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

Stauft, Charles B.; Shen, Sam H.; Song, Yinglin; Gorbatsevych, Oleksandr; Asare, Emmanuel; Futcher, Bruce; Mueller, Steffen; Payne, Anne F.; Brecher, Michael; Kramer, Laura D.; Wimmer, Eckard

doi:10.1101/365189

Cited by 4 publications

(3 citation statements)

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“…In all of our experiments described here, we have used our synthetic DENV type 2 clone based on 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 non-functional cap (GpppAN…, non-methylated), or a functional cap (m 7 GpppAN…).…”

Section: Resultsmentioning

confidence: 99%

Dengue and Zika virus 5’-UTRs harbor IRES functions

Song

Mugavero

et al. 2019

Preprint

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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...

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Section: Resultsmentioning

confidence: 99%

Dengue and Zika virus 5’-UTRs harbor IRES functions

Song

Mugavero

et al. 2019

Preprint

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“…Influenza, respiratory syncytial virus, and DENV-2 vaccines based on this technology have documented both safety and immunogenicity in animal models. [113][114][115] The University of Hong Kong is developing an intranasal vaccine using an attenuated influenza virus (similar to what is in FluMist [AstraZeneca]) expressing the SARS-CoV2 S protein.…”

Section: Live Virus Vaccines and Whole Inactivated Vaccinesmentioning

confidence: 99%

SARS-CoV-2 Vaccine Development: Current Status

Poland

Ovsyannikova

Crooke

et al. 2020

Mayo Clinic Proceedings

106

113

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In the midst of the severe acute respiratory syndrome coronavirus 2 pandemic and its attendant morbidity and mortality, safe and efficacious vaccines are needed that induce protective and long-lived immune responses. More than 120 vaccine candidates worldwide are in various preclinical and phase 1 to 3 clinical trials that include inactivated, live-attenuated, viral-vectored replicating and nonreplicating, protein-and peptide-based, and nucleic acid approaches. Vaccines will be necessary both for individual protection and for the safe development of population-level herd immunity. Publicprivate partnership collaborative efforts, such as the Accelerating COVID-19 Therapeutic Interventions and Vaccines mechanism, are key to rapidly identifying safe and effective vaccine candidates as quickly and efficiently as possible. In this article, we review the major vaccine approaches being taken and issues that must be resolved in the quest for vaccines to prevent coronavirus disease 2019. For this study, we scanned the PubMed database from 1963 to 2020 for all publications using the following search terms in various combinations: SARS, MERS, COVID-19, SARS-CoV-2, vaccine, clinical trial, coronavirus, pandemic, and vaccine development. We also did a Web search for these same terms. In addition, we examined the World Health Organization, Centers for Disease Control and Prevention, and other public health authority websites. We excluded abstracts and all articles that were not written in English.

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“…We generated four RSVs with up to nine CPD ORFs. Replication of each of the four CPD RSV viruses was strikingly temperature sensitive, a phenotype also observed in other CPD viruses (13). These viruses exhibited a range of increasing attenuation in vitro and in vivo (12), providing new live-attenuated RSV vaccine Significance Synonymous recoding of viral genomes by codon-pair deoptimization (CPD) provides live-attenuated vaccine candidates expected to be genetically stable.…”

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Rescue of codon-pair deoptimized respiratory syncytial virus by the emergence of genomes with very large internal deletions that complemented replication

Nouën

McCarty

Yang

et al. 2021

Proc. Natl. Acad. Sci. U.S.A.

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Recoding viral genomes by introducing numerous synonymous but suboptimal codon pairs—called codon-pair deoptimization (CPD)—provides new types of live-attenuated vaccine candidates. The large number of nucleotide changes resulting from CPD should provide genetic stability to the attenuating phenotype, but this has not been rigorously tested. Human respiratory syncytial virus in which the G and F surface glycoprotein ORFs were CPD (called Min B) was temperature-sensitive and highly restricted in vitro. When subjected to selective pressure by serial passage at increasing temperatures, Min B substantially regained expression of F and replication fitness. Whole-genome deep sequencing showed many point mutations scattered across the genome, including one combination of six linked point mutations. However, their reintroduction into Min B provided minimal rescue. Further analysis revealed viral genomes bearing very large internal deletions (LD genomes) that accumulated after only a few passages. The deletions relocated the CPD F gene to the first or second promoter-proximal gene position. LD genomes amplified de novo in Min B–infected cells were encapsidated, expressed high levels of F, and complemented Min B replication in trans. This study provides insight on a variation of the adaptability of a debilitated negative-strand RNA virus, namely the generation of defective minihelper viruses to overcome its restriction. This is in contrast to the common “defective interfering particles” that interfere with the replication of the virus from which they originated. To our knowledge, defective genomes that promote rather than inhibit replication have not been reported before in RNA viruses.

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

Cited by 4 publications

References 58 publications

Dengue and Zika virus 5’-UTRs harbor IRES functions

Dengue and Zika virus 5’-UTRs harbor IRES functions

SARS-CoV-2 Vaccine Development: Current Status

Rescue of codon-pair deoptimized respiratory syncytial virus by the emergence of genomes with very large internal deletions that complemented replication

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