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Mechanism of tripartite RNA genome packaging in Rift Valley fever virus.

ABSTRACT: The Bunyaviridae family includes pathogens of medical and veterinary importance. Rift Valley fever virus (RVFV), a member in the Phlebovirus genus of the family Bunyaviridae, is endemic to sub-Saharan Africa and causes a mosquito-borne disease in ruminants and humans. Viruses in the family Bunyaviridae carry a tripartite, single-stranded, negative-sense RNA genome composed of L, M, and S RNAs. Little is known about how the three genomic RNA segments are copackaged to generate infectious bunyaviruses. We explored the mechanism that governs the copackaging of the three genomic RNAs into RVFV particles. The expression of viral structural proteins along with replicating S and M RNAs resulted in the copackaging of both RNAs into RVFV-like particles, while replacing M RNA with M1 RNA, lacking a part of the M RNA 5' UTR, abrogated the RNA copackaging. L RNA was efficiently packaged into virus particles released from cells supporting the replication of L, M, and S RNAs, and replacing M RNA with M1 RNA abolished the packaging of L RNA. Detailed analyses using various combinations of replicating viral RNAs suggest that M RNA alone or a coordinated function of M and S RNAs exerted efficient L RNA packaging either directly or indirectly. Collectively, these data are consistent with the possibility that specific intermolecular interactions among the three viral RNAs drive the copackaging of these RNAs to produce infectious RVFV.

SUBMITTER: Terasaki K

PROVIDER: S-EPMC3021031 | biostudies-literature | 2011 Jan

REPOSITORIES: biostudies-literature

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Mechanism of tripartite RNA genome packaging in Rift Valley fever virus.

Terasaki Kaori K Murakami Shin S Lokugamage Kumari G KG Makino Shinji S

Proceedings of the National Academy of Sciences of the United States of America 20101227 2

The Bunyaviridae family includes pathogens of medical and veterinary importance. Rift Valley fever virus (RVFV), a member in the Phlebovirus genus of the family Bunyaviridae, is endemic to sub-Saharan Africa and causes a mosquito-borne disease in ruminants and humans. Viruses in the family Bunyaviridae carry a tripartite, single-stranded, negative-sense RNA genome composed of L, M, and S RNAs. Little is known about how the three genomic RNA segments are copackaged to generate infectious bunyavir ...[more]

PMID: 21187405

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Project description:Bunyavirus genomes comprise a small (S), a medium (M), and a large (L) RNA segment of negative polarity. Although the untranslated regions have been shown to comprise signals required for transcription, replication, and encapsidation, the mechanisms that drive the packaging of at least one S, M, and L segment into a single virion to generate infectious virus are largely unknown. One of the most important members of the Bunyaviridae family that causes devastating disease in ruminants and occasionally humans is the Rift Valley fever virus (RVFV). We studied the flexibility of RVFV genome packaging by splitting the glycoprotein precursor gene, encoding the (NSm)GnGc polyprotein, into two individual genes encoding either (NSm)Gn or Gc. Using reverse genetics, six viruses with a segmented glycoprotein precursor gene were rescued, varying from a virus comprising two S-type segments in the absence of an M-type segment to a virus consisting of four segments (RVFV-4s), of which three are M-type. Despite that all virus variants were able to grow in mammalian cell lines, they were unable to spread efficiently in cells of mosquito origin. Moreover, in vivo studies demonstrated that RVFV-4s is unable to cause disseminated infection and disease in mice, even in the presence of the main virulence factor NSs, but induced a protective immune response against a lethal challenge with wild-type virus. In summary, splitting bunyavirus glycoprotein precursor genes provides new opportunities to study bunyavirus genome packaging and offers new methods to develop next-generation live-attenuated bunyavirus vaccines.Rift Valley fever virus (RVFV) causes devastating disease in ruminants and occasionally humans. Virions capable of productive infection comprise at least one copy of the small (S), medium (M), and large (L) RNA genome segments. The M segment encodes a glycoprotein precursor (GPC) protein that is cotranslationally cleaved into Gn and Gc, which are required for virus entry and fusion. We studied the flexibility of RVFV genome packaging and developed experimental live-attenuated vaccines by applying a unique strategy based on the splitting of the GnGc open reading frame. Several RVFV variants, varying from viruses comprising two S-type segments to viruses consisting of four segments (RVFV-4s), of which three are M-type, could be rescued and were shown to induce a rapid protective immune response. Altogether, the segmentation of bunyavirus GPCs provides a new method for studying bunyavirus genome packaging and facilitates the development of novel live-attenuated bunyavirus vaccines.

Project description:The emerging bunyavirus Rift Valley fever virus (RVFV) is transmitted to humans and livestock by a large number of mosquito species. RNA interference (RNAi) has been characterized as an important innate immune defense mechanism used by mosquitoes to limit replication of positive-sense RNA flaviviruses and togaviruses; however, little is known about its role against negative-strand RNA viruses such as RVFV. We show that virus-specific small RNAs are produced in infected mosquito cells, in Drosophila melanogaster cells, and, most importantly, also in RVFV vector mosquitoes. By addressing the production of small RNAs in adult Aedes sp. and Culex quinquefasciatus mosquitoes, we showed the presence of virus-derived Piwi-interacting RNAs (piRNAs) not only in Aedes sp. but also in C. quinquefasciatus mosquitoes, indicating that antiviral RNA interference in C. quinquefasciatus mosquitoes is similar to the described activities of RNAi in Aedes sp. mosquitoes. We also show that these have antiviral activity, since silencing of RNAi pathway effectors enhances viral replication. Moreover, our data suggest that RVFV does not encode a suppressor of RNAi. These findings point toward a significant role of RNAi in the control of RVFV in mosquitoes. IMPORTANCE Rift Valley fever virus (RVFV; Phlebovirus, Bunyaviridae) is an emerging zoonotic mosquito-borne pathogen of high relevance for human and animal health. Successful strategies of intervention in RVFV transmission by its mosquito vectors and the prevention of human and veterinary disease rely on a better understanding of the mechanisms that govern RVFV-vector interactions. Despite its medical importance, little is known about the factors that govern RVFV replication, dissemination, and transmission in the invertebrate host. Here we studied the role of the antiviral RNA interference immune pathways in the defense against RVFV in natural vector mosquitoes and mosquito cells and draw comparisons to the model insect Drosophila melanogaster. We found that RVFV infection induces both the exogenous small interfering RNA (siRNA) and piRNA pathways, which contribute to the control of viral replication in insects. Furthermore, we demonstrate the production of virus-derived piRNAs in Culex quinquefasciatus mosquitoes. Understanding these pathways and the targets within them offers the potential of the development of novel RVFV control measures in vector-based strategies.

Dataset Information

Mechanism of tripartite RNA genome packaging in Rift Valley fever virus.

Publications

Mechanism of tripartite RNA genome packaging in Rift Valley fever virus.

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