A paramyxovirus-like model for Ebola virus bipartite promoters

Abstract

Paramyxo- and filovirus nucleocapsids (NCs) have bipartite promoters at their 3' ends to initiate RNA synthesis. The 2 elements, promoter element 1 (PE1) and promoter element 2 (PE2), are separated by a spacer region that must be exactly a multiple of 6 nucleotides (nt) long. Paramyxovirus NCs have 13 nucleoprotein (NP) subunits/turn, such that PE1 and PE2 are juxtaposed on the same face of the NC helix, for concerted recognition by the viral polymerase. Ebola virus (EBOV) NCs, in contrast, have 25 to 28 subunits/turn, meaning that PE1 and PE2 cannot be juxtaposed. However, there is evidence that the number of subunits/turn at the 3' end of the EBOV NC is variable. We propose a paramyxovirus-like model for EBOV explaining why there are 8 contiguous copies of the PE2 repeat when 3 are sufficient, why expanding this run to 13 further improves minigenome performance, and why there is a limit to the number of hexa-nt that can be inserted in the spacer region.

Fig 1. Filovirus bipartite promoters.

This alignment of the EBOV/U7 genome 3′ ends is based on the premise that genomes are assembled directly from their 5′ ends, 6 nt/NP protomer, generating genome hexamer phase where only nt in hexamer positions 3, 4, and 5 point away from the protein core and can readily interact with the polymerase (underlined below). At least for ZEBOV, sequences upstream of the editing site will have different hexamer phases, e.g., 3′ NNNURR for EBOV/U7 and 3′ NNURRN for EBOV/U8 for the PE2 repeats. The conserved 3′ URR of contiguous PE2 repeats (shaded in blue) is capitalized. The conserved U of the repeat is highlighted in red and that of G75U is circled, and asterisks below the EBOV sequences indicate their overall sequence conservation. MARV, aligned below, does not appear to have any spacer region between gs (indicated by bent arrow) and the 3 PE2 repeats. Only the ZEBOV and MARV PE2 repeats have been experimentally verified [23]. BDBV, Bundibugyo virus; BOMV, Bombali virus; EBOV, Ebola virus; gs, gene start; MARV, Marburg virus; NP, nucleoprotein; nt, nucleotides; PE1, promoter element 1; PE2, promoter element 2; RESTV, Reston virus; SUDV, Sudan virus; TAFV, Tai Forest virus; ZEBOV, EBOV Zaire.

Fig 2. A paramyxovirus-like model for EBOV bipartite promoters, based on a variable and dynamic 3′ end of the NC helix.

The G75U and wt NC helices are schematically shown as 2-dimensional chains of NP subunits (green spheres), numbered from their 3′ ends, whose first turn is either 10 (panel A), 13 (panel B), or 18 subunits long (panels C and D). The open spheres in panel D represent hexa-nt insertions in the wt minigenome. Their genome RNAs are shown as a wavy line within their NP RNA-binding grooves (darker green). PE1, modeled as the first 3 subunits, is highlighted in yellow. Subunits containing contiguous PE2 hexamer repeats (3′ NNURRN for EBOV/8U and 3′ NNNURR for EBOV/7U) are highlighted in red. The polymerase (L) is shown in gray, interacting with PE1 and the minimum PE2 tripartite repeat needed for the initiation of RNA synthesis. For further detail, see text. EBOV, Ebola virus; NC, nucleocapsid; NP, nucleoprotein; nt, nucleotides; PE1, promoter element 1; PE2, promoter element 2; wt, wild-type.