Bunyamwera bunyavirus RNA synthesis requires cooperation of 3'- and 5'-terminal sequences

Abstract

Bunyamwera virus (BUNV) is the prototype of both the Orthobunyavirus genus and the Bunyaviridae family of segmented negative-sense RNA viruses. The tripartite BUNV genome consists of small (S), medium (M), and large (L) segments that are each transcribed to yield a single mRNA and are replicated to generate an antigenome that acts as a template for synthesis of further genomic strands. As for all negative-sense RNA viruses, the 3'- and 5'-terminal nontranslated regions (NTRs) of the BUNV S, M, and L segments exhibit nucleotide complementarity and, except for one conserved U-G pairing, this complementarity extends for 15, 18, and 19 nucleotides, respectively. We investigated whether the complementarity of 3' and 5' NTRs reflected a functional requirement for terminal cooperation to promote BUNV RNA synthesis or, alternatively, was a consequence of genomic and antigenomic NTRs having similar functions requiring sequence conservation. We show that cooperation between 3'- and 5'-NTR sequences is required for BUNV RNA synthesis, and our results suggest that this cooperation is due to nucleotide complementarity allowing 3' and 5' NTRs to associate through base-pairing interactions. To examine the importance of complementarity in promoting BUNV RNA synthesis, we utilized a competitive replication assay able to examine the replication ability of all possible combinations of interacting nucleotides within a defined region of BUNV 3' and 5' NTRs. We show here that maximal RNA replication was signaled when sequences exhibiting perfect complementarity within 3' and 5' NTRs were selected.

FIG. 1.

(A) Schematic of the BUNV S, M, and L genomic RNAs. Only the first 25 nt of both 3′- and 5′-terminal regions of BUNV segments are shown. Potential to form Watson-Crick base pairs (✽) or noncanonical U-G pairings (•) is indicated. The first 11 nt of both 3′ and 5′ termini are conserved between all segments and comprise the conserved complementary region (boxed sequence). The variable complementary region of each segment is shown (shaded sequence). (B) Schematic of plasmids pBUN-S(ren), pBUN-M(ren), and pBUN-L(ren) designed to generate BUNV S-, M-, and L-segment-specific templates BUN-S(ren), BUN-M(ren), and BUN-L(ren), respectively. The nucleotides that comprise the entire 3′ and 5′ NTRs of each genomic RNA are specified above each corresponding NTR. The entire 3′- and 5′-NTR sequences from each BUNV segment (S, white; M, black; L, gray) were placed flanking a cDNA encoding a heterologous sequence (Renilla luciferase [ren]). NTRs were flanked by the T7 RNA polymerase promoter and the self-cleaving hepatitis deltavirus ribozyme, such that the primary transcript was of antigenomic polarity and contained two additional G residues at its 5′ terminus. The positions of the StuI and XbaI restriction enzyme recognition sites common to all three genome analog-expressing plasmids and used to generate exchanged templates are shown.

FIG. 2.

Schematic of exchanged genome analog templates and analysis of the RNAs they generate. (A) The entire 3′ and 5′ NTRs of parental genome analogs BUN-S(ren), BUN-M(ren), and BUN-L(ren) were exchanged to generate six genome analogs having all possible combinations of S-, M-, and L-segment 3′ and 5′ NTRs. (B) The RNA synthesis characteristics of exchanged genome analogs were compared to the parental genome analogs by direct visualization of metabolically labeled actinomycin D-resistant RNAs after agarose-urea gel electrophoresis and autoradiography. The replication and transcription products generated from BUN-S(ren) are marked. The RNAs generated by BUN-M(ren) and BUN-L(ren) are less well resolved due to their migration characteristics in this gel system. (C) Positive-sense RNAs were also analyzed by primer extension analysis with negative-sense oligonucleotide 3′RENSEQ(−). RNAs were harvested from vTF7-3-infected BHK-21 cells transfected with cDNAs expressing a parental or exchanged genome analog and either BUNV S and L support plasmids (+) or the BUNV S support plasmid alone (−). The lanes of the primer extension gel are positioned below the lanes of the corresponding template on the agarose-urea gel. Bands representing mRNAs (vertical bars), T7 RNA polymerase transcripts (white arrowheads), and antigenomic RNA (black arrowheads) generated by each parental template are marked adjacent to the lanes, as are the T7 RNA transcripts made by the exchanged templates. The cDNA expressing BUN-S(ren) was sequenced by using oligonucleotide 3′RENSEQ(−) to act as size marker, and the antigenomic terminal nucleotide is also marked (✽).

FIG. 3.

Schematic of genome analog templates with restored complementarity within the variable complementary region, and analysis of the RNAs they generate. (A) Exchanged genome analogs BUN-S/M(ren), BUN-M/L(ren), and BUN-L/S(ren) were altered within their 5′ NTRs to generate the corresponding templates S/M-comp, M/L-comp, and L/S-comp. These alterations are marked (arrowheads), and the variable complementary regions are shaded. Only the first 25 nt of both 3′- and 5′-terminal regions of the genomic strand are shown, and the conserved complementary region is boxed. The potential to form Watson-Crick base pairs (✽) or noncanonical U-G pairings (•) is indicated. (B) The RNA synthesis characteristics of S/M-comp, M/L-comp, and L/S-comp were analyzed by direct visualization of metabolically labeled actinomycin D-resistant RNAs. RNA synthesis activity of parental genome analogs BUN-S(ren), BUN-M(ren), and BUN-L(ren) are shown alongside for comparison, and BUN-S(ren)-specific RNAs are indicated by arrowheads.

FIG.4.

Schematic of genome analog templates with alterations within the variable complementary region and analysis of the RNAs they generate. (A) The parental genome analog BUN-S(ren) was altered to generate the templates 3′+5′-comp, 3′-comp, and 5′-comp by making alterations within nt 12 to 15 (arrowheads). The conserved (boxed sequences) and variable complementary regions (shaded sequences) of these templates are shown. Only the first 25 nt of both 3′- and 5′-terminal regions of the genomic strand are shown, and their potential to form Watson-Crick base pairs (✽) or noncanonical U-G pairings (•) is indicated. (B) The RNA synthesis characteristics of 3′+5′-comp, 3′-comp, and 5′-comp were analyzed by direct visualization of metabolically labeled actinomycin D-resistant RNAs. Replication and transcription products are identified (arrowheads). (C) Positive-sense RNAs were also analyzed by primer extension analysis with negative-sense oligonucleotide 3′RENSEQ(−). RNAs were harvested from vTF7-3-infected BHK-21 cells transfected with a parental or altered genome analog expressing cDNA, and either BUNV S and L support plasmids (+), or BUNV S support plasmid alone (−). The cDNA expressing BUN-S(ren) was sequenced with oligonucleotide 3′RENSEQ(−) to act as size marker, and the antigenomic terminal nucleotide is marked (✽).

FIG. 5.

Identification of sequences within the variable complementary region that signal most active genome replication. (A) Schematic representation of the procedure used to generate and analyze the replication ability of genome analogs with randomized nucleotides within the variable complementary region. (B) Five different cDNA pools were generated that transcribed genome analogs with defined nt 12 to 14 of the 3′ genomic NTR, and randomized nucleotides at the corresponding positions 12 to 14 at the 5′ genomic NTR. The nucleotide sequence of each pool of cDNA templates was determined to confirm that randomization had been successful (left boxes). After transfection of randomized cDNAs into BHK cells, the sequence of the same nucleotide region of the corresponding genome analogs was determined to identify sequences that signaled most active RNA replication (right boxes).