Computing the Structural Dynamics of RVFV L Protein Domain in Aqueous Glycerol Solutions.

Many biological and biotechnological processes are controlled by protein-protein and protein-solvent interactions. In order to understand, predict, and optimize such processes, it is important to understand how solvents affect protein structure during protein-solvent interactions. In this study, all-atom molecular dynamics are used to investigate the structural dynamics and energetic properties of a C-terminal domain of the Rift Valley Fever Virus L protein solvated in glycerol and aqueous glycerol solutions in different concentrations by molecular weight. The Generalized Amber Force Field is modified by including restrained electrostatic potential atomic charges for the glycerol molecules. The peptide is considered in detail by monitoring properties like the root-mean-squared deviation, root-mean-squared fluctuation, radius of gyration, hydrodynamic radius, end-to-end distance, solvent-accessible surface area, intra-potential energy, and solvent-peptide interaction energies for hundreds of nanoseconds. Secondary structure analysis is also performed to examine the extent of conformational drift for the individual helices and sheets. We predict that the peptide helices and sheets are maintained only when the modeling strategy considers the solvent with lower glycerol concentration. We also find that the solvent-peptide becomes more cohesive with decreasing glycerol concentrations. The density and radial distribution function of glycerol solvent calculated when modeled with the modified atomic charges show a very good agreement with experimental results and other simulations at 298.15K.

The Change P82L in the Rift Valley Fever Virus NSs Protein Confers Attenuation in Mice.

Rift Valley fever virus (RVFV) is a mosquito-borne bunyavirus that causes an important disease in ruminants, with great economic losses. The infection can be also transmitted to humans; therefore, it is considered a major threat to both human and animal health. In a previous work, we described a novel RVFV variant selected in cell culture in the presence of the antiviral agent favipiravir that was highly attenuated in vivo. This variant displayed 24 amino acid substitutions in different viral proteins when compared to its parental viral strain, two of them located in the NSs protein that is known to be the major virulence factor of RVFV. By means of a reverse genetics system, in this work we have analyzed the effect that one of these substitutions, P82L, has in viral attenuation in vivo. Rescued viruses carrying this single amino acid change were clearly attenuated in BALB/c mice while their growth in an interferon (IFN)-competent cell line as well as the production of interferon beta (IFN-ß) did not seem to be affected. However, the pattern of nuclear NSs accumulation was modified in cells infected with the mutant viruses. These results highlight the key role of the NSs protein in the modulation of viral infectivity.

NSs Filament Formation Is Important but Not Sufficient for RVFV Virulence In Vivo.

Rift Valley fever virus (RVFV) is a mosquito-borne phlebovirus that represents as a serious health threat to both domestic animals and humans. The viral protein NSs is the key virulence factor of RVFV, and has been proposed that NSs nuclear filament formation is critical for its virulence. However, the detailed mechanisms are currently unclear. Here, we generated a T7 RNA polymerase-driven RVFV reverse genetics system based on a strain imported into China (BJ01). Several NSs mutations (T1, T3 and T4) were introduced into the system for investigating the correlation between NSs filament formation and virulence in vivo. The NSs T1 mutant showed distinct NSs filament in the nuclei of infected cells, the T3 mutant diffusively localized in the cytoplasm and the T4 mutant showed fragmented nuclear filament formation. Infection of BALB/c mice with these NSs mutant viruses revealed that the in vivo virulence was severely compromised for all three NSs mutants, including the T1 mutant. This suggests that NSs filament formation is not directly correlated with RVFV virulence in vivo. Results from this study not only shed new light on the virulence mechanism of RVFV NSs but also provided tools for future in-depth investigations of RVFV pathogenesis and anti-RVFV drug screening.

Modeling the Tertiary Structure of the Rift Valley Fever Virus L Protein.

A tertiary structure governs, to a great extent, the biological activity of a protein in the living cell and is consequently a central focus of numerous studies aiming to shed light on cellular processes central to human health. Here, we aim to elucidate the structure of the Rift Valley fever virus (RVFV) L protein using a combination of in silico techniques. Due to its large size and multiple domains, elucidation of the tertiary structure of the L protein has so far challenged both dry and wet laboratories. In this work, we leverage complementary perspectives and tools from the computational-molecular-biology and bioinformatics domains for constructing, refining, and evaluating several atomistic structural models of the L protein that are physically realistic. All computed models have very flexible termini of about 200 amino acids each, and a high proportion of helical regions. Properties such as potential energy, radius of gyration, hydrodynamics radius, flexibility coefficient, and solvent-accessible surface are reported. Structural characterization of the L protein enables our laboratories to better understand viral replication and transcription via further studies of L protein-mediated protein-protein interactions. While results presented a focus on the RVFV L protein, the following workflow is a more general modeling protocol for discovering the tertiary structure of multidomain proteins consisting of thousands of amino acids.

A glycerophospholipid-specific pocket in the RVFV class II fusion protein drives target membrane insertion.

The Rift Valley fever virus (RVFV) is transmitted by infected mosquitoes, causing severe disease in humans and livestock across Africa. We determined the x-ray structure of the RVFV class II fusion protein Gc in its postfusion form and in complex with a glycerophospholipid (GPL) bound in a conserved cavity next to the fusion loop. Site-directed mutagenesis and molecular dynamics simulations further revealed a built-in motif allowing en bloc insertion of the fusion loop into membranes, making few nonpolar side-chain interactions with the aliphatic moiety and multiple polar interactions with lipid head groups upon membrane restructuring. The GPL head-group recognition pocket is conserved in the fusion proteins of other arthropod-borne viruses, such as Zika and chikungunya viruses, which have recently caused major epidemics worldwide.

Rift Valley fever phlebovirus NSs protein core domain structure suggests molecular basis for nuclear filaments.

Rift Valley fever phlebovirus (RVFV) is a clinically and economically important pathogen increasingly likely to cause widespread epidemics. RVFV virulence depends on the interferon antagonist non-structural protein (NSs), which remains poorly characterized. We identified a stable core domain of RVFV NSs (residues 83-248), and solved its crystal structure, a novel all-helical fold organized into highly ordered fibrils. A hallmark of RVFV pathology is NSs filament formation in infected cell nuclei. Recombinant virus encoding the NSs core domain induced intranuclear filaments, suggesting it contains all essential determinants for nuclear translocation and filament formation. Mutations of key crystal fibril interface residues in viruses encoding full-length NSs completely abrogated intranuclear filament formation in infected cells. We propose the fibrillar arrangement of the NSs core domain in crystals reveals the molecular basis of assembly of this key virulence factor in cell nuclei. Our findings have important implications for fundamental understanding of RVFV virulence.

Four-segmented Rift Valley fever virus-based vaccines can be applied safely in ewes during pregnancy.

Rift Valley fever virus (RVFV) causes severe and recurrent outbreaks on the African continent and the Arabian Peninsula and continues to expand its habitat. This mosquito-borne virus, belonging to the genus Phlebovirus of the family Bunyaviridae contains a tri-segmented negative-strand RNA genome. Previously, we developed four-segmented RVFV (RVFV-4s) variants by splitting the M-genome segment into two M-type segments each encoding one of the structural glycoproteins; Gn or Gc. Vaccination/challenge experiments with mice and lambs subsequently showed that RVFV-4s induces protective immunity against wild-type virus infection after a single administration. To demonstrate the unprecedented safety of RVFV-4s, we here report that the virus does not cause encephalitis after intranasal inoculation of mice. A study with pregnant ewes subsequently revealed that RVFV-4s does not cause viremia and does not cross the ovine placental barrier, as evidenced by the absence of teratogenic effects and virus in the blood and organs of the fetuses. Altogether, these results show that the RVFV-4s vaccine virus can be applied safely in pregnant ewes.

NSs Virulence Factor of Rift Valley Fever Virus Engages the F-Box Proteins FBXW11 and ß-TRCP1 To Degrade the Antiviral Protein Kinase PKR.

UNLABELLED: Rift Valley fever virus (RVFV, family Bunyaviridae, genus Phlebovirus) is a relevant pathogen of both humans and livestock in Africa. The nonstructural protein NSs is a major virulence factor known to suppress the type I interferon (IFN) response by inhibiting host cell transcription and by proteasomal degradation of a major antiviral IFN effector, the translation-inhibiting protein kinase PKR. Here, we identified components of the modular SCF (Skp1, Cul1, F-box protein)-type E3 ubiquitin ligases as mediators of PKR destruction by NSs. Small interfering RNAs (siRNAs) against the conserved SCF subunit Skp1 protected PKR from NSs-mediated degradation. Consequently, RVFV replication was severely reduced in Skp1-depleted cells when PKR was present. SCF complexes have a variable F-box protein subunit that determines substrate specificity for ubiquitination. We performed an siRNA screen for all (about 70) human F-box proteins and found FBXW11 to be involved in PKR degradation. The partial stabilization of PKR by FBXW11 depletion upregulated PKR autophosphorylation and phosphorylation of the PKR substrate eIF2α and caused a shutoff of host cell protein synthesis in RVFV-infected cells. To maximally protect PKR from the action of NSs, knockdown of structurally and functionally related FBXW1 (also known as ß-TRCP1), in addition to FBXW11 deletion, was necessary. Consequently, NSs was found to interact with both FBXW11 and ß-TRCP1. Thus, NSs eliminates the antiviral kinase PKR by recruitment of SCF-type E3 ubiquitin ligases containing FBXW11 and ß-TRCP1 as substrate recognition subunits. This antagonism of PKR by NSs is essential for efficient RVFV replication in mammalian cells. IMPORTANCE: Rift Valley fever virus is a pathogen of humans and animals that has the potential to spread from Africa and the Arabian Peninsula to other regions. A major virulence mechanism is the proteasomal degradation of the antiviral kinase PKR by the viral protein NSs. Here, we demonstrate that NSs requires E3 ubiquitin ligase complexes of the SCF (Skp1, Cul1, F-box protein) type to destroy PKR. SCF-type complexes can engage variant ubiquitination substrate recognition subunits, and we found the F-box proteins FBXW11 and ß-TRCP1 to be relevant for the action of NSs against PKR. Thus, we identified the host cell factors that are critically needed by Rift Valley fever virus to uphold its replication against the potent antiviral kinase PKR.

The use of Nanotrap particles in the enhanced detection of Rift Valley fever virus nucleoprotein.

BACKGROUND: Rift Valley fever virus (RVFV) is a highly pathogenic arthropod-borne virus that has a detrimental effect on both livestock and human populations. While there are several diagnostic methodologies available for RVFV detection, many are not sensitive enough to diagnose early infections. Furthermore, detection may be hindered by high abundant proteins such as albumin. Previous findings have shown that Nanotrap particles can be used to significantly enhance detection of various small analytes of low abundance. We have expanded upon this repertoire to show that this simple and efficient sample preparation technology can drastically improve the detection of the RVFV nucleoprotein (NP), the most abundant and widely used viral protein for RVFV diagnostics. RESULTS: After screening multiple Nanotrap particle architectures, we found that one particle, NT45, was optimal for RVFV NP capture, as demonstrated by western blotting. NT45 significantly enhanced detection of the NP at levels undetectable without the technology. Importantly, we demonstrated that Nanotrap particles are capable of concentrating NP in a number of matrices, including infected cell lysates, viral supernatants, and animal sera. Specifically, NT45 enhanced detection of NP at various viral titers, multiplicity of infections, and time points. Our most dramatic results were observed in spiked serum samples, where high abundance serum proteins hindered detection of NP without Nanotrap particles. Nanotrap particles allowed for sample cleanup and subsequent detection of RVFV NP. Finally, we demonstrated that incubation of our samples with Nanotrap particles protects the NP from degradation over extended periods of time (up to 120 hours) and at elevated temperatures (at 37ºC). CONCLUSION: This study demonstrates that Nanotrap particles are capable of drastically lowering the limit of detection for RVFV NP by capturing, concentrating, and preserving RVFV NP in clinically relevant matrices. These studies can be extended to a wide range of pathogens and their analytes of diagnostic interest.

Characterization of RNA aptamers directed against the nucleocapsid protein of Rift Valley fever virus.

Nucleocapsid protein (N) is an essential RNA binding protein in many RNA viruses. During replication, N protein encapsidates viral genomic and antigenomic RNA, but not viral mRNA or other cellular RNAs. To discriminate between different species of RNA in a host cell, it is likely that N interacts with specific sequences and/or secondary structures on its target RNA. In this study, we explore the RNA binding properties of N using both natural and artificially selected RNAs as ligands. We found that N binds to RNAs that resemble the terminal panhandle structures of RVFV genomic and antigenomic RNA. Furthermore, we used SELEX to isolate RNA aptamers that bound N with high affinity and determined that N specifically recognizes and binds to GAUU and pyrimidine/guanine motifs. Interestingly, BLAST analysis revealed the presence of these motifs within the coding region of the viral genome, suggesting that N may interact with non-terminal viral RNA sequences during replication. Finally, the aptamer RNAs were used to construct a sensitive fluorescence based sensor of N binding with potential applications for drug screening and imaging methodologies.

The hexamer structure of Rift Valley fever virus nucleoprotein suggests a mechanism for its assembly into ribonucleoprotein complexes.

Rift Valley fever virus (RVFV), a Phlebovirus with a genome consisting of three single-stranded RNA segments, is spread by infected mosquitoes and causes large viral outbreaks in Africa. RVFV encodes a nucleoprotein (N) that encapsidates the viral RNA. The N protein is the major component of the ribonucleoprotein complex and is also required for genomic RNA replication and transcription by the viral polymerase. Here we present the 1.6 Å crystal structure of the RVFV N protein in hexameric form. The ring-shaped hexamers form a functional RNA binding site, as assessed by mutagenesis experiments. Electron microscopy (EM) demonstrates that N in complex with RNA also forms rings in solution, and a single-particle EM reconstruction of a hexameric N-RNA complex is consistent with the crystallographic N hexamers. The ring-like organization of the hexamers in the crystal is stabilized by circular interactions of the N terminus of RVFV N, which forms an extended arm that binds to a hydrophobic pocket in the core domain of an adjacent subunit. The conformation of the N-terminal arm differs from that seen in a previous crystal structure of RVFV, in which it was bound to the hydrophobic pocket in its own core domain. The switch from an intra- to an inter-molecular interaction mode of the N-terminal arm may be a general principle that underlies multimerization and RNA encapsidation by N proteins from Bunyaviridae. Furthermore, slight structural adjustments of the N-terminal arm would allow RVFV N to form smaller or larger ring-shaped oligomers and potentially even a multimer with a super-helical subunit arrangement. Thus, the interaction mode between subunits seen in the crystal structure would allow the formation of filamentous ribonucleocapsids in vivo. Both the RNA binding cleft and the multimerization site of the N protein are promising targets for the development of antiviral drugs.

Comparative production analysis of three phlebovirus nucleoproteins under denaturing or non-denaturing conditions for crystallographic studies.

Nucleoproteins (NPs) encapsidate the Phlebovirus genomic (-)RNA. Upon recombinant expression, NPs tend to form heterogeneous oligomers impeding characterization of the encapsidation process through crystallographic studies. To overcome this problem, we set up a standard protocol in which production under both non-denaturing and denaturing/refolding conditions can be investigated and compared. The protocol was applied for three phlebovirus NPs, allowing an optimized production strategy for each of them. Remarkably, the Rift Valley fever virus NP was purified as a trimer under native conditions and yielded protein crystals whereas the refolded version could be purified as a dimer. Yields of trimeric Toscana virus NP were higher from denaturing than from native condition and lead to crystals. The production of Sandfly Fever Sicilian virus NP failed in both protocols. The comparative protocols described here should help in rationally choosing between denaturing or non-denaturing conditions, which would finally result in the most appropriate and relevant oligomerized protein species. The structure of the Rift Valley fever virus NP has been recently published using a refolded monomeric protein and we believe that the process we devised will contribute to shed light in the genome encapsidation process, a key stage in the viral life cycle.

Structure of the Rift Valley fever virus nucleocapsid protein reveals another architecture for RNA encapsidation.

Rift Valley fever virus (RVFV) is a negative-sense RNA virus (genus Phlebovirus, family Bunyaviridae) that infects livestock and humans and is endemic to sub-Saharan Africa. Like all negative-sense viruses, the segmented RNA genome of RVFV is encapsidated by a nucleocapsid protein (N). The 1.93-A crystal structure of RVFV N and electron micrographs of ribonucleoprotein (RNP) reveal an encapsidated genome of substantially different organization than in other negative-sense RNA virus families. The RNP polymer, viewed in electron micrographs of both virus RNP and RNP reconstituted from purified N with a defined RNA, has an extended structure without helical symmetry. N-RNA species of approximately 100-kDa apparent molecular weight and heterogeneous composition were obtained by exhaustive ribonuclease treatment of virus RNP, by recombinant expression of N, and by reconstitution from purified N and an RNA oligomer. RNA-free N, obtained by denaturation and refolding, has a novel all-helical fold that is compact and well ordered at both the N and C termini. Unlike N of other negative-sense RNA viruses, RVFV N has no positively charged surface cleft for RNA binding and no protruding termini or loops to stabilize a defined N-RNA oligomer or RNP helix. A potential protein interaction site was identified in a conserved hydrophobic pocket. The nonhelical appearance of phlebovirus RNP, the heterogeneous approximately 100-kDa N-RNA multimer, and the N fold differ substantially from the RNP and N of other negative-sense RNA virus families and provide valuable insights into the structure of the encapsidated phlebovirus genome.

Rift valley fever virus L protein forms a biologically active oligomer.

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.

Laboratory safe detection of nucleocapsid protein of Rift Valley fever virus in human and animal specimens by a sandwich ELISA.

A safe laboratory procedure, based on a sandwich ELISA (sAg-ELISA), was developed and evaluated for the detection of nucleocapsid protein (NP) of Rift Valley fever virus (RVFV) in specimens inactivated at 56 degrees C for 1h in the presence of 0.5% Tween-20 (v/v) before testing. Polyclonal capture and detection immune sera were generated respectively in sheep and rabbits immunized with recombinant NP antigen. The assay was highly repeatable and specific; it detected strains of RVFV from the entire distributional range of the disease, isolated over a period of 53 years; no cross-reactivity with genetically related African phleboviruses or other members of the family Bunyaviridae was observed. In specimens spiked with RVFV, including human and animal sera, homogenates of liver and spleen tissues of domestic ruminants, and Anopheles mosquito homogenates, the sAg-ELISA detection limit ranged from log(10)10(2.2) to 10(3.2) TCID(50)/reaction volume. The ELISA detected NP antigen in spiked bovine and sheep liver homogenates up to at least 8 days of incubation at 37 degrees C whereas infectious virus could not be detected at 48h incubation in these adverse conditions. Compared to virus isolation from sera from RVF patients and sheep infected experimentally, the ELISA had 67.7% and 70% sensitivity, and 97.97% and 100% specificity, respectively. The assay was 100% accurate when testing tissues of various organs from mice infected experimentally and buffalo foetuses infected naturally. The assay was able to detect NP antigen in infective culture supernatants 16-24h before cytopathic effects were observed microscopically and as early as 8h after inoculation with 10(5.8) TCID(50)/ml of RVFV. This ability renders the assay for rapid identification of the virus when its primary isolation is attempted in vitro. As a highly specific, safe and simple assay format, the sAg-ELISA represents a valuable diagnostic tool for use in less equipped laboratories in Africa, and for routine differential diagnosis of viral hemorrhagic fevers.

Synthesis, proteolytic processing and complex formation of N-terminally nested precursor proteins of the Rift Valley fever virus glycoproteins.

The genomic M RNA segment of Rift Valley fever virus is transcribed to produce a single mRNA with multiple translation initiation sites. The products of translation are an N-terminal nested series of polyproteins. These polyproteins enter the secretory system of the host cell and are proteolytically processed to yield the mature virion glycoproteins, Gn and Gc, and two non-structural glycoproteins. By means of pulse-chase immune precipitation experiments we identify the Gn and Gc precursor molecules and also show that signal peptidase cleavage is required for mature Gn and Gc production. We also demonstrate that a hydrophobic domain at the N-terminus of Gn acts as a signal peptide only in the context of the polyprotein precursors that initiate at the second, fourth or fifth AUGs. In addition, we document that formation of Gn/Gc heteromeric complexes occur rapidly (<5 min) and can occur prior to signal peptidase processing of Gn, suggesting that this complex forms in the endoplasmic reticulum. Interestingly, Gc can form a complex with a glycoprotein that has been considered nonstructural, a discovery that has implications for both the topology and potential packaging of this glycoprotein.

The N terminus of Rift Valley fever virus nucleoprotein is essential for dimerization.

Rift Valley fever virus (RVFV) is a Phlebovirus in the Bunyaviridae family. The nucleoprotein N is the most abundant component of the virion; numerous copies of N associate with the viral RNA genome and form pseudohelicoidal ribonucleoproteins (RNPs) circularized by a panhandle structure formed by the base-paired RNA sequences at the 3' and 5' termini. These structures play a central role in transcription and replication. We investigated the intermolecular interactions of the RVFV N protein and found that after chemical cross-linking treatment, the nucleoprotein from purified RNPs migrates mainly as dimers. The N-N interaction was studied using the yeast two-hybrid system, the GST pull-down method, and mutational analysis. We demonstrated that the N terminus from residue 1 to 71, and particularly Tyr 4 and Phe 11, which are conserved among phlebovirus N sequences, are involved in the interaction. The C-terminal region did not seem to be essential for the N-N interaction. Moreover, we showed that N(TOS), the N protein of the related Toscana phlebovirus, interacts with itself and forms heterodimers with N(RVF), suggesting that the dimeric form of N may be a conserved feature in phlebovirus RNPs.

The carboxy-terminal acidic domain of Rift Valley Fever virus NSs protein is essential for the formation of filamentous structures but not for the nuclear localization of the protein.

The ambisense S segment of Rift Valley fever (RVF) virus (a phlebovirus in the Bunyaviridae family) codes for two proteins: the viral complementary-sense RNA for the N nucleoprotein and the genomic-sense RNA for the nonstructural protein NSs. Except for the fact that the NSs protein is phosphorylated and forms filamentous structures in the nuclei of infected cells (R. Swanepoel and N. K. Blackburn, J. Gen. Virol. 34:557-561, 1977), its role is poorly understood, especially since the replication cycle of all these viruses takes place in the cytoplasm. To investigate the mechanisms involved in filament formation, we expressed NSs in mammalian cells via a recombinant Semliki Forest virus and demonstrated that the protein alone was able to form structures similar to those observed in RVF virus-infected cells, indicating that the presence of other RVF virus proteins is not required for filament formation. The yeast two-hybrid system was used to show that the protein interacts with itself and to map the interacting domains. Various deletion and substitution mutants were constructed, and the mutant proteins were analyzed by immunoprecipitation, Western blotting and immunofluorescence. These experiments indicated that the 10 to 17 amino acids of the carboxy-terminal domain were involved in self-association of the protein and that deletion of this acidic carboxy-terminal domain prevents the protein from forming filaments but does not affect its nuclear localization. The role of two phosphorylation sites present in this domain was also investigated, but they were not found to have a major influence on the formation of the nuclear filament.

Mapping of the mutations present in the genome of the Rift Valley fever virus attenuated MP12 strain and their putative role in attenuation.

The MP12 attenuated strain of Rift Valley fever virus was obtained by 12 serial passages of a virulent isolate ZH548 in the presence of 5-fluorouracil (Caplen et al., 1985. Mutagen-directed attenuation of Rift Valley fever virus as a method for vaccine development. J. Gen. Virol., 66, 2271-2277). The comparison of the M segment of the two strains has already been reported by Takehara et al. (Takehara et al., 1989. Identification of mutations in the M RNA of a candidate vaccine strain of Rift Valley fever virus. Virology 169, 452-457). We have completed the comparison and found that altogether a total of nine, 12 and four nucleotides were changed in the L, M and S segments of the two strains, respectively. Three mutations induced amino acid changes in the L protein but none of them was located in the recognized motifs conserved among RNA dependent polymerases. In the S segment, a single change modified an amino acid in the NSs protein and in the M segment, seven of the mutations resulted in amino acid changes in each of the four encoded G1, G2, 14 kDa and 78 kDa proteins. Characterization of the MP12 virus indicated that determinants for attenuation were present in each segment and that they were introduced progressively during the 12 passages in the presence of the mutagen (Saluzzo and Smith, 1990. Use of reassortant viruses to map attenuating and temperature-sensitive mutations of the Rift Valley fever virus MP-12 vaccine. Vaccine 8, 369-375). Passages 4 and 7-9 were found to be essential for introduction of temperature-sensitive lesions and attenuation. In an attempt to correlate some of the mutations with the attenuated or temperature-sensitive phenotypes, we determined by sequencing the passage level at which the different mutations appeared. This work should help to address the question of the role of the viral gene products in Rift Valley fever pathogenesis.