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.

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-β 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 α-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)2α 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 eIF2α accumulation. These data suggested that a combination of RVFV replication and NSs-induced host transcriptional suppression induces PKR-mediated eIF2α phosphorylation, while the NSs facilitates efficient viral translation by downregulating PKR and inhibiting PKR-mediated eIF2α 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.

Rift Valley fever virus (RVFV) is a member of the genus Rift Valley fever virus (RVFV), a member of the genusPhlebovirus within the family Bunyaviridae, causes periodic outbreaks among livestock and humans in sub-Saharan African countries (42). RVFV infection results in severe hepatic diseases, with high mortality and abortion rates, in domestic ruminants and also causes an acute febrile myalgic syndrome, a hemorrhagic syndrome, ocular disease, and encephalitis in humans (3, 39). The most recent outbreak was reported in Kenya and resulted in a high reported case-fatality ratio for infected humans (12). RVFV is transmitted primarily by mosquitoes or through direct contact with infected animal blood. Even though the natural infectious cycle of RVFV has been linked to mosquitoes of Aedes species, many other mosquito species can be infected and subsequently transmit the virus (16), implying that these mosquito species can increase the possibility of RVFV becoming epidemic and perhaps endemic outside of its traditional area.RVFV has a single-stranded, tripartite RNA genome composed of the L, M, and S segments. The L segment is of negative polarity and encodes the RNA-dependent RNA polymerase (L). The S segment uses an ambisense strategy for gene expression; a nonstructural protein, NSs, is translated from viral-sense mRNA, while the nucleocapsid (N) protein is expressed from anti-viral-sense mRNA (42). The NSs protein has been known as an interferon antagonist due to its shutting off of host transcription (5, 34). The anti-viral-sense M segment encodes two envelope glycoproteins, Gn and Gc, and two accessory proteins, the 14-kDa NSm protein and the 78-kDa protein.The M gene open reading frame (ORF) in M mRNA contains five in-frame translation initiation codons within the preglycoprotein (pre-Gn) region, which is located upstream of the Gn and Gc genes (see Fig. 1A) (20,26,48). The 78-kDa protein is translated from the first AUG codon of the ORF, and its coding sequence includes the entire NSm and Gn coding sequences. NSm is translated from the region from the second AUG codon to the end of the pre-Gn region (see Fig. 1). The NSm and 78-kDa proteins are not essential for RVFV replication in cell culture (18, 51), whereas all RVFVs thus far sequenced carry the pre-Gn region (7), strongly suggesting that there is selection pressure(s) to retain an RNA element(s) in the pre-Gn region and/or proteins encoded (fully or partially) by the pre-Gn region, i.e., NSm and the 78-kDa protein. Currently, the biological functions of the NSm and 78-kDa proteins of RVFV and proteins encoded by the pre-Gn regions of other phleboviruses are totally unclear, though a mutant RVFV lacking both NSm and 78-kDa protein expression showed attenuated virulence in rats (6), implying a possible role of the RVFV NSm and/or 78-kDa protein in viral pathogenesis.In the present study, we have generated a deletion mutant of an attenuated MP-12 strain of RVFV, which expresses neither the NSm protein nor the 78-kDa protein, due to a large deletion in the...

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 competitionpropagation assay revealed that the parental virus was slightly more fit than the mutant virus lacking expression of both proteins.Rift Valley fever virus (RVFV) (of the genus Phlebovirus, family Bunyaviridae), which is the cause of severe epidemics in ruminants of sub-Saharan African, is also recognized as a human pathogen that can cause a syndrome with fever and myalgia, a hemorrhagic syndrome, ocular disease, and encephalitis (1, 9, 10). RVFV has a single-stranded, tripartite RNA genome composed of the L, M, and S segments. The L segment encodes the L protein, an RNA-dependent RNA polymerase. Using an ambisense strategy, the S segment expresses the N protein and the nonstructural NS proteins (12).The RVFV M segment encodes four proteins, two major envelope glycoproteins, Gn (or G2) and Gc (or G1), which most probably bind to an as-yet-unknown viral receptor molecule to initiate virus infection, and two minor proteins, the 14-kDa nonstructural NSm protein (7) and the 78-kDa protein, which is reported to be a structural protein (15). The biological functions of the NSm and 78-kDa proteins are totally unknown, but they probably do not have a role in viral RNA synthesis; RVFV minigenome RNA replication and transcription occur efficiently in the absence of expression of the NSm, 78-kDa, and Gn and Gc proteins (2). The region upstream from the Gn gene (pre-Gn region) contains five in-frame AUG codons (Fig. 1), and it appears that each of these five AUGs serves as an initiation codon of different proteins. The first and second AUGs serve as initiation codons for the 78-kDa and NSm proteins, respectively, while the third, fourth, and fifth AUGs initiate the Gn-Gc fusion protein (6, 13, 16). The 78-kDa protein consists of pre-Gn and Gn regions. NSm contains the region that starts from the second AUG to the end of the pre-Gn region. A precursor of the Gn-Gc fusion protein is translated from the third to the fifth AUG, and then it undergoes protein processing to generate Gn and Gc proteins. Gn and Gc protein synthesis still occurs in the absence of the first and second AUGs (7, 16).To know whether the NSm and 78-kDa proteins are required for RVFV replication, we attempted to generate mutant viruses lacking the expression of one or both proteins by using a reverse-genetics system of an attenuated vaccine candidate of RVFV, namely MP-12 (3). Previously, we reported that MP-12 recovered by using a reverse-genetics system carries an XhoI site in each RNA segment (3), yet the rescued parental ...

Analysis of purified Rift Valley fever virus (RVFV) particles demonstrated the presence of three negativesense 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.Viruses belonging to the Bunyaviridae family carry three single-stranded RNAs, designated L, M, and S. Viral RNA synthesis occurs in the cytoplasm, and the host mRNA-derived cap structure is used as a primer for viral mRNA synthesis (16). Host protein synthesis is essential for viral RNA replication (2,5,6,15,18) and is also important for viral mRNA synthesis in mammalian cells; viral mRNA elongation, but not initiation, is suppressed in the presence of protein synthesis inhibitors (2,15,18). How the host protein synthesis machinery facilitates viral mRNA elongation is unknown; it has been suggested that ribosome binding to nascent viral mRNA prevents its annealing to the template RNA and allows mRNA elongation (2).Rift Valley fever virus (RVFV) (family Bunyaviridae, genus Phlebovirus) causes severe epidemics among ruminants in the sub-Saharan African and has spread to Egypt, Yemen, and Saudi Arabia. It is also an important human pathogen that causes a syndrome of fever and myalgia, a hemorrhagic syndrome, ocular disease, and encephalitis (1, 13). The anti-viralsense L segment encodes L protein, a viral RNA polymerase, and the anti-viral-sense M segment encodes two structural glycoproteins, G1 and G2, NSm protein, and a 78-kDa protein.As in other viruses of the genus Phlebovirus, RVFV S segment uses an ambisense strategy to express N and NSs proteins, and the viral-sense S and anti-viral-sense S segments serve as templates for N mRNA and NSs mRNA, respectively (16). It has been believed that NSs mRNA is produced only after viralsense RNA has been copied to anti-viral-sense RNA; therefore, the NSs protein would appear at a later stage in infection than the structural proteins (6, 16).RVFV NSs protein plays an important role in RVFV pathogenesis and replication. This protein inhibits host mRNA synthesis, including alpha/beta interferon (IFN-␣/␤) mRNAs (3, 10), hence suppressing host innate immune responses to viral invasion, and it is a major virus virulence factor (4). We showed that coexpression of RVFV NSs protein with N and L proteins enhances viral RNA accumulation in the RVFV minigenome system (7). Because this effect appears to be independent of the NSs-mediated inhibition of IFN-␣/␤ produc...

The effects of the cellular environment on innate immunity remain poorly characterized. Here, we show that in Drosophila ATP-sensitive potassium channels (K(ATP)) mediate resistance to a cardiotropic RNA virus, Flock House virus (FHV). FHV viral load in the heart rapidly increases in K(ATP) mutant flies, leading to increased viremia and accelerated death. The effect of K(ATP) channels is dependent on the RNA interference genes Dcr-2, AGO2, and r2d2, indicating that an activity associated with this potassium channel participates in this antiviral pathway in Drosophila. Flies treated with the K(ATP) agonist drug pinacidil are protected against FHV infection, thus demonstrating the importance of this regulation of innate immunity by the cellular environment in the heart. In mice, the Coxsackievirus B3 replicates to higher titers in the hearts of mayday mutant animals, which are deficient in the Kir6.1 subunit of K(ATP) channels, than in controls. Together, our data suggest that K(ATP) channel deregulation can have a critical impact on innate antiviral immunity in the heart.

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-β mRNAs. Here we demonstrated that the NSs protein induced post-transcriptional downregulation of dsRNA-dependent protein kinase, PKR, to prevent phosphorylation of eIF2α 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.

Rift Valley fever virus (RVFV) (genus