Here, we describe a highly efficient reverse genetics system that does not require infectious CCHFV and allows genetic modification of the virus L and N proteins in addition to the RNA template, permitting precise studies of virus mechanisms such as transcription, replication, and packaging. Although such systems are available for viruses of other genera of the Bunyaviridae
family, this is the first such system available for a member of the Nairovirus
genus. A previous report describing the generation of CCHFV carrying either S-GFP or S-chloramphenicol acetyltransferase minigenomes, confirmed that the S segment UTRs were sufficient for transcription, replication, and packaging (15
). Unfortunately, this system was rather limited by the fact that it required live CCHFV to drive minigenome replication; thus, its use was restricted to a BSL-4 laboratory, and the viral L and N genes could not be manipulated for functional studies.
To assay the functions of the L protein, cells were transfected with S-, M-, and L-segment minigenomes and subsequently infected with CCHFV. Passage of virus particles containing the S, M, or L minigenomes was demonstrated, indicating that the mature T7-derived primary transcripts were replicated, transcribed, and packaged by CCHFV. In addition, nonvirus templated nucleotides (5′ GG), introduced to improve T7 transcription, were found to be removed after replication, further demonstrating the ability of these transcripts to initiate replication after their T7 transcription. The S, M, or L segment UTRs contributed to significant differences in the expression of two different reporters on passage of transfected cell supernatants to new cell monolayers, with the L UTR being the most efficient and S the least (L > M > S). Similar observations in S, M, and L UTR efficiencies were previously reported for Bunyamwera and Uukuniemi virus minigenome systems (14
). However, superinfection experiments should be interpreted with caution as the reporter activity measured is the result of the combination of transcription, replication, encapsidation, and packaging. The results of the Bunyamwera and Uukuniemi virus experiments suggested that the packaging signals within their L UTRs were the strongest while the S segment UTRs were the weakest of the three (L > M > S). In contrast, approximately equivalent levels of activity were observed with CCHFV S, M, and L UTRs in our minigenome replication experiments, where replication occurred with coexpressed L and N protein as opposed to infection of a helper virus.
These findings suggest that the differences observed in the virus superinfection and passage experiments may be the result of differences in packaging efficiency rather than replication promoter efficiency, with UTR packaging signal strengths being L > M > S. Analysis of passage efficiency of infectious virus-like particles with the different UTRs could more precisely address this question as the current observation involves comparison of a helper-virus-driven system relative to a plasmid-driven system. It is interesting that the gradation observed in packaging efficiency of RNA segment UTRs (L > M > S) appears to be a broad feature of bunyaviruses, given that this gradation has now been seen with Bunyamwera virus (genus Orthobunyavirus
), Uukuniemi virus (genus Phlebovirus
), and CCHFV (genus Nairovirus
). The packaging strength is inversely proportional to the size of the RNA segment, perhaps compensating for the increased difficulty in packaging larger RNPs into virion particles so that the small RNA does not outcompete the larger RNAs during virus passage. This hypothesis could be tested by examining passage efficiencies of L UTR-containing minigenomes of increasing size, varying from authentic S to L segment size (~1.6 to 13 kb).
We demonstrated that the expressed recombinant CCHFV nucleoprotein localized to the perinuclear region of transfected cells (Fig. ) and closely resembles nucleoprotein localization in CCHFV-infected cells, a pattern dependent on the cellular actin network (3
). By tagging V5 on the N terminus of the L protein, we show the interaction of L protein with N when the proteins are coexpressed in transfected cells. When expressed independently, L was detected throughout the cytoplasm but colocalized with N in the perinuclear regions in close proximity to the Golgi cisternae, where CCHFV assembly is reported to occur. This recruitment of L protein by N was independent of L polymerase or OTU protease function as their colocalization was not altered when these functions were abolished. How L protein and N interact with actin filaments and whether actin is required for formation of virus replication complexes are areas for future research.
The L proteins encoded by the viruses of the Nairovirus
genus are among the largest of the negative-strand RNA viruses and are almost twice the size of those encoded by most viruses of other genera in the Bunyaviridae
family. Sequence comparison of the known RdRp motifs suggests that the additional size of these L proteins is due to the presence of extra N-terminal domains in the nairovirus L proteins. One domain includes an OTU protease. These features have led to the speculation that the large L protein is further cleaved and that the N terminus harbors additional functions not present in other viruses of the family Bunyaviridae
). Recent work from Frías-Stäheli et al. has confirmed that the L-OTU domain is indeed a functional cysteine protease that is capable of deconjugating ubiquitin and ISG15 from cellular proteins (17
). The OTU domain of the positive-stranded RNA arterivirus porcine respiratory and reproductive syndrome virus undergoes cleavage which activates the virus polymerase (17
). In addition, herpes simplex virus type 1 encodes a ubiquitin-specific cysteine protease within the amino terminus of the large (3,161 amino acids) tegument protein. Interestingly, it has been suggested, based on ubiquitin suicide inhibitor probe studies, that the protein may require posttranslational cleavage to generate the active form of the ubiquitin-specific protease (25
). Such observations led to considerable interest in determining whether the CCHFV L protein was proteolytically cleaved and whether this was a result of autocatalytic cleavage activity of the OTU domain.
Western blot analysis of the V5-tagged WT L protein revealed a ~450-kDa band, which corresponds with the predicted size (448 kDa) of the full-length L protein. Smaller proteins sharing the amino terminus V5 epitope were also detected and could be cleavage products. However, these smaller proteins were also detected in cells expressing the mutant L protein lacking L-OTU activity (L-C40A) and therefore do not appear to be the result of OTU protease activity. Some minor N-tagged L protein bands appeared reduced with the mutant L protein lacking OTU activity, but the significance of these and the question of whether these faster-migrating bands represent authentic posttranslational products or an artifact of L overexpression remain an open question.
In the course of establishing the CCHFV minigenome system, we determined that the transfection of minigenome RNA, as opposed to expression from a DNA plasmid, provided the most robust system, with signal-to-noise ratios approaching 5,000-fold above background. It is unclear why such high background signals were seen when plasmid DNA constructs were transfected, but this finding strongly suggests the presence of mammalian cryptic promoter(s). The same V0.0/B plasmid backbone had been used with Rift Valley fever virus reverse genetics experiments without excessive background issues (data not shown), suggesting that the CCHFV-specific inserts were enhancing the cryptic promoter activity in some way. Transfecting in vitro synthesized CCHFV S minigenome RNA instead of plasmid DNA greatly improved the signal-to-noise ratio, similar to results in the previously reported Lassa virus minigenome system (20
We show that CCHFV minigenome replication is inhibited by ribavirin, illustrating that this system can be used for antiviral drug screening. The ribavirin mechanism of action appears complex and varies according to the viruses studied (39
). The first proposed mode of action of this drug is mediated by inhibition of the inositide-5′-monophosphate dehydrogenase, which depletes intracellular GTP levels and interferes with a subset of virus polymerases. The second is through the stimulation of the virus mutation rate, which results in lethal error catastrophe. Finally, direct interaction with virus polymerase (39
) or mimicry of the m7
G cap can also contribute to ribavirin activity (26
). Here, we found inhibition of minigenome replication at a ribavirin dose of approximately 2 μg/ml, with maximal inhibition of reporter activity at 25 μg/ml. This inhibitory effect is in a similar range to that observed with infectious virus assays (38
). For instance, Watts et al. have shown that ribavirin doses as low as 5 μg/ml caused a transient reduction of virus yields, and a rapid and sustained reduction of virus to undetectable levels was seen at doses of 50 μg/ml or greater (43
). Here, we showed that ribavirin preferentially inhibited the synthesis of minigenome reporter protein over cellular RNA polymerase activity (as measured by AP activity), demonstrating that the effect of ribavirin is specific for CCHFV. One of the main advantages of this method over the use of live CCHFV (38
) is that it does not require a BSL-4 laboratory.
The identification of a putative L-OTU protease domain in the L protein N terminus, together with the unusually large size of the protein, had led to earlier speculation that the L protein may undergo autocatalytic processing (21
). However, no evidence of OTU protease domain-specific proteolytic processing of L was detected. The L-RdRp activity of the CCHFV L protein defective in RdRp or OTU protease activity further illustrates the use of this reverse genetics system for CCHFV structure-function studies. In order to address the role of the L-OTU protease activity on the L-RdRp activity, we compared the rate of replication of an L-OTU inactive mutant (L-C40A) to the WT L protein. Interestingly, the inactivation of L-OTU protease yielded comparable RdRp activity to the WT L protein, thus providing direct evidence that the L-OTU activity is dispensable for L-RdRp function. Taken as a whole, our results suggest that the L-segment-encoded protease has a role in another step of the virus life cycle such as immune evasion (17
) or at another uncharacterized step. If the L-OTU activity is not required for virus replication, as suggested by the minigenome data presented here, it may well be possible to engineer a CCHFV mutant with no L-OTU activity once a full CCHFV reverse genetics system capable of generating infectious virus has been developed. Such an OTU-deficient CCHFV would help to precisely clarify the L-OTU function in its most relevant biological context.
The current study lays the foundation for building an infectious clone system. We demonstrated that T7 and murine PolI vectors both yield vRNA templates which are replicated by CCHFV viral proteins or recombinant L protein and nucleoprotein in BSR-T7 cells which stably express T7. This cell line has been successfully used in past reverse genetics systems to rescue RNA viruses, and CCHFV infection of these cells does support growth of virus to decent titers. Efficient rescue systems have been developed for bunyaviruses and arenaviruses by reducing the number of plasmids needed to initiate virus rescue to only the T7-expressed full-length antigenome RNA segments and omitting any support plasmids (containing the individual N and L ORFs) (1
). Apparently, translation of these uncapped virus segment antigenome RNAs is sufficient to launch virus replication. Given the demonstrated efficiency of the CCHFV minigenome system, such an approach should be promising for the future development of an infectious CCHFV rescue system.