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

Abstract

Dengue virus (DENV), an arthropod-borne (“arbovirus”) virus, causes a range of human maladies ranging from self-limiting dengue fever to the life-threatening dengue shock syndrome and proliferates well in two different taxa of the Animal Kingdom, mosquitoes and primates. Mosquitoes and primates show taxonomic group-specific intolerance to certain codon pairs when expressing their genes by translation. This is called “codon pair bias”. By necessity, dengue viruses evolved to delicately balance this fundamental difference in their open reading frames (ORFs). We have undone the evolutionarily conserved genomic balance in the DENV2 ORF sequence and specifically shifted the encoding preference away from primates. However, this recoding of DENV2 raised concerns of ‘gain-of-function,’ namely whether recoding could inadvertently increase fitness for replication in the arthropod vector. Using mosquito cell lines and two strains of Aedes aegypti we did not observe any increase in fitness in DENV2 variants codon pair deoptimized for humans. This ability to disrupt and control DENV2’s host preference has great promise towards developing the next generation of synthetic vaccines not only for DENV but for other emerging arboviral pathogens such as chikungunya virus and Zika virus.

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

“…In all of our experiments described here, we have used our synthetic DENV type 2 clone based on wild-type (wt) DENV2 strain 16681 (8). This plasmid was equipped with a T7 phi2.5 promoter that synthesizes RNA starting with adenosine, the first base (following the cap) of the wt DENV genome (9, 10). Using phage T7 RNA polymerase, we synthesized transcript RNAs and modified the transcripts with one of three different 5′ termini: cap unrelated (pppAN…), a nonfunctional cap (GpppAN…, nonmethylated), or a functional cap (m 7 GpppAN…).…”

Effects of Arbovirus Multi-Host Life Cycles on Dinucleotide and Codon Usage Patterns

“…Arboviruses have also been engineered with altered codons, these include DENV [19,98], ZIKV [99], VSV [100], CHIKV [101], and tick-borne encephalitis (TBEV) [20] viruses. A summary of all codon altered arbovirus experiments can be found in Table 1.…”

“…To investigate CPB the envelope (env), helicase (NS3), and RNA-dependent RNA polymerase (RdRp)/methyltransferase (Mtase) (NS5); the env and NS1; or the RdRp (L gene) respectively were recoded. For DENV and ZIKV recoding was designed to minimize the use of codon pairs that matched those used by humans but maintain a similar CPB to that preferred in mosquitoes, all while maintaining the global codon frequency of WT viruses [19,98,99]. For both DENV and ZIKV, shifting CPB away from the human preference resulted in attenuation in vertebrate cell culture and in vivo systems [19,99].…”

“…For both DENV and ZIKV, shifting CPB away from the human preference resulted in attenuation in vertebrate cell culture and in vivo systems [19,99]. ZIKV resulted in a slight decrease in titers in mosquito cell culture [99], but codon pair recoding did not result in any changes to the replication of DENV in mosquito cell culture or in vivo [98]. It is important to note however, when codon pair usage is altered it routinely alters dinucleotide usage making it difficult to determine if CPB or a change in dinucleotide usage is the cause of attenuation [102,103].…”

Selective Targeting of Virus Replication by Proton Pump Inhibitors

“…Cells were incubated for 6 hr at room temperature while being rocked gently. Following treatment, the medium was aspirated and replaced with 250 µL containing 100 FFU (MOI = 0.0001) or 10,000 FFU DENV2 syn (MOI = 0.01) as described 45 . The plates were rocked at room temperature for 30 minutes, then 250 µL of 5% BCS DMEM was added to a total volume of 500 µL.…”