Rescue of Infectious Rotavirus Reassortants by a Reverse Genetics System Is Restricted by the Receptor-Binding Region of VP4

Figure 1. Chimeric simian/turkey VP4. (a) Schematic representation of the crystal structure of the rotavirus outer capsid layer based on PDB 4V7Q. The amino acid residues located in cavity formed by VP6 and VP7 are shown in red. (b) Amino acid sequence alignment of VP4 from simian SA11 (VP4-Simian) and turkey 2E10 (VP4-Turkey) in comparison to the sequence of the chimeric VP4 construct (VP4-Si-Tu-Si). The red boxes correspond to the amino acid residues shown in red in (a). (c) Schematic representation of VP4-encoding genome segments used for rescue experiments. N/Head: N-terminus and head region, VP4-Simian: red, VP4-Turkey: dark gray.

Figure 2. Chimeric chicken/turkey VP4. (a) Chimeric VP4-encoding genome segments used for rescue experiments. N/Head: N-terminus and head region, VP4-Chicken: blue, VP4-Turkey: dark gray. (b) Agarose gel analysis of a restriction enzyme digestion of RT-PCR products from VP4-Chicken and VP4-Tu-A. The primer-binding sites and restriction enzyme sites are indicated. (c) Amino acid sequence alignment of the C-terminal part of the VP4 head region. The amino acid residues in VP4-Tu-A that were substituted with the corresponding residues of VP4-Chicken are highlighted in blue. The black line indicates the position of a known sialic acid-binding site for simian RRV [38]. (d) Location of amino acid substitutions (highlighted in cyan) present in the head region of VP4-Tu-A as projected into the 3D structure of simian RRV VP4 (PDB 4V7Q).

Figure 3. Chimeric bat/human VP4 constructs. N/Head: N-terminus and head region, Bat: orange, human Wa: dark gray, human Moz60a: light gray, human Moz308: white. Successful rescue was confirmed by the presence of a CPE and the detection of viral RNA by qRT-PCR. Negative rescue was confirmed by the absence of a CPE and no detection of viral RNA by qRT-PCR. (a) Chimeric genome segments with nucleotides encoding the head region derived from VP4-Bat and the foot region derived from human strains. (b) VP4-encoding genome segments with sequences from VP4-Bat and VP4-Human Moz60a (c) Genome segments encoding VP4 with different combinations of the N-terminus and foot region.

Figure 4. Chimeric simian/human VP4. N/Head: N-terminus and head region, human Wa: dark gray, simian SA11: red. (a) Agarose gel analysis of a restriction enzyme digestion of RT-PCR amplicons from VP4-Wa-A and VP4-Simian. The primer-binding sites and restriction sites are indicated left in the schematic overview of the different constructs. (b) Amino acid sequence alignment of the substituted head region. The sequences of simian RRV and a human Indian strain are included as their glycan-binding site are known. The known glycan-binding sites of simian RRV VP4 [38] and human Indian VP4 [14] are highlighted in green and purple, respectively.

Figure 5. Replication kinetics of the cell culture-adapted parent viruses and their rescued reassortants. MA-104 or BSR-T7/5 cells were infected with 2 × 104 TCID50. The TCID50 was determined in triplicates at the indicated time points. (a) Growth curves of cell-culture adapted viruses. Data are means plus/minus SD (b) Growth curves of reassortants. Data are means plus/minus SD. (c) Comparison of titer changes of parent viruses and reassortants in BSR-T7/5 cells. The TCID50 was normalized to the value from day 0 and the fold change was calculated. TCID50 values at day 0 were: 1.94 log10 for simian SA11, 2.83 log10 for human Wa, 2.17 log10 for VP4-Wa-A, 2.28 log10 for turkey 2E10, 1.94 log10 for chicken 2G3, and 1.72 log10 for VP4-Tu-A. Data are means plus/minus SD from three independent experiments. ** p < 0.01 for human Wa vs. VP4-Wa-A.