Replication of alphaviruses requires a pseudoknot that involves the poly(A) tail

Figures

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  FIGURE 1.. (A) Alignment of CSE from all known alphaviruses to date for which the 3′-end is complete. (*) CHIKV NC_004162, Mayaro NC_003417, VEEV Venezuelan Equine Encephalitis virus NC_001449, Aura NC_003900, EEEV Eastern Equine Encephalitis virus NC_003899, Madariaga NC_023812, Mucambo NC_038672, Mwinilunga LC361437, Tai Forest NC_032681, Fort Morgan NC_013528, Cabassou NC_038670, Rio Negro NC_038674, Mosso das Pedras NC_038857, Pixuna NC_038673, Tonate NC_038675, Everglades NC_038671, O'nyong nyong NC_001512, Igbo Ora AF079457, SINV NC_001547, WEEV Western equine encephalitis virus NC_003908, Highlands J NC_012561, SESV Southern elephant seal virus HM147990, Babanki HM147984, Whataroa NC_016961, Ockelbo M69205, SINV MF589985, MF409177. (**) Ross River MH987781 plus others, Barmah Forest MN115377 plus others, Alphavirus M1 EF011023, Getah NC_006558, Sagiyama AB032553. (B) Putative hairpin structures formed by the CSE in diverse alphaviruses. (SDV) Sleeping disease virus. (C) Putative pseudoknot structures involving the poly(A) tail.

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  FIGURE 2.. Effect of poly(A) tail length on replication and translation. (A) GFP expression in BHK21J cells transfected with SINV replicon RNAs harboring different sizes of the poly(A) tail. Images show a representative area of a 48-well plate and are intentionally overexposed to show the density of cells present in this area of the plate. (B) Luciferase expression in BHK21J cells transfected with Renilla luciferase mRNAs harboring the 3′-UTR of SINV with the indicated length of the poly(A) tail. Error bars indicate standard deviation of two independent experiments.

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  FIGURE 3.. Role of S1 length and stability for replication. The minimum free energy for each S1 was calculated using Mfold (Zuker 2003). Percentages were calculated from at least two independent transfections using wild-type SinA as 100% control.

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  FIGURE 4.. Stem 2 and Loop 1 mutants. Percentages were calculated from at least two independent transfections using wild-type SinA as 100% control.

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  FIGURE 5.. (A) Putative base triples in the SINV pseudoknot, adopting either a KSHV-like conformation (top) or a human telomerase pseudoknot-like conformation (bottom). Red dotted lines indicate Hoogsteen base-pairing of U with an AU base pair. (B) Testing the KSHV-like pseudoknot conformation by mutating a putative base triple between U−3, A+13, and U−12 using the SINV replicon.

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  FIGURE 6.. Disruption and restoration of base triples in SINV pseudoknot. (A) GFP expression in BHK21J cells transfected with SINV replicons carrying the indicated changes in the pseudoknot. (B) Relative nanoluciferase expression of BHK21J cells transfected with SINV-Nanoluc replicons. Error bars indicate standard deviation of two experiments.

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  FIGURE 7.. Native gel-electrophoresis at pH 8.3 and 5.5 of SINV pseudoknot RNAs with wild-type (SINA) or mutant U−3A+12U−11 base triples (SINB, SINC). RNAs were visualized by Stains-All (left gel) or Ethidium bromide (right gel).

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  FIGURE 8.. Native gel-electrophoresis at pH 8.3 and 5.5 of MIDV pseudoknot RNAs with wild-type (MidA) or mutant U−3A+12U−11 base triples (MidB, MidC). RNAs were visualized by Stains-All (upper gel) or Ethidium bromide (lower gel).

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  FIGURE 9.. Data from in vitro (−) RNA synthesis of Hardy and Rice (2005) in relation to the pseudoknot structure.

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  FIGURE 10.. Comparison of alphavirus and telomerase pseudoknots. (A) Similarities between SINV and human and tetrahymena telomerase pseudoknots. Green dotted lines indicate A-minor interactions, red dotted lines indicate Hoogsteen base pairs, and black dashes Watson–Crick base pairs. The interactions in T. pigmentosa are drawn by analogy to the T. thermophila pseudoknot (Cash and Feigon 2017). (B) Detailed models of the MIDV and SINV pseudoknots are drawn to resemble the human telomerase pseudoknot (Theimer et al. 2005).