Generation and Analysis of Infectious Virus-Like Particles of Uukuniemi Virus (
            <i>Bunyaviridae</i>
            ): a Useful System for Studying Bunyaviral Packaging and Budding

Self Cite

121

117

Rift Valley fever virus (RVFV) is a member of the genusPhlebovirus within the family Bunyaviridae. It is a mosquito-borne zoonotic agent that can cause hemorrhagic fever in humans. The enveloped RVFV virions are known to be covered by capsomers of the glycoproteins G N and G C , organized on a T‫21؍‬ icosahedral lattice. However, the structural units forming the RVFV capsomers have not been determined. Conflicting biochemical results for another phlebovirus (Uukuniemi virus) have indicated the existence of either G N and G C homodimers or G N -G C heterodimers in virions. Here, we have studied the structure of RVFV using electron cryo-microscopy combined with three-dimensional reconstruction and single-particle averaging. The reconstruction at 2.2-nm resolution revealed the organization of the glycoprotein shell, the lipid bilayer, and a layer of ribonucleoprotein (RNP). Five-and six-coordinated capsomers are formed by the same basic structural unit. Molecular-mass measurements suggest a G N -G C heterodimer as the most likely candidate for this structural unit. Both leaflets of the lipid bilayer were discernible, and the glycoprotein transmembrane densities were seen to modulate the curvature of the lipid bilayer. RNP densities were situated directly underneath the transmembrane densities, suggesting an interaction between the glycoprotein cytoplasmic tails and the RNPs. The success of the single-particle averaging approach taken in this study suggests that it is applicable in the study of other phleboviruses, as well, enabling higher-resolution description of these medically important pathogens.

Section: Discussionmentioning

confidence: 60%

Section: Discussionmentioning

confidence: 99%

Electron Cryo-Microscopy and Single-Particle Averaging of Rift Valley Fever Virus: Evidence for G _N -G _C Glycoprotein Heterodimers

Huiskonen

Överby

Weber

et al. 2009

Self Cite

121

117

“…ift Valley fever virus (RVFV) (the genus Phlebovirus, family Bunyaviridae) carries three single-stranded, negative-sense RNA segments, L, M, and S. The viral RNA-dependent RNA polymerase (L protein), envelope Gn/Gc glycoproteins, and N protein, all of which are essential for virus replication, are encoded in L, M, and S RNAs, respectively; hence, copackaging of the three genomic RNA segments into a virus particle is necessary for the generation of infectious RVFV, yet our understanding of bunyavirus RNA packaging mechanisms is still in its infancy (2,4,11,15,19,23,26). Using RVFV, we address several unexplored questions in bunyavirus genome packaging, including the biological activities of the noncoding regions (NCRs) of each viral RNA segment for RNA packaging, the identification of RNA packaging signals, and a possible role(s) of the coding regions in viral RNA packaging.…”

mentioning

confidence: 99%

Roles of the Coding and Noncoding Regions of Rift Valley Fever Virus RNA Genome Segments in Viral RNA Packaging

Murakami

Terasaki

Narayanan

et al. 2012

We characterized the RNA elements involved in the packaging of Rift Valley fever virus RNA genome segments, L, M, and S. The 5=-terminal 25 nucleotides of each RNA segment were equally competent for RNA packaging and carried an RNA packaging signal, which overlapped with the RNA replication signal. Only the deletion mutants of L RNA, but not full-length L RNA, were efficiently packaged, implying the possible requirement of RNA compaction for L RNA packaging. Rift Valley fever virus (RVFV) (the genus Phlebovirus, family Bunyaviridae) carries three single-stranded, negative-sense RNA segments, L, M, and S. The viral RNA-dependent RNA polymerase (L protein), envelope Gn/Gc glycoproteins, and N protein, all of which are essential for virus replication, are encoded in L, M, and S RNAs, respectively; hence, copackaging of the three genomic RNA segments into a virus particle is necessary for the generation of infectious RVFV, yet our understanding of bunyavirus RNA packaging mechanisms is still in its infancy (2,4,11,15,19,23,26). Using RVFV, we address several unexplored questions in bunyavirus genome packaging, including the biological activities of the noncoding regions (NCRs) of each viral RNA segment for RNA packaging, the identification of RNA packaging signals, and a possible role(s) of the coding regions in viral RNA packaging.RVFV M RNA as well as S RNA is efficiently packaged, in the absence of any other viral RNA segment, into virus-like particles (VLPs), released from cells expressing the viral structural proteins and harboring the replicating M RNA and S RNA, respectively (26). However, RVFV L RNA is not packaged efficiently into VLPs in the absence of other viral RNA segments; both M and S RNAs are required for efficient L RNA packaging (26). To test the possibility that L RNA lacks a packaging signal, BSR-T7/5 cells stably expressing T7 RNA polymerase (3) were cotransfected with a plasmid expressing T7 polymerase-driven anti-viral-sense L RNA-derived L-SacI RNA or L-NcoI RNA, each carrying a large internal deletion within the L gene (Fig. 1A), along with the plasmids expressing L, Gn/Gc, and N proteins (26). As a control, we used a plasmid expressing the fulllength L RNA. At 3 days posttransfection, cell extracts were collected and the VLPs released into the supernatant were purified by ultracentrifugation (10, 26). The intracellular accumulations and the incorporations of Gn/Gc and N proteins into VLPs were similar among all three samples (Fig. 1B), suggesting the production of similar levels of VLPs. The intracellular accumulation of full-length viral-sense L RNA was appreciably lower than that of the two deletion mutants (Fig. 1C, left panel); the L RNA deletion mutants most probably replicate faster than the full-length L RNA due to their shorter lengths, resulting in higher intracellular accumulation of the L RNA deletion mutants. The amount of the full-length L RNA in VLPs was also substantially lower than that of the deletion mutants (Fig. 1C, right panel). Comparing the band intensities of the ...

“…Previous work on Uukuniemi phlebovirus (38,39) and BUNV orthobunyavirus (42) indicated that the cytoplasmic tails of the viral glycoproteins, particularly that of Gn, were indispensable for viral replication. This was confirmed by Piper et al (40), who showed that the cytoplasmic tail of RVFV Gn recruits the L protein, nucleocapsid protein, and the genomic RNA into virions.…”

Section: Discussionmentioning

confidence: 99%

“…We show here that a recombinant RVFV can maintain a viral-like segment expressing a foreign gene without any selective pressure or apparent advantage to the virus. This system thus provides a valuable tool to facilitate the further exploration of genomic segment packaging in the context of a fully functional virus, rather than VLP assays as have been previously used (14,20,38). It also provides the potential for RVFV to act as a vector for other foreign gene sequences.…”

Section: Discussionmentioning

confidence: 99%

Creation of a Recombinant Rift Valley Fever Virus with a Two-Segmented Genome

Brennan

Welch

McLees

et al. 2011

Rift Valley fever virus (RVFV; family Bunyaviridae) is a clinically important, mosquito-borne pathogen of both livestock and humans, which is found mainly in sub-Saharan Africa and the Arabian Peninsula. RVFV has a trisegmented single-stranded RNA (ssRNA) genome. The L and M segments are negative sense and encode the L protein (viral polymerase) on the L segment and the virion glycoproteins Gn and Gc as well as two other proteins, NSm and 78K, on the M segment. The S segment uses an ambisense coding strategy to express the nucleocapsid protein, N, and the nonstructural protein, NSs. Both the NSs and NSm proteins are dispensable for virus growth in tissue culture. Using reverse genetics, we generated a recombinant virus, designated r2segMP12, containing a two-segmented genome in which the NSs coding sequence was replaced with that for the Gn and Gc precursor. Thus, r2segMP12 lacks an M segment, and although it was attenuated in comparison to the three-segmented parental virus in both mammalian and insect cell cultures, it was genetically stable over multiple passages. We further show that the virus can stably maintain an M-like RNA segment encoding the enhanced green fluorescent protein gene. The implications of these findings for RVFV genome packaging and the potential to develop multivalent live-attenuated vaccines are discussed. Rift Valley fever virus (RVFV)is an emerging pathogen capable of causing serious epidemics among livestock and humans. Originally isolated in sub-Saharan Africa, the virus has spread northward to Egypt and the Arabian peninsula (43) and has resulted in huge economic losses of domesticated animals as well as more than a thousand human deaths. In ruminants, particularly sheep and cattle, RVFV disease is characterized by fetal deformities, abortion, and high rates of mortality among young animals (44). In humans, the disease can progress from self-limiting febrile illness to hemorrhagic fever, encephalitis, or retinal vasculitis. RVFV is predominantly transmitted by mosquitoes, and the virus has been isolated from more than 40 species in nature, while under laboratory conditions many different arthropods are capable of transmitting the virus (reference 46 and references therein). In an era of climate change, the spread of competent insect vectors into "virgin" territories is a major public health concern, making RVF a potential worldwide human, animal, and economic threat (12).RVFV is a member of the Phlebovirus genus in the Bunyaviridae family and contains a tripartite single-stranded RNA (ssRNA) genome comprising two negative-sense and one ambisense segment (reviewed in reference 7). The large (L) segment (approximately 6.4 kb) codes for the viral RNA-dependent RNA polymerase (L protein). The medium (M) segment (approximately 3.8 kb) encodes four proteins in a single open reading frame (ORF): the precursor to the virion envelope glycoproteins Gn and Gc and two other proteins called NSm (or NSm2) and 78K (or NSm1), depending on which of five methionine codons that translation initiates f...