The translational landscape of Zika virus during infection of mammalian and insect cells

Nerea Irigoyen; Adam M. Dinan; Luke W. Meredith; Ian Goodfellow; Ian Brierley; Andrew E. Firth

RPFs, indicating that they are bona fide ribosome footprints (S1 Fig). A prominent peak of RPF 87 density occurred at nucleotide 25 of the 5′ UTR in Vero cells, coinciding with a non-canonical 88 (CUG) initiation codon (Fig. 2B) . RPFs mapped in-frame along the length of the associated 29-89 codon upstream ORF (uORF1), which ends at nucleotide 111 (4th nucleotide of the polyprotein 90 ORF) (Fig. 2B-C) . Additionally, RPFs mapped in-frame to a second uORF (uORF2), associated 91 with a non-canonical (UUG) initiation codon at nucleotide 80 ( Fig. 2B-C) . This uORF2, 77 92 codons in length, extends 202 nucleotides into the polyprotein ORF, and is generally conserved 93 across ZIKV isolates ( Fig. 1C; Frame +2) . Both uORFs were also occupied by ribosomes during 94 infection of C6/36 cells, although uORF2 was more prominent in this case (Fig. 2B) . The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/112904 doi: bioRxiv preprint 5 uORFs are often non-functional (15). In some instances, ribosomes reinitiate at downstream AUG 103 codons after translating short uORFs (16); however this appears unlikely in ZIKV given that both 104 uORFs extend beyond the polyprotein AUG codon. Additional work in animal infection models is 105 required to assess the functional significance of these uORFs. 106 107 Few RPFs were present in the 3′ UTR, consistent with a lack of translation of this region. In Vero 108 cell infections, 3′ UTR RNA-Seq density was on average 68% higher than in the rest of the gRNA. (Fig. 2D) . This location is one nucleotide upstream of the predicted 5′ end 116 of RNA "stem-loop 2" (SL2). In contrast, while Xrn1 halts preferentially at the adjacent SL1 in 117 ZIKV strain PRVABC59 (17), we found only a much more modest peak in read 5′ end mappings 118 at the SL1 site (nucleotide position 10394). Illumina smallRNA v2 to allow multiplexing, as described previously (21, 22) . The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/112904 doi: bioRxiv preprint 7 Ribosomal RNA was depleted using the "Human, Mouse, Rat" Ribo-Zero kit (Illumina) which is 154 also recommended for insect rRNA depletion. Due to poor Ribo-Zero depletion of rRNA in the 155 C6/36 Ribo-Seq samples, additional aliquots of the two biological repeats were further treated with 156 duplex-specific nuclease (DSN; Evrogen) as described previously (21) The order of mapping was tested to check that host-derived reads were not accidentally mis-mapped 169 to the virus genome, or vice versa. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/112904 doi: bioRxiv preprint 8 To compare the 5′ UTRs of ZIKV strains, the complete set of available ZIKV genomes was 180 retrieved from NCBI via a tblastn search, using the ZIKV PE243 polyprotein sequence as the query. 181