Transcriptional analysis of viral mRNAs reveals common transcription patterns in cells infected by five different filoviruses

Abstract

Filoviruses are notorious viral pathogens responsible for high-consequence diseases in humans and non-human primates. Transcription of filovirus mRNA shares several common features with transcription in other non-segmented negative-strand viruses, including differential expression of genes located across the viral genome. Transcriptional patterns of Ebola virus (EBOV) and Marburg virus (MARV) have been previously described using traditional, laborious methods, such as northern blots and in vivo labeling of viral mRNAs. More recently, however, the availability of the next generation sequencing (NGS) technology has offered a more straightforward approach to assess transcriptional patterns. In this report, we analyzed the transcription patterns of four ebolaviruses-EBOV, Sudan (SUDV), Bundibugyo (BDBV), and Reston (RESTV) viruses-in two different cell lines using standard NGS library preparation and sequencing protocols. In agreement with previous reports mainly focused on EBOV and MARV, the remaining filoviruses used in this study also showed a consistent transcription pattern, with only minor variations between the different viruses. We have also analyzed the proportions of the three mRNAs transcribed from the GP gene, which are characteristic of the genus Ebolavirus and encode the glycoprotein (GP), the soluble GP (sGP), and the small soluble GP (ssGP). In addition, we used NGS methodology to analyze the transcription pattern of two previously described recombinant MARV. This analysis allowed us to correct our construction design, and to make an improved version of the original MARV expressing reporter genes.

Fig 1

(A) Schematic representation of the genome of EBOV Makona variant (GenBank #KT589389), depicted in the viral complementary sense, with the seven genes shown in the 5′ to 3′ orientation. The lengths of the 5′ and 3′ untranslated regions (UTR), the intergenic regions (IGR), and the glycoprotein (GP) editing site are also indicated. (B) Graphical depiction of the mapped reads corresponding to EBOV mRNAs. Huh7 cells were infected with EBOV at moi = 0.1. Total RNA was harvested 2 days post infection, and purified mRNAs were used to make NGS libraries. (C and D) Graphical depiction and table showing the RPKM analysis of EBOV gene levels. The RPKM index standardizes the number of mapped reads to each gene, normalizing for gene length and for the total mapped reads of the library. “Cells” indicates mRNA isolated from cell lysates; “Sup.” refers to total RNA harvested from supernatants of infected cells. (E) Expression ratio of each viral gene relative to the nucleoprotein (NP) mRNA levels in Huh7 cells infected with EBOV, SUDV, BDBV, or RESTV.

Fig 2

(A and B) Graphical depiction and table showing the RPKM analysis of EBOV gene levels in infected Huh7 and human macrophage (Mpg) cells. (C) Expression of viral genes during propagation of EBOV, SUDV, BDBV, or RESTV in Huh7 or Mpg cells. Both cell types were infected at moi = 0.1, and harvested at 1, 2, or 3 dpi. For each virus, 3 columns are shown, representing each harvesting day. NGS libraries were designed as in Fig 1.

Fig 3. Effects of incubation temperature on viral mRNA levels.

Vero-E6 cells were infected with EBOV, SUDV, BDBV, or RESTV at moi ≥ 2. After 1 h of virus adsorption at 37°C, infected cell cultures were incubated at 37°C or 40°C. Infected cells were harvested at 1 dpi. NGS libraries and RPKM analysis were done as in Fig 1.

Fig 4

(A) Schematic representation of recombinant MARV genomes, depicted in the viral complementary sense. (B) Coverage analysis across the genome of rMARV-GFP. The area of unusually high coverage in the second IGR is indicated with an arrow. (C) Average coverage in each intergenic region of the three recombinant MARV viruses. (D) MARV mRNA levels in human macrophage (Mpg) cells infected with wild-type recombinant MARV (rMARV), rMARV expressing GFP (rMARV/GFP, previously created), or rMARV expressing ZsGreen (rMARV/ZsG, designed in this study). Cells were infected with the indicated viruses at moi = 1. Total RNA was harvested 1 dpi and NGS libraries were created as in Fig 1. (E) Cellular antiviral response. Human macrophages were infected with the indicated viruses, and gene expression was measured using a standard qRT-PCR array. Data from representative interferon-related genes were plotted as fold induction over mock-infected cells. (F) Growth kinetics of recombinant MARV. Human macrophages were infected with rMARV, rMARV/GFP, or rMARV-ZsG, and viral titers were determined by TCID₅₀ assay in Vero-E6 cells.