Nucleotide sequence analysis of the large (L) genomic RNA segment of Bunyamwera virus, the prototype of the family Bunyaviridae (original) (raw)
Related papers
Journal of Virology, 2000
The genome of Bunyamwera virus (BUN) (family Bunyaviridae, genus Bunyavirus) comprises three negativesense RNA segments which act as transcriptional templates for the viral polymerase only when encapsidated by the nucleocapsid protein (N). Previous studies have suggested that the encapsidation signal may reside within the 5 terminus of each segment. The BUN N protein was expressed as a 6-histidine-tagged fusion protein in Escherichia coli and purified by metal chelate chromatography. An RNA probe containing the 5-terminal 32 and 3-terminal 33 bases of the BUN S (small) genome segment was used to investigate binding by the N protein in vitro using gel mobility shift and filter binding assays. On acrylamide gels a number of discrete RNA-N complexes were resolved, and analysis of filter binding data indicated a degree of cooperativity in N protein binding. RNA-N complexes were resistant to digestion with up to 1 g of RNase A per ml. Competition assays with a variety of viral and nonviral RNAs identified a region within the 5 terminus of the BUN S segment for which N had a high preference for binding. This site may constitute the signal for initiation of encapsidation by N.
Attenuation of Bunyavirus Replication by Rearrangement of Viral Coding and Noncoding Sequences
Journal of Virology, 2005
Bunyaviridae. BUN has a tripartite negativesense RNA genome comprising 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, while the internal regions are unique. The UTRs direct replication and transcription of viral RNA and are sufficient to allow encapsidation of viral RNA into ribonucleoprotein complexes. To investigate the segment-specific functions of the UTRs, we have used reverse genetics to recover a recombinant virus (called BUN MLM) in which the L segment open reading frame (ORF)
Analyses of the mRNA transcription processes of snowshoe hare bunyavirus S and M RNA species
Journal of Virology
The time course of synthesis of snowshoe hare bunyavirus small (S)- and medium (M)-sized viral RNA (vRNA), viral cRNA (vcRNA), and mRNA species was analyzed by using single-stranded DNA probes representing the S- and M-coded gene products. In the presence of puromycin, an inhibitor of protein synthesis, the subgenomic S mRNA species were detected, but not full-length S vcRNA or S vRNA species. No M-related RNA species were identified in puromycin-treated cells. In the absence of puromycin, full-length M and S vRNA, S vcRNA, and subgenomic S mRNA species were observed, as well as apparently full-length M vcRNA species, presumably including the approximately similar-sized M mRNA species. The 5' ends of the S and M mRNA species have been shown to be heterogeneous and some 12 to 17 bases longer than the ends of their corresponding presumptive replicative vcRNA species, in agreement with an earlier report that they represent nonviral primer sequences (D. H. L. Bishop, M. E. Gay, and ...
The Bunyamwera Virus Nonstructural Protein NSs Inhibits Viral RNA Synthesis in a Minireplicon System
Virology, 2001
The small (S) genomic segment of Bunyamwera virus (family Bunyaviridae, genus Bunyavirus) encodes the nucleocapsid protein, N, and a nonstructural protein, NSs, in overlapping reading frames. In order to elucidate the function of NSs, we established a plasmid-based minireplicon system using mammalian cells that express large amounts of T7 RNA polymerase. Expression of N, the viral polymerase protein (L), and a minireplicon containing a reporter gene was sufficient to reconstitute functional virus nucleocapsids. Coexpression of NSs, however, led to a dose-dependent decrease in reporter activity without affecting expression of controls. The inhibition could not be reversed by overexpression of N, L or the minireplicon, indicating that the NSs effect was not caused by a reduction in virus gene expression. The NSs proteins of two other members of the Bunyavirus genus, Guaroa virus and Lumbo virus, were also inhibitory in our system. The intracellular localisation of Bunyamwera virus NSs was investigated and found to be predominantly cytoplasmic, but intranuclear inclusion was also detected. Taken together, these data suggest that, in mammalian cells, the bunyavirus NSs protein controls the activity of the viral polymerase by a highly conserved mechanism.
Nucleotide sequence and expression of the small (S) RNA segment of Maguari bunyavirus
Virology, 1989
The small (S) RNA segment of the Maguari bunyavirus genome has been cloned as cDNA and its nucleotide sequence determined. The nucleocapsid protein, N, (NI, 26K) and a nonstructural protein, NSs, (M, 11 K), are encoded in overlapping reading frames, similar to other bunyavirus S RNA segments. In addition, a third AUG-initiated open reading frame encoding a 9.3K protein was observed. All three polypeptides were translated in cell free systems programmed with RNA transcribed in vitro from the cDNA subcloned downstream of a bacteriophage T7 promoter. The effects on expression of subcloning parts of the cDNA and by site-specific mutagenesis are discussed in relation to the scanning model of initiation of translation. A recombinant baculovirus has been constructed to express the Maguari virus S segment gene products. The N protein was efficiently expressed in infected cells, and a significant amount was in a soluble form. We could not detect the synthesis of NSs nor the 9.3K protein, and the reasons for this are discussed. The 9.3K protein has not been found in Maguari virus-infected cells and so the question of its functional significance remains open.