The generation of a reverse genetics system for ... - Loewen Labs

Virus Research 163 (2012) 431–438

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The generation of a reverse genetics system for Kyasanur Forest Disease Virus and the ability to antagonize the induction of the antiviral state in vitro Bradley W.M. Cook a,b , Todd A. Cutts a , Deborah A. Court b , Steven Theriault a,b,∗ a b

Canadian Science Center for Human and Animal Health, Public Health Agency of Canada, 1015 Arlington Street, Winnipeg, Manitoba, Canada R3E 3P6 The University of Manitoba, Department of Microbiology, Winnipeg, Manitoba, Canada R3T 2N2

a r t i c l e

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Article history: Received 20 July 2011 Received in revised form 2 November 2011 Accepted 4 November 2011 Available online 12 November 2011 Keywords: Kyasanur Forest Disease Virus Flavivirus Tick-Borne flavivirus Reverse genetics system Interferon

a b s t r a c t Kyasanur Forest Disease Virus (KFDV) is a tick-borne, hemorrhagic fever-causing member of the Flaviviridae virus family. With infections annually ranging from 50 to 1000 people in south-west India and the lack of effective treatments, a better understanding of this virus is needed. The development of a reverse genetics system (RGS) for KFDV would provide the opportunity to address these issues. The KFDV genome sequence was elucidated and the RGS was created. Utilizing this system, live infectious KFDV particles were produced from mammalian cell culture, thereby validating the success of the RGS. Flaviviruses have the ability to suppress the type 1 interferon response and indications are that the non structural (NS) proteins serve this role. Using luciferase bioassays, the NS5 protein of KFDV was determined to be the primary antagonist of the IFN response when compared to the other NS proteins, specifically NS4B and NS4B-2k. Moreover, our results indicate that this is attributed to a region, beginning before and including the RNA-dependent RNA polymerase (RdRp). With evasion of the interferon response by KFDV established, the further implementation of the reverse genetics system will enable investigation into pathogenesis and disease progression of KFDV with respect to the innate immune response, at the IFN and the NS5 protein levels. Crown Copyright © 2011 Published by Elsevier B.V. All rights reserved.

1. Introduction Kyasanur Forest Disease Virus (KFDV) is an arbovirus, transmitted primarily by Ixodidae ticks. This member of the Tick-Borne Encephalitis serocomplex of the Flaviviridae (Calisher et al., 1989) was discovered in 1957 within the Shimoga district in south west India with an epizootic in monkeys, then a following outbreak in humans (Howley and Knipe, 2007; Work et al., 1959). Since the initial outbreak in 1957–1958 (Work et al., 1959), subsequent epizootic mortality of monkeys, the black-faced langur (Presbytis entellus) and red-faced bonnet (Macaca radiate), are thought to be closely linked to the human acquisitions of KFDV (Upadhyaya et al., 1975). Human infections may range from 50 to 1000 cases reported annually (Goodman et al., 2005; Pattnaik, 2006; Upadhyaya et al., 1975; Venugopal et al., 1994). KFDV is transmitted to incidental hosts, humans and monkeys, primarily by the Ixodes tick Haemaphysalis spinigera, which breaks the forest maintenance cycle (Dobler, 2010; Goodman et al., 2005; Pattnaik, 2006). The clinical

∗ Corresponding author at: 1015 Arlington Street, Winnipeg, Manitoba, Canada R3E 3P6. Tel.: +1 204 784 7505; fax: +1 204 784 5991. E-mail address: [email protected] (S. Theriault).

symptoms of a KFDV infection follow a biphasic course of illness. Beginning with a febrile period which includes “flu-like” signs, over half of those infected may progress into a second phase of illness (Goodman et al., 2005; Grard et al., 2007; Pattnaik, 2006). This phase can be comprised of central nervous system disorders and hemorrhagic fever manifestations (Adhikari Prabha et al., 1993; Howley and Knipe, 2007). Mortality rates from KFDV infections vary from 2 to 10% in humans (Pattnaik, 2006) and 85% in monkeys (Dobler, 2010). KFDV being a member of the Flaviviridae family has a singlestranded, RNA genome of 10,774 nucleotides (nt) in size with positive-sensed polarity, encased in an icosahedral nuclocapsid and surrounded by a lipid bilayer with two surface proteins. The genome encodes a polyprotein that is cleaved by viral and cellular proteases into three structural (C, prM/M and E) and seven nonstructural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) (Harris et al., 2006; Howley and Knipe, 2007). RNA secondary structures appear to be vital for successful translation and replication of the genome in the hosts’ cytoplasm (reviewed in references) (Davies and Kaufman, 1992; Harris et al., 2006; Kofler et al., 2006; Villordo and Gamarnik, 2009). Reverse genetics systems (RGS) for RNA viruses have allowed the rescue of RNA viruses from cloned cDNA. RGSs for Flaviviruses

0168-1702/$ – see front matter. Crown Copyright © 2011 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.virusres.2011.11.002

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B.W.M. Cook et al. / Virus Research 163 (2012) 431–438

involved rescue of viruses upon transfection of in vitro transcribed RNA from Yellow Fever Virus (Rice et al., 1989), Dengue Virus type 4 (Lai et al., 1991) and Japanese Encephalitis Virus (Yun et al., 2003). RGS systems for negative-stranded viruses including: Zaire ebolavirus (Volchkov et al., 2001), Rift Valley Fever Virus (Billecocq et al., 2008) and Influenza Viruses (Kawaoka, 2004), required the transfection of helper plasmids. In the case of Zaire ebolavirus, the helper plasmids provide the expression of the viral proteins (RNP complex) that are essential for replication and transcription, and T7 DNA-dependent RNA polymerase which is required for transcription of the cDNA genome in vivo. Utilizing the same methodologies and principles of the RGS for negative-stranded RNA viruses, we developed a similar system for KFDV. Interferon was first described for its’ ability to interfere with virus infection in 1957 by Isaacs and Lindenmann (Isaacs and Lindenmann, 1957; Isaacs et al., 1957). Upon infection by viruses, the cellular pathogen-recognition receptors (PRRs) recognize specific viral constituents (pathogen-associated molecular patterns or PAMPs) and express products such as, type 1 IFN and proinflammatory cytokines (Takeuchi and Akira, 2009). Type 1 IFN cytokines, as part of the cellular response to viral infection, confers an antiviral state. The antiviral state provides the surrounding, naïve cells to become resistant to viral infection (Hanley and Weaver, 2010; Pestka, 2007). Initiation of this state by IFN binding to cellular surface receptors, results in the subsequent induction of ISGs (IFN-stimulated genes) through the Jak/STAT pathway in a positive feedback mechanism. Expression of the ISGs requires the ISGF3 (IFN-stimulated growth factor 3) complex binding to an ISRE (IFN-stimulated response element). The ISRE enhancer region is present in the promoters of the ISGs, increasing IFN-induced expression of these genes which play roles in: stopping cell differentiation, halting cellular activities (translation, mRNA levels), apoptosis induction and antigen presentation (immunomodulation) (Meager, 2002; Randall and Goodbourn, 2008; Wathelet et al., 1998). Flaviviruses exploit the innate immune system by suppressing the type 1 IFN response via the non structural (NS) proteins. However the particular NS protein(s) involved and the mechanism of IFN-mediated signalling obstruction appear to be unique for each Flavivirus (Robertson et al., 2009). The virus and NS protein combinations studied thus far include: NS5 in Langat Virus (Best et al., 2005), Tick-Borne Encephalitis Virus, Japanese Encephalitis Virus, Dengue Virus and West Nile Virus (Laurent-Rolle et al., 2010), NS4B in Dengue Virus (effectiveness increased with the additions of NS2A and NS4A) (Munoz-Jordan et al., 2005) and NS2A in West Nile Virus (Liu et al., 2006). Moreover, for Dengue, West Nile and Yellow Fever viruses, the NS4B protein requires its N-terminal leader peptide (2k) (or pre-cleavage intermediate) for optimum suppression (Laurent-Rolle et al., 2010; Munoz-Jordan et al., 2005). The cessation of this level of the innate immune response appears vital to the sustainability of a Flavivirus infection. This idea is strengthened through the knowledge that IFN is a potent inhibitor of the replication capabilities of Flaviviruses (Best et al., 2005). Impairment of the type 1 IFN response leads to robust replication and increased tissue tropism in IFN ␣/␤−/− receptor mouse models infected with WNV (NY2000), including the central nervous system (Samuel and Diamond, 2005). In this report, we describe the generation of a RGS for KFDV and the examination of the abilities of this virus to block the cellular antiviral state. The function of the RGS in cell culture was confirmed by using reverse-transcription polymerase chain reaction (RT-PCR) and immunofluorescence assays. Investigation into the roles of the KFDV NS proteins, specifically the NS5 and its regions, in limiting the induction of the type 1 IFN response was determined in vitro with a luciferase bioassay. These results will pave the way for future research into the mechanism of KFDV evasion using the RGS as a tool, in vivo.

2. Materials and methods 2.1. KFDV infection, RNA harvest and reverse transcription KFDV P9605 strain (MOI 0.1) was propagated in VeroE6 cells within a Containment Level 4 (CL-4) laboratory. Infected cells were harvested at 72 h post infection when 70–80% cytopathic effect (CPE) was established. The supernatant from the harvest was concentrated on a 20% sucrose cushion at 50,000 g and extracted using TRIzolLS reagent kit (Invitrogen, Burlington, Ont., Canada), according to the manufacturer’s protocol. Samples were removed from CL-4 for RT-PCR under CL-3+ conditions. The sequencing of the full-length KFDV sequence involved primers developed using the polyprotein sequence on the NCBI website [Accession number: AY323490]. The polyprotein sequence was divided into four overlapping fragments and the 5 and 3 UTR regions, Fig. 1A. The single-stranded (ss) cDNA was generated using Thermoscript (Invitrogen) in a two-step reverse transcription reaction using the manufacturer’s protocol and subsequent incubation with RNaseH enzyme (New England Biolabs, Pickering, Ont., Canada) for 30 min at 37 ◦ C on the KFDV RNA template. Synthesis of the 3 UTR cDNA required the addition of a RNA adapter (5 P-dT(25)dd(quencher)3 ) (New England BioLabs) using T4 RNA ligase. The 5 UTR cDNA was amplified following the ligation of the 5 UTR to the 3 UTR using T4 RNA ligase I (New England BioLabs) following cap removal with Tobacco Acid Pyrophosphatase (TAP) (Epicentre Biotechnologies, Markham, Ont., Canada). All of the nascent cDNAs were used for the PCR reactions designed for genome sequencing and the construction of the RGS and helper proteins. 2.2. Sequencing of the full-length KFDV genome All PCR reactions were performed in a total volume of 50 ␮l, using iproof High-Fidelity DNA polymerase (Bio-Rad Laboratories, Mississauga, Ont., Canada), 10 ␮l 5× iproof HF buffer (1× final concentration), 1 ␮l dNTP mixture (200 ␮M each nucleotide final concentration), 0.5 ␮l 100 ␮M sense primer and 0.5 ␮l 100 ␮M antisense primer (1 ␮M final concentration each), 1 ␮l iproof HF DNA polymerase (2 Units/␮l final concentration), 1 ␮l cDNA template (RT mixture) and 36 ␮l sterile water (Sambrook and Russell, 2001). The cycling conditions were in accordance with the manufacturer’s conditions. The six amplified fragments encompassing the entire KFDV genome were blunt-end cloned into pCR4-TOPO sequencing vectors (Invitrogen) and transformed into Escherichia coli Top10 cloning strain (Invitrogen). Colony-PCR, plasmid propagation, plasmid harvesting and digestion of clones were performed using standard molecular techniques (Sambrook et al., 1989). Sequencing of each fragment was permitted by the fragment and internal primers. 2.3. Generation of the KFDV reverse genetics system Expression of the KFDV genome was designed for the eukaryotic expression vector, pTM1, in which we modified the multiple cloning site for our cloning purposes (Moss et al., 1990). Construction of the full-length cDNA RGS was performed in a step-wise manner building the entire genome, fragment by fragment using unique restriction sites in between each section. Each segment was PCR amplified from their pCR4-TOPO sequencing vector template with a restriction site on the 5 end of each respective primer, as outlined in Fig. 1A. The final step was the addition of the HDV ribozyme (Ciesiolka et al., 2001; Ebihara et al., 2005; Kawaoka, 2004). The beginning steps of the KFDV RGS-ribo construction was transformed into E. coli Top10 (Invitrogen), the latter fragments were transformed into E. coli XL10-Gold (Stratagene, Cedar


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Fig. 1. RGS cloning strategy of the KFDV full-length genome. (A) Development of KFDV RGS-ribo construct: KFDV full-length genome cloned into pTM1 expression vector under T7 DNA-dependent RNA polymerase control. (B) RT-PCR amplification of tissue culture supernatants to confirm KFDV rescue. Lane 1: KFDV RGS-ribo and T7-pCAGGS transfected tissue culture supernatant sample. Lane 2: negative control: non-transfected tissue culture supernatant sample. Lane 3: PCR positive control (KFDV RGS template). Lane 4: 1 kb+ DNA ladder (Invitrogen) on a 1% agarose gel. Predicted band size is 793 bp in length as visualized from the DNA marker. The color change seen in the gel is a by-product of image cropping. (C) Indirect immunofluoresence of KFDV virions. Ten independent transfections were performed in a 50:50 ratio of HEK293T and VeroE6 cells, blind passaged in BHK-21 cells, fresh BHK-21 cells were infected with rescued KFDV and fixed at 2 days post-infection. 1. Native KFDV infection control, 2. Infection negative control, 3. Positive KFDV rescue, 4. Negative KFDV rescue.

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Creek, TX, USA) due to low plasmid yield and all followed standard cloning procedures (Sambrook et al., 1989). Digestion and sequencing were performed at each cloning stage, for verification. 2.4. KFDV helper proteins and NS5 mutants Construction of the ten virally encoded proteins clones of KFDV and the NS4B-2k protein began, first with the mapping of each gene based on previously proposed protease recognition sites (Grard et al., 2007). Amplification of each gene was performed using primer sets synthesized with restriction sites on the 5 ends from the KFDV cDNA template. Accompanying the restriction sites was the addition of either a start (ATG) or stop (TAA) codon in order to complete the open reading frame when cloned into pCAGGS-MCS eukaryotic expression vector. The clones were confirmed by digestion and sequencing. The NS5 mutants were generated by site-directed mutagenesis using NS5-pCAGGS as a template. Five mutations corresponding to the published polyprotein sequence [AY323490/AAQ91607] which indicated the MTase and RdRp regions (Grard et al., 2007). The mutants are numbered in accordance with our full-length sequenced genome (GenBank accession number: HM055369). The mutants: 6 maintained the native viral serine protease cut site, then had amino acids 6–54 of the NS5 open reading frame (7686–7832 base pairs) deleted while being in frame with the NS5-pCAGGS start and stop codons, 55 MTase region deletion corresponded to amino acids 55–222 (7833–8336), 223 removed the amino acid region of 223–431 which is in between MTase and the RdRp (8337–8963), 432 was without the RdRp region (amino acids 432–742) (8964–9896) and 743 the 743–903 amino acid region after the RdRp and before the stop codon was deleted (9897–10,379). 2.5. KFDV rescues Rescues were set up with a 50:50 ratio of HEK293T (Human embryonic kidney) and VeroE6 (African green monkey kidney) tissue culture monolayers split into 6 well plates with the intent of having ∼80% confluency, 24 h before the intended rescue. All transfection steps were the same in terms of media and transfection reagent for each sample which included the KFDV RGS-ribo clone and T7 DNA-dependent RNA polymerase expression clone. Briefly, sterile tubes, 100 ␮l of serum-free media Opti-MEM (Gibco, Burlington, Ont., Canada), a 2 ␮l aliquot of TransIT-LT1 (Mirus, Ottawa, Ont., Canada) transfection reagent was added and incubated for 5 min at room temperature. 1 ␮g of each DNA plasmid construct was added, followed by 30 min incubation at room temperature. A subsequent 900 ␮l addition of Opti-MEM completed the preparation of the DNA complexes for transfection. The growth medium from each well was removed and the transfection mixtures were then applied to their respective wells, one for RT-PCR and ten for IFA analysis. The tissue culture plates were transferred in the CL-4 suite and subjected to an overnight incubation at 37 ◦ C with 5% CO2 . After incubation but, before 24 h the transfection mixture for each well was replaced with 4 ml of Dulbecco’s Modified Eagle Medium (DMEM) (Gibco) supplemented with 2% Fetal Bovine Serum (FBS) (Gibco) and 1% Penicillin–Streptomycin (PenStrep) (Gibco). The plates were reincubated for an additional 4 days under the same conditions in CL-4, after which, each well was blind passaged in T 25 tissue culture flasks of BHK-21 (Baby hamster kidney) cells, prepared for 80% confluency after 24 h incubation. Confirmation of a successful rescue was performed when significant CPE developed.

2.6. Confirmation of KFDV rescues 2.6.1. RT-PCR Once a significant level of CPE was viewed from the blind passage, tissue culture media was collected, inactivated with RLT buffer from the TRIzolLS reagent kit (Invitrogen) and safely removed from the CL-4 suite. While in CL-3+ laboratory conditions, the KFDV genomic RNA was harvested with RNeasy kit (Qiagen) and subjected to RT-PCR using Qiagen OneStep RT-PCR (Qiagen) kit using manufacturer’s protocols and an internal primer set specific to KFDV. These contents were mixed and subjected to the following cycling conditions: 50 ◦ C for 30 min, 95 ◦ C 15 min, 28 cycles of 95 ◦ C for 1 min, 58 ◦ C for 1 min and 72 ◦ C for 1 min, respectively, a final extension of 72 ◦ C for 10 min. The completed reactions were treated with RNAse H enzyme (New England Biolabs) and then run on a 1% agarose gel. 2.6.2. Indirect immunofluorescence assay BHK-21 cells were grown in 6 well plates were infected with 1.5 ml of rescued KFDV from the blind passage and 1 ml native KFDV (5.4 logs TCID50 ) for 30 min at 37 ◦ C with 5% CO2 . The media was replaced with fresh DMEM, supplemented with 2% FBS and 1% antibiotics. After an incubation period of 2 days, the cells were fixed and virus was inactivated with 10% formalin for 2 days with one fixative exchange. The cells were then removed from containment and washed three times with Phosphate buffered saline (PBS). Permeabilization with 0.1% Triton X-100 in PBS for 30 min at room temperature was immediately followed with PBS washing. The cells were blocked for 1 h at 37 ◦ C with 5% goat serum (Sigma–Aldrich, St. Louis, USA) in PBS. The primary antibody (1:250 dilution in PBS with 5% goat serum) was a polyclonal mouse-derived anti-KFDV ascitic fluid, kindly provided by the World Reference Center for Emerging Viruses and Arboviruses (WRCEVA) in association with the National Institute of Allergy and Infectious Diseases (NIAID). After an incubation of 1 h at 37 ◦ C, the cells were washed with PBS. The secondary antibody was a goat-derived fluorescein isothiocyanate (FITC) labeled anti-mouse conjugate (a 1:250 dilution in PBS and 5% goat serum), incubated for 1 h at 37 ◦ C and subsequently washed with PBS. The labeled cells were analyzed using an Axioplan 2 Fluorescence microscope (Zeiss, Germany). 2.7. Interferon assays HEK293T cells were passaged into 24 well tissue culture plates for 80% confluency, 24 h prior to the transfections. The transfections using Attractine transfection reagent (Qiagen) were performed according to the manufacturer’s protocols with Opti-MEM media (Gibco). Each of the KFDV NS proteins, including NS4B-2k and the NS5 mutants, previously cloned into pCAGGS-MCS was assessed individually and in combination with the ISRE-lucifease reporter (SA Biosciences). After an incubation period of 24 h, the transfection mixtures were removed, washed with PBS and replaced with DMEM containing 10% serum with or without 1000 U/ml of Universal type 1 IFN ␣ (a recombinant human IFN hybrid) (PBL Interferon Source) supplemented. The cells were washed with PBS and harvested following the 18 h incubation at 37 ◦ C and 5% CO2 . The Luciferase activity was examined using the Dual-Luciferase Reporter (DLR) assay kit (Promega) with a luminometer (Tecan Group Ltd.) using 96 well white flat-bottom plates (Fisher Scientific, Canada) and normalized with internal Renilla luciferase control. 3. Results and discussion Emerging infectious diseases, like KFDV, are an ever increasing threat to the global population. Viral Hemorrhagic fever (VHF) viruses may have extremely high case fatality rates of up to 90%,


as in the case of Zaire ebolavirus (Ebihara et al., 2005). The risk posed by these pathogens’ possible global dissemination should not be ignored, as travel and global shipment have increased (Peters, 2006). Increased dispersal of Flaviviruses can be seen in Powassan Virus (1958) and West Nile Virus (1999) being recently introduced into North America (Hinten et al., 2008; Howley and Knipe, 2007), Alkhurma Hemorrhagic Fever Virus in Egypt in 2010 (Carletti et al., 2010; Ravanini et al., 2011) and Kyasanur Forest Disease Virus variant Nanjianyin virus in China during the 1980s (Wang et al., 2009), this isolate has been called into question (Mehla et al., 2009). Another concern is the development of an “urban cycle” involving human reservoirs as seen in Dengue Virus, potentially alleviating the requirement of a “forest cycle” (Hanley and Weaver, 2010; Howley and Knipe, 2007). The creation of the RGS for KFDV may help research directions in relation to VHF, widespread transmission, human reservoir potential and developing new platforms to replace the current failing vaccine in India (Pattnaik, 2006). The design of the KFDV reverse genetics system (RGS) involved dividing the published polyprotein sequence [AY323490] into four overlapping fragments and separately cloning 5 and 3 UTRs (Fig. 1A). The resulting six fragments were sequenced and cloned into the pTM1 mammalian vector, under T7 DNA dependent RNA polymerase promoter control. The genomic ends were subjected to RT-PCR after self-ligation and adaptor addition steps were performed, as described in Section 2. The sequencing of the KFDV genome by our group, to our knowledge, produced the first annotated full-length KFDV virus sequence [GenBank accession number: HM055369] which is 10,774 nt in length. Comparison with the published polyprotein sequence [AY323490] of 10,376 nt in length exposed ten base polymorphisms. In total, there are five transitions, four transversions and one deletion: the transitions were at nucleotide positions: C 3489 T, C 4962 T, A 6559 G, G 6910 A and A 10,029 G, i.e. position 3489 differed between AY323490 and our strain. Transversions were noted at positions: C 3488 G, T 5528 A, C 9147 A and C 9150 G, the T 10,309 – was deleted in our strain. However, this deletion was downstream of the NS5 gene sequence and thus outside of the open reading frame of the KFDV polypeptide. At the amino acid level, only four differences were identified within the coding region of KFDV: T 1163 S (C 3488 G and C 3489 T), T 2187 A (A 6559 G), A 2304 T (G 6910 A) and V 1843 E (T 5528 A). Perhaps these differences are due, in part to tissue culture adaptation. RNA secondary structures or stem-loop structures have been previously described in the genomes of Tick-Borne and MosquitoBorne Flaviviruses, aiding in replication and translation of their genomes (Davies and Kaufman, 1992; Harris et al., 2006; Kofler et al., 2006; Villordo and Gamarnik, 2009). KFDV being a member of the Tick-Borne flaviviruses appears to be no different with respect to possessing the suggested secondary structures within the 5 and 3 UTR regions as compared to Tick-Borne Encephalitis VirusNeudoerfl strain, as predicted by the default settings of the RNAfold WebServer program (data not shown) (Gruber et al., 2008). Phylogenetic analysis of the full-length KFDV P9605 genome in relation to other Flaviviruses, both Mosquito-Borne (MB) and Tick-Borne (TB), using the DAMBE program (Xia and Xie, 2001) and ClustalW alignment and Maximum Likelihood (ML) approaches (Baxevanis and Ouelette, 2001) indicated that KFDV: clusters within the TB Flavivirus cluster (TBE serocomplex) as expected and forms a distinct clade with AHFV (Alkhurma Hemorrhagic Fever Virus) (data not shown). Our result is similar to the described evolution of Flaviviruses described in the literature (Charrel et al., 2005; Grard et al., 2007; Howley and Knipe, 2007; Lin et al., 2003; Wang et al., 2009). The KFDV RGS was based on a previous model for Zaire ebolavirus RGS using a transcription mechanism for rescue attempts in the eukaryotic host’s cytoplasm using RNA polymerase II and T7 promoter-driven clones (Ebihara et al., 2005; Neumann et al., 2002; Theriault et al., 2004; Volchkov et al., 2001). The

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transfection procedure was set up as described in Section 2. About 4–5 days post transfection, if present, rescued virions will have egressed into the extracellular environment. The blind passage procedure will remove these infectious KFDV particles from their respective wells and used to infect a fresh monolayer of BHK-21 cells in the T-25 culture flasks. Incubation for up to 14 days may be necessary to see any signs of significant CPE (Theriault et al., 2004). Approximately 60 percent CPE was seen, up to and after 14 days post infection (data not shown due to technical limitation in CL-4), after which each T-25 flask was processed for RNA harvesting from the culture media and to re-infect fresh BHK-21 cells for the indirect immunofluorescence assays. The successful detection of the rescued KFDV in the tissue culture media from the blind passage procedure was detected genetically. Extracted viral RNA was subjected to RT-PCR using internal KFDV primers within the NS3 gene (spanning fragmets two and three). Positive reactions produced an amplicon of approximately 793 bp (Fig. 1B). The existence of KFDV infectious virus particles were found by immunofluorescence assays of freshly infected tissue culture (naïve BHK-21 cells) which were incubated with post blind passage tissue culture media for approximately 30 min, to initiate infection. After 48 h, cells were fixed, followed by permeabilization of KFDV-infected cells. The cells were incubated with anti-KFDV polyclonal sera obtained from mice and anti-mouse FITC-conjugated monoclonal antibodies, provided the identification of infectious virus particles as seen in Fig. 1C. Out of the ten transfections, eight were positive and two were negative, giving an overall efficiency of 80% for the RGS. Upon blind passage, both samples lacked any distinguishable CPE from the negative control (data not shown). However, these samples were treated the same as the other samples which displayed significant CPE. The RT-PCR and immunofluorescence assays results, validate the RGS’ ability to produce infectious KFDV particles entirely from cloned cDNA in vivo. Many full-length cDNA clones created for Flavivirus have been previously reported as being problematic in E. coli cloning strains, resulting in non-sense mutations and low plasmid yield (Hurrelbrink et al., 1999; Rice et al., 1989; Sumiyoshi et al., 1992; Yun et al., 2003). During assembly of our full-length RGS for KFDV, low plasmid yields were observed within the Top10 cloning E. coli strain with the addition of the third fragment. This fragment comprises the latter 1.1 kb of the NS3 gene up to the first 1.2 kb of the NS5 gene. Perhaps, the low yields were due to the plasmid size. To overcome this, switching cloning strains to XL10-Gold for the latter clones allowed for greater bacterial growth and plasmid harvesting to be attained. Therefore, it is reasonable to assume that the low plasmid yields were not due to toxic viral protein products, expressed from a cryptic bacterial promoter located within the KFDV polyprotein. This appears different from what was seen in the Dengue Virus cDNA infectious clone and perhaps these cryptic promoters are present solely, in the 5 UTRs of Mosquito-Borne Flaviviruses, as insinuated by Li et al. (2011). However, our low plasmid harvests were not evident until the third fragment of the full-length genome was cloned. We cannot rule out the possibility of cryptic promoters within this region, acting in a non-canonical fashion with respect to prokaryotic transcription. In regards to genetic stability within the polyprotein area, mutations were not seen with either Top10 or XL10-Gold cloning strains. Flaviviruses have been reported to inhibit the hosts’ type 1 IFN response via their non structural (NS) proteins and halt the cellular antiviral state (Laurent-Rolle et al., 2010; Robertson et al., 2009). The mosquito-borne group: Japanese Encephalitis Virus (JEV), Dengue Virus (DV) and West Nile Virus (WNV) (LaurentRolle et al., 2010; Liu et al., 2006; Munoz-Jordan et al., 2005) and the tick-borne group: Langat Virus (Best et al., 2005), Tick-Borne Encephalitis Virus (TBEV) (Laurent-Rolle et al., 2010). However, the NS proteins of KFDV have not been investigated in the same context. An in vitro system was used to mimic the induction of the

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E VP24 NS5 NS4B-2k (2A/4A) NS4B (2A/4A) NS4B-2k NS4B NS4A NS2A IFN Induction Induction Control Negative Control Positive Control 0

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Fig. 2. Luciferase bioassay to assess the capabilities of KFDV NS proteins to inhibit the antiviral state induced by Universal Type 1 IFN ␣ on ISRE-luciferase reporter using HEK293T cells. Top: Positive induction control: individual Firefly and Renilla luciferase constructs under non-IFN inducible CMV promoters. Negative induction control: promoter-less Firefly luciferase and Renilla luciferase construct under CMV promoter excluding the IFN-mediated enhancer. Induction control and IFN induction: Firefly luciferase under basal promoter element (TATA box) and tandem repeats of IFN-inducible ISRE enhancer with and without IFN added, and a Renilla luciferase construct under a CMV promoter without IFN-mediated enhancer. The induction control constructs were added to all subsequent transfections. Ebola Virus-Zaire VP24 clone acted as a positive control for anti IFN-mediated signalling. In frame KFDV NS protein clones were transfected and assessed for luciferase induction with IFN supplemented. The KFDV E clone was used as a control for non anti-IFN activity. Average RLU units were determined from normalized luciferase activity in triplicate experiments. Bottom: Fold induction of luciferase activity. Average normalized RLU for each sample was divided by the IFN induction sample (100%) and converted to a percentage.

type 1 IFN response using an ISRE-luciferase reporter system (SA Biosciences). Expression of luciferase is powered by the addition of the exogenously added human IFN ␣ (PBL Interferon Source) causing the functional Jak/STAT signalling cascade to act on the ISRE of the luciferase construct and inducing expression of endogenous IFN from HEK293T. Thus, amplifying, not only more IFN, but also the subsequent expression of luciferase. Upon co-transfection with the KFDV NS protein constructs (under the chicken beta-actin mammalian promoter control) both individually and in combination (NS2A, NS4A and NS4B and/or NS4B-2k), analogous to those reported for West Nile, Dengue and Yellow Fever Viruses, if the Jak/STAT pathway is negatively impacted, this will be reflected in the blocking of the induction of the reporter when IFN ␣ is supplied. A published type 1 IFN antagonist, VP24 of Zaire ebolavirus (Reid et al., 2006) served as a positive control for comparison. The E protein of KFDV was included as a negative control because there has been no reported anti-IFN activity associated with the structural proteins of Flaviviruses (Fig. 2). Based on this assay, the NS5 protein of KFDV appears to act as the strongest repressor of luciferase expression, a decline of 9.1 RLU (98%) when compared to the total amount IFN induction (9.3 RLU or 100% induction). Such a result suggests a hindrance of the antiviral state, when compared to the positive control and published IFN-mediated signalling antagonist,

VP24, a decrease of 9.0 RLU (97%) was observed. The other NS proteins may play secondary roles in inhibition, NS4B with and without the 2k peptide (in combination with NS2A and NS4A) causing a lessening of 7.8 RLU (83%) and 7.0 RLU (75%), respectively, as seen in Fig. 2. Additionally, the NS2A protein demonstrated stronger activities (7.2 RLU/77%) than NS4B (5.6 RLU/60%) and NS4B-2k (6.2 RLU/67%). Interference of IFN ␤ transcription and IFN-mediated signalling by the NS2A of both WNV (Liu et al., 2006) and DV-2 (Munoz-Jordan et al., 2003) has been previously described. Perhaps the NS2A of KFDV plays the secondary role in Jak/STAT pathway inhibition which may or may not include the NS4A and/or NS4B (2k) proteins acting in concert. However, the evidence suggests that these proteins are minor players, at least with respect to this in vitro investigation. What remains to be investigated, is the manner in which the Jak/STAT cascade is affected by KFDVs NS proteins, some of these mechanisms have already been addressed. The VP24 protein of Zaire ebolavirus is a structural protein which besides IFN suppression, is involved in nucleocapsid formation, assembly and virion release. The anti-IFN action has been shown to affect the translocation of the phosphorylated STAT1:STAT2 heterodimer (Reid et al., 2006). In contrast, Flaviviruses appear to use their replication-based, non structural proteins for IFN-mediated signalling suppression. For Dengue and West Nile viruses, the dual actions of NS5 and the partnership of NS2A, NS4A and NS4B-2k, target STAT2 for degradation and arrest the activation of STAT1 (phosphorylation), respectively (Hanley and Weaver, 2010; Laurent-Rolle et al., 2010; Munoz-Jordan et al., 2005). The NS5 protein has two main functions: the amino terminus has methyltransferase (MTase) and guanyltransferase activities, adding a guanosine mono-phosphate (GMP) cap to the nascent genomic RNA and to perform two subsequent methylation events (Gehrke et al., 2003; Howley and Knipe, 2007; Issur et al., 2009). The carboxyl terminus houses the RNA dependent RNA polymerase (RdRp), which is essential for genome replication (Howley and Knipe, 2007). The latter region contains the amino acid region(s) responsible for the blocking of STAT1 phosphorylation in both Dengue and West Nile viruses (F653) (Laurent-Rolle et al., 2010). For Langat Virus, two sites within the 355–735 residue region of the RdRp, harbours the still unknown action(s) of Jak/STAT pathway interference (Park et al., 2007). These two sites located in the amino (374–380) and carboxyl-termini (624–647) which orientate towards each other potentially acting in concert, to modulate the IFN response (Park et al., 2007). In an attempt to narrow-down the region of the KFDV NS5 protein that is responsible, we deleted the regions outside of and including the MTase (methyl-tranferase) and the RdRp (RNA-dependent RNA polymerase). Consecutive regions of the NS5 were deleted, while maintaining transcriptional codons and the polyprotein cleavage sequence for the viral serine protease. Mutants: 6, 55, 223, 432, 743 deleted the region before the MTase, the MTase, in between MTase and RdRp, the RdRp and after the RdRp, respectively. These clones were assayed for their Jak/STAT pathway hindrance using the same luciferase bioassay conditions (Fig. 3), mentioned previously. Our results would suggest that the RdRp of KFDV also carries the role for the inhibition of IFN-mediated signalling. This region(s) appears to span from just prior to the RdRp and through the RdRp, amino acid regions 223–431 and 432–742, respectively. This became evident as luciferase induction was not diminished by either of the 223 or the 432 mutants. What remains to be elucidated is the manner at which this occurs and if this activity is linked to the replication capabilities of KFDV. It has been noted that these two functions may or may not be, in fact linked (Park et al., 2007). It should be noted that there appears to be a reduction in luciferase activity seen with the pCAGGS vector. With multiple repetitions, along with other mammalian expression vectors (pBK-CMV and pTM1), these reductions were maintained (data


6

55 (MTase)

223

432 (RdRp)

RGS. Perhaps, the interruption of the IFN-mediated signalling in mice may correlate to human infections.

743

pCAGGS

4. Conclusions

743 432 223 55 6 IFN Induction Induction Control Negative Control Positive Control 0

437

4.00

8.00

12.00

16.00

20.00

Reverse Genetic Systems offer a wide variety of research directions in the realm of public health. Such a system for KFDV will enable our laboratory to study the pathogenesis and tissue tropism at the molecular level. Investigation into the roles of the innate immune response and replication competence, specifically the KFDV RdRp and its interaction with IFN ␣/␤-mediated signalling, may provide insight which may lead to novel therapeutics and more effective treatment strategies.

Average Normalized LuciferaseActivity (RLU)

Acknowledgements pCAGGS

This work was supported by funds from the University of Manitoba. We would like to thank the Canadian Science Center of Human and Animal Health’s support staff for oligo synthesis and sequencing, and the Special Pathogens program. Thank you to the World Reference Center for Emerging Viruses and Arboviruses (WRCEVA)/National Institute of Allergy and Infectious Diseases (NIAID) for the primary antibody.

743 432 223 55 6 IFN Induction Induction Control Negative Control

References

Positive Control

0

25

50

75

100

Fold Induction (Percent)

Fig. 3. Luciferase bioassay to determine which regions of the NS5 protein of KFDV is responsible for limiting the antiviral state induced by Universal Type 1 IFN ␣ on ISRE-luciferase reporter using HEK293T cells. Top: Positive induction control: individual Firefly and Renilla luciferase constructs under non-IFN inducible CMV promoters. Negative induction control: promoter-less Firefly luciferase and Renilla luciferase construct under CMV promoter excluding the IFN-mediated enhancer. Induction control: Firefly luciferase under basal promoter element (TATA box) and tandem repeats of IFN-inducible ISRE enhancer with and without IFN added, and a Renilla luciferase construct under a CMV promoter without IFN-mediated enhancer. The induction control constructs were added to all subsequent transfections. The pCAGGS (empty) vector was added as a control for non anti-IFN activity. Average RLU units were determined from normalized luciferase activity in triplicate experiments. In frame NS5 region deletions were transfected and assessed for luciferase induction with IFN supplemented. Bottom: Fold induction of luciferase activity. Average normalized RLU for each sample was divided by the IFN induction sample (100%) and converted to a percentage.

not shown). Nevertheless with respect to the NS5 and the differences with its mutations, show distinct variations in the lessening of IFN-induced luciferase activity. Thus, an additional role in host innate immune suppression can be supported by the fact that the NS5 protein, has been found to be associated not only with the RC, but also in the cytoplasm and the nucleus (Davidson, 2009). One thought is, that the NS5 associates with the intracellular side of the cellular membrane in order to counteract the phosphorylation of STAT1. Perhaps this is possible through association with the IFNAR2 receptor (Best et al., 2005) and guided by the host protein, Scribble (hScrib) interacting with the MTase domain of NS5 (Werme et al., 2008). From an infection perspective, the potential for Flaviviruses to oppose the antiviral state has been shown to be detrimental to mouse survivability (Hanley and Weaver, 2010; Samuel and Diamond, 2005). The lack of a type 1 IFN response in mouse models, demonstrates high virus titres and expanded tissue tropism, including CNS (central nervous system) tissues in mice which have the IFN ␣/␤ receptor deleted (Daffis et al., 2007, 2009; Robertson et al., 2009; Samuel and Diamond, 2005). Thus, the KFDV NS proteins, specifically the NS5s RdRp, may give further insight into the pathogenesis of KFDV through mutational analysis and subsequent recombinant KFDV rescue using the

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