The Experts below are selected from a list of 318 Experts worldwide ranked by ideXlab platform
Gholamreza Darai - One of the best experts on this subject based on the ideXlab platform.
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Stable Expression of Nucleocapsid Proteins of Puumala and Hantaan Virus in Mammalian Cells
Virus genes, 1998Co-Authors: Tania Mara Welzel, Roland Kehm, Christian A. Tidona, Walter Muranyi, Gholamreza DaraiAbstract:The development of an in vitro-system for the stable expression and the analysis of native hantavirus Proteins using hantaviral cDNA is of particular interest. As a first step the expression of the hantavirus Nucleocapsid (N) Proteins in mammalian cells was studied in more detail. The cDNA of the S-RNA segment of Puumala virus strain CG-1820 and Hantaan virus strain 76-118 was used for the construction of eucaryotic expression vectors that allow the generation and selection of mammalian cells harboring and expressing the N protein genes of hantaviruses. A variety of conventional and novel expression vectors as well as different mammalian cell lines were screened. The expression of the N protein of Puumala virus using the pGRE5-1 vector in which the transcription is under control of inducible glucocorticoid responsive elements (GRE) revealed that the Puumala virus N protein can be expressed in Vero E6 cells efficiently without any detectable cell toxicity. From the variety of expression vectors tested, it was found that pCR3.1 is the vector of choice for stable expression of hantavirus N Proteins. The successful establishment of different mammalian cell lines expressing considerable amounts of Puumala and Hantaan virus N protein indicates that the stable and efficient expression of this particular viral protein in the cell lines of three evolutionary distinct species (human, monkey, and mouse) is possible. The system described here represents the experimental basis for further studies of hantavirus infection, replication, and pathogenesis using a reverse genetics approach.
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A major antigenic domain of hantaviruses is located on the aminoproximal site of the viral Nucleocapsid protein.
Virus genes, 1997Co-Authors: Peter Go¨tt, Gholamreza Darai, Lothar Zo¨ller, Ekkehard K. F. BautzAbstract:Hantavirus Nucleocapsid protein has recently been shown to be an immunodominant antigen in hemorrhagic with renal syndrome (HFRS) inducing an early and long-lasting immune response. Recombinant Proteins representing various regions of the Nucleocapsid Proteins as well as segments of the G1 and the G2 glycoProteins of hantavirus strains CG18-20 (Puumala serotype) and Hantaan 76-118 have been expressed in E. coli. The antigenicity of these Proteins was tested in enzyme immunoassays and immunoblots. These studies revealed that human IgG immune response is primarily directed against epitopes located within the amino acid residues 1 to 119 of the amino terminus of viral Nucleocapsid Proteins. This fragment was recognized by all HFRS patient sera tested (n=128). The corresponding enzyme immunoassays proved to be more sensitive than the indirect immunofluorescence assays. Furthermore, the majority of bank vole monoclonal antibodies raised against Puumala virus reacted specifically with this site. A recombinant G1 protein (aa 59 to 401) derived from the CG 18-20 strain was recognized by 19 out of 20 sera from HFRS patients.
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rna binding of recombinant Nucleocapsid Proteins of hantaviruses
Virology, 1993Co-Authors: Peter Gött, Gholamreza Darai, Ralf Stohwasser, Paul Schnitzler, Ekkehard K. F. BautzAbstract:Genes encoding the Nucleocapsid (N) Proteins of two hantaviruses, Hantaan virus strain 76-118 (HTN) and Puumala virus strain CG 18-20 (PUU), were expressed in Escherichia coli as histidine-tagged Proteins. They were purified by metal-chelate affinity chromatography under native or denaturing conditions to near homogeneity. The soluble form of HTN N protein was associated with RNA of E. coli. Renatured N Proteins were shown to bind in vitro transcribed RNA representing the hantaviral small genomic (S) RNA segment. RNA binding was shown by affinity to filter-immobilized N Proteins and by gel mobility shift assays. Competition experiments using tRNA, poly(U) and poly(A)+ U indicated that binding of RNA by the N protein is nonspecific. However, direct binding of ds-RNA resulted in efficient formation of large complexes suggesting that double-stranded nucleic acids are bound preferentially. Carboxyterminal fragments of HTN and PUU N Proteins containing about 100 amino acids of the carboxy termini retained full binding capacity indicating that RNA binding occurs via a carboxyterminal domain.
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use of recombinant Nucleocapsid Proteins of the hantaan and nephropathia epidemica serotypes of hantaviruses as immunodiagnostic antigens
Journal of Medical Virology, 1993Co-Authors: Lothar Zöller, Peter Gött, Ekkehard K. F. Bautz, S Yang, Gholamreza DaraiAbstract:Hantavirus Nucleocapsid protein has previously been identified as the major antigen recognized by the humoral immune response in hemorrhagic fever with renal syndrome (HFRS). It was therefore considered to be a suitable antigen for the development of rapid and reliable immunodiagnostic assays. Genes encoding the Nucleocapsid Proteins of two Hantavirus strains, one of the Puumala serotype [nephropathia epidemica virus (NEV)] and the other of the Hantaan serotype were expressed in E. coli, and the expression products were used as diagnostic antigens in solid-phase enzyme immunoassays. The assays were used to detect IgG- and IgM-antibodies in sera of HFRS patients originating from different geographic regions (China, Germany, Greece, Yugoslavia, Scandinavia). ELISA was highly sensitive and proved to be superior to the indirect immunofluorescence assay. Both antigens were necessary to diagnose all HFRS cases originating from the different countries. Most of the sera revealed a predominant reactivity with either 1 of the 2 antigens, allowing the characterization of the etiologic virus as Hantaan-like or NEV-like. The results of the analysis of sera obtained from China and Greece suggested that the Hantaviruses prevalent in these countries are closely related to the Hantaan serotype. In contrast, an NEV-like reactivity was observed in Central and Northern European patients. In the sera of Yugoslav patients both reactivity patterns were found, suggesting that both virus types occur in the Balkan region.
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Antigenicity of hantavirus Nucleocapsid Proteins expressed in E. coli.
Virus Research, 1991Co-Authors: Peter Gött, Lothar Zöller, Ralf Stohwasser, Ekkehard K. F. Bautz, Gholamreza DaraiAbstract:Abstract DNA clones representing the small genomic segment of Nephropathia epidemica virus strain Hallnas B1 (NEV) and Hantaan virus strain 76–118 (HTV) encoding their Nucleocapsid Proteins were inserted into the E. coli vector pIN-III-ompA for secretion of Proteins into the periplasmic space. The complete HTV and NEV Nucleocapsid Proteins and two truncated versions of the NEV Nucleocapsid Proteins were expressed as fusion Proteins. Unexpectedly, all products accumulated as insoluble aggregates. Most of the ompA signal peptide remained uncleaved. However, Nucleocapsid fusion Proteins could be purified from the insoluble fraction by extraction with 8 M urea followed by separation on SDS-PAGE and electroelution. Rabbits were immunized with the eluted Proteins and the resulting antibodies reacted specifically with authentic viral Nucleocapsid Proteins of HTV and NEV. The recombinant Nucleocapsid Proteins were found to react specifically with various hantavirus-immune sera, but not with human control sera, indicating their suitability as potential diagnostic antigens. This is the first report on the expression of a protein of a NEV serotype strain of hantaviruses by use of recombinant DNA techniques.
Stephan Becker - One of the best experts on this subject based on the ideXlab platform.
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establishment and application of an infectious virus like particle system for marburg virus
Journal of General Virology, 2010Co-Authors: Jorg Wenigenrath, Thomas Hoenen, Evamaria Mittler, Larissa Kolesnikova, Stephan BeckerAbstract:The highly pathogenic Marburg virus (MARV) can only be investigated in high containment laboratories, which is time consuming and expensive. To investigate the MARV life cycle under normal laboratory conditions, an infectious virus-like particle (VLP) system was developed. The infectious VLP system is based on the T7-polymerase driven synthesis of a MARV-specific minigenome that encodes luciferase and is transcribed and replicated by the simultaneously expressed MARV Nucleocapsid Proteins NP, VP35, L and VP30. Transcription of the minigenome resulted in luciferase activity and replication resulted in encapsidated minigenomes. The encapsidated minigenomes, together with the viral matrix Proteins VP40 and VP24 and the surface glycoprotein (GP), formed VLPs at the plasma membrane. Among the released pleomorphic VLPs, filamentous particles of 200–400 nm in length showed the highest capacity to induce reporter activity upon infection of target cells. To characterize the infectious VLP system, the intracellular concentration of one of the components was titrated, while all others were held constant. Intracellular concentrations of Nucleocapsid Proteins that resulted in highest replication and transcription activities also yielded VLPs with the highest ability to induce luciferase activity in target cells. High intracellular levels of VP40 maximized the release of VLPs, but reduced their ability to induce luciferase activity in target cells. The intracellular concentration of GP positively correlated with its incorporation into VLPs and their infectivity. Finally, we demonstrated that the infectious VLP system was suitable for rapid screening of neutralizing antibodies directed against MARV.
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inhibition of marburg virus protein expression and viral release by rna interference
Journal of General Virology, 2005Co-Authors: Trent Fowler, Hans-dieter Klenk, Stephan Becker, Sandra Bamberg, Peggy Moller, Thomas F Meyer, Thomas RudelAbstract:High mortality rates and lack of an available vaccine against Marburg haemorrhagic fever (MHF) highlight the need for a defensive therapy against MHF and greater knowledge of the causative agent, the Marburg virus (MARV). Here, RNA interference (RNAi) is employed to destroy MARV transcripts, disrupting replication and allowing analysis of various roles of MARV Proteins. Small interfering RNAs (siRNAs) homologous to three MARV transcripts (NP, VP35 and VP30) were co-transfected into cells with plasmids encoding the corresponding Nucleocapsid Proteins. The resulting decrease in MARV Nucleocapsid-protein levels was shown to be specific, as siRNA that was not homologous to the MARV genome did not decrease the levels of viral Nucleocapsid Proteins. Additionally, transcript levels of double-stranded RNA (dsRNA)-sensor Proteins, the dsRNA-activated protein kinase and 2′,5′-oligoadenylate synthetase 1 remained unchanged, suggesting that the decrease in viral Proteins was not a result of activation of the antiviral properties of the interferon system. Subsequently, siRNAs were shown to reduce intracellular viral Proteins in MARV-infected cells and viral material released into the medium. Targeted reduction of VP30 downregulated the intracellular levels of all other viral Proteins, suggesting that VP30 plays an essential role for transcription/replication. The efficient reduction of MARV replication also suggests that RNAi may provide an agent against MHF.
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three of the four Nucleocapsid Proteins of marburg virus np vp35 and l are sufficient to mediate replication and transcription of marburg virus specific monocistronic minigenomes
Journal of Virology, 1998Co-Authors: Elke Mühlberger, Hans-dieter Klenk, Beate Lotfering, Stephan BeckerAbstract:This paper describes the first reconstituted replication system established for a member of the Filoviridae, Marburg virus (MBGV). MBGV minigenomes containing the leader and trailer regions of the MBGV genome and the chloramphenicol acetyltransferase (CAT) gene were constructed. In MBGV-infected cells, these minigenomes were replicated and encapsidated and could be passaged. Unlike most other members of the order Mononegavirales, filoviruses possess four Proteins presumed to be components of the Nucleocapsid (NP, VP35, VP30, and L). To determine the protein requirements for replication and transcription, a reverse genetic system was established for MBGV based on the vaccinia virus T7 expression system. Northern blot analysis of viral RNA revealed that three Nucleocapsid Proteins (NP, VP35, and L) were essential and sufficient for transcription as well as replication and encapsidation. These data indicate that VP35, rather than VP30, is the functional homologue of rhabdo- and paramyxovirus P Proteins. The reconstituted replication system was profoundly affected by the NP-to-VP35 expression ratio. To investigate whether CAT gene expression was achieved entirely by mRNA or in part by full-length plus-strand minigenomes, a copy-back minireplicon containing the CAT gene but lacking MBGV-specific transcriptional start sites was employed in the artificial replication system. This construct was replicated without accompanying CAT activity. It was concluded that the CAT activity reflected MBGV-specific transcription and not replication.
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interactions of marburg virus Nucleocapsid Proteins
Virology, 1998Co-Authors: Stephan Becker, C Rinne, U Hofsas, Hans-dieter Klenk, Elke MühlbergerAbstract:Abstract In this study, the components of Marburg virus Nucleocapsid complex were determined, and interactions between the compounds were investigated. Using salt dissociation of isolated virions, four Proteins (NP, VP35, VP30, and L) remained attached to the core complex. Same Proteins were detected intracellularly to be localized in MBGV-induced inclusion bodies, which are presumed to represent areas of Nucleocapsid formation. To investigate interactions between the four Proteins, immunofluorescence analysis of coexpressed Proteins was carried out. Complexes between NP-VP35 and NP-VP30 were formed, which was demonstrated by redistribution of VP35 and VP30 into NP-induced inclusion bodies. Furthermore, complexes between L and VP35 were detected by coimmunoprecipitation. Using deletion mutants of L, the binding site of VP35 on L could be restricted to the N-terminal 530 amino-acid residues. Coexpression of NP, VP35, and L led to the formation of a triple complex where VP35 linked NP and L. The detected complexes are presumed to represent the key components of the MBGV transcription and replication machinery.
Andrew H J Wang - One of the best experts on this subject based on the ideXlab platform.
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identification of the Nucleocapsid tegument and envelope Proteins of the shrimp white spot syndrome virus virion
Journal of Virology, 2006Co-Authors: Jyhming Tsai, Andrew H J Wang, Han Ching Wang, Jiann Horng Leu, Ying Zhuang, Peter J Walker, Guang Hsiung KouAbstract:The protein components of the white spot syndrome virus (WSSV) virion have been well established by proteomic methods, and at least 39 structural Proteins are currently known. However, several details of the virus structure and assembly remain controversial, including the role of one of the major structural Proteins, VP26. In this study, Triton X-100 was used in combination with various concentrations of NaCl to separate intact WSSV virions into distinct fractions such that each fraction contained envelope and tegument Proteins, tegument and Nucleocapsid Proteins, or Nucleocapsid Proteins only. From the protein profiles and Western blotting results, VP26, VP36A, VP39A, and VP95 were all identified as tegument Proteins distinct from the envelope Proteins (VP19, VP28, VP31, VP36B, VP38A, VP51B, VP53A) and Nucleocapsid Proteins (VP664, VP51C, VP60B, VP15). We also found that VP15 dissociated from the Nucleocapsid at high salt concentrations, even though DNA was still present. These results were confirmed by CsCl isopycnic centrifugation followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and liquid chromatography-nanoelectrospray ionization-tandem mass spectrometry, by a trypsin sensitivity assay, and by an immunogold assay. Finally, we propose an assembly process for the WSSV virion.
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biochemical and immunological studies of Nucleocapsid Proteins of severe acute respiratory syndrome and 229e human coronaviruses
Proteomics, 2005Co-Authors: Tswenkei Tang, Shuitsung Chen, Minghon Hou, Minghsiang Hong, Fuming Pan, Jennhan Chen, Chenwen Yao, Andrew H J WangAbstract:Severe acute respiratory syndrome (SARS) is a serious health threat and its early diagnosis is important for infection control and potential treatment of the disease. Diagnostic tools require rapid and accurate methods, of which a capture ELISA method may be useful. Toward this goal, we have prepared and characterized soluble full-length Nucleocapsid Proteins (N protein) from SARS and 229E human coronaviruses. N Proteins form oligomers, mostly as dimers at low concentration. These two N Proteins degrade rapidly upon storage and the major degraded N protein is the C-terminal fragment of amino acid (aa) 169–422. Taken together with other data, we suggest that N protein is a two-domain protein, with the N-terminal aa 50–150 as the RNA-binding domain and the C-terminal aa 169–422 as the dimerization domain. Polyclonal antibodies against the SARS N protein have been produced and the strong binding sites of the anti-Nucleocapsid protein (NP) antibodies produced were mapped to aa 1–20, aa 150–170 and aa 390–410. These sites are generally consistent with those mapped by sera obtained from SARS patients. The SARS anti-NP antibody was able to clearly detect SARS virus grown in Vero E6 cells and did not cross-react with the NP from the human coronavirus 229E. We have predicted several antigenic sites (15–20 amino acids) of S, M and N Proteins and produced antibodies against those peptides, some of which could be recognized by sera obtained from SARS patients. Antibodies against the NP peptides could detect the cognate N protein clearly. Further refinement of these antibodies, particularly large-scale production of monoclonal antibodies, could lead to the development of useful diagnostic kits for diseases associated with SARS and other human coronaviruses.
Juozas Staniulis - One of the best experts on this subject based on the ideXlab platform.
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Synthesis of recombinant human parainfluenza virus 1 and 3 Nucleocapsid Proteins in yeast Saccharomyces cerevisiae.
Virus research, 2008Co-Authors: Mindaugas Juozapaitis, Aurelija Zvirbliene, Rimantas Slibinskas, Indre Kucinskaite, Indre Sezaite, Mayte Coiras, Fernando De Ory Manchon, María Rosa López-huertas, Pilar Pérez-breña, Juozas StaniulisAbstract:Human parainfluenza virus types 1 and 3 (HPIV1 and HPIV3, respectively), members of the virus family Paramyxoviridae, are common causes of lower respiratory tract infections in infants, young children, the immunocompromised, the chronically ill, and the elderly. In order to synthesize recombinant HPIV1 and HPIV3 Nucleocapsid Proteins, the coding sequences were cloned into the yeast Saccharomyces cerevisiae expression vector pFGG3 under control of GAL7 promoter. A high level of recombinant virus Nucleocapsid Proteins expression (20-24 mg l(-1) of yeast culture) was obtained. Electron microscopy demonstrated the assembly of typical herring-bone structures of purified recombinant Nucleocapsid Proteins, characteristic for other paramyxoviruses. These structures contained host RNA, which was resistant to RNase treatment. The Nucleocapsid Proteins were stable in yeast and were easily purified by caesium chloride gradient ultracentrifugation. Therefore, this system proved to be simple, efficient and cost-effective, suitable for high-level production of parainfluenza virus Nucleocapsids as Nucleocapsid-like particles. When used as coating antigens in an indirect ELISA, the recombinant N Proteins reacted with sera of patients infected with HPIV1 or 3. Serological assays to detect HPIV-specific antibodies could be designed on this basis.
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Generation of henipavirus Nucleocapsid Proteins in yeast Saccharomyces cerevisiae.
Virus Research, 2006Co-Authors: Mindaugas Juozapaitis, Linfa Wang, Brian J. Shiell, Andrius Serva, Aurelija Zvirbliene, Rimantas Slibinskas, Juozas Staniulis, Kestutis Sasnauskas, Wojtek P. MichalskiAbstract:Abstract Hendra and Nipah viruses are newly emerged, zoonotic viruses and their genomes have nucleotide and predicted amino acid homologies placing them in the family Paramyxoviridae . Currently these viruses are classified in the new genus Henipavirus , within the subfamily Paramyxovirinae , family Paramyxoviridae . The genes encoding HeV and NiV Nucleocapsid Proteins were cloned into the yeast Saccharomyces cerevisiae expression vector pFGG3 under control of GAL7 promoter. A high level of expression of these Proteins (18–20 mg l −1 of yeast culture) was obtained. Mass spectrometric analysis confirmed the primary structure of both Proteins with 92% sequence coverage obtained using MS/MS analysis. Electron microscopy demonstrated the assembly of typical herring-bone structures of purified recombinant Nucleocapsid Proteins, characteristic for other paramyxoviruses. The Nucleocapsid Proteins revealed stability in yeast and can be easily purified by cesium chloride gradient ultracentrifugation. HeV Nucleocapsid protein was detected by sera derived from fruit bats, humans, horses infected with HeV, and NiV Nucleocapsid protein was immunodetected with sera from, fruit bats, humans and pigs. The development of an efficient and cost-effective system for generation of henipavirus Nucleocapsid Proteins might help to improve reagents for diagnosis of viruses.
Mark C Williams - One of the best experts on this subject based on the ideXlab platform.
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nucleic acid binding kinetics of hiv 1 Nucleocapsid Proteins from single molecule dna stretching
Biophysical Journal, 2013Co-Authors: Robert J Gorelick, Ioulia Rouzina, Mark C WilliamsAbstract:The human immunodeficiency virus type 1 (HIV-1) Gag protein is essential for retroviral assembly. During viral maturation, Gag is processed to form matrix (MA), capsid (CA), and Nucleocapsid (NC). Mature NCp7 is derived from processing of NCp15 and NCp9. NCp7 functions as a nucleic acid chaperone during retroviral replication, in which it rearranges nucleic acids to facilitate reverse transcription and recombination. In this work, we used single-molecule DNA stretching to probe the interactions of these Proteins with DNA. Using this technique, we have previously shown that NCp7 destabilizes DNA with rapid kinetics, yielding almost reversible force-extension curves. NCp7 intercalates into dsDNA to keep the two single strands close together while destabilizing them. Surprisingly, multiple stretch and release cycles of DNA in the presence of NCp7 yields changing force-extension curves on the time scale of tens of minutes. If the NCp7 solution is rinsed from the buffer surrounding the DNA molecule, we find that some fraction of the bound protein does not dissociate. The protein only dissociates completely when competitor DNA is introduced to the solution surrounding the stretched DNA molecule. Thus, NCp7 exhibits binding modes on multiple time scales, including both rapid microscopic and slow macroscopic dissociation rates. To test the origin of this surprising behavior and understand how NC-DNA interactions are regulated, we will also apply these methods to NCp9 and NCp15 interactions with DNA. Further studies will elucidate the kinetics of these protein-DNA interactions. This work was funded in part by Federal Funds from NCI, NIH under contract HHSN261200800001E (RJG).
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dna interactions kinetics of hiv 1 Nucleocapsid Proteins by single molecule dna stretching
Biophysical Journal, 2012Co-Authors: Robert J Gorelick, Karin Musierforsyth, Ioulia Rouzina, Mark C WilliamsAbstract:The human immunodeficiency virus type 1 (HIV-1) Gag protein is essential for retroviral assembly. During viral maturation, Gag is processed to form matrix (MA), capsid (CA), and Nucleocapsid (NC). NC is initially cleaved into NCp15, then NCp9, and finally NCp7. NCp7 functions as a nucleic acid chaperone during retroviral replication, in which it rearranges nucleic acids to facilitate reverse transcription and recombination. The role of Gag cleavage intermediates in facilitating nucleic acid remodeling is not well understood, although it is likely that they also function as chaperones during viral assembly and early reverse transcription steps. We use single molecule stretching to probe the DNA interactions of these three NC protein forms. In the presence of NCp7, the DNA elongates at a lower force almost reversibly, demonstrating that NCp7 facilitates structural transitions in DNA with rapid kinetics. To quantitatively probe the DNA annealing kinetics after the DNA is destabilized by force, we monitor the DNA length at constant force as it relaxes towards equilibrium. The time scale of annealing is found to be tens of seconds, suggesting that, while the kinetics of NCs interactions are faster than many single-stranded DNA binding Proteins, the final structural rearrangement is only moderately fast. These results also suggest NCp7 acts as a weak intercalator, allowing the two DNA strands to remain together as the dsDNA structure is destabilized, likely facilitating nucleic acid rearrangements. Somewhat different behavior is observed when DNA is stretched and released in the presence of NCp9 and NCp15, and we will apply this method to quantitatively determine the differences in DNA interaction kinetics for all three HIV-1 NC Proteins. This work was funded in part by Federal Funds from NCI, NIH under contract HHSN261200800001E (RJG).
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nucleic acid chaperone activity of wild type and mutant fiv Nucleocapsid Proteins
Biophysical Journal, 2012Co-Authors: Micah J Mccauley, Robert J Gorelick, Karin Musierforsyth, Ioulia Rouzina, Mark C WilliamsAbstract:The feline immunodeficiency virus (FIV) Nucleocapsid (NC) protein contains 66 amino acids. It has similar composition to HIV-1 NC, with a high number of positively charged amino acids and two zinc fingers, each having a single aromatic residue (F12 and W44). However, there are some differences between the structures of these two Proteins, which likely contribute to their different nucleic acid chaperone properties. In addition to a short N-terminal basic domain, FIV NC has a C-terminal positively charged tail. Moreover, whereas HIV-1 NC’s two aromatic residues are located on the same side of each zinc finger domain, FIV NC’s aromatic residues are located on opposite sides of the zinc fingers. It is known that HIV-1 NC has optimal chaperone activity, including the ability to strongly aggregate nucleic acids, destabilize nucleic acid secondary structure and facilitate rapid protein-nucleic acid interaction kinetics. We use single molecule experiments to measure the characteristics of wild type and mutant FIV NC. By stretching single DNA molecules in the presence of these Proteins, we measure their ability to induce aggregation, stabilize or destabilize dsDNA, and facilitate nucleic acid annealing. Our results show that wild type FIV NC induces significant DNA aggregation, but instead of destabilizing double-stranded DNA, it appears to stabilize the DNA. FIV NC variants containing mutations in the aromatic residues, basic residues, and zinc finger residues possess strongly altered nucleic acid chaperone properties. This work allows us to directly relate FIV NC structure with its function. The single molecule experiments are also compared to ensemble nucleic acid binding and chaperone studies. Taken together, this study sheds new light on the mechanism by which specific types and locations of residues contribute to NC’s nucleic acid chaperone activity and retroviral replication in these systems.
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retroviral Nucleocapsid Proteins display nonequivalent levels of nucleic acid chaperone activity
Journal of Virology, 2008Co-Authors: Kristen M Stewartmaynard, Robert J Gorelick, Margareta Cruceanu, Fei Wang, Mark C Williams, Ioulia Rouzina, Karin MusierforsythAbstract:Human immunodeficiency virus type 1 (HIV-1) Nucleocapsid protein (NC) is a nucleic acid chaperone that facilitates the remodeling of nucleic acids during various steps of the viral life cycle. Two main features of NC’s chaperone activity are its abilities to aggregate and to destabilize nucleic acids. These functions are associated with NC’s highly basic character and with its zinc finger domains, respectively. While the chaperone activity of HIV-1 NC has been extensively studied, less is known about the chaperone activities of other retroviral NCs. In this work, complementary experimental approaches were used to characterize and compare the chaperone activities of NC Proteins from four different retroviruses: HIV-1, Moloney murine leukemia virus (MLV), Rous sarcoma virus (RSV), and human T-cell lymphotropic virus type 1 (HTLV-1). The different NCs exhibited significant differences in their overall chaperone activities, as demonstrated by gel shift annealing assays, decreasing in the order HIV-1 RSV > MLV HTLV-1. In addition, whereas HIV-1, RSV, and MLV NCs are effective aggregating agents, HTLV-1 NC, which exhibits poor overall chaperone activity, is unable to aggregate nucleic acids. Measurements of equilibrium binding to single- and double-stranded oligonucleotides suggested that all four NC Proteins have moderate duplex destabilization capabilities. Single-molecule DNAstretching studies revealed striking differences in the kinetics of nucleic acid dissociation between the NC Proteins, showing excellent correlation between nucleic acid dissociation kinetics and overall chaperone activity.
