Mutations in the rotavirus spike protein VP4 reduce trypsin sensitivity but not viral spread

Abstract

Infectious entry of the nonenveloped rotavirus virion requires proteolysis of the spike protein VP4 to mediate conformational changes associated with membrane penetration. We sequenced and characterized an isolate that was cultured in the absence of trypsin and found that it is more resistant to proteolysis than WT virus. A substitution mutation abrogates one of the defined trypsin-cleavage sites, suggesting that blocking proteolysis at this site reduces the overall kinetics of proteolysis. Kinetic analysis of the membrane penetration-associated conformational change indicated that the ‘fold-back’ of the mutant spike protein is slower than that of WT. Despite these apparent biochemical defects, the mutant virus replicates in an identical manner to the WT virus. These findings enhance an understanding of VP4 functions and establish new strategies to interrogate rotavirus cell entry.

Rotavirus is the most frequently identified cause of severe gastroenteritis and dehydrating diarrhoea in infants and young children (Parashar et al., 2009). The virion is nonenveloped and encapsidates an 11-segment dsRNA genome (Trask et al., 2012). Similar to other Reoviridae, rotavirus must deliver a large subviral particle into the cytoplasm to replicate (Trask et al., 2012). Enterocytes of the small intestine are the primary sites of infection (Greenberg & Estes, 2009).

Virions become fully infectious only after release from an infected cell and cleavage of the spike protein, VP4, by enteric trypsin-like proteases. Entry, particularly membrane penetration, appears to specifically require a properly cleaved spike (Trask et al., 2010). Failure to cleave the spike or cleavage with another protease (e.g. chymotrypsin), results in minimal infectivity and limits cell-to-cell spread (Estes et al., 1981; Trask et al., 2010). The sites of VP4 cleavage are three well-defined, conserved Arg residues (R231, R241 and R247). Proteolysis results in two VP4 fragments that remain noncovalently associated: VP8* (the N-terminal fragment) and the larger VP5* (the C-terminal fragment) (Arias et al., 1996; López et al., 1985) form an asymmetrical spike complex (Crawford et al., 2001; Settembre et al., 2011). Membrane penetration is mediated by membrane binding and refolding of VP5* into a stable trimer (Dormitzer et al., 2004; Kim et al., 2010; Wolf et al., 2011). It is thought that there is an ordered ‘cleavage cascade’ from the most sensitive of these sites (R241) to the least sensitive (R247) to generate fully infectious rotavirus (Arias et al., 1996). Concordantly, cleavage after R247 is essential for syncytium formation in a model system, suggesting that this cleavage is also essential for membrane penetration during entry (Gilbert & Greenberg, 1998).

In a previous effort to understand the functions of VP4 proteolysis, the SA11 H96 (herein SA11) strain was serially passaged (as a persistently infected culture) in the absence of trypsin (Mrukowicz et al., 1998). We now have molecularly characterized one of the viruses, strain D/128, which has several mutations in the VP4 protein. D/128 requires trypsin for cell-to-cell spread, indicating that it is not trypsin independent, and one of the mutations in VP4 renders it significantly more resistant to trypsin proteolysis than SA11 VP4. Despite this apparent defect, D/128 is fully infectious, suggesting that one or more other mutations in VP4 are compensatory and allow the virus to infect with a reduced complement of cleaved VP5*.

Sequencing of the D/128 gene encoding VP4 showed several differences from the SA11 strain (Fig. 1a). The most prominent of these is a unique R241T mutation that removes one of the three trypsin-cleavage sites in VP4. Another mutation in D/128 VP4 not previously reported is N477S, at a site near the base of the β-barrel domain of VP5* (Fig. 1b, c). Three other mutations were observed (P157S, A187G and Y332S), but were synonymous with changes seen in other SA11 isolates (e.g. SA11 S1), indicating that these mutations are functionally equivalent variants (Fig. 1a). We also noted two sites of conservative variation between SA11 and SA11-S1, M72T and M366V, which are also indicated in Fig. 1a. Passage and plaque purification of D/128 in the presence of trypsin (0.5 µg ml⁻¹; 14 712 U mg⁻¹; Sigma) did not result in reversion, suggesting that the mutations are stable (data not shown). The genes encoding the proteins that interact with VP4 on the particle, VP6 and VP7, were also sequenced, but showed no nucleotide changes relative to SA11 (data not shown).

Fig. 1.

Sequence and location of mutations within D/128 VP4. (a) Sequence alignment of SA11 and D/128 VP4 proteins. The underlined sequence was determined by sequencing primer; yellow text indicates nonunique changes among SA11 isolates and red text indicates a unique change. GenBank accession numbers: D/128 (AFR67535.1), SA11 (H96) (DQ841262.1) and SA11 S1 (BAA03850.1). (b) Location of mutations within the known structures of VP4. Sites of mutation are labelled and coloured as in (a) on one of the VP4/VP5* monomers (blue). Left, the virion-associated spike conformation (PDB ID# 3IYU); right, the ‘post-penetration’ conformation of VP5* (PDB ID# 1SLQ). Note that amino acid 241 (within the trypsin-cleavage region) has been proteolytically removed in both structures. (c) Multiple sequence alignment of VP4 proteins in regions of unique D/128 mutations. The canonical trypsin-cleavage sites (231, 241 and 247) for many mammalian (mam) group A rotaviruses are highlighted in red [cleavage sites in avian (avi) group A and group C VP4 proteins have not been formally identified]. The unique mutations observed in D/128 VP4 are highlighted in blue. N477 (green) is conserved in all strains shown with the exception of D/128. The consensus sequence represents absolute conservation (capital letter), ≥50 % conservation (lower case letter) and <50 % (blank). Sequences were aligned using MacVector 12.5.0 with the default settings. GenBank accession numbers: Wa (ACR22783.1), KU (AB222784.1), DS-1 (BAC82358.1), TB-Chen (AAV65734.1), Au-1 (BAA01747.1), T152 (BAB88672.1), RF (AAB07453.1), UK (AFC40989.1), Gottfried (AAA47095.1), OSU (P11114.1), RRV (M18736.1), ETD (ACY95263.1), EDIM (AAB94758.2), PO-13 (BAA24149.1), AvRV-2 (JQ085405.1), Shintoku (AAB01672.1), Bristol (YP_392514.1).

Mutation of one of the Arg residues involved in VP4 priming hinted that D/128 has altered protease sensitivity. To test this hypothesis, we infected MA104 cells with either SA11 or D/128 at an m.o.i. of 0.05 p.f.u. per cell and cultured the viruses with 0–1.0 µg ml⁻¹ of trypsin (14 712 U mg⁻¹; Sigma) for 48 h. Under all conditions, an initial release of virus was detected by 12 h p.i. (Fig. 2a). At 0 and 0.01 µg ml⁻¹ trypsin, no further release of infectious virus was observed for either SA11 or D/128, indicating a failure to spread through the culture. Conversely, at higher concentrations of trypsin (0.1 and 1.0 µg ml⁻¹), both viruses continued to replicate between 12 and 24 h p.i. with nearly identical kinetics. Thus, despite the conditions under which D/128 was selected, the virus appears to require trypsin for efficient cell-to-cell spread, and it does so with kinetics that are identical to the parental virus under all conditions tested. Furthermore, plaque assay in the presence of trypsin indicated that the D/128 isolate forms slightly larger plaques than SA11 H96 (2.6±0.6 mm vs 1.8±0.6 mm, respectively), suggesting an enhanced capacity for spread (Fig. 2b). Given the possibility that mutations in the remaining seven unsequenced genes could contribute to the enhanced spread of D/128, we elected to focus specifically on the biochemical properties of D/128 VP4 during entry-associated functions.

Fig. 2.

Replication of SA11 and D/128. (a) Multicycle replication kinetics of SA11 H96 and D/128 at varying trypsin concentrations. MA104 cells were infected at an m.o.i. of 0.05 p.f.u. per cell and monitored for virus titre over a 48 h period. A dashed line indicates a titre of 10⁷ p.f.u. ml⁻¹ in each graph. Error bars represent the sd of three independently titrated samples. (b) Plaque morphologies of SA11 H96 and D/128 in monolayers of MA104 cells cultured in the presence of 0.5 µg ml⁻¹ trypsin. An unpaired t test was used to compare plaque diameters. All mathematical analyses were performed using GraphPad Prism v5.0d.

We inquired if the D/128 VP4 protein has altered kinetics of trypsin proteolysis. SA11 and D/128 were cultured in the presence of aprotinin (0.5 µg ml⁻¹; Sigma) to recover virions with intact VP4 (Crawford et al., 2001). The aprotinin was removed by ultracentrifugation of virions through a 35 % (w/v) sucrose cushion (Arnold et al., 2009). Particles were treated with sequencing-grade trypsin (>5000 U mg⁻¹; Promega) for 1 h at 37 °C; the reaction was quenched with 1 mM PMSF (Sigma). Proteolysis was monitored by SDS-PAGE, followed by immunoblotting with mAb HS2 (Padilla-Noriega et al., 1993), which recognizes both VP4 and VP5* (Fig. 3a). Immunoblots of multiple experiments permitted quantification of the data using a LI-COR Odyssey infrared imager and determination of the rate of VP4 proteolysis relative to the concentration of trypsin ([T]):

Fig. 3.

Kinetics of trypsin proteolysis of virion-associated D/128 VP4 and of in vitro VP5* conformational change. (a) Immunoblots of VP4 and VP5*. SA11 and D/128 virions were digested with trypsin, separated by SDS-PAGE (10 % acrylamide), transferred to nitrocellulose and incubated with anti-VP4 mAb HS2. (b) Quantification of VP4 proteolysis kinetics as a function of trypsin concentration. Points represent the empirical data derived from two independent immunoblots, and lines indicate the curve fit using the equation reported in the text. The coefficient of determination (R²) is reported for each fit, and the K_m values are reported below the abscissa. (c) Representative immunoblot of SA11 and D/128 VP5* after virion uncoating and electrophoresis in the absence of heating. (d) Kinetic analysis of VP5* refolding. Two independent immunoblots were quantified and then fit using GraphPad’s ‘Plateau followed by one phase decay’ function, as indicated in the text. All mathematical analyses were performed using Microsoft Excel v14.2.4 and GraphPad Prism v5.0c and 5.0d.

I_VP5*/(I_VP4+I_VP5*) = F_VP5* = V_max[T]/(K_m+[T])

where I_VP4 and I_VP5* are the respective intensities of VP4 and VP5* bands, F_VP5* is the fraction of VP4 cleaved to VP5* in 1 h and V_max was set to 1. This formula allowed fitting of the data and identification of K_m values consistent with empirical data (Fig. 3b), which indicated that D/128 VP4 is approximately 10-fold less sensitive to trypsin proteolysis than SA11 VP4. This phenotype is most likely due to the R241T mutation, which eliminates the most sensitive VP4 cleavage site and confirms aspects of the ‘cascade’ model, in which proteolysis at R241 promotes subsequent cleavage at R231 and R247 (Arias et al., 1996). Blocking proteolysis at R241 (by mutation in D/128 VP4) appears to inhibit overall cleavage of VP4 into VP5* and VP8*.

How does D/128 VP4 mediate infectious entry when only a fraction of VP4 molecules have been proteolytically converted into VP5*? We hypothesized that mutations in D/128 VP4 could enhance membrane penetration activity, thereby allowing the virus to retain infectivity despite a reduced complement of VP5*. In vitro uncoating of the rotavirus virion triggers VP5* to undergo a conformational change that is comparable to that observed during rotavirus entry (Wolf et al., 2011) and is thought to drive membrane penetration (Dormitzer et al., 2004). This conformational change can be performed in association with lipid bilayers or in solution (Trask et al., 2010). Previous analysis of in vitro VP5* refolding revealed end-state reactions, typically after 30 min incubations (Kim et al., 2010; Trask et al., 2010). To investigate the possibility of energetic changes in D/128 VP5* refolding, we modified the assay system to monitor kinetics. SA11 and D/128 virions were produced in the presence of 1 µg ml⁻¹ trypsin, further digested with trypsin (5 µg ml⁻¹, 37 °C, 1 h) to ensure complete proteolysis to VP5*, treated with 0.1 mM PMSF for 30 min at 4 °C and purified through a 35 % sucrose cushion. VP5* refolding was triggered through the addition of EDTA to 5 mM and protease inhibitor cocktail (Roche), followed by incubation at 32 °C. When incubated at 37 °C, VP5* refolding was essentially complete after 1 min (not shown); thus, incubation at 32 °C allowed more precise measurement of refolding kinetics. To halt refolding at the specified time points, samples were quickly moved to 25 °C and mixed with reducing SDS-PAGE sample buffer. Samples were then separated on 4–20 % polyacrylamide gels and transferred to nitrocellulose. Immunoblot analysis with antibody HS2 permitted identification of VP5* species and quantitative evaluation of refolding kinetics (Fig. 3c). As previously observed, in vitro refolding results in the conversion of approximately 50 % of VP5* to an SDS-resistant band that migrates at 220 kDa (Trask et al., 2010; Dormitzer et al., 2004; Dormitzer et al., 2001; Yoder et al., 2009). For both proteins, kinetics appeared to follow delayed one-phase kinetics (Fig. 3d):

At t<t₀, F_trimer = 0

At t≥t₀, F_trimer = F_max − F_max e^{−k(t − t}₀⁾

The period of delay (t₀) was longer for D/128 VP5* (2.4 min) than for SA11 VP5* (1.0 min). D/128 VP5* refolding also occurred at a slower rate (k) than that of SA11 VP5*: 0.27 min⁻¹ and 0.50 min⁻¹, respectively. Finally, the total fraction of refolded VP5* (F_max) was also reduced for D/128 (0.40) as compared to SA11 (0.54). Compared to the fusogenic refolding of enveloped virus fusion proteins, in vitro VP5* refolding appears to operate on a slower timescale. Kinetic analysis of individual influenza virions fusing with a lipid bilayer indicates that fusion is complete by approximately 1 min at room temperature (Costello et al., 2012; Floyd et al., 2008). Given that the precise order and number of steps that occur during rotavirus membrane penetration – including uncoating, VP5* refolding and dissociation of VP5* from the virion – the kinetics reported here may not be directly comparable to those of influenza HA refolding. Additional kinetic studies of VP5* refolding, including mutant proteins and the inclusion of liposomes, using the methodologies established here should significantly enhance knowledge of VP5* function and rotavirus membrane penetration.

This report identifies unique VP4 mutations, R241T and N477S, which have not been observed in any other rotavirus strain (Fig. 1c). The mutant VP4 of the D/128 strain is substantially more resistant to trypsin proteolysis than the WT protein (Fig. 3a, b). Previous analyses of VP4 indicated that trypsin cleavage at R241 facilitates proteolysis at the adjacent R231 and R247 sites, the latter of which is linked to peak virus infectivity (Arias et al., 1996). Our findings support this model, as mutating 241 to abrogate trypsin cleavage reduces proteolysis kinetics. We devised a method to assess the kinetics of VP5* conformational change in vitro and found that D/128 VP5* is somewhat slower than the WT protein to undergo penetration-associated refolding (Fig. 3c, d). Despite these biochemical defects, the mutant virus replicates with WT-like kinetics (Fig. 2). Further analysis of the unique mutations identified in this study could provide new mechanistic insight into the functions of VP4 during rotavirus entry.

We thank Philip R. Dormitzer and Kristen M. Ogden for helpful discussions and scientific advice. This work was supported by Public Health Service award Z01 AI000788 from the Intramural Research Program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health (S. D. T. and J. T. P.), R01 AI32539 (T. S. D.) from the National Institute of Allergy and Infectious Diseases, National Institutes of Health and the Elizabeth B. Lamb Center for Pediatric Research.