Successful Propagation of Flavivirus Infectious cdnas by a Novel Method to Reduce the

Transcription

1 JVI Accepts, published online ahead of print on 1 January 0 J. Virol. doi:./jvi.01- Copyright 0, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. 1 Successful Propagation of Flavivirus Infectious cdnas by a Novel Method to Reduce the Cryptic Bacterial Promoter Activity of Virus Genomes Szu-Yuan Pu 1,, Ren-Huang Wu 1,, Chi-Chen Yang 1,, Tzu-Ming Jao 1, Ming-Han Tsai 1, Jing-Chyi Wang 1, Hui-Mei Lin 1,Yu-Sheng Chao 1, and Andrew Yueh 1,* 1 Division of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Miaoli 0, Taiwan, R.O.C. Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan, R.O.C. * Corresponding author andrewyeuh@nhri.org.tw Phone: ---1 ext.1. Fax: ---. Both authors contributed equally to this work Running title: A novel way to clone flavivirus infectious cdnas 1

2 Abstract Reverse genetics is a powerful tool to study single-stranded RNA viruses. Despite tremendous efforts made to improve the methodology for constructing flavivirus cdnas, the cause of toxicity of flavivirus cdnas in bacteria remains unknown. Here we performed mutational analysis studies to identify E. coli promoter (ECP) sequences within nucleotide (nt) of the dengue virus type (DENV) and Japanese Encephalitis virus (JEV) genomes. Eight and four active ECPs were demonstrated within nt of DENV and JEV genome, respectively, using fusion constructs containing DENV or JEV segments and empty vector reporter gene Renilla luciferase. Full-length DENV and JEV cdnas were obtained by inserting mutations reducing their ECP activity in bacteria without altering amino acid sequences. Severe cytopathic effect occurred when BHK1 cells were transfected with in-vitro transcribed RNAs either from a DENV cdna clone with multiple silent mutations within prm-e-ns1 region of dengue genome or a JEV cdna clone with an A-to-C mutation at nt 0 of JEV genome. The virions derived from the DENV or JEV cdna clone exhibited similar infectivity as their parental viruses in C/ and BHK1 cells. A cis-acting element essential for virus replication was revealed by introducing silent mutations into the central portion (nt -) of core gene of DENV infectious cdna or subgenomic DENV replicon clone. This novel strategy constructing DENV and JEV infectious clones could be applied to other flaviviruses or pathogenic RNA viruses to facilitate research in virology, viral pathogenesis, and vaccine development.

3 Introduction The Flavivirus genus consists of more than 0 members that are categorized into several antigenic groups (). Most flaviviruses are transmitted by mosquito or tick vectors and cause serious human and animal diseases (). They include dengue virus (DENV), Japanese encephalitis virus (JEV), West Nile virus (WNV), yellow fever virus (YFV), and tick-borne encephalitis virus (TBEV). DENV and JEV cause some of the most serious arthropod-borne viral illnesses. There are four different serotypes of dengue virus, DENV1, DENV, DEN, and DEN. DENV cases have been reported in over 0 countries, and an estimated. billion people live in areas in which dengue is epidemic (,, ). DENV infection often leads to dengue fever, dengue hemorrhagic fever, and dengue shock syndrome (,, ). JEV transmission has been observed in the southern hemisphere and has the potential to become a worldwide public health threat. JEV can cause permanent neuropsychiatric sequelae and is sometimes fatal in children (, 0, 1). Flaviviruses are enveloped RNA viruses that consist of single-stranded, positive-sense,.- to -kb genomic RNA. The genome is associated with multiple copies of capsid proteins that are translated as a single polyprotein. After entering a host cell, the translated polyprotein is then cleaved into three structural proteins (C, prm, and E) and seven non-structural proteins (NS1, NSA, NSB, NS, NSA, NSB, and NS) by host proteases and a single virus-encoded protease to initiate viral replication (1, 1). Introduction of flavivirus genomic RNA into susceptible cell lines can result in the production of infectious virus particles (1). This phenomenon has prompted the study of flavivirus virology via introduction of flavivirus genomic RNA that has been transcribed in vitro from full-length flavivirus infectious cdna. Reverse genetics is a powerful method for studying the viral replication of positive-strand RNA viruses (). Unfortunately, the instability of full-length flavivirus cdna in E. coli has been a major hurdle in a attempt to construct flavivirus cdnas (reviewed in (,, )). Several strategies have been developed to avoid or overcome the instability of infectious flavivirus cdna. The instability of

4 plasmids containing full-length YFV was avoided using an in vitro ligation approach derived from two plasmids (). The in vitro ligation method has also been successfully applied to other flaviviruses, such as JEV (), DENV (1), DEN (), and TBEV (0). A second approach involves a special E. coli strain to construct stable full-length DEN cdna (0). This method has been used to construct cdnas of a DENV1- chimera (), DENV (), DEN () and TBE (0), but it has failed in the preparation of DEN cdna (1). A third approach involves the use of medium or low copy-number plasmids to stabilize full-length flavivirus cdnas. This strategy has been employed with different strains of DENV cdnas (,, 1, 1), West Nile virus cdna (, ), Kunjin virus cdna (, ), and TBEV cdna (). Two other modified methods have been derived from this strategy to facilitate the construction of flavivirus cdnas. One is based on homologous recombination of yeast for the assembly of full-length DENV (1), DENV1 (), and DEN ()cdnas. The other modification uses low copy-number vectors to construct TBE-infectious clones in AbleK cells by ligating an kb PCR fragment of full-length TBEV cdna into a vector (). Despite reports of successful construction of flavivirus cdnas from the above strategies, many of these plasmids are still deleterious for E. coli and result in slow growth and low yield of cdnas (reviewed in (,, )). Several attempts have failed to construct genetically stable JEV cdnas using low, medium, and high copy-numbers of vectors or special bacterial hosts (,, ). One special design inserts an intron containing a stop codon into the C terminus of a JEV core gene that markedly stabilizes the JEV infectious cdna clone in bacteria (). In another approach, full-length JEV cdnas were successfully harbored by cloning the JEV genome into bacterial artificial chromosome (BAC) vectors (0). YFV (), DENV1 (), and DENV () infectious cdnas can be assembled into BAC vectors, which suggests that bacteria can withstand any toxicity that arises from flavivirus cdna cloned into a BAC vector. Similarly, the BAC vector has also been used to construct other RNA virus cdnas, e.g. transmissible gastroenteritis coronavirus (TGEV) cdna () with poison

5 sequences that make the infectious cdna clone plasmid unstable. However, the genetic stability of TGEV cdna in a BAC vector lasts for about 0 generations in E. coli (0). This indicates that the intact full-length TGEV cdnas in the BAC vector still possess some intrinsic toxicity to E. coli cells. The fact that BAC plasmid was not commonly used for cloning most of coronavirus infectious cdna clones, e.g. TGEV (), avian infectious bronchitis virus (), mouse hepatitis virus (), severe acute respiratory syndrome coronavirus (), suggesting that the BAC vector is not so feasible to overcome the intrinsic toxicity in the sequences of most coronavirus genome. Little is known about what causes the toxicity of flavivirus cdnas in E. coli. We sought to develop a reliable and convenient method for constructing stable full-length flavivirus or other RNA virus cdna by reducing the intrinsic toxicity of the viral genome sequence in E. coli. In the present study, we took a novel approach to assembling infectious DENV and JEV cdna clones by introducing silent mutations into the putative E. coli promoter (ECP) sequences within the DNEV and JEV genomes. A cis-acting element located at the central portion of DENV core gene was revealed to be for essential DENV replication. Thus, a feasible reverse genetics method was established to prepare full-length infectious DENV and JEV cdna clones. This method will greatly facilitate the manipulation, e.g. sited-directed mutagenesis, deletion, and insertion, of difficultly manipulated pathogenic flavivirus or other RNA virus cdna clones. The feasibility and convenience of handling toxic flavivirus cdna clones in bacteria by our methodology will speed up the study in virology, virus pathogenesis and vaccine development.

6 Materials and Methods Reagents. Dulbecco s modified Eagle s medium (DMEM), Alpha-modified minimal essential medium (alpha-mem), fetal bovine serum (FBS), and RPMI were obtained from Invitrogen (Carlsbad, CA). Unless otherwise specified, all chemicals were purchased from Sigma (Poole, UK). Cell lines and virus strains. The baby hamster kidney (BHK1) clone 1 cells were kindly provided by Dr. Beatty (Department of Molecular and Cell Biology, University of California at Berkeley, California, USA) and cultured in alpha-mem supplemented with % FBS at C in a % CO incubator. Aedes albopictus C/ cells (ATCC CRL-) were cultured in RPMI supplemented with % FBS and 1% non-essential amino acids at C in a % CO incubator. Virus-infected BHK1 cells were grown in their respective medium supplemented with % FBS. DENV (Taiwanese PL0 strain) (1) and JEV (RP- strain) (1) were both kindly provided by C.L. Liao (Institute of Biomedical Sciences, National Defense Medical Center, Taiwan). A virus stock was prepared in C/ cells by infecting at an appropriate multiplicity of infection (MOI) in RPMI medium containing % FBS and incubated at C until the appearance of cytopathic effects. The supernatant was then harvested and stored in 0% FBS at -0 C. Virus titers were determined using a plaque-forming assay in BHK1 clone 1 cells. E. coli, yeast methods, and strains. Frozen, competent E. coli strain C1, a derivative of BL1(DE)(), was purchased from OverExpress Inc. C1 was cultured in YT (1 g of Bacto Trypton, g of Yeast extract and g of NaCl in 1 L distilled water, ph.) medium or agar plate. Standard yeast medium and methods were used (). Saccharomyces cerevisiae YPH was purchased from ATCC. The genotype YPH is MATα ade-1 lys-01 ura- trp1- HIS CAN1 his- 00 leu- 1 cyh. Competent yeast cells were prepared using the lithium acetate procedure (). Plaque-forming assay. BHK1 clone 1 cells were plated at a density of cells per well in 1-well plates containing 1 ml of medium and cultured overnight. Then, 0.1 ml of serially diluted

7 virus solution was added to ~0-0% confluent BHK1 cells. After a -h adsorption period, the virus solutions were replaced with either 0.% methyl cellulose (Sigma, M-01)-containing DMEM and % FBS for DENV-infected cells or 1.% methyl cellulose-containing DMEM and % FBS for JEV-infected cells. On the fifth or third day after Dengue or JEV infection, respectively, the methyl cellulose solution was removed from the wells and the cells were fixed and stained with a crystal violet solution (1% crystal violet, 0.% NaCl and % formaldehyde) (). The plaque forming unit (PFU) per ml of DENV or JEV was then determined. E. coli promoter prediction in the DENV and JEV genome. To search for potential E. coli promoter regions within the DENV or JEV genomes, the web site ( /promoter.html) search program (Neural Network promoter prediction) from the Berkeley Drosophila Genome Project was used to analyze the complete DENV or JEV genome sequence. Preparation of viral RNAs and viral cdnas by reverse transcription. To prepare viral RNAs, DENV and JEV viruses were grown and amplified in C/ cells. The amplified DENV or JEV viruses were harvested and measured using a plaque assay to determine the titers. We applied approximately 00 µl PL0 or RP virus (1 X PFU/ml) to purify viral RNA using the Qiagen RNeasy Kit, as described in the manufacturer s protocol. Viral RNAs served as templates for the reverse transcription (RT) of viral RNAs using the Transcriptor first strand cdna synthesis kit (Roche Biochemicals) with primer -AGAACCTGTTGATTCAACA- for DENV or primer -AGATCCTGTGTTCTTCCT- JEV, according to the manufacturer s protocol. The RT products of the DENV and JEV viral RNAs served as templates for the synthesis of viral cdnas by PCR. Construction of DEN-Luc, JEV-Luc, DENV mutant, JEV-A0C and DENV and mutant replicon clones. The detailed methods regarding the construction of all clones are described in the Supplementary Materials and Methods. In vitro transcription and transfection. Full-length DENV and JEV cdna clones were linearized with XbaI or KpnI, respectively, extracted with phenol-chloroform, ethanol precipitated,

8 and re-dissolved in RNase-free water. The in vitro transcription reaction mixtures consisted of µg linearized DNA, 0. mm (each) ATP, CTP, and UTP, 0.1mM GTP, 0. mm cap analog mg( )ppp( )G (Ambion), mm DTT, 0 U RNasin, 0 U SP RNA polymerase, and 1X SP RNA polymerase buffer in a total volume of 0 µl. The reaction mixtures were incubated at 0 for h, and µl was loaded in an agarose gel for electrophoresis to determine if the reaction was working. The remaining reaction mixture was aliquoted and stored at -0. The typical yields of the transcribed RNAs were approximately µg. To transfect the viral RNA into BHK1 cells, approximately 0. µg full length in-vitro-transcribed DENV or JEV viral RNA was incubated with 1 µl Lipofectamine 000 (Invitrogen) in µl Opti-MEM medium. The mixture was then added to BHK1 cells in 1-well plates and incubated at for h. The Lipofectamine-RNA mixture was removed, and the cells were fed with alpha-mem maintenance media containing % FBS. The synthesized virus particles were harvested from the supernatant of the transfected BHK1 cells post-transfection. The harvested viral particles were amplified in C/ cells for two passages, and then applied to naïve BHK1 cells to test for virus titer. Indirect immunofluorescence assay (IFA) to detect viral antigens. The BHK1 cells were cultured and manipulated in 1-well plates. The BHK1 cells transfected with in-vitro-transcribed DENV or JEV RNAs were fixed with 1% paraformaldehyde for 1 h at C, and then permeabilized with methanol for 1 min at -0 C. The cells were blocked with % horse serum, and each well was stained with 0. µg/ml anti-den prm protein antibody (mouse monoclonal, HB-, ATCC) () or with a 1:,000 dilution of anti-jev envelope protein antibody (mouse monoclonal, G, ATCC)(). The cells were then incubated with an AlexaFlour anti-mouse IgG antibody (Molecular Probes, Invitrogen). The nuclei were stained with DAPI, and images were acquired using a Leica DMIRB microscope equipped with CoolSNAP TM Cooled CCD cameras and Empix Northern Ellipse image software. Replicon RNA transcription and transfection. Replicon plasmids were linearized by XbaI

9 for DENV. DNA was phenol-chloroform extracted, precipitated and used as a template for in vitro transcription using a SP Message mmachine kit (Ambion). The RNA was quantified by spectrophotometer and stored at -0 C. RNAs were transfected into BHK1 cells by LipofectamineTM 000 (Invitrogen) according to the manufacturer s protocol. Transient dengue replicon assay. The transient replicon assay was performed for quantification of viral translation and viral RNA synthesis. BHK1 cells were seeded in -wells plates (1 cells per well) and incubated overnight. 0. µg of RNA was transfected for each well. Duplicated wells were lysed for luminometry at,, 1,,,, 0,, and h post-transfection. Renilla Luciferase Assay Kit (Promega) was used to analyze dengue replicon activity. Quantitative RT-PCR. To quantify replicated viral positive-strand RNA in replicon transfected BHK1 cells, the RNA was isolated from the above-mentioned cell samples using the Qiagen RNeasy Kit as described in manufacturer s protocol. The primers used for quantitative RT-PCR are listed in Supplementary Table. Equal quantities of viral RNAs were reverse transcribed to cdna using the Invitrogen Thermoscript RT Kit with specific primers DV.PS-R for detection of positiveand negative-strand viral RNA basing on the manufacturer s protocol and as described (), with slight modification. Real-time quantitative PCR reaction was used to measure the viral RNA as described by (0). The reaction was conducted in 0 µl volume comprising µl of cdna, 1 x LightCycler TaqMan Master Mix (Roche), 00 nm of each previous described primers DV.U-F and DV.L1-R, and 0 nm hydrolysis probe DV.P1 (TIB MOLBIOL) levels. The LightCycler TaqMan Master Kit (Roche Biochemicals) and LightCycler 1. Instrument (Roche Biochemicals) were utilized in this study under the following conditions: pre-incubated at C for min followed by cycles of three-step incubations at C for 1 s (denaturation), 0 C for 0 s (annealing and elongation), and C for 1 s (complete elongation with a single fluorescence measurement). A linear relationship was established between RNA copy numbers per milliliter and corresponding threshold cycle (CT) value over seven logs of RNA concentration (correlation coefficient, r = 0.). The copy

10 number of RNA was determined by using standards that were measured by spectrophotometer. Growth curve of virus replication. C/ or BHK1 cells were infected with DENV PL0 strain parental virus or with transcript-derived virus at an MOI of 0.01 or 0.1 PFU/cell in six-well plates. The supernatant of infected cells was removed daily and stored at -0 C. DENV titers in each collected sample were determined through serial titration on BHK1 cells. Statistical analysis Data was analyzed with SPSS Statistics, version 1. ECP activities of wild-type and silent mutant fragments of DENV or JEV were tested for significant differences by using t-test. Comparison of viral RNA levels between cells transfected with wild-type and mutant replicons were also tested for significant differences by t-test. Downloaded from on October, 01 by guest

11 Results Identification of active E. coli promoter sequences within the DENV genome. Due to the known toxicity of flavivirus cdna to E. coli (), we initially decided to use either low-copy vectors (pbr) for cloning in E. coli or a yeast shuttle vector (prs1) for cloning in yeast cells to reduce the toxicity derived from the DENV (PL0 strain), and JEV (RP strain) genomes. Unfortunately, we were not able to obtain full-length DENV and JEV cdna clones using both approaches. We therefore hypothesized that the toxicity of the DENV and JEV cdnas originates from the cryptic expression of viral proteins in E. coli. To examine the possibility of cryptic expression of DENV and JEV viral proteins in E. coli, a DNA fragment corresponding to core-prm-e-ns1 gene (nt 1-000) of DENV or JEV was used. The DENV or JEV cdna fragment (nt 1-000) was synthesized by RT-PCR derived from parental DENV (PL0 strain) or JEV (RP strain) virus stocks. The RT-PCR synthesized DENV or JEV cdna fragment (nt 1-000) was subjected to sequencing and analyzed for the prediction of putative ECPs. Surprisingly, 1 putative ECPs with score higher than 0. were found in the DENV genome sequences using the Neural Network promoter program from the Berkeley Drosophila Genome Project ( (data not shown). In contrast, only four putative ECPs with score higher than 0. were predicted in the JEV genome sequences by the Neural Network promoter program (Table 1). The prediction of 1 putative ECPs with score higher than 0. within nt of DENV genome lead us to determine if the nt of DENV genome has active ECPs in E. coli. Segments of DENV nt were fused to Renilla luciferase and evaluated for the fusion protein expression in E. coli. The DENV cdna fragment (nt 1-000) was divided into fragments (nt 1-00, 01-00, 01-00, 01-0, 1-0, 1-0, 1-0, 1-00, 01-00, and ). Each fragment was fused in-frame with Renilla luciferase gene and inserted into prs1 plasmid through homologous recombination. Individual construct containing

12 DENV-Luciferase gene was transformed into E. coli C1 cells. The C1 cells harboring DENV-Luciferase gene were cultured, harvested and cell extracts were subjected to measure luciferase enzymatic activity. As expected, several DENV-Luc constructs (1-00, 01-00, 01-00, 01-0, 1-0, 1-00, 01-00) have relatively higher luciferase enzymatic activity in bacteria compared to control plasmid prs-luc, which contain only luciferase gene without DENV sequence (Fig. 1A). To predict the location of active ECPs within the DENV segments of DENV-Luc constructs (1-00, 01-00, 01-00, 01-0, 1-0, 1-00 and 01-00), we used Neural Network promoter program to look for ECPs with higher score (>0.) within the DENV segments of DENV-Luc constructs with luciferase activity. Also, we focused on the ECPs that have an optimal start codon located downstream from the ECP initiation site and in-frame with the virus coding region (). We predicted ECP1 (nt -0) and ECP (nt 1-) within the DENV segment nt 1-00; ECP (nt -1) within the DENV segment nt 01-00; ECP (nt -) within the DENV segment nt 01-00; ECP (nt -0) within the DENV segment nt 01-0; ECP (nt -1) within the DENV segment nt 1-00; ECP (nt -) and ECP (1-0) within the DENV segment nt (Table 1). Since the luciferase activity of DENV-Luc (0-0) is low and there is no optimal start codon (in frame with dengue coding region) downstream of the only predicted ECP (nt 1-1), we decided to ignore the region of nt 0-0 of DENV genome. To reduce the likelihood that these putative E. coli promoters were contained in the DENV (nt 1-000) genomes, silent mutations were created in the predicted DENV ECP1- (Table 1) to reduce the scores without altering the amino acid sequences. The effects of all possible silent mutations on the E. coli promoter prediction scores of the DENV were then evaluated by the Neural Network promoter program. The silent mutations predicted to severely reduce the score of ECPs were then chosen for creating DENV-Luc mutant constructs (Table 1). 1

13 To further determine if the chosen silent mutations that predicted to reduce E. coli promoter activity of ECP1- of DENV genome (Table 1) indeed affect E. coli promoter activity in bacteria, the silent mutations were individually introduced into the wild-type DENV-Luc constructs (1-00, 01-00, 01-00, 01-0, 1-0, 1-00, and 01-00). When compared to the luciferase activity of the various wild-type DENV-Luc constructs, most of the silent mutations significantly decreased the luciferase activity of all DENV-Luc constructs in E. coli (Fig. 1B). In addition to luciferase activity, Western blots with anti-renilla luciferase monoclonal antibody demonstrated DENV-Luc fusion protein expression. As expected, there was no band appearing in the lane of RS-Luc control even after longer exposure of Western blot (Fig. 1C). However, apparent expression of the DENV-Luc proteins was found in the wild-type DENV-Luc constructs, e.g. 1-00, and Moderate expression of luciferase protein was shown in the wild-type DENV-Luc constructs, e.g and Several DENV-Luc constructs with silent mutations exhibited substantially lower DENV-Luc fusion protein expression (e.g., 1-00, 01-00, 01-00, 01-0, and 1-00), which is consistent with the observation that the luciferase activity of DENV-Luc constructs in E. coli was significantly reduced by the silent mutations introduced into ECP1- (Fig. 1B). Identification of active E. coli promoter sequences within the JEV genome. To search for putative ECPs within nt of JEV genome, we used the Neural Network promoter program to predict ECPs with score higher than 0.. In contrast to the 1 putative ECPs within nt of DENV genome, only four ECPs (nt 0-, -, -1 and 1-1) with score higher than 0. were found (Table 1). Consequently, three JEV-Luc constructs (1-, 00-, 0-1) were directly constructed to determine if the predicted ECP-1 are active and synthesize JEV-Luc fusion proteins in E. coli. Similar to the construction of DENV-Luc constructs, three JEV segments, nt 1- containing 1

14 ECP and ECP, nt 00- containing ECP and nt 0-1 containing ECP 1, were fused to the empty vector reporter gene (Renilla luciferase) to determine if there was JEV-Luc fusion protein expression in E. coli. Those DNA segments of JEV were inserted in-frame with the downstream sequence of the Renilla luciferase gene. Luciferase enzymatic activity was used to measure the promoter activity of various JEV-Luc constructs in E. coli. Wild-type JEV-Luc construct (1-) displayed strong luciferase activity, indicating strong intrinsic E. coli promoter activity in E. coli (Fig. A). In contrast, much lower luciferase activity was observed in the other two JEV-Luc constructs (00- and 0-1). Since JEV-Luc (1-) construct displayed strong promoter activity in bacteria compared to JEV-Luc (00-) and (0-1), we therefore focused on ECP and ECP, which are located at nt 1- of JEV genome. To reduce the prokaryotic promoter activity of JEV-Luc (1-) construct and determine whether ECP or/and ECP is responsible for the strong prokaryotic promoter activity of JEV-Luc (1-) construct in bacteria, several mutations were designed by the Neural Network promoter program and introduced into ECP or/and ECP of JEV-Luc constructs (Table 1). The luciferase activity was greatly reduced when an A-to-C mutation at nt 0 was introduced into ECP within nt 1- of JEV-Luc (1-) construct (Fig. B). The designed silent mutations (M1) predicted to reduce E. coli promoter activity of ECP were also able to significantly reduce the luciferase activity of JEV-Luc (1-). The luciferase activity was abolished to undetectable level when mutations were introduced into both ECP and ECP within JEV-Luc (1-) construct. Western blotting was performed to determine if the JEV-Luc (1-) fusion protein was expressed. Consistent with the enzymatic luciferase activity of JEV-Luc (1-) wild-type and various mutant constructs in bacteria, introduction of the A0C mutation into ECP or mutations into both ECP and ECP of JEV-Luc (1-) construct decreased JEV-Luc (1-) fusion protein expression (Fig. C). 1

15 Stabilizing full-length DENV cdna with silent mutations that reduce E. coli promoter activity within the DENV genome. To prepare DENV cdna fragments for the construction of full-length DENV cdna, we first synthesized three fragments of DENV cdnas (nt 1-000, and 000-) derived from RT-PCR of DENV viral RNAs. The three individual DENV cdna fragments covered the whole DENV genome and were sequenced directly, serving as a reference for later cloning steps. Instead of using conventional molecular cloning technology, we took advantage of homologous recombination mechanism in yeast cells to clone full-length DENV and JEV cdnas. Initially, we used yeast cells to assemble full-length DENV PL0 cdna without introducing any mutations in the L fragment of the DENV genome (Fig. ). We were not able to obtain correct full-length prs/flden plasmid purified from HIS+ yeast colonies in E. coli. After encountering the difficulty faced by most labs in cloning flavivirus cdnas, we decided to determine if lowering E. coli promoter activity in the viral genome by inserting silent mutations (Table 1) could stabilize DENV and JEV cdna clones in bacteria. Since it has been shown that the N-terminus of dengue core gene affects virus replication via functioning as a cis element that enhances recognition of the core start codon and efficient RNA synthesis (1, 1), we suspected that the silent mutations within ECP1 and ECP may affect virus replication, as they are located in the middle of DENV core coding region. Thus, we decided to construct four different full-length DENV cdna mutants. One mutant, designated as DENV-M, contains eight sets of silent mutations within ECP1-ECP of DENV genome. Six sets of silent mutations were introduced into ECP - of the DENV genome to construct the other mutant, DENV-M, The other two mutants, designated as DENV-M-1 and DENV-M-, contain silent mutations within ECP (1 and -) and ECP-, respectively. To construct the prs/denl* as shown in Figure A-D, six to eight sets of silent mutations were sequentially introduced into the L fragment of dengue genome. In general, E. coli colonies with the correct prs/denl* plasmid exhibited a relatively normal grow rate, indicating that the DENV L* 1

16 fragment had no apparent toxicity. In contrast, E. coli colonies harboring prs/denl grew very slowly and no correct prs/denl plasmid can be steadily obtained. We continued to use the prs/denl* plasmid to finish assembling full-length DNEV cdna clones with the R fragment (nt 000-) and M fragment (nt 0-000) of the DENV genome (Fig. E-J). Yeast colonies carrying the full-length DENV cdna clone, prs/flden (DENV-M, DENV-M-1, DENV-M-, and DENV-M), were identified by colony PCR to detect the M fragment, and all six yeast colonies were positive. After the purified yeast DNA was transformed into C1, the C1 colonies carrying full-length DENV cdna clones (DENV-M, DENV-M-1, DENV-M-, and DENV-M) were generally homogeneous in size, unlike the prs/denl plasmid, which resulted in various sizes. Four out of six positive yeast DNAs were chosen and transformed into C1. The DENV-M, DENV-M-1, DENV-M-, and DENV-M plasmids purified from the C1 colonies displayed good DNA yields and correct restriction enzyme-digestion patterns. The DNA yield of the DENV-M ( ng/ cells) construct were slightly better than those of the DENV-M ( ng/ cells), DENV-M-1 ( ng/ cells) and DENV-M- ( ng/ cells) constructs in bacteria. Since we were not able to obtain wild type DENV cdna clone, it is impossible to quantitatively show the plasmid stability between wild type and various mutant DENV cdna clones. Instead, we used a prs1 vector (same vector to harbor DENV mutant clone) to harbor widely regarded stable Hepatitis C virus 1b replicon reporter sequence () to construct prs1-hcv1b clone, which has similar size to DNEV mutant clones. The plasmid stability of HCV1b replicon construct was used to compare with that of various DENV mutant clones. In general, bacteria colony formation number represents the degree of toxicity or stability of plasmids in bacteria. Thus, bacteria colony formation number was used to correlate to plasmid stability of DENV mutant constructs. As shown in Table, 1 ng of prs1-hcv1b plasmid transformed into C1 bacteria cells resulted in colonies. 1 colonies were detected in the bacteria transformed 1

17 with 1ng of DENV-M plasmid. 1,, and 00 colonies were found in the bacteria transformed with 1ng of DENV-M-1, DENV-M-, and DENV-M plasmid, respectively. The colony formation efficiency indicates that silent mutations indeed increase plasmid stability of DENV mutant plasmid in bacteria. The stability of cloned DENV-M infectious cdnas was further accessed by repeated subculture in E. coli strain C1. C1 was transformed with DENV-M and the grown colonies were selected on YT agar plate containing ampicillin ( µg/ml). colonies were picked, mixed and incubated at 0 C in liquid YT medium containing ampicillin ( µg/ml) for another 1 day. The saturated YT medium containing bacteria harboring DENV-M plasmids was re-streaked onto YT-ampicillin agar plate. colonies grown on agar plate were picked, mixed for the same growth testing cycle for times. After growth cycles, clones containing DENV-M plasmid was test and confirmed the integrity by using two sets of digestion by restriction enzyme to make sure no transposon insertion and DNA rearrangement occurred during the passages. The integrity of DENV-M could be maintained for at least passages of subcultures. DENV-M, DENV-M-1, DENV-M-, and DENV-M plasmids obtained from C1 colonies were verified by DNA sequencing to check the integrity of the cloned DENV genomic cdna and correct plasmids are subjected to further analyses. Previously, we intended to directly assemble full-length DENV cdna in bacteria without doing any mutagenesis within DENV genome. We failed to get correct clone but obtained wrong full-length DENV plasmid, DENV-def (defective cdna clone), with several mutations within the poison DNA fragment, core-prm-e-ns (data not shown). After we successfully assembled DENV-M infectious cdna clones in yeast cells and amplified in bacteria, we like to know if our previously failed cloning strategy (direct cloning in bacteria) can assemble the poison region containing sets of silent mutations to get correct full-length infectious clone. We then digested DENV-def with SacI (right upstream of SP promoter sequence) and SmaI (nt within DENV genome) restriction enzymes to remove the DNA fragment (nt 1-) containing poison sequences 1

18 to serve as a vector to assemble the corresponding DNA fragment (nt 1-) with sets of silent mutations by ligation in bacteria. As expected, we steadily obtained correct DENV-M clones out of bacteria colonies. sets of silent mutation were verified in those correct clones by DNA sequencing analyses. Thus, we can get the DENV-M infectious cdna clone either by assembling in yeast or bacteria cells. Assembly of a full-length JEV cdna by reducing E. coli promoter activity within JEV genome. As we had successfully cloned the full-length DENV cdna, we used a similar cloning strategy to construct a full-length JEV cdna clone (Fig. ) by introducing silent mutations into ECP and (Table 1). The result shown in Fig. B indicated that moderate luciferase activity was found in the JEV-Luc (00-) and (0-1) compared to strong luciferase activity of JEV-Luc (1-). Thus, we decided to determine if the mutations introduced into ECP, ECP or ECP+ stabilize the full-length JEV cdnas in E. coli. In general, the strategy consisted of assembling three JEV cdna fragments, fragment L, R, and M, into a prs1 shuttle vector through homologous recombination (Fig. ). Since ECP is located at the well conserved non-coding region of the JEV genome, introduction of mutations into ECP may impair virus replication. Thus, we decided to construct three types of JEV mutations by introducing mutations into ECP or ECP alone or into both ECP and ECP. To introduce those three types of silent mutations within the L fragment, site-directed mutagenesis was performed to generate the L* fragment (Fig. H). Yeast colonies that harbored the assembled full-length JEV cdna clones containing the L fragment had a much slower growth phenotype than those containing the L* fragment. Yeast colonies containing prs/jevfl (L* or L) were chosen, amplified, and transformed into bacteria. After several attempts, we were not able to obtain the correct full-length JEV cdna clones containing the L fragment from E. coli. The phenomenon was similar to the construction of the full-length DENV cdna clones with no mutations within ECPs of the DENV 1

19 genome. We also observed that C1 cells harboring the full-length JEV cdna clone with silent mutations within ECP alone were not able to propagate. In contrast, the full-length JEV cdna clones with silent mutations within either at ECP alone or at both ECP and, designated as JEV-A0C or JEV-DM, respectively, were easily obtained, amplified and purified from C1 cells. After restriction digestion pattern and sequencing analyses, most of colonies carrying the JEV-A0C and JEV-DM plasmids were found to be correct and have decent DNA yields although the DNA yields of JEV-DM cdna clone is slightly better than those of JEV-A0C cdna clone. DNA sequencing analyses revealed that all JEV-A0C and JEV-DM cdna clones have the same nucleotide sequences except the JEV-DM clone has additional mutations within ECP of JEV genome. The stability of cloned JEV-A0C infectious cdnas was accessed by repeated passage in E. coli strain C1. C1 was transformed with JEV-A0C and the grown colonies were selected on YT agar plate containing ampicillin ( µg/ml). colonies were picked, mixed, and re-streaked on YT agar plate. The passage cycle for growing C1 cells harboring JEV-A0C plasmid was repeated for times. clones containing JEV-A0C plasmid was test and confirmed the integrity by using two sets of restriction enzyme digestion to make sure no transposon insertion and DNA rearrangement occurred during the passages. The integrity of JEV-A0C could be maintained for at least passages of cultures on agar plate. To show JEV-A0C plasmid can be assembled and easily manipulated in bacteria as shown in the DNA manipulation of DENV-M plasmid in bacteria, we like to know if the poison region (core-prm-e-ns1) containing A0C silent mutation can be cloned into plasmid by direct cloning in bacteria. Previously, we failed to get correct full-length JEV cdna clone by direct cloning in bacteria. We often got JEV cdna clones with 1 nt deletion, e.g. JEV-dl01 (1 nt deletion at nt 01). We digested JEV-dl01 clone with BssHII (~1 kb upstream of SP promoter sequence) and BspEI (nt of JEV genome) restriction enzymes to remove the region, nt 1-, to serve as a 1

20 vector to assemble the corresponding DNA fragment (nt 1-) with A0C mutation. As expected, we steadily obtained correct JEV-A0C clones out of colonies, which were verified by DNA sequencing analyses. Thus, we can get the JEV-A0C infectious cdna clone either by assembling in yeast or bacteria cells Silent mutations introducing into the central portion of DENV core gene affect the infectivity of DENV. To determine if full-length DENV cdna clones harboring different sets of silent mutations are infectious, full-length DENV cdna clones were used as templates to produce full-length synthetic RNAs by in vitro transcription and transfected into BHK1 cells. There was substantial cytopathic effect (CPE) as a result of efficient DENV viral infection three days after transfection into BHK1 cells with RNA transcripts derived from the DENV-M and DENV-M- cdna clones with silent mutations introduced into the ECP - and ECP-, respectively. There was no apparent CPE in the BHK1 cells even five days after transfected with RNA transcripts derived from the DENV-M and DENV-M-1 cdna clone with silent mutations within ECP1- and ECP(1,-), respectively. To further confirm the infectivity of the various DENV mutant viruses derived from DENV-M, DENV-M-1, DENV-M- and DENV-M cdna clones, indirect immunofluorescence assay (IFA) was used to detect cells expressing DENV prm protein in transfected BHK1 cells. DENV prm proteins could be detected in cells days after transfection with in-vitro transcribed DENV-M or DENV-M- RNAs (Fig. A). More cells with strong fluorescence staining were observed from cells transfected with DENV-M RNA transcripts than those transfected with DENV-M- RNA transcripts three days after transfection. There was apparent decrease of cell population with strong IFA staining in the cells transfected with DENV-M transcripts four days after transcription due to cell death resulting from CPE caused by virus infection. Almost every cell is immunopositive on day five after transfection with DENV-M 0

21 or DENV-M- RNA transcripts although more severe CPE effect (0% of BHK1 cells remaining) was seen in cells transfected with DENV-M compared to DENV-M-. Therefore, the transfected cells with DENV-M or DENV-M- synthetic RNA transcripts were efficiently expressed, and the virus progeny were able to efficiently replicate and infect cells. In contrast, DENV prm was detected in more than 0% of BHK1 cells only four days after transfection with DENV-M or DENV-M RNA transcripts. Cells with strong immuno-staining with prm antibody occurred days after transfection with DENV-M or DENV-M RNA transcripts. To further quantitatively evaluate the difference in virus spreading activity between various full-length DENV mutant cdna clones harboring silent mutations, the titers of virions synthesized from BHK1 cells transfected with various DENV mutant RNAs were measured by plaque formation assay. The titers of virions derived from the DENV-M cdna clone reached highest titer (around 1 x PFU/ml) on day three after transfection (Fig. B). A dramatic drop in the titers (from 1 x to 1 x PFU/ml) of virions derived from the DENV-M cdna clone five days after transfection, which is consistent with the observation that only around 0% of BHK1 cells were alive five days after transfection with DENV-M RNA transcripts. A delay of timing reaching virus titers of 1 x PFU/ml occurred in the cells five days after transfection with DENV-M- RNA transcripts. The virions derived from DENV-M or DENV-M slowly reached around virus titers of 1 x PFU/ml five days after transfection. A cis-acting element essential for virus replication of DENV is located at the central portion of DENV core gene Since the silent mutations introduced into the DENV core gene affected virus spreading of viruses derived from DENV-M-1, DENV-M-, and DENV-M RNA transcripts (Fig. ), we decided to utilize sub-genomic dengue replicon to further determine the effect of those silent mutations on the translation or replication capacity of dengue replicon RNAs in BHK1 cells. The same silent mutations introduced to ECP1 or/and ECP within core gene of full-length DENV 1

22 cdna clone were constructed into the core gene of wild-type sub-genomic dengue replicon with reporter luciferase gene, designated as mecp1, mecp and mecp1+ (Fig. A). We transfected BHK1 cells with wild-type or various DENV mutant replicons (Fig. A), mecp1, mecp, and mecp1+, RNAs and monitored the luciferase signal at different time points. The luciferase activity of wild-type and various DENV replicons are similar at, and 1 h post transfection, indicating the mutations within ECP1, and 1+ did not apparently affect translation efficiency of viral RNAs (Fig. B). However, mecp1 and mecp1+ replicons showed lower luciferase activity h post transfection compared to wild-type and mecp replicon. The dramatic difference in luciferase activity between wild-type dengue replicon and mecp1 or mecp1+ was observed after h post transfection. A slight reduction of luciferase activity was found in the mecp replicon after h post transfection compared to wild-type dengue replicon. To further determine if the mutations on dengue core gene affect the level of viral RNAs of dengue replicon, the viral RNAs were measured by RT-PCR derived from cells transfected wild-type or various dengue mutant replicons either or h post transfection. Consistent with the results from the reporter luciferase activity, there is also significant reduction in the level of positive-stranded viral RNAs in mecp1 and mecp1+ dengue replicons compared to wild-type dengue replicon. A slight reduction in the level of positive-stranded viral RNAs derived from mecp dengue replicon compared to wild-type dengue replicon either or h post transfection. Virions derived from DENV-M cdna clone exhibit similar plaque morphology and growth curve to DENV parental virus. Since the silent mutations introduced into central portion of core gene caused slower replication phenotype in the virus spreading of DENV-M-1, DENV-M- and DENV-M cdna-derived viruses, we decided to determine if the virions derived from DENV-M cdna clone (with six sets of silent mutations within ECP-) possess similar virus spreading activity to parental DENV virus stock. First, we compared the plaque morphology of

23 viruses derived from DENV-M to parental DENV virus stocks. As shown in Figure A, the size of plaque in BHK1 cells infected with DENV-M virus is quite similar to that of plaque from parental DENV viruses. To evaluate the effect of the silent mutations introduced to the ECP- of DENV genome on DENV virus growth, the growth kinetics of synthetic DENV viruses in BHK1 cells were analyzed and compared to those of DENV parental virus stocks. BHK1 cells were infected at the same MOI (either 0.01 or 0.1) of DENV viruses derived from the DENV-M cdna clone and DENV parental virus stocks. The supernatants of infected BHK1 cells were removed daily and the titers of viruses were subsequently determined. The growth curves of viruses derived from the DENV-M cdna clone and DENV parental viruses were indistinguishable in BHK1 cells (Fig. B). To further determine if the grow curve of viruses derived from DENV-M in BHK1 cells is similar to that in mosquito cells, C/ cells. The growth kinetics of synthetic DENV viruses in C/ cells were analyzed and compared to those of DENV parental virus stocks. C/ cells were infected at the same MOI (either 0.01 or 0.1) of DENV viruses derived from the DENV-M cdna clone and DENV parental virus stocks. The supernatants of infected C/ cells were removed daily and the titers of viruses were subsequently determined. The growth curves of viruses derived from the DENV-M cdna clone and DENV parental viruses were indistinguishable (Fig. C). These results suggest that the promoter silencing mutations introduced into ECP- of the DENV genome did not perturb the normal functions of the DENV viral proteins, and that the cloned full-length DENV cdna retained its integrity in our cloning strategy. Virions derived from JEV-A0C cdna clone exhibit similar phenotypes in the plaque morphology and growth curve to JEV parental virus. Since it has been shown that an A-to-C mutation at nt 0 of JEV genome reduced the promoter activity of ECP and stabilized the JEV-A0C plasmid in bacteria (Figs. B and C), we sought to evaluate if the JEV-A0C cdna clone is an infectious cdna clone and recombinant JEV-A0C viruses have similar infectivity to

24 1 1 parental JEV viruses. First, virus infectivity of recombinant JEV-A0C virions was determined by IFA staining to detect the JEV envelope protein expression in BHK1 cells transfected with JEV-A0A RNA transcripts. IFA staining of JEV E protein was detected in transfected BHK1 cells two days after transfection (Fig. A). Strong IFA staining and severe CPE were observed in transfected BHK1 cells three days after transfection. Next, the plaque forming ability was also used to compare the virus replication capacity between parental JEV and recombinant JEV-A0C viruses. Similar plaque morphology was shown in BHK1 cells infected either by JEV-A0A recombinant viruses or parental JEV viruses (Fig. B). The kinetics of virus growth derived from parental JEV or recombinant JEV-A0C viruses was also compared. As shown in Figure C, there is no apparent difference in the growth curve of virus replication in BHK1 cells infected by parental JEV and recombinant JEV-A0C viruses at a MOI of 0.01 or 0.1. Similarly, no apparent difference in the growth curve of virus replication in C/ cells infected with parental JEV and recombinant JEV-A0C viruses at a MOI of 0.01 or 0.1 (Fig. D). Downloaded from on October, 01 by guest

25 Discussion The novel methods described here are convenient approaches for cloning full-length DENV and JEV cdna clones with little toxicity in E. coli. For years, attempts have been made to develop a feasible method for constructing full-length flavivirus cdna clones, which are essential to a successful reverse genetics system. Most previous attempts encountered the intrinsic toxicity of the flavivirus genome sequences in E. coli. We first demonstrated that the instability of the DENV and JEV cdna clones in bacteria is due to the toxicity of the cryptic expression of viral proteins by multiple ECP sequences that are embedded in the viral genome. Furthermore, we showed that silent mutations, which were introduced to reduce ECP activities, stabilized the full-length DENV and JEV cdna clones in E. coli. The infectious virions were efficiently produced in cells transfected with in vitro-transcribed RNAs derived from the full-length DENV and JEV cdna clones with mutations. Interestingly, a cis-acting element essential for DENV replication was discovered and located at the central portion (nt -) of core gene within DENV genome. Thus, the methods described in this paper provide a feasible way to construct full-length flavivirus cdna clones. Our methodology should greatly facilitate the study of the molecular mechanism of flavivirus replication and pathogenesis, as well as future vaccine development. We primarily used the homologous recombination cloning approach, or recombineering, in yeast cells to construct unstable full-length DENV and JEV cdna clones because recombineering not only has a much higher cloning efficiency, but it is also more tolerant than bacteria to the toxicity of poisonous sequences. Yeast cells can easily assemble full-length DENV cdna clones through homologous recombination, without the tedious conventional cloning process. Unlike the vulnerability of bacteria to the poisonous flavivirus sequences, yeast cells serve as perfect hosts for cloning DENV (NGC strain) (1) and DENV1 (Western Pacific strain) () infectious cdna clones because they are more tolerant than bacteria to the toxicity of DEN cdna. The full-length DENV (NGC strain) infectious clone was successfully obtained from E. coli, but the DENV cdna toxicity

26 still persisted, resulting in slow bacterial growth. Using a similar strategy, we were able to construct and obtain full-length DENV (PL0 strain) cdna clones from yeast cells without any modification of the DENV genome. However, we were not able to amplify full-length DENV (PL0 strain) cdna plasmids in bacteria, indicating that yeast cells can endure the toxicity of DENV cdna as they presumably promote less cryptic viral protein expression than bacteria. We suspect that our failure to obtain the full-length DENV (PL0 strain) cdna clone from E. coli may have stemmed from differences in the genomic sequences between DENV NGC and PL0 strains. Furthermore, we observed that yeast cells are able to carry full-length DENV cdna plasmids from another strain (DENV ), although they grew very slowly (data not shown). Like the DENV PL0 strain, we were not able to recover the full-length DENV ( strain) cdna clones in E. coli. This observation is consistent with the speculation that varying degrees of toxicity are derived from sequence variations among the different DENV strains (NGC, PL0 and ). This speculation may also explain why infectious cdna clones were obtained only for certain strains of DENV. Interestingly, unlike constructing the DENV cdna clone, assembling the full-length JEV (RP strain) cdna clone in yeast cells was not successful when no silent mutations were introduced into the JEV genome. Full-length JEV cdna clones (JEV-A0C and JEV-DM) were obtained from yeast and bacteria when the silent mutations were introduced into ECP and ECP+ of the JEV genome, respectively (Table 1). This clearly suggests that yeast cells were not able to circumvent the JEV cdna toxicity that results from inappropriate expression of JEV cdnas, even though they were able to tolerate the toxicity due to DENV (NGC, PL0 and strains) cdnas. Several previous reports have speculated that cryptic expression of viral proteins occurs in bacteria harboring flavivirus cdna. The aberrant expression of flavivirus proteins in bacteria is thought to be responsible for their toxicity in E. coli. First, insertions and deletions are usually found and mapped to the E-NS1-NSA region during the construction of a DNE infectious clone, which

27 may be due to the secondary structure or the adventitious expression of some toxic product (1). Furthermore, the introduction of a stop codon at residue 1 of the NS1 protein stabilizes DEN/ and DENV1/ chimera infectious cdnas, which clearly indicates that the toxicity is derived from the spurious expression of dengue structural genes (core-prm-e-ns1)(). Moreover, spontaneous nonsense mutations often occur during cloning of the JEV half plasmid (, ). It is likely that spurious transcription is initiated from a prokaryotic promoter-like sequence that is located somewhere in the JEV E gene, as nonsense mutations (e.g., a stop codon) prevent the expression of the problem genome region at the level of translation (). Two of our own observations also support this speculation. One observation is that deletion (either a large deletion or a single nucleotide deletion), insertion (transposon insertion), and premature stop codons frequently occurred in the region of the DENV (PL0 strain) and JEV (RP strain) structural genes when the full-length DENV or JEV cdna clone was constructed without any silent mutations introduced into the ECP1- shown in Table 1 (data not shown). Taken together, our observations are consistent with several previous findings (, 1,, ) demonstrating that the well-known toxicity of flavivirus cdnas may arise from inappropriate expression of the flavivirus genome in bacteria. An important phenomenon disclosed by this report is that the cryptic expression of the DENV and JEV viral proteins, which accounted for the instability, was initiated from multiple ECPs within the DENV and JEV genomes. Active ECPs within the DENV and JEV genomes were demonstrated by viral sequence-driven DENV- or JEV-luciferase fusion protein expression and luciferase enzyme activities (Figs. 1 and ). The activity of ECPs in bacteria was further evidenced by the fact that promoter-silencing mutations could reduce DENV and JEV cdna fragment-reporter expression in bacteria (Figs. 1 and ). The observation that DENV-M (silent mutations within ECP-) and DEN-M (silent mutations within ECP1-) cdna clones were steadily constructed in bacteria supports the instability of DENV cdna clones in bacteria results from multiple active ECPs within DENV genome. In contrast, four sets of silent mutations

28 introduced either into ECP1,,, and or ECP- of the DENV genome were not able to circumvent the instability of full-length DENV cdna in bacteria (data not shown), supporting the toxicity of DENV cdna resulting from multiple active ECP in bacteria. Another line of evidence supporting that more silent mutations inserted into ECP1 or and ECP of DENV-M infectious cdna increase its plasmid stability comes from the result that bacteria colony numbers of DENV-M-1, -M-, and -M were better than those of DENV-M (Table ). Similarly, ECP-1 within JEV genome were shown to be active in bacteria (Fig. A). As expected, we were not able to construct and get a full-length JEV cdna clone without inserting silent mutations into JEV genome. However, full-length JEV cdna clones, JEV-A0C and JEV-DM, were obtained steadily when the silent mutations were inserted into ECP alone and ECP+ of the JEV genome, respectively. Insertion of silent mutations at ECP alone within JEV genome was not able to stabilize the stability of JEV cdna in bacteria although silent mutations inserted into the ECP of JEV-Luc (1-) significantly reduced promoter activity in bacteria (Fig. B). The results clearly indicated that the ECP is the key element responsible for the instability of full-length JEV cdna in bacteria although the promoter activity of ECP-1 can be detected in bacteria. The other line of evidence supporting the multiple-ecp viral protein expression of DENV or JEV cdna in E. coli came from our successful cloning of full-length DENV1 and DEN cdnas using the same rationale by introducing multiple silent mutations into the predicted ECPs (different from the ECPs within the DENV genome) within the DENV1 and DENV genomes (nt 1-000) (unpublished results). Our results strongly suggest that multiple, active E. coli promoters exist within nt of DENV1,,, and JEV genomes although the ECPs among DENV1,, and are all different. Therefore, the toxicity of the DEN 1,,, and JEV cdnas in bacteria is very likely due to the cryptic viral protein expression that is initiated from multiple ECPs of DEN and JEV genome in bacteria. The involvement of ECPs within the DENV and JEV genomes in the toxicity to E. coli not only occurs in flavivirus cdnas, but also in the other non-prokaryotic genes. It has been noted that

29 some non-prokaryotic genes are difficult to clone into plasmids, presumably due to the toxicity derived from those genes. This includes the human cystic fibrosis transmembrane conductance regulator (hctfr) (1), human growth hormone (hgh) receptor (), and potato virus X gene (). In these cases, the cryptic promoter activity of hctfr or potato virus X in E. coli can be detected by either the CAT reporter assay or by northern blot. The instability and toxicity of hctfr, hgh receptor, and potato virus X full-length clones in E. coli are greatly reduced by the site-directed mutagenesis of one cryptic promoter sequence within those full-length clones, which decreases the prokaryotic promoter activity. The reports described above are in agreement with our results (Figs. 1 and ) that the stability of the DENV and JEV cdna clones increases by reducing the activity of multiple prokaryotic promoters in bacteria. Interestingly, most of the toxic genes mentioned above encode membrane proteins. The toxic sequences within the flavivirus cdnas are mostly located in the viral gene region that encodes membrane proteins, core-prm-e-ns1. Therefore, it follows that our novel method for cloning DNE and JEV cdnas may provide a convenient way to construct toxic genes encoding membrane proteins in E. coli. The introduction of silent mutations into ECP- of DENV genome increased the stability of DENV cdna clone in bacteria and did not apparently affect the transmission of virus from cells transfected with RNA transcripts derived from DENV-M cdna clones (Figs. A and ). IFA was detected very rapidly (two days after transfection) and virus titers reached around 1 X PFU/ml (three days after transfection) in BHK1 cells transfected with DENV-M RNA transcripts. The virions derived from DENV-M RNA transcripts replicated as efficiently as parental virus stocks either in C/ cells or BHK1 cells (Figs. A and B), which strongly indicates the silent mutations within ECP- did not affect virus replication. In contrast, a delay in the IFA staining and reduction of virus titers (-0 fold reduction) was observed in BHK1 cells transfected with DENV-M-1 or DENV-M- RNA transcripts (Fig. ), which contain additional silent mutations either within ECP1 or ECP of DENV-M RNA transcripts, respectively. It suggested that the silent mutations

30 introduced into ECP1 (nt -0) or ECP (nt 1-) located at the dengue core gene but not the ECP- within prm-e-ns1 of DENV genome affect virus replication. DENV reporter replicon experiments further showed that the mutations either at ECP1 or ECP reduce the viral replication of dengue replicon but have no apparent effect on the translation of viral RNAs (Figs. A and B). We suspected that the silent mutations introduced into either ECP1 or ECP within the middle region of DENV core gene may interfere with DENV replication by disrupting the cis-acting element located at the core coding region essential for virus replication. It has been reported that a core hairpin (chp) (nt -1) within the N-terminus of dengue core coding region serves as a cis-acting element essential for virus replication (1, 1). The hairpin structure chp affects virus life cycle by enhancing recognition of the core start codon (1) and efficient RNA synthesis (1). It was proposed that chp may stabilize the overall - panhandle structure or participate in recruitment of factors associated with the replicase machinery (1). The other element, downstream AUG region (nt 0-), located at the N-terminus of core coding region was also found to affect virus replication by functioning as a cis-acting element that possibly involves in genome circularization (1). Taken together, we uncovered a cis-acting element located at the central portion (nt -) of core gene, which extends our understanding in the role of DENV core gene serving a cis-acting element essential for virus replication. Detailed mechanism by which nt - of core gene serves as a cis-element is needed to investigate if the region interacts with end of dengue genome and participates in genome circularization. A full-length JEV cdna was steadily obtained when the site-directed mutagenesis was performed to introduce a mutation (A-to-C mutation) at nt 0 within ECP of untranslated region ( UTR) of JEV genome, which reduced the toxicity resulting from cryptic expression of JEV viral proteins in bacteria (Fig. ). Severe CPE was observed in BHK1 cells three days after transfection with JEV-A0C RNA transcripts (Fig. A), suggesting that JEV-A0C cdna clone is a JEV infectious cdna clone. Since the A0C mutation is located at very well conserved UTR of JEV 0

31 genome, there is a concern that the produced virions derived from JEV-A0C RNA transcripts may have a reversion (C-to-A reversion) at nt 0 of virus genome. The amplified virions from virions synthesized from cells transfected with JEV-A0C RNA transcripts were subjected to do RT-PCR and the A0C mutation was found to be in the viral RNAs of amplified virions (data not shown). Amplified JEV-A0C virions containing an A-to-C mutation at nt 0 of JEV genome were shown to have similar infectivity as parental JEV viruses, demonstrating a single mutation (A0C) made the manipulation of a notoriously unstable JEV infectious cdna clone much convenient and feasible. The novel approach presented herein will facilitate basic and applied viral research fields. First, our method for cloning DENV and JEV cdna clones demonstrates that silent mutations in multiple ECPs stabilize the DENV and JEV cdna clones in bacteria and explains for the first time the long-standing unsolved mystery of the toxic nature of flavivirus cdnas in E. coli. This knowledge can be applied to other RNA viruses or any unstable cdna. Second, an in-depth understanding of flavivirus virology at the molecular level has not been achieved due to difficulties in manipulating flavivirus infectious cdna clones. Easy manipulation of stable, infectious cdna clones will be immensely useful for providing insights into viral replication at the molecular level. Third, it is known that the ease of handling infectious cdna clones is important to provide a simple, reliable, and cost-effective method for maintaining viruses and vaccine depositories (, ). In particular, large scales of live attenuated vaccine cdna clones can be easily obtained by our approach and test the efficacy of vaccine candidates. Our method will therefore facilitate vaccine development. Studies are in progress to improve our established methodology, including designing stable DNA-based infectious cdna clones (eukaryotic promoter-driven) and other ways to reduce the intrinsic toxicity of flavivirus cdna and stabilize flavivirus cdna clones. Those improvements will further facilitate our manipulation of flavivirus infectious cdna clones and broaden our understanding of flavivirus replication and pathogenesis. 1

32 Acknowledgements We thank Drs. Stephen P. Goff, Chung-Pu Wu, and Jyh-Lyh Juang for critical reading of early drafts. We are grateful to Dr. Ching-Len Liao for kindly providing DENV and JEV viruses and helpful advices, and Dr. P. Robert Beatty for generously providing BHK1/clone 1 cells. We thank Pei-Sun Wu for technical support. This work was supported by the National Health Research Institutes (Grant No. BP-0-PP-0) and the National Science Council of the Republic of China (Grant No, NSC -0-B MY). Downloaded from on October, 01 by guest

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40 Virus Res 1:-. 0

41 TABLE 1. Predicted E. coli promoters within nt of DENV and JEV genome E. coli promoter DEN sequences a Scores b ECP1 WT...ctgacaaagagattctcact MT...ctgacGaagCgGttctcact...0 n.d. c ECP WT 1...ggaccattaaaactgttcat MT 1...ggaccaCtGaaGctgttcat... n.d. ECP WT...actgcaggcatgatcattat MT...actgcaggcCtgatcattat...1 n.d. ECP WT...ccacatgggtaacttatggg MT...ccacatgggtGacttatggg... n.d. ECP WT...ccaaacaacctgccactcta MT...ccaaGcaacctgccacCcta...0 n.d. ECP WT...tctatcggcaaaatgcttga MT...tctatcggcaGaatgcttga...1 n.d. ECP WT...acaagactggaaaacctgat MT...acaagactggaGaacctgat... n.d. ECP WT 1...acaccagaattgaatcacat MT 1...acaccagaGCtgaaCcacat...0 n.d. JEV sequences a ECP WT 0...aacggaagataaccatgact MT 0...aacggaagCtaaccatgacg... n.d. ECP WT...catgactaaaaaaccaggag MT...catgacGaaGaaGccaggag... n.d. ECP WT...ctacgtccaatatggacggt MT...ctacgtccaGtaCggacggt...1 n.d. ECP1 WT 1..ttgggagaacaatccagccagaaaacatcaaat MT 1..tCgggagaacaatccagccagaaaacatcaaGt..1 n.d. a Mutations introduced into ECPs are indicated by upper case and underline. b The score of predicted prokaryotic promoter was calculated by the web site ( search program (Neural Network promoter prediction) from Berkeley Drosophia Genome Project. c n.d.: not detectable. 1

42 TABLE. Colony formation from bacteria transformed with different plasmids. Plasmids Number of colonies per ng a DENV-M 1±0 DENV-M-1 1± DENV-M- ±0 DENV-M 00± HCV1b replicon ±1 a Each value represents the mean±s.e.m. of three independent experiments.

43 Figure Legends Figure 1. Identification of functional E. coli promoters within the DENV genome in E. coli. (A) Nucleotides (nt) of DENV posses different prokaryotic promoter activities. An empty vector plasmid, prs1, was used to harbor DENV-Luc fusion constructs or Luc alone construct, RS-Luc. Ten fragments corresponding to nt 1-00, 01-00, 01-00, 01-0, 1-0, 1-0, 1-0, 1-00, 01-00, and of the DENV genome were fused in-frame with the Renilla luciferase gene and transformed into E. coli. Cell lysates from E. coli transformed with variant DENV-Luc constructs or the control plasmid, RS-Luc, were tested for luciferase activity. The error bars represent the S.E.M. from four independent experiments (n=). (B) The silent mutations affect the luciferase activity of DENV-Luc constructs. Six fragments corresponding to nt 1-00, 01-00, 01-00, 01-0, 1-00, and of the DENV genome were fused in-frame with the Renilla luciferase gene and transformed into E. coli. Cell lysates from E. coli transformed with variant DENV-Luc constructs or the control plasmid, RS-Luc, were tested for luciferase activity. Closed bars represent wild-type fragments that harbor no mutations. Open bars represent the fragments with mutations. RS-Luc plasmid was served as an empty vector control construct. The error bars represent the S.E.M. from four independent experiments (n=). (*p 0.0; **p 0.01; ***p 0.001) (C) DENV-Luc fusion protein expression in E. coli cells. Lysates from E. coli cells transformed with variant DENV-Luc constructs with or without mutations were separated by SDS-PAGE, transferred to a PVDF membrane, and detected using a monoclonal antibody against Renilla luciferase. Figure. Identification of functional E. coli promoters within the JEV genome in E. coli. (A) Nucleotides (nt) of JEV posses different prokaryotic promoter activities. An empty vector plasmid, prs1, was used to harbor JEV-Luc fusion constructs or Luc alone construct, RS-Luc. Three fragments corresponding to nt 1-, 00-, and 0-1 of the JEV genome were fused

44 in-frame with the Renilla luciferase gene and transformed into E. coli. Cell lysates from E. coli transformed with variant JEV-Luc constructs or the control plasmid, RS-Luc, were tested for luciferase activity. Open bars represent wild-type fragments that harbor no mutations. RS-Luc plasmid was served as an empty vector control construct. The error bars represent the S.E.M. from five independent experiments (n=). (B) The effect of silent mutations on the luciferase activity of the JEV-Luc (1-) construct. Three types of silent mutations, A0C, M1, and DM, were introduced to ECP, ECP, and ECP+ within JEV-Luc (1-) construct, respectively. Cell lysates from E. coli transformed with wild-type JEV-Luc (1-), variant JEV-Luc (1-) mutant constructs and the control plasmid, RS-Luc, were tested for luciferase activity. Open bars represent wild-type fragments that harbor no mutations. RS-Luc plasmid was served as an empty vector control construct. The error bars represent the S.E.M. from five independent experiments (n=). (**p 0.01; ***p 0.001) (C) The fusion protein expression of wild-type JEV-Luc (1-) and various JEV-Luc (1-) mutants in E. coli cells. Lysates from E. coli cells transformed with the wild-type JEV-Luc (1-) and variant JEV-Luc (1-) mutant constructs were separated by SDS-PAGE, transferred to a PVDF membrane, and detected using a monoclonal antibody against Renilla luciferase. Figure. Construction of a full-length DENV cdna clone with the yeast shuttle vector prs1. (A) The yeast shuttle vector contained a bacterial replication origin (ori), a selection marker (Amp r ), a yeast replication origin (CEN), and selection maker (His). (B) The DENV cdna fragment L was amplified from DENV viral RNA by RT-PCR, and eight sets of silent mutations were sequentially introduced to fragment L to create fragment L*. A fragment L* possessed short overlaps with the termini of the linearized prs1 vector. (C) SacI-linearized prs1 was mixed with fragment L* and used to transform yeast strain YPH to His+. In yeast, recombination occurred between the short homologous regions at the termini of the polylinker of prs1 with fragment L to generate the prs/denl plasmid. (D, E, F, G) The XhoI-linearized prs/denl* was

45 incubated with fragment R synthesized from DENV viral RNA and transformed into yeast cells to generate the prs/denl*r plasmid through homologous recombination between the termini of fragment R and the linear prs/denl*. (H, I, J) The prs/denl*r plasmid was linearized using NotI and mixed with fragment M (obtained by RT-PCR of DENV viral RNA). It was then transformed into yeast cells to generate full-length prs/flden cdna. Regions of crossing over (X) are indicated. Figure. Construction of a full-length JEV cdna clone with the yeast shuttle vector prs1. (A) The yeast shuttle vector contained a bacterial replication origin (ori) and a selection marker (Amp r ), a yeast replication origin (CEN), and selection maker (His). (B) JEV cdna fragment M was amplified from JEV viral RNA by RT-PCR. Fragment M possesses short overlaps with the termini of the linearized prs1 vector. (C) BamHI linearized prs1 was mixed with fragment M and used to transform yeast strain YPH to His+. (D) In yeast, recombination occurred between the short homologous regions at the termini of the polylinker of prs1 with fragment M to generate the prs/jevm plasmid. (E, F, G) ClaI-linearized prs/jevm was incubated with the R fragment synthesized by RT-PCR from viral RNA and transformed into yeast cells to generate the prs/jevmr plasmid through homologous recombination. (H, I, J) The L fragment synthesized from viral RNA by RT-PCR was first mutated to create the L* fragment. The L* fragment was incubated with the XhoI-linearized prs/jevmr plasmid and transformed into yeast cells to generate the full-length prs/fljev infectious cdna. Regions of crossing over (X) are indicated. Figure. DENV infectivity is affected by the silent mutations inserted into the central region of core gene within DENV genome. (A) Immunofluorescence analysis from cells transfected with variant DENV RNA transcripts. The BHK1 cells were transfected in vitro with transcripts derived from DENV-M, DENV-M-1, DENV-M-, DENV-M plasmid or were mock transfected as

46 a control. Monoclonal antibody against the DENV prm antigen was used to detect the infected cells by indirect immunofluorescence at two, three, four and five days after transfection with in vitro-derived transcripts. (B) Titer determination of virions from cells transfected with variant DENV mutant RNA transcripts. The BHK1 cells were transfected in vitro with transcripts derived from DENV-M, DENV-M-1, DENV-M-, DENV-M plasmid. Plaque assay was performed to determine the virus titer derived from the supernatants of BHK1 cells transfected with in vitro-derived transcripts at one, two, three, four, and five days after transfection. The error bars represent the S.E.M. from three independent experiments. Figure. DENV replicon activity is affected by the silent mutations inserted into the central region of core gene within DENV genome. (A) Schematic diagram of the DENV reporter replicon. Marks are the UTR (black line), N-terminal amino acids of the C protein (C), the Renilla luciferase gene (Rluc), the FMDVA cleavage site (black box), the neomycin resistance gene (Neo), an EMCV IRES element (gray box), the C-terminal amino acids of E (E), the entire NS regions (NS1~NS), and the UTR (black line). The replicon was used to quantify the effects of silent mutations on viral RNA replication of DENV. (B) Kinetic of transient expression of wild-type and variant dengue mutant replicons, mecp1, mecp and mecp1+, in BHK1 cells. The luciferase activity at different time points,,, 1,,,, 0,, and h, was measured in cytoplasmic extracts prepared from BHK1 cells transfected with wild-type or variant dengue mutant replicon RNAs. (C) Quantitation of variant dengue mutant replicon RNAs by real time RT-PCR. Positive-strand viral RNA copy numbers are shown at and h after transfection of BHK1 cells with wild type or variant dengue mutant replicon RNAs. The error bars represent the S.E.M. from three independent experiments (n=). (*p 0.0; **p 0.01) Figure. Plaque morphology and growth curve of DENV-M transcript-derived viruses are

47 similar to those of parental DENV stocks. (A) Plaque morphology of virus derived from DENM transcript and parental DENV. BHK1 cells were infected with the parental DENV PL0 strain viruses, or infected with DENV-M transcript-derived viruses, overlaid with methyl-cellulose, and stained days with crystal violet. (B) Growth kinetics of virions derived from DENV-M RNA transcripts and parental DENV in BHK1 cells. BHK1 cells were infected with parental DENV virus stock or with DENV-M transcript-derived viruses at MOI of 0.01 and 0.1 PFU/cell. Virus samples from the medium of infected BHK1 cells were harvested daily, and the viral titer of each sample was determined in BHK1 cells. (C) Growth kinetics of virions derived from DENV-M RNA transcripts and parental DENV in mosquito cells, C/ cells. C/ cells were infected with parental DENV virus stock or with DENV-M transcript-derived DENV at MOI of 0.01 and 0.1 PFU/cell. Virus samples from the medium of infected C/ cells were harvested daily, and the viral titer of each sample was determined in BHK1 cells. The error bars represent the S.E.M. from three independent experiments. Figure. Plaque morphology and growth curve of JEV-A0C transcript-derived viruses are similar to those of parental JEV stocks. (A) Immunofluorescence analysis from cells transfected with JEV-A0C RNA transcript. The BHK1 cells were transfected in vitro with transcripts derived from JEV-A0C plasmid were mock transfected as a control. Monoclonal antibody against the JEV envelope antigen was used to detect the infected cells by indirect immunofluorescence at one, two, three and four days after transfection with in vitro-derived transcripts. (B) Plaque morphology of virus derived from JEV-A0C transcript and parental JEV. BHK1 cells were infected with the parental JEV RP strain viruses, or infected with JEV-A0C transcript-derived viruses, overlaid with methyl-cellulose, and stained days with crystal violet. (C) Growth kinetics of virions derived from JEV-A0C RNA transcripts and parental JEV in BHK1 cells. BHK1 cells were infected with parental JEV virus stock or with JEV transcript-derived viruses at MOI of 0.01 and 0.1 PFU/cell.

48 Virus samples from the medium of infected BHK1 cells were harvested daily, and the viral titer of each sample was determined in BHK1 cells. (D) Growth kinetics of virions derived from JEV-A0C RNA transcripts and parental JEV in mosquito cells, C/ cells. C/ cells were infected with parental JEV virus stock or with JEV-A0C transcript-derived JEV at MOI of 0.01 and 0.1 PFU/cell. Virus samples from the medium of infected C/ cells were harvested daily, and the viral titer of each sample was determined in BHK1 cells. The error bars represent the S.E.M. from three independent experiments. Downloaded from on October, 01 by guest

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