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)

The internal structural proteins of retroviruses are proteolytically processed from the Gag polyprotein, which alone is able to assemble into virus-like particles when expressed in cells. All Gag proteins contain domains corresponding to the three structural proteins MA, CA, and NC. We have expressed the CA and NC domains together as a unit in Escherichia coli, both for Rous sarcoma virus (RSV) and for human immunodeficiency virus type 1 (HIV-1). We also expressed a similar HIV-1 protein carrying the C-terminal p6 domain. RSV CA-NC, HIV-1 CA-NC, and HIV-1 CA-NC-p6 were purified in native form by classic methods. After adjustment of the pH and salt concentration, each of these proteins was found to assemble at a low level of efficiency into structures that resembled circular sheets and roughly spherical particles. The presence of RNA dramatically increased the efficiency of assembly, and in this case all three proteins formed hollow, cylindrical particles whose lengths were determined by the size of the RNA. The optimal pH at which assembly occurred was 5.5 for the RSV protein and 8.0 for the HIV-1 proteins. The treatment of the RSV CA-NC cylindrical particles with nonionic detergent, with ribonuclease, or with viral protease caused disassembly. These results suggest that RNA plays an important structural role in the virion and that it may initiate and organize the assembly process. The in vitro system described should facilitate the dissection of assembly pathways in retroviruses.
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PMID:Self-assembly in vitro of purified CA-NC proteins from Rous sarcoma virus and human immunodeficiency virus type 1. 766 50

We constructed a human immunodeficiency virus (HIV) matrix (MA) deletion mutant by deletion of about 80% of the HIV type 1 Gag MA domain but retaining myristylation and proteolytic processing signals. The effects of this deletion matrix (dl.MA) mutant on HIV particle assembly, processing, and infectivity were analyzed. Surprisingly, the dl.MA mutant still could assemble and process virus particles, had a wild-type (wt) retrovirus particle density, and possessed wt reverse transcriptase activity. RNase protection experiments showed that dl.MA mutant particles preferentially packaged viral genomic RNA. When both mutant and wt particles were pseudotyped with an amphotropic murine leukemia virus envelope protein, mutant infectivity was about 10% of wt level. In contrast, infectivity of the dl.MA mutant was 1,000-fold less than that of wild-type when mutant and wt particles were pseudotyped with the HIV envelope protein. Protein analyses of pseudotyped virions indicated that there were no major differences between mutant and wt viruses in the efficiency of amphotropic murine leukemia virus envelope protein incorporation. In contrast, there was a reduction in the amount of mutant particle-associated HIV envelope protein gp120. Our results suggest that an intact HIV matrix domain is not absolutely required for reverse transcription, nuclear localization, or integration but is necessary for appropriate HIV envelope protein function.
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PMID:Conditional infectivity of a human immunodeficiency virus matrix domain deletion mutant. 769 66

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 antiviral strategy of capsid-targeted viral inactivation (CTVI) was designed to disable newly produced virions by fusing a Gag or Gag-Pol polyprotein to a degradative enzyme (e.g., a nuclease or protease) that would cause the degradative enzyme to be inserted into virions during assembly. Several new experimental approaches have been developed that increase the antiviral effect of the CTVI strategy on retroviral replication in vitro. A Moloney murine leukemia virus (Mo-MLV) Gag-Escherichia coli RNase HI fusion has a strong antiviral effect when used prophylactically, inhibiting the spread of Mo-MLV and reducing virus titers 1,500- to 2,500-fold. A significant (approximately 100-fold) overall improvement of the CTVI prophylactic antiviral effect was produced by a modification in the culture conditions which presumably increases the efficiency of delivery and expression of the Mo-MLV Gag fusion polyproteins. The therapeutic effect of Mo-MLV Gag-RNase HI polyproteins is to reduce the production of infectious Mo-MLV up to 18-fold. An Mo-MLV Gag-degradative enzyme fusion junction was designed that can be cleaved by the Mo-MLV protease to release the degradative enzyme.
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PMID:Expression of a murine leukemia virus Gag-Escherichia coli RNase HI fusion polyprotein significantly inhibits virus spread. 906 Jul 1

GST-Gag(p55) binds specifically to HIV-1 RNA sequences 1-406, in vitro, with a Kd of about 50 nM. This RNA transcript contains a number of stem loop (SL) structures. The binding is due to the Gag moiety of the fusion protein, not GST. There is a high affinity binding site for Gag in an RNA containing nucleotides 325-362. SL4 is predicted by both biochemical studies and computer folding to be located between nucleotides 335 and 358. An RNA transcript ending at nucleotide 335 does not bind Gag. The deletion of nucleotides 334-358 from HIV-1 RNAs does not affect Gag binding. Digestions with RNase V1 and T1 show that nucleotides 297-300 in SL2, 310, 312, 313, 315, 317, 318, 325 in SL3, and 342 and 343 in SL4 are protected in the presence of Gag. The cleavage of nucleotides 348-351 in SL4 by RNAse V1 is enhanced by Gag binding. At least two Gag binding sites are therefore located in the leader RNA. Those located 5' of nucleotide 335 require the presence of additional 3' sequences.
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PMID:HIV-1 Gag binds specifically to RNA stem-loops in the 5' leader sequence. 965 23

Previous work has shown that spleen necrosis virus (SNV) long terminal repeats (LTRs) are associated with Rex/Rex-responsive element-independent expression of bovine leukemia virus RNA and supports the hypothesis that SNV RNA contains a cis-acting element that interacts with cellular Rex-like proteins. To test this hypothesis, the human immunodeficiency virus type 1 (HIV) Rev/RRE-dependent gag gene was used as a reporter to analyze various SNV sequences. Gag enzyme-linked immunosorbent assay and Western blot analyses reveal that HIV Gag production is enhanced at least 20, 000-fold by the 5' SNV LTR in COS, D17, and 293 cells. Furthermore, SNV RU5 in the sense but not the antisense orientation is sufficient to confer Rev/RRE-independent expression onto a cytomegalovirus-gag plasmid. In contrast, the SNV 3' LTR and 3' untranslated sequence between env and the LTR did not support Rev-independent gag expression. Quantitative RNase protection assays indicate that the SNV 5' RNA terminus enhances cytoplasmic accumulation and polysome association of HIV unspliced and spliced transcripts. However, comparison of the absolute amounts of polysomal RNA indicates that polysome association is not sufficient to account for the significant increase in Gag production by the SNV sequences. Our analysis reveals that the SNV 5' RNA terminus contains a unique cis-acting posttranscriptional control element that interacts with hypothetical cellular Rev-like proteins to facilitate HIV RNA transport and efficient translation.
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PMID:The 5' RNA terminus of spleen necrosis virus contains a novel posttranscriptional control element that facilitates human immunodeficiency virus Rev/RRE-independent Gag production. 1023 46

Human immunodeficiency virus type 1 (HIV-1) gag-encoded proteins play key functions at almost all stages of the viral life cycle. Since these functions may require association with cellular factors, the HIV-1 matrix protein (MA) was used as bait in a yeast two-hybrid screen to identify MA-interacting proteins. MA was found to interact with elongation factor 1-alpha (EF1alpha), an essential component of the translation machinery that delivers aminoacyl-tRNA to ribosomes. EF1alpha was then shown to bind the entire HIV-1 Gag polyprotein. This interaction is mediated not only by MA, but also by the nucleocapsid domain, which provides a second, independent EF1alpha-binding site on the Gag polyprotein. EF1alpha is incorporated within HIV-1 virion membranes, where it is cleaved by the viral protease and protected from digestion by exogenously added subtilisin. The specificity of the interaction is demonstrated by the fact that EF1alpha does not bind to nonlentiviral MAs and does not associate with Moloney murine leukemia virus virions. The Gag-EF1alpha interaction appears to be mediated by RNA, in that basic residues in MA and NC are required for binding to EF1alpha, RNase disrupts the interaction, and a Gag mutant with undetectable EF1alpha-binding activity is impaired in its ability to associate with tRNA in cells. Finally, the interaction between MA and EF1alpha impairs translation in vitro, a result consistent with a previously proposed model in which inhibition of translation by the accumulation of Gag serves to release viral RNA from polysomes, permitting the RNA to be packaged into nascent virions.
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PMID:Translation elongation factor 1-alpha interacts specifically with the human immunodeficiency virus type 1 Gag polyprotein. 1036 86

The human immunodeficiency virus type 1 (HIV-1) Gag polyprotein directs the formation of virions from productively infected cells. Many gag mutations disrupt virion assembly, but little is known about the biochemical effects of many of these mutations. Protein-protein interactions among Gag monomers are believed to be necessary for virion assembly, and data suggest that RNA may modify protein-protein interactions or even serve as a bridge linking Gag polyprotein monomers. To evaluate the primary sequence requirements for HIV-1 Gag homomeric interactions, a panel of HIV-1 Gag deletion mutants was expressed in bacteria and evaluated for the ability to associate with full-length Gag in vitro. The nucleocapsid protein, the major RNA-binding domain of Gag, exhibited activity comparable to that of the complete polyprotein. In the absence of the nucleocapsid protein, relatively weak activity was observed that was dependent upon both the capsid-dimer interface and basic residues within the matrix domain. The relevance of the in vitro findings was confirmed with an assay in which nonmyristylated mutant Gags were assessed for the ability to be incorporated into virions produced by wild-type Gag expressed in trans. Evidence of the importance of RNA for Gag-Gag interaction was provided by the demonstration that RNase impairs the Gag-Gag interaction and that HIV-1 Gag interacts efficiently with Gags encoded by distantly related retroviruses and with structurally unrelated RNA-binding proteins. These results are consistent with models in which Gag multimerization involves indirect contacts via an RNA bridge as well as direct protein-protein interactions.
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PMID:Human immunodeficiency virus type 1 Gag polyprotein multimerization requires the nucleocapsid domain and RNA and is promoted by the capsid-dimer interface and the basic region of matrix protein. 1048 6

The encapsidation signal of the yeast L-A virus contains a 24-nucleotide stem-loop structure with a 5-nucleotide loop and an A bulged at the 5' side of the stem. The Pol part of the Gag-Pol fusion protein is responsible for encapsidation of viral RNA. Opened empty viral particles containing Gag-Pol specifically bind to this encapsidation signal in vitro. We found that binding to empty particles protected the bulged A and the flanking-two nucleotides from cleavage by Fe(II)-EDTA-generated hydroxyl radicals. The five nucleotides of the loop sequence ((4190)GAUCC(4194)) were not protected. However, T1 RNase protection and in vitro mutagenesis experiments indicated that G(4190) is essential for binding. Although the sequence of the other four nucleotides of the loop is not essential, data from RNase protection and chemical modification experiments suggested that C(4194) was also directly involved in binding to empty particles rather than indirectly through its potential base pairing with G(4190). These results suggest that the Pol domain of Gag-Pol contacts the encapsidation signal at two sites: one, the bulged A, and the other, G and C bases at the opening of the loop. These two sites are conserved in the encapsidation signal of M1, a satellite RNA of the L-A virus.
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PMID:Recognition of RNA encapsidation signal by the yeast L-A double-stranded RNA virus. 1095 12


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