Cell Cycle Arrest is a Conserved Function of Norovirus VPg Proteins

Figures

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  Figure 1. Conservation of a G0/G1 phase cell cycle arrest among norovirus genogroups. RAW-Blue cells were transfected with 4–5 µg of in vitro transcript RNA corresponding to enhanced green fluorescent protein (EGFP), murine norovirus (MNV) VPg, as well as genogroup (G) GI VPg, GII VPg, GIII VPg, or GIV VPg. Mock transfected (MT) cells were seeded at the time of transfection as a negative control. At 12 hours post-transfection (hpt), cells were harvested for fluorescence-activated cell sorting (FACS) analysis of the cell cycle. (A) Representative FACS histograms from one of three experiments. (B) The histograms were analysed using MODfit LT 3.0, and the percentage of cells in each phase of the cell cycle are shown. The results present the mean and SD from three independent experiments. Statistical significance was determined for comparisons between transfected cells and mock transfected cells, using a one-way ANOVA with Dunnett’s post-test. * p ≤ 0.05, ** p ≤ 0.01 and **** p ≤ 0.0001.

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  Figure 2. Human sapovirus (HuSV) VPg and rabbit hemorrhagic disease virus (RHDV) VPg induce a G0/G1 phase cell cycle arrest. RAW-Blue cells were transfected with 4–5 µg of in vitro transcript RNA corresponding to EGFP, MNV VPg, HuSV VPg, RHDV VPg, feline calicivirus (FCV) VPg, or Newbury1 VPg. Mock transfected (MT) cells were seeded at the time of transfection as a negative control. At 12 hpt, cells were harvested for FACS analysis of the cell cycle. (A) Representative FACS histograms from one of three experiments. (B) The histograms were analysed using MODfit LT 3.0, and the percentage of cells in each phase of the cell cycle are shown. The results present the mean and SD from three independent experiments. Statistical significance was determined for comparisons between transfected cells and mock transfected cells using a one-way ANOVA with Dunnett’s post-test. **** p ≤ 0.0001.

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  Figure 3. Expression of VPg 1–62 induces a G0/G1 phase cell cycle arrest. RAW-Blue cells were transfected with 4–5 µg of in vitro transcript RNA corresponding to MNV VPg, VPg 1–62, VPg 11–124, or VPg 11–107. Mock transfected (MT) cells were seeded at the time of transfection as a negative control. At 12 hpt, cells were harvested for western blot and FACS analysis of the cell cycle. (A) Schematic of full-length MNV VPg and synthetic constructs. Numbers indicate the amino acid position within key structural elements. (B) Representative western blot of full length MNV VPg and VPg truncations. Histograms (C) from FACS analysis were analysed on MODfit LT 3.0, and the percentage of cells in each phase of the cell cycle graphed (D). The results present the mean and SD from three independent experiments. Statistical significance was determined for comparisons between transfected cells and mock transfected cells using a one-way ANOVA with Dunnett’s post-test. *** p ≤ 0.001.

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  Figure 4. Alignment of the first 62 amino acids of VPg from different norovirus genogroups and representative members of the Caliciviridae family. An asterisk (*) indicates identical residues. A colon (:) indicates residues that are identical in >50% of sequences. Conserved amino acids of the N-terminal region are shown in red, and in bold red are MNV VPg residues used for point mutations. The tyrosine residue (Y) corresponding to Y26 of MNV VPg is shown in bold. A conserved leucine residue used as the junction point for the construction of chimeric VPg proteins is shown in blue. The represented genogroups are GI Norwalk virus VPg, GII Sydney 2012 VPg, GIII Jena virus VPg, and GIV Lake Macquarie VPg.

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  Figure 5. Mutation of positively charged amino acids within the N-terminal region of MNV VPg reduces the G0/G1 cell cycle arrest. RAW-Blue cells were transfected with 4–5 µg of in vitro transcript RNA, corresponding to MNV VPg or single-point mutant versions K5A, K7A, G9A, or R10A. Mock transfected (MT) cells were seeded at the time of transfection as a negative control. At 12 hpt, cells were harvested for western blot and FACS analysis of the cell cycle. (A) Expression of MNV VPg and point mutants was determined by western blot with actin as a loading control. (B) Representative FACS histograms from one of three experiments. (C) The histograms were analysed using MODfit LT 3.0, and the percentage of cells in each phase of the cell cycle are shown. The results present the mean and SD from three independent experiments. Statistical significance was determined for comparisons between mutanted VPg transfections and MNV VPg transfected cells using a one-way ANOVA with Dunnett’s post-test. * p ≤ 0.05 and ** p ≤ 0.01.

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  Figure 6. Chimeric MNV VPg and Newbury 1 VPg proteins do not induce a cell cycle arrest. RAW-Blue cells were transfected with 4–5 µg of in vitro transcript RNA corresponding to EGFP, MNV VPg, or chimeric VPg constructs. Mock transfected (MT) cells were seeded at the time of transfection as a negative control. At 12 hpt, cells were harvested for western blot and FACS analysis of the cell cycle. (A) Schematic for the design of chimeric VPg constructs. Chimeric constructs were designed around a conserved leucine reside of MNV VPg and Newbury 1 VPg to exchange the N-terminal region. (B) Expression of MNV VPg and chimeric VPg proteins was determined by purification of Strep-tag II protein on beads followed by western blot. (C) Representative FACS histograms from one of three experiments. (D) The histograms were analysed using MODfit LT 3.0 and the percentage of cells in each phase of the cell cycle are shown. The results present the mean and SD from three independent experiments. Statistical significance was determined for comparisons between transfected cells and mock transfected cells using a one-way ANOVA with Dunnett’s post-test. * p ≤ 0.05.

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  Figure 7. A chimeric protein M20/FCV VPg induces a G0/G1 arrest. RAW-Blue cells were transfected with 4–5 µg of in vitro transcript RNA corresponding to EGFP, MNV VPg, or chimeric VPg constructs. Mock transfected (MT) cells were seeded at the time of transfection as a negative control. At 12 hpt, cells were harvested for western blot and FACS analysis of the cell cycle. (A) Schematic for the design of chimeric VPg constructs. Chimeric constructs were designed around a conserved leucine reside of MNV VPg and FCV VPg to exchange the N-terminal region. (B) Expression of MNV VPg and chimeric VPg proteins was determined by purification of Strep-tag II protein on beads followed by western blot. (C) Representative FACS histograms from one of three experiments. (D) The histograms were analysed using MODfit LT 3.0, and the percentage of cells in each phase of the cell cycle are shown. The results present the mean and SD from three independent experiments. Statistical significance was determined for comparisons between transfected cells and mock transfected cells using a one-way ANOVA with Dunnett’s post-test. * p ≤ 0.05 and **** p ≤ 0.0001.