The general features of RVFV transcription and replication are similar to those of other negative stranded RNA viruses [35Elliott RM. The Bunyaviridae. New York: Plenum Press 1996.]. During the replication cycle, each segment is transcribed into a mRNA and is replicated through a process which involves the synthesis of the exact copy of the genome, called complementary RNA (cRNA) or antigenome. For Phleboviruses, and RVFV in particular, the cRNA representing the copy of the S ambisense segment serves as template for the synthesis of the NSs mRNA. Since some cRNA is also present in the input virus [33Ikegami T, Won S, Peters CJ, Makino S. Rift Valley fever virus NSs mRNA is transcribed from an incoming anti-viral-sense S RNA segment J Virol 2005; 79: 12106-1.], the virulence factor NSs is expressed immediately after the virus has entered the host cell. Bunyaviral mRNA synthesis in general is initiated through a cap-snatching mechanism by which host mRNAs are cleaved through an endonuclease activity of the L protein [36Patterson JL, Holloway B, Kolakofsky D. La Crosse virions contain a primer-stimulated RNA polymerase and a methylated cap-dependent endonuclease J Virol 1984; 52: 215-2.] to obtain 10 to 18 nucleotide-long primers for transcription [36Patterson JL, Holloway B, Kolakofsky D. La Crosse virions contain a primer-stimulated RNA polymerase and a methylated cap-dependent endonuclease J Virol 1984; 52: 215-2.-40Simons JF, Pettersson RF. Host-derived 5' ends and overlapping complementary 3' ends of the two mRNAs transcribed from the ambisense S segment of Uukuniemi virus J Virol 1991; 65: 4741-8.]. Synthesis of cRNA and vRNA does not require an oligonucleotide primer and is directly initiated with 5’ nucleoside triphosphates. cRNA represents a complete copy of the vRNA, whereas mRNAs terminate before the 5’ end of the template. The mechanism of the switch between transcription and replication remains ill-characterized. Moreover, although RNA-dependent polymerase consensus motifs are present in the RVFV L protein [41Muller R, Poch O, Delarue M, Bishop DH, Bouloy M. Rift Valley fever virus L segment: correction of the sequence and possible functional role of newly identified regions conserved in RNA-dependent polymerases J Gen Virol 1994; 75(Pt 6): 1345-52.], the domains responsible for the different activities have not been defined precisely. However, minigenome systems have been helpful to analyze some steps in RNA synthesis i.e. transcription, replication, transcription termination, and packaging. Minigenomes are similar to viral genome segments but the viral ORF is replaced by a reporter gene. Minigenomes are usually expressed either from T7- or from Pol I-based promoter plasmids, or transcribed in vitro and transfected as RNA. Early on, studies with minigenomes have established that transcription and replication of the bunyaviral RNA requires the N and L proteins [42Jin H, Elliott RM. Expression of functional Bunyamwera virus L protein by recombinant vaccinia viruses J Virol 1991; 65: 4182-9., 43Lopez N, Muller R, Prehaud C, Bouloy M. The L protein of Rift Valley fever virus can rescue viral ribonucleoproteins and transcribe synthetic genome-like RNA molecules J Virol 1995; 69: 3972-9.]. Together with the vRNA, L and N are needed to reconstitute RNPs since, as for all negative stranded genomes, naked vRNA cannot be transcribed. Analysis of the RVFV RNPs showed that the N protein is able to establish intermolecular interactions through several amino-acids located in the N terminal region of the protein which are conserved among phlebovirus N sequences [44Le May N, Gauliard N, Billecocq A, Bouloy M. The N terminus of Rift Valley fever virus nucleoprotein is essential for dimerization J Virol 2005; 79: 11974-80.]. The ability of N to form oligomers may be a conserved feature in bunyaviruses [45Alfadhli A, Love Z, Arvidson B, Seeds J, Willey J, Barklis E. Hantavirus nucleocapsid protein oligomerization J Virol 2001; 75: 2019-3.-48Mir MA, Panganiban AT. Trimeric hantavirus nucleocapsid protein binds specifically to the viral RNA panhandle J Virol 2004; 78: 8281-.]. The L protein, probably in association with N, is able to perform both transcription and replication, excluding the possibility that the L protein had to be modified by a third viral factor to function as a replicase. For RVFV, the viral protein NSs was reported to promote viral RNA replication and transcription in a minigenome system [49Ikegami T, Peters CJ, Makino S. Rift valley fever virus nonstructural protein NSs promotes viral RNA replication and transcription in a minigenome system J Virol 2005; 79: 5606-15.]. However, these experiments were performed in human 293T cells expressing T7 polymerase. RNAs produced by the T7 polymerase contain 5’ triphosphate ends which are strong inducers of the antiviral type I interferon (IFN) response [50Hornung V, Ellegast J, Kim S, et al. 5'-Triphosphate RNA Is the Ligand for RIG-I Science 2006; 314: 994-7., 51Pichlmair A, Schulz O, Tan CP, et al. RIG-I-mediated antiviral responses to single-stranded RNA bearing 5'-phosphates Science 2006; 314: 997-1001., 51aPlumet S, Herschke F, Bourhis JM, Valentin H, Longhi S, Gerlier D. Cytosolic 5'-triphosphate ended viral leader transcript of measles virus as activator of the RIG I-mediated interferon response PLoS Onel 2007; 2(3): e279.]. 293T cells, like most other cells, are capable to respond to 5’triphosphate RNAs, whereas the BHK-derived cells used by most researchers to express T7 polymerase are unresponsive due to a defect in the RIG-I pattern recognition pathway [52Habjan M, Penski N, Spiegel M, Weber F. T7 RNA polymerase-dependent and -independent systems for cDNA-based rescue of Rift Valley fever virus J Gen Virol 2008; 89: 2157-66.]. Moreover, the stimulatory effect of NSs was not confirmed in BHK cells (MB, unpublished data) but rather inhibited minireplicon activity in a manner similar to the NSs of the related viruses Bunyamwera [53Weber F, Dunn EF, Bridgen A, Elliott RM. The Bunyamwera virus nonstructural protein NSs inhibits viral RNA synthesis in a minireplicon system Virology 2001; 281: 67-74.] and La Crosse [54Blakqori G, Kochs G, Haller O, Weber F. Functional L polymerase of La Crosse virus allows in vivo reconstitution of recombinant nucleocapsids J Gen Virol 2003; 84(Pt 5): 1207-4.]. In line with this, when infectious RVFV particles were produced by T7-based systems in BHK-derived cells, RVFV NSs was shown to be either inhibitory [55Ikegami T, Won S, Peters CJ, Makino S. Rescue of infectious rift valley fever virus entirely from cDNA, analysis of virus lacking the NSs gene, and expression of a foreign gene J Virol 2006; 80: 2933-40.], or irrelevant for generating recombinant viruses [52Habjan M, Penski N, Spiegel M, Weber F. T7 RNA polymerase-dependent and -independent systems for cDNA-based rescue of Rift Valley fever virus J Gen Virol 2008; 89: 2157-66., 56Billecocq A, Gauliard N, Le May N, Elliott RM, Flick R, Bouloy M. RNA polymerase I-mediated expression of viral RNA for the rescue of infectious virulent and avirulent Rift Valley fever viruses Virology 2008; 378: 377-84.]. It is thus conceivable that the observed stimulatory effect of RVFV NSs is indirect, namely through the well-established ability to suppress the antiviral IFN response (see below).

When compared to each other, the L, M and S segment-based minigenomes do not express identical levels of reporter gene. This indicates differential promoter activities associated with the UTRs [57Gauliard N, Billecocq A, Flick R, Bouloy M. Rift Valley fever virus noncoding regions of L, M and S segments regulate RNA synthesis Virology 2006; 351: 170-9.]. Although the mutagenesis analysis was not as extensive as the one carried out for Uukuniemi virus [58Flick R, Elgh F, Pettersson RF. Mutational analysis of the Uukuniemi virus (Bunyaviridae family) promoter reveals two elements of functional importance J Virol 2002; 76: 10849-60.], it appeared that some of the conserved UTR nucleotides play an important role in promoter activity and the regulation of gene expression [59Prehaud C, Lopez N, Blok MJ, Obry V, Bouloy M. Analysis of the 3' terminal sequence recognized by the Rift Valley fever virus transcription complex in its ambisense S segment Virology 1997; 227: 189-97.].

Bunyavirus mRNAs (except for Sin Nombre hantavirus [60Hutchinson KL, Peters CJ, Nichol ST. Sin Nombre virus mRNA synthesis Virology 1996; 224: 139-49.]) are not polyadenylated. They have a shortened 3’ end relative to the full-length cRNA, suggesting that a specific termination signal is recognized during transcription. Preliminary data had indicated that a conserved motif is present in the intergenic region of the S segment of the phleboviruses Toscana (TOSV), Sandfly fever Sicilian (SFSV), and RVFV [32Giorgi C, Accardi L, Nicoletti L, et al. Sequences and coding strategies of the S RNAs of Toscana and Rift Valley fever viruses compared to those of Punta Toro, Sicilian Sandfly fever, and Uukuniemi viruses Virology 1991; 180: 738-53., 38Gro MC, Di Bonito P, Accardi L, Giorgi C. Analysis of 3' and 5' ends of N and NSs messenger RNAs of Toscana Phlebovirus Virology 1992; 191: 435-8.]. Albarino et al. [61Albarino CG, Bird BH, Nichol ST. A shared transcription termination signal on negative and ambisense RNA genome segments of rift valley Fever, sandfly Fever sicilian, and toscana viruses J Virol 2007; 81: 5246-6.] mapped precisely the 3’ ends of the L, M, N and NSs mRNAs of TOSV, SFSV and RVFV, and identified a sequence motif 3’-C_1-3GUCG/A-5’ which is conserved on the M and S segments of phleboviruses. The phleboviral L segment mRNA, by contrast, was found to have a full-length 3’ end. Ikegami et al. [62Ikegami T, Won S, Peters CJ, Makino S. Characterization of rift valley fever virus transcriptional terminations J Virol 2007; 81: 8421-38.] came to similar conclusions for the termination of the M and S segment mRNAs of RVFV, but claimed that the L mRNA lacks the last 16-41 nucleotides when compared to the full-length cRNA copy, and that the termination signal corresponds to two 13-nucleotide-long complementary sequences present in the 5’ non-coding region of the L genomic segment.

The RVFV envelope glycoproteins mediate particle entry into cells through receptors which, like for most bunyaviruses, remain to be identified. RVFV entry is predicted to employ a class II fusion mechanism that is activated by low pH following endocytosis of the virion [63Filone CM, Heise M, Doms RW, Bertolotti-Ciarlet A. Development and characterization of a Rift Valley fever virus cell-cell fusion assay using alphavirus replicon vectors Virology 2006; 356: 155-64.]. The RVFV M segment encodes Gn (encoded by amino-terminal sequences) and Gc (encoded by carboxy-terminal sequences) as well as two nonstructural proteins NSm1 (78kDa) and NSm2 (14kDa). The NSm1 and NSm2 proteins are produced by alternative use of the first and second of the 5 in-frame AUG codons, respectively, which are present at the 5’ end of the M mRNA i.e. upstream of the Gn sequence (Fig. 1B). The role of these proteins will be discussed further below.

Like all bunyaviruses, RVFV particles usually bud into the lumen of the Golgi apparatus [35Elliott RM. The Bunyaviridae. New York: Plenum Press 1996.]. Budding at the plasma membrane was however observed in rat hepatocytes [64Anderson GW Jr, Smith JF. Immunoelectron microscopy of Rift Valley fever viral morphogenesis in primary rat hepatocytes Virology 1987; 161: 91-100.]. Both RVFV Gn and Gc localize to the Golgi apparatus when expressed together from polyprotein-expressing plasmids [65Wasmoen TL, Kakach LT, Collett MS. Rift Valley fever virus M segment: cellular localization of M segment-encoded proteins Virology 1988; 166: 275-80.]. When expressed alone, Gn could be transported to the Golgi apparatus due to a specific Golgi localization motif, whereas Gc localized to the endoplasmic reticulum (ER) due to the presence of a specific ER retention signal [66Gerrard SR, Nichol ST. Characterization of the Golgi retention motif of Rift Valley fever virus G(N) glycoprotein J Virol 2002; 76: 12200-0.]. This suggests that, similar to other bunyaviruses [67Shi X, Elliott RM. Golgi localization of Hantaan virus glycoproteins requires coexpression of G1 and G2 Virology 2002; 300: 31-8., 68Shi X, Lappin DF, Elliott RM. Mapping the Golgi targeting and retention signal of Bunyamwera virus glycoproteins J Virol 2004; 78: 10793-802.], Gc moves to the Golgi apparatus via its physical association with Gn.

The minigenome systems expressing a reporter gene were also useful to investigate viral packaging activity. In that case, the glycoproteins were co-expressed with the transcription machinery to allow formation of viral like particles (VLPs) released into the medium [69Habjan M, Penski N, Wagner V, et al. Efficient production of Rift Valley fever virus-like particles: The antiviral protein MxA can inhibit primary transcription of bunyaviruses Virology 2009; 385: 400-8.]. When observed by electron microscopy and negative staining, these particles resembled RVFV particles in size and morphology. They were able to infect naïve cells and to undergo the first step of the replication cycle, i. e. primary transcription. If both L and N were provided in trans, replication of minigenomes also occured [69Habjan M, Penski N, Wagner V, et al. Efficient production of Rift Valley fever virus-like particles: The antiviral protein MxA can inhibit primary transcription of bunyaviruses Virology 2009; 385: 400-8.]. Using these VLPs, it was shown that the interferon-induced antiviral protein MxA inhibits both primary and secondary transcription of RVFV. Interestingly, similar RVFV VLPs were produced in insect cells infected with a dual baculovirus vector expressing Gn/Gc and N [70Liu L, Celma CC, Roy P. Rift Valley fever virus structural proteins: expression, characterization and assembly of recombinant proteins Virol J 2008; 5: 82.]. Another vector expressing N and Gc only resulted in particles which were more pleiomorphic than full-fledged VLPs and RVFV particles, suggesting that both Gn and Gc contribute to the assembly process and likely interact with N.

Reverse genetics systems allow to freely manipulate the viral genome and to dissect individual steps in the viral multiplication cycle. The minireplicon and VLP systems mentioned above were proven useful to study primary transcription, cell-autonomous antiviral responses, genome replication, and RNP packaging [49Ikegami T, Peters CJ, Makino S. Rift valley fever virus nonstructural protein NSs promotes viral RNA replication and transcription in a minigenome system J Virol 2005; 79: 5606-15., 57Gauliard N, Billecocq A, Flick R, Bouloy M. Rift Valley fever virus noncoding regions of L, M and S segments regulate RNA synthesis Virology 2006; 351: 170-9., 69Habjan M, Penski N, Wagner V, et al. Efficient production of Rift Valley fever virus-like particles: The antiviral protein MxA can inhibit primary transcription of bunyaviruses Virology 2009; 385: 400-8.]. Fully infectious, recombinant virus particles can be recovered from cloned cDNA plasmids if the minireplicon is replaced by constructs for the full-length viral RNA segments. This technique was first established for Bunyamwera virus, the prototype of the Bunyaviridae [71Bridgen A, Elliott RM. Rescue of a segmented negative-strand RNA virus entirely from cloned complementary DNAs Proc Natl Acad Sci USA 1996; 93: 15400-4., 72Lowen AC, Noonan C, McLees A, Elliott RM. Efficient bunyavirus rescue from cloned cDNA Virology 2004; 330: 493-500.], followed by the related La Crosse virus [73Blakqori G, Weber F. Efficient cDNA-based rescue of La Crosse bunyaviruses expressing or lacking the nonstructural protein NSs J Virol 2005; 79: 10420-8.]. These rescue systems were based on T7-transgenic cells to express the constructs for the viral genome segments and the helper proteins N and L. T7 systems were also used to recover recombinant RVFV [15Gerrard SR, Bird BH, Albarino CG, Nichol ST. The NSm proteins of Rift Valley fever virus are dispensable for maturation, replication and infection Virology 2007; 359: 459-65., 55Ikegami T, Won S, Peters CJ, Makino S. Rescue of infectious rift valley fever virus entirely from cDNA, analysis of virus lacking the NSs gene, and expression of a foreign gene J Virol 2006; 80: 2933-40.], but promoters for the cellular pol I could also be used to express the genome segments [52Habjan M, Penski N, Spiegel M, Weber F. T7 RNA polymerase-dependent and -independent systems for cDNA-based rescue of Rift Valley fever virus J Gen Virol 2008; 89: 2157-66., 56Billecocq A, Gauliard N, Le May N, Elliott RM, Flick R, Bouloy M. RNA polymerase I-mediated expression of viral RNA for the rescue of infectious virulent and avirulent Rift Valley fever viruses Virology 2008; 378: 377-84.]. This indicates that RVFV, a cytoplasmic virus, does not contain critical sequences e.g. cryptic splice sites, which could interfere with nuclear transcription of the genome. In fact, T7- and Pol I/Pol II-driven systems were shown to be of similar efficiency [52Habjan M, Penski N, Spiegel M, Weber F. T7 RNA polymerase-dependent and -independent systems for cDNA-based rescue of Rift Valley fever virus J Gen Virol 2008; 89: 2157-66.]. It should be noted that the Pol I system requires co-transfection of the N and L helper plasmids, whereas in the T7 system helper plasmids are either not necessary or even inhibitory. However, in T7-driven systems the T7 polymerase has to be provided in trans, usually by stably expressing cell lines. As outlined above, the cell lines used for T7 expression [55Ikegami T, Won S, Peters CJ, Makino S. Rescue of infectious rift valley fever virus entirely from cDNA, analysis of virus lacking the NSs gene, and expression of a foreign gene J Virol 2006; 80: 2933-40., 74Buchholz UJ, Finke S, Conzelmann KK. Generation of bovine respiratory syncytial virus (BRSV) from cDNA: BRSV NS2 is not essential for virus replication in tissue culture, and the human RSV leader region acts as a functional BRSV genome promoter J Virol 1999; 73: 251-9.] are deficient in the RIG-I pathway [52Habjan M, Penski N, Spiegel M, Weber F. T7 RNA polymerase-dependent and -independent systems for cDNA-based rescue of Rift Valley fever virus J Gen Virol 2008; 89: 2157-66.] which would normally respond to the 5’ triphosphorylated T7 transcripts with the production of the antiviral IFNs.

Depending on the laboratory which developed the rescue system, the genetic backbone of the recombinant virus was based either on the attenuated strain MP12 [55Ikegami T, Won S, Peters CJ, Makino S. Rescue of infectious rift valley fever virus entirely from cDNA, analysis of virus lacking the NSs gene, and expression of a foreign gene J Virol 2006; 80: 2933-40., 56Billecocq A, Gauliard N, Le May N, Elliott RM, Flick R, Bouloy M. RNA polymerase I-mediated expression of viral RNA for the rescue of infectious virulent and avirulent Rift Valley fever viruses Virology 2008; 378: 377-84.], or the virulent strains ZH501 [15Gerrard SR, Bird BH, Albarino CG, Nichol ST. The NSm proteins of Rift Valley fever virus are dispensable for maturation, replication and infection Virology 2007; 359: 459-65.] or ZH548 [52Habjan M, Penski N, Spiegel M, Weber F. T7 RNA polymerase-dependent and -independent systems for cDNA-based rescue of Rift Valley fever virus J Gen Virol 2008; 89: 2157-66., 56Billecocq A, Gauliard N, Le May N, Elliott RM, Flick R, Bouloy M. RNA polymerase I-mediated expression of viral RNA for the rescue of infectious virulent and avirulent Rift Valley fever viruses Virology 2008; 378: 377-84.].

NSs of RVFV forms a ribbon-like filament in the nucleus [12Struthers JK, Swanepoel R, Shepherd SP. Protein synthesis in Rift Valley fever virus-infected cells Virology 1984; 134: 118-24.] (Fig. 2). This feature is unexpected for a virus replicating in the cytoplasm. Moreover, it is particular to RVFV and not shared with NSs proteins of other bunyaviruses. NSs was characterized as a major virulence factor counteracting the antiviral IFN system [16Vialat P, Billecocq A, Kohl A, Bouloy M. The S segment of rift valley fever phlebovirus (Bunyaviridae) carries determinants for attenuation and virulence in mice J Virol 2000; 74: 1538-43., 75Billecocq A, Spiegel M, Vialat P, et al. NSs protein of Rift Valley Fever Virus blocks interferon production by inhibiting host gene transcription J Virol 2004; 78: 9798-806., 76Bouloy M, Janzen C, Vialat P, et al. Genetic evidence for an interferon-antagonistic function of rift valley fever virus nonstructural protein NSs J Virol 2001; 75: 1371-7.]. It has become clear meanwhile that NSs is a multifunctional protein inhibiting cellular transcription in general and suppressing two different arms of the IFN response, namely (i) IFN induction by a specific, early, mechanism (ii) the antiviral protein PKR.

With respect to specific suppression of IFN induction, the cellular protein SAP30 was identified as interacting with NSs. SAP30 belongs to the Sin3A/NCoR/HDAC repressor complexes intervening in gene transcription regulation [77Le May N, Mansuroglu Z, Leger P, et al. A SAP30 complex inhibits IFN-beta expression in Rift Valley fever virus infected cells PLoS Pathog 2008; 4: e13.]. Moreover, it is known that SAP30 interacts directly with YY1 [78Huang NE, Lin CH, Lin YS, Yu WC. Modulation of YY1 activity by SAP30 Biochem Biophys Res Commun 2003; 306: 267-75.], a transcription factor involved in the regulation of expression of numerous genes, including IFN-β [79Weill L, Shestakova E, Bonnefoy E. Transcription factor YY1 binds to the murine beta interferon promoter and regulates its transcriptional capacity with a dual activator/repressor role J Virol 2003; 77: 2903-14.]. Through a series of co-immunoprecipitation, confocal microscopy and chromatin immunoprecipitations, it was demonstrated that NSs, SAP30, YY1 and Sin3A-associated corepressors are recruited on the IFN-β promoter inhibiting CBP recruitment, histone acetylation and transcriptional activation [77Le May N, Mansuroglu Z, Leger P, et al. A SAP30 complex inhibits IFN-beta expression in Rift Valley fever virus infected cells PLoS Pathog 2008; 4: e13.]. Using reverse genetics, a recombinant ZH548 in which NSs was deleted of the SAP30 interaction domain was produced. In contrast with the wt virus, this mutant, ZH548-NSs∆210-230, induced IFN-β expression and was unable to kill mice. Although this mutant protein was still located in the nucleus, it was not able to form filaments. Further analyses on the wt NSs filament indicated that, even though cellular DNA is mostly excluded from the filament, heterochromatin clusters of pericentromeric gamma satellite sequences appeared intimately associated with NSs [79aMansuroglu Z, Josse T, Gilleron J, et al. Nonstructural NSs protein of rift valley fever virus interacts with pericentromeric DNA sequences of the host cell, inducing chromosome cohesion and segregation defects J Virol 2010; 84: 928-39.]. This results in a high incidence of nuclear anomalies, translating chromosome cohesion and segregation defects which could contribute to RVFV pathology.

NSs also suppresses cellular gene expression by a more general mechanism which relies on the interaction with the host cell protein p44. p44 is a subunit of the TFIIH basal transcription factor and becomes sequestered into the NSs filamentous structure characteristic of RVFV infection [80Le May N, Dubaele S, De Santis LP, Billecocq A, Bouloy M, Egly JM. TFIIH transcription factor, a target for the Rift Valley hemorrhagic fever virus Cell 2004; 116: 541-0.]. As a consequence, TFIIH cannot assemble and its concentration drops rapidly, explaining the drastically reduced transcriptional activity of cells infected with RVFV. Interestingly, NSs thus functions as a general inhibitor of Pol II as well as Pol I, since TFIIH is involved in transcription by both these host polymerases. Inhibition of TFIIH and hence general transcription is a relatively late event occurring after 8 h of infection, whereas the specific inhibition of IFN-β transcription (mediated by SAP30 recruitment) is in place as early as 3-4 h after infection.

Very recently, a novel function of NSs was described, the targeting of the antiviral, IFN-induced protein kinase PKR [81Habjan M, Pichlmair A, Elliott RM, et al. NSs protein of rift valley fever virus induces the specific degradation of the double-stranded RNA-dependent protein kinase J Virol 2009; 83: 4365-75., 82Ikegami T, Narayanan K, Won S, Kamitani W, Peters CJ, Makino S. Rift Valley fever virus NSs protein promotes post-transcriptional downregulation of protein kinase PKR and inhibits eIF2alpha phosphorylation PLoS Pathog 2009; 5: e1000287.]. PKR is a serine-threonine kinase activated by viral RNAs and mediating a stop of translation [83Sadler AJ, Williams BR. Interferon-inducible antiviral effectors Nat Rev Immunol 2008; 8: 559-68.]. NSs-dependent degradation is specific for PKR and occurs through the proteasome. Hence, cells infected with RVFV are devoid of PKR, whereas PKR was clearly detected and activated by infection with NSs-deleted virus strains e.g. Clone 13. This anti-PKR activity is important for viral replication and pathogenesis, since mice expressing PKR clear infection by Clone 13, whereas knockout mice lacking PKR succumb to Clone 13 and develop high virus titers in the liver [81Habjan M, Pichlmair A, Elliott RM, et al. NSs protein of rift valley fever virus induces the specific degradation of the double-stranded RNA-dependent protein kinase J Virol 2009; 83: 4365-75.]. The fact that NSs itself provokes a PKR-knockout-like situation is likely to contribute to the pathogenicity of wt RVFV. Interestingly, it was also observed that this activity is specific to RVFV NSs and not shared with other Phleboviruses like Sandfly fever Sicilian virus or the orthobunyavirus La Crosse [81Habjan M, Pichlmair A, Elliott RM, et al. NSs protein of rift valley fever virus induces the specific degradation of the double-stranded RNA-dependent protein kinase J Virol 2009; 83: 4365-75.].

Altogether, it appears that NSs has multiple functions to counteract the IFN system, either at the transcriptional level or at the translational level by degrading PKR.

The biological function of the NSm1 and NSm2 proteins was addressed by producing RVFV mutants with either a deletion in the pre-Gn region [15Gerrard SR, Bird BH, Albarino CG, Nichol ST. The NSm proteins of Rift Valley fever virus are dispensable for maturation, replication and infection Virology 2007; 359: 459-65., 84Won S, Ikegami T, Peters CJ, Makino S. NSm protein of Rift Valley fever virus suppresses virus-induced apoptosis J Virol 2007; 81: 13335-45.] or point mutations in the start codons of the two respective reading frames [17Won S, Ikegami T, Peters CJ, Makino S. NSm and 78-kilodalton proteins of Rift Valley fever virus are nonessential for viral replication in cell culture J Virol 2006; 80: 8274-.]. In all cases, virus growth in cell culture was similar to the parental viruses. However, when the pre-Gn deletion was introduced into the MP12 strain, plaques were larger than for the parental MP12 strain and a more extensive apoptosis due to elevated caspase activity was observed [84Won S, Ikegami T, Peters CJ, Makino S. NSm protein of Rift Valley fever virus suppresses virus-induced apoptosis J Virol 2007; 81: 13335-45.]. Curiously, a similar deletion introduced into the virulent strain ZH501 did not show any differences in plaque size, and an increase in apoptosis was not reported [15Gerrard SR, Bird BH, Albarino CG, Nichol ST. The NSm proteins of Rift Valley fever virus are dispensable for maturation, replication and infection Virology 2007; 359: 459-65.]. Moreover, if the AUG-inactivating point mutations in the pre-Gn region, which abrogated the NSm1 and NSm2 expression, were introduced into the MP12 background, they also led to a plaque phenotype indistinguishable from the parental virus [17Won S, Ikegami T, Peters CJ, Makino S. NSm and 78-kilodalton proteins of Rift Valley fever virus are nonessential for viral replication in cell culture J Virol 2006; 80: 8274-.]. The differences between the pre-Gn deletion and the AUG point mutant of MP12 can be explained by residual expression of a truncated version of the NSm1 protein, the 73-kDa protein [84Won S, Ikegami T, Peters CJ, Makino S. NSm protein of Rift Valley fever virus suppresses virus-induced apoptosis J Virol 2007; 81: 13335-45.]. Why this is not happening for ZH501 remains to be investigated. In any case, all available data are congruent in indicating that the NSm1 and NSm2 proteins of RVFV are dispensable for virus growth in cell culture.