Secretory pathway antagonism by calicivirus homologues of Norwalk virus nonstructural protein p22 is restricted to noroviruses
© Sharp et al.; licensee BioMed Central Ltd. 2012
Received: 15 November 2011
Accepted: 22 August 2012
Published: 3 September 2012
Our previous report that the Norwalk virus nonstructural protein p22 is an antagonist of the cellular secretory pathway suggests a new aspect of norovirus/host interaction. To explore conservation of function of this highly divergent calicivirus protein, we examined the effects of p22 homologues from four human and two murine noroviruses, and feline calicivirus on the secretory pathway.
All human noroviruses examined induced Golgi disruption and inhibited protein secretion, with the genogroup II.4 Houston virus being the most potent antagonist. Genogroup II.6 viruses have a conserved mutation in the mimic of an Endoplasmic Reticulum export signal (MERES) motif that is highly conserved in human norovirus homologues of p22 and is critical for secretory pathway antagonism, and these viruses had reduced levels of Golgi disruption and inhibition of protein secretion. p22 homologues from both persistent and nonpersistent strains of murine norovirus induced Golgi disruption, but only mildly inhibited cellular protein secretion. Feline calicivirus p30 did not induce Golgi disruption or inhibit cellular protein secretion.
These differences confirm a norovirus-specific effect on host cell secretory pathway antagonism by homologues of p22, which may affect viral replication and/or cellular pathogenesis.
KeywordsCalicivirus Norovirus p22 Secretory pathway
Cuernavaca 2007 Virus
Membrane Association Domain
- MERES motif:
Mimic of an Endoplasmic Reticulum Export Signal
Secreted Alkaline Phosphatase.
The Caliciviridae consists of five genera of (+)ssRNA viruses. The genus Norovirus contains the human noroviruses (NoV) that are the predominant cause of gastroenteritis in the US  and globally . Norovirus is divided into five genogroups (GI-GV) and most human NoV fall within GI and GII . Genogroups are further subdivided into genotypes; genotype GII.4 NoV are currently the most frequently detected in humans . Genogroup V contains murine norovirus (MNV), which is lethal to STAT1−/− mice . The genus Vesivirus contains feline calicivirus (FCV), a cause of severe respiratory disease in cats.
The nonstructural protein p22 from Norwalk virus (NV), the prototype NoV, encodes a novel and well-conserved motif that mimics a traditional di-acidic ER export signal . This mimic of an Endoplasmic Reticulum (ER) export signal (MERES) motif allows p22 to gain access to COPII vesicles and is necessary but not sufficient to antagonize COPII vesicle trafficking to induce Golgi disassembly and inhibit cellular protein secretion. Since NV, MNV and FCV all induce Golgi disruption [6–8], we compared calicivirus homologues of p22 for the following: sequence similarity, including conserved motifs; cellular distribution and location following expression; ability to induce Golgi disassembly; and inhibition of cellular protein secretion. We examined human NoV homologues of p22, referred to as “p22-like proteins” (p22L), from the GI.1 NV, the GII.4 Houston virus (HOV) , the GII.3 Saitama U201 , and the GII.6 Cuernavaca 2007 (CNV07) . We also examined MNV p18 from persistent (CR6 ) and non-persistent (CW3 ) strains (cDNA provided by “Skip” Virgin of Washington University), and FCV p30 (cDNA provided by John Parker of Cornell University), which are all homologues of p22 .
Of seven identified human NoV that lack the full MERES motif, four belong to GII.6 isolated from different individuals during a single outbreak  and encode either YRVSSDG or YRVSSNG in the MERES motif (Figure 1C). The fifth is an untyped GII NoV that also encodes YRVSSDG, the sixth is a GII.8 NoV that encodes YRISSDG, and finally a GII.16 NoV encodes FDLESDG. We sequenced the p22L of two additional GII.6 NoV, CNV07  and TCH97 E99-13646, the latter of which was isolated from an infant male hospitalized with gastroenteritis at Texas Children’s Hospital in 1997. Both viruses encode the same YRVSSNG sequence as other GII.6 NoV, demonstrating retention of the E68S MERES motif mutation in GII.6 NoV from at least 1997 through 2007.
Due to inefficient transfection of macrophages, MNV p18 was expressed in 293T cells to evaluate p18 localization and effect on the Golgi. p18 from MNV strains CW3 and CR6 induced Golgi disruption in 94% and 97% of cells, respectively (Figure 2F). In contrast to p22, MNV p18 puncta co-localized with the peri-nuclear remnants of the Golgi, suggesting that p18 associates with the Golgi despite disruption.
p18 from persistent (CR6) and nonpersistent (CW3) MNV strains both inhibited protein secretion significantly less (10% compared to GFP) than p22 (p ≤ 0.0002). FCV p30 did not reduce SEAP secretion in CRFK cells at 24 hpt compared to GFP alone (p = 0.44), although PV 3A efficiently inhibited SEAP secretion (p = 0.00002) (Figure 4B); CRFK cells did not express p22 in sufficient quantity to allow quantitation of SEAP secretion (data not shown).
These results demonstrate that all NoV p22L examined inhibit cellular protein secretion, but to different extents and with differences in cellular localization. Most interesting is the reduction of inhibition seen for CNV07 p22L, which has a natural mutation in the MERES motif. Inhibition of SEAP secretion similarly occurs at intermediate levels when a E68A mutation is made in NV p22 , suggesting a causal relationship between this mutation and reduced inhibition of SEAP secretion by CNV07 p22L. Nonetheless, the biological significance of secretory pathway antagonism by NoV p22L, as well as the differences between them in level of inhibition, remains to be elucidated.
Future studies to examine the 20 amino acids conserved between all NoV p22L that are not part of the MERES motif or MAD may assist in identifying the second factor required to mediate secretory pathway antagonism . Additional information may be obtained by comparing NoV p22L with MNV p18, as p18 does not encode the MERES motif but still localizes to the Golgi and disrupts it ( and Figure 2). However, lack of the MERES motif in p18 correlates with reduced efficiency of protein secretion inhibition, although Golgi disruption still occurs. This suggests that the MERES motif assists in secretory pathway antagonism by p22L, whereas Golgi disruption is mediated by a region of p22L outside of this motif. This relationship also holds true for FCV p30, which lacks the MERES motif and does not inhibit protein secretion or induce Golgi disruption. Comparison of residues conserved in p22L from NoV and MNV that are absent in FCV will assist in identifying the residues responsible for Golgi disruption.
MNV p18 from persistent and nonpersistent strains had a similar effect on the secretory pathway, suggesting that persistence is not attributable to differences in secretory pathway antagonism by p18. Our result that p18 induces Golgi disruption in human 293T cells is consistent with a report that MNV-infected murine macrophages lack a discernable Golgi , and retention of p18 at Golgi remnants suggests antagonism by interaction with a Golgi-localized protein. Indeed, the MNV replication complex is localized peri-Golgi , suggesting a role for p18 in its formation.
Since HOV p22L was the most potent secretory pathway antagonist examined, it may be that increased inhibition of protein secretion is beneficial to NoV infectivity or replication. This is supported by the observation that GII.4 p22L undergo positive selection . This coupled with the observation that NV-infected individuals make antibodies specific to p22 ( and unpublished observation) suggests that the high variability of p22L may be due to evasion of immuno-recognition.
In summary, secretory pathway antagonism by calicivirus homologues of NV p22 correlates with preservation of the MERES motif, and many p22 homologues affect Golgi phenotype. All homologues examined herein are predicted to be membrane associated, suggesting that despite varying effects on the secretory pathway they may have a conserved role in viral replication. Conservation of alternate and additional functions for p22 homologues should therefore be explored to more fully elucidate how caliciviruses interact with their host cells.
We thank David Strong and “Skip” Virgin for helpful comments and discussion.
This work was funded by the Public Health Service grants from the National Institute of Health P01 AI057788 (MKE) and T32 AI04741 (TMS), and by the East Asia and Pacific Summer Institute grant 7316 (TMS) from the National Science Foundation. Microscopy equipment support was from the John S. Dunn Gulf Coast Consortium for Chemical Genomics (MA Mancini). Additional funding for imaging was provided by SCCPR U54 HD-007495 (BW O’Malley), P30 DK-56338 (MK Estes), P30 CA-125123 (CK Osborne), and the Dan L. Duncan Cancer Center of Baylor College of Medicine.
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