A phylogenetically conserved sequence within viral 3' untranslated RNA pseudoknots regulates translation

1993 ◽  
Vol 13 (9) ◽  
pp. 5331-5347
Author(s):  
V Leathers ◽  
R Tanguay ◽  
M Kobayashi ◽  
D R Gallie
Keyword(s):  
Mutational Analysis ◽  
Order Structure ◽  
Primary Sequence ◽  
Base Region ◽  
Suspension Cells ◽  
Conserved Sequence ◽  
Binding Data ◽  
Rna Pseudoknots ◽  
Efficient Level ◽  
Necessary And Sufficient

Both the 68-base 5' leader (omega) and the 205-base 3' untranslated region (UTR) of tobacco mosaic virus (TMV) promote efficient translation. A 35-base region within omega is necessary and sufficient for the regulation. Within the 3' UTR, a 52-base region, composed of two RNA pseudoknots, is required for regulation. These pseudoknots are phylogenetically conserved among seven viruses from two different viral groups and one satellite virus. The pseudoknots contained significant conservation at the secondary and tertiary levels and at several positions at the primary sequence level. Mutational analysis of the sequences determined that the primary sequence in several conserved positions, particularly within the third pseudoknot, was essential for function. The higher-order structure of the pseudoknots was also required. Both the leader and the pseudoknot region were specifically recognized by, and competed for, the same proteins in extracts made from carrot cell suspension cells and wheat germ. Binding of the proteins is much stronger to omega than the pseudoknot region. Synergism was observed between the TMV 3' UTR and the cap and to a lesser extent between omega and the 3' UTR. The functional synergism and the protein binding data suggest that the cap, TMV 5' leader, and 3' UTR interact to establish an efficient level of translation.

scholarly journals A phylogenetically conserved sequence within viral 3' untranslated RNA pseudoknots regulates translation.

1993 ◽  
Vol 13 (9) ◽  
pp. 5331-5347 ◽  
Author(s):  
V Leathers ◽  
R Tanguay ◽  
M Kobayashi ◽  
D R Gallie
Keyword(s):  
Mutational Analysis ◽  
Order Structure ◽  
Primary Sequence ◽  
Base Region ◽  
Suspension Cells ◽  
Conserved Sequence ◽  
Binding Data ◽  
Rna Pseudoknots ◽  
Efficient Level ◽  
Necessary And Sufficient

Both the 68-base 5' leader (omega) and the 205-base 3' untranslated region (UTR) of tobacco mosaic virus (TMV) promote efficient translation. A 35-base region within omega is necessary and sufficient for the regulation. Within the 3' UTR, a 52-base region, composed of two RNA pseudoknots, is required for regulation. These pseudoknots are phylogenetically conserved among seven viruses from two different viral groups and one satellite virus. The pseudoknots contained significant conservation at the secondary and tertiary levels and at several positions at the primary sequence level. Mutational analysis of the sequences determined that the primary sequence in several conserved positions, particularly within the third pseudoknot, was essential for function. The higher-order structure of the pseudoknots was also required. Both the leader and the pseudoknot region were specifically recognized by, and competed for, the same proteins in extracts made from carrot cell suspension cells and wheat germ. Binding of the proteins is much stronger to omega than the pseudoknot region. Synergism was observed between the TMV 3' UTR and the cap and to a lesser extent between omega and the 3' UTR. The functional synergism and the protein binding data suggest that the cap, TMV 5' leader, and 3' UTR interact to establish an efficient level of translation.

scholarly journals Mutational Analysis of an RNA Polymerase II Elongation Factor in Drosophila melanogaster

2005 ◽  
Vol 25 (17) ◽  
pp. 7803-7811 ◽  
Author(s):  
Mark A. Gerber ◽  
Ali Shilatifard ◽  
Joel C. Eissenberg
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Cultured Cells ◽  
Mutational Analysis ◽  
Elongation Factor ◽  
Regulatory Function ◽  
Polymerase Binding ◽  
Necessary And Sufficient ◽  
Elongation Activity

ABSTRACT The ELL family of proteins function in vitro as elongation factors for RNA polymerase II. Deletion studies have defined domains in mammalian ELL required for transcription elongation activity and RNA polymerase binding in vitro, for transformation of cultured cells when overexpressed, and for leukemogenesis and cell proliferation as part of a leukemic fusion protein. The goal of this study was to identify domains required for chromosome targeting and viability in the unique Drosophila ELL (dELL) protein. Here, we show that an N-terminal domain of dELL is necessary and sufficient for targeting to transcriptionally active puff sites in chromatin, supporting a role for this domain in recruiting dELL to elongating RNA polymerase II. We demonstrate that a central domain of dELL is required for rapid mobilization of ELL during the heat shock response, suggesting a regulatory function for this domain. Unexpectedly, transgenic dELL in which the N-terminal chromosome binding domain is deleted can complement the recessive lethality of mutations in ELL, suggesting that Drosophila ELL has an essential activity in development distinct from its role as an RNA polymerase II elongation factor.

scholarly journals The Hantavirus Nucleocapsid Protein Recognizes Specific Features of the Viral RNA Panhandle and Is Altered in Conformation upon RNA Binding

2005 ◽  
Vol 79 (3) ◽  
pp. 1824-1835 ◽  
Author(s):  
M. A. Mir ◽  
A. T. Panganiban
Keyword(s):  
Rna Binding ◽  
Viral Rna ◽  
Order Structure ◽  
Primary Sequence ◽  
Nucleotide Substitutions ◽  
Viral Rnas ◽  
N Protein ◽  
High Affinity ◽  
Molecular Events ◽  
Rna Interaction

ABSTRACT Hantaviruses are tripartite negative-sense RNA viruses and members of the Bunyaviridae family. The nucleocapsid (N) protein is the principal structural component of the viral capsid. N forms a stable trimer that specifically recognizes the panhandle structure formed by the viral RNA termini. We used trimeric glutathione S-transferase (GST)-N protein and small RNA panhandles to examine the requirements for specific recognition by Sin Nombre hantavirus N. Trimeric GST-N recognizes the panhandles of the three viral RNAs (S, M, and L) with high affinity, whereas the corresponding plus-strand panhandles of the complementary RNA are recognized with lower affinity. Based on analysis of nucleotide substitutions that alter either the higher-order structure of the panhandle or the primary sequence of the panhandle, both secondary structure and primary sequence are necessary for stable interaction with N. A panhandle 23 nucleotides long is necessary and sufficient for high-affinity binding by N, and stoichiometry calculations indicate that a single N trimer interacts with a single panhandle. Surprisingly, displacement of the panhandle structure away from the terminus does not eliminate recognition by N. The binding of N to the panhandle is an entropy-driven process resulting in initial stable N-RNA interaction followed by a conformational change in N. Taken together, these data provide insight into the molecular events that take place during interaction of N with the panhandle and suggest that specific high-affinity interaction between an RNA binding domain of trimeric N and the panhandle is required for encapsidation of the three viral RNAs.

Mutational analysis of two conserved sequence motifs in HIV-1 reverse transcriptase

FEBS Letters ◽  
1991 ◽  
Vol 282 (2) ◽  
pp. 231-234 ◽  
Author(s):  
Denise M. Lowe ◽  
Vanita Parmar ◽  
Sharon D. Kemp ◽  
Brendan A. Larder
Keyword(s):  
Reverse Transcriptase ◽  
Mutational Analysis ◽  
Sequence Motifs ◽  
Conserved Sequence ◽  
Conserved Sequence Motifs ◽  
Hiv 1

scholarly journals Alterations to both the Primary and Predicted Secondary Structure of Stem-Loop IIIc of the Hepatitis C Virus 1b 5′ Untranslated Region (5′UTR) Lead to Mutants Severely Defective in Translation Which Cannot Be Complemented intrans by the Wild-Type 5′UTR Sequence

1999 ◽  
Vol 73 (3) ◽  
pp. 2359-2364 ◽  
Author(s):  
Shixing Tang ◽  
Adam J. Collier ◽  
Richard M. Elliott
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Untranslated Region ◽  
Mutational Analysis ◽  
Single Point ◽  
Primary Sequence ◽  
Wild Type ◽  
Entry Site ◽  
Transient Expression Assay ◽  
Stem Loop

ABSTRACT Cap-independent translation of the hepatitis C virus (HCV) genomic RNA is mediated by an internal ribosome entry site (IRES) within the 5′ untranslated region (5′UTR) of the virus RNA. To investigate the effects of alterations to the primary sequence of the 5′UTR on IRES activity, a series of HCV genotype 1b (HCV-1b) variant IRES elements was generated and cloned into a bicistronic reporter construct. Changes from the prototypic HCV-1b 5′UTR sequence were identified at various locations throughout the 5′UTR. The translation efficiencies of these IRES elements were examined by an in vivo transient expression assay in transfected BHK-21 cells and were found to range from 0.4 to 95.8% of the activity of the prototype HCV-1b IRES. Further mutational analysis of the three single-point mutants most severely defective in activity, whose mutations were all located in or near stem-loop IIIc, demonstrated that both the primary sequence and the maintenance of base pairing within this stem structure were critical for HCV IRES function. Complementation studies indicated that defective mutants containing either point mutations or major deletions within the IRES elements could not be complemented in trans by a wild-type IRES.

Generalization of Hölder's Theorem to Ordered Modules

1969 ◽  
Vol 21 ◽  
pp. 149-157 ◽  
Author(s):  
T. M. Viswanathan
Keyword(s):  
Sufficient Conditions ◽  
Open Interval ◽  
Order Structure ◽  
Real Numbers ◽  
Ordered Group ◽  
Quotient Field ◽  
Interval Topology ◽  
Ordered Field ◽  
Topological Completion ◽  
Necessary And Sufficient

Hölder's theorem on archimedean groups states:An ordered (abelian) group G is order isomorphic to an ordered subgroup of the ordered group R of real numbers if and only if it is archimedean.We comprehend this theorem in the following setting: G is a Z-module and Ris the completion with respect to the open interval topology of the ordered field Q; Qitself is the ordered quotient field of the ordered domain Z.Rephrasing the situation, we raise the following question: We start with a fully ordered domain A,let Kbe its ordered quotient field. We endow Kwith the open interval topology and consider , the topological completion of K. Is it possible to impose a compatible order structure on and if this can be done, when can we say that an ordered A-module Mis order isomorphic to an ordered A-submodule of ? In Theorem 3.1, we obtain a set of necessary and sufficient conditions for this isomorphism to hold.

scholarly journals Structure-Based Mutational Analysis of the Hepatitis C Virus NS3 Helicase

2001 ◽  
Vol 75 (17) ◽  
pp. 8289-8297 ◽  
Author(s):  
Chun-Ling Tai ◽  
Wen-Ching Pan ◽  
Shwu-Huey Liaw ◽  
Ueng-Cheng Yang ◽  
Lih-Hwa Hwang ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Atpase Activity ◽  
Rna Binding ◽  
Mutational Analysis ◽  
Nonstructural Protein ◽  
Rna Helicase ◽  
Helicase Domain ◽  
Helicase Activity ◽  
Conserved Sequence

ABSTRACT The carboxyl terminus of the hepatitis C virus (HCV) nonstructural protein 3 (NS3) possesses ATP-dependent RNA helicase activity. Based on the conserved sequence motifs and the crystal structures of the helicase domain, 17 mutants of the HCV NS3 helicase were generated. The ATP hydrolysis, RNA binding, and RNA unwinding activities of the mutant proteins were examined in vitro to determine the functional role of the mutated residues. The data revealed that Lys-210 in the Walker A motif and Asp-290, Glu-291, and His-293 in the Walker B motif were crucial to ATPase activity and that Thr-322 and Thr-324 in motif III and Arg-461 in motif VI significantly influenced ATPase activity. When the pairing between His-293 and Gln-460, referred to as gatekeepers, was replaced with the Asp-293/His-460 pair, which makes the NS3 helicase more like the DEAD helicase subgroup, ATPase activity was not restored. It thus indicated that the whole microenvironment surrounding the gatekeepers, rather than the residues per se, was important to the enzymatic activities. Arg-461 and Trp-501 are important residues for RNA binding, while Val-432 may only play a coadjutant role. The data demonstrated that RNA helicase activity was possibly abolished by the loss of ATPase activity or by reduced RNA binding activity. Nevertheless, a low threshold level of ATPase activity was found sufficient for helicase activity. Results in this study provide a valuable reference for efforts under way to develop anti-HCV therapeutic drugs targeting NS3.

scholarly journals Binding of Acf1 to DNA Involves a WAC Motif and Is Important for ACF-Mediated Chromatin Assembly

2002 ◽  
Vol 22 (18) ◽  
pp. 6344-6353 ◽  
Author(s):  
Dmitry V. Fyodorov ◽  
James T. Kadonaga
Keyword(s):  
Dna Binding ◽  
Chromatin Remodeling ◽  
Atp Hydrolysis ◽  
Mutational Analysis ◽  
Large Subunit ◽  
Deposition Process ◽  
Chromatin Assembly ◽  
Sequence Motifs ◽  
Binding Region ◽  
Conserved Sequence

ABSTRACT ACF is a chromatin-remodeling complex that catalyzes the ATP-dependent assembly of periodic nucleosome arrays. This reaction utilizes the energy of ATP hydrolysis by ISWI, the smaller of the two subunits of ACF. Acf1, the large subunit of ACF, is essential for the full activity of the complex. We performed a systematic mutational analysis of Acf1 to elucidate the functions of specific subregions of the protein. These studies revealed DNA- and ISWI-binding regions that are important for the chromatin assembly and ATPase activities of ACF. The DNA-binding region of Acf1 includes a WAC motif, which is necessary for the efficient binding of ACF complex to DNA. The interaction of Acf1 with ISWI requires a DDT domain, which has been found in a variety of transcription and chromatin-remodeling factors. Chromatin assembly by ACF is also impaired upon mutation of an acidic region in Acf1, which may interact with histones during the deposition process. Lastly, we observed modest chromatin assembly defects on mutation of other conserved sequence motifs. Thus, Acf1 facilitates chromatin assembly via an N-terminal DNA-binding region with a WAC motif, a central ISWI-binding segment with a DDT domain, and a C-terminal region with an acidic stretch, a WAKZ motif, PHD fingers, and bromodomain.

scholarly journals Role of the zinc-finger and basic motifs of chrysanthemum virus B p12 protein in nucleic acid binding, protein localization and induction of a hypersensitive response upon expression from a viral vector

2009 ◽  
Vol 90 (3) ◽  
pp. 723-733 ◽  
Author(s):  
N. I. Lukhovitskaya ◽  
I. V. Ignatovich ◽  
E. I. Savenkov ◽  
J. Schiemann ◽  
S. Yu. Morozov ◽  
...  
Keyword(s):  
Dna Binding ◽  
Zinc Finger ◽  
Nuclear Localization ◽  
Hypersensitive Response ◽  
Systemic Acquired Resistance ◽  
Mutational Analysis ◽  
Viral Vector ◽  
Binding Capacity ◽  
Protein Localization ◽  
Conserved Sequence

The genomes of carlaviruses encode cysteine-rich proteins (CRPs) of unknown function. The 12 kDa CRP of chrysanthemum virus B (CVB), p12, has been shown previously to induce a hypersensitive response (HR) when expressed from potato virus X (PVX). This study demonstrated that a p12-induced HR was preceded by induction of a number of genes related to pathogenesis, stress and systemic acquired resistance. p12 localized predominantly to the nucleus. Interestingly, it was found that p12 bound both RNA and DNA in vitro, but notably exhibited a preference for DNA in the presence of Zn2+ ions. Mutational analysis of the p12 conserved sequence motifs demonstrated that the basic motif is required for p12 translocation to the nucleus, thus representing part of the protein nuclear localization signal, whereas the predicted zinc finger motif is needed for both Zn2+-dependent DNA binding and eliciting an HR in PVX-infected leaves. Collectively, these results link, for the first time, nuclear localization of the protein encoded by a cytoplasmically replicating virus and its DNA-binding capacity with HR induction. Furthermore, these data suggest that p12 may mediate induction of the host genes by binding to the plant genomic DNA, and emphasize that CVB p12 is functionally distinct from other known nuclear-localized proteins encoded by the plant positive-stranded RNA viruses.

scholarly journals A conserved acidic cluster motif in SERINC5 confers resistance to antagonism by HIV-1 Nef

2019 ◽  
Author(s):  
Charlotte A. Stoneham ◽  
Peter W. Ramirez ◽  
Rajendra Singh ◽  
Marissa Suarez ◽  
Andrew Debray ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
Mutational Analysis ◽  
Cellular Membrane ◽  
Cellular Protein ◽  
Intrinsic Activity ◽  
Antiviral Protein ◽  
Cytoplasmic Loop ◽  
Relative Resistance ◽  
Conserved Sequence ◽  
Hiv 1

AbstractThe cellular protein SERINC5 inhibits the infectivity of diverse retroviruses and is counteracted by the glycoGag protein of MLV, the S2 protein of EIAV, and the Nef protein of HIV-1. Determining regions within SERINC5 that provide restrictive activity or Nef-sensitivity should inform mechanistic models of the SERINC5/HIV-1 relationship. Here, we report that deletion of the highly conserved sequence EDTEE, which is located within a cytoplasmic loop of SERINC5 and is reminiscent of an acidic cluster membrane trafficking signal, increases the sensitivity of SERINC5 to antagonism by Nef while having no effect on the intrinsic activity of the protein as an inhibitor of infectivity. The effects on infectivity correlated with enhanced removal of the ΔEDTEE mutant relative to wild type SERINC5 from the cell surface and with enhanced exclusion of the mutant protein from virions by Nef. Mutational analysis revealed that the acidic residues, but not the threonine, within the EDTEE motif are important for the relative resistance to Nef. Deletion of the EDTEE sequence did not increase the sensitivity of SERINC5 to antagonism by the glycoGag protein of MLV, suggesting that its virologic role is Nef-specific. These results are consistent with the reported mapping of the cytoplasmic loop that contains the EDTEE sequence as a general determinant of Nef-responsiveness, but they further indicate that sequences inhibitory to as well as supportive of Nef-activity reside in this region. We speculate that the EDTEE motif might have evolved to mediate resistance against retroviruses that use Nef-like proteins to antagonize SERINC5.ImportanceCellular membrane proteins in the SERINC family, especially SERINC5, inhibit the infectivity of retroviral virions. This inhibition is counteracted by retroviral proteins, specifically HIV-1 Nef, MLV glycoGag, and EIAV S2. One consequence of such a host-pathogen “arms race” is compensatory change in the host antiviral protein as it evolves to escape the effects of the viral antagonist. This is often reflected in a genetic signature, positive selection, which is conspicuously missing inSERINC5. Here we show that despite this lack of genetic evidence, a sequence in SERINC5 nonetheless provides relative resistance to antagonism by HIV-1 Nef.

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