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In JoVE (1)
- Using Reverse Genetics to Manipulate the NSs Gene of the Rift Valley Fever Virus MP-12 Strain to Improve Vaccine Safety and Efficacy
Other Publications (26)
- Journal of Clinical Microbiology
- Journal of Clinical Microbiology
- Experimental Animals / Japanese Association for Laboratory Animal Science
- Microbiology and Immunology
- Experimental Animals / Japanese Association for Laboratory Animal Science
- Clinical and Diagnostic Laboratory Immunology
- Journal of Virological Methods
- Clinical and Diagnostic Laboratory Immunology
- Journal of Virology
- Journal of Virology
- Journal of Virology
- Clinical and Vaccine Immunology : CVI
- Journal of Virology
- Proceedings of the National Academy of Sciences of the United States of America
- Journal of Virology
- Journal of Virology
- Journal of Virology
- PLoS Pathogens
- Annals of the New York Academy of Sciences
- Journal of Virology
- PloS One
- Journal of Virology
- Emerging Infectious Diseases
Articles by Tetsuro Ikegami in JoVE
Using Reverse Genetics to Manipulate the NSs Gene of the Rift Valley Fever Virus MP-12 Strain to Improve Vaccine Safety and Efficacy
Birte Kalveram, Olga Lihoradova, Sabarish V. Indran, Tetsuro Ikegami
Department of Pathology, University of Texas Medical Branch
The reverse genetics system for the Rift Valley fever virus MP-12 vaccine strain is a useful tool for creating additional MP-12 mutants with increased attenuation and immunogenicity. We describe the protocol to generate and characterize NSs mutant strains.
Other articles by Tetsuro Ikegami on PubMed
Immunofluorescence Technique Using HeLa Cells Expressing Recombinant Nucleoprotein for Detection of Immunoglobulin G Antibodies to Crimean-Congo Hemorrhagic Fever Virus
Journal of Clinical Microbiology. Feb, 2002 | Pubmed ID: 11825944
A HeLa cell line continuously expressing recombinant nucleoprotein (rNP) of the Crimean-Congo hemorrhagic fever virus (CCHFV) was established by transfection with an expression vector containing the cDNA of CCHFV NP (pKS336-CCHFV-NP). These cells were used as antigens for indirect immunofluorescence (IF) to detect immunoglobulin G antibodies to CCHFV. The sensitivity and specificity of this IF technique were examined by using serum samples and were compared to those of the IF technique using CCHFV-infected Vero E6 cells (authentic antigen). Staining of the CCHFV rNP expressed in HeLa cells showed a unique granular pattern similar to that of CCHFV-infected Vero E6 cells. Positive staining could easily be distinguished from a negative result. All 13 serum samples determined to be positive by using the authentic antigen were also determined to be positive by using CCHFV rNP-expressing HeLa cells (recombinant antigen). The 108 serum samples determined to be negative by using the authentic antigen were also determined to be negative by using the recombinant antigen. Thus, both the sensitivity and the specificity of this IF technique were 100% compared to the IF with authentic antigen. The novel IF technique using CCHFV rNP-expressing HeLa cells can be used not only for diagnosis of CCHF but also for epidemiological studies on CCHFV infections.
Recombinant Nucleoprotein-based Enzyme-linked Immunosorbent Assay for Detection of Immunoglobulin G Antibodies to Crimean-Congo Hemorrhagic Fever Virus
Journal of Clinical Microbiology. May, 2002 | Pubmed ID: 11980926
The full-length nucleoprotein of Crimean-Congo hemorrhagic fever virus (CCHFV; 482 amino acid residues) was expressed as a His-tagged recombinant protein (His-CCHFV rNP) in the baculovirus system. The His-CCHFV rNP was efficiently expressed in insect cells and purified by Ni(2+) column chromatography. Using this substrate, an immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) was developed. We evaluated the sensitivity and specificity of the IgG ELISA, using serum samples previously determined to be antibody positive or negative by immunofluorescence tests on CCHFV-infected Vero E6 cells. We found very good correlation between the two tests: 87% for the positive sera (13 of 15) and 99% for the negative sera (107 of 108). These results indicate that the new IgG ELISA using His-CCHFV rNP has high sensitivity and specificity for detecting CCHFV antibodies. The CCHF patients' sera with high titers reacted only with the NP fragment containing amino acid residues between 201 and 306 in Western blotting. It is known that amino acid homologies are high in this region among various isolates. Thus, it is expected that this ELISA can detect antibodies not only for Chinese strains of CCHFV but also for other strains circulating in the world. These results suggest that the IgG ELISA system developed with the recombinant CCHFV NP is a valuable tool for diagnosis and epidemiological investigations of CCHFV infections.
Chronological and Spatial Analysis of the 1996 Ebola Reston Virus Outbreak in a Monkey Breeding Facility in the Philippines
Experimental Animals / Japanese Association for Laboratory Animal Science. Apr, 2002 | Pubmed ID: 12012728
To describe the transmission pattern of natural infection with Ebola Reston (EBO-R) virus in a breeding colony, the chronological and spatial analysis of mortality during the 1996 EBO-R virus outbreak was done in this study. The EBO-R virus infection among monkeys in the facility was widespread. Over a period of 3 months, 14 out of 21 occupied units were contaminated with antigen positive animals. A large number of wild-caught monkeys were involved in this outbreak suggesting that wild-caught monkeys have a high susceptibility to EBO-R virus infection. In this outbreak, morbidity patterns for individual animal units were very different regardless of the type and size of cages, individual or gang cages. The results suggest that not only the cage size but also poor animal husbandry practices may be risk factors for the spread of EBO-R infection.
Development of an Immunofluorescence Method for the Detection of Antibodies to Ebola Virus Subtype Reston by the Use of Recombinant Nucleoprotein-expressing HeLa Cells
Microbiology and Immunology. 2002 | Pubmed ID: 12437031
An indirect immunofluorescent assay (IFA) to detect Ebola virus subtype Reston (EBO-R) antibodies was developed by the use of a HeLa cell line stably expressing EBO-R nucleoprotein (NP). This IFA has a high specificity for the detection of EBO-R IgG antibodies in both hyperimmune rabbit sera and monkey sera collected during an EBO-R outbreak in the Philippines in 1996. Furthermore, this IFA showed a higher sensitivity for the detection of EBO-R antibodies than did the IFA using HeLa cells expressing the NP of Ebola virus subtype Zaire. These results suggest that this new IFA is useful for seroepidemiological studies of EBO-R infection among monkeys.
Histopathology of Natural Ebola Virus Subtype Reston Infection in Cynomolgus Macaques During the Philippine Outbreak in 1996
Experimental Animals / Japanese Association for Laboratory Animal Science. Oct, 2002 | Pubmed ID: 12451705
We investigated the livers, spleens, kidneys and lungs collected from 24 cynomolgus macaques (Macaca fascicularis) naturally infected with Ebola virus subtype Reston (EBO-R) during the Philippine outbreak in 1996, in order to reveal the histopathologic findings. These macaques showed necrotic hepatocytes with inclusions, slight to massive fibrin deposition in splenic cords, depletion of lymphoid cells in the white pulp of the spleen, and fibrin thrombi in some organs. Immunohistochemical analysis using anti-leukocyte antigen L1 antibody revealed an increase in blood-derived macrophages/monocytes in the livers, kidneys and lungs of EBO-R infected macaques. EBO-R NP antigens were detected in the macrophages/monocytes, endothelial cells and fibroblasts in the liver, spleen, kidney and lung. These results indicate that EBO-R infection is characterized by systemic coagulopathy and an increase in blood-derived macrophages/monocytes in accordance with the EBO-R propagation in macrophages/monocytes.
Analysis of Linear B-cell Epitopes of the Nucleoprotein of Ebola Virus That Distinguish Ebola Virus Subtypes
Clinical and Diagnostic Laboratory Immunology. Jan, 2003 | Pubmed ID: 12522044
Ebola virus consists of four genetically distinguishable subtypes. We developed monoclonal antibodies (MAbs) to the nucleoprotein (NP) of Ebola virus Zaire subtype and analyzed their cross-reactivities to the Reston and Sudan subtypes. We further determined the epitopes recognized by these MAbs. Three MAbs reacted with the three major subtypes and recognized a fragment containing 110 amino acids (aa) at the C-terminal extremity. They did not show specific reactivities to any 10-aa short peptides in Pepscan analyses, suggesting that these MAbs recognize conformational epitope(s) located within this region. Six MAbs recognized a fragment corresponding to aa 361 to 461 of the NP. Five of these six MAbs showed specific reactivities in Pepscan analyses, and the epitopes were identified in two regions, aa 424 to 430 and aa 451 to 455. Three MAbs that recognized the former epitope region cross-reacted with all three subtypes, and one that recognized the same epitope region was Zaire specific. One MAb, which recognized the latter epitope region, was reactive with Zaire and Sudan subtypes but not with the Reston subtype. These results suggest that Ebola virus NP has at least two linear epitope regions and that the recognition of the epitope by MAbs can vary even within the same epitope region. These MAbs showing different subtype specificities might be useful reagents for developing an immunological system to identify Ebola virus subtypes.
Detection of Immunoglobulin G to Crimean-Congo Hemorrhagic Fever Virus in Sheep Sera by Recombinant Nucleoprotein-based Enzyme-linked Immunosorbent and Immunofluorescence Assays
Journal of Virological Methods. Mar, 2003 | Pubmed ID: 12565161
Crimean-Congo hemorrhagic fever virus is a tick-borne virus that causes severe hemorrhagic symptoms with an up to 50% mortality rate in humans. Wild and domestic animals, such as sheep, cattle and goats, are the reservoirs. The recombinant nucleoprotein-based Crimean-Congo hemorrhagic fever virus antibody detection systems for sheep sera were developed by enzyme-linked immunosorbent assay (ELISA) and an indirect immunofluorescence assay techniques. The samples used for evaluation were 80 sera collected from sheep in a Crimean-Congo hemorrhagic fever-endemic area (western part of the Xinjiang Uygur Autonomous Region) and 39 sera collected from sheep in a disease-free region (Shandong province, eastern China). The ELISA and indirect immunofluorescence assay using recombinant nucleoprotein of the virus proved to have high sensitivity and specificity for detecting the immunoglobulin G antibodies to the virus in sheep sera. Within this limited number of samples, the recombinant nucleoprotein-based ELISA and indirect immunofluorescence assay are considered to be useful tools for seroepidemiological study of virus infections in sheep sera.
Antigen Capture Enzyme-linked Immunosorbent Assay for Specific Detection of Reston Ebola Virus Nucleoprotein
Clinical and Diagnostic Laboratory Immunology. Jul, 2003 | Pubmed ID: 12853385
Antigen capture enzyme-linked immunosorbent assay (ELISA) is one of the most useful methods to detect Ebola virus rapidly. We previously developed an antigen capture ELISA using a monoclonal antibody (MAb), 3-3D, which reacted not only to the nucleoprotein (NP) of Zaire Ebola virus (EBO-Z) but also to the NPs of Sudan (EBO-S) and Reston Ebola (EBO-R) viruses. In this study, we developed antigen capture ELISAs using two novel MAbs, Res2-6C8 and Res2-1D8, specific to the NP of EBO-R. Res2-6C8 and Res2-1D8 recognized epitopes consisting of 4 and 8 amino acid residues, respectively, near the C-terminal region of the EBO-R NP. The antigen capture ELISAs using these two MAbs detected the EBO-R NP in the tissues from EBO-R-infected cynomolgus macaques. The antigen capture ELISAs using Res2-6C8 and Res2-1D8 are useful for the rapid detection of the NP in EBO-R-infected cynomolgus macaques.
Uirusu. Dec, 2004 | Pubmed ID: 15745161
Rift Valley fever virus (RVFV) causes massive mosquito-borne epidemics among humans and decimates ruminants in which the mortality rate is about 1% and 10-30%, respectively. Morbidity in RVFV-infected humans is high largely due to the effects of hemorrhagic fever and encephalitis. This virus is native to sub-Saharan Africa; yet if this virus is introduced into the environment, virus transmission appears to occur whenever sheep and cattle are present with abundant mosquito populations. RVFV is a negative-strand RNA virus which belongs to the family Bunyaviridae, genus Phlebovirus, and contains tripartite-segmented genomes (S, M, and L). S-segment is the ambisense genome, where N and NSs genes are coded in an antiviral-sense and viral sense S-segment, respectively. The inhibition of host mRNA synthesis, which is induced by the binding of NSs protein to RNA polymerase II transcription factor TFIIH, is the primary reason for the host-protein shut-off in RVFV-infected cells. Development of a RVFV reverse genetics system, which has not been accomplished yet, is important for the study of viral replication mechanisms, host virus interaction, viral pathogenicity as well as vaccine evaluation and development.
Rift Valley Fever Virus Nonstructural Protein NSs Promotes Viral RNA Replication and Transcription in a Minigenome System
Journal of Virology. May, 2005 | Pubmed ID: 15827175
Rift Valley fever virus (RVFV), which belongs to the genus Phlebovirus, family Bunyaviridae, has a tripartite negative-strand genome (S, M, and L segments) and is an important mosquito-borne pathogen for domestic animals and humans. We established an RVFV T7 RNA polymerase-driven minigenome system in which T7 RNA polymerase from an expression plasmid drove expression of RNA transcripts for viral proteins and minigenome RNA transcripts carrying a reporter gene between both termini of the M RNA segment in 293T cells. Like other viruses of the Bunyaviridae family, replication and transcription of the RVFV minigenome required expression of viral N and L proteins. Unexpectedly, the coexpression of an RVFV nonstructural protein, NSs, with N and L proteins resulted in a significant enhancement of minigenome RNA replication. Coexpression of NSs protein with N and L proteins also enhanced minigenome mRNA transcription in the cells expressing viral-sense minigenome RNA transcripts. NSs protein expression increased the RNA replication of minigenomes that originated from S and L RNA segments. Enhancement of minigenome RNA synthesis by NSs protein occurred in cells lacking alpha/beta interferon (IFN-alpha/beta) genes, indicating that the effect of NSs protein on minigenome RNA replication was unrelated to a putative NSs protein-induced inhibition of IFN-alpha/beta production. Our finding that RVFV NSs protein augmented minigenome RNA synthesis was in sharp contrast to reports that Bunyamwera virus (genus Bunyavirus) NSs protein inhibits viral minigenome RNA synthesis, suggesting that RVFV NSs protein and Bunyamwera virus NSs protein have distinctly different biological roles in viral RNA synthesis.
Journal of Virology. Sep, 2005 | Pubmed ID: 16140788
Analysis of purified Rift Valley fever virus (RVFV) particles demonstrated the presence of three negative-sense RNA genomes, plus three anti-viral-sense RNA segments. The virion-associated anti-viral-sense S segment served as a template for the synthesis of NSs mRNA immediately after infection. NSs protein synthesis also occurred early in infection, suggesting that NSs protein produced early in infection probably has biologically significant roles in virus replication and/or survival in the host. Translation inhibitor treatment of mammalian cells infected with viruses belonging to the Bunyaviridae family generally inhibits viral mRNA synthesis. However, RVFV NSs mRNA synthesis, but not N mRNA synthesis, was resistant to puromycin treatment during primary transcription, pointing to the uniqueness of RVFV NSs mRNA synthesis.
Rescue of Infectious Rift Valley Fever Virus Entirely from CDNA, Analysis of Virus Lacking the NSs Gene, and Expression of a Foreign Gene
Journal of Virology. Mar, 2006 | Pubmed ID: 16501102
Rift Valley fever virus (RVFV) (genus Phlebovirus, family Bunyaviridae) has a tripartite negative-strand genome, causes a mosquito-borne disease that is endemic in sub-Saharan African countries and that also causes large epidemics among humans and livestock. Furthermore, it is a bioterrorist threat and poses a risk for introduction to other areas. In spite of its danger, neither veterinary nor human vaccines are available. We established a T7 RNA polymerase-driven reverse genetics system to rescue infectious clones of RVFV MP-12 strain entirely from cDNA, the first for any phlebovirus. Expression of viral structural proteins from the protein expression plasmids was not required for virus rescue, whereas NSs protein expression abolished virus rescue. Mutants of MP-12 partially or completely lacking the NSs open reading frame were viable. These NSs deletion mutants replicated efficiently in Vero and 293 cells, but not in MRC-5 cells. In the latter cell line, accumulation of beta interferon mRNA occurred after infection by these NSs deletion mutants, but not after infection by MP-12. The NSs deletion mutants formed larger plaques than MP-12 did in Vero E6 cells and failed to shut off host protein synthesis in Vero cells. An MP-12 mutant carrying a luciferase gene in place of the NSs gene replicated as efficiently as MP-12 did, produced enzymatically active luciferase during replication, and stably retained the luciferase gene after 10 virus passages, representing the first demonstration of foreign gene expression in any bunyavirus. This reverse genetics system can be used to study the molecular virology of RVFV, assess current vaccine candidates, produce new vaccines, and incorporate marker genes into animal vaccines.
Laboratory Diagnostic Systems for Ebola and Marburg Hemorrhagic Fevers Developed with Recombinant Proteins
Clinical and Vaccine Immunology : CVI. Apr, 2006 | Pubmed ID: 16603611
NSm and 78-kilodalton Proteins of Rift Valley Fever Virus Are Nonessential for Viral Replication in Cell Culture
Journal of Virology. Aug, 2006 | Pubmed ID: 16873285
Rift Valley fever viruses carrying mutations of the M gene preglycoprotein region, one lacking NSm protein expression, one lacking 78-kDa protein expression, and one lacking expression of both proteins, were compared in cell culture. All of the mutants and their parent virus produced plaques with similar sizes and morphologies in Vero E6 cells and had similar growth kinetics in Vero, C6/36, and MRC5 cells, demonstrating that the NSm and 78-kDa proteins were not needed for the virus to replicate efficiently in cell culture. A competition-propagation assay revealed that the parental virus was slightly more fit than the mutant virus lacking expression of both proteins.
Severe Acute Respiratory Syndrome Coronavirus Nsp1 Protein Suppresses Host Gene Expression by Promoting Host MRNA Degradation
Proceedings of the National Academy of Sciences of the United States of America. Aug, 2006 | Pubmed ID: 16912115
Severe acute respiratory syndrome (SARS) coronavirus (SCoV) causes a recently emerged human disease associated with pneumonia. The 5' end two-thirds of the single-stranded positive-sense viral genomic RNA, gene 1, encodes 16 mature proteins. Expression of nsp1, the most N-terminal gene 1 protein, prevented Sendai virus-induced endogenous IFN-beta mRNA accumulation without inhibiting dimerization of IFN regulatory factor 3, a protein that is essential for activation of the IFN-beta promoter. Furthermore, nsp1 expression promoted degradation of expressed RNA transcripts and host endogenous mRNAs, leading to a strong host protein synthesis inhibition. SCoV replication also promoted degradation of expressed RNA transcripts and host mRNAs, suggesting that nsp1 exerted its mRNA destabilization function in infected cells. In contrast to nsp1-induced mRNA destablization, no degradation of the 28S and 18S rRNAs occurred in either nsp1-expressing cells or SCoV-infected cells. These data suggested that, in infected cells, nsp1 promotes host mRNA degradation and thereby suppresses host gene expression, including proteins involved in host innate immune functions. SCoV nsp1-mediated promotion of host mRNA degradation may play an important role in SCoV pathogenesis.
Journal of Virology. Aug, 2007 | Pubmed ID: 17537856
Rift Valley fever virus (RVFV) (genus Phlebovirus, family Bunyaviridae) has a tripartite negative-strand genome and causes a mosquito-borne disease among humans and livestock in sub-Saharan African and Arabian Peninsula countries. Phlebovirus L, M, and N mRNAs are synthesized from the virus-sense RNA segments, while NSs mRNA is transcribed from the anti-virus-sense S segment. The present study determined the 3' termini of all RVFV mRNAs. The 3' termini of N and NSs mRNAs were mapped within the S-segment intergenic region and were complementary to each other by 30 to 60 nucleotides. The termini of M and L mRNAs were mapped within 122 to 107 nucleotides and 16 to 41 nucleotides, respectively, from the 5' ends of their templates. Viral RNA elements that control phlebovirus transcriptional terminations are largely unknown. Our studies suggested the importance of a pentanucleotide sequence, CGUCG, for N, NSs, and M mRNA transcription terminations. Homopolymeric tracts of C sequences, which were located upstream of the pentanucleotide sequence, promoted N and M mRNA terminations. Likewise, the homopolymeric tracts of G sequences that are found upstream of the pentanucleotide sequence promoted NSs mRNA termination. The L-segment 5'-untranslated region (L-5' UTR) had neither the pentanucleotide sequence nor homopolymeric sequences, yet replacement of the S-segment intergenic region with the L-5' UTR exerted N mRNA termination in an infectious virus. The L-5' UTR contained two 13-nucleotide-long complete complementary sequences, and their sequence complementarities were important for L mRNA termination. A computer-mediated RNA secondary structure analysis further suggested that RNA secondary structures formed by the sections of the two 13-nucleotide-long sequences and by the sequence between them may have a role in L mRNA termination. Our data demonstrated that viral RNA elements that govern L mRNA termination differed from those that regulate mRNA terminations in the M and S segments.
Journal of Virology. Dec, 2007 | Pubmed ID: 17913816
Rift Valley fever virus (RVFV) is a member of the genus Phlebovirus within the family Bunyaviridae. It can cause severe epidemics among ruminants and fever, myalgia, a hemorrhagic syndrome, and/or encephalitis in humans. The RVFV M segment encodes the NSm and 78-kDa proteins and two major envelope proteins, Gn and Gc. The biological functions of the NSm and 78-kDa proteins are unknown; both proteins are dispensable for viral replication in cell cultures. To determine the biological functions of the NSm and 78-kDa proteins, we generated the mutant virus arMP-12-del21/384, carrying a large deletion in the pre-Gn region of the M segment. Neither NSm nor the 78-kDa protein was synthesized in arMP-12-del21/384-infected cells. Although arMP-12-del21/384 and its parental virus, arMP-12, showed similar growth kinetics and viral RNA and protein accumulation in infected cells, arMP-12-del21/384-infected cells induced extensive cell death and produced larger plaques than did arMP-12-infected cells. arMP-12-del21/384 replication triggered apoptosis, including the cleavage of caspase-3, the cleavage of its downstream substrate, poly(ADP-ribose) polymerase, and activation of the initiator caspases, caspase-8 and -9, earlier in infection than arMP-12. NSm expression in arMP-12-del21/384-infected cells suppressed the severity of caspase-3 activation. Further, NSm protein expression inhibited the staurosporine-induced activation of caspase-8 and -9, demonstrating that other viral proteins were dispensable for NSm's function in inhibiting apoptosis. RVFV NSm protein is the first identified Phlebovirus protein that has an antiapoptotic function.
Severe Acute Respiratory Syndrome Coronavirus Nsp1 Suppresses Host Gene Expression, Including That of Type I Interferon, in Infected Cells
Journal of Virology. May, 2008 | Pubmed ID: 18305050
The severe acute respiratory syndrome coronavirus (SARS-CoV) nsp1 protein has unique biological functions that have not been described in the viral proteins of any RNA viruses; expressed SARS-CoV nsp1 protein has been found to suppress host gene expression by promoting host mRNA degradation and inhibiting translation. We generated an nsp1 mutant (nsp1-mt) that neither promoted host mRNA degradation nor suppressed host protein synthesis in expressing cells. Both a SARS-CoV mutant virus, encoding the nsp1-mt protein (SARS-CoV-mt), and a wild-type virus (SARS-CoV-WT) replicated efficiently and exhibited similar one-step growth kinetics in susceptible cells. Both viruses accumulated similar amounts of virus-specific mRNAs and nsp1 protein in infected cells, whereas the amounts of endogenous host mRNAs were clearly higher in SARS-CoV-mt-infected cells than in SARS-CoV-WT-infected cells, in both the presence and absence of actinomycin D. Further, SARS-CoV-WT replication strongly inhibited host protein synthesis, whereas host protein synthesis inhibition in SARS-CoV-mt-infected cells was not as efficient as in SARS-CoV-WT-infected cells. These data revealed that nsp1 indeed promoted host mRNA degradation and contributed to host protein translation inhibition in infected cells. Notably, SARS-CoV-mt infection, but not SARS-CoV-WT infection, induced high levels of beta interferon (IFN) mRNA accumulation and high titers of type I IFN production. These data demonstrated that SARS-CoV nsp1 suppressed host innate immune functions, including type I IFN expression, in infected cells and suggested that SARS-CoV nsp1 most probably plays a critical role in SARS-CoV virulence.
Rift Valley Fever Virus NSs Protein Promotes Post-transcriptional Downregulation of Protein Kinase PKR and Inhibits EIF2alpha Phosphorylation
PLoS Pathogens. Feb, 2009 | Pubmed ID: 19197350
Rift Valley fever virus (RVFV) (genus Phlebovirus, family Bunyaviridae) is a negative-stranded RNA virus with a tripartite genome. RVFV is transmitted by mosquitoes and causes fever and severe hemorrhagic illness among humans, and fever and high rates of abortions in livestock. A nonstructural RVFV NSs protein inhibits the transcription of host mRNAs, including interferon-beta mRNA, and is a major virulence factor. The present study explored a novel function of the RVFV NSs protein by testing the replication of RVFV lacking the NSs gene in the presence of actinomycin D (ActD) or alpha-amanitin, both of which served as a surrogate of the host mRNA synthesis suppression function of the NSs. In the presence of the host-transcriptional inhibitors, the replication of RVFV lacking the NSs protein, but not that carrying NSs, induced double-stranded RNA-dependent protein kinase (PKR)-mediated eukaryotic initiation factor (eIF)2alpha phosphorylation, leading to the suppression of host and viral protein translation. RVFV NSs promoted post-transcriptional downregulation of PKR early in the course of the infection and suppressed the phosphorylated eIF2alpha accumulation. These data suggested that a combination of RVFV replication and NSs-induced host transcriptional suppression induces PKR-mediated eIF2alpha phosphorylation, while the NSs facilitates efficient viral translation by downregulating PKR and inhibiting PKR-mediated eIF2alpha phosphorylation. Thus, the two distinct functions of the NSs, i.e., the suppression of host transcription, including that of type I interferon mRNAs, and the downregulation of PKR, work together to prevent host innate antiviral functions, allowing efficient replication and survival of RVFV in infected mammalian hosts.
Dual Functions of Rift Valley Fever Virus NSs Protein: Inhibition of Host MRNA Transcription and Post-transcriptional Downregulation of Protein Kinase PKR
Annals of the New York Academy of Sciences. Sep, 2009 | Pubmed ID: 19751406
Rift Valley fever virus (RVFV), which belongs to the genus Phlebovirus, family Bunyaviridae, is a negative-stranded RNA virus carrying a single-stranded, tripartite RNA genome. RVFV is an important zoonotic pathogen transmitted by mosquitoes and causes large outbreaks among ruminants and humans in Africa and the Arabian Peninsula. Human patients develop an acute febrile illness, followed by a fatal hemorrhagic fever, encephalitis, or ocular diseases. A viral nonstructural protein, NSs, is a major viral virulence factor. Past studies showed that NSs suppresses the transcription of host mRNAs, including interferon-beta mRNAs. Here we demonstrated that the NSs protein induced post-transcriptional downregulation of dsRNA-dependent protein kinase (PKR), to prevent phosphorylation of eIF2alpha and promoted viral translation in infected cells. These two biological activities of the NSs most probably have a synergistic effect in suppressing host innate immune functions and facilitate efficient viral replication in infected mammalian hosts.
Journal of Virology. Dec, 2009 | Pubmed ID: 19812169
Rift Valley fever virus (RVFV) (genus Phlebovirus, family Bunyaviridae) causes mosquito-borne epidemic diseases in humans and livestock. The virus carries three RNA segments, L, M, and S, of negative or ambisense polarity. L protein, an RNA-dependent RNA polymerase, encoded in the L segment, and N protein, encoded in the S segment, exert viral RNA replication and transcription. Coexpression of N, hemagglutinin (HA)-tagged L, and viral minigenome resulted in minigenome replication and transcription, a finding that demonstrated HA-tagged L was biologically active. Likewise L tagged with green fluorescent protein (GFP) was biologically competent. Coimmunoprecipitation analysis using extracts from cells coexpressing HA-tagged L and GFP-tagged L showed the formation of an L oligomer. Bimolecular fluorescence complementation analysis and coimmunoprecipitation studies demonstrated the formation of an intermolecular L-L interaction through its N-terminal and C-terminal regions and also suggested an intramolecular association between the N-terminal and C-terminal regions of L protein. A biologically inactive L mutant, in which the conserved signature SDD motif was replaced by the amino acid residues GNN, exhibited a dominant negative phenotype when coexpressed with wild-type L in the minigenome assay system. Expression of this mutant L also inhibited viral gene expression in virus-infected cells. These data provided compelling evidence for the importance of oligomerization of RVFV L protein for its polymerase activity.
Vaccine. Nov, 2009 | Pubmed ID: 19837291
Rift Valley fever virus (RVFV), which belongs to the genus Phlebovirus, family Bunyaviridae, is a negative-stranded RNA virus carrying a tripartite RNA genome. RVFV is transmitted by mosquitoes and causes large outbreaks among ruminants and humans in Africa and the Arabian Peninsula. Human patients develop an acute febrile illness, followed by a fatal hemorrhagic fever, encephalitis or ocular diseases, whereas ruminants experience abortions during outbreak. Effective vaccination of both humans and ruminants is the best approach to control Rift Valley fever. This article summarizes the development of inactivated RVFV vaccine, live attenuated vaccine, and other new generation vaccines.
Rapid Accumulation of Virulent Rift Valley Fever Virus in Mice from an Attenuated Virus Carrying a Single Nucleotide Substitution in the M RNA
PloS One. 2010 | Pubmed ID: 20376320
Rift Valley fever virus (RVFV), a member of the genus Phlebovirus within the family Bunyaviridae, is a negative-stranded RNA virus with a tripartite genome. RVFV is transmitted by mosquitoes and causes fever and severe hemorrhagic illness among humans, while in livestock it causes fever and high abortion rates.
NSs Protein of Rift Valley Fever Virus Promotes Posttranslational Downregulation of the TFIIH Subunit P62
Journal of Virology. Jul, 2011 | Pubmed ID: 21543505
Rift Valley fever virus (RVFV; family Bunyaviridae, genus Phlebovirus) is an important emerging pathogen of humans and ruminants. Its NSs protein has previously been identified as a major virulence factor that suppresses host defense through three distinct mechanisms: it directly inhibits beta interferon (IFN-β) promoter activity, it promotes the degradation of double-stranded RNA-dependent protein kinase (PKR), and it suppresses host transcription by disrupting the assembly of the basal transcription factor TFIIH through sequestration of its p44 subunit. Here, we report that in addition to PKR, NSs also promotes the degradation of the TFIIH subunit p62. Infection of cells with the RVFV MP-12 vaccine strain reduced p62 protein levels to below the detection limit early in the course of infection. This NSs-mediated downregulation of p62 was posttranslational, as it was unaffected by pharmacological inhibition of transcription or translation and MP-12 infection had no effect on p62 mRNA levels. Treatment of cells with proteasome inhibitors but not inhibition of lysosomal acidification or nuclear export resulted in a stabilization of p62 in the presence of NSs. Furthermore, p62 could be coprecipitated with NSs from lysates of infected cells. These data suggest that the RVFV NSs protein is able to interact with the TFIIH subunit p62 inside infected cells and promotes its degradation, which can occur directly in the nucleus.
Viruses. May, 2011 | Pubmed ID: 21666766
Rift Valley fever (RVF) is an emerging zoonotic disease distributed in sub-Saharan African countries and the Arabian Peninsula. The disease is caused by the Rift Valley fever virus (RVFV) of the family Bunyaviridae and the genus Phlebovirus. The virus is transmitted by mosquitoes, and virus replication in domestic ruminant results in high rates of mortality and abortion. RVFV infection in humans usually causes a self-limiting, acute and febrile illness; however, a small number of cases progress to neurological disorders, partial or complete blindness, hemorrhagic fever, or thrombosis. This review describes the pathology of RVF in human patients and several animal models, and summarizes the role of viral virulence factors and host factors that affect RVFV pathogenesis.
Emerging Infectious Diseases. Aug, 2011 | Pubmed ID: 21801651