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1.
Sci Rep ; 4: 3775, 2014 Jan 20.
Article in English | MEDLINE | ID: mdl-24441019

ABSTRACT

Innovative vaccine platforms are needed to develop effective countermeasures against emerging and re-emerging diseases. These platforms should direct antigen internalization by antigen presenting cells and promote immunogenic responses. This work describes an innovative systems approach combining two novel platforms, αGalactose (αGal)-modification of antigens and amphiphilic polyanhydride nanoparticles as vaccine delivery vehicles, to rationally design vaccine formulations. Regimens comprising soluble αGal-modified antigen and nanoparticle-encapsulated unmodified antigen induced a high titer, high avidity antibody response with broader epitope recognition of antigenic peptides than other regimen. Proliferation of antigen-specific CD4(+) T cells was also enhanced compared to a traditional adjuvant. Combining the technology platforms and augmenting immune response studies with peptide arrays and informatics analysis provides a new paradigm for rational, systems-based design of next generation vaccine platforms against emerging and re-emerging pathogens.


Subject(s)
Immunity, Innate , Nanoparticles/chemistry , Vaccines/immunology , alpha-Galactosidase/immunology , Adjuvants, Immunologic , Antigen-Presenting Cells/immunology , Antigens/immunology , Antigens/therapeutic use , CD4-Positive T-Lymphocytes/immunology , Epitopes/chemistry , Epitopes/immunology , Humans , Nanoparticles/therapeutic use , Peptides/chemistry , Peptides/immunology , Systems Biology , alpha-Galactosidase/therapeutic use
2.
Virus Res ; 167(1): 118-23, 2012 Jul.
Article in English | MEDLINE | ID: mdl-22808531

ABSTRACT

The aim of this study was to evaluate the contribution of positively charged amino acid residues for the Uukuniemi virus (UUKV) N protein functionality. Based on phlebovirus nucleocapsid (N) protein alignments and 3D-structure predictions of UUKV N protein, 14 positively charged residues were chosen as targets for the mutagenesis. The impact of mutations to the N protein functionality was analyzed using minigenome-, virus-like particle-, and mammalian two-hybrid-assays. Seven of the mutations affected the functional competence in all three assays, while others had milder impact or no impact at all. In the 3D-model of UUKV N protein, five of the affected residues, R61, R64, R73, R98 and R115, were located either within or in close proximity to the central cavity that could potentially bind RNA.


Subject(s)
Mutation , Nucleocapsid Proteins/genetics , Uukuniemi virus/genetics , Amino Acid Sequence , Amino Acids/chemistry , Amino Acids/genetics , Amino Acids/metabolism , DNA Mutational Analysis , Molecular Sequence Data , Nucleocapsid Proteins/chemistry , Nucleocapsid Proteins/metabolism , Phlebovirus/chemistry , Phlebovirus/genetics , Phlebovirus/metabolism , Sequence Alignment , Uukuniemi virus/chemistry , Uukuniemi virus/metabolism
3.
Virol J ; 7: 187, 2010 Aug 10.
Article in English | MEDLINE | ID: mdl-20698970

ABSTRACT

BACKGROUND: Uukuniemi virus (UUKV) belongs to the Phlebovirus genus in the family Bunyaviridae. As a non-pathogenic virus for humans UUKV has served as a safe model bunyavirus in a number of studies addressing fundamental questions such as organization and regulation of viral genes, genome replication, structure and assembly. The present study is focused on the oligomerization of the UUKV nucleocapsid (N) protein, which plays an important role in several steps of virus replication. The aim was to locate the domains involved in the N protein oligomerization and study the process in detail. RESULTS: A set of experiments concentrating on the N- and C-termini of the protein was performed, first by completely or partially deleting putative N-N-interaction domains and then by introducing point mutations of amino acid residues. Mutagenesis strategy was based on the computer modeling of secondary and tertiary structure of the N protein. The N protein mutants were studied in chemical cross-linking, immunofluorescence, mammalian two-hybrid, minigenome, and virus-like particle-forming assays. The data showed that the oligomerization ability of UUKV-N protein depends on the presence of intact alpha-helices on both termini of the N protein molecule and that a specific structure in the N-terminal region plays a crucial role in the N-N interaction(s). This structure is formed by two alpha-helices, rich in amino acid residues with aromatic (W7, F10, W19, F27, F31) or long aliphatic (I14, I24) side chains. Furthermore, some of the N-terminal mutations (e.g. I14A, I24A, F31A) affected the N protein functionality both in mammalian two-hybrid and minigenome assays. CONCLUSIONS: UUKV-N protein has ability to form oligomers in chemical cross-linking and mammalian two-hybrid assays. In mutational analysis, some of the introduced single-point mutations abolished the N protein functionality both in mammalian two-hybrid and minigenome assays, suggesting that especially the N-terminal region of the UUKV-N protein is essential for the N-N interaction.


Subject(s)
Nucleocapsid Proteins/metabolism , Protein Interaction Mapping , Protein Multimerization , Uukuniemi virus/physiology , Virus Assembly , Amino Acid Substitution , Animals , Cell Line , Cricetinae , Humans , Models, Molecular , Nucleocapsid Proteins/genetics , Point Mutation , Protein Binding , Protein Interaction Domains and Motifs , Protein Structure, Secondary , Protein Structure, Tertiary , Sequence Deletion , Spodoptera
4.
J Virol Methods ; 169(2): 259-68, 2010 Nov.
Article in English | MEDLINE | ID: mdl-20655330

ABSTRACT

Rift Valley fever virus (RVFV) is an arthropod-borne pathogen that often results in severe morbidity and mortality in both humans and livestock. As its geographic range continues to expand, it presents a real threat to naïve populations around the world by accidental introduction (e.g., the result of increased travel) or intentional release (e.g., a bioterror event). While there is a clear need for a safe and efficacious vaccine against this emerging and re-emerging pathogen, no FDA-approved vaccine is currently available. This need was addressed by the establishment of novel mammalian and insect suspension cell line systems for the efficient production of RVF virus-like particle (VLP)-based vaccine candidates. A direct comparison of the production of RVF VLPs in these systems was performed. Optimization and characterization resulted in a production platform suitable for scale-up. Furthermore, RVF VLP-based vaccines were tested in a lethal challenge model and showed full protection, demonstrating that RVF VLPs present promising RVFV vaccine candidates.


Subject(s)
Rift Valley fever virus/growth & development , Viral Vaccines/biosynthesis , Animals , Cell Culture Techniques/methods , Cell Line , Disease Models, Animal , Humans , Rats , Rats, Inbred WF , Rift Valley Fever/immunology , Rift Valley Fever/prevention & control , Rift Valley fever virus/genetics , Spodoptera , Survival Analysis , Vaccines, Virosome/biosynthesis , Vaccines, Virosome/genetics , Viral Vaccines/genetics
5.
Hum Vaccin ; 6(7): 597-601, 2010 Jul.
Article in English | MEDLINE | ID: mdl-20421731

ABSTRACT

Rift Valley fever virus (RVFV) is an arthropod-borne pathogen that often results in severe morbidity and mortality in both humans and livestock. As its geographic range continues to spread, it presents a real threat to naïve populations around the world by accidental introduction (e.g., the result of increased world travel) or a bioterror event. The lack of prophylactic and therapeutic measures, the potential for human-to-human transmission, and the significant threat to livestock associated with RVFV make infection with these pathogens a serious public health concern. Rift Valley fever epizootics and epidemics might rapidly overwhelm the capacities of the public health and veterinary medical communities to provide rapid diagnostic testing, distribution of countermeasures and adequate medical care.


Subject(s)
Communicable Diseases, Emerging/epidemiology , Rift Valley Fever/epidemiology , Rift Valley Fever/veterinary , Rift Valley fever virus/isolation & purification , Animals , Communicable Diseases, Emerging/virology , Humans , Zoonoses/epidemiology , Zoonoses/transmission
6.
Virology ; 397(1): 187-98, 2010 Feb 05.
Article in English | MEDLINE | ID: mdl-19932911

ABSTRACT

Virus-like particles (VLPs) present viral antigens in a native conformation and are effectively recognized by the immune system and therefore are considered as suitable and safe vaccine candidates against many viral diseases. Here we demonstrate that chimeric VLPs containing Rift Valley fever virus (RVFV) glycoproteins G(N) and G(C), nucleoprotein N and the gag protein of Moloney murine leukemia virus represent an effective vaccine candidate against Rift Valley fever, a deadly disease in humans and livestock. Long-lasting humoral and cellular immune responses are demonstrated in a mouse model by the analysis of neutralizing antibody titers and cytokine secretion profiles. Vaccine efficacy studies were performed in mouse and rat lethal challenge models resulting in high protection rates. Taken together, these results demonstrate that replication-incompetent chimeric RVF VLPs are an efficient RVFV vaccine candidate.


Subject(s)
Rift Valley Fever/immunology , Rift Valley fever virus/immunology , Vaccines, Virosome/immunology , Virus Replication , Animals , Antibodies, Neutralizing/blood , Antibodies, Viral/blood , Cytokines/metabolism , Female , Mice , Mice, Inbred BALB C , Microscopy, Electron, Transmission , Moloney murine leukemia virus/genetics , Rats , Rats, Inbred WF , Rift Valley Fever/prevention & control , Rift Valley fever virus/physiology , Survival Analysis , T-Lymphocytes/immunology , Vaccines, Virosome/genetics , Viral Proteins/genetics , Virosomes/ultrastructure
7.
Antiviral Res ; 84(2): 101-18, 2009 Nov.
Article in English | MEDLINE | ID: mdl-19682499

ABSTRACT

The advent of reverse genetics technology has revolutionized the study of RNA viruses, making it possible to manipulate their genomes and evaluate the effects of these changes on their biology and pathogenesis. The fundamental insights gleaned from reverse genetics-based studies over the last several years provide a new momentum for the development of designed therapies for the control and prevention of these viral pathogens. This review summarizes the successes and stumbling blocks in the development of reverse genetics technologies for Rift Valley fever virus and their application to the further dissection of its pathogenesis and the design of new therapeutics and safe and effective vaccines.


Subject(s)
Antiviral Agents , Genetic Techniques , RNA, Viral/genetics , Rift Valley fever virus/genetics , Viral Vaccines , Animals , Bioterrorism/prevention & control , Cell Line , Cricetinae , DNA, Complementary/genetics , Humans , Mice , RNA, Viral/biosynthesis , Rift Valley Fever/drug therapy , Rift Valley Fever/prevention & control , Rift Valley Fever/virology , Rift Valley fever virus/immunology , Rift Valley fever virus/pathogenicity , Viral Vaccines/genetics
8.
J Virol ; 83(19): 10176-86, 2009 Oct.
Article in English | MEDLINE | ID: mdl-19625394

ABSTRACT

To explore mechanisms of entry for Ebola virus (EBOV) glycoprotein (GP) pseudotyped virions, we used comparative gene analysis to identify genes whose expression correlated with viral transduction. Candidate genes were identified by using EBOV GP pseudotyped virions to transduce human tumor cell lines that had previously been characterized by cDNA microarray. Transduction profiles for each of these cell lines were generated, and a significant positive correlation was observed between RhoC expression and permissivity for EBOV vector transduction. This correlation was not specific for EBOV vector alone as RhoC also correlated highly with transduction of vesicular stomatitis virus GP (VSVG) pseudotyped vector. Levels of RhoC protein in EBOV and VSV permissive and nonpermissive cells were consistent with the cDNA gene array findings. Additionally, vector transduction was elevated in cells that expressed high levels of endogenous RhoC but not RhoA. RhoB and RhoC overexpression significantly increased EBOV GP and VSVG pseudotyped vector transduction but had minimal effect on human immunodeficiency virus (HIV) GP pseudotyped HIV or adeno-associated virus 2 vector entry, indicating that not all virus uptake was enhanced by expression of these molecules. RhoB and RhoC overexpression also significantly enhanced VSV infection. Similarly, overexpression of RhoC led to a significant increase in fusion of EBOV virus-like particles. Finally, ectopic expression of RhoC resulted in increased nonspecific endocytosis of fluorescent dextran and in formation of increased actin stress fibers compared to RhoA-transfected cells, suggesting that RhoC is enhancing macropinocytosis. In total, our studies implicate RhoB and RhoC in enhanced productive entry of some pseudovirions and suggest the involvement of actin-mediated macropinocytosis as a mechanism of uptake of EBOV GP and VSVG pseudotyped viral particles.


Subject(s)
Ebolavirus/enzymology , Genetic Vectors , Vesiculovirus/metabolism , rho GTP-Binding Proteins/metabolism , Animals , COS Cells , Cell Line , Cell Line, Tumor , Chlorocebus aethiops , Clostridioides difficile , Humans , Microscopy, Fluorescence/methods , Models, Biological , Plasmids/metabolism , Vero Cells
9.
Virology ; 378(2): 377-84, 2008 Sep 01.
Article in English | MEDLINE | ID: mdl-18614193

ABSTRACT

Rift Valley fever virus (RVFV, Bunyaviridae, Phlebovirus) is a mosquito-transmitted arbovirus that causes human and animal diseases in sub-Saharan Africa and was introduced into the Arabian Peninsula in 2000. Here, we describe a method of reverse genetics to recover infectious RVFV from transfected plasmids based on the use of the cellular RNA polymerase I promoter to synthesize viral transcripts. We compared its efficiency with a system using T7 RNA polymerase and found that both are equally efficient for the rescue of RVFV generating titers of approx. 10(7) to 10(8) pfu/ml. We used the RNA polymerase I-based system to rescue both attenuated MP12 and virulent ZH548 strains as well as chimeric MP12-ZH548 viruses, and in addition RVFV expressing reporter proteins.


Subject(s)
RNA Polymerase I/metabolism , RNA, Viral/biosynthesis , Rift Valley fever virus/growth & development , Virology/methods , Virus Replication , Animals , Chlorocebus aethiops , Mice , Rift Valley Fever/virology , Rift Valley fever virus/genetics , Rift Valley fever virus/pathogenicity , Survival Analysis , Vero Cells , Viral Plaque Assay , Virulence
10.
PLoS Pathog ; 4(1): e13, 2008 Jan.
Article in English | MEDLINE | ID: mdl-18225953

ABSTRACT

Rift Valley fever virus (RVFV) nonstructural protein NSs acts as the major determinant of virulence by antagonizing interferon beta (IFN-beta) gene expression. We demonstrate here that NSs interacts with the host protein SAP30, which belongs to Sin3A/NCoR/HDACs repressor complexes and interacts with the transcription factor YY1 that regulates IFN-beta gene expression. Using confocal microscopy and chromatin immunoprecipitation, we show that SAP30, YY1, and Sin3A-associated corepressor factors strongly colocalize with nuclear NSs filaments and that NSs, SAP30 and Sin3A-associated factors are recruited on the IFN-beta promoter through YY1, inhibiting CBP recruitment, histone acetylation, and transcriptional activation. To ascertain the role of SAP30, we produced, by reverse genetics, a recombinant RVFV in which the interacting domain in NSs was deleted. The virus was unable to inhibit the IFN response and was avirulent for mice. We discuss here the strategy developed by the highly pathogenic RVFV to evade the host antiviral response, affecting nuclear organization and IFN-beta promoter chromatin structure.


Subject(s)
Histone Deacetylases/metabolism , Interferon-beta/metabolism , Repressor Proteins/metabolism , Rift Valley fever virus/physiology , Viral Nonstructural Proteins/metabolism , YY1 Transcription Factor/metabolism , Animals , Cell Nucleus/metabolism , Cells, Cultured , Chlorocebus aethiops , Gene Expression Regulation, Viral , Histone Deacetylases/genetics , Interferon-beta/genetics , Mice , Microscopy, Confocal , Mutation , Sin3 Histone Deacetylase and Corepressor Complex , Two-Hybrid System Techniques , Vero Cells , Viral Nonstructural Proteins/genetics , Virulence
11.
Virology ; 370(1): 33-44, 2008 Jan 05.
Article in English | MEDLINE | ID: mdl-17904180

ABSTRACT

In this study we report the development and optimization of two minigenome rescue systems for Nipah virus, a member of the Paramyxoviridae family. One is mediated by the T7 RNA polymerase supplied either by a constitutively expressing cell line or by transfection of expression plasmids and is thus independent from infection with a helper virus. The other approach is based on RNA polymerase I-driven transcription, a unique approach for paramyxovirus reverse genetics technology. Minigenome rescue was evaluated by reporter gene activities of (i) the two different minigenome transcription systems, (ii) genomic versus antigenomic-oriented minigenomes, (iii) different ratios of the viral protein expression plasmids, and (iv) time course experiments. The high efficiency and reliability of the established systems allowed for downscaling to 96-well plates. This served as a basis for the development of a high-throughput screening system for potential antivirals that target replication and transcription of Nipah virus without the need of high bio-containment. Using this system we were able to identify two compounds that reduced minigenome activity.


Subject(s)
DNA-Directed RNA Polymerases/metabolism , Genome, Viral/genetics , Nipah Virus/physiology , Plasmids/genetics , RNA Polymerase I/metabolism , RNA, Viral/metabolism , Viral Proteins/metabolism , Antiviral Agents/pharmacology , Cell Line , Chloramphenicol O-Acetyltransferase/metabolism , DNA-Directed RNA Polymerases/genetics , HeLa Cells , Humans , Microbial Sensitivity Tests/methods , Nipah Virus/drug effects , Nipah Virus/genetics , Paramyxoviridae/genetics , Paramyxoviridae/metabolism , RNA Polymerase I/genetics , RNA, Viral/drug effects , RNA, Viral/genetics , Transcription, Genetic/drug effects , Transfection , Viral Proteins/genetics , Virus Replication/drug effects
12.
Virology ; 351(1): 170-9, 2006 Jul 20.
Article in English | MEDLINE | ID: mdl-16630639

ABSTRACT

Rift Valley fever virus (RVFV) (Phlebovirus, Bunyaviridae) possesses a genome composed of three negative-stranded RNA molecules. Each segment contains 3' and 5' noncoding regions with terminal complementary sequences forming a panhandle structure. We showed that transcription-replication of the L, M and S segments is regulated, and we established a minigenome rescue system expressing a CAT reporter to investigate the role of the noncoding regions in this process. The L, M and S segment-based minigenomes were shown to drive bona fide transcription and replication and to express variable levels of CAT reporter, indicating differential promoter activities within the noncoding sequences. In addition, we found a good correlation between the relative promoter strength and the abundance of viral RNA species in RVFV-infected cells. Altogether, these results show that RVFV minigenomes are powerful tools to study transcription and replication and constitute a valuable basis to rescue infectious virus from cDNAs.


Subject(s)
DNA, Intergenic/genetics , Gene Expression Regulation, Viral , RNA, Viral/biosynthesis , RNA, Viral/genetics , Rift Valley fever virus/genetics , Transcription, Genetic/genetics , Animals , Cell Line , Cricetinae , Genome, Viral
13.
PLoS Pathog ; 2(2): e7, 2006 Feb.
Article in English | MEDLINE | ID: mdl-16477309

ABSTRACT

EphrinB2 was recently discovered as a functional receptor for Nipah virus (NiV), a lethal emerging paramyxovirus. Ephrins constitute a class of homologous ligands for the Eph class of receptor tyrosine kinases and exhibit overlapping expression patterns. Thus, we examined whether other ephrins might serve as alternative receptors for NiV. Here, we show that of all known ephrins (ephrinA1-A5 and ephrinB1-B3), only the soluble Fc-fusion proteins of ephrinB3, in addition to ephrinB2, bound to soluble NiV attachment protein G (NiV-G). Soluble NiV-G bound to cell surface ephrinB3 and B2 with subnanomolar affinities (Kd = 0.58 nM and 0.06 nM for ephrinB3 and B2, respectively). Surface plasmon resonance analysis indicated that the relatively lower affinity of NiV-G for ephrinB3 was largely due to a faster off-rate (K(off) = 1.94 x 10(-3) s(-1) versus 1.06 x 10(-4) s(-1) for ephrinB3 and B2, respectively). EphrinB3 was sufficient to allow for viral entry of both pseudotype and live NiV. Soluble ephrinB2 and B3 were able to compete for NiV-envelope-mediated viral entry on both ephrinB2- and B3-expressing cells, suggesting that NiV-G interacts with both ephrinB2 and B3 via an overlapping site. Mutational analysis indicated that the Leu-Trp residues in the solvent exposed G-H loop of ephrinB2 and B3 were critical determinants of NiV binding and entry. Indeed, replacement of the Tyr-Met residues in the homologous positions in ephrinB1 with Leu-Trp conferred NiV receptor activity to ephrinB1. Thus, ephrinB3 is a bona fide alternate receptor for NiV entry, and two residues in the G-H loop of the ephrin B-class ligands are critical determinants of NiV receptor activity.


Subject(s)
Ephrin-B3/chemistry , Ephrin-B3/metabolism , Nipah Virus/metabolism , Receptors, Virus/metabolism , Animals , Binding Sites , CHO Cells , Cricetinae , Cricetulus , Ephrin-B2/genetics , Ephrin-B2/metabolism , Ephrin-B3/genetics , Henipavirus Infections/virology , Humans , Leucine , Nipah Virus/physiology , Tryptophan
14.
Curr Mol Med ; 5(8): 753-60, 2005 Dec.
Article in English | MEDLINE | ID: mdl-16375710

ABSTRACT

Crimean-Congo hemorrhagic fever virus (CCHFV) is an important human pathogen, which is the cause of a tick-borne illness occurring in many areas of Africa, Asia, and Europe. CCHF is characterized by a sudden onset of high fever, chills, and severe headache. Other symptoms can include gastrointestinal disorders, such as nausea, vomiting, and diarrhea. In severe cases, hemorrhagic manifestations can occur and often present as large areas of ecchymosis, rather than frank bleeding. Exposure to ticks, particularly those in the genus Hyalomma, or direct contact with virus-infected animals or people are considered the major risk factors. Studies on CCHFV are impeded by the biocontainment needed for their manipulation. However, the increasing worldwide medical awareness, the enormous interest of the media in hemorrhagic fever diseases, and their potential to be used as a bioweapon, have greatly spurred on research on this important virus, as evidenced by many new developments including the development of a reverse genetics system which should greatly enhance future research with this virus.


Subject(s)
Hemorrhagic Fever Virus, Crimean-Congo/metabolism , Hemorrhagic Fever, Crimean/diagnosis , Hemorrhagic Fever, Crimean/therapy , Hemorrhagic Fever Virus, Crimean-Congo/genetics , Hemorrhagic Fever Virus, Crimean-Congo/pathogenicity , Hemorrhagic Fever, Crimean/epidemiology , Humans , Prognosis
15.
Curr Mol Med ; 5(8): 827-34, 2005 Dec.
Article in English | MEDLINE | ID: mdl-16375716

ABSTRACT

Rift Valley fever is considered to be one of the most important viral zoonoses in Africa. In 2000, the Rift valley fever virus spread to the Arabian Peninsula and caused two simultaneous outbreaks in Yemen and Saudi Arabia. It is transmitted to ruminants and to humans by mosquitoes. The viral agent is an arbovirus, which belongs to the Phlebovirus genus in the Bunyaviridae family. This family of viruses comprises more than 300 members grouped into five genera: Orthobunyavirus, Phlebovirus, Hantavirus, Nairovirus, and Tospovirus. Several members of the Bunyaviridae family are responsible for fatal hemorrhagic fevers: Rift Valley fever virus (Phlebovirus), Crimean-Congo hemorrhagic fever virus (Nairovirus), Hantaan, Sin Nombre and related viruses (Hantavirus), and recently Garissa, now identified as Ngari virus (Orthobunyavirus). Here are reviewed recent advances in Rift Valley fever virus, its epidemiology, molecular biology and focus on recent data on the interactions between viral and cellular proteins, which help to understand the molecular mechanisms utilized by the virus to circumvent the host cellular response.


Subject(s)
Rift Valley Fever/virology , Rift Valley fever virus/pathogenicity , Animals , Disease Vectors , Humans , Immunity , Public Health , Rift Valley Fever/diagnosis , Rift Valley Fever/therapy , Rift Valley fever virus/classification , Rift Valley fever virus/metabolism
16.
Virol J ; 2: 42, 2005 Apr 25.
Article in English | MEDLINE | ID: mdl-15850490

ABSTRACT

BACKGROUND: Crimean-Congo Hemorrhagic Fever virus (CCHFV), a member of the genus Nairovirus, family Bunyaviridae, is a tick-borne pathogen causing severe disease in humans. To better understand the CCHFV life cycle and explore potential intervention strategies, we studied the biosynthesis and intracellular targeting of the glycoproteins, which are encoded by the M genome segment. RESULTS: Following determination of the complete genome sequence of the CCHFV reference strain IbAr10200, we generated expression plasmids for the individual expression of the glycoproteins GN and GC, using CMV- and chicken beta-actin-driven promoters. The cellular localization of recombinantly expressed CCHFV glycoproteins was compared to authentic glycoproteins expressed during virus infection using indirect immunofluorescence assays, subcellular fractionation/western blot assays and confocal microscopy. To further elucidate potential intracellular targeting/retention signals of the two glycoproteins, GFP-fusion proteins containing different parts of the CCHFV glycoprotein were analyzed for their intracellular targeting. The N-terminal glycoprotein GN localized to the Golgi complex, a process mediated by retention/targeting signal(s) in the cytoplasmic domain and ectodomain of this protein. In contrast, the C-terminal glycoprotein GC remained in the endoplasmic reticulum but could be rescued into the Golgi complex by co-expression of GN. CONCLUSION: The data are consistent with the intracellular targeting of most bunyavirus glycoproteins and support the general model for assembly and budding of bunyavirus particles in the Golgi compartment.


Subject(s)
Glycoproteins/metabolism , Hemorrhagic Fever Virus, Crimean-Congo/metabolism , Viral Nonstructural Proteins/metabolism , Animals , Cell Line , Chlorocebus aethiops , Cricetinae , Cytoplasm , Endoplasmic Reticulum , Gene Expression Regulation, Viral , Glycoproteins/genetics , Golgi Apparatus , Humans , Immunohistochemistry , Viral Nonstructural Proteins/genetics
17.
J Virol ; 79(7): 4425-33, 2005 Apr.
Article in English | MEDLINE | ID: mdl-15767442

ABSTRACT

In general, Ebola viruses are well known for their ability to cause severe hemorrhagic fever in both human and nonhuman primates. However, despite substantial sequence homology to other members of the family Filoviridae, Reston ebolavirus displays reduced pathogenicity for nonhuman primates and has never been demonstrated to cause clinical disease in humans, despite its ability to cause infection. In order to develop a tool to explore potential roles for transcription and replication in the reduced pathogenicity of Reston ebolavirus, we developed an RNA polymerase I (Pol I)-driven minigenome system. Here we demonstrate successful Reston ebolavirus minigenome rescue, including encapsidation, transcription, and replication, as well as the packaging of minigenome transcripts into progeny particles. The Pol I-driven Reston ebolavirus minigenome system provides a higher signal intensity with less background (higher signal-to-noise ratio) than a comparable T7-driven Reston ebolavirus minigenome system which was developed simultaneously. Successful Reston ebolavirus minigenome rescue was also achieved by the use of helper plasmids derived from the closely related Zaire ebolavirus or the more distantly related Lake Victoria marburgvirus. The use of heterologous helper plasmids in the Reston ebolavirus minigenome system yielded levels of reporter expression which far exceeded the level produced by the homologous helper plasmids. This comparison between minigenomes and helper plasmids from different filovirus species and genera indicates that inherent differences in the transcription and/or replication capacities of the ribonucleoprotein complexes of pathogenic and apathogenic filoviruses may exist, as these observations were confirmed in a Lake Victoria marburgvirus minigenome system.


Subject(s)
Ebolavirus/genetics , Ebolavirus/physiology , RNA Polymerase I/metabolism , RNA, Viral/physiology , Transcription, Genetic , Virus Replication/physiology , Artificial Gene Fusion , Chloramphenicol O-Acetyltransferase/analysis , Chloramphenicol O-Acetyltransferase/genetics , Genes, Reporter , Genome, Viral , Helper Viruses/genetics , Marburgvirus/genetics , RNA, Viral/genetics , Virus Assembly/physiology
18.
FEMS Microbiol Lett ; 244(1): 9-18, 2005 Mar 01.
Article in English | MEDLINE | ID: mdl-15727815

ABSTRACT

The advent of reverse genetics technology has revolutionized the field of RNA viruses. It is now possible to manipulate even negative-stranded RNA viruses at will, and evaluate the effects of these changes on the biology and pathogenesis of these viruses. The fundamental insights gleaned from the reverse genetics-based studies over the last several years have provided a new momentum for the development of designed therapies for the control and prevention of these viral pathogens. The recombinant viruses have been exploited also as vectors for devising targeted therapies for non-viral diseases such as malignancies, and in gene therapy for inherited disorders. This review provides a brief summary of the stumbling blocks and the successes in the development of the technology for the negative-stranded RNA viruses. The many and varied applications of the recombinant vectors are also outlined.


Subject(s)
RNA Viruses/genetics , Animals , DNA, Complementary/genetics , DNA, Viral/genetics , Genetic Techniques , Genetic Vectors , Humans , RNA Viruses/growth & development , RNA Viruses/physiology , RNA, Viral/biosynthesis , RNA, Viral/genetics , Recombination, Genetic , Vaccines, Attenuated/genetics , Viral Vaccines/genetics , Virus Replication
19.
J Virol ; 78(21): 11726-38, 2004 Nov.
Article in English | MEDLINE | ID: mdl-15479814

ABSTRACT

The role of the variable portion of the noncoding regions (NCRs) of the three Bunyaviridae RNA segments (L, M, S) in transcription, replication, and packaging was studied using the recently developed plasmid-driven RNA polymerase I minigenome system for Uukuniemi (UUK) virus, genus Phlebovirus (11), as a model. Comparison of the different segments showed that all NCRs were sufficient to mediate transcription/replication of a minigenome but demonstrated decreased promoter strength in the order M > L > S. Chimeric minigenomes with flanking NCRs from different genome segments revealed that the number of total base pairs within the inverted, partially complementary ends was important for transcription and replication. Point mutations increasing the base-pairing potential produced increased reporter expression, indicating that complementarity between the 5' and 3' ends is crucial for promoter activity. The role of the intergenic region (IGR) located between the two open reading frames of the ambisense UUK virus S segment was analyzed by inserting this sequence element downstream of the reporter genes. The presence of the IGR was found to enhance reporter expression, demonstrating that efficient transcription termination, regulated by the IGR, is important for optimal minigenome mRNA translation. Finally, genome packaging efficacy varied for different NCRs and was strongest for L followed by M and S. Strong reporter gene activity was still observed after seven consecutive cell culture passages, indicating a selective rather than random genome-packaging mechanism. In summary, our results demonstrate that the NCRs from all three segments contain the necessary signals to initiate transcription and replication as well as packaging. Based on promoter strength, M-segment NCRs may be the preferred choice for the development of reverse genetics and minigenome rescue systems for bunyaviruses.


Subject(s)
Bunyaviridae/genetics , RNA, Untranslated/physiology , RNA, Viral/physiology , Animals , Base Sequence , Cricetinae , Genome, Viral , Molecular Sequence Data , Promoter Regions, Genetic , Recombination, Genetic , Virus Assembly
20.
Virology ; 321(1): 23-8, 2004 Mar 30.
Article in English | MEDLINE | ID: mdl-15033561

ABSTRACT

Crimean-Congo hemorrhagic fever (CCHF) virus is highly pathogenic for humans and remains the only Category A virus for which full sequence information is currently unavailable. In this study we completed CCHF genome characterization by determining the L segment sequence using Dugbe and CCHF virus-specific oligonucleotides. Sequence alignments revealed the presence of four previously described conserved regions in all Bunyaviridae polymerases. Interestingly, additional regions containing putative Ovarian Tumor (OTU)-like cysteine protease and helicase domains were identified in the L segments of CCHF and Dugbe viruses, suggesting an autoproteolytic cleavage process for nairovirus L proteins.


Subject(s)
Genome, Viral , Hemorrhagic Fever Virus, Crimean-Congo/genetics , RNA-Dependent RNA Polymerase/genetics , Amino Acid Sequence , Cloning, Molecular , Cysteine Endopeptidases/genetics , Hemorrhagic Fever Virus, Crimean-Congo/classification , Hemorrhagic Fever Virus, Crimean-Congo/enzymology , Molecular Sequence Data , Open Reading Frames , Protein Structure, Tertiary/genetics , RNA Helicases/genetics , Sequence Alignment
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