Successful Propagation of Flavivirus Infectious cDNAs by a Novel Method To Reduce the Cryptic Bacterial Promoter Activity of Virus Genomes

Abstract

Reverse genetics is a powerful tool to study single-stranded RNA viruses. Despite tremendous efforts having been 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 Escherichia coli promoter (ECP) sequences within nucleotides (nt) 1 to 3000 of the dengue virus type 2 (DENV2) and Japanese encephalitis virus (JEV) genomes. Eight and four active ECPs were demonstrated within nt 1 to 3000 of the DENV2 and JEV genomes, respectively, using fusion constructs containing DENV2 or JEV segments and empty vector reporter gene Renilla luciferase. The Flavivirus genus consists of more than 70 members that are categorized into several antigenic groups (46). Most flaviviruses are transmitted by mosquito or tick vectors and cause serious human and animal diseases (46). 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, DENV2, DENV3, and DENV4. Dengue cases have been reported in over 100 countries, and an estimated 2.5 billion people live in areas in which dengue is epidemic (26, 27, 49). DENV infection often leads to dengue fever, dengue hemorrhagic fever, and dengue shock syndrome (24,28,48). 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 (56,60,61).Flaviviruses are enveloped RNA viruses that consist of single-stranded, positive-sense, 10.5-to 11-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 nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) by host proteases and a single virus-encoded protease to initiate viral replication (12,18). 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 fulllength flavivirus infectious cDNA.Reverse genetics is a powerful method for studying the viral replication of positive-strand RNA viruses (8). Unfortunately, the instability of full-length flavivirus cDNA in Escherichia coli has been a major hurdle in a attempt to construct flavivirus cDNAs (reviewed in references 4, 54, and 63). Several strategies have been developed to avoid or overcome the instability of infectious flavivirus cDNA. The instability of plasmids containing full-length YFV was avoided using an in vitro ligation approach involving two plasmids (5...

Novel cis -Acting Element within the Capsid-Coding Region Enhances Flavivirus Viral-RNA Replication by Regulating Genome Cyclization

“…The vRNA level of 91.93 also showed a moderate reduction of ca. 50% at 48 to 72 h, which is in agreement with previous findings (28). The viral loads in the supernatants of the WT-, 75.78-, and S3X-transfected BHK-21 cells were compared.…”

“…Enhancer-like elements (DB1 and DB2) had only been found in the 3=UTR (24,26) until recently, when a cis-acting enhancer element, SL6, was discovered in the capsid-coding region of tickborne flavivirus (27). The results from different groups (28)(29)(30) also indicate that the region downstream of the DENV 5=CS regulates vRNA replication, but the mechanism by which this occurs is unknown. In this study, using a combination of bioinformatic, biochemical, and reverse genetic approaches, we identified a novel cis-acting element, the downstream of 5=CS pseudoknot (DCS-PK), in the capsid-coding region, which is conserved in mosquitoborne flaviviruses.…”

Development and Characterization of Recombinant Virus Generated from a New World Zika Virus Infectious Clone

“…Preliminary studies indicated that full-length infectious clones were unstable in Escherichia coli (data not shown), likely due to cryptic prokaryotic promoters within the E and NS1 coding sequences (18). Therefore, we developed a two-plasmid system that would allow the 5= and 3= ends of the viral genome to be joined through unique ApaLI and SapI restriction enzyme sites in NS1 (Fig.…”

Characterization of cis -Acting RNA Elements of Zika Virus by Using a Self-Splicing Ribozyme-Dependent Infectious Clone

“…In general, cloning of the full-length cDNA of flavivirus downstream of either a T7/SP6 promoter or a eukaryotic RNA polymerase II promoter is sufficient to enable the recovery of progeny virus from the corresponding in vitro transcribed RNA or plasmid DNA. However, the cDNAs of flavivirus genomes are known to be toxic and unstable in common Escherichia coli hosts (16)(17)(18)(19)(20), which can be primarily attributed to the leaky expression of toxic viral proteins from the cryptic prokaryotic promoters in viral cDNA (19,20). This phenomenon has significantly hindered the development of flavivirus infectious clones (16)(17)(18)21).…”