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Journal of Virology, May 2005, p. 5606-5615, Vol. 79, No. 9
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.9.5606-5615.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Rift Valley Fever Virus Nonstructural Protein NSs Promotes Viral RNA Replication and Transcription in a Minigenome System

Tetsuro Ikegami,1 C. J. Peters,1,2 and Shinji Makino1*

Departments of Microbiology and Immunology,1 Pathology, University of Texas Medical Branch, Galveston, Texas 77555-10192

Received 1 September 2004/ Accepted 23 December 2004


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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}/ß) genes, indicating that the effect of NSs protein on minigenome RNA replication was unrelated to a putative NSs protein-induced inhibition of IFN-{alpha}/ß 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.


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INTRODUCTION
 
The family Bunyaviridae is comprised of five genera: Bunyavirus, including Bunyamwera virus (BUNV) and La Crosse virus (LACV); Phlebovirus, including Rift Valley fever virus (RVFV), Punta Toro virus, and Uukuniemi virus (UUKV); Hantavirus, including Hantaan virus and Sin Nombre virus; Nairovirus, including Crimean-Congo hemorrhagic fever virus; and Tospovirus (28). The viral genome of this family is comprised of three molecules of negative or ambi-sense, single-stranded RNA, designated L, M, and S.

RVFV causes severe epidemics among ruminants, such as sheep, goats, and cattle, in the sub-Saharan area of the African continent, Egypt, Yemen, and Saudi Arabia, and it is also recognized as a human pathogen that causes a syndrome of fever and myalgia, a hemorrhagic syndrome, ocular disease, and encephalitis (2, 7, 8, 25). Viral transmission is primarily mosquito borne or due to direct contact with infected animal blood. The RVFV L segment (6,404 nucleotides [nt]) encodes an RNA-dependent RNA polymerase, the M segment (3,885 nt) encodes two structural glycoproteins (G1 and G2) and two proteins of unknown function (NSm and 78-kDa protein), and the S segment (1,690 nt) encodes a nucleoprotein (N) in the anti-viral sense and a nonstructural protein (NSs) in the viral sense (28). The NSs gene is found in the S segment of viruses in the genuses Bunyavirus, Phlebovirus, and Tospovirus, and viruses belonging to the latter two genuses use an ambi-sense strategy to express the N and NSs proteins (28).

Contemporary reverse genetics systems of RNA viruses have revolutionized the study of viral replication mechanisms, host cell-virus interactions, and viral pathogenicity, as well as the potential for vaccine evaluation and development. Among the family Bunyaviridae, the establishment of a reverse genetics system has been reported only for BUNV (5), while minigenome (or minireplicon) systems have been developed in BUNV (13), LACV (3), Crimean-Congo hemorrhagic fever virus (15), Hantaan virus (14), UUKV (16), and RVFV (1, 21, 26). In a typical minigenome system, virus-like RNA (minigenome) transcripts contain an internal open reading frame (ORF) of a reporter gene in place of a viral ORF sandwiched by untranslated regions (UTR) of viral RNA termini. The expressed minigenome RNA transcripts undergo RNA replication and transcription in the presence of coexpressed viral proteins or coinfected helper virus; the levels of reporter expression are a measure of the efficiency of minigenome RNA replication and transcription. Minigenome RNA transcripts are expressed by either host RNA polymerase I or T7 RNA polymerase, the latter of which is often provided by vaccinia virus (1, 3, 13) or Sindbis virus (31). The expression of viral proteins is accomplished by transfecting either an expression plasmid that carries a eukaryotic promoter sequence followed by a viral structural gene or a T7 expression plasmid carrying a T7 promoter sequence in place of the eukaryotic promoter of the former; in the latter case, vaccinia virus expressing T7 RNA polymerase is frequently used to provide T7 RNA polymerase and capping of expressed RNA transcripts. Another method for viral protein expression is to infect cells with recombinant vaccinia viruses, each of which encodes a virus protein. Studies using minigenomes and other systems revealed that the coexpression of N and L proteins was required for the RNA synthesis of viruses that belong to the Bunyaviridae family (1, 3, 13, 14, 16, 21).

Recent studies on the NSs protein of bunyaviruses have uncovered its biological functions. Expression studies indicated that BUNV NSs protein promotes apoptosis and/or inhibits cellular translation (10). Analysis of a BUNV lacking the NSs gene demonstrated that NSs protein expression counteracts interferon (IFN) regulatory factor 3-mediated induction of early cell death (19). RVFV NSs, which is known to have an IFN-antagonistic function (4), interacts with the p44 subunit of TFIIH transcription factor and inhibits the function of host RNA polymerase II (20); host mRNA synthesis, including that of IFN mRNAs, is inhibited in cells expressing NSs protein. In addition, NSs protein of BUNV (31) and LACV (3) inhibits RNA synthesis by minigenomes, whereas reports from studies of RVFV and UUKV minigenome systems demonstrated that NSs protein of both viruses has no effect on minigenome RNA synthesis (16, 21).

The present study describes the establishment of a new RVFV minigenome system. Our study confirmed that N and L proteins were necessary for RVFV minigenome replication and transcription. Furthermore, we found that coexpression of NSs protein with L and N proteins substantially enhanced minigenome replication and transcription, suggesting that RVFV NSs protein plays a critical role in RVFV RNA synthesis.


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MATERIALS AND METHODS
 
Media, cells, and viruses. 293T cells and Vero E6 cells were maintained in Dulbecco's modified minimum essential medium (Invitrogen, Carlsbad, CA) containing 10% fetal calf serum (Invitrogen). Penicillin (100 U/ml) and streptomycin (100 µg/ml; Invitrogen) were added to the medium. RVFV vaccine strain MP12 was grown in 293T cells and assayed for its infectivity by plaque assay in Vero E6 cells. MP12-infected 293T cells were used to prepare cell extracts for Western blot analysis.

Plasmid constructions. Lysates of Vero E6 cells infected with wild-type (wt) RVFV, strain ZH501, in TRIzol reagent (Invitrogen) were kindly provided by S. T. Nichol (Centers for Disease Control and Prevention, Atlanta, GA). Using random hexamer (Amersham Biosciences, Piscataway, NJ), first-strand cDNA was prepared by using Ready-To-Go You-Prime First-Strand beads (Amersham Biosciences) according to the manufacturer's instructions. The first-strand cDNA was used for the construction of expression plasmids and minigenomes. The ORFs of N, L, and NSs were amplified by PCR and were individually cloned into a modified pBL-EMCV plasmid (pBluescript plasmid origin), pT7-IRES, yielding pT7-IRES-N, pT7-IRES-L, and pT7-IRES-NSs, respectively. The Photinus (firefly) luciferase (fLuc) ORF was amplified by PCR from plasmid pRLHL (32) and cloned into a pT7-IRES plasmid, designated pT7-IRES-fLuc. In pT7-IRES-N, pT7-IRES-L, pT7-IRES-NSs, and pT7-IRES-fLuc, an encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) sequence was placed between the T7 promoter and the inserted ORF. Plasmids expressing minigenome RNA transcripts were constructed to carry a T7 promoter sequence, minigenome sequence, hepatitis delta virus (HDV) ribozyme (18), and a T7 terminator sequence in pT7 vector, which was derived from pBL-EMCV plasmid. The minigenome sequence in each expression plasmid was as follows: pT7-M-rLuc(+), the 5' UTR of the anti-viral-sense M segment of RVFV wt strain ZH501, a Renilla luciferase (rLuc) ORF, and the 3' UTR of the anti-viral-sense ZH501 M segment; pT7-M-rLuc(-), the 5' UTR of the viral-sense M segment, an antisense rLuc ORF, and the 3' UTR of the viral-sense M segment; pT7-S-rLuc(-), the 5' UTR of the viral-sense S segment, antisense rLuc ORF, and the 3' UTR of the viral-sense S segment; pT7-L-rLuc(-), the 1,084 nt at the 5' of the viral sense L segment, the region from nt 2070 nt to nt 2736 of the viral-sense L segment, an antisense rLuc ORF, and the 3' UTR of the viral-sense L segment. A hammerhead ribozyme sequence (~53-nt length) (18) was inserted between the T7 promoter and the 5' UTR of the viral-sense M segment of pT7-M-rLuc(-) to generate pT7-HH-M-rLuc(-). Insertion of the same hammerhead ribozyme sequence between the T7 promoter and the 5' UTR of the anti-viral-sense M segment of pT7-M-rLuc(+) resulted in pT7-HH-M-rLuc(+). All of the constructs were confirmed to have the expected sequences.

Minigenome reporter assay. Subconfluent monolayers of 293T cells in six-well plates were cotransfected with a mixture of 1 µg of pCT7pol (23), 2 µg of pT7-M-rLuc(+), 0.4 µg of pT7-IRES-fLuc, and 4.5 µg of pT7-IRES using TransIT-293 (Mirus, Madison, WI) in a control group. In experimental groups, pT7-IRES-N alone, pT7-IRES-L alone, pT7-IRES-NSs alone, a mixture of pT7-IRES-N and pT7-IRES-L, or a mixture of pT7-IRES-N, pT7-IRES-L, and pT7-IRES-NSs was added to the mixture of pCT7pol, pT7-M-rLuc(+), pT7-IRES-fLuc, and pT7-IRES; 2.66 µg of pT7-IRES-N, 1.33 µg of pT7-IRES-L, and 0.5 µg of pT7-IRES-NSs was used. 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 after transfection, cells were lysed in 500 µl of Dual Luciferase passive lysis buffer (Promega Corporation, Madison, WI). Aliquots of 20 µl of lysate were assayed for fLuc and rLuc activities according to the manufacturer's instructions. fLuc activities were measured by using 1/10 of the samples that were used for rLuc activity measurements.

Preparation of strand-specific RNA probes. A PCR fragment (primers GCT AGG TAC CAT GAC TTC GAA AGT TTA TGA TCC and TAA CAA GCT TTA CAA CGT CAG GTT TAC CAC CT) from the pRLHL plasmid was subcloned into the KpnI-HindIII fragment of pSPT18 (DIG RNA labeling kit; Roche Applied Science), yielding pSPT18-rLuc. pSPT18-rLuc was digested with KpnI and then filled in using T4 DNA polymerase or was digested with HindIII. The linearized DNA was transcribed in vitro using SP6 RNA polymerase or T7 RNA polymerase for sense and antisense probes, respectively. Anti-viral-sense and viral-sense rLuc RNA probes were made by using a DIG RNA labeling kit (Roche Applied Science, Penzberg, Germany) according to the manufacturer's instructions.

Northern (RNA) blotting. Total intracellular RNA was extracted at 48 h after transfection using TRIzol reagent. In some experiments RNAs were extracted at 24, 36, 48, and 60 h posttransfection. Intracellular RNA (2 µg) was denatured and separated on 1.5% denaturing agarose-formaldehyde gels and transferred onto a nylon membrane or a BIOTRANS nylon membrane (ICN Biomedicals, Inc., Costa Mesa, CA). Northern blot analysis was performed with strand-specific RNA probes for sense rLuc ORF or antisense rLuc ORF using a DIG system (Roche Applied Science) according to the manufacturer's protocol.

Western blot analysis. Transfected cells were lysed with sample buffer and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Western blot analyses were performed as described previously (9). Briefly, the separated proteins were transferred onto polyvinylidene difluoride membranes (Amersham Biosciences). The membranes were blocked in blocking solution (0.5% bovine serum albumin, 1% Tween 20, 75 mM NaCl, and 10 mM Tris-HCl; pH 7.6) for 1 h at room temperature. The membranes were incubated with anti-RVFV mouse polyclonal antibody or antiactin goat polyclonal antibody (I-19; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) overnight at 4°C and with secondary antibodies for 1 h at room temperature. The membranes were developed with an enhanced chemiluminescence kit (Amersham Biosciences) according to the manufacturer's instructions. As a positive control, MP12-infected 293T cell lysates were used; 293T cells were infected with MP-12 at a multiplicity of infection of 1, and cell extracts were harvested at 24 h postinfection.


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RESULTS
 
Outline of a T7 RNA polymerase-driven RVFV minigenome system. As a first step for the development of an RVFV reverse genetics system and understanding of RVFV replication mechanisms, we aimed to establish an RVFV minigenome system. For the expression of anti-viral-sense minigenome RNA transcripts, we constructed plasmid pT7-M-rLuc(+), which contains a T7 promoter sequence, the 5' UTR of the anti-viral-sense M segment of RVFV wt strain ZH501, rLuc ORF, the 3' UTR of the anti-viral-sense M segment, HDV ribozyme, and T7 terminator sequence (Fig. 1A). After cotransfection of pT7-M-rLuc(+) and pCT7pol (23), which encodes T7 RNA polymerase downstream of the chicken ß-actin promoter, into mammalian cells, we expected synthesis of anti-viral-sense M segment-like minigenome RNA transcripts, which would contain an extra two G residues at the 5' end and the authentic viral 3'-end sequence (Fig. 1A). We also constructed minigenome plasmids pT7-M-rLuc(-), pT7-S-rLuc(-), and pT7-L-rLuc(-), which expressed viral-sense M segment-like RNA transcripts (Fig. 1A), viral-sense S segment-like RNA, and viral-sense L segment-like RNA, respectively (Fig. 1B).



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  		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.

In past studies by others, it was reported that the expression of viral L and N proteins is necessary and sufficient for viral RNA replication and transcription of bunyaviruses (1, 3, 13, 14, 16, 21). In order to drive RNA synthesis of the expressed minigenome RNA transcripts, plasmid pT7-IRES-N and plasmid pT7-IRES-L, which express ZH501 strain N protein and L protein, respectively, were constructed. Plasmid pT7-IRES-NSs was also constructed to express the NSs protein of ZH501. We expected the synthesis of the RNA transcripts carrying the EMCV IRES, followed by the viral ORF after cotransfection of one of these protein-expressing plasmids with pCT7pol plasmid and the subsequent expression of the expected viral protein by using a cap-independent translation mechanism. Accordingly, cotransfection of the pCT7pol plasmid, pT7-IRES-N, pT7-IRES-L, and a plasmid encoding one of the minigenomes into mammalian cells would result in minigenome RNA transcript expression, accumulation of N and L proteins, and minigenome RNA replication and transcription. To monitor transfection efficiency, a fixed amount of pT7-IRES-fLuc plasmid, which contained the T7 promoter, EMCV IRES, and fLuc ORF, was added to a mixture of the plasmids. Because RNA transcripts from pT7-IRES-fLuc would not be amplified in transfected cells, fLuc activity would indicate DNA transfection efficiency.

Assessment of minigenome RNA replication and transcription based on rLuc activity. We performed the luciferase assays as described in Materials and Methods to learn whether the T7 RNA polymerase-driven RVFV minigenome system worked. We used the ratio of pT7-IRES-N and pT7-IRES-L of 2:1, because our preliminary studies showed that this was the optimal ratio for the highest rLuc activity (data not shown). At 48 h posttransfection, cell extracts were prepared and fLuc and rLuc activities were measured (Fig. 2); the data shown in Fig. 2 were the result of three independent experiments. In the cells expressing the anti-viral-sense M segment minigenomes (Fig. 2A), a similar level of rLuc activities was detected in the control group and in the cells expressing N protein alone, L protein alone, or NSs protein alone, while in the cells expressing N and L proteins, rLuc activities were 2.6 times higher than those of the control group, indicating that coexpressed N and L proteins had driven minigenome amplification and mRNA synthesis, as was expected. Surprisingly, rLuc activity that was about six times greater than that of the control group was obtained in the cells expressing N, L, and NSs proteins. A substantial increase in rLuc activity in the presence of L, N, and NSs proteins indicated that NSs protein further enhanced the production of minigenome mRNA and/or translation of rLuc. A similar trend was also observed in the cells expressing viral-sense minigenomes (Fig. 2B); very low levels of rLuc were detected in the control group and in the cells expressing N protein alone, L protein alone, or NSs protein alone, whereas coexpression of N and L proteins and that of N, L, and NSs proteins increased the rLuc activity by about 18 times and 122 times, respectively. In each set of the experimental group, fLuc levels were similar among the control group, cells expressing N protein alone, those expressing L protein alone, and those expressing both N and L proteins, while fLuc activities in the cells expressing NSs protein alone and N, L, and NSs proteins were higher than those in other samples (Fig. 2).



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  		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.

Effect of NSs protein expression on minigenome RNA synthesis. The effects of NSs protein on minigenome RNA synthesis were further examined by Northern blot analyses, where strand-specific RNA probes identified anti-viral-sense RNA (Fig. 3A, first and third panels) and viral-sense RNA (Fig. 3A, second and fourth panels). Expression of anti-viral-sense minigenome RNA transcripts from pT7-M-rLuc(+) plasmid in 293T cells resulted in the synthesis of primary RNA transcripts of a larger size and minigenome RNA transcripts of the expected size (Fig. 3A, first panel, lanes 2 to 7); judging from the size of these RNAs, it appeared that HDV ribozyme-mediated RNA cleavage did not occur in the former but did take place in the latter. Accumulation of viral-sense minigenomes occurred in the cells coexpressing N and L proteins (Fig. 3A, second panel, lane 6), but not in the cells expressing only N protein (lane 3), only L protein (lane 4), or only NSs protein (lane 5), demonstrating that both N and L proteins were necessary for minigenome replication. It is evident that coexpression of NSs protein with L and N proteins substantially increased the amount of minigenome RNA in both the viral and anti-viral sense (Fig. 3A, first and second panels, lane 7). Similarly, in the cells expressing the viral-sense minigenome RNA transcripts as primary transcripts, accumulation of anti-viral-sense minigenome RNA occurred in the cells coexpressing N and L proteins but not in those expressing either N protein, L protein, or NSs protein alone, and coexpression of NSs protein with N and L proteins significantly boosted the level of anti-viral-sense minigenome RNA (Fig. 3A, third and fourth panels). These data demonstrated that coexpression of N and L proteins was necessary for minigenome RNA replication and that NSs expression with N and L proteins significantly augmented minigenome RNA replication.



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  		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.

To further confirm and understand the NSs protein-mediated minigenome replication amplification, efficiencies of minigenome RNA replication were examined in the cells expressing various amounts of L and N proteins and the fixed amount of NSs protein (Fig. 3B). Western blot analysis using anti-RVFV mouse polyclonal antibody demonstrated an accumulation of L, N, and NSs proteins in MP12-infected 293T cells and not in mock-infected cells (Fig. 3B, top and second panels, lanes 1 and 2). Cell extracts were prepared after transfection of 293T cells with a mixture of 1 µg of pCT7pol, 2 µg of pT7-M-rLuc(+), and various amounts of pT7-IRES-N and pT7-IRES-L in the fixed ratio of the former to the latter of 2:1 in one group (Fig. 3B, lanes 3 to 8) and the same mixture of plasmids plus 0.1 µg of pT7-IRES-NSs in the other group (Fig. 3B, lanes 9 to 14). Signals for N and L proteins in the transfected cells roughly correlated with the amounts of transfected pT7-IRES-N and pT7-IRES-L plasmids (Fig. 3B, top two panels). The amount of viral-sense minigenome increased when the cells expressed increased amounts of L and N proteins, suggesting that the higher amounts of N and L proteins increased minigenome replication (Fig. 3B, sixth panel, lanes 3 to 8). Under these experimental conditions, NSs protein accumulation was substantially lower in transfected cells than in MP12-infected cells; in transfected cells, an NSs protein signal was detected after long exposure of the membrane (Fig. 3B, third panel), and it was masked by N protein signals in the cells expressing large amounts of N protein (Fig. 3B, third panel, lanes 12 to 14). It appears that NSs protein expression did not significantly alter the amounts of N and L proteins. In spite of a low level of NSs protein, NSs protein expression increased minigenome RNA replication (Fig. 3B, bottom two panels, lanes 9 to 14). These data demonstrated that NSs protein promoted efficient minigenome RNA replication without significantly altering the amount of N and L proteins. We also noticed that the amount of viral-sense minigenome in the cells transfected with 0.33 µg of pT7-IRES-N, 0.17 µg of pT7-IRES-L, and 0.1 µg of pT7-IRES-NSs (Fig. 3B, lane 10) was clearly higher than that in cells transfected with eight-times-higher amounts of pT7-IRES-N and pT7-IRES-L (Fig. 3B, lane 8), while the amounts of L and N proteins were lower in the former than the latter, demonstrating that amounts of L and N proteins were not the sole determinant for the minigenome RNA replication efficiency.

We next tested whether NSs protein expression affected the optimal ratio of pT7-IRES-N and pT7-IRES-L for minigenome replication by examining minigenome RNA accumulation in the cells that were transfected with different ratios of pT7-IRES-N and pT7-IRES-L in the presence and absence of pT7-IRES-NSs (Fig. 3C). In all cases, the expression of NSs protein substantially augmented minigenome RNA replication, while the optimal ratio of pT7-IRES-N and pT7-IRES-L was about 2:1 to 4:1, both in cells expressing NSs and those not expressing NSs. These data suggested that NSs protein did not alter the optimal ratio of N and L proteins for minigenome RNA replication.

NSs protein-mediated augmentation of minigenome RNA replication in cells lacking IFN genes. RVFV NSs protein inhibits the synthesis of host mRNAs, including IFN mRNAs, by binding to the p44 subunit of TFIIH transcription factor (20). This unique property of NSs protein led us to investigate the possibility that efficient minigenome RNA replication in the cells expressing NSs protein was due to NSs protein-mediated inhibition of IFN-{alpha}/ß production. Namely, in the absence of NSs protein expression, minigenome RNA replication may induce IFN-{alpha}/ß production in 293T cells and the released IFNs subsequently may suppress minigenome RNA amplification, whereas minigenome RNA replication in the cells expressing NSs protein may fail to produce IFN-{alpha}/ß, allowing efficient minigenome RNA amplification. To determine whether inhibition of putative IFN-{alpha}/ß production by NSs protein was the primary reason for the NSs protein-mediated minigenome RNA replication amplification, the effect of NSs protein on minigenome RNA replication was tested in the Vero E6 cell line (ATCC C1008), which is derived from the Vero cell line and lacks IFN-{alpha}/ß genes (11, 12, 22, 30). As shown in Fig. 4, NSs protein expression enhanced minigenome RNA accumulation in the cells expressing the anti-viral-sense minigenome from pT7-M-rLuc(+) as well as in the cells expressing the viral-sense minigenome from pT7-M-Luc(-) (compare lanes 5 and 6), demonstrating that NSs-mediated augmentation of minigenome RNA amplification occurred in the cells lacking IFN-{alpha}/ß genes and that NSs protein enhanced RNA replication from the viral-sense minigenome RNA transcripts as well as from the anti-viral-sense minigenome RNA transcripts. These data suggested that NSs protein enhances the RNA-dependent RNA polymerase activities of L and N proteins to promote efficient virus RNA replication.



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  		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.

Optimization and further characterization of the minigenome system. To further optimize the minigenome system, we examined minigenome RNA replication efficiency in the presence of various amounts of NSs protein. Six different amounts of pT7-IRES-NSs (0 µg to 4 µg) were added with the mixture of pT7-IRES-N (0.66 µg), pT7-IRES-L (0.33 µg), pCT7pol (1 µg), and pT7-rLuc(+) or pT7-rLuc(-) (2 µg), and then the mixture was transfected into 293T cells. Western blot analysis demonstrated that accumulation of NSs protein roughly correlated with the amounts of pT7-IRES-NSs in the inoculum (Fig. 5A, fifth panel). Minigenome RNA replication was hardly detectable in the cells transfected with less than 0.01 µg of pT7-IRES-NSs (Fig. 5A, lanes 1 to 3), whereas it was clearly enhanced in the presence of 0.1 µg of pT7-IRES-NSs (Fig. 5A, lane 4). It was evident that transfection of 2 or 4 µg of pT7-IRES-NSs substantially enhanced minigenome RNA replication from the viral-sense minigenome RNA transcripts as well as from the anti-viral-sense minigenome RNA transcripts (Fig. 5A, lanes 5 and 6), demonstrating that higher amounts of NSs protein drastically increased minigenome RNA replication.



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  		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.

We noticed a substantial increase in the amounts of minigenome mRNA in the cells expressing the viral-sense RNA transcripts with 2 µg or 4 µg of pT7-IRES-NSs (Fig. 5A, third panel, lanes 5 and 6), whereas this effect was not evident in the cells expressing anti-viral-sense RNA transcripts (Fig. 5A, top panel, lanes 5 and 6). In addition, in the cells expressing viral-sense RNA transcripts (Fig. 5A, third and fourth panels), similar amounts of primary transcripts and replicating minigenome RNA were detected between the cells transfected with 2 µg of pT7-IRES-NSs and those transfected with 4 µg of pT7-IRES-NSs, whereas the amount of minigenome mRNA was higher in the latter cells, indicating that increased amounts of NSs protein expression promoted minigenome RNA transcription. RNA replication of minigenome RNA was needed prior to minigenome mRNA transcription in cells expressing anti-viral-sense RNA transcripts, while expressed viral-sense RNA could serve as the template for minigenome mRNA in cells expressing viral-sense RNA (Fig. 1A). Accordingly, minigenome mRNA synthesis from a different-sense template may have different kinetics, and analysis of viral RNAs at only 48 h posttransfection might have caused us to miss a possible NSs-mediated accumulation of minigenome mRNA in the cells expressing anti-viral-sense minigenome RNA transcripts. We next examined the accumulation of minigenome RNA and mRNA at various times posttransfection (Fig. 5B). Intracellular RNAs were extracted at 24, 36, 48, and 60 h posttransfection from 293T cells that were cotransfected with the mixture of pT7-IRES-N, pT7-IRES-L, pT7-IRES-NSs, pCT7pol, and pT7-M-rLuc(+) or pT7-M-rLuc(-). Accumulation of mRNA was observed from 24 to 60 h posttransfection in the cells expressing viral-sense minigenome RNA transcripts. In contrast, the cells expressing anti-viral-sense minigenome RNA transcripts revealed very low levels of minigenome mRNA accumulation. These data demonstrated that NSs-mediated enhancement of minigenome mRNA transcription occurred in the cells expressing viral-sense minigenome RNA transcripts but not in the cells expressing anti-viral-sense minigenome RNA transcripts.

Minigenome RNA transcripts from pT7-rLuc(+) or pT7-rLuc(-) were expected to contain two nonviral G residues at their 5' end. To know whether these extra nonviral nucleotides affected minigenome RNA synthesis, we examined minigenome RNA synthesis in the cells that expressed RNA transcripts not carrying these extra nonviral sequences. To this end, 293T cells were cotransfected with the mixture of pT7-IRES-N, pT7-IRES-L, pCT7pol, pT7-HH-M-rLuc(+) or pT7-HH-M-rLuc(-), and various amounts of pT7-IRES-NSs; pT7-HH-M-rLuc(+) and pT7-HH-M-rLuc(-) had an insertion of the hammerhead ribozyme sequence between T7 promoter and the viral sequence of pT7-M-Luc(+) and pT7-M-Luc(-), respectively. As shown in Fig. 5C, minigenome RNA replication from the expressed RNA transcripts from pT7-HH-M-rLuc(+) and pT7-HH-M-rLuc(-) was barely detectable in the absence of NSs protein expression, whereas coexpression of NSs protein significantly enhanced the replication of both of the minigenome RNAs. Furthermore, NSs protein expression promoted minigenome mRNA transcription in pT7-HH-M-rLuc(-)-transfected cells (Fig. 5B, third panel). Overall, there were no substantial differences in minigenome RNA replication and mRNA transcription between the cells expressing RNA transcripts containing the authentic viral 5' end and those expressing transcripts with the 5' two nonviral G residues.

Effects of NSs protein expression on minigenomes derived from L and S segments. All of the experiments described above used M segment-like minigenome RNAs. We examined whether NSs protein expression also stimulates the replication of S segment-like minigenome RNA and L segment-like minigenome RNA by analyzing minigenome-derived RNAs in 293T cells that were cotransfected with the mixture of pT7-IRES-N, pT7-IRES-L, pCT7pol, pT7-S-rLuc(-) or pT7-L-rLuc(-), and various amounts of pT7-IRES-NSs. We were unable to convincingly demonstrate RNA replication of expressed viral-sense S and L segment-like minigenomes in the cells expressing N and L proteins (Fig. 6, lane 1), whereas minigenome RNA replication was evident in the presence of the NSs protein (Fig. 6, lanes 2 to 3). NSs-mediated stimulation of RNA replication of S and L segment-like minigenomes also occurred in 293T cells expressing anti-viral-sense, S segment-like minigenome RNA and in those expressing anti-viral-sense, L segment-like minigenome RNA (data not shown). We concluded that the coexpression of NSs protein with N and L proteins substantially enhanced the replication of minigenome RNAs that were derived from all three viral segments.



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  		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.


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DISCUSSION
 
We presented here the establishment of a T7 RNA polymerase-driven RVFV minigenome system in which viral minigenome RNA transcripts and viral proteins were initially synthesized from transfected plasmids by using cotransfected pCT7pol-derived T7 RNA polymerase. Expressed minigenome RNA transcripts that contain the rLuc ORF between both termini of the M RNA segment in the anti-viral sense, as well as in the viral sense, underwent RNA replication and transcription in the cells coexpressing N and L proteins. To our surprise, coexpression of NSs protein with N and L proteins substantially enhanced minigenome RNA replication and transcription. NSs protein expression also augmented the synthesis of the S segment-like minigenome RNA and L segment-like minigenome RNA. Analysis of minigenome RNA replication in Vero E6 cells suggested that the inhibition of putative IFN-{alpha}/ß production by NSs protein was an unlikely reason for the NSs protein-mediated minigenome RNA replication enhancement. Our present study predicts that expression of the NSs protein will be critical for the successful development of an RVFV reverse genetics system.

Our reporter assay showed that fLuc activities were similar among cells expressing the N protein only, L protein only, or both N and L proteins, while they were about two times higher in the cells expressing NSs protein alone and in those expressing N, L, and NSs proteins (Fig. 2), demonstrating that NSs protein expression somehow resulted in higher fLuc activities. Because the NSs protein suppresses host mRNA synthesis (20), the amounts of mature host mRNAs were likely low in the cells expressing NSs protein. The translation efficiency of RNA transcripts from pT7-IRES-fLuc might be increased in the NSs protein-expressing cells because there were lesser amounts of host mRNAs that could compete with the T7 RNA transcripts for the host translational machinery. Accordingly, the putative decrease in the amount of mature host mRNAs may have contributed to the efficient translation of virus-specific proteins whose synthesis is independent of host polymerase II function. Expressed NSs protein probably also inhibited transcription from pCT7pol, leading to reduction of T7 polymerase accumulation in NSs-expressing cells. However, our Northern blot data revealed that the amounts of primary transcripts of minigenome RNAs were not significantly different between the cells expressing NSs protein and those not expressing NSs protein (Fig. 3, 4, 5, and 6), indicating that a possible reduction of the amount of T7 polymerase in NSs protein-expressing cells did not affect the synthesis of T7 RNA transcripts from transfected plasmids.

The present study convincingly demonstrated that NSs protein expression augmented minigenome RNA synthesis, but the mechanism of augmentation is not yet clear. We observed that NSs protein could not be replaced with the N protein in viral RNA synthesis, because minigenome RNA replication and transcription did not occur in the cells coexpressing only L and NSs proteins when viral-sense or anti-viral-sense M segment-like minigenome RNA transcripts were expressed (data not shown). As shown in Fig. 3B, viral RNA synthesis levels were higher in the cells expressing the NSs protein with small amounts of the N and L proteins than in those not expressing the NSs protein and with higher amounts of the L and N proteins (compare lanes 10 and 8). One possible explanation of this finding is that the NSs protein might stimulate the activities of the viral replication complex, which presumably consisted of the L protein, viral RNA template encapsidated by N protein, elongating nascent RNA molecules, and unidentified host factors, leading to a higher level of viral RNA synthesis than the viral replication complex with larger amounts of the N and L proteins yet lacking NSs protein stimulation. The fact that minigenome RNA replication increased as the amounts of the NSs protein increased (Fig. 5A) implies that this putative NSs protein-mediated enhancement of the activities of the viral replication complex is not catalytic; NSs protein may be continuously associated with the viral replication complex to stimulate its functions.

Substantial NSs protein-mediated enhancement of minigenome mRNA transcription occurred in the cells expressing viral-sense minigenome RNA transcripts, but not in the cells expressing anti-viral-sense minigenome RNA transcripts (Fig. 5). One possible reason may be that minigenome mRNA was transcribed from two different templates in the cells expressing viral-sense minigenome RNA transcripts: one is viral-sense minigenome RNA, which underwent RNA replication, and the other is the viral-sense primary transcripts. The accumulation of minigenome mRNA was less efficient in the cells expressing anti-viral-sense minigenome RNA transcripts, because these cells lacked viral-sense primary transcripts.

The current dogma of Phlebovirus RNA synthesis states that NSs mRNA is produced only after viral-sense RNA has been copied to anti-viral-sense RNA; therefore, the NSs protein would appear at a later stage in infection than the structural proteins (17, 28). If the NSs protein is indeed important for efficient RVFV RNA synthesis, then its accumulation later in the infection process may not be optimal for efficient replication. We confirmed previous reports (24) that the NSs protein is not a virus structural protein (T. Ikegami, C. J. Peters, and S. Makino, unpublished data), suggesting that the NSs protein cannot be provided to infected cells from the input virus. Surprisingly, in addition to three viral-sense RNA segments, the anti-viral sense of S segment RNA was packaged into MP12 particles (Ikegami et al., unpublished); a possibility emerged that RVFV mRNA encoding NSs protein could be synthesized from the virion-associated anti-viral-sense S segment RNA immediately after infection. The anti-viral-sense S segment RNA was also detected in purified LACV (27) and UUKV (29). Studies are in progress to test a possibility that mRNA encoding NSs protein is synthesized from the virion-associated anti-viral-sense S segment RNA after RVFV infection.

The first RVFV minigenome system was described by Lopez et al. (21). They demonstrated that transfection of CV1 cells with CsCl gradient-purified ribonucleoprotein complexes extracted from RVFV and infection of vaccinia virus encoding L protein result in viral mRNA synthesis. Furthermore, recombinant vaccinia virus-mediated coexpression of L and N proteins and transfection of an S-like genome containing the chloramphenicol acetyltransferase (CAT) gene result in CAT expression, indicating mRNA transcription of an S-like genome. Those authors also reported that coinfection of recombinant vaccinia virus encoding NSs protein with virus expressing L and N proteins does not alter CAT activities, suggesting that NSs protein does not augment mRNA transcription of the S-like segment; their data differ from our data shown in the present study. Because NSs protein expression was not examined in the studies by Lopez et al. (21), expression levels of NSs protein might not have been optimized in their study. Also, they used vaccinia viruses to express virus proteins; vaccinia virus-mediated alteration of the cellular environment may have affected the outcome of the results.

Expression of the NSs protein inhibits minigenome RNA synthesis in two members of the genus Bunyavirus, BUNV (31) and LACV (3), rather than enhancing it, as in our findings. Although functions of a protein between two genera in the same family might be conserved, this is not necessarily so. In the Bunyavirus genus, the NSs gene is smaller (10 to 13 kDa versus 31 kDa for Phlebovirus) (24, 28) and is coded in the anti-viral sense rather than the viral sense, as in Phlebovirus. Thus, it is not surprising that a mutant BUNV lacking NSs (6) replicates efficiently in cell culture, suggesting that in the Bunyavirus genus NSs is not critical for viral RNA synthesis.


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ACKNOWLEDGMENTS
 
We thank R. B. Tesh at the University of Texas Medical Branch (UTMB) for anti-RVFV mouse polyclonal antibody, S. T. Nichol (Centers for Disease Control and Prevention) for cell lysate of RVFV ZH501-infected cells, T. Takimoto (St. Jude Children's Research Hospital, Memphis, Tennessee) for pCT7pol plasmid, and I. Frolov (UTMB) and R. Rijnbrand (UTMB) for various plasmids.

This work was supported by grants from the National Institute of Allergy and Infectious Diseases (S.M. and C.J.P.) through the Western Regional Center of Excellence for Biodefense and Emerging Infectious Diseases Research, National Institutes of Health grant number U54 AI057156.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX 77555-1019. Phone: (409) 772-2323. Fax: (409) 772-5065. E-mail: shmakino{at}utmb.edu. Back


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Journal of Virology, May 2005, p. 5606-5615, Vol. 79, No. 9
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.9.5606-5615.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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