Attenuation of Bunyamwera Orthobunyavirus Replication by Targeted Mutagenesis of Genomic Untranslated Regions and Creation of Viable Viruses with Minimal Genome Segments

Abstract

Bunyamwera virus (BUNV) is the prototype virus for both the genus Orthobunyavirus and the family Bunyaviridae. BUNV has a tripartite, negative-sense RNA genome. The coding region of each segment is flanked by untranslated regions (UTRs) that are partially complementary. The UTRs play an important role in the virus life cycle by promoting transcription, replication, and encapsidation of the viral genome. Using reverse genetics, we generated recombinant viruses that contained deletions within the 3= and/or 5= UTRs of the L or M segments to determine the minimal UTRs competent for virus viability. We then generated viruses carrying deleted UTRs in all three segments. These viruses were grossly attenuated in tissue culture, being significantly impaired in their ability to produce plaques in BHK cells, and had a reduced capacity to cause host cell protein shutoff. After serial passage in tissue culture, some viruses partially recovered fitness, generating higher titers and producing larger plaques. We determined the complete nucleotide sequence for each virus. The deleted UTR sequences were maintained, and no amino acid changes were observed in the nonstructural proteins (NSs and NSm), the nucleocapsid protein (N), or the Gn glycoprotein. One virus had a single amino acid substitution in Gc. Three viruses contained amino acid changes in the viral polymerase that mostly occurred in the C-terminal domain of the L protein. Although the role of this domain remains unknown, we suggest that those changes might be involved in the evolution of the polymerase to recognize the deleted UTRs more efficiently. T he genusOrthobunyavirus and the family Bunyaviridae take their name from Bunyamwera virus (BUNV), the prototype bunyavirus, which was isolated from mosquitoes of Aedes species in the Semliki Forest in Uganda (25). The BUNV genome is composed of three single-stranded RNA segments of negative polarity, named large (L), medium (M), and small (S), which encode four structural proteins. The L segment codes for the RNA-dependent RNA polymerase or L protein; the M segment codes for the two envelope glycoproteins, Gn and Gc; and the S segment codes for the nucleocapsid protein (N). Nonstructural proteins are encoded on the M and S segment and are named NSm and NSs, respectively (reviewed in reference 10). The NSm protein was shown to be involved in virion assembly (24), while NSs plays a role in counteracting the host cell innate immune response through a general block in transcription and translation (4,16,26,27). Each genome segment is encapsidated by numerous copies of the N protein and is associated with a few copies of the L protein to form ribonucleoprotein (RNP) complexes that are the functional templates for both transcription and replication.The coding regions are flanked by untranslated regions (UTRs), whose size and sequence vary greatly between segments, but as a general rule, the 3= UTR is shorter than the 5= UTR. BUNV 3= and 5= UTRs on the L and M segments are about 50 nucleotides (nt) and 100 nt, respectively, whil...

Evolution of the Bunyamwera Virus Polymerase To Accommodate Deletions within Genomic Untranslated Region Sequences

“…Previous studies on BUNV showed that the internal parts of the UTRs are nonessential for virus viability but play an important role during virus replication (5,6). Indeed recombinant viruses carrying deletions in their UTRs are grossly attenuated in growth in cultured cells compared to wild-type (wt) BUNV, and the bigger the deletion the more attenuation the viruses display (5,6).…”

“…These minimal UTRs were defined by creating recombinant viruses that carried one deleted segment and two wt segments. However, a virus containing all minimal UTRs was not viable, but recombinant viruses carrying smaller deletions on all three segments were created; such viruses proved to be grossly attenuated (6). Serial passage of these viruses in BHK-21 cells allowed them to partially recover fitness.…”

“…Interestingly, regain of fitness did not occur through modification of the UTR sequences. Instead, fullgenome sequencing revealed amino acid substitutions within the L protein (6).…”

“…The UTRs contain the N protein encapsidation signal, viral promoters, mRNA transcription termination signals, and (probably) RNP-packaging signals, and thus it is remarkable that the UTRs show such considerable sequence variation and yet carry out the same functions (3). Previous studies on BUNV showed that the internal parts of the UTRs are nonessential for virus viability but play an important role during virus replication (5,6). Indeed recombinant viruses carrying deletions in their UTRs are grossly attenuated in growth in cultured cells compared to wild-type (wt) BUNV, and the bigger the deletion the more attenuation the viruses display (5,6).…”

“…The minimal UTRs required for virus replication were defined as follows: 29 and 112 nt for the S segment (5), 39 and 38 nt for the L segment, and either 33 nt in the 3= UTR with the full-length 5= UTR or 44 nt in the 5= UTR with the full-length 3=UTR for the M segment (6). These minimal UTRs were defined by creating recombinant viruses that carried one deleted segment and two wt segments.…”