Characterization of Toscana Virus-Defective Interfering Particles Generatedin Vivo

We have analyzed the importance of specific amino acids in the cytoplasmic tail of the glycoprotein G N for packaging of ribonucleoproteins (RNPs) into virus-like particles (VLPs) of Uukuniemi virus (UUK virus), a member of the Bunyaviridae family. In order to study packaging, we added the G N /G C glycoprotein precursor (p110) to a polymerase I-driven minigenome rescue system to generate VLPs that are released into the supernatant. These particles can infect new cells, and reporter gene expression can be detected. To determine the role of UUK virus glycoproteins in RNP packaging, we performed an alanine scan of the glycoprotein G N cytoplasmic tail (amino acids 1 to 81). First, we discovered three regions in the tail (amino acids 21 to 25, 46 to 50, and 71 to 81) which are important for minigenome transfer by VLPs. Further mutational analysis identified four amino acids that were important for RNP packaging. These amino acids are essential for the binding of nucleoproteins and RNPs to the glycoprotein without affecting the morphology of the particles. No segment-specific interactions between the RNA and the cytoplasmic tail could be observed. We propose that VLP systems are useful tools for analyzing protein-protein interactions important for packaging of viral genome segments, assembly, and budding of other members of the Bunyaviridae family.

Section: Discussionmentioning

confidence: 98%

The Glycoprotein Cytoplasmic Tail of Uukuniemi Virus ( Bunyaviridae ) Interacts with Ribonucleoproteins and Is Critical for Genome Packaging

Överby

Pettersson

Neve

2007

“…Most likely, multiply passage of RVFV-4s in BSR cells results in the accumulation of defective RNAs. The accumulation of defective RNAs of the L segment upon sequential passaging is commonly observed in bunyavirus research (44)(45)(46). Fortunately, we found that passage of the same viruses in Vero-E6 cells did not result in detectable levels of defective RNAs, and we have therefore selected these cells for future vaccine production.…”

Section: Discussionmentioning

confidence: 92%

Creation of Rift Valley Fever Viruses with Four-Segmented Genomes Reveals Flexibility in Bunyavirus Genome Packaging

Schreur

Oreshkova

Moormann

et al. 2014

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. IMPORTANCE 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.

“…The generation of deletions through illegitimate recombination mediated by short direct repeats has been described for numerous prokaryote or eukaryote genomes (16,29). In two recent studies, defective interfering particles of RNA viruses, Toscana virus and tomato spotted wilt virus, were shown to be generated by this mechanism (27,23). Recombination through copy choice can also occur during PCR amplification by Thermus aquaticus DNA polymerase I (42).…”

Section: Discussionmentioning

confidence: 99%

Cloning and Sequencing of Defective Particles Derived from the Autonomous Parvovirus Minute Virus of Mice for the Construction of Vectors with Minimal cis -Acting Sequences

Clément

Avalosse

Bakkouri

et al. 2001

The production of wild-type-free stocks of recombinant parvovirus minute virus of mice [MVM(p)] is difficult due to the presence of homologous sequences in vector and helper genomes that cannot easily be eliminated from the overlapping coding sequences. We have therefore cloned and sequenced spontaneously occurring defective particles of MVM(p) with very small genomes to identify the minimal cis-acting sequences required for DNA amplification and virus production. One of them has lost all capsid-coding sequences but is still able to replicate in permissive cells when nonstructural proteins are provided in trans by a helper plasmid. Vectors derived from this particle produce stocks with no detectable wild-type MVM after cotransfection with new, matched, helper plasmids that present no homology downstream from the transgene.The autonomous parvovirus minute virus of mice [MVM(p)] is a lytic virus that replicates as an episome, preferentially in transformed cells. Given its oncotropism but also its innocuousness in the adult host and its resistance to extreme pH and temperatures, MVM(p) has been proposed as a vector for the gene therapy of cancer (34). The 5-kb single-stranded DNA genome of MVM(p) is organized into two overlapping transcription units. The early P4 promoter controls transcription of the nonstructural proteins, NS1 and NS2. The pleiotropic NS1 protein is responsible for the cytotoxic activity of MVM(p) in transformed cells (6, 10), is required for viral DNA amplification, and can positively or negatively affect the activity of homologous or heterologous promoters (26). NS1 also transactivates the second promoter, P38, thus allowing the synthesis of capsid proteins VP1 and VP2 at the end of the viral cycle (15).We have previously derived a recombinant parvovirus from MVM(p) that transfers the cDNA for human interleukin-2 (IL-2). This virus is defective since part of the genes coding for capsid proteins (VP) is deleted. Transducing recombinant virus can, however, be produced if VP proteins are provided in trans by a cotransfected helper plasmid (35) or from genes integrated into the host chromosome (packaging cells) (7, 17). However, these stocks are contaminated by wild-type (WT) virus, which renders impossible their amplification through serial infections. The WT virus is generated through homologous recombination between vector and helper DNAs. Ideally, removing all sequence homology between these DNA sequences should prevent the formation of WT virus. However, data from Astell et al. suggest that a cis-acting element, necessary for DNA replication, overlaps the capsid-coding genes (2). On the other hand, data obtained from the study of defective particles indicate that particles as small as 15% of the size of WT MVM(p) are able to replicate. These defective particles are generated spontaneously during high-multiplicity infections (19) and are selectively amplified during serial infections. They vary in size from 15 to 70% of that of WT MVM(p), but they always retain the two terminal palindromes of 1...