, and N373K that rescued a deleterious mutation R362A located at the C-terminus of CA. A238T is located within the NTD of CA, G358S and N373K are positioned proximal to R362A. One of the mechanisms underlying this compensation event is correction of reduced packaging of viral RNA into the R362A mutated viruses, as shown by the results of RNase protection assays, native Northern blots experiments as well as filter-binding assays. These data suggest that one potential function for the C-terminal disordered sequence of CA in HIV-1 replication is to regulate HIV-1 RNA packaging.
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PMID:The R362A mutation at the C-terminus of CA inhibits packaging of human immunodeficiency virus type 1 RNA. 1618 96

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