Abstract
The L protein of Bunyamwera virus (BUNV; family Bunyaviridae) is an RNA-dependent RNA polymerase, 2238 aa in length, that catalyses transcription and replication of the negative-sense, tripartite RNA genome. To learn more about the molecular interactions of the L protein and to monitor its intracellular distribution we inserted a 14 aa V5 epitope derived from parainfluenza virus type 5, against which high-affinity antibodies are available, into different regions of the protein. Insertion of the epitope at positions 1935 or 2046 resulted in recombinant L proteins that retained functionality in a minireplicon assay. Two viable recombinant viruses, rBUNL4V5 and rBUNL5V5, expressing the tagged L protein were rescued by reverse genetics, and characterized with respect to their plaque size, growth kinetics and protein synthesis profile. The recombinant viruses behaved similarly to wild-type (wt) BUNV in BHK-21 cells, but formed smaller plaques and grew to lower titres in Vero E6 cells compared with wt BUNV. Immunofluorescent staining of infected cells showed the L protein to have a punctate to reticular distribution in the cytoplasm, and cell fractionation studies indicated that the L protein was present in both soluble and microsomal fractions. Co-immunoprecipitation and confocal microscopic assays confirmed an interaction between BUNV L and N proteins. The recombinant viruses expressing tagged L protein will be highly valuable reagents for the detailed dissection of the role of the BUNV L protein in virus replication.
Highlights
The family Bunyaviridae contains more than 300 members and is divided into five genera, Orthobunyavirus, Hantavirus, Nairovirus, Phlebovirus and Tospovirus
The Bunyamwera virus (BUNV) L protein plays a key role in viral replication and transcription, it is the least characterized of all the structural proteins
We described the effects of some specific mutations on the activity of the BUNV L protein (Dunn et al, 1995; Jin & Elliott, 1992), and produced monospecific antibodies that recognized the L protein in immunoprecipitation and Western blotting procedures (Jin & Elliott, 1992)
Summary
The family Bunyaviridae contains more than 300 members and is divided into five genera, Orthobunyavirus, Hantavirus, Nairovirus, Phlebovirus and Tospovirus. Several members are important human pathogens, such as La Crosse orthobunyavirus (LACV), Hantaan hantavirus, Rift Valley fever phlebovirus and Crimean-Congo hemorrhagic fever nairovirus. Bunyamwera virus (BUNV) is the prototype of both the genus Orthobunyavirus and the family. Like other members of the family Bunyaviridae, BUNV possesses a tripartite, negative-sense RNA genome that encodes four structural proteins. The largest segment (L) codes for the L protein, an RNA-dependent RNA polymerase (RdRp); the medium segment (M) codes for a precursor that is co-translationally cleaved into the two glycoproteins (Gn and Gc) and a non-structural protein (NSm); and the small segment (S) codes for the nucleoprotein (N) and a second non-structural protein (NSs) in overlapping reading frames (Elliott, 1996; Nichol et al, 2005; Schmaljohn & Hooper, 2001).
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