Caspase-mediated cleavage of Murine Norovirus NS1/2 potentiates apoptosis and is required for persistent infection

Human norovirus (HuNoV) is the leading cause of acute gastroenteritis and is spread by fecal shedding that can often persist for weeks to months after infection. Murine norovirus (MNV) is also shed persistently in the feces and provides a tractable model to study molecular mechanisms of enteric persistence. Previous studies have identified non-structural protein 1 (NS1) from the persistent MNV strain CR6 as critical for persistent infection in intestinal epithelial cells (IECs), but its mechanism of action remains unclear. We now find that the function of NS1 in promoting persistence is regulated by apoptosis. Following induction of apoptosis in infected cells, a minority of NS1 is cleaved from the precursor NS1/2 protein, and this cleavage is prevented by mutation of caspase target motifs. MNV strain CR6 with these mutations (CR6∆casp) is profoundly compromised in infection of IECs and persistence in the intestine. Conversely, replication in tissues outside of the intestine, or in a cultured macrophage cell line, is unchanged, indicating that the requirement of NS1/2 cleavage is intestine-specific. Intriguingly, we also find that cleavage of CR6 NS1/2 potentiates apoptosis, suggesting that regulation of cell death is a novel function of this viral protein. Together, these data indicate that the ability of NS1 to promote MNV persistence in IECs is regulated by host caspases, and suggest that potentiation of apoptosis plays a role in viral tropism in the intestine. Author Summary Human Norovirus infection is highly contagious and the most common cause of acute gastroenteritis. Norovirus can persist and be shed for months after infection, leading to continued outbreaks. There are many unanswered questions as to host and viral components of norovirus pathogenesis that can be addressed within the murine norovirus (MNV) model system. We previously identified a critical role for a viral protein, NS1, for intestinal persistence. Herein we describe how the regulation of NS1 is critical for persistent infection in intestinal epithelial cells, but is not required for acute infection of non-epithelial cells, or infection of tissues outside of the gut. Additionally, we demonstrate that NS1 is both regulated by the host cell death machinery, and also reciprocally regulates that machinery to promote cell death during MNV infection, and found that this is specific to persistent strain of MNV. Altogether these data identify a role for how NS1 in a new pathway involved in establishing a persistent norovirus infection in the intestine.


Abstract 16
Human norovirus (HuNoV) is the leading cause of acute gastroenteritis and is spread by fecal shedding that 17 can often persist for weeks to months after infection. Murine norovirus (MNV) is also shed persistently in 18 the feces and provides a tractable model to study molecular mechanisms of enteric persistence. Previous 19 studies have identified non-structural protein 1 (NS1) from the persistent MNV strain CR6 as critical for 20 persistent infection in intestinal epithelial cells (IECs), but its mechanism of action remains unclear. We 21 now find that the function of NS1 in promoting persistence is regulated by apoptosis. Following induction 22 of apoptosis in infected cells, a minority of NS1 is cleaved from the precursor NS1/2 protein, and this 23 cleavage is prevented by mutation of caspase target motifs. MNV strain CR6 with these mutations 24 (CR6Dcasp) is profoundly compromised in infection of IECs and persistence in the intestine. Conversely, 25 replication in tissues outside of the intestine, or in a cultured macrophage cell line, is unchanged, indicating 26 that the requirement of NS1/2 cleavage is intestine-specific. Intriguingly, we also find that cleavage of CR6 27 NS1/2 potentiates apoptosis, suggesting that regulation of cell death is a novel function of this viral protein. Together, these data indicate that the ability of NS1 to promote MNV persistence in IECs is regulated by 29 host caspases, and suggest that potentiation of apoptosis plays a role in viral tropism in the intestine. Human norovirus (HuNoV) is the most common cause of epidemic gastroenteritis, and can be particularly dangerous for infants and the elderly [1,2]. Persistent viral shedding often occurs for weeks to 48 months after acute infection, even in asymptomatic individuals [3,4], and is a potential source for continued 49 outbreaks. Despite recent advances in culture systems to study HuNoV [5,6], there is still not an efficient 50 in vitro or small animal system for investigating the molecular components of HuNoV infection. 51 Fortunately, HuNoV and murine norovirus (MNV) share genotypic (ssRNA, positive-sense, ~7.5kb 52 genome) and phenotypic (fecal-oral transmission, persistence in intestinal epithelial cells (IECs), and 53 shedding in the feces) similarities, and utilization of the MNV model system has considerably advanced 54 our molecular understanding of norovirus pathogenesis and persistence [7][8][9]. 55 All noroviruses encode six non-structural proteins (NS1/2, NS3, NS4, NS5, NS6, and NS7) 56    Fig 1A), however, the necessity and utility of these sites during viral infection was unknown. Therefore, 126 to assess the role of NS1/2 cleavage during viral persistence, we mutated these sites (D121G and D131G) 127 within the persistent MNV strain, CR6, and named this virus CR6Dcasp (Fig 1B). Analyses of viral protein 128 expression during MNV infection of BV2 cells demonstrates cleavage of a minority of total NS1/2 protein 129 at late times post-infection with both an acute (CW3) and persistent (CR6) strain of MNV ( Fig 1C). 130 Cleavage of NS1/2 did not occur in CR6Dcasp-infected cells, demonstrating that it is dependent on the caspase 131 motifs (Fig 1C), and, importantly, these mutations did not disrupt overall expression of full-length NS1/2 132 protein. To determine whether host caspases are responsible for NS1/2 cleavage, cells were treated at 10h 133 post-infection (PI) with a pan-caspase inhibitor, ZVAD-fmk, and indeed, the ~28kDa NS2 protein was no 134 longer detected in infected cells at late times post-infection ( Fig 1D). The ~18kDa NS1 protein was 135 inconsistently detected in cells via western blot and may be a technical result of the small amounts of overall 136 NS1/2 that get cleaved during infection. Together, these data demonstrate that a small portion of total NS1/2 137 protein is cleaved by host caspases at relatively late times post-infection with MNV. 138 139 Cleavage of NS1/2 is not required for viral replication in vitro. The abundance of full-length NS1/2 140 following infection with CR6Dcasp indicated that viral protein production was not dependent on these 141 cleavage motifs (Fig 1C, D). To further assess whether NS1/2 cleavage is necessary for production of 142 infectious virus, we compared plaque forming units (PFU) produced from cell cultures infected with CR6 143 vs. CR6Dcasp over a 48h time course. Inhibition of NS1/2 cleavage did not significantly impair CR6 144 replication in BV2 cells (Fig 2A), or within wildtype (WT) bone-marrow derived macrophages (BMDCs) 145 ( Fig 2C). 146 Our in vivo analyses discussed in the following section aimed to assess the role of NS1/2 cleavage 147 on systemic replication as well as intestinal replication of MNV; therefore, we generated CR6 and CR6Dcasp 148 viral strains that could spread systemically due to chimeric expression of VP1 CW3 (Fig 1B) [15]. We 149 performed growth curves using these capsid chimeras in parallel with growth curves for CR6 and CR6Dcasp 150 described above. In vitro growth kinetics in BV2 cells and WT BMDCs displayed minimal differences 151 between CR6-VP1 CW3 and CR6Dcasp-VP1 CW3 (Fig 2B, D). The minor, yet significant, decrease in peak titers 152 of CR6Dcasp-VP1 CW3 compared to CR6-VP1 CW3 in WT BMDCs but not in BV2 cells indicated NS1/2 153 cleavage may serve a critical function specific to BMDC cultures. Recently published data suggests a role 154 for NS1 CR6 in IFN antagonism, therefore, we assessed whether NS1/2 cleavage affected growth in BMDC 155 cultures that are not responsive to IFN (Stat1 -/-BMDCs). All the MNV strains we analyzed demonstrated 156 higher peak titers in Stat1 -/-BMDCs compared to WT BMDCs (Fig 2C-F), confirming the known inhibitory 157 effect of IFNs on MNV replication [19], and the decreased replicative capacity of CR6Dcasp-VP1 CW3 was no 158 longer evident (compare Fig 2D to 2F). Altogether, these data indicate that NS1/2 cleavage is not necessary 159 for viral replication but may play a role in counteracting the IFN response. of persistence (Fig 3A, B). Consistent with the high titers in intestinal tissue, we also detected infectious 167 virus shed in the feces of mice infected with CR6 and CR6-VP1 CW3 at 3dpi (Fig 3C, D). However, in mice 168 infected with CR6Dcasp or CR6Dcasp-VP1 CW3 , we rarely detected infectious virus in intestinal tissues (Fig 3A,  169 B), nor being shed in the feces (Fig 3C, D), indicating that caspase cleavage of NS1/2 is required for 170 promoting early infection of the intestine. 171 Although production of infectious virus was severely compromised in intestinal tissues of MNV-172 infected mice in the absence of NS1/2 cleavage, titers in the spleen were only modestly reduced and not 173 significantly different at 3dpi in the absence of NS1/2 cleavage (Fig 3B), indicating that NS1/2 cleavage is 174 not required for systemic spread. Similarly, we were able to detect MNV RNA within the Peyer's patches 175 of animals infected with CR6Dcasp-VP1 CW3 using the ultra-sensitive in situ RNA hybridization platform, 176 RNAscope (Fig 3F), and this was not visibly different from CR6-VP1 CW3 -infected mice (Fig 3E-G). 177 Together, these data suggest no requirement for NS1/2 cleavage during the initial infection in Peyer's 178 patches or for subsequent systemic spread (Fig 3B, spleen). 179 To further verify that NS1/2 cleavage is not required for systemic MNV infection and pathogenesis, 180 Accordingly, we found that peroral challenge with CR6-VP1 CW3 resulted in lethal MNV infection in Stat1 -183 /mice, and although NS1/2 cleavage is critical for efficient infection in the intestine, CR6Dcasp-VP1 CW3 184 infection also resulted in 100% mortality in Stat1 -/mice ( Fig 3H). As a control, we infected Stat1 -/mice 185 with CR6, which persists, but does not result in lethal infection ( Fig 3H) [15]. Overall, our characterization 186 of acute infection demonstrates that NS1/2 cleavage is not required for systemic spread of MNV and is not 187 necessary for lethal pathology in Stat1 -/mice. However, NS1/2 cleavage is specifically required for 188 efficient MNV replication in the ileum (outside of Peyer's patches) and colon, the source of virus shed in 189 the stool and the site of eventual persistence. 190

Due to limited detection of virus at 3dpi in the intestinal tissues of mice infected with CR6Dcasp and 191
CR6Dcasp-VP1 CW3 , we predicted that these mutants would also fail to persist. Indeed, whereas CR6 or CR6-192 VP1 CW3 persisted in the ileum and colon at 21dpi (Fig 4A, B), and were shed in the stool (Fig 4C, D), we 193 rarely detected persistent virus in any tissues from mice infected with CR6Dcasp or CR6Dcasp-VP1 CW3 (Fig 4). 194 Altogether, these findings support a role for NS1/2 cleavage specifically within the intestinal tissue, and 195 temporally link the activity of NS1/2 cleavage to the first 3 days post-infection. Thus, NS1/2 cleavage is 196 required for establishing an infection in the intestine and subsequent persistence. or Ifnlr1 -/mice with CR6 or CR6Dcasp and monitored shedding in the stool over time (Fig 5A, B), and 203 persistence in the colon at 21dpi (Fig 5C, D). Additionally, to determine whether an early barrier to MNV 204 infection could be overcome with increased infectious dose, we included an experimental group with 10-205 fold more CR6Dcasp (1e7 PFU) in the inoculum. MNV was shed in all WT mice infected with CR6 beginning 206 on day 3 and continuing until the experimental endpoint, day 21. Consistent with the protective role of IFN-207 l, Ifnlr1 -/mice infected with CR6 shed greater than 10-fold more virus relative to WT mice (Fig 5A, B), 208 and CR6 was detected in colon tissue at levels commensurate with fecal shedding at 21dpi (Fig 5C, D). In 209 contrast, we were unable to detect shedding of CR6Dcasp at any time from WT or Ifnlr1 -/mice (Fig 5A, B). 210 These data indicate that NS1/2 CR6 cleavage promotes persistent infection via a novel mechanism separate 211 from IFN antagonism. 212 213

Cleavage of NS1/2 is critical for MNV infection of intestinal epithelial cells. Our preceding data 214
demonstrated that disrupting NS1/2 cleavage leads to an overall reduction of infectious virus produced in 215 intestinal tissues of either WT (Figs 3, 4) or Ifnlr1 -/- (Fig 5) mice. Therefore, to attain a more thorough 216 understanding of the role of NS1/2 cleavage on cellular tropism, we infected WT or knockout mice with 217 CR6 or CR6Dcasp and analyzed infection of IECs at 3dpi by flow cytometry and RNAscope (Fig 6). We 218 concurrently quantified MNV infection of Peyer's patches by qPCR to enable contextualization of the IEC 219 infection data. We were able to detect viral RNA in Peyer's patches in the majority of CR6-infected mice 220 and ~50% of CR6Dcasp-infected mice (Fig 6A). Although CR6Dcasp infection of Peyer's patches was less 221 robust than that of CR6, these data confirm that NS1/2 cleavage is not strictly required for initial infection 222 of non-epithelial cells during acute infection (Figs 3E-F, 6A). This detectable but reduced infection by 223 CR6Dcasp was observed in mice deficient in IFN-l receptor (Ifnlr1 -/-; LRKO), doubly-deficient in IFN-l and 224 IFN-a/b receptors (Ifnlr1 -/-/Ifnar1 -/-; DKO), as well as receptor-sufficient controls (WT) (Fig 6A), 225 suggesting NS1/2 cleavage amplifies infection in Peyer's Patches. 226 To assess the requirement of NS1/2 cleavage in IEC infection, intestinal tissue devoid of Peyer's 227 patches was mechanically and enzymatically stripped to obtain a single cell suspension and stained for 228

CD45 and EpCAM as markers of immune cells and epithelial cells, respectively. The presence of MNV 229
infection was detected via dual intracellular staining using two distinct antibodies to NS1/2 in mice infected 230 with CR6 or CR6Dcasp (Fig 6B, C). We analyzed IEC infection in the same mice as in Fig 6A, including 231 knockouts and littermate controls. Regardless of genotype, we only detected IEC infection when mice were 232 infected with CR6, but not CR6Dcasp (Fig 6B, C). Additionally, tissue sections were examined via 233 RNAscope, and we reproducibly detected CR6 infection of IECs (Fig 6D, E), as well as non-epithelial cells 234 in the lamina propria ( Fig 6E) but were unable to find evidence for CR6Dcasp infection of IECs at 3dpi (Fig  235   6F). These data demonstrate the necessity of NS1/2 cleavage for CR6 infection of IECs, the cellular 236 reservoir of MNV persistence. Furthermore, the profound absence of CR6Dcasp in IECs would translate to 237 a loss of IEC-dependent viral amplification and may explain the reduced titers in Peyer's patches (Fig 6A)  238 and MLN (Fig 3A, B) during acute infection. Altogether, our data demonstrate that eliminating IFN 239 signaling is not sufficient to rescue CR6Dcasp infection of IECs (Figs 5 and 6), indicating that cleavage of 240 NS1/2 functions to promote persistent infection of IECs by a mechanism independent of the IFN-l 241 response. 242 243 NS1/2 cleavage promotes MNV-induced apoptosis. We next sought to identify an IFN-l-independent 244 cellular response altered by NS1/2 cleavage. Because apoptotic-associated caspases have been implicated 245 in cleavage of NS1/2 (Fig 1) [10], and NS1/2 cleavage is critical for developing a persistent MNV infection 246 (Figs. 4, 5), we postulated that apoptosis may be altered by NS1/2 cleavage to promote persistent infection 247 in the gut. Consistent with this hypothesis, we found that CR6 induced significantly more apoptosis in BV2 248 cells compared to CR6Dcasp (Fig 7). This included an increase in the active form of caspase 3 (Fig 7A, B) 249 and an increase in PARP cleavage (Fig 7A), a known target of activated caspase 3 [44]. The increase in 250 caspase 3 activity at 18hpi (Fig 7B) was not simply due to increased infection in CR6-infected cultures, as 251 there were similar levels of ProPol detected in all infection conditions (Fig 7C). We also measured a 252 significant increase in cells expressing phosphatidylserine on the external plasma membrane via Annexin 253 V staining in CR6-infected cells (Fig 7E-G). Similarly, there was also an increase in these apoptotic 254 markers following infection with CR6 -VP1 CW3 compared to CR6Dcasp-VP1 CW3 (Fig 7D, E-H). Furthermore, 255 in support of the hypothesis that NS1/2 CR6 cleavage promotes apoptosis in a manner that is specific for 256 persistence, the acute strain of MNV, CW3, induced less apoptosis in all our analyses (Fig 7), despite 257 demonstrating NS1/2 cleavage (Fig 1C). Interestingly, CR6-VP1 CW3 infection resulted in increased cell 258 permeability and death relative to CR6 at 17hpi (Fig 7E, H), consistent with our separate findings that 259 VP1 CW3 drives lytic cell death and inflammatory cytokine production (Jacob VanWinkle, et al, manuscript 260 under review). However, even in the context of increased cell lysis associated with VP1 CW3 , we still 261 observed a significant decrease in Annexin V-positivity in the absence of NS1/2 CR6 cleavage (Fig 7F, H), 262 suggesting NS1/2 cleavage is still promoting apoptosis even in the context of lysis and inflammation driven 263 by VP1 CW3 . Together with the preceding data, these findings suggest that potentiation of apoptotic cell 264 death by caspase-mediated cleavage of NS1/2 is critical for determining IEC tropism and persistence.  Interestingly, similar to NS1/2 cleavage promoting apoptosis during infection, some Bcl-2 proteins (e.g. 315 Bid) are also regulated via cleavage, in which the truncated version is the active, pro-apoptotic form of the 316 protein [57]. It will be interesting to examine whether NS1/2 interactions with the apoptotic machinery 317 plays a role in maintaining IEC infection. 318 Death by apoptosis could also function to avoid immune detection and to limit inflammation 319 associated with cell lysis. Virus production that results from cell lysis would promote inflammatory signals 320 to alert the host of an infection. Thus, promotion of an apoptotic cell death in myeloid cells, mediated by 321 NS1/2 CR6 cleavage (Fig 7), would limit inflammation and benefit persistence. In addition to professional 322 phagocytes, phagocytosis by non-professional phagocytes (e.g. epithelial cells) is critical for efficient 323 clearance of apoptotic cells, and for limiting inflammation in tissues with high cellular turnover [58]. were frozen, thawed, vortexed, and spun at 3,000xg to remove large debris. Supernatants were expanded 359 by two passages at MOI <0.05 in BV2 cells, and p2 virus supernatant was filtered (0.45um), concentrated 360 by ultra-centrifugation through a 30% sucrose cushion, and titered via plaque assay. 361 Plaque assays were performed in BV2 cells, similar to previously described methods [15]. Briefly, 362 BV2 cells were grown in 6well plates, infected with serial dilutions of each sample (500ul per well, 1h, RT, 363 on a rocking platform), after which the inoculum was removed and cells were overlaid with 1% 364 methylcellulose in complete DMEM. At 2-3d post-overlay, cells were fixed and stained with 20% EtOH / 365 0.1% crystal violet. 366 367

Mice, infections, and tissue collection 368
Stat1 -/-(Stat1 tm1Dlv ) mice were originally obtained from the Jackson Laboratories (stock #012606). All mice (7-9weeks old) were perorally infected with 1e6 PFU in a 25µL volume administered by 375 pipet, with the exception of experiments in Figure 6 where 1e6 or 1e7 PFU (as indicated in figure) was 376 administered by oral gavage in a 100µL volume. An equal proportion of males and females were 377 maintained among experimental groups to mitigate results due to any unidentified sex-dependent variables. 378 Same-sex mice, infected with the same MNV strain, were often co-housed for acute infection analyses (less 379 than three days), but singly-housed for extended experiments to prevent continued transmission between 380

Ethics Statement 387
All mice were bred on C57BL/6 background and maintained in specific-pathogen-free barrier