Rift valley fever virus nonstructural protein NSs promotes viral RNA replication and transcription in a minigenome system

Abstract

Rift Valley fever virus (RVFV), which belongs to the genus Phlebovirus, family Bunyaviridae, has a tripartite negative-strand genome (S, M, and L segments) and is an important mosquito-borne pathogen for domestic animals and humans. We established an RVFV T7 RNA polymerase-driven minigenome system in which T7 RNA polymerase from an expression plasmid drove expression of RNA transcripts for viral proteins and minigenome RNA transcripts carrying a reporter gene between both termini of the M RNA segment in 293T cells. Like other viruses of the Bunyaviridae family, replication and transcription of the RVFV minigenome required expression of viral N and L proteins. Unexpectedly, the coexpression of an RVFV nonstructural protein, NSs, with N and L proteins resulted in a significant enhancement of minigenome RNA replication. Coexpression of NSs protein with N and L proteins also enhanced minigenome mRNA transcription in the cells expressing viral-sense minigenome RNA transcripts. NSs protein expression increased the RNA replication of minigenomes that originated from S and L RNA segments. Enhancement of minigenome RNA synthesis by NSs protein occurred in cells lacking alpha/beta interferon (IFN-alpha/beta) genes, indicating that the effect of NSs protein on minigenome RNA replication was unrelated to a putative NSs protein-induced inhibition of IFN-alpha/beta production. Our finding that RVFV NSs protein augmented minigenome RNA synthesis was in sharp contrast to reports that Bunyamwera virus (genus Bunyavirus) NSs protein inhibits viral minigenome RNA synthesis, suggesting that RVFV NSs protein and Bunyamwera virus NSs protein have distinctly different biological roles in viral RNA synthesis.

FIG. 1.

Outline of the T7 RNA polymerase-driven RVFV minigenome system (A) and structure of minigenome RNAs (B). (A) Plasmid pT7-M-rLuc(+) contains a T7 promoter sequence (black arrow), the 5′ UTR of the anti-viral-sense M segment (black box), rLuc ORF, 3′ UTR of the anti-viral-sense M segment (box with a gradient tone), HDV ribozyme, and T7 terminator sequence (white triangle). The structure of pT7-M-rLuc(-) is also shown at the bottom. Cotransfection of pT7-M-rLuc(+) or pT7-M-rLuc(-) with pCT7pol that encodes T7 RNA polymerase resulted in the synthesis of primary transcripts, followed by processing of the 3′ end of the transcripts by the HDV ribozyme. GG represents the expected two additional G residues at the 5′ end of the RNA transcripts. In the presence of the N and L proteins, it is expected that processed minigenome RNA transcripts undergo RNA replication and mRNA transcription. (B) Minigenome RNAs derived from M (M-vRNA and M-cRNA), S (S-vRNA), and L (L-vRNA) segments. S segment-like minigenome expressed from pT7-S-rLuc(-) contains the 5′ UTR of the viral-sense S segment, antisense rLuc ORF, and the 3′ UTR of the viral-sense S segment, while the L segment-like minigenome expressed from L-vRNA contains the 5′ UTR of the viral-sense L segment, the region from nt 108 to nt 1084 of the L segment, the region from nt 2070 to nt 2736 of the L segment, antisense rLuc ORF, and the 3′ UTR of the viral-sense L segment. Restriction sites used to construct the L segment-like minigenome are shown as arrowheads.

FIG. 2.

Minigenome reporter assay. (A) 293T cells were transfected with a mixture of pCT7pol, pT7-M-rLuc(+), pT7-IRES-fLuc, and pT7-IRES (control). In experimental groups, pT7-IRES-N alone (N), pT7-IRES-L alone (L), pT7-IRES-NSs alone (NSs), a mixture of pT7-IRES-N and pT7-IRES-L (N and L), or that of pT7-IRES-N, pT7-IRES-L, and pT7-IRES-NSs (N, L and NSs) was added to the mixture of pCT7pol, pT7-M-rLuc(+), pT7-IRES-fLuc, and pT7-IRES. The amount of pT7-IRES in each sample was adjusted so that the same amount of DNA was included in all samples. Forty-eight hours posttransfection, cell extracts were prepared and fLuc and rLuc activities were measured. fLuc activities were measured by using 1/10 of the samples that were used for rLuc activity determinations. The values represent three independent experiments with standard deviation bars. Plasmid pT7-M-Luc(-) was used in the place of pT7-M-Luc(+) in panel B.

FIG. 3.

Effect of NSs protein expression on M segment-like minigenome RNA synthesis. 293T cells were cotransfected with mixtures of different combinations of 1 μg of pCT7pol, 2 μg of pT7-M-rLuc(+) or pT7-M-rLuc(-), pT7-IRES-N, pT7-IRES-L, and pT7-IRES-NSs; the amounts of the plasmids are shown at the top of the figure. A plasmid, pT7-IRES, was added to the mixture of plasmids so that the same amount of DNA was used for each transfection. At 48 h posttransfection, intracellular RNAs were extracted. Northern blot analyses were performed using an RNA probe that specifically recognizes the antisense and sense sequence of rLuc ORF; the former probe detects anti-viral-sense RNA (Antisense), and the latter probe detects viral-sense RNA (Sense). MG, viral-sense minigenome; a-MG, anti-viral-sense minigenome. (A) Northern blot analyses of RNA extracted from the cells expressed anti-viral-sense (upper two panels) and viral-sense minigenomes (bottom two panels). (B) Western blot analyses using anti-RVFV mouse polyclonal antibody demonstrated the accumulation of L, N, and NSs proteins (top three panels). Actin protein was detected by anti-actin antibody (fourth panel). Lanes 1 and 2 represent cell extracts from MP12-infected cells and mock-infected cells, respectively. The third panel represents a longer exposure of the second panel to demonstrate accumulation of NSs protein (shown by asterisks). The bottom two panels demonstrate Northern blot analyses with rLuc strand-specific probes. (C) Minigenome RNA synthesis in the presence and absence of NSs protein expression with varying ratios of pT7-IRES-N and pT7-IRES-L.

FIG. 4.

Effect of NSs protein expression on minigenome RNA replication in Vero E6 cells. Vero E6 cells were transfected with 1 μg of pCT7pol, 2 μg of pT7-M-rLuc(+) (top two panels) or pT7-M-Luc(-) (bottom two panels), pT7-IRES-N, pT7-IRES-L, and pT7-IRES-NSs; the amounts of the latter three plasmids are shown in the figure. At 48 h posttransfection, intracellular RNAs were extracted and Northern blot analyses were performed to detect viral-sense RNAs and anti-viral-sense RNAs.

FIG. 5.

Effect of various amounts of NSs protein expression on minigenome RNA synthesis and kinetics of minigenome RNA synthesis. 293T cells were transfected with 1 μg of pCT7pol, 2 μg of pT7-M-rLuc(+), pT7-M-rLuc(-), pT7-HH-M-rLuc (+), or pT7-HH-M-rLuc(-), 0.33 μg of pT7-IRES-L, 0.66 μg of pT7-IRES-N, and various amounts of pT7-IRES-NSs. At 48 h (A and C) or at 24, 36, 48, and 60 h posttransfection (B), intracellular RNAs were extracted and Northern blot analyses were performed to detect viral-sense RNAs or anti-viral-sense RNAs. mRNA, minigenome mRNA. (A) Effects of various amounts of pT7-IRES-NSs on minigenome RNA synthesis (top four panels). Western blot analysis revealed amounts of NSs protein (fifth panel) and actin protein (sixth panel). (B) Kinetics of minigenome RNA synthesis. (C) Minigenome RNA synthesis from expressed RNA transcripts not carrying 5′ nonviral extra nucleotide.

FIG. 6.

Effect of NSs protein expression on RNA synthesis of S segment-like minigenome and L segment-like minigenome. 293T cells were transfected with 1 μg of pCT7pol, 2 μg of pT7-S-rLuc(-) (top two panels) or pT7-L-rLuc(-) (bottom two panels), pT7-IRES-N, pT7-IRES-L, and pT7-IRES-NSs; the amounts of the latter three plasmids are shown in the figure. At 48 h posttransfection intracellular RNAs were extracted and Northern blot analyses were performed to detect minigenome RNAs.