Efficient bunyavirus rescue from cloned cDNA

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The NSs protein of Bunyamwera virus (Bunyaviridae) is an antiapoptotic interferon antagonist involved in silencing host protein expression by interfering with mRNA synthesis. Here, we show that the ability to inhibit both host transcription and the interferon response is linked to interaction of NSs with the MED8 component of Mediator, a protein complex necessary for mRNA production. The interacting domain on NSs was mapped to the C-terminal region, which contains amino acids conserved among orthobunyavirus NSs proteins. A recombinant virus in which the interacting domain in NSs was deleted had strongly reduced ability to inhibit host protein expression and was unable to inhibit the interferon response. This study provides further information on the mechanisms by which bunyavirus nonstructural proteins are involved in pathogenesis.

Section: Methodsmentioning

confidence: 99%

Interaction of Bunyamwera Orthobunyavirus NSs Protein with Mediator Protein MED8: a Mechanism for Inhibiting the Interferon Response

Léonard

Kohl

Hart

et al. 2006

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“…Recombinant viruses were produced using the recently described three-plasmid system (26). Briefly, subconfluent BSR-T7/5 cells (10 6 cells per 60-mm-diameter petri dish) were transfected with 1 g each pT7riboBUNM(ϩ), pT7riboBUNL(ϩ), and pT7ribo BUNS(ϩ) (for wild-type virus), or the appropriate mutant pT7riboBUNS(ϩ) construct, using Fugene 6 transfection reagent (Roche), according to the manufacturer's protocol.…”

Section: Methodsmentioning

confidence: 99%

Mutational Analyses of the Nonconserved Sequences in the Bunyamwera Orthobunyavirus S Segment Untranslated Regions

Elliott

2005

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Bunyamwera virus (BUNV) is the prototype of the genus Orthobunyavirus and the family Bunyaviridae. BUNV has a tripartite genome of negative-sense RNA composed of small (S), medium (M), and large (L) segments. Partially complementary untranslated regions (UTRs) flank the coding region of each segment. The terminal 11 nucleotides of these UTRs are conserved between the three segments and throughout the genus, while the internal regions are unique to each segment and largely nonconserved between different viruses. To investigate the functions of the UTR sequences, we constructed a series of BUNV S segment cDNA clones with deletions in the 3 and/or 5 UTR and then attempted to rescue these segments into recombinant viruses. We found that the genomic 5 UTR was much more sensitive to mutation than the 3 UTR and, in general, sequences proximal to the termini were more important than those flanking the coding region. Northern blot analyses of infectedcell RNA showed that the internal, nonconserved sequences of the S segment 3 UTR play a role in the regulation of transcription and replication and the balance between these two processes. In contrast, deletions in the 5 UTR caused attenuation of the recombinant virus but did not specifically affect levels of S segment RNAs or the encoded nucleocapsid protein. Thus, the internal regions of both UTRs are functional: most of the 5 UTR is essential to viral growth, and, while nonessential, the internal 3 UTR is important to the regulation of viral RNA synthesis.

“…We further utilize this system to study the role of the genomic 5Ј and 3Ј noncoding regions (NCRs) of LASV in in vitro growth and in vivo virulence. This system relies on the transcription of full-length viral complementary genomes (antigenomes) on a T7 RNA polymerase-based platform that has been well de- scribed previously (1,7,18). Briefly, we amplified the S and L genomic RNAs of LASV (Josiah strain) by reverse transcription (RT)-PCR and cloned them into a T7 transcription vector.…”

mentioning

confidence: 99%

Efficient Rescue of Recombinant Lassa Virus Reveals the Influence of S Segment Noncoding Regions on Virus Replication and Virulence

Albariño

Bird

Chakrabarti

et al. 2011

Lassa virus (LASV), is a significant cause of severe, often fatal, hemorrhagic fever in humans throughout western Africa, with an estimated 100,000 infections each year. No vaccines are commercially available. We report the development of an efficient reverse genetics system to rescue recombinant LASV and to investigate the contributions of the long 5 and 3 noncoding regions (NCRs) of the S genomic segment to in vitro growth and in vivo virulence. This work demonstrates that deletions of large portions of these NCRs confer an attenuated phenotype and are a first step toward further insights into the high virulence of LASV.