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Previous Article | Next Article 
Journal of Virology, September 2005, p. 12106-12111, Vol. 79, No. 18
0022-538X/05/$08.00+0 doi:10.1128/JVI.79.18.12106-12111.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Departments of Microbiology and Immunology,1 Pathology, The University of Texas Medical Branch at Galveston, Galveston, Texas2
Received 7 April 2005/ Accepted 28 June 2005
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Rift Valley fever virus (RVFV) (family Bunyaviridae, genus Phlebovirus) causes severe epidemics among ruminants in the sub-Saharan African and has spread to Egypt, Yemen, and Saudi Arabia. It is also an important human pathogen that causes a syndrome of fever and myalgia, a hemorrhagic syndrome, ocular disease, and encephalitis (1, 13). The anti-viral-sense L segment encodes L protein, a viral RNA polymerase, and the anti-viral-sense M segment encodes two structural glycoproteins, G1 and G2, NSm protein, and a 78-kDa protein. As in other viruses of the genus Phlebovirus, RVFV S segment uses an ambisense strategy to express N and NSs proteins, and the viral-sense S and anti-viral-sense S segments serve as templates for N mRNA and NSs mRNA, respectively (16). It has been believed 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 (6, 16).
RVFV NSs protein plays an important role in RVFV pathogenesis and replication. This protein inhibits host mRNA synthesis, including alpha/beta interferon (IFN-
/ß) mRNAs (3, 10), hence suppressing host innate immune responses to viral invasion, and it is a major virus virulence factor (4). We showed that coexpression of RVFV NSs protein with N and L proteins enhances viral RNA accumulation in the RVFV minigenome system (7). Because this effect appears to be independent of the NSs-mediated inhibition of IFN-/ß production, NSs protein most likely augments viral RNA synthesis in the minigenome system (7) and probably during viral infection. The effect of NSs on viral RNA synthesis led us to speculate that RVFV NSs protein might be synthesized early in infection to promote viral RNA synthesis. The present study tested this possibility. Our data indeed support this supposition and showed unexpected properties of NSs mRNA synthesis.
We first examined the possibility of anti-viral-sense S segment incorporation into RVFV particles; if this occurs, the incoming anti-viral-sense S segment may serve as a template for NSs mRNA synthesis immediately after infection. A vaccine strain of RVFV, MP12, which has multiple mutations (19), including the NSs gene, compared with wild-type RVFV, was used as the source of infectious RVFV. MP12 was propagated at 37°C in various mammalian cells, including 293T, Vero E6, and BHK-21 cells, and in mosquito C6/36 cells at 28°C after inoculation at a multiplicity of infection (MOI) of 0.1. At 48 h postinfection (p.i.), culture fluid was collected and clarified by low-speed centrifugation. Then virus particles were partially purified by two subsequent ultracentrifugations on a discontinuous sucrose gradient consisting of 60, 50, 30, and 20% sucrose using a Beckman SW28 rotor (9, 11); the sample was first centrifuged at 28,000 rpm for 3 h, and the virus particles at the interface of 30 and 50% sucrose were further centrifuged at 28,000 rpm for 18 h. The virus particles at the interface of 30 and 50% sucrose were collected, diluted, and then further applied on a continuous sucrose gradient of 20 to 60% sucrose. The samples were centrifuged at 28,000 rpm for 18 h. Subsequently, 10 fractions were collected, and the sucrose density in each fraction was measured. Virus particles in the each fraction were pelleted through a 20% sucrose cushion at 38,000 rpm for 2 h using a Beckman SW 41 rotor (8) and subjected to further analysis. Western blot analysis of the virus sample propagated in Vero E6 cells using anti-RVFV mouse antibody (7) detected the peak viral protein signals, L, G1, G2, and N, at a sucrose density of approximately 1.16 g/cm3 (Fig. 1A, upper panel). The buoyant densities of other virus preparations were from 1.16 to 1.18 g/cm3 (data not shown), similar to previously reported data (16). Northern blot analyses were performed to identify virus-specific RNAs in the purified virion with buoyant densities from 1.16 to 1.18 g/cm3. A total of six digoxigenin-labeled (Roche Applied Science), strand-specific riboprobes were independently synthesized in vitro and grouped into two sets; a mixture of three probes, each of which binds to the viral-sense S, M, or L segments, was designated as probe set A1, while probe set B contained a mixture of three probes, each of which binds to anti-viral-sense S, M, or L segments (Fig. 1B). To confirm the probe specificities, a mixture of the same amounts of in vitro-transcribed, full-length viral-sense S-, M-, and L-segment RNA transcripts, as well as anti-viral-sense transcripts, was separated by agarose gel electrophoresis, and then Northern blot analysis was performed using probe set A1 and probe set B (Fig. 1C, right two panels), which were specifically hybridized to viral-sense RNA transcripts and anti-viral-sense RNA transcripts, respectively, establishing the probe specificities. However, the intensity of each band differed, indicating that the efficiencies of hybridization of each probe to its target RNA transcripts were not the same. A band that migrated between the viral-sense L and M segments probably represented L RNA segment transcripts with a premature termination. Northern blot analysis of virion RNA from purified MP12 that was propagated in 293T cells, Vero E6 cells, BHK-21 cells or C6/36 cells is shown in the two left panels of Fig. 1C. As expected, probe set A1 detected three viral-sense RNA segments in the purified viruses. Unexpectedly, probe set B also demonstrated all three anti-viral-sense RNA segments. Northern blot analysis of intracellular RNA species (see Fig. 2C) showed an accumulation of NS and N mRNAs in infected cells, whereas both were absent in the purified virus sample (Fig. 1C), suggesting that detection of anti-viral-sense RNA segments in the purified RVFV was not due to merely contamination of the purified virion sample with intracellular RNAs.
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To test the possibility that the incoming anti-viral-sense S segment serves as the template RNA for NSs mRNA synthesis early in infection, we employed an RNase protection assay to examine the synthesis of N and NSs mRNAs. The feasibility of an RNase protection assay was tested first. The 32P-labeled, 389-nt-long RNA probe (N probe) and the 32P-labeled, 336-nt-long RNA probe (NSs probe) were independently synthesized by in vitro transcription (Fig. 2A); the former contained a 27-nt nonviral sequence at the 5' end and binds to the region from nucleotide (nt) 631 to 990 from the 5' end of the anti-viral-sense S segment and to the 3' region of N mRNA, while the latter contained a 27-nt nonviral sequence at the 5'end and binds to the region from nt 736 to 1036 from the 5' end of the viral-sense S segment and to the 3' region of NSs mRNA (Fig. 2A). Each probe was mixed with intracellular RNAs extracted from MP12-infected Vero E6 cells at 6 h p.i. or purified virion RNAs. An RNase protection assay was performed using an RNase protection assay kit (BD Biosciences) according to the manufacturer's protocol, and the RNase-resistant RNA fragments were separated on a 5% Tris-borate-EDTA-urea gel. As expected, an N probe revealed the virion-associated anti-viral-sense S segment, the intracellular anti-viral-sense S segment, and N mRNA, while an NSs probe detected the virion-associated viral-sense S segment, intracellular viral-sense S segment, and NSs mRNA (Fig. 2B). The synthesis of N and NSs mRNAs early in infection was examined next. To synchronize virus infection, Vero E6 cells were inoculated with MP12 at an MOI of 5, and the cells were kept at 0°C for 1 h. After virus adsorption, cells were washed with chilled medium to remove unadsorbed viruses and then incubated with prewarmed (37°C) medium to promote synchronized virus penetration. At 0, 20, 40, 60, or 120 min after incubation, total intracellular RNAs were extracted. The RNase protection assay detected signals of both incoming viral-sense and anti-viral-sense S segments in the 0-min sample, and their signal intensities somewhat decreased during 20 min to 60 min incubation (Fig. 2D, left two panels). At 120 min after incubation, both signals increased slightly, indicating that RNA replication was initiated between 60 and 120 min of incubation. Signals of both N mRNA and NSs mRNA were absent in the 0-min sample, yet accumulation of both mRNAs was evident as early as 20 min of incubation, and their amounts increased consistently thereafter. Intracellular RNAs from mock-infected cells and yeast tRNAs showed no RNA signals. These data demonstrated that the synthesis of N and NSs mRNAs occurred immediately after infection and strongly suggested that N mRNA and NSs mRNA were transcribed from the virion-associated viral-sense S segment and the virion-associated anti-viral-sense S segment, respectively, during primary transcription and prior to viral RNA replication.
To further establish NSs mRNA synthesis during primary transcription, we examined the synthesis of N and NSs mRNAs in the presence of puromycin, a translation inhibitor. It has been reported that bunyavirus genome RNA replication does not occur in the presence of translational inhibitors (2, 5, 6, 15, 18). Accordingly, if NSs mRNA synthesis occurs in the presence of puromycin immediately after infection, then the data would conclusively demonstrate that NSs mRNA synthesis occurred during primary transcription from the incoming anti-viral-sense S segment. To determine the appropriate concentration of puromycin to inhibit viral RNA synthesis, Vero E6 cells were incubated with 0, 0.5, 5.0, 25, 50, or 100 µg/ml of puromycin for 1 h prior to virus adsorption and during virus adsorption (MOI = 5) and incubation of infected cells. Northern blot analysis of intracellular RNAs, which were extracted at 6 h p.i., demonstrated that treatment with 25 to 100 µg/ml of puromycin completely inhibited viral RNA synthesis (Fig. 2C). Accordingly, 100 µg/ml of puromycin was used to examine NSs mRNA synthesis. Vero E6 cells were incubated with puromycin for 1 h, and then virus inoculum containing puromycin was added at an MOI of 5. After 1 h incubation at 0°C and removal of unadsorbed viruses, infected cells were incubated in the presence of puromycin. An RNase protection assay was performed using intracellular RNAs that were extracted at 0, 20, 40, 60, or 120 min incubation. As expected, viral-sense and anti-viral-sense S-segment RNAs did not accumulate (Fig. 2D, right two panels), demonstrating that puromycin treatment inhibited viral RNA replication. Significantly, NSs mRNA synthesis (Fig. 2D, right bottom panel), but not N mRNA synthesis (Fig. 2D, right top panel), occurred in the presence of puromycin. These data conclusively established that NSs mRNA synthesis occurred during primary transcription from the incoming anti-viral-sense S segment. Furthermore, RVFV NSs mRNA synthesis, which was resistant to treatment with the protein synthesis inhibitor puromycin, differed from that in previous reports of mRNA synthesis of other bunyaviruses, in which host translation machineries appeared to be required for viral mRNA transcription (2, 15, 18).
We next examined NSs protein synthesis early in infection. To efficiently detect NSs protein synthesis, we made anti-NSs peptide (N-EESDDDGFVEVD-C) rabbit polyclonal antibody (ProSci Inc, Poway, CA) and tested its reactivity. Western blot analysis of cell extracts from MP12-infected 293T cells using anti-RVFV antibody demonstrated an accumulation of NSs and N proteins, while anti-NSs antibody detected only NSs protein (Fig. 3A, lane 1); neither antibody detected host protein signals in the mock-infected cell extracts (Fig. 3A, lane 4). To further establish the anti-NSs antibody specificity, 293T cells were independently transfected with a mixture of pCT7pol (12), which expresses T7 RNA polymerase, and pT7-IRES-NSs (7) or that of pCT7 pol and pT7-IRES-N (7); pT7-IRES-NSs and pT7-IRES-N express NSs protein and N protein, respectively, by using a cap-independent translation mechanism (7). Forty-eight hours after transfection, cell extracts were prepared and used for Western blot analysis (Fig. 3A, lanes 2 and 3). Anti-RVFV antibody detected both expressed NSs and N proteins, while anti-NSs antibody detected expressed NSs protein but not N protein, demonstrating that anti-NSs antibody specifically recognized NSs protein. Also, anti-NSs antibody was substantially more sensitive than was anti-RVFV antibody in detecting intracellular NSs protein (Fig. 3A). Western blot analysis of intracellular proteins early in RVFV infection demonstrated that anti-RVFV antibody detected the incoming N protein in the 0-min sample (Fig. 3B). In the presence of puromycin treatment, the N protein signals clearly decreased subsequent to incubation at 37°C, implying degradation of the incoming N protein. In the absence of puromycin treatment, the N protein signal in the samples from 20 to 120 min incubation was only slightly lower than that of the 0-min sample, and it clearly increased after 180 min incubation (Fig. 3B). The NSs protein signal was at background level during the first 40 min of incubation, and yet it increased gradually after about 60 to 80 min incubation. Accumulation of NSs protein was quite evident after 180 min incubation and detectable by anti-RVFV antibody. These data demonstrated that NSs protein synthesis was detectable as early as 60 to 80 min p.i.
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Synthesis of N mRNA and NSs mRNA was detected as early as 20 min p.i. (Fig. 2D), suggesting translation of both N and NSs proteins early in infection. Analysis of intracellular N protein early infection suggested that in the absence of N mRNA synthesis, the incoming N protein underwent substantial degradation (Fig. 3B). In contrast, only a modest reduction in the amounts of N protein occurred after 20 to 120 min incubation in the absence of puromycin. A straightforward interpretation of these data is that N protein synthesis occurred as early as 20 min from newly synthesized N mRNA (Fig. 2D). In contrast, NSs protein accumulation was evident only after 60 to 80 min p.i. (Fig. 3B). Although it is unclear why we were unable to detect NSs protein accumulation during first 40 min p.i., it is conceivable that the amount of NSs mRNA would be substantially lower than that of N mRNA immediately after infection, because the amounts of anti-viral-sense S segment in the virus particles were only 1 to 20% of the viral-sense S segment. Also, the sensitivity of anti-NSs antibody for detecting NSs protein might be lower than that of anti-RVFV antibody for detecting N protein.
Because NSs mRNA synthesis occurred in the absence of viral RNA replication (Fig. 2D, right panel), NSs mRNA that was accumulated early in infection should have been transcribed from the incoming anti-viral-sense S segment. Virions containing an anti-viral-sense S segment were a minor population in the virus progeny from our cell culture, suggesting that NSs protein synthesis early in infection occurs in only a fraction of cells infected with RVFV. We speculate that the immediate expression of the NSs protein with its inhibition of host mRNA transcription, particularly of the interferon genes, may provide a selective advantage during viral infection. This "head start" may be quite important in particular cells or organs or at a point early in infection at which innate immunity plays a more important role in host resistance. The need for this selective viral advantage may be responsible for the evolutionary retention of the ability of the virions to include subpopulations with diploid genomes. It will be of interest to study the proportions of diploid genomes under different circumstances of passage in vitro and in vivo in interferon-incompetent systems or highly susceptible hosts.
Previous observations that host translation is required for viral mRNA synthesis of viruses of the Bunyaviridae family (2, 15, 18) led to the hypothesis that nascent bunyavirus mRNA needs to bind to ribosomes to prevent premature transcription termination; without this binding, the nascent mRNA may interact with its template and stop mRNA elongation (2). Like other bunyaviruses, puromycin treatment inhibited RVFV N mRNA synthesis during primary transcription (Fig. 2D). Unexpectedly, NSs mRNA synthesis occurred in the presence of puromycin. Note that the probes used for the RNase protection assay hybridize only with mature N and NSs mRNAs (Fig. 2A); hence, this assay detected mature mRNAs and not premature transcription products. The simplest explanation for the puromycin-resistant NSs mRNA synthesis is that nascent NSs mRNA forms stable secondary or ternary RNA structures and does not bind to the template RNA, preventing premature transcription termination. Obviously, further studies will be needed to determine the mechanism of puromycin-resistant NSs mRNA synthesis.
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ACKNOWLEDGMENTS |
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This work was supported by grants from NIAID to S.M. and C.J.P. through the Western Regional Center of Excellence for Biodefense and Emerging Infectious Diseases Research, NIH grant number U54 AI057156.
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