Rotavirus Rearranged Genomic RNA Segments Are Preferentially Packaged into Viruses Despite Not Conferring Selective Growth Advantage to Viruses

The rotavirus (RV) genome consists of 11 double-stranded RNA segments. Sometimes, partial sequence duplication of an RNA segment leads to a rearranged RNA segment. To specify the impact of rearrangement, the replication efficiencies of human RV with rearranged segments 7, 11 or both were compared to these of the homologous human wild-type RV (wt-RV) and of the bovine wt-RV strain RF. As judged by viral growth curves, rotaviruses with a rearranged genome (r-RV) had no selective growth advantage over the homologous wt-RV. In contrast, r-RV were selected over wt-RV during competitive experiments (i.e mixed infections between r-RV and wt-RV followed by serial passages in cell culture). Moreover, when competitive experiments were performed between a human r-RV and the bovine wt-RV strain RF, which had a clear growth advantage, rearranged segments 7, 11 or both always segregated in viral progenies even when performing mixed infections at an MOI ratio of 1 r-RV to 100 wt-RV. Lastly, bovine reassortant viruses that had inherited a rearranged segment 7 from human r-RV were generated. Although substitution of wt by rearranged segment 7 did not result in any growth advantage, the rearranged segment was selected in the viral progenies resulting from mixed infections by bovine reassortant r-RV and wt-RV, even for an MOI ratio of 1 r-RV to 107 wt-RV. Lack of selective growth advantage of r-RV over wt-RV in cell culture suggests a mechanism of preferential packaging of the rearranged segments over their standard counterparts in the viral progeny

One-step viral growth curves of bovine reassortant viruses RF7R and RF7RΔ.

<p>For each virus (RF, RF7R and RF7RΔ), confluent MA-104 cells were inoculated at the same MOI of 0.5 PFU/cell. The infected cell cultures were harvested at 2, 6, 8, 10, and 18 hours post-infection and virus titers were determined by plaque assay in MA-104 cells. The rearranged segment 7 of bovine reassortant viruses RF7R and RF7RΔ derives from human r-RV M1 and M3, respectively.</p

Competitive experiments between human r-RV and bovine wt-RV at an MOI ratio of 1∶100.

<p>Competitive experiments were performed by co-infecting MA-104 cells by 0.003 PFU/cell of r-RV M1 or M3 and 0.3 PFU/cell of bovine wt-RV RF (1∶100 MOI ratio). RNA profiles of viral progenies resulting from mixed infections by wt-bovine RF virus and human r-RV M1 (A) or M3 (B). Pn indicate the passage number; numbers indicate the location of RNA segments; arrows indicate rearranged segments 7R and 7RΔ. (C) Western-blot detection of the NSP3 protein expressed by the viral progenies resulting from the M3+RF co-infection (1∶100 MOI ratio) at passage 3 (P3) and 9 (P9). Arrows indicate the NSP3 and the modified mNSP3 proteins expressed by wt-RV RF and r-RV M3, respectively. Numbers indicate molecular size, in kilodaltons.</p

Competitive experiments between bovine wt-RV and human r-RV.

<p>Competitive experiments were performed by co-infecting MA-104 cells by the bovine wt-RV RF and one of the human r-RV at an MOI ratio of 1∶1 (0.3 PFU/cell for each virus). The resulting cell culture lysates were serially propagated in MA-104 cells. RNA profiles of viral progenies resulting from mixed infections by wt-bovine RF virus and human r-RV M1 (A), M3 (B), M4 (C), or M2 (D). RNA profiles of wt-bovine RF and human r-RV show differences of mobility for 8 RNA segments (segments 1, 4–6, 8–11). I and Pn indicate the initial inoculum used for mixed infections and the passage number, respectively. Numbers indicate the location of RNA segments. Arrows indicate rearranged segments 7R, 7RΔ, and 11R.</p

Competitive experiments between human wt-RV M0 and r-RV M1 or M3.

<p>Competitive experiments were performed by co-infecting MA-104 cells by wt-RV and r-RV at an MOI ratio of 1∶1 (0.3 PFU/cell for each virus). The resulting cell culture lysates were serially propagated in MA-104 cells. RNA profiles of viral progenies resulting from mixed infections by wt-RV M0 and r-RV M1 (A) or M3 (B) are shown. I and Pn indicate the initial inoculum used for mixed infections and the passage number, respectively. Numbers indicate the location of RNA segments. Arrows indicate the rearranged segment 7R from M1 and 7RΔ from M3. (C) Western-blot detection of the NSP3 protein expressed by the viral progenies resulting from the M0+M3 co-infection at passage 1(P1) and 3 (P3). Arrows indicate the NSP3 and the modified mNSP3 proteins encoded by wt segment 7 (M0 virus) and rearranged segment 7RΔ (M3 virus), respectively. Numbers indicate molecular size, in kilodaltons.</p

Competitive experiments between human wt-RV M0 and r-RV M4 or M2.

<p>Competitive experiments were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020080#pone-0020080-g003" target="_blank">figure 3</a>. RNA profiles of viral progenies resulting from mixed infections by wt-RV M0 and r-RV M4 (A) or M2 (B). Pn indicates the passage number. Numbers indicate the location of RNA segments. Arrows indicate rearranged segment 11R (M4 and M2 viruses) and 7R (M2).</p

One-step viral growth curves of human wt-RV and r-RV.

<p>For each virus, confluent MA-104 cells were inoculated at the same MOI of 0.5 PFU/cell. The infected cell cultures were harvested at 2, 6, 8, 10, and 18 hours post-infection and virus titers were determined by plaque assay in MA-104 cells. Human wt-RV M0 and r-RV M1 to M4 share the same genetic background. The rearranged segments of r-RV M1 to M4 are indicated in brackets. The bovine RV strain RF was used as control.</p