VP4 Mutation Boosts Replication of Recombinant Human/Simian Rotavirus in Cell Culture

Abstract

Rotavirus A (RVA) is the leading cause of diarrhea requiring hospitalization in children and causes over 100,000 annual deaths in Sub-Saharan Africa. In order to generate next-generation vaccines against African RVA genotypes, a reverse genetics system based on a simian rotavirus strain was utilized here to exchange the antigenic capsid proteins VP4, VP7 and VP6 with those of African human rotavirus field strains. One VP4/VP7/VP6 (genotypes G9-P[6]-I2) triple-reassortant was successfully rescued, but it replicated poorly in the first cell culture passages. However, the viral titer was enhanced upon further passaging. Whole genome sequencing of the passaged virus revealed a single point mutation (A797G), resulting in an amino acid exchange (E263G) in VP4. After introducing this mutation into the VP4-encoding plasmid, a VP4 mono-reassortant as well as the VP4/VP7/VP6 triple-reassortant replicated to high titers already in the first cell culture passage. However, the introduction of the same mutation into the VP4 of other human RVA strains did not improve the rescue of those reassortants, indicating strain specificity. The results show that specific point mutations in VP4 can substantially improve the rescue and replication of recombinant RVA reassortants in cell culture, which may be useful for the development of novel vaccine strains.

Keywords: Sub-Saharan Africa; cell culture; next-generation sequencing; point mutation; replication kinetics; reverse genetics system; rotavirus; triple-reassortant.

Figure 1

Generation of SA11 triple-reassortants containing VP4, VP7 and VP6 from three African human RVA strains. (a) Overview of the developed cytopathic effect (CPE) upon passaging in MA-104 cells. (b) Analyses of the freeze–thaw supernatants by qRT-PCR after the indicated passages in MA-104 cells. (c) Overview of the VP7 (G-type), VP4 (P-type) and VP6 (I-type) genotypes and the number of successful rescue experiments. The first rescue experiment was performed in duplicates but counted as one experiment. Mock = Mock-infected cells; rSA11 = Recombinant SA11; rSA11/triple-GR10924, rSA11/triple-Moz60a and rSA11/triple-Moz308 = Recombinant rotaviruses carrying segment 4 (VP4), segment 9 (VP7) and segment 6 (VP6) from the indicated human RVA strain in the backbone of SA11; P1–10 = Passages 1–10; red minus = No CPE; yellow O = Mild CPE; green plus = Strong CPE; NA = Not analyzed; GCEs = Genome copy equivalents.

Figure 2

Sequence analyses of rSA11/triple-GR10924 and the duplicate. (a) Nucleotide (Nt) and amino acid (Aa) substitutions in the open reading frames of the eleven rotavirus genome segments identified by next-generation sequencing after passage 10. (b) Sanger sequencing analyses of the VP4-encoding genome segment from the rSA11/triple-GR10924 reassortant that replicated to a higher titer in early passages. Respective sequencing chromatograms of passage 4 and 5 are shown. The red circle marks nucleotide position 797 in the VP4-encoding genome segment from human RVA strain GR10924. The green line below the chromatograms indicates that the probability for a wrong base call was equal to or less than 1 in 1000. rSA11/triple-GR10924 = Recombinant rotavirus carrying segment 4 (VP4), segment 9 (VP7) and segment 6 (VP6) from human RVA strain GR10924 in the backbone of SA11.

Figure 3

Rescue of SA11 mono- and triple-reassortants containing VP4 from human RVA strain GR10924 with and without the mutation A797G in the VP4-encoding genome segment. (a) Cytopathic effect (CPE) upon passage in MA-104 cells. (b) Determined number of genome copy equivalents (GCEs)/mL in freeze–thaw supernatant after each passage. (c) Detection of VP4- and VP7-encoding genome segments from rSA11 and rescued reassortants via RT-PCR using strain- and genome segment-specific primer pairs followed by agarose gel electrophoresis analysis. (d) Detection of VP6-encoding genome segments from rSA11 and rescued reassortants via RT-PCR using VP6-specific primer pairs followed by Sanger sequencing. The black squares mark nucleotide differences between the VP6-encoding genome segment from SA11 and GR10924. The green line below the chromatograms indicates that the probability for a wrong base call was equal to or less than 1 in 1000. rSA11 = Recombinant SA11; rSA11/triple-GR10924 = Recombinant rotavirus carrying segment 4 (VP4), segment 9 (VP7) and segment 6 (VP6) from human RVA strain GR10924 in the backbone of SA11; rSA11/triple-GR10924_E263G = rSA11/triple-GR10924 with VP4-E263G; rSA11/mono-GR10924 = Recombinant rotavirus carrying segment 4 (VP4) from human RVA strain GR10924 in the backbone of SA11; rSA11/mono-GR10924_E263G = rSA11/mono-GR10924 with VP4-E263G; gs = genome segment; P1–4 = Passages 1–4; red minus = No CPE; yellow O = Mild CPE; green plus = Strong CPE; NA = Not analyzed; GCEs = Genome copy equivalents.

Figure 4

Replication kinetics in MA-104 cells. Cells were infected with 2 × 10⁴ genome copy equivalents (GCEs) corresponding to 0.04 GCEs/cell and the number of GCEs in culture supernatants was determined by qRT-PCR at the indicated time points post-infection. Data are means ± standard deviation from three independent experiments. rSA11 = Recombinant SA11; rSA11/triple-GR10924 = Recombinant rotavirus carrying segment 4 (VP4), segment 9 (VP7) and segment 6 (VP6) from human RVA strain GR10924 in the backbone of SA11; rSA11/triple-GR10924_E263G = rSA11/triple-GR10924 with VP4-E263G; rSA11/mono-GR10924 = Recombinant rotavirus carrying segment 4 (VP4) from human RVA strain GR10924 in the backbone of SA11; rSA11/mono-GR10924_E263G = rSA11/mono-GR10924 with VP4-E263G; ** p < 0.01 for rSA11/triple-GR10924_E263G versus rSA11/triple-GR10924 on day 3. * p < 0.05 for rSA11/mono-GR10924_E263G versus rSA11/mono-GR10924 on day 3.

Figure 5

Location of E263 in VP4. (a) Schematic of VP4. VP4 is cleaved by trypsin into VP8* and VP5*. The location of the trypsin cleavage site is indicated. VP8* is composed of an α-helix at the N-terminus followed by the head region. The N-terminal helix interacts with the foot region of VP5* and the head region contains the putative receptor-binding site. VP5* contains the body and stalk region at the N-terminus and the foot region at the C-terminus. The location of E263G is shown in red. (b) Three-dimensional structure of VP4 on the basis of the atomic model of an infectious rhesus rotavirus (RRV) particle (PDB 4v7q, chain BX). VP8* is colored in magenta and VP5* in blue. Predicted hydrogen bonds are in cyan. The location of E263 in VP4 from GR10924 corresponding to E264 in VP4 from RRV is highlighted in red. R368 in VP4 from GR10924 corresponding to R369 in VP4 from RRV is shown in orange.