- Open Access
Rift Valley fever virus NSs protein functions and the similarity to other bunyavirus NSs proteins
Virology Journalvolume 13, Article number: 118 (2016)
Rift Valley fever is a mosquito-borne zoonotic disease that affects both ruminants and humans. The nonstructural (NS) protein, which is a major virulence factor for Rift Valley fever virus (RVFV), is encoded on the S-segment. Through the cullin 1-Skp1-Fbox E3 ligase complex, the NSs protein promotes the degradation of at least two host proteins, the TFIIH p62 and the PKR proteins. NSs protein bridges the Fbox protein with subsequent substrates, and facilitates the transfer of ubiquitin. The SAP30-YY1 complex also bridges the NSs protein with chromatin DNA, affecting cohesion and segregation of chromatin DNA as well as the activation of interferon-β promoter. The presence of NSs filaments in the nucleus induces DNA damage responses and causes cell-cycle arrest, p53 activation, and apoptosis. Despite the fact that NSs proteins have poor amino acid similarity among bunyaviruses, the strategy utilized to hijack host cells are similar. This review will provide and summarize an update of recent findings pertaining to the biological functions of the NSs protein of RVFV as well as the differences from those of other bunyaviruses.
Rift Valley fever (RVF) is a zoonotic viral disease transmitted by mosquitoes. It was first identified in Kenya in 1930 , and has been endemic in sub-Saharan Africa for more than 80 years . Outbreaks of RVF have also occurred in Madagascar, Egypt, Saudi Arabia, and Yemen, probably through infected animals or mosquitoes [2–6]. The largest recorded RVF outbreak occurred in Egypt back in 1977–78, where there were an estimate of 20,000 to 200,000 human cases and roughly 600 confirmed deaths . The disease is caused by the Rift Valley fever virus (RVFV), which belongs to the genus Phlebovirus within the family Bunyaviridae . Pregnant cattle, goats, and especially sheep, are highly susceptible to RVFV, resulting in fetal malformation (e.g., hydrop amnii, arthrogryposis, scoliosis, hydraencephaly, cerebellar hypoplasia) and abrupt abortion in 40 to 100 % of pregnant ewes [2, 8]. Newborn lambs are also highly susceptible to RVFV, and the mortality rate is estimated to be 95 to 100 % . In adult ruminants, transient fever and viremia are generally displayed and the mortality rate of infected sheep has been documented to be up to 20 % . Most human cases are characterized with self-limiting febrile illness, and less than 8 % of patients develop a more severe form of the disease, such as hemorrhagic fever, encephalitis, or retinitis. The overall mortality rate for RVF patients is 0.5 to 1 % [10–12]. Through vertical transmission, flood water Aedes mosquitoes play an important role in the maintenance of RVFV in nature [13, 14]. Periods of heavy rainfall generates ideal conditions for the hatching of infected Aedes eggs , thus triggering the transmission of RVFV to animals. Transmission of the disease to humans (e.g., veterinarian, farmers, or abattoir workers) can then be accomplished through direct contact with bodily fluids derived from infected animals. However, during an outbreak, infected mosquito vectors, such as Culex pipiens, could also play an important role in RVFV transmission to humans [14, 15]. Potential spread of RVFV, either naturally or intentionally, into non-endemic countries is a major public health concern. Due to a concern towards bioterrorism in the U.S., RVFV is classified as Category A Priority Pathogen (NIAID/NIH), and an overlap select agent (U.S. Department of Human Health Services, and U.S. Department of Agriculture). A strict quarantine system is important to prevent the importation of RVFV infected animals . However, the case is not the same for humans, because infected patients can travel by airplanes, and may bring RVFV from endemic countries into non-endemic area . Fortunately, human to human transmission has not been documented and uninfected individuals are unlikely able to trigger RVF outbreaks. Currently, there are no licensed RVF vaccines for humans, and only a few licensed vaccines are available for animals , such as the live-attenuated MP-12 vaccine, which is conditionally licensed in the U.S. . No effective therapeutic is available for RVF patients. Reduction of viral load with increased survival rate was shown in mice or nonhuman primates infected with RVFV through daily post-exposure treatment with ribavirin (a guanosine analog), Poly (ICLC) (a polyriboinosinic-polyribocytidylic acid stabilized with poly-L-lysine and carboxymethyl cellulose), recombinant IFN-αA or human IFN-α [20–25]. However, both ribavirin and IFN-α have side effects associated with therapeutic use . Improvement of antivirals that is not only effective for RVFV, but for other pathogenic bunyaviruses, could be important countermeasures against the threat that bunyaviruses impose. Currently, the challenge lies within the development of broadly-active antivirals or vaccines that can encompass the large genetic diversity of bunyaviruses.
The bunyavirus genome is comprised of three negative-sense single stranded RNA segments; Large (L)-, Medium (M)-, and Small (S)-segments. The L-segment encodes the RNA-dependent RNA polymerase (L protein). The M-segment encodes a single open reading frame (ORF) for glycoprotein precursor gene that produces the envelope glycoproteins; Gn and Gc. The S-segment encodes for the nucleoprotein (N protein). A nonstructural protein, NSm, is encoded in the M-segment of orthobunyaviruses, nairoviruses, tospoviruses, or sandfly or mosquito-borne phleboviruses, while NSs protein is encoded in the S-segment of orthobunyaviruses, phlebovirues, nairovirus, or hantaviruses that are transmitted by Arvicolinae or Sigmodontinae rodents, but not by murinae rodents [7, 27–31].
Termini on the viral genome RNA are complementary and forms a pan-handle structure, which serves as signals for N protein encapsidation and RNA synthesis . Bunyavirus L proteins cleave capped host mRNA to initiate the synthesis of viral mRNA [32–35]. This cap-snatching mechanism is also commonly used in other segmented negative-stranded RNA viruses (e.g., influenza viruses, and arenaviruses). The transcription of bunyavirus mRNA is terminated within the untranslated region (UTR) [36–40], while L protein do not recognize the termination signal during the mode of viral genomic RNA synthesis. The mechanism of transcription and genome replication, however, remains largely unknown.
RVFV M-segment encodes two precursor proteins, 78kD-Gc and NSm-Gn-Gc, for the expression of 78kD, NSm, Gn, and Gc proteins through co-translational cleavage of precursor protein via signal peptidases . The 78kD protein is a structural protein that is incorporated into virions matured from mosquito cells, but not those from Vero cells . The 78kD protein is important for efficient viral dissemination in mosquitoes [43–45], while the NSm protein localizes to the mitochondrial outer membrane, and delays apoptosis in mammalian cells [46, 47].
Genetic diversity among bunyaviruses
The Bunyaviridae family consists of more than 350 distinct viruses, and belongs to the genus Orthobunyavirus, Phlebovirus, Nairovirus, Hantavirus, and Tospovirus . Phleboviruses consists of at least 70 named viruses, and is divided into two antigenic groups; the sandfly fever group, and the Uukuniemi group, which is based on the presence of the preglycoprotein coding region in the M-segment . Traditionally phlebovirus species were defined by the distinct antigenic cross-reactivity, however, some phleboviruses do not form clear plaques in mammalian cells, and the antigenic cross-reactivity has not been well characterized. A number of viruses have not been approved as phlebovirus species partly due to such technical inconvenience. Phleboviruses are currently comprised of the following genetically distinct groups (Fig. 1); (i) sandfly or mosquito-borne phleboviruses; Rift Valley fever group, Sandfly fever Naples group (e.g., Toscana virus: TOSV), Sandfly fever Sicilian group (e.g., Sandfly fever Sicilian virus: SFSV), Joa group (e.g., Frijoles virus: FRIV), Salobo group (e.g., Icoaraci virus: ICOV, Belterra virus), Candiru group (e.g., Candiru virus, Alenquer virus, Itaituba virus), Punta Toro group (e.g., Punta Toro virus: PTV), Salehabad group (e.g., Salehabad virus, Arumowot virus), and Aguacate group (e.g., Aguacate virus), (ii) tick-borne phleboviruses; Uukuniemi group (e.g., Uukuniemi virus: UUKV), Bhanja group (e.g., Bhanja virus, Kismayo virus), Kaisodi group (e.g., Kaisodi virus, Lanjan virus), and Severe Fever with Thrombocytopenia Syndrome (SFTS) and Heartland group (e.g., SFTSV, Heartland virus). Sandfly fever is characterized by a self-limiting febrile illness in humans, whereas TOSV is known to cause aseptic meningitis or meningoencephalitis [49, 50]. Most sandfly fever viruses are transmitted by sandfly species within genus Phlebotomus (old world sandflies: Sandfly fever Naples group, Sandfly fever Sicilian group, and Salehabad group) or genus Lutzomya (new world sandflies: Punta Toro group, Joa group, Salobo group, Aguacate group, Joa group, and Candiru group). On the other hand, RVFV, ICOV, Arumowot virus, and Itaporanga virus (this virus still remains to be grouped) transmitted by mosquitoes . SFTSV is transmitted by ticks (Haemaphysalis longicornis or Rhipicephalus microplus) in China, and the disease is characterized by an acute onset of fever, accompanied by thrombocytopenia, leukopenia, and diarrhea in humans, with the mortality rate of 12 to 30 % [52, 53]. Patients with SFTSV were also identified in Korea and Japan [52–55]. Heartland virus is transmitted by lone star ticks (Amblymma americanum) in the U.S., and the disease is characterized with the onset of fevers with thrombocytopenia and leukopenia .
The NSs protein is considered to be the major virulence factor for bunyaviruses, and plays an important role in viral evasion from the host innate immunity. It is important to characterize common biological functions of bunyavirus NSs proteins toward the development of effective therapeutic regimens and vaccines. However, NSs proteins exhibit a wide diversity of amino acid sequences among sandfly fever phleboviruses [57, 58]. For example, the amino acid identities of NSs proteins among RVFV, SFSV, TOSV, PTV, ICOV, and FRIV are low, and range from 7.5 to 28.6 % . Although phleboviruses are genetically diverse, identification of common mechanisms utilized by phlebovirus NSs proteins may lead to the development of broadly-active antiviral molecules. We will describe the current understanding of RVFV NSs functions, as well as discuss the similarities and differences observed in NSs proteins of other bunyaviruses in following sections.
Inhibition of host general transcription by bunyavirus NSs proteins
RVFV NSs proteins are present in both the nucleus and cytoplasm of infected cells, and it forms a filamentous structure in nucleus . The NSs protein of RVFV is comprised of 265 amino acids (aa.) and is estimated to be 31kD in size . The 17aa. at the C-terminus is highly acidic, and is responsible for the formation of the filamentous structure seen in the nucleus . Currently, no nuclear localization signal for RVFV NSs protein has been identified, however, nuclear accumulation of RVFV NSs protein has been shown to be dependent on PXXP motifs (P: proline, X: any amino acid) . RVFV NSs protein also encodes the FVEV (ΩXaV motif: Ω, omega: Trp or Phe, X: any amino acid, a: Asp or Glu, V: Val) within the last 17 amino acids of the C-terminus. Recombinant RVFV encoding NSs lacking the ΩXaV motif, failed to form NSs filaments in the nucleus, indicating the role of ΩXaV motif in filament formation .
Le May et al. demonstrated that RVFV NSs protein inhibits cellular general transcription activity by directly interacting with transcription factor (TF) IIH components . The TFIIH is comprised of ten subunits; i.e., (i) the core complex: XPB, XPD, p52, p44, p62, p34, and p8, and (ii) the cyclin activating kinase sub-complexes: cdk7, cyclin H, and MAT1. As one of the basal transcription factors, TFIIH along with TFIIA, TFIIB, TFIID, TFIIE, and TFIIF, forms the transcription preinitiation complex for RNA polymerase II. TFIIH is involved in promoter melting and allowing Pol-II to escape from the promoter to transition from the initiation complex to the elongation complex via cyclin-dependent phosphorylation of the Pol-II large subunit C-terminal domain (CTD). TFIIH is also required for RNA polymerase I-mediated transcription . Synthesis of both poly-A(+) and poly-A(−) RNA was decreased in the presence of RVFV NSs protein. The XPB and p44 subunit was shown to co-localize with RVFV NSs filaments, unlike p62, p52, cdk7, and XPD, which did not. It was also observed that XPD and p62 proteins were decreased in the presence of NSs proteins, whereas the p44, XPB, and TBP (TATA-binding protein) did not. NSs proteins bound to p44, but not directly to XPB. The NSs protein did not interact with p44 subunits that had been already incorporated into the TFIIH complex. From what had been gathered, it was hypothesized that RVFV NSs protein competes for the binding between XPD and p44, thus preventing the assembly of the TFIIH complex in the nucleus.
In later studies, Kalveram et al. demonstrated that RVFV NSs protein interacts with TFIIH p62, and subsequently promotes its post-translational degradation . The p62 protein was stabilized in the presence of proteasome inhibitors, MG132 or lactacystin, and degradation occurred even in the presence of a nuclear export inhibitor, leptomycin B, or a lysosomal inhibitor, chloroquine. These studies demonstrated that RVFV NSs protein induces host transcriptional shutoff, by two potentially redundant mechanisms: (1) interruption of TFIIH complex assembly by NSs-p44 binding, and (2) post-translational degradation of TFIIH p62 subunit.
Cellular protein degradation occurs mainly through the ubiquitin (Ub)-proteasome system, where polyubiquitination of proteins with chains of at least four Lys 48-linked Ubs leads to degradation through the 26S proteasome [65, 66]. To ubiquitinate proteins, three sequential steps are necessary; (i) Ub-binding to E1 (the Ub activating enzyme), (ii) transfer of Ub to E2 (the Ub conjugating enzyme) and (iii) transfer of Ub from E2 to a substrate protein in the E3 ligase complex [66–68]. To promote the transfer of Ub to TFIIH p62, it was hypothesized that RVFV NSs protein serves as an adaptor protein in the E3 ligase complex . The human cell encodes 8 different cullins (CUL1, 2, 3, 4A, 4B, 5, 7, and 9) . As a scaffold protein, cullin bridges either the Rbx1 or Rbx2 proteins, which encodes for the RING domain. The ligase activity of the E3 ligase complex is dependent on the covalent modification of cullin with the ubiquitin-like modifier NEDD8 . Different cullins bind to their respective adaptor proteins; e.g., CUL1 or CUL7 binds to Skp1 protein, CUL2 or CUL5 binds to elongin BC complex, CUL3 binds to BTB-domain proteins, and CUL4A binds to DNA-damage-binding protein-1 (DDB1) . Substrate receptor proteins bridge the E3 ligase with specific substrate. For example, the CUL1-Skp1 or CUL7-Skp1 complex binds to substrate receptors that encodes an F-box motif, the CUL2-elongin BC or CUL5-elongin BC complex binds to those encoding a suppressor of cytokine signaling (SOCS) box motif, and the CUL3-BTB-domain complex binds to their substrates via the C-terminus. Many viral proteins bridges the cullin complex with their specific substrate proteins: e.g., paramyxovirus V proteins, human immunodeficiency virus Vif and Vpr proteins, adenovirus E1b55K and E4orf6 proteins [67, 72–78]. Therefore, unique specificity of virulence factors to substrate proteins may contribute to the pathogenesis of viruses in animal hosts.
To characterize the mechanism of TFIIH p62 protein degradation induced by RVFV NSs protein, proteomic screening using recombinant RVFV encoding NSs with a C-terminal TAP tag was accomplished. This resulted in the identification of the F-box protein (FBXO3) as an interacting partner protein for RVFV NSs protein . Knockdown of FBXO3 or Skp1, inhibited p62 degradation from RVFV NSs protein, however knockdown of Rbx, CUL1 or CUL7 did not. The study also showed that the interaction of RVFV NSs protein with CUL1 most likely occurs through FBXO3 and Skp1 (Fig. 2). However, the ubiquitination of p62 could not be demonstrated. The FBXO3 (471 aa) has a full-length form (FBXO3/1) and a C-terminus truncated short form (FBXO3/2: 415 aa). Though FBXO3/1 is expressed in both the nucleus and cytoplasm, the amount of FBXO3/1 proteins, but not FBXO3/2 proteins, was shown to be decreased in cells infected with RVFV. This in turn indicates the alteration of protein turnover in FBXO3/1 proteins, which is due to its incorporation into the E3 ligase complex.
Unlike RVFV NSs proteins, TOSV, SFTSV, or UUKV NSs proteins localize in the cytoplasm only, and not the nucleus [79–83]. The localization of other phlebovirus NSs is currently unknown. The use of click chemistry, which covalently links fluorescent dye to nascent RNA, allows for the measurement of newly synthesized host RNA in infected cells . Cells that were infected with RVFV, showed decreased incorporation of 5-ethynyluridine (5-EU), a nucleoside analog, into newly synthesized nascent host mRNA. Decreased 5-EU labeling also occurred in cells infected with recombinant RVFV MP-12 strain (rMP-12) encoding PTV NSs, in the place of MP-12 NSs . However, this was not the case with cells infected with rMP-12 encoding SFSV NSs or TOSV NSs [85–87]. Though the mechanism has yet to be defined, PTV NSs proteins may decrease host transcriptional activities. Transcriptional regulations induced by phlebovirus NSs proteins, other than RVFV NSs protein, will need to be further characterized toward better understanding of NSs proteins.
Bunyamwera virus (BUNV), which is a member of the genus Orthobunyavirus (Bunyamwera serogroup), expresses NSs proteins (101 amino acids) from the +1 ORF that overlaps the N ORF. The NSs proteins of BUNV is also presented in both in the cytoplasm and nucleus . The NSs proteins of BUNV target cellular RNA polymerase II, by inhibiting the phosphorylation of the C-terminal domain (CTD), YSPTSPS repeats, of which various serine residues are phosphorylated to regulate transcription . Serine at the 2nd or 5th position plays a role in mRNA elongation and transcriptional initiation, respectively. BUNV NSs protein inhibits CTD serine phosphorylation at the 2nd, and not the 5th position, thus blocking the transition from transcriptional initiation to mRNA elongation. BUNV NSs protein also interacts with the mediator of RNA polymerase II transcription subunit 8 (MED8) protein, which regulates the transcriptional function of RNA polymerase II . Intriguingly, RNA polymerase II protein level was shown to be decreased in BUNV infected cells, whereas this reduction did not occur when BUNV NSs protein lacked the MED8-binding domain . Léonard et al. proposed that BUNV NSs protein mediates the degradation of RNA polymerase II through the E3 ligase complex, because the MED8 is also incorporated into the Rbx1-CUL2-elongin BC complex [90, 91]. However, the role of BUNV NSs protein or MED8 in the putative E3 ligase complex in the degradation of RNA polymerase II remains unknown. La Crosse virus (LACV), which is a member of the California serogroup of the genus Orthobunyavirus, also expresses NSs protein (92 amino acids). The NSs protein of LACV has a 38 % amino acid identity with BUNV NSs protein . Although LACV NSs protein does not bind to MED8, it also induces the inhibition of cellular RNA polymerase II CTD serine phosphorylation at the 2nd, and not at the 5th position . Furthermore, LACV NSs protein also induces selective degradation of the hyperphosphorylated IIO form of the RNA polymerase II RPB1 subunit via the proteasome pathway. This in turn does not directly have any effects with the hypophosphorylated IIA form . The degradation of RPB1 subunit occurs in cells when the RNA polymerase II is stalled during elongation . However, it remains unknown whether LACV NSs protein interacts with the E3 ligase complex to specifically target RPB1 or not.
RVFV and TOSV NSs protein promotes posttranslational degradation of dsRNA-dependent protein kinase (PKR)
It is known that RVFV can replicate in the presence of host transcriptional shutoff induced by actinomycin D (ActD) . To determine the role of RVFV NSs protein in the presence of host transcriptional shutoff, viral replication was characterized in cells treated with ActD. MP-12 replication was not decreased when infected Vero cells were treated with ActD. However, recombinant MP-12 encoding Renilla Luciferase (rLuc) in the place of NSs (rMP12-rLuc), showed more than a 2 log reduction in viral titer when infected Vero cells were treated with ActD . This indicated that RVFV NSs protein is capable of promoting viral replication in the presence of host transcriptional shutoff. Eukaryotic initiation factor (eIF) 2α was shown to be highly phosphorylated in cells infected with rMP12-rLuc and treated with ActD, whereas eIF2α phosphorylation was minimal in cells infected with parental MP-12 and treated with ActD .
Subsequently, it was also demonstrated that RVFV NSs protein promotes the degradation of PKR [96, 97]. PKR (551 amino acids; human) is a serine-threonine protein kinase that is ubiquitously expressed in mammalian cells [98–100]; the majority of PKR is found in the cytoplasm and less than 20 % is found in the nucleus . The degradation of PKR in RVFV-infected cells occurred in both the cytoplasm and nucleus . Proteasomal inhibitors, MG132 or lactacystin, stabilized PKR in the presence of RVFV NSs protein. The accumulation of phosphorylated eIF2α did not occur in ActD treated cells infected with recombinant MP-12 encoding a dominant-negative PKR (PKR∆E7). Since eIF2α phosphorylation leads to translation initiation shutoff, RVFV NSs protein serves a role in facilitating efficient viral translation through blocking the PKR-mediated eIF2α phosphorylation.
Using reverse genetics, a number of NSs point mutants were subsequently screened for functional phenotypes. Among those that were screened, the R173A mutant did not promote PKR degradation, yet was able to still induce transcriptional shutoff and inhibit IFN-β mRNA upregulation . Thus phosphorylation of eIF2α was induced in cells infected with RVFV expressing NSs R173A mutant without ActD treatment. Although the mechanism to induce eIF2α phosphorylation by RVFV NSs R173A mutant is unknown, the study showed that RVFV NSs mediated transcription suppression and IFN-β gene suppression occur independently from PKR degradation. The R173A mutant did not interact with PKR, thus indicating the need of the NSs-PKR interaction to trigger PKR degradation.
For NSs-mediated PKR degradation, the specific E3 ligase complex has been further characterized [102, 103] (Fig. 3). As described above, NEDD8 covalently binds to cullin (NEDDylation), to recruit E2 to E3 ligase. Thus the NEDD8 activating enzyme (NAE1) is required for the NEDDylation. As a small molecule inhibitor, MLN4924 functions as a potent and selective inhibitor for NAE1 . As a result, in a dose-dependent manner, MLN4924 inhibited RVFV replication and increased the phosphorylation of PKR and eIF2α. RVFV NSs protein was also screened for potential interaction with CUL 1, 2, 3, 4A, 4B, 5, 7, or 9. Of which, RVFV NSs protein was found to only interact with CUL1. However, CUL1 mutants, ∆N53 or Y42A/M43A, which cannot interact with Skp1, was shown to be poorly bound to NSs protein. Therefore Skp1 mediates the interaction between NSs protein and CUL1. To identify specific F-box protein, which can interact with the CUL1-Skp1, 70 different F-box genes were screened via siRNA assay. As a result, the FBXW11 was screened out from siRNA pools from the reduction of RVFV replication  or PKR degradation . Since BTRC (also known as βTrCP1) and FBXW11 (also known as βTrCP2) are functional paralogs of the BTRC gene (also known as βTrCP gene: Beta-transducin repeat containing protein), further protein analysis was subsequently carried out for both. The siRNA knockdown of both BTRC and FBXW11 mRNA inhibited RVFV replication significantly more than the single knockdown of FBXW11 . Similarly, siRNA knockdown of BTRC gene expression in FBXW11 knockout cells also completely abolished PKR degradation by RVFV NSs protein . The study also demonstrated that the C-terminal WD40 domain repeat of FBXW11 or BTRC, interacts with the C-terminal of NSs protein, which contains the “degron” sequence DDGFVE. Thus, RVFV NSs protein was identified as the adaptor protein connecting the CUL1-Skp1-FBXW11 or the CUL1-Skp1-BTRC complex with PKR. The ubiquitination of PKR could not be demonstrated, however, this may be due to low endogenous expression of PKR or high proteasomal activity.
TOSV NSs (316 aa.), but not that of PTV or SFSV, was also shown to promote the degradation of PKR similar to that of RVFV . Though RVFV and TOSV share similar NSs functions, the amino acid sequence similarity is low, with no shared motifs identified. To better understand the potential mechanism commonly used in PKR degradation, it is important to characterize the role of TOSV NSs protein in the putative E3 ligase complex. This may be accomplished through identifying PKR binding motifs, and the type of cullin-RING complex used. It is also important to recognize whether phleboviruses, other than RVFV and TOSV, encode functions to facilitate viral translation without affecting the PKR.
NSs proteins from RVFV, BUNV, and LACV localize in both the nucleus and cytoplasm, and can induce a strong shutoff of host protein synthesis in a NSs-dependent manner [96, 105, 106]. Tick-borne UUKV or SFTSV expresses cytoplasmic NSs and does not induce host translational shutoff [107–109]. Although host translation is inhibited, RVFV, BUNV, or LACV can maintain viral protein synthesis during infection. Most bunyavirus mRNAs are unique, because they lack the polyA tail at the 3′ terminus. The polyadenylate binding protein 1 (PABP1) binds to the polyA tail and the cap of mRNA via eIF4G, and mRNA forms a circular structure for efficient translation . RVFV NSs protein did not directly bind to PABP1, however, PABP1 was sequestered to nuclear speckles in cells expressing RVFV NSs protein [111, 112]. Nuclear retention of PABP also occurred in BUNV-infected cells at late stage of infection, and it probably due to decreased host mRNA in cytoplasm during NSs-mediated transcriptional shutoff . Thus, RVFV and BUNV NSs proteins facilitate selective inhibition of host protein synthesis through transcription shutoff. The knockdown of eIF4G, but not PABP, decreased the translation of reporter gene flanked by BUNV 5′-UTR and 3′-UTR, which lacks a polyA tail . Hantaviruses, on the other hand, do not induce host translational shutoff and some do not even encode the NSs gene. The N protein of hantaviruses functions as a surrogate for the eIF4F complex (eIF4A, eIF4G, and eIF4F), and thus recruits capped mRNA from the host to initiate viral translation . Further study will be required to understand bunyavirus NSs functions regulating host and viral protein synthesis.
Suppression of interferon (IFN)-β gene up-regulation by NSs protein of RVFV and other bunyaviruses
Type-I, II, and III interferons (IFNs) play important roles in the host defense against pathogens. IFNs exert their biological functions through the stimulation of target cells via specific IFN receptors, using autocrine and paracrine signaling processes. Through type-I IFN receptors (IFNAR), Type-I IFNs stimulate various types of cells, including lymphocytes, myeloid cells, epithelial cells, and stromal cells. Type-III IFNs show limited target specificity, such as epithelial cells, through type-III IFN receptor (IFNλR) [115–117]. Type-II IFN is dominantly expressed from T cells and NK cells, thus playing major roles in immunomodulation and antiviral activities through lymphocytes . Type-I IFNs plays a major role in innate immunity during viral infections. Cells stimulated through IFNAR induce various IFN-stimulated genes (ISGs) to repress viral replication. IFNB gene promoter activation is induced via the coordinated binding of the homodimer of interferon regulatory factor 3 (IRF3) (or the heterodimer of IRF3 and IRF7), nuclear translocation of NFkB, and ATF2/cJun (AP1) to the positive regulatory cis-elements for IFNB gene . The activation of these transcription factors are triggered by the recognition of replicating viral RNA from pattern recognition receptors (PRRs), such as retinoic acid-inducible gene I (RIG-I), melanoma differentiation-associated gene 5 (MDA5), or endosomal viral RNA through Toll-like receptors (TLRs) [115, 119]. Binding of viral RNA to RIG-I or MDA5 leads to their interaction with mitochondrial antiviral signaling protein (MAVS), and activation of TBK1 [tumor necrosis-receptor associated factor (TRAF) family member associated NFkB activator-binding kinase 1] . Activated TBK1 induces phosphorylation and dimerization of IRF3 and IRF7.
RVFV NSs protein is a type-I IFN antagonist, and thus supports efficient viral replication in type-I IFN-competent cells or immune-competent hosts [120, 121]. A natural isolate, RVFV Clone 13 strain (C13), encodes an in-frame truncation of the NSs ORF from aa.16 to 198 (69 % of NSs ORF). The IFN-β mRNA can be upregulated as early as 3–6 h post infection with the C13 strain . Bouloy et al. analyzed the virulence of reassortant RVFV generated between C13 and ZH548 strains in inbred 129/SvPaslco mice. They confirmed the attenuation of RVFV encoding C13 S-segment, and the rapid induction of type-I IFNs in the absence of intact NSs . Therefore, the NSs is considered to be the major virulence factor for RVFV. Cells infected with RVFV strain encoding intact NSs protein did not upregulate IFN-β mRNA, but still activated RIG-I, dimerization and nuclear translocation of IRF3, and nuclear translocation of NFkB and AP1 [60, 122]. Thus, RVFV NSs protein inhibits the upregulation of IFN-β promoter downstream of the activation of transcription factors. Le May et al.  demonstrated that NSs protein binds to Sin3A Associated Protein 30 (SAP30) and subsequently prevents the activation of IFN-β promoter via interaction with a transcription factor, Yin Yang 1 (YY1) protein in mouse cells . In the presence of NSs-SAP30-YY1 complex, the CREB-binding protein was not recruited to the IFN-β promoter, and subsequent acetylation of histone K8H4 or K14H3 did not occur. The RVFV NSs protein aa.210-230 was mapped to bind to SAP30 protein, and replication of recombinant RVFV encoding NSs∆210-230 induced IFN-β mRNA in infected cells. In addition, the NSs∆210-230 protein did not form NSs filament, or interact with SAP30 or IFN-β promoter. Thus, this study indicated that IFN-β promoter activation is inhibited from the interaction between RVFV NSs protein and SAP30 protein.
On the other hand, 11 different RVFV NSs mutants, encoding serial in-frame 25 aa truncation, did not suppress IFN-β gene upregulation, regardless of the location of truncations . This indicated that RVFV NSs mutants failed to exert biological functions due to structural changes or alteration of subcellular localizations. Another study demonstrated that the TFIIH inhibition may also be responsible for the IFN-β promoter suppression. The siRNA knockdown of FBXO3 mRNA (E3 ligase for p62) allowed induction of IFN-β mRNA during RVFV infection . The gaps in biological phenotypes should be further investigated for a better understanding of the mechanism.
TOSV NSs protein localizes to the cytoplasm, and inhibits nuclear localization of IRF3 . In addition, TOSV NSs protein interacted with the N-terminal CARD domain of RIG-I, and promoted the degradation of RIG-I via the proteasome . The inhibition of IFN-β clearly occurred when TOSV NSs was expressed from plasmid DNA or recombinant RVFV, however, it was weak in cells infected with authentic TOSV. This may most likely be due to the low expression level of NSs [79, 86, 126]. Later on, it was suggested that TOSV isolates from the Spanish lineage inhibits IFN-β gene more efficiently than those from the Italian lineage .
SFSV NSs protein inhibits the induction of IFN-β gene, however, the inhibition mechanism of type-I IFN suppression remains unknown [85, 97]. In addition, SFSV NSs protein induces neither host general transcription shutoff, nor PKR degradation [85, 97]. However, cells transfected with a reporter plasmid expressing Renilla luciferase under constitutively active SV40 promoter, showed an increase in reporter activity when SFSV NSs proteins were co-expressed . Thus, SFSV NSs protein probably encodes a biological function upregulating host gene expression either via transcriptional or translational level, with unknown mechanism.
PTV NSs protein also inhibits IFN-β gene induction. The PTV Adames strain is highly pathogenic in hamsters and mice, whereas the PTV Balliet strain does not [128–130]. Similar to TOSV infection, authentic PTV did not completely inhibit the induction of IFN-β in infected cells. When infected with the PTV Balliet strain, type-I IFN was induced more abundantly in hamster embryonic fibroblast cells or mouse primary macrophages than with the Adames strain [130, 131]. In addition, PTV NSs protein expressed from plasmids or recombinant RVFV exerted a more efficient biological function than that expressed from PTV. In a reporter assay, PTV Balliet strain NSs protein inhibited Sendai virus-mediated IFN-β promoter activation less efficiently than PTV Adames strain NSs protein (~4-fold) . Recombinant RVFV MP-12 strain encoding PTV Adames strain NSs was able to completely inhibit the up-regulation of mouse IFN-β mRNA and ISG56 mRNA . It remains unknown how PTV NSs protein targets type-I IFN induction pathway, or how the replications of PTV Adames and PTV Balliet strains differentially induce type-I IFNs in the presence of NSs protein.
Tick-borne SFTSV NSs protein forms cytoplasmic inclusion bodies, binds to IRF3 via TBK1 (or IKKε) and then relocates them to cytoplasmic inclusion bodies. This in turn inhibits the induction of IFN-β gene, since IRF3 is being sequestered to cytoplasmic inclusion bodies [82, 83, 132]. In inclusion bodies, the SFTSV NSs-TBK1 complex was also associated with RIG-I and the E3 ligase TRIM25 . Interestingly, SFTSV NSs inclusion bodies sequestered the cellular signal transducer and activator of transcription 2 (STAT2) and relocated both STAT1 and STAT2 to cytoplasmic inclusion bodies . Thus, SFTSV NSs inclusion bodies play a key role in the inhibition of type-I IFN induction and signaling pathways. Other studies also indicated that SFTSV NSs protein forms viroplasm-like structure, containing NSs, N, dsRNA, and lipid droplets, suggesting the role of SFTSV NSs as a scaffold protein for viral RNA replication .
BUNV NSs protein inhibits IFN-β gene upregulation [105, 134] but does not inhibit the homodimerization or nuclear translocation of IRF3 . Thus, the suppression of IFN-β promoter by BUNV NSs occurs downstream of the IRF3 activation. BUNV NSs protein may also inhibit IFN-β gene upregulation directly via blocking mRNA elongation of host RNA polymerase II .
Similarly to the IFN-β promoter suppression mechanism through SAP30-NSs binding, RVFV NSs protein also alters the process of mitosis in infected cells. RVFV-infected cells showed abnormal nuclei, such as lobulated nuclei, intranuclear DNA bridge, or micronuclei, in an NSs-dependent manner . Through the SAP30-binding domain (amino acid 210–230), RVFV NSs filament interacted with pericentromeric major γ-satellite sequence, but not centromeric minor α-satellite sequence. It was also indicated that YY1 may mediate the interaction between the NSs-SAP30 complex and the γ-satellite sequence DNA. Since RVFV NSs protein accumulates in the nucleus, host cells also induce DNA damage signaling, including ataxia-telangiectasia mutated (ATM), checkpoint kinase 2 (Chk.2), and p53 . RVFV NSs protein also triggers cell cycle arrests, either at the S phase (MP-12 strain) or the G0/G1 phase (ZH548 strain) . Abnormal replication of infected cells, through NSs-mediated DNA damages, affects normal tissue differentiation, and may play a part in the pathogenesis of fetal malformation in infected ruminants.
Although RVFV NSs proteins shutoff host general transcription, including IFN-β gene, the host immune response to RVFV infected cells in vivo is unpredictable. Cytokine profiles were analyzed from human sera: six fatal and 20 non-fatal cases in Saudi Arabia , or 19 fatal and 85 non-fatal cases in South Africa . Fatal cases showed elevated IL-10 (suppression of cell-mediated immune responses), IL-1RA (antagonist for IL-1), IP-10, MCP-1, CXCL9, or IL-8, indicating that fatal cases of RVF failed to mount a systemic pro-inflammatory cytokine response. Mitochondrial antiviral signaling (Mavs) gene knockout mice are a highly lethal RVFV infection model using MP-12 strain, and MCP-1, IL-10, IL-6, and CXCL9 were also significantly increased during RVFV infection . The transcriptional profile of RVFV infected mouse cells was characterized at a relatively early stage of infection (8 hpi) using Chromatin immunoprecipitation (ChIP) and a mouse promoter array . RVFV ZH548 strain showed significant downregulation of ten genes, including Fbox3, Mapk8ip3, Stat2, IL3, IL10rb, Tyk2, Casp9, Phf21, Ncoa3, and Notch4, when compared to recombinant ZH548 lacking NSs (ZH∆NSs). This indicated the impact of NSs expression on apoptosis, innate immunity, and other immunological pathways. Accordingly, cytokine profiles can be important markers to predict the severity of RVFV infection, and will thus be useful to evaluate the effect of antiviral treatments.
Genetically, bunyaviruses are highly diverse, and some species can cause lethal infections in humans or livestock. RVFV is one of the most important bunyaviruses, and has caused large outbreaks in both ruminants and humans, resulting in devastating economic loss in affected regions. The major virulence factor NSs, exerts several different biological functions to hijack infected cells. Each mechanism is apparently similar to those used by other bunyavirus NSs proteins, as summarized in Table 1. The degradation of PKR is induced by RVFV and TOSV NSs proteins. RVFV NSs protein also promotes the degradation of TFIIH p62 protein, whereas TOSV NSs protein triggers the degradation of RIG-I. Since RVFV NSs protein functions as viral adaptor protein to form the E3 ligase complex for p62 or PKR degradation, similar mechanisms may be utilized by TOSV NSs protein for PKR or RIG-I degradation. Host general transcription suppression is induced by RVFV, BUNV, and LACV NSs proteins. These NSs proteins induce DNA damage responses, and trigger host translational shutoff. The expression of IFN-β gene is suppressed at the transcriptional level, which is in sharp contrast with other NSs proteins, as they only localize in the cytoplasm (e.g., TOSV, SFTSV). TOSV or SFTSV NSs proteins inhibit specific proteins in the IFN-β gene induction pathway (e.g., RIG-I, TBK-1). Further grouping of phleboviruses and orthobunyaviruses based on NSs protein functions will lead to effective strategies geared towards combating pathogenic bunyaviruses. Many bunyaviruses are neglected from funding agencies or public health policies, due to restricted viral distributions in specific arthropod vectors (e.g., sandfly, ticks), as well as limited marketability of vaccines or antivirals. Thus, further understanding of the unique biological function of the NSs protein in viruses that are currently uncharacterized, will be important in uncovering viral strategies used to adapt to both arthropod insects and vertebrates.
ActD, actinomycin D; AP1, activating transcription factor 2/cJun; APOBEC3G, the apolipoprotein B mRNA editing enzyme catalytic polypeptide 3G; ATM, ataxia-telangiectasia mutated; ATR, ataxia-telangiectasia mutated and Rad3-related kinase; BC, elongin BC; BTB, broad-complex, tramtrack and bric a brac; BTRC, beta-transducin repeat containing E3 ubiquitin protein kinase; BUNV, bunyamwera virus; Cdk7, cyclin-dependent kinase 7; ChIP, chromatin immunoprecipitation; Chk.2, checkpoint kinase 2; CTD, C-terminal domain; CUL, cullin; CXCL9, chemokine (C-X-C motif) ligand 9; dsRBMs, dsRNA-binding motifs; eIF2α, eukaryotic initiation factor 2α; eIF4F, eukaryotic initiation factor 4 F; eIF4G, eukaryotic initiation factor 4G; FRIV, Frijoles virus; HIV, human immunodeficiency virus; ICOV, Icoaraci virus; IFN, interferon; IFNAR, interferon-α/β receptor; IRF3, interferon regulatory factor 3; IRF7, interferon regulatory factor 7; ISGs, IFN-stimulated genes; LACV, La Crosse virus; L-segment, large-segment; MAT1, menage a trois 1; MAVS, mitochondrial antiviral signaling; MCP-1, monocyte chemoattractant protein-1; MDA5, melanoma differentiation-associated gene 5; M-segment, medium-segment; NAE1, NEDD8 activating enzyme 1; NEDD8, neural precursor cell expressed developmentally downregulated protein 8; NIAID, National Institute of Allergy and Infectious Diseases; NIH, National Institutes of Health; NS, nonstructural protein; NSs, nonstructural protein on the S segment; ORF, open reading frame; PABP1, polyadenylate binding protein 1; PKR, dsRNA-dependent protein kinase; PRRs, pattern recognition receptors; PTV, Punta Toro virus; RIG-I, retinoic acid-inducible gene 1; RING, really interesting new gene; rLuc, Renilla luciferase; RVF, Rift Valley fever; RVFV, Rift Valley fever virus; SAP30, Sin3A-associated 30-kD protein; SFSV, sandfly fever sicilian virus; SFTS, severe fever with thrombocytopenia syndrome; SFTSV, severe fever with thrombocytopenia syndrome virus; Skp1, S-phase kinase-associated protein 1; SOCS, suppressor of cytokine signaling; S-segment, small-segment; STATs, signal transducers and activators of transcription; TBP, TATA-binding protein; TFIIH, transcription factor IIH; TLRs, toll-like receptors; TOSV, toscana virus; TRAF, tumor necrosis-receptor associated factor; Ub, ubiquitin; UUKV, uukuniemi virus; XPB, xeroderma pigmentosum type B; XPD, xeroderma pigmentosum group D; YY1, Yin Yang 1; βTrCP, Beta-transducin repeat containing protein
Daubney R, Hudson JR. Enzootic hepatitis or Rift Valley fever: An undescribed virus disease of sheep cattle and man from east Africa. J Pathol Bacteriol. 1931;34:545–79.
Swanepoel R, Coetzer JAW. Rift Valley fever. In: Coetzer JAW, Tustin RC, editors. Infectious diseases of livestock with special reference to southern Africa. 2nd ed. Cape Town: Oxford University Press; 2004. p. 1037–70.
Shoemaker T, Boulianne C, Vincent MJ, Pezzanite L, Al-Qahtani MM, Al-Mazrou Y, et al. Genetic analysis of viruses associated with emergence of Rift Valley fever in Saudi Arabia and Yemen, 2000–01. Emerg Infect Dis. 2002;8:1415–20.
Carroll SA, Reynes JM, Khristova ML, Andriamandimby SF, Rollin PE, Nichol ST. Genetic evidence for Rift Valley fever outbreaks in Madagascar resulting from virus introductions from the East African mainland rather than enzootic maintenance. J Virol. 2011;85:6162–7.
Meegan JM. The Rift Valley fever epizootic in Egypt 1977–78. 1. Description of the epizzotic and virological studies. Trans R Soc Trop Med Hyg. 1979;73:618–23.
Bird BH, Khristova ML, Rollin PE, Ksiazek TG, Nichol ST. Complete genome analysis of 33 ecologically and biologically diverse Rift Valley fever virus strains reveals widespread virus movement and low genetic diversity due to recent common ancestry. J Virol. 2007;81:2805–16.
Schmaljohn C, Nichol ST. Bunyaviridae. In: Knipe DM, Howley PM, Griffin DE, Lamb RA, Martin MA, Roizman B, Straus SE, editors. Fields Virology. 5th ed. Philadelphia: Lippincott, Williams & Wilkins; 2007. p. 1741–89.
Coetzer JA, Barnard BJ. Hydrops amnii in sheep associated with hydranencephaly and arthrogryposis with wesselsbron disease and rift valley fever viruses as aetiological agents. Onderstepoort J Vet Res. 1977;44:119–26.
Easterday BC. Rift valley fever. Adv Vet Sci. 1965;10:65–127.
Ikegami T, Makino S. The Pathogenesis of Rift Valley Fever. Viruses. 2011;3:493–519.
CDC. Rift Valley fever outbreak--Kenya, November 2006-January 2007. MMWR Morb Mortal Wkly Rep. 2007;56:73–6.
Bird BH, Ksiazek TG, Nichol ST, Maclachlan NJ. Rift Valley fever virus. J Am Vet Med Assoc. 2009;234:883–93.
Linthicum KJ, Davies FG, Kairo A, Bailey CL. Rift Valley fever virus (family Bunyaviridae, genus Phlebovirus). Isolations from Diptera collected during an inter-epizootic period in Kenya. J Hyg (Lond). 1985;95:197–209.
Pepin M, Bouloy M, Bird BH, Kemp A, Paweska J. Rift Valley fever virus (Bunyaviridae: Phlebovirus): an update on pathogenesis, molecular epidemiology, vectors, diagnostics and prevention. Vet Res. 2010;41:61.
Turell MJ, Dohm DJ, Fonseca DM. Comparison of the Potential for Different Genetic Forms in the Culex pipiens Complex in North America to Transmit Rift Valley Fever Virus. J Am Mosq Control Assoc. 2014;30:253–9.
Balenghien T, Cardinale E, Chevalier V, Elissa N, Failloux AB, Jean Jose Nipomichene TN, et al. Towards a better understanding of Rift Valley fever epidemiology in the south-west of the Indian Ocean. Vet Res. 2013;44:78.
CDC. Rift Valley fever with retinopathy-Canada. MMWR Morb Mortal Wkly Rep. 1979;28:607–8.
Mansfield KL, Banyard AC, McElhinney L, Johnson N, Horton DL, Hernandez-Triana LM, et al. Rift Valley fever virus: A review of diagnosis and vaccination, and implications for emergence in Europe. Vaccine. 2015;33:5520–31.
Miller MM, Bennett KE, Drolet BS, Lindsay R, Mecham JO, Reeves WK, et al. Evaluation of the efficacy, potential for vector transmission, and duration of immunity of MP-12, an attenuated Rift Valley fever virus vaccine candidate, in sheep. Clin Vaccine Immunol. 2015;22:930–7.
Peters CJ, Reynolds JA, Slone TW, Jones DE, Stephen EL. Prophylaxis of Rift Valley fever with antiviral drugs, immune serum, an interferon inducer, and a macrophage activator. Antiviral Res. 1986;6:285–97.
Kende M, Alving CR, Rill WL, Swartz Jr GM, Canonico PG. Enhanced efficacy of liposome-encapsulated ribavirin against Rift Valley fever virus infection in mice. Antimicrob Agents Chemother. 1985;27:903–7.
Huggins JW. Prospects for treatment of viral hemorrhagic fevers with ribavirin, a broad-spectrum antiviral drug. Rev Infect Dis. 1989;11 Suppl 4:S750–61.
Kende M, Lupton HW, Rill WL, Levy HB, Canonico PG. Enhanced therapeutic efficacy of poly(ICLC) and ribavirin combinations against Rift Valley fever virus infection in mice. Antimicrob Agents Chemother. 1987;31:986–90.
Kende M. Prophylactic and therapeutic efficacy of poly(I, C)-LC against Rift Valley fever virus infection in mice. J Biol Response Mod. 1985;4:503–11.
Morrill JC, Jennings GB, Cosgriff TM, Gibbs PH, Peters CJ. Prevention of Rift Valley fever in rhesus monkeys with interferon-alpha. Rev Infect Dis. 1989;11 Suppl 4:S815–25.
Manns MP, Wedemeyer H, Cornberg M. Treating viral hepatitis C: efficacy, side effects, and complications. Gut. 2006;55:1350–9.
Elliott RM. Orthobunyaviruses: recent genetic and structural insights. Nat Rev Microbiol. 2014;12:673–85.
Jaaskelainen KM, Kaukinen P, Minskaya ES, Plyusnina A, Vapalahti O, Elliott RM, et al. Tula and Puumala hantavirus NSs ORFs are functional and the products inhibit activation of the interferon-beta promoter. J Med Virol. 2007;79:1527–36.
Altamura LA, Bertolotti-Ciarlet A, Teigler J, Paragas J, Schmaljohn CS, Doms RW. Identification of a novel C-terminal cleavage of Crimean-Congo hemorrhagic fever virus PreGN that leads to generation of an NSM protein. J Virol. 2007;81:6632–42.
Kormelink R, Storms M, Van Lent J, Peters D, Goldbach R. Expression and subcellular location of the NSM protein of tomato spotted wilt virus (TSWV), a putative viral movement protein. Virology. 1994;200:56–65.
Barnwal B, Karlberg H, Mirazimi A, Tan YJ. The Non-structural Protein of Crimean-Congo Hemorrhagic Fever Virus Disrupts the Mitochondrial Membrane Potential and Induces Apoptosis. J Biol Chem. 2016;291:582–92.
Reguera J, Weber F, Cusack S. Bunyaviridae RNA polymerases (L-protein) have an N-terminal, influenza-like endonuclease domain, essential for viral cap-dependent transcription. PLoS Pathog. 2010;6:e1001101.
Garcin D, Lezzi M, Dobbs M, Elliott RM, Schmaljohn C, Kang CY, et al. The 5' ends of Hantaan virus (Bunyaviridae) RNAs suggest a prime-and-realign mechanism for the initiation of RNA synthesis. J Virol. 1995;69:5754–62.
Klemm C, Reguera J, Cusack S, Zielecki F, Kochs G, Weber F. Systems to establish bunyavirus genome replication in the absence of transcription. J Virol. 2013;87:8205–12.
Morin B, Coutard B, Lelke M, Ferron F, Kerber R, Jamal S, et al. The N-terminal domain of the arenavirus L protein is an RNA endonuclease essential in mRNA transcription. PLoS Pathog. 2010;6:e1001038.
Ikegami T, Won S, Peters CJ, Makino S. Characterization of Rift Valley fever virus transcriptional terminations. J Virol. 2007;81:8421–38.
Albarino 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;81:5246–56.
Lara E, Billecocq A, Leger P, Bouloy M. Characterization of wild-type and alternate transcription termination signals in the Rift Valley fever virus genome. J Virol. 2011;85:12134–45.
Barr JN. Bunyavirus mRNA synthesis is coupled to translation to prevent premature transcription termination. RNA. 2007;13:731–6.
Barr JN, Rodgers JW, Wertz GW. Identification of the Bunyamwera bunyavirus transcription termination signal. J Gen Virol. 2006;87:189–98.
Gerrard SR, Nichol ST. Synthesis, proteolytic processing and complex formation of N-terminally nested precursor proteins of the Rift Valley fever virus glycoproteins. Virology. 2007;357:124–33.
Weingartl HM, Zhang S, Marszal P, McGreevy A, Burton L, Wilson WC. Rift Valley fever virus incorporates the 78 kDa glycoprotein into virions matured in mosquito C6/36 cells. PLoS ONE. 2014;9:e87385.
Kreher F, Tamietti C, Gommet C, Guillemot L, Ermonval M, Failloux AB, et al. The Rift Valley fever accessory proteins NSm and P78/NSm-Gn are determinants of virus propagation in vertebrate and invertebrate hosts. Emerg Microbes Infect. 2014;3:e71.
Kading RC, Crabtree MB, Bird BH, Nichol ST, Erickson BR, Horiuchi K, et al. Deletion of the NSm virulence gene of Rift Valley fever virus inhibits virus replication in and dissemination from the midgut of Aedes aegypti mosquitoes. PLoS Negl Trop Dis. 2014;8:e2670.
Crabtree MB, Kent Crockett RJ, Bird BH, Nichol ST, Erickson BR, Biggerstaff BJ, et al. Infection and transmission of Rift Valley fever viruses lacking the NSs and/or NSm genes in mosquitoes: potential role for NSm in mosquito infection. PLoS Negl Trop Dis. 2012;6:e1639.
Won S, Ikegami T, Peters CJ, Makino S. NSm protein of Rift Valley fever virus suppresses virus-induced apoptosis. J Virol. 2007;81:13335–45.
Terasaki K, Won S, Makino S. The C-terminal region of Rift Valley fever virus NSm protein targets the protein to the mitochondrial outer membrane and exerts anti-apoptotic function. J Virol. 2013;87:676–82.
Plyusnina A, Beaty BJ, Elliott RM, Goldbach R, Kormelink R, Lundkvist A, et al. Family Bunyaviridae. In: King AMQ, Adams MJ, Carsten EB, Lefkowitz EJ, editors. Virus taxonomy: ninth report of the International Committee on Taxonomy of Viruses. 1st ed. London: Elsevier; 2011. p. 725–41.
Dionisio D, Esperti F, Vivarelli A, Valassina M. Epidemiological, clinical and laboratory aspects of sandfly fever. Curr Opin Infect Dis. 2003;16:383–8.
Sabin AB. Experimental studies on Phlebotomus (pappataci, sandfly) fever during World War II. Arch Gesamte Virusforsch. 1951;4:367–410.
Tesh RB. The genus Phlebovirus and its vectors. Annu Rev Entomol. 1988;33:169–81.
Liu S, Chai C, Wang C, Amer S, Lv H, He H, et al. Systematic review of severe fever with thrombocytopenia syndrome: virology, epidemiology, and clinical characteristics. Rev Med Virol. 2014;24:90–102.
Liu Q, He B, Huang SY, Wei F, Zhu XQ. Severe fever with thrombocytopenia syndrome, an emerging tick-borne zoonosis. Lancet Infect Dis. 2014;14:763–72.
Saito T, Fukushima K, Umeki K, Nakajima K. Severe fever with thrombocytopenia syndrome in Japan and public health communication. Emerg Infect Dis. 2015;21:487–9.
Yun SM, Lee WG, Ryou J, Yang SC, Park SW, Roh JY, et al. Severe fever with thrombocytopenia syndrome virus in ticks collected from humans, South Korea, 2013. Emerg Infect Dis. 2014;20:1358–61.
Vasconcelos PF, Calisher CH. Emergence of Human Arboviral Diseases in the Americas, 2000–2016. Vector Borne Zoonotic Dis. 2016;16:295–301.
Xu F, Chen H, Travassos da Rosa AP, Tesh RB, Xiao SY. Phylogenetic relationships among sandfly fever group viruses (Phlebovirus: Bunyaviridae) based on the small genome segment. J Gen Virol. 2007;88:2312–9.
Giorgi C, Accardi L, Nicoletti L, Gro MC, Takehara K, Hilditch C, 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.
Yadani FZ, Kohl A, Prehaud C, Billecocq A, Bouloy M. The carboxy-terminal acidic domain of Rift Valley Fever virus NSs protein is essential for the formation of filamentous structures but not for the nuclear localization of the protein. J Virol. 1999;73:5018–25.
Billecocq A, Spiegel M, Vialat P, Kohl A, Weber F, Bouloy M, et al. NSs protein of Rift Valley fever virus blocks interferon production by inhibiting host gene transcription. J Virol. 2004;78:9798–806.
Cyr N, de la Fuente C, Lecoq L, Guendel I, Chabot PR, Kehn-Hall K, et al. A OmegaXaV motif in the Rift Valley fever virus NSs protein is essential for degrading p62, forming nuclear filaments and virulence. Proc Natl Acad Sci U S A. 2015;112:6021–6.
Le May N, Dubaele S, Proietti De Santis L, Billecocq A, Bouloy M, Egly JM. TFIIH transcription factor, a target for the Rift Valley hemorrhagic fever virus. Cell. 2004;116:541–50.
Iben S, Tschochner H, Bier M, Hoogstraten D, Hozak P, Egly JM, et al. TFIIH plays an essential role in RNA polymerase I transcription. Cell. 2002;109:297–306.
Kalveram B, Lihoradova O, Ikegami T. NSs Protein of Rift Valley Fever Virus Promotes Post-Translational Downregulation of the TFIIH Subunit p62. J Virol. 2011;85:6234–43.
Sorokin AV, Kim ER, Ovchinnikov LP. Proteasome system of protein degradation and processing. Biochemistry (Mosc). 2009;74:1411–42.
Hershko A, Ciechanover A. The ubiquitin system. Annu Rev Biochem. 1998;67:425–79.
Randow F, Lehner PJ. Viral avoidance and exploitation of the ubiquitin system. Nat Cell Biol. 2009;11:527–34.
Komander D, Rape M. The ubiquitin code. Annu Rev Biochem. 2012;81:203–29.
Kainulainen M, Habjan M, Hubel P, Busch L, Lau S, Colinge J, et al. Virulence factor NSs of rift valley fever virus recruits the F-box protein FBXO3 to degrade subunit p62 of general transcription factor TFIIH. J Virol. 2014;88:3464–73.
Petroski MD, Deshaies RJ. Function and regulation of cullin-RING ubiquitin ligases. Nat Rev Mol Cell Biol. 2005;6:9–20.
Deshaies RJ, Joazeiro CA. RING domain E3 ubiquitin ligases. Annu Rev Biochem. 2009;78:399–434.
Chen M, Gerlier D. Viral hijacking of cellular ubiquitination pathways as an anti-innate immunity strategy. Viral Immunol. 2006;19:349–62.
Li T, Chen X, Garbutt KC, Zhou P, Zheng N. Structure of DDB1 in complex with a paramyxovirus V protein: viral hijack of a propeller cluster in ubiquitin ligase. Cell. 2006;124:105–17.
Wolfe LS, Stanley BJ, Liu C, Eliason WK, Xiong Y. Dissection of the HIV Vif interaction with human E3 ubiquitin ligase. J Virol. 2010;84:7135–9.
Schrofelbauer B, Yu Q, Zeitlin SG, Landau NR. Human immunodeficiency virus type 1 Vpr induces the degradation of the UNG and SMUG uracil-DNA glycosylases. J Virol. 2005;79:10978–87.
Belzile JP, Duisit G, Rougeau N, Mercier J, Finzi A, Cohen EA. HIV-1 Vpr-mediated G2 arrest involves the DDB1-CUL4AVPRBP E3 ubiquitin ligase. PLoS Pathog. 2007;3:e85.
Le Rouzic E, Belaidouni N, Estrabaud E, Morel M, Rain JC, Transy C, et al. HIV1 Vpr arrests the cell cycle by recruiting DCAF1/VprBP, a receptor of the Cul4-DDB1 ubiquitin ligase. Cell Cycle. 2007;6:182–8.
Dallaire F, Blanchette P, Groitl P, Dobner T, Branton PE. Identification of integrin alpha3 as a new substrate of the adenovirus E4orf6/E1B 55-kilodalton E3 ubiquitin ligase complex. J Virol. 2009;83:5329–38.
Gori Savellini G, Weber F, Terrosi C, Habjan M, Martorelli B, Cusi MG. Toscana virus induces interferon although its NSs protein reveals antagonistic activity. J Gen Virol. 2011;92:71–9.
Simons JF, Persson R, Pettersson RF. Association of the nonstructural protein NSs of Uukuniemi virus with the 40S ribosomal subunit. J Virol. 1992;66:4233–41.
Wu X, Qi X, Liang M, Li C, Cardona CJ, Li D, et al. Roles of viroplasm-like structures formed by nonstructural protein NSs in infection with severe fever with thrombocytopenia syndrome virus. FASEB J. 2014;28:2504–16.
Wu X, Qi X, Qu B, Zhang Z, Liang M, Li C, et al. Evasion of antiviral immunity through sequestering of TBK1/IKKepsilon/IRF3 into viral inclusion bodies. J Virol. 2014;88:3067–76.
Santiago FW, Covaleda LM, Sanchez-Aparicio MT, Silvas JA, Diaz-Vizarreta AC, Patel JR, et al. Hijacking of RIG-I signaling proteins into virus-induced cytoplasmic structures correlates with the inhibition of type I interferon responses. J Virol. 2014;88:4572–85.
Kalveram B, Lihoradova O, Indran SV, Head JA, Ikegami T. Using click chemistry to measure the effect of viral infection on host-cell RNA synthesis. J Vis Exp. 2013;78:50809.
Lihoradova OA, Indran SV, Kalveram B, Lokugamage N, Head JA, Gong B, et al. Characterization of Rift Valley Fever Virus MP-12 Strain Encoding NSs of Punta Toro Virus or Sandfly Fever Sicilian Virus. PLoS Negl Trop Dis. 2013;7:e2181.
Indran SV, Lihoradova OA, Phoenix I, Lokugamage N, Kalveram B, Head JA, et al. Rift Valley fever virus MP-12 vaccine encoding Toscana virus NSs retains neuroinvasiveness in mice. J Gen Virol. 2013;94:1441–50.
Kalveram B, Ikegami T. Toscana Virus NSs Protein Promotes Degradation of Double-Stranded RNA-Dependent Protein Kinase. J Virol. 2013;87:3710–8.
Weber 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.
Thomas D, Blakqori G, Wagner V, Banholzer M, Kessler N, Elliott RM, et al. Inhibition of RNA polymerase II phosphorylation by a viral interferon antagonist. J Biol Chem. 2004;279:31471–7.
Leonard VH, Kohl A, Hart TJ, Elliott RM. Interaction of Bunyamwera Orthobunyavirus NSs protein with mediator protein MED8: a mechanism for inhibiting the interferon response. J Virol. 2006;80:9667–75.
Brower CS, Sato S, Tomomori-Sato C, Kamura T, Pause A, Stearman R, et al. Mammalian mediator subunit mMED8 is an Elongin BC-interacting protein that can assemble with Cul2 and Rbx1 to reconstitute a ubiquitin ligase. Proc Natl Acad Sci U S A. 2002;99:10353–8.
Dunn EF, Pritlove DC, Elliott RM. The S RNA genome segments of Batai, Cache Valley, Guaroa, Kairi, Lumbo, Main Drain and Northway bunyaviruses: sequence determination and analysis. J Gen Virol. 1994;75:597–608.
Verbruggen P, Ruf M, Blakqori G, Overby AK, Heidemann M, Eick D, et al. Interferon antagonist NSs of La Crosse virus triggers a DNA damage response-like degradation of transcribing RNA polymerase II. J Biol Chem. 2011;286:3681–92.
Somesh BP, Reid J, Liu WF, Sogaard TM, Erdjument-Bromage H, Tempst P, et al. Multiple mechanisms confining RNA polymerase II ubiquitylation to polymerases undergoing transcriptional arrest. Cell. 2005;121:913–23.
Swanepoel R, Blackburn NK. Demonstration of nuclear immunofluorescence in Rift Valley fever infected cells. J Gen Virol. 1977;34:557–61.
Ikegami 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.
Habjan M, Pichlmair A, Elliott RM, Overby AK, Glatter T, Gstaiger M, 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.
Gale Jr M, Tan SL, Katze MG. Translational control of viral gene expression in eukaryotes. Microbiol Mol Biol Rev. 2000;64:239–80.
Langland JO, Cameron JM, Heck MC, Jancovich JK, Jacobs BL. Inhibition of PKR by RNA and DNA viruses. Virus Res. 2006;119:100–10.
Garcia MA, Gil J, Ventoso I, Guerra S, Domingo E, Rivas C, et al. Impact of protein kinase PKR in cell biology: from antiviral to antiproliferative action. Microbiol Mol Biol Rev. 2006;70:1032–60.
Kalveram B, Lihoradova O, Indran SV, Lokugamage N, Head JA, Ikegami T. Rift Valley fever virus NSs inhibits host transcription independently of the degradation of dsRNA-dependent protein kinase PKR. Virology. 2013;435:415–24.
Mudhasani R, Tran JP, Retterer C, Kota KP, Whitehouse CA, Bavari S. Protein Kinase R Degradation Is Essential for Rift Valley Fever Virus Infection and Is Regulated by SKP1-CUL1-F-box (SCF)FBXW11-NSs E3 Ligase. PLoS Pathog. 2016;12:e1005437.
Kainulainen M, Lau S, Samuel CE, Hornung V, Weber F. NSs virulence factor of Rift Valley fever virus engages the F-box proteins FBXW11 and beta-TRCP1 to degrade the antiviral protein kinase PKR. J Virol. 2016;90:6140–7.
Soucy TA, Smith PG, Rolfe M. Targeting NEDD8-activated cullin-RING ligases for the treatment of cancer. Clin Cancer Res. 2009;15:3912–6.
Bridgen A, Weber F, Fazakerley JK, Elliott RM. Bunyamwera bunyavirus nonstructural protein NSs is a nonessential gene product that contributes to viral pathogenesis. Proc Natl Acad Sci U S A. 2001;98:664–9.
Blakqori G, Weber F. Efficient cDNA-based rescue of La Crosse bunyaviruses expressing or lacking the nonstructural protein NSs. J Virol. 2005;79:10420–8.
Pettersson RF. Effect of Uukuniemi virus infection on host cell macromolecule synthesis. Med Biol. 1974;52:90–7.
Brennan B, Li P, Zhang S, Li A, Liang M, Li D, et al. Reverse genetics system for severe fever with thrombocytopenia syndrome virus. J Virol. 2015;89:3026–37.
Rezelj VV, Overby AK, Elliott RM. Generation of mutant Uukuniemi viruses lacking the nonstructural protein NSs by reverse genetics indicates that NSs is a weak interferon antagonist. J Virol. 2015;89:4849–56.
Eliseeva IA, Lyabin DN, Ovchinnikov LP. Poly(A)-binding proteins: structure, domain organization, and activity regulation. Biochemistry (Mosc). 2013;78:1377–91.
Copeland AM, Altamura LA, Van Deusen NM, Schmaljohn CS. Nuclear relocalization of polyadenylate binding protein during rift valley fever virus infection involves expression of the NSs gene. J Virol. 2013;87:11659–69.
Copeland AM, Van Deusen NM, Schmaljohn CS. Rift Valley fever virus NSS gene expression correlates with a defect in nuclear mRNA export. Virology. 2015;486:88–93.
Blakqori G, van Knippenberg I, Elliott RM. Bunyamwera orthobunyavirus S-segment untranslated regions mediate poly(A) tail-independent translation. J Virol. 2009;83:3637–46.
Hussein IT, Mir MA. How hantaviruses modulate cellular pathways for efficient replication? Front Biosci (Elite Ed). 2013;5:154–66.
McNab F, Mayer-Barber K, Sher A, Wack A, O'Garra A. Type I interferons in infectious disease. Nat Rev Immunol. 2015;15:87–103.
Witte K, Witte E, Sabat R, Wolk K. IL-28A, IL-28B, and IL-29: promising cytokines with type I interferon-like properties. Cytokine Growth Factor Rev. 2010;21:237–51.
de Weerd NA, Samarajiwa SA, Hertzog PJ. Type I interferon receptors: biochemistry and biological functions. J Biol Chem. 2007;282:20053–7.
Farrar MA, Schreiber RD. The molecular cell biology of interferon-gamma and its receptor. Annu Rev Immunol. 1993;11:571–611.
Honda K, Taniguchi T. IRFs: master regulators of signalling by Toll-like receptors and cytosolic pattern-recognition receptors. Nat Rev Immunol. 2006;6:644–58.
Muller R, Saluzzo JF, Lopez N, Dreier T, Turell M, Smith J, et al. Characterization of clone 13, a naturally attenuated avirulent isolate of Rift Valley fever virus, which is altered in the small segment. Am J Trop Med Hyg. 1995;53:405–11.
Bouloy M, Janzen C, Vialat P, Khun H, Pavlovic J, Huerre M, et al. Genetic evidence for an interferon-antagonistic function of rift valley fever virus nonstructural protein NSs. J Virol. 2001;75:1371–7.
Ermler ME, Yerukhim E, Schriewer J, Schattgen S, Traylor Z, Wespiser AR, et al. RNA helicase signaling is critical for type i interferon production and protection against Rift Valley fever virus during mucosal challenge. J Virol. 2013;87:4846–60.
Le May N, Mansuroglu Z, Leger P, Josse T, Blot G, Billecocq A, et al. A SAP30 complex inhibits IFN-beta expression in Rift Valley fever virus infected cells. PLoS Pathog. 2008;4:e13.
Weill 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.
Head JA, Kalveram B, Ikegami T. Functional analysis of Rift Valley fever virus NSs encoding a partial truncation. PLoS ONE. 2012;7:e45730.
Gori-Savellini G, Valentini M, Cusi MG. Toscana virus NSs protein inhibits the induction of type I interferon by interacting with RIG-I. J Virol. 2013;87:6660–7.
Brisbarre NM, Plumet S, de Micco P, Leparc-Goffart I, Emonet SF. Toscana virus inhibits the interferon beta response in cell cultures. Virology. 2013;442:189–94.
Pifat DY, Smith JF. Punta Toro virus infection of C57BL/6 J mice: a model for phlebovirus-induced disease. Microb Pathog. 1987;3:409–22.
Anderson Jr GW, Slayter MV, Hall W, Peters CJ. Pathogenesis of a phleboviral infection (Punta Toro virus) in golden Syrian hamsters. Arch Virol. 1990;114:203–12.
Perrone LA, Narayanan K, Worthy M, Peters CJ. The S segment of Punta Toro virus (Bunyaviridae, Phlebovirus) is a major determinant of lethality in the Syrian hamster and codes for a type I interferon antagonist. J Virol. 2007;81:884–92.
Mendenhall M, Wong MH, Skirpstunas R, Morrey JD, Gowen BB. Punta Toro virus (Bunyaviridae, Phlebovirus) infection in mice: strain differences in pathogenesis and host interferon response. Virology. 2009;395:143–51.
Ning YJ, Wang M, Deng M, Shen S, Liu W, Cao WC, et al. Viral suppression of innate immunity via spatial isolation of TBK1/IKKepsilon from mitochondrial antiviral platform. J Mol Cell Biol. 2014;6:324–37.
Ning YJ, Feng K, Min YQ, Cao WC, Wang M, Deng F, et al. Disruption of type I interferon signaling by the nonstructural protein of severe fever with thrombocytopenia syndrome virus via the hijacking of STAT2 and STAT1 into inclusion bodies. J Virol. 2015;89:4227–36.
Weber F, Bridgen A, Fazakerley JK, Streitenfeld H, Kessler N, Randall RE, et al. Bunyamwera bunyavirus nonstructural protein NSs counteracts the induction of alpha/beta interferon. J Virol. 2002;76:7949–55.
Kohl A, Clayton RF, Weber F, Bridgen A, Randall RE, Elliott RM. Bunyamwera virus nonstructural protein NSs counteracts interferon regulatory factor 3-mediated induction of early cell death. J Virol. 2003;77:7999–8008.
Mansuroglu Z, Josse T, Gilleron J, Billecocq A, Leger P, Bouloy M, et al. Non structural NSs protein of Rift Valley Fever Virus interacts with pericentromeric DNA sequences of the host cell inducing chromosome cohesion and segregation defects. J Virol. 2009;84:928–39.
Baer A, Austin D, Narayanan A, Popova T, Kainulainen M, Bailey C, et al. Induction of DNA damage signaling upon Rift Valley fever virus infection results in cell cycle arrest and increased viral replication. J Biol Chem. 2012;287:7399–410.
McElroy AK, Nichol ST. Rift Valley fever virus inhibits a pro-inflammatory response in experimentally infected human monocyte derived macrophages and a pro-inflammatory cytokine response may be associated with patient survival during natural infection. Virology. 2012;422:6–12.
Jansen van Vuren P, Shalekoff S, Grobbelaar AA, Archer BN, Thomas J, Tiemessen CT, et al. Serum levels of inflammatory cytokines in Rift Valley fever patients are indicative of severe disease. Virol J. 2015;12:159.
Benferhat R, Josse T, Albaud B, Gentien D, Mansuroglu Z, Marcato V, et al. Large-scale chromatin immunoprecipitation with promoter sequence microarray analysis of the interaction of the NSs protein of Rift Valley fever virus with regulatory DNA regions of the host genome. J Virol. 2012;86:11333–44.
Palacios G, Tesh R, Travassos da Rosa A, Savji N, Sze W, Jain K, et al. Characterization of the Candiru antigenic complex (Bunyaviridae: Phlebovirus), a highly diverse and reassorting group of viruses affecting humans in tropical America. J Virol. 2011;85:3811–20.
Palacios G, Wiley MR, Travassos da Rosa AP, Guzman H, Quiroz E, Savji N, et al. Characterization of the Punta Toro species complex (genus Phlebovirus, family Bunyaviridae). J Gen Virol. 2015;96:2079–85.
Palacios G, Savji N, Travassos da Rosa A, Desai A, Sanchez-Seco MP, Guzman H, et al. Characterization of the Salehabad virus species complex of the genus Phlebovirus (Bunyaviridae). J Gen Virol. 2013;94:837–42.
Palacios G, Tesh RB, Savji N, Travassos da Rosa AP, Guzman H, Bussetti AV, et al. Characterization of the Sandfly fever Naples species complex and description of a new Karimabad species complex (genus Phlebovirus, family Bunyaviridae). J Gen Virol. 2014;95:292–300.
Palacios G, Savji N, Travassos da Rosa A, Guzman H, Yu X, Desai A, et al. Characterization of the Uukuniemi virus group (Phlebovirus: Bunyaviridae): evidence for seven distinct species. J Virol. 2013;87:3187–95.
Matsuno K, Weisend C, Travassos da Rosa AP, Anzick SL, Dahlstrom E, Porcella SF, et al. Characterization of the Bhanja serogroup viruses (Bunyaviridae): a novel species of the genus Phlebovirus and its relationship with other emerging tick-borne phleboviruses. J Virol. 2013;87:3719–28.
Xu F, Liu D, Nunes MR, DA Rosa AP, Tesh RB, Xiao SY. Antigenic and genetic relationships among Rift Valley fever virus and other selected members of the genus Phlebovirus (Bunyaviridae). Am J Trop Med Hyg. 2007;76:1194–200.
Palacios G, da Rosa AT, Savji N, Sze W, Wick I, Guzman H, et al. Aguacate virus, a new antigenic complex of the genus Phlebovirus (family Bunyaviridae). J Gen Virol. 2011;92:1445–53.
We thank for educational and research supports through the Sealy Center for Vaccine Development at The University of Texas Medical Branch at Galveston (UTMB). This review was partly supported by the National Institute of Health (grant number R01 AI087643-01A1).
This work was partly supported by the National Institute of Health (grant number R01 AI087643-01A1) (T.I.), and the funding from the Sealy Center for Vaccine Development at The University of Texas Medical Branch at Galveston (UTMB) (TI).
Availability of data and materials
HJL and TI drafted the manuscript and designed artworks. Both authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Ethics approval and consent to participate