Replication Element Research Articles

The Salmonella enterica Plasmidome as a Reservoir of Antibiotic Resistance.

The emergence of multidrug-resistant bacterial strains worldwide has become a serious problem for public health over recent decades. The increase in antimicrobial resistance has been expanding via plasmids as mobile genetic elements encoding antimicrobial resistance (AMR) genes that are transferred vertically and horizontally. This study focuses on Salmonella enterica, one of the leading foodborne pathogens in industrialized countries. S. enterica is known to carry several plasmids involved not only in virulence but also in AMR. In the current paper, we present an integrated strategy to detect plasmid scaffolds in whole genome sequencing (WGS) assemblies. We developed a two-step procedure to predict plasmids based on i) the presence of essential elements for plasmid replication and mobility, as well as ii) sequence similarity to a reference plasmid. Next, to confirm the accuracy of the prediction in 1750 S. enterica short-read sequencing data, we combined Oxford Nanopore MinION long-read sequencing with Illumina MiSeq short-read sequencing in hybrid assemblies for 84 isolates to evaluate the proportion of plasmid that has been detected. At least one scaffold with an origin of replication (ORI) was predicted in 61.3% of the Salmonella isolates tested. The results indicated that IncFII and IncI1 ORIs were distributed in many S. enterica serotypes and were the most prevalent AMR genes carrier, whereas IncHI2A/IncHI2 and IncA/C2 were more serotype restricted but bore several AMR genes. Comparison between hybrid and short-read assemblies revealed that 81.1% of plasmids were found in the short-read sequencing using our pipeline. Through this process, we established that plasmids are prevalent in S. enterica and we also substantially expand the AMR genes in the resistome of this species.

Nuclear Egress Complexes of HCMV and Other Herpesviruses: Solving the Puzzle of Sequence Coevolution, Conserved Structures and Subfamily-Spanning Binding Properties.

Herpesviruses uniquely express two essential nuclear egress-regulating proteins forming a heterodimeric nuclear egress complex (core NEC). These core NECs serve as hexameric lattice-structured platforms for capsid docking and recruit viral and cellular NEC-associated factors that jointly exert nuclear lamina as well as membrane-rearranging functions (multicomponent NEC). The regulation of nuclear egress has been profoundly analyzed for murine and human cytomegaloviruses (CMVs) on a mechanistic basis, followed by the description of core NEC crystal structures, first for HCMV, then HSV-1, PRV and EBV. Interestingly, the highly conserved structural domains of these proteins stand in contrast to a very limited sequence conservation of the key amino acids within core NEC-binding interfaces. Even more surprising, although a high functional consistency was found when regarding the basic role of NECs in nuclear egress, a clear specification was identified regarding the limited, subfamily-spanning binding properties of core NEC pairs and NEC multicomponent proteins. This review summarizes the evolving picture of the relationship between sequence coevolution, structural conservation and properties of NEC interaction, comparing HCMV to α-, β- and γ-herpesviruses. Since NECs represent substantially important elements of herpesviral replication that are considered as drug-accessible targets, their putative translational use for antiviral strategies is discussed.

Minimization of Elements for Isothermal DNA Replication by an Evolutionary Approach.

DNA replication is one of the central functions of the cell. The complexity of modern DNA replication systems raises a question: is it possible to achieve a simpler continuous isothermal DNA replication using fewer proteins? Here, we searched such replication using an evolutionary approach. Through a long-term serial dilution experiment with phi29 DNA polymerase, we found that large repetitive DNAs spontaneously appear and continuously replicate. The repetitive sequence is critical for replication. Arbitrary sequences can replicate if they contain many repeats. We also demonstrated continuous DNA replication using expressed polymerase from the DNA for 10 rounds. This study revealed that continuous isothermal DNA replication can be achieved in a scheme simpler than that employed by modern organisms, providing an alternative strategy for simpler artificial cell synthesis and a clue to possible primitive forms of DNA replication.

SARS-CoV-2 proteins (version 2020.2) in the IUPHAR/BPS Guide to Pharmacology Database

Coronaviruses are large, often spherical, enveloped, single-stranded positive-sense RNA viruses, ranging in size from 80-220 nm. Of the four structural proteins encoded in the viral genome, the RNA winds around the highly basic nucleocapsid (N) protein. The three other structural proteins, envelope (E), membrane (M) and spike (S), are transmembrane proteins. The E protein is a small (9-12 kDa) single transmembrane domain protein, which enables virus assembly with the M protein, a larger (23-35 kDa) 3TM protein. Coronaviruses are named for the crown-shaped appearance of the virus due to the large (120+ kDa) S spike 1TM glycoprotein, which forms extended homotrimers. The spike protein binds to the animal host cell by interacting with specific anchoring proteins, typically proteinases, such as angiotensin-converting enzyme 2 or aminopeptidase N. This binding facilitates viral entry into the cell and the release of the genome. Aside from the four structural proteins, the remainder of the genome encodes accessory or non-structural proteins and includes proteinases to cleave the two encoded polyproteins. The remainder of the genome encodes elements for viral replication, assembly and release, as well as proteins which manipulate the host's innate immune system.

Hepatitis C Virus Translation Regulation.

Translation of the hepatitis C virus (HCV) RNA genome is regulated by the internal ribosome entry site (IRES), located in the 5’-untranslated region (5′UTR) and part of the core protein coding sequence, and by the 3′UTR. The 5′UTR has some highly conserved structural regions, while others can assume different conformations. The IRES can bind to the ribosomal 40S subunit with high affinity without any other factors. Nevertheless, IRES activity is modulated by additional cis sequences in the viral genome, including the 3′UTR and the cis-acting replication element (CRE). Canonical translation initiation factors (eIFs) are involved in HCV translation initiation, including eIF3, eIF2, eIF1A, eIF5, and eIF5B. Alternatively, under stress conditions and limited eIF2-Met-tRNAiMet availability, alternative initiation factors such as eIF2D, eIF2A, and eIF5B can substitute for eIF2 to allow HCV translation even when cellular mRNA translation is downregulated. In addition, several IRES trans-acting factors (ITAFs) modulate IRES activity by building large networks of RNA-protein and protein–protein interactions, also connecting 5′- and 3′-ends of the viral RNA. Moreover, some ITAFs can act as RNA chaperones that help to position the viral AUG start codon in the ribosomal 40S subunit entry channel. Finally, the liver-specific microRNA-122 (miR-122) stimulates HCV IRES-dependent translation, most likely by stabilizing a certain structure of the IRES that is required for initiation.

The Emerging Roles of Fox Family Transcription Factors in Chromosome Replication, Organization, and Genome Stability.

The forkhead box (Fox) transcription factors (TFs) are widespread from yeast to humans. Their mutations and dysregulation have been linked to a broad spectrum of malignant neoplasias. They are known as critical players in DNA repair, metabolism, cell cycle control, differentiation, and aging. Recent studies, especially those from the simple model eukaryotes, revealed unexpected contributions of Fox TFs in chromosome replication and organization. More importantly, besides functioning as a canonical TF in cell signaling cascades and gene expression, Fox TFs can directly participate in DNA replication and determine the global replication timing program in a transcription-independent mechanism. Yeast Fox TFs preferentially recruit the limiting replication factors to a subset of early origins on chromosome arms. Attributed to their dimerization capability and distinct DNA binding modes, Fkh1 and Fkh2 also promote the origin clustering and assemblage of replication elements (replication factories). They can mediate long-range intrachromosomal and interchromosomal interactions and thus regulate the four-dimensional chromosome organization. The novel aspects of Fox TFs reviewed here expand their roles in maintaining genome integrity and coordinating the multiple essential chromosome events. These will inevitably be translated to our knowledge and new treatment strategies of Fox TF-associated human diseases including cancer.

Structural and phenotypic analysis of Chikungunya virus RNA replication elements

Chikungunya virus (CHIKV) is a re-emerging, pathogenic Alphavirus transmitted to humans by Aedes spp. mosquitoes. We have mapped the RNA structure of the 5′ region of the CHIKV genome using selective 2′-hydroxyl acylation analysed by primer extension (SHAPE) to investigate intramolecular base-pairing at single-nucleotide resolution. Taking a structure-led reverse genetic approach, in both infectious virus and sub-genomic replicon systems, we identified six RNA replication elements essential to efficient CHIKV genome replication - including novel elements, either not previously analysed in other alphaviruses or specific to CHIKV. Importantly, through a reverse genetic approach we demonstrate that the replication elements function within the positive-strand genomic copy of the virus genome, in predominantly structure-dependent mechanisms during efficient replication of the CHIKV genome. Comparative analysis in human and mosquito-derived cell lines reveal that a novel element within the 5′UTR is essential for efficient replication in both host systems, while those in the adjacent nsP1 encoding region are specific to either vertebrate or invertebrate host cells. In addition to furthering our knowledge of fundamental aspects of the molecular virology of this important human pathogen, we foresee that results from this study will be important for rational design of a genetically stable attenuated vaccine.

Characterization of Mutational Tolerance of a Viral RNA-Protein Interaction.

Replication of RNA viruses is generally markedly error-prone. Nevertheless, these viruses usually retain their identity under more or less constant conditions due to different mechanisms of mutation tolerance. However, there exists only limited information on quantitative aspects of the mutational tolerance of distinct viral functions. To address this problem, we used here as a model the interaction between a replicative cis-acting RNA element (oriL) of poliovirus and its ligand (viral protein 3CD). The mutational tolerance of a conserved tripeptide of 3CD, directly involved in this interaction, was investigated. Randomization of the relevant codons and reverse genetics were used to define the space of viability-compatible sequences. Surprisingly, at least 11 different amino acid substitutions in this tripeptide were not lethal. Several altered viruses exhibited wild-type-like phenotypes, whereas debilitated (but viable) genomes could increase their fitness by the acquisition of reversions or compensatory mutations. Together with our study on the tolerance of oriL (Prostova et al., 2015), the results demonstrate that at least 42 out of 51 possible nucleotide replacements within the two relevant genomic regions are viability-compatible. These results provide new insights into structural aspects of an important viral function as well as into the general problems of viral mutational robustness and evolution.

A Direct RNA-to-RNA Replication System for Enhanced Gene Expression in Bacteria.

A long-standing objective of metabolic engineering has been to exogenously increase the expression of target genes. In this research, we proposed the permanent RNA replication system using DNA as a template to store genetic information in bacteria. We selected Qβ phage as the RNA replication prototype and made many improvements to achieve target gene expression enhancement directly by increasing mRNA abundance. First, we identified the endogenous gene Rnc, the knockout of which significantly improved the RNA replication efficiency. Second, we elucidated the essential elements for RNA replication and optimized the system to make it more easily applicable. Combined with optimization of the host cell and the system itself, we developed a stable RNA-to-RNA replication tool to directly increase the abundance of the target mRNA and subsequently the target protein. Furthermore, it was proven efficient in enhancing the expression of specific proteins and was demonstrated to be applicable in metabolic engineering. Our system has the potential to be combined with any of the existing methods for increasing gene expression.

The mitochondrial R-loop.

The DNA in mitochondria contributes essential components of the organelle’s energy producing machinery that is essential for life. In 1971, many mitochondrial DNA molecules were found to have a third strand of DNA that maps to a region containing critical regulatory elements for transcription and replication. Forty-five years later, a third strand of RNA in the same region has been reported. This mitochondrial R-loop is present on thousands of copies of mitochondrial DNA per cell making it potentially the most abundant R-loop in nature. Here, I assess the discovery of the mitochondrial R-loop, discuss why it remained unrecognized for almost half a century and propose for it central roles in the replication, organization and expression of mitochondrial DNA, which if compromised can lead to disease states.

Molecular Mechanisms of DNA Replication and Repair Machinery: Insights from Microscopic Simulations.

Reproduction, the hallmark of biological activity, requires making an accurate copy of the genetic material to allow the progeny to inherit parental traits. In all living cells, the process of DNA replication is carried out by a concerted action of multiple protein species forming a loose protein-nucleic acid complex, the replisome. Proofreading and error correction generally accompany replication but also occur independently, safeguarding genetic information through all phases of the cell cycle. Advances in biochemical characterization of intracellular processes, proteomics and the advent of single-molecule biophysics have brought about a treasure trove of information awaiting to be assembled into an accurate mechanistic model of the DNA replication process. In this review, we describe recent efforts to model elements of DNA replication and repair processes using computer simulations, an approach that has gained immense popularity in many areas of molecular biophysics but has yet to become mainstream in the DNA metabolism community. We highlight the use of diverse computational methods to address specific problems of the fields and discuss unexplored possibilities that lie ahead for the computational approaches in these areas.

The HCV genome domains 5BSL3.1 and 5BSL3.3 act as managers of translation

The RNA genome of the hepatitis C virus (HCV) encodes a single open reading frame (ORF) containing numerous functional elements. Among these, the cis-acting replication element (CRE) at the 3′ end of the viral ORF, has become of increasing interest given its dual role as a viral translation repressor and replication enhancer. Long-range RNA-RNA contacts mediated by the CRE build the structural scaffold required for its proper functioning. The recruitment of different cellular factors, many related to the functioning of the translation machinery, might aid in the CRE-exerted downregulation of viral translation. The present data show that the CRE promotes a defect in polysome production, and hinders the assembly of the 80S complex, likely through the direct, high affinity recruitment of the 40S ribosomal subunit. This interaction involves the highly conserved 5BSL3.1 and 5BSL3.3 domains of the CRE, and is strictly dependent on RNA-protein contacts, particularly with the ribosomal proteins RPSA and RPS29. These observations support a model in which the CRE-mediated inhibition of viral translation is a multifactorial process defined by the establishment of long-range RNA-RNA interactions between the 5′ and 3′ ends of the viral genome, the sequestration of the 40S subunit by the CRE, and the subsequent stalling of polysome elongation at the 3′ end of the ORF, all governed by the highly stable hairpin domains 5BSL3.1 and 5BSL3.3. The present data thus suggest a new managerial role in HCV translation for these 5BSL3.1 and 5BSL3.3 domains.

Exploring the Remote Ties between Helitron Transposases and Other Rolling-Circle Replication Proteins.

Rolling-circle replication (RCR) elements constitute a diverse group that includes viruses, plasmids, and transposons, present in hosts from all domains of life. Eukaryotic RCR transposons, also known as Helitrons, are found in species from all eukaryotic kingdoms, sometimes representing a large portion of their genomes. Despite the impact of Helitrons on their hosts, knowledge about their relationship with other RCR elements is still elusive. Here, we compared the endonuclease domain sequence of Helitron transposases with the corresponding region from RCR proteins found in a wide variety of mobile genetic elements. To do that, we used a stepwise alignment approach followed by phylogenetic and multidimensional scaling analyses. Although it has been suggested that Helitrons might have originated from prokaryotic transposons or eukaryotic viruses, our results indicate that Helitron transposases share more similarities with proteins from prokaryotic viruses and plasmids instead. We also provide evidence for the division of RCR endonucleases into three groups (Y1, Y2, and Yx), covering the whole diversity of this protein family. Together, these results point to prokaryotic elements as the likely closest ancestors of eukaryotic RCR transposons, and further demonstrate the fluidity that characterizes the boundaries separating viruses, plasmids, and transposons.

A novel method for transforming the thermophilic bacterium Geobacillus kaustophilus

BackgroundBacterial strains of the genus Geobacillus grow at high temperatures of 50–75 °C and could thus be useful for biotechnological applications. However, genetic manipulation of these species is difficult because the current techniques for transforming Geobacillus species are not efficient. In this study, we developed an easy and efficient method for transforming Geobacillus kaustophilus using the conjugative plasmid pLS20cat.ResultsWe constructed a transformation system comprising (i) a mobilizable Bacillus subtilis–G. kaustophilus shuttle plasmid named pGK1 that carries the elements for selection and replication in Geobacillus, and (ii) a pLS20cat-harboring B. subtilis donor strain expressing the dam methylase gene of Escherichia coli and the conjugation-stimulating rapLS20 gene of pLS20cat. This system can be used to efficiently introduce pGK1 into G. kaustophilus by mobilization in a pLS20cat-dependent way. Whereas the thermostable kanamycin marker and Geobacillus replication origin of pGK1 as well as expression of dam methylase in the donor were indispensable for mobilization, ectopic expression of rapLS20 increased its efficiency. In addition, the conditions of the recipient influenced mobilization efficiency: the highest mobilization efficiencies were obtained using recipient cells that were in the exponential growth phase. Furthermore, elimination of the origin of transfer from pLS20cat enhanced the mobilization.ConclusionsWe describe a novel method of plasmid mobilization into G. kaustophilus recipient from B. subtilis donor depending on the helper function of pLS20cat, which enables simple, rapid, and easy transformation of the thermophilic Gram-positive bacterium.

Development and characterization of DNAzyme candidates demonstrating significant efficiency against human rhinoviruses

Infections with human rhinoviruses (RVs) are responsible for millions of common cold episodes and the majority of asthma exacerbations, especially in childhood. No drugs specifically targeting RVs are available. We sought to identify specific anti-RV molecules based on DNAzyme technology as candidates to a clinical study. A total of 226 candidate DNAzymes were designed against 2 regions of RV RNA genome identified to be sufficiently highly conserved between virus strains (ie, the 5'-untranslated region and cis-acting replication element) by using 3 test strains: RVA1, RVA16, and RVA29. All DNAzymes were screened for their cleavage efficiency against invitro-expressed viral RNA. Those showing any catalytic activity were subjected to bioinformatic analysis of their reverse complementarity to 322 published RV genomic sequences. Further molecular optimization was conducted for the most promising candidates. Cytotoxic and off-target effects were excluded in HEK293 cell-based systems. Antiviral efficiency was analyzed in infected human bronchial BEAS-2B cells and exvivo-cultured human sinonasal tissue. Screening phase-generated DNAzymes characterized by either good catalytic activity or by high RV strain coverage but no single molecule represented a satisfactory combination of those 2 features. Modifications in length of the binding domains of 2 lead candidates, Dua-01(-L12R9) and Dua-02(-L10R11), improved their cleavage efficiency to an excellent level, with no loss in eminent strain coverage (about 98%). Both DNAzymes showed highly favorable cytotoxic/off-target profiles. Subsequent testing of Dua-01-L12R9 in BEAS-2B cells and sinonasal tissue demonstrated its significant antiviral efficiency. Effective and specific management of RV infections with Dua-01-L12R9 might be useful in preventing asthma exacerbations, which should be verified by clinical trials.

Core elements of the vegetative replication control of the Inc1 plasmid pO104_90 of Escherichia coli O104:H4 also regulate its transfer frequency

The highly virulent Escherichia coli O104:H4 isolate that caused a large outbreak in 2011 carries three plasmids. Out of these, only one, the IncI plasmid pO104_90 that encodes two extended spectrum beta-lactamases, can transfer itself by conjugation. Considering its potential contribution to the emergence and virulence of the outbreak strain, we aimed to get a closer insight into pO104_90 transfer efficiency and control. We tested the host spectrum of the plasmid and observed transmission into Enterobactericeae including clinically relevant enterobacterial pathogens like Salmonella typhimurium and Shigella flexneri. However, we found that this plasmid did not transfer into E. coli strains that kill the donor strain due to bacteriocin production, e.g. the probiotic E. coli Nissle 1917. Under the same conditions, the highly transmittable control plasmid RP4 was efficiently transferred into all these recipients. Therefore we hypothesized that the failure of transfer of pO104_90 was simply due to the generally much lower transmission rates of this IncI plasmid and we decided to screen for factors that negatively affect the transfer of the plasmid by an in vivo deletion analysis. Our attempts to delete larger regions of the plasmid resulted in cells containing both a truncated plasmid (Δ50 kb and Δ75 kb) and a wild type copy of pO104_90. When used as donors in conjugation experiments, these cells transferred the wild type plasmid at dramatically increased rates. This indicated that the relatively limited region shared by both plasmids contained an activator of transfer. We therefore analyzed its transcriptional organization, dissected the candidate region by subcloning and showed that additional copies of repY/INC were sufficient to increase the transfer frequency of pO104_90 to the observed level. To our knowledge, this is the first evidence for a direct regulatory cross talk between core control elements of the vegetative replication and the transfer functions of an IncI1 plasmid.

Discovery of Enterovirus A71-like nonstructural genomes in recent circulating viruses of the Enterovirus A species

Enterovirus A71 (EV-A71) is an important nonpolio enterovirus that causes severe neurological complications. In 1998, Taiwan experienced an EV-A71 outbreak that caused 78 deaths. Since then, periodic epidemics of EV-A71 associated with newly emerging strains have occurred. Several of these strains are known to be recombinant; however, how these strains arose within such a short period of time remains unknown. Here, we sequenced 64 full-length genomes from clinical isolates collected from 2005 to 2016 and incorporated all 91 Taiwanese genomes downloaded from the Virus Pathogen Resource to extensively analyze EV-A71 recombination in Taiwan. We found that the B3 subgenotype was a potential recombinant parent of the EV-A71 C2-like and C4 strains by intratypic recombination. Such B3-similar regions were also found in many cocirculating coxsackieviruses belonging to Enterovirus A species (EV-A) through a series of intertypic recombinations. Therefore, locally enriched outbreaks of cocirculating viruses from different genotypes/serotypes may facilitate recombination. Most recombination breakpoints we found had nonrandom distributions and were located within the region spanning from the boundary of P1 (structural gene) and P2 (nonstructural) to the cis-acting replication element at P2, indicating that specific genome reassembly of structural and nonstructural genes may be subject to natural selection. Through intensive recombination, 11 EV-A71-like signatures (including one in 3A, two in 3C, and eight in 3D) were found to be present in a variety of recently cocirculating EV-A viruses worldwide, suggesting that these viruses may be targets for wide-spectrum antiviral development.

Bacterial CRISPR Regions: General Features and their Potential for Epidemiological Molecular Typing Studies.

Introduction:CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) loci as novel and applicable regions in prokaryotic genomes have gained great attraction in the post genomics era.Methods:These unique regions are diverse in number and sequence composition in different pathogenic bacteria and thereby can be a suitable candidate for molecular epidemiology and genotyping studies. Results:Furthermore, the arrayed structure of CRISPR loci (several unique repeats spaced with the variable sequence) and associated cas genes act as an active prokaryotic immune system against viral replication and conjugative elements. This property can be used as a tool for RNA editing in bioengineering studies.Conclusion:The aim of this review was to survey some details about the history, nature, and potential applications of CRISPR arrays in both genetic engineering and bacterial genotyping studies.

The 5BSL3.2 Functional RNA Domain Connects Distant Regions in the Hepatitis C Virus Genome.

Viral genomes are complexly folded entities that carry all the information required for the infective cycle. The nucleotide sequence of the RNA virus genome encodes proteins and functional information contained in discrete, highly conserved structural units. These so-called functional RNA domains play essential roles in the progression of infection, which requires their preservation from one generation to the next. Numerous functional RNA domains exist in the genome of the hepatitis C virus (HCV). Among them, the 5BSL3.2 domain in the cis-acting replication element (CRE) at the 3′ end of the viral open reading frame has become of particular interest given its role in HCV RNA replication and as a regulator of viral protein synthesis. These functionalities are achieved via the establishment of a complex network of long-distance RNA–RNA contacts involving (at least as known to date) the highly conserved 3′X tail, the apical loop of domain IIId in the internal ribosome entry site, and/or the so-called Alt region upstream of the CRE. Changing contacts promotes the execution of different stages of the viral cycle. The 5BSL3.2 domain thus operates at the core of a system that governs the progression of HCV infection. This review summarizes our knowledge of the long-range RNA–RNA interaction network in the HCV genome, with special attention paid to the structural and functional consequences derived from the establishment of different contacts. The potential implications of such interactions in switching between the different stages of the viral cycle are discussed.

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

Zika virus (ZIKV) has caused significant outbreaks and epidemics in the Americas recently, raising global concern due to its ability to cause microcephaly and other neurological complications. A stable and efficient infectious clone of ZIKV is urgently needed. However, the instability and toxicity of flavivirus cDNA clones in Escherichia coli hosts has hindered the development of ZIKV infectious clones. Here, using a novel self-splicing ribozyme-based strategy, we generated a stable infectious cDNA clone of a contemporary ZIKV strain imported from Venezuela to China in 2016. The constructed clone contained a modified version of the group II self-splicing intron P.li.LSUI2 near the junction between the E and NS1 genes, which were removed from the RNA transcripts by an easy-to-establish in vitro splicing reaction. Transfection of the spliced RNAs into BHK-21 cells led to the production of infectious progeny virus that resembled the parental virus. Finally, potential cis-acting RNA elements in ZIKV genomic RNA were identified based on this novel reverse genetics system, and the critical role of 5'-SLA promoter and 5'-3' cyclization sequences were characterized by a combination of different assays. Our results provide another stable and reliable reverse genetics system for ZIKV that will help study ZIKV infection and pathogenesis, and the novel self-splicing intron-based strategy could be further expanded for the construction of infectious clones from other emerging and reemerging flaviviruses.IMPORTANCE The ongoing Zika virus (ZIKV) outbreaks have drawn global concern due to the unexpected causal link to fetus microcephaly and other severe neurological complications. The infectious cDNA clones of ZIKV are critical for the research community to study the virus, understand the disease, and inform vaccine design and antiviral screening. A panel of existing technologies have been utilized to develop ZIKV infectious clones. Here, we successfully generated a stable infectious clone of a 2016 ZIKV strain using a novel self-splicing ribozyme-based technology that abolished the potential toxicity of ZIKV cDNA clones to the E. coli host. Moreover, two crucial cis-acting replication elements (5'-SLA and 5'-CS) of ZIKV were first identified using this novel reverse genetics system. This novel self-splicing ribozyme-based reverse genetics platform will be widely utilized in future ZIKV studies and provide insight for the development of infectious clones of other emerging viruses.