Synthetic Biology Approaches for Vaccine Development

Mugavero

Stauft

et al. 2019

mBio

Self Cite

The Flavivirus genus of the Flaviviridae family encompasses numerous enveloped plus-strand RNA viruses. Dengue virus (DENV), a flavivirus, is the leading cause of serious arthropod-borne disease globally. The genomes of DENV, like the genomes of yellow fever virus (YFV), West Nile fever virus (WNV), or Zika virus (ZIKV), control their translation by a 5′-terminal capping group. Three other genera of Flaviviridae are remarkable because their viruses use internal ribosomal entry sites (IRESs) to control translation, and they are not arthropod transmitted. In 2006, E. Harris’ group published work suggesting that DENV RNA does not stringently need a cap for translation. They proposed that instead DENV translation is controlled by an interplay between 5′ and 3′ termini. Here we present evidence that the DENV or ZIKV 5′ untranslated regions (5′-UTRs) alone have IRES competence. This conclusion is based, first, on the observation that uncapped monocistronic mRNAs 5′ terminated with the DENV or ZIKV 5′-UTRs can efficiently direct translation of a reporter gene in BHK and C6/36 cells and second, that either 5′-UTR placed between two reporter genes can efficiently induce expression of the downstream gene in BHK cells but not in C6/36 cells. These experiments followed observations that uncapped DENV/ZIKV genomic transcripts, 5′ terminated with pppAN… or GpppAN…, can initiate infections of mammalian (BHK) or mosquito (C6/36) cells. IRES competence of the 5′-UTRs of DENV/ZIKV raises many open questions regarding the biology and control, as well as the evolution, of insect-borne flaviviruses. IMPORTANCE Members of the genus Flavivirus of Flaviviridae are important human pathogens of great concern because they cause serious diseases, sometimes death, in human populations living in tropical, subtropical (dengue virus [DENV], Zika virus [ZIKV], and yellow fever virus), or moderate climates (West Nile virus). Flaviviruses are known to control their translation by a cap-dependent mechanism. We have observed, however, that the uncapped genomes of DENV or ZIKV can initiate infection of mammalian and insect cells. We provide evidence that the short 5′ untranslated region (5′-UTR) of DENV or ZIKV genomes can fulfill the function of an internal ribosomal entry site (IRES). This strategy frees these organisms from the cap-dependent mechanism of gene expression at an as yet unknown stage of proliferation. The data raise new questions about the biology and evolution of flaviviruses, possibly leading to new controls of flavivirus disease.

Section: Methodsmentioning

confidence: 99%

Dengue and Zika Virus 5′ Untranslated Regions Harbor Internal Ribosomal Entry Site Functions

Mugavero

Stauft

et al. 2019

mBio

Self Cite

“…The infectious dengue virus serotype 2 clone DENV2 syn was synthesized and assembled in our lab (42). An infectious cDNA ZIKV clone (strain FSS13025, GenBank number KU955593.1) developed by Dr. Pei-Yong Shi’s laboratory at University of Texas Medical Branch (UTMB), Galveston, TX was kindly gifted to us (43).…”

Section: Methodsmentioning

confidence: 99%

Dengue and Zika virus 5’-UTRs harbor IRES functions

Mugavero

et al. 2019

Preprint

Self Cite

18Members of Flavivirus, a genus of Flaviviridae, encompass numerous enveloped plus strand 19 RNA viruses, of which globally dengue virus (DENV) is the leading cause of serious arthropod-20 borne disease. The genomes of DENV, just as those of yellow fever virus (YFV), West Nile 21 2 fever virus (WNV), or Zika virus (ZIKV), control their translation by a 5'-terminal capping 22 group. Three other genera of Flaviviridae are remarkable because their viruses use internal 23 ribosomal entry sites (IRESs) to control translation and they are not arthropod transmitted. In 24 2006 E. Harris' group published work suggesting that DENV RNA does not stringently need a 25 109 virus at the end point of experiments roughly equal to that of the transcripts with the functional 110 m 7 G cap (Fig. 1). As we have observed previously (8), infectious virus yields were always 111 6slightly higher in C6/36 cells than in BHK cells. This observation suggests that DENV infection 112 is more productive in mosquito cells compared to mammalian cells. 113Both the m 7 GpppA-capped and unmethylated GpppA-capped DENV transcripts produced 114 similar viral titers on BHK cells post transfection (Fig. 1). Therefore, we carried out a detailed 115 assay of infectivity with the two uncapped pppAN-and GpppAN-DENV genome transcripts 116 wondering if we would find major differences in specific infectivity of the in vitro RNAs. 117Samples were harvested daily (up to day 5) from medium and used for focus forming assays in 118 Vero cells (see Materials and Methods). The result showed that the pppA-DENV2 genome RNA 119 produced viral titers that were 2-3 log10 lower than those obtained with the unmethylated 120 GpppA-5'modified RNAs (Fig. S1). This observation is in accordance with a previous report 121 obtained with transcripts of yellow fever virus cDNA (11). We assume that the viruses harvested 122 at the end of the incubation contain genomes with DENV-specific capping groups. The reason is 123 that the viral replication machinery, that was newly assembled in the course of the replication 124 cycle, will provide newly synthesized genomes with the 5' terminal modification (12)(13)(14). 125Experiments to test this hypothesis are currently in progress. 126As was mentioned in the Introduction, there are different possibilities to explain the infectivity of 127 uncapped DENV transcripts (see Discussion). In the following we report our experiments to test 128 the DENV and ZIKV 5' UTRs for IRES competence. 129 Translation of mono-cistronic mRNAs under the control of DENV 5'-UTR variants in 130 mammalian cells 131To test whether the uncapped transcripts of the DENV cDNA harbor an activity that allows cap-132 independent translation we designed mono-cistronic mRNAs 5' terminated with either pppAN, 133 GpppAN or m 7 GpppAN, followed by the 96-nt-long DENV 5'-UTR sequence (D5; Fig. 2) and 134 7 the Gluc ORF (15). We chose two different 3' termini, the DENV 3'-UTR (D3) because of its 135 possible role in DENV translation (16) or the polyadenylated 3'-UTR (P) of poli...

“…Synthetic biology has the potential to revolutionize the rapid development of vaccines to treat infectious diseases as the research paradigm shifts from empirical to rational design [1,2]. Since the first demonstration of an infectious fully synthetic virus in 2002 (7.5 kb) [3] and the ensuing initial societal concerns[4], rapid advances in DNA synthesis including decreased cost [5], has led to the general acceptance of synthetic organisms as a research tool[1,6,7].…”

Section: Introductionmentioning

confidence: 99%

“…There are multiple methods of recoding a viral genome to achieve attenuation including the introduction of random point mutations [18], scrambling of codons while maintaining natural biases [19], reduction of codon bias for the host organism [20], and, as described here, changing of codon pair bias (CPB) to negative values[2,21]. Previously, our laboratory has exploited the universal phenomenon of CPB [22,23], whereby codons are prone to pair more or less frequently than expected with one another, independently of individual codon bias.…”

Section: Introductionmentioning

confidence: 99%

Extensive recoding of dengue virus type 2 specifically reduces replication in primate cells without gain-of-function in Aedes aegypti mosquitoes

Stauft

Shen

et al. 2018

Preprint

Self Cite

Abstract18 Dengue virus (DENV), an arthropod-borne ("arbovirus") virus causing a range of 19 human maladies ranging from self-limiting dengue fever to the life-threatening 20 dengue shock syndrome, proliferates well in two different taxa of the Animal 21 Kingdom, mosquitoes and primates. Unexpectedly, mosquitoes and primates have 22 distinct preferences when expressing their genes by translation, e.g. members of 23 these taxa show taxonomic group-specific intolerance to certain codon pairs. This is 24 called "codon pair bias". By necessity, arboviruses evolved to delicately balance this 25 fundamental difference in their ORFs. Using the mosquito-borne human pathogen 26 DENV we have undone the evolutionarily conserved genomic balance in its ORF 27 sequence and specifically shifted the encoding preference away from primates.28 However, this recoding of DENV raised concerns of 'gain-of-function,' namely 29 whether recoding could inadvertently increase fitness for replication in the 30 arthropod vector. Using mosquito cell cultures and two strains of Aedes aegypti we 31 did not observe any increase in fitness in DENV2 variants codon pair deoptimized . CC-BY 4.0 International license It is made available under a was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.