Flexibility of the Rotavirus NSP2 C-Terminal Region Supports Factory Formation via Liquid-Liquid Phase Separation

Fig. 1. Sequence conservation and structure of NSP2 CTR. (A) Cartoon schematic of NSP2 (317 amino acids in length). The core of the protein is made up of residues 1-290 (grey). The C-terminal region (CTR) is comprised of a linker (yellow; residues 291-300), a C-terminal a-helix (cyan; residues 301-313), and an unstructured region (white; residues 314-317). An amino acid sequence alignment of the NSP2 CTR from a variety of human and animal strains is shown below the schematic. Non-conserved residues are colored grey and lysine 294 is colored red. The truncation location of a previously described CTR mutant (ΔCTR, missing residues 293-317) is indicated. (B) The octameric structure of strain SA11 NSP2 modeled in Chimera software (PDB#4G0A) is colored as in panel A (32). The CTR exhibits open (domain-swapping) and closed (non-domaining swapping) conformations. (C) Zoom view of super-positioned, open and closed conformations of the NSP2 CTR. Linker residues M293, K294, and P295 are shown in stick representation and labeled (open residues are colored green, and closed residues are colored red).

Fig. 2. In silico molecular dynamics simulations of NSP2 protein structures. (A) Average B-factors are shown for either NSP2WT (black line) or a molecular model of NSP2K294E (grey line). Residue position numbers are shown on the x-axis. (B) Ribbon structure of the NSP2WT showing regions of dynamic change. The CTR is colored as in Fig. 1, and an N-terminal loop (residues 61-68) is colored green. The dominant trajectory models of these regions are shown as insets with NSP2WT in black and NSP2K294E in grey. Residues with B-factors that differ between NSP2WT and NSP2K294E are colored red. (C) Average B-factors for residues 61-68. Colors are the same as in panel A. Error bars represent standard error from the mean following three independent simulations. Statistical significance was determined using a two-tailed T test. Asterisks (*) indicate statistically significant differences (p<0.05). ns=not significant. (D) Average B-factors for CTR residues 287-306. Colors are the same as in panel A. Error bars represent standard error from the mean following three independent simulations. Statistical significance was determined using a two-tailed T test. Asterisks (*) indicate statistically…

Fig. 3. Reverse genetics rescue and replication of rRV-NSP2K294E. (A) Sequence analysis of the rRV-NSP2K294E working stock. The NSP2-coding gene of rRV-NSP2K294E was amplified by RT-PCR and subjected to Sanger sequencing. The chromatograms of two independent sequencing reactions (read 1 and read 2) are shown across the engineered lesion only. Nucleotide and amino acid sequences of the unmodified wildtype (WT) pT7-NSP2-SA11 plasmid are shown above the consensus mutant virus sequence. The engineered codon is highlighted yellow. (B) Electropherotyping. Viral dsRNA was extracted from laboratory strain SA11-4F (4F), rRV-WT, or rRV-NSP2κ294E working viral stock and visualized following SDS-PAGE and SYBER Gold staining. The positions of the 11 viral genome segments (g1-g11) are labeled to the right of the gel. (C) Single-cycle replication assay. MA104 cells were infected at an MOI of 3 PFU per cell with either rRV-WT (white) or rRV-NSP2K294E (grey). Viral titers were determined by plaque assay. Error bars represent standard error from the mean. Statistical significance was determined using a two-tailed, unpaired T-test. Asterisks (**) indicate p<0.001. (C) Images of representative plaque assay…

Fig. 4. In vitro NTPase activity of NSP2WT and NSP2K294E proteins. (A) Representative TLC plate images showing hydrolysis of Pi from [α32P]-ATP to produce [α 32P]-ADP in the presence of various cHis tagged or untagged (UT) NSP2 proteins. No protein was added in the negative control reactions. (B) Quantification of ATP hydrolysis and release of Pi by various NSP2 proteins as described in Material and Methods. Error bars represent standard error from the mean.

Fig. 5. Confocal imaging of viroplasms in rRV-WT- or rRV-NSP2K294E-infected cells. MA104 cells on glass coverslips were infected with rRV-WT or rRV-NSP2K294E at an MOI of 3 PFU per cell for 4, 8, 12, 16 or 24 h. (A and B) Cells were fixed with methanol and stained using αNSP2 and an Alexa-546 conjugated secondary antibody. Nuclei were stained using Hoechst. Confocal microscopy was used to determine the localization of NSP2 (561-nm; red) and nuclei (405-nm; blue). Scale bar indicates 10 μm. (C and D) Cells were fixed with methanol and stained using αVP1 or αVP2 and an Alexa-546 conjugated secondary antibody. Nuclei were stained using Hoechst. Confocal microscopy was used to determine the localization of VP1 or VP2 (561-nm; red) and nuclei (405-nm; blue). Scale bar indicates 10 μm.

Fig. 6. Quantification of viroplasm number and size. (A) The number of viroplasms per cell for rRV-WT (white) or rRV-NSP2K294E (grey) was quantified in blinded αNSP2-stained micrographs at each time point. The mean is shown as a dashed line and the median is shown as a solid line in the box and whiskers plot. Statistical significance was determined using the negative binomial model. Asterisks (****) indicate p<0.0001. (B) The data for viroplasm numbers at the 8 and 24 h timepoints were binned using a custom R script. Each bin represents the percentage of cells with the viroplasm numbers according to the legend. (C) The diameter of viroplasms for rRV-WT (white) or rRV-NSP2K294E (grey) was quantified in blinded αNSP2-stained micrographs at each time point. The mean is shown as a dashed line and the median is shown as a solid line in the box and whiskers plot. Statistical significance was determined using the Box-cox transformation followed by a linear regression. Asterisks (****) indicate p<0.0001. (D) The data for viroplasm diameters at the 8 and 24 h timepoints were binned using a custom R script. Each bin represents the percentage of cells containing viroplasms with the size range…

Fig. 7. Western blot of NSP2 and NSP5. MA104 cells were either mock infected or infected with rRV-WT or rRV-NSP2K294E at an MOI of 3 PFU per cell for 8 or 24 h. Lysates were subjected to western blot using αNSP2, αNSP5 aVP1/VP2, or αtubulin as a loading control. Secondary antibodies conjugated to HRP were used for detection. Molecular weight markers (in kDa) are shown to the left of the blots, and the locations of protein of interest are labeled on the right. Hyper-phosphorylated NSP5 (NSP5-p).

Fig. 8. Live-cell imaging of viroplasms in NSP2wτ:mCherry expressing MA104 cells. MA104 cells stably expressing NSP2WT :mCherry were infected with rRV-WT (A) or rRV-NSP2K294E (B) at an MOI of 3 PFU per cell for 16 h. Cells were imaged every 10 min using confocal microscopy. Representative still images of two regions of interest (ROI 1 and ROI 2) are shown at 16 h p.i. Viroplasms were tracked as described in the Materials and Methods to measure the time to fusion and average speed. (C) Time to fusion. Numbers are shown in h. The mean is shown as a dashed line and the median is shown as a solid line in the box and whiskers plot. A total of 20 individual fusion events were analyzed for each virus. Statistical significance was determined using a two-tailed, unpaired T-test. The asterisk (*) indicates p<0.05. (D) Viroplasm speed. Numbers are shown in nm/sec. A total of 90 viroplasms were tracked per virus. Statistical significance was determined using a two-tailed, unpaired T-test. Error bars represent the standard error from the mean. ns=not significant.

Fig. 9. Perinuclear localization of viroplasms. MA104 cells on glass coverslips were infected with rRV-WT or rRV-NSP2K294E at an MOI of 3 PFU per cell for 8 or 24 h. Cells were fixed with methanol and stained using αNSP2 and an Alexa-546 conjugated secondary antibody. Nuclei were stained using Hoechst. Confocal microscopy was used to determine the localization of NSP2 (561-nm; red) and nuclei (405-nm; blue). Perinuclear condensation [V/C ratio] was determined as described in the Methods. Statistical significance was determined by a two-tailed, unpaired T-test. ns=not significant. Asterisks (**) indicate p<0.01.

Fig. 10. VLS formation by NSP2 CTR mutants. Cos-7 cells on glass coverslips were transfected with plasmids expressing untagged NSP5 and either GFP only (A) or the following NSP2-GFP fusion proteins: NSP2WT(B), NSP2ΔCTR (C), NSP2κ294E (D), NSP2κ294A (E), or NSP2κ294R (F). At 48 h post-transfection, cells were fixed with methanol and stained using αNSP5 and an Alexa-546 conjugated secondary antibody. Confocal microscopy was used to determine the localization of GFP signal (488-nm; green) relative to αNSP5 staining (561-nm; red). Co-localization of green and red is shown as yellow in the merged images. The location of punctate VLSs formed of NSP2 and NSP5 are indicated with closed arrowheads, while aggregates of NSP2 fusion proteins are indicated with open arrowheads. Scale bar indicates μm.

Fig. 11. In vitro LLPS droplet formation. Phase diagrams generated through fluorescent imaging of recombinant unlabeled NSP5 mixed with 488Atto-labeled NSP2WT (A) or NSP2K249E (B). NSP2 and NSP5 protein samples were mixed at varying concentrations (5-20 μM) as shown on both axes. Droplets formed upon the addition of NSP5 and were imaged following a 5 min incubation. Scale bar=10 μm. Droplet formation assays and sizing of NSP5/NSP2 condensates. 15 μM of the recombinant 488Atto-labeled NSP2WT or the NSP2K294E was mixed with 15 μM of NSP5. Droplet formation was imaged directly after the proteins were mixed (not shown), and 15 min following the incubation upon their formation to allow them to fuse (C). Five microfluidic wells are shown for each protein. Scale bar=10 μm. (D) Droplet sizes were quantified immediately after their formation and (E) 15 min after their formation. Droplets were detected and counted as described in Materials and Methods. ns=not significant for t = 0 min. Statistical significance was determined using a two-tailed, unpaired T-test. The asterisks (**** represent p< 0.0001. Red lines denote the mean, black lines show the quartiles.