INTRODUCTION
Caliciviruses are a family of positive-sense, single-stranded RNA viruses that contain the
Vesivirus,
Sapovirus,
Lagovirus, and
Norovirus genera. They infect a wide variety of mammals, causing an array of different diseases. For example, feline calicivirus (FCV), a vesivirus, is associated with a range of conditions, including upper respiratory tract disease in cats (
1), while noroviruses and sapoviruses cause gastroenteritis. Human noroviruses represent a major health problem: they are estimated to be responsible for 21 million cases of gastroenteritis in the United States every year and 200,000 deaths among children in developing countries (
2,
3).
The study of human noroviruses (HuNV) has been impeded by the absence of a viable cell culture system for this pathogen and the fact that baby gnotobiotic pigs are the only available small-animal model (
4,
5). As a result, other caliciviruses have often been used as surrogate models for the investigation of HuNV biology (
6). FCV and murine norovirus (MNV) have been widely used to investigate general features of calicivirus replication since they both grow in tissue culture and have workable reverse genetics systems (
7–11). MNV is emerging as a popular model system since it can cause systemic disease or gastroenteritis in immunocompromised mice that resembles human norovirus infections (
12,
13).
The ∼7.5-kb calicivirus genome contains 2 to 4 open reading frames (ORFs) depending on the genus. The major (VP1; ORF2) and minor (VP2; ORF3) capsid proteins of caliciviruses are usually translated from a subgenomic RNA (as is the protein derived from ORF4 in MNV [
14]). ORF1 encodes a large polyprotein precursor that is cleaved into mature nonstructural proteins (NS1 to NS7) by the virus-encoded protease NS6
pro (
15–18). The mature proteins include an RNA-dependent RNA polymerase (RdRp) (NS7
pol), an ATPase (NS3), and proteins that disrupt cellular trafficking (NS1-2 and NS4) (
18–23).
In addition, proteolytic cleavage of the ORF1 polyprotein releases the NS5 protein, which is also known as VPg (viral protein genome linked). The caliciviral VPg is a 13- to 15-kDa protein that is found covalently attached to the 5′ terminus of the genomic and subgenomic RNAs (
24–26). Covalently attached terminal proteins are also found at the 5′ ends of the genomes of other positive-sense RNA viruses, including the
Picornaviridae and
Astroviridae, which are mammalian viruses, and several plant virus families, such as the
Potyviridae,
Comoviridae, and
Nepoviridae. They also occur in DNA viruses in the
Adenoviridae and in members of the bacteriophage
Podoviridae (
27–34).
The VPg proteins of different viruses vary in size and sequence. For example, within the positive-sense RNA viruses,
Comoviridae and
Picornaviridae VPgs are ∼3 kDa, the
Potyviridae VPg is ∼24 kDa, and the
Calicivirus VPg is 13 to 15 kDa. The calicivirus VPg proteins are more similar to the VPgs found in
Astroviridae, which are predicted to be around 11 kDa in size and have 20 to 27% amino acid sequence identity with their calicivirus counterparts (
28).
VPg is essential for translation initiation from genomic and subgenomic RNAs and appears to act as a cap substitute for ribosomal recruitment (
8,
25). The molecular basis for this function is now emerging. The VPg proteins from both FCV and MNV have been found to interact with the cap-binding protein eIF4E (
35,
36). In addition, an interaction has been demonstrated between HuNV VPg and purified eIF3 complex (
37). These studies suggest that VPg may recruit the ribosome to the genomic RNA through interaction with the canonical translation initiation factors. A similar role has also been described for the VPg proteins from the
Nepoviridae and
Potyviridae (
38–41). Moreover, the observations that protease K treatment to remove VPg from astrovirus RNA renders the viral RNA noninfectious and that infectivity can be restored to RNA transcripts by addition of a 5′ m
7G cap are consistent with a role for astroviral VPg in translation (
28,
42).
VPg is also important for genome replication. The caliciviral VPg can be nucleotidylated by the viral polymerase via a phosphodiester linkage to a conserved tyrosine residue (
43)—Y24, Y26, and Y27 in the VPg of FCV, MNV, and human norovirus (HuNV), respectively (
43–45). Nucleotidylated VPg can be extended in a template-dependent manner to produce RNA-linked VPg (
46). While the mechanism of caliciviral RNA replication has not been fully characterized, it is likely that VPg becomes covalently attached to the genomic RNA by acting as a primer for positive-strand RNA synthesis. Yeast two-hybrid data also suggest that the FCV VPg protein interacts with the major capsid protein VP1, indicative of a possible role in the selective encapsidation of VPg-linked viral RNA (
47).
The solution structures of the 22-amino-acid VPg from poliovirus, a picornavirus, have been determined using nuclear magnetic resonance (NMR) spectroscopy for both the native peptide and VPg that has been nucleotidylated on its acceptor Tyr 3 residue (
48,
49). In its native form, poliovirus VPg appears to be highly flexible; a defined conformation was observed only in the presence of high concentrations (1 M) of the organic solvent trimethylamine
N-oxide (TMAO). Intriguingly, the nucleotide-linked poliovirus VPg-pU appeared more stable and exhibited a defined, globular conformation in an aqueous solution that lacked TMAO (
48). However, the physiological relevance of this structure is uncertain; although the poliovirus VPg-pU structure may be computationally docked with the viral three-dimensional (3D) polymerase, the resulting model did not position the uridine in VPg close enough to the active-site residues of the polymerase to plausibly account for the nucleotidylation reaction. Moreover, this model does not take into account the conformational changes that might be induced by association with the polymerase and is inconsistent with the extended conformation of VPg-pU observed in the crystal structure of the 3D
pol-VPg-pU complex for the closely related picornavirus foot-and-mouth disease virus (FMDV) (
50).
Both the potyviral and noroviral VPgs have been predicted to be largely disordered (
51). In the case of potyviruses, circular dichroism and thermal stability measurements and tryptic digest assays all pointed to the presence of some structured elements within the protein (
52–54). But although NMR analyses have detected several aromatic shifted methyl peaks in 1D and
1H-
13C heteronuclear single quantum coherence (HSQC) spectra for potyvirus VPg, suggesting that the protein is at least partially folded (
54), it is commonly regarded as “intrinsically disordered” (
55).
We report here the determination of the solution structures of two calicivirus VPgs using NMR spectroscopy. We find that the FCV and MNV VPg proteins both possess a compact helical core that is flanked by unstructured N- and C-terminal tails. Intriguingly, the MNV VPg core contains only the first two of the three helices present in the core of FCV VPg. For both proteins, the nucleotidylated Tyr residue projects into solvent from the center of the first helix in the protein core. The core structure facilitated the design of mutagenesis experiments to investigate the role of the protein structure in RNA replication and infectivity. The results showed that mutations that disrupt the fold of the helical core generally impair the ability of VPg to support virus replication and nucleotidylation by the MNV VPg. The structured core therefore appears to be functionally important for interaction with the MNV RdRp.
MATERIALS AND METHODS
VPg cloning and expression.
MNV VPg 11-62, 11-85, and 1-124 (full length) were amplified from the MNV-1 infectious clone pT7:MNV 3′Rz (NCBI accession number
DQ285629.1) and ligated into the expression vector pETM11 (
7,
56). FCV VPg 1-111 (full length) was amplified from a full-length FCV VPg clone of the F9 strain (NCBI accession number
M86379) and ligated into pETM11. FCV VPg 9-79 was amplified from the same template and ligated into the expression vector pQE30 (Qiagen). Both pQE30 and pETM11 encode N-terminal 6-histidine tags. Processing of proteins expressed from the pETM11 vector with tobacco etch virus (TEV) NIa protease removed the tag, leaving GAM and G residues on the N termini of MNV and FCV VPg, respectively. The pQE30 vector adds a noncleavable polyhistidine tag (MRGSHHHHHHGS) to the N terminus of the expressed protein.
QuikChange mutagenesis (Stratagene) was performed on pETM11 FCV VPg 1-111 and MNV VPg 1-124 constructs to produce the following mutants for NMR analyses: FCV VPg Y24A, F43A, R47E, F62A, and R69E and MNV VPg Y26A, Y26F, F29A, R32D, Y40A, Y40F, Y45A, Y45F, D48R, and the R32D D48R double mutant. The sequences of primers used for mutagenesis can be found at
https://doi.org/10.6084/m9.figshare.155873.
All isotopically labeled MNV VPg proteins were expressed in Escherichia coli (DE3) Rosetta cells for 16 to 18 h at 20°C by addition of isopropyl-β-d-thiogalactopyranoside (IPTG) to a final concentration of 1 mM. The MNV VPg 11-85 construct used for structure determination was expressed using E. coli OD 2 13C, 15N-rich medium (Silantes), whereas 15N-labeled MNV VPg 1-124 and 11-62 were expressed in minimal medium containing 0.2% (wt/vol) 15NH4Cl.
FCV VPg 1-111 protein for backbone assignment and
1H-
15N heteronuclear nuclear Overhauser effect (hetNOE) analysis was expressed in
E. coli BL21(DE3) cells for 4 h in
E. coli OD 1
13C
15N-rich medium (Silantes) at 22°C with 1 mM final IPTG. The FCV VPg 11-79 protein for the structure determination was expressed overnight in
E. coli M15 in minimal medium containing 0.1% (wt/vol)
15NH
4Cl and 0.4% (wt/vol)
13C
6 glucose at 18°C with 0.5 mM final IPTG. Unlabeled MNV VPg 1-124 and FCV VPg 1-111 and mutant variants of these proteins used in 1D NMR experiments were produced in
E. coli (DE3) Rosetta cells for 4 h in lysogeny broth (LB) (
57) at 20°C by addition of 1 mM IPTG.
Protein purification.
Isotopically labeled MNV VPg proteins were purified by affinity chromatography using Talon resin (Clontech). The polyhistidine tag was then removed by overnight incubation with approximately 1 mg of polyhistidine-tagged TEV NIa protease per 15 mg of VPg in a buffer typically made up of 50 mM sodium phosphate (pH 6.5), 300 mM NaCl, and 1 mM dithiothreitol (DTT). TEV NIa protease and cleaved tags were removed from VPg by reapplication of the cleaved VPg to Talon. Final purification of MNV VPg 11-85 for the structure determination and MNV VPg 1-124 used in NaCl, pH, and temperature titrations was performed by size exclusion chromatography on an S75 16/60 column (GE Healthcare) with a buffer containing 50 mM sodium phosphate (pH 6.5), 300 mM NaCl, and 1 mM DTT.
FCV VPg 1-111 used in backbone assignments and hetNOE analysis was purified using the same protocol as for MNV VPg 11-82. FCV VPg 9-79 was purified by one-step affinity chromatography to Ni-nitrilotriacetic acid (NTA) agarose resin (Qiagen). MNV VPg 1-124 and FCV VPg 1-111 and mutant proteins for use in 1D NMR were purified by one-step affinity chromatography using Talon resin.
MNV and FCV VPg assignment and structure determination.
All NMR spectra were recorded on Bruker 500-, 600-, or 800-MHz spectrometers. Backbone assignments of FCV VPg 1-111 were obtained using the following 3D heteronuclear experiments: HNCA, CBCA(CO)NH, HNCACB, HNCO, HN(CA)CO, HBHA(CBCACO)NH, and 1H-15N NOESY (NNOESY) HSQC. Additionally, hetNOE data for FCV VPg 1-111 were recorded. All these experiments were done using FCV VPg at a concentration of 0.5 to 1.0 mM in buffer composed of 50 mM sodium phosphate (pH 6.5), 200 mM NaCl, 1 mM EDTA, 1 mM NaN3, and 1 mM β-mercaptoethanol at 298 K. Data were analyzed using NMRView version 4.1.3 (One Moon Scientific).
For both MNV VPg 11-85 and FCV VPg 9-79, the proteins used in structure determination and backbone and aliphatic side chain assignments were completed using an in-house NMRView-based assignment protocol (
58). This was initially used in combination with MARS to establish sequential connectivity, after which assignments were completed (
59). The experiments used in the backbone assignment were HNCACB, CBCA(CO)NH, HNCO, and HN(CA)CO. Aliphatic side chain assignment was completed primarily using the HBHA(CBCA)NH, H(C)CH TOCSY, and (H)CCH TOSCY experiments; in addition, the H(CC)(CO)NH and (H)CC(CO)NH TOCSY experiments were used in MNV VPg 11-85 for these assignments. Aromatic side chain assignments were made primarily using NOE correlation of aromatic side groups to the assigned H
β and an aromatic
1H-
13C HSQC. In addition, (HB)CB(CGCD)HD and (HB)CB(CGCDCE)HE experiments were used in the MNV VPg 11-85 aromatic assignment. NOE distance restraints were derived using the NNOESY HSQC and
1H-
13C NOESY (CNOESY) HMQC experiments. NOESY mixing times for FCV and MNV proteins were 100 ms and 125 ms, respectively. In all structure determination experiments, FCV VPg 9-79 was at a concentration of 100 to 200 μM in 20 mM HEPES (pH 7), 300 mM NaCl, and 1 mM NaN
3. MNV VPg 11-85 was at a concentration of 590 μM in 50 mM sodium phosphate (pH 6.5), 300 mM NaCl, and 1 mM DTT for all but the
1H-
13C NOESY-HMQC, H(C)CH-TOCSY, (HB)CB-(CGCD)HD, (HB)CB(CGCDCE)HE, and (H)CCH-TOSCY experiments. For these experiments, the protein was at 710 μM in 60 mM sodium phosphate (pH 6.5), 360 mM NaCl, 1 mM DTT, 0.1% (wt/vol) NaN
3, and 1× Complete protease inhibitor cocktail (Roche). Despite buffer differences, the NOESY and side chain experiments, recorded in different buffers, correlate very well with each other. Experiments on MNV VPg 11-85 and FCV VPg 9-79 were recorded at 303 K and 283 K, respectively.
NOEs were assigned and structures were calculated using the ARIA protocol and CNS, including a final water refinement (
60–62). TALOS+ was used to generate dihedral angle restraints that were also implemented in the structure calculation (
63). In the case of FCV VPg 9-79,
1H-
15N residual dipolar coupling (RDC) restraints were also applied. In-phase/anti-phase (IPAP)
1H-
15N HSQC spectra were recorded under conditions described previously and with 15 mg/ml PF1 filamentous phage as alignment medium (ASLA Biotech). Model validation of FCV and MNV VPg structures was performed using PSVS (
64).
Validation of the MNV VPg structure.
1H-15N HSQC spectra of MNV VPg 1-124 and 11-62 for structure validation purposes were recorded at 240 μM and 550 μM protein concentrations, respectively, in 50 mM sodium phosphate (pH 6.5), 300 mM NaCl, and 1 mM DTT. These experiments were performed at 303 K and compared to the reference 1H-15N HSQC of MNV VPg 11-85 obtained using 590 μM protein in 50 mM sodium phosphate (pH 6.5), 300 mM NaCl, and 1 mM DTT. Heteronuclear NOE experiments were performed using both MNV VPg 11-85 and MNV VPg 1-124 at concentrations of 590 μM and 420 μM, respectively. The buffer conditions were the same as those for the MNV VPg 1-124 1H-15N HSQC, with the exception that 0.1% NaN3 and 1× Complete protease inhibitor cocktail were present in the VPg 11-85 sample.
1D NMR of VPg mutants.
The 1D NMR spectra of wild-type (WT) and mutant MNV VPg 1-124 proteins were typically recorded at 298 K in 50 mM Tris (pH 7), 300 mM NaCl, and 2 mM β-mercaptoethanol. These conditions were also used for FCV VPg 1-111 and mutants thereof, with the exception that 150 mM NaCl was used. The protein concentration used was ∼45 μM for FCV VPg and between 90 and 170 μM for MNV VPg.
MNV and FCV viability assays.
For FCV, the QuikChange II XL site-directed mutagenesis kit (Agilent Technologies, Inc.) and set of mutagenic primers were used to modify the FCV full-length cDNA clone pQ14 (
8) to introduce amino acid changes into the FCV VPg sequence at positions 30 (H to A, I), 43 (F to A), 47 (R to E, G), 62 (F to A), 65 (W to A), 66 (W to A), and 69 (R to E). The modified clones were screened by sequencing, and plasmids containing desired mutations were selected for further experiments. The recovery of infectious FCV from the selected full-length cDNA clones was conducted using capped
in vitro-transcribed RNA as described previously (
8,
65) but with the minor modification of using enzymatically capped RNA transcripts as described previously (
11). CRFK (Crandell-Rees feline kidney) cells were transfected with capped genomic RNA transcripts synthesized from the corresponding plasmid DNA templates by
in vitro transcription using T7 RNA polymerase. Cells were transfected with 1 μg of RNA using Lipofectamine 2000 (Invitrogen) and incubated at 37°C for 24 h. Cells were freeze-thawed at −80°C to release virus particles, and viral titer was determined using the 50% tissue culture infective dose (TCID
50) on CRFK cells.
For MNV, mutations in VPg were introduced by overlapping PCR mutagenesis, with the mutagenized region being inserted back into the MNV-1 full-length infectious clone pT7:MNV 3′Rz. The complete sequence of the mutagenized region was determined in all cases prior to use. The effect of the various VPg mutations on virus recovery was determined using the reverse genetics system based on recombinant Fowlpox expressing T7 RNA polymerase as previously described (
7). Briefly, BSRT7 cells were infected with fowlpox virus expressing T7 RNA polymerase (based on the virus titer in chicken embryo fibroblasts) at a multiplicity of infection (MOI) of 0.5 PFU per cell. After 2 h, 1 μg of each MNV cDNA construct was transfected using Lipofectamine 2000 according to the manufacturer's instructions. At 24 h after transfection of DNA, the cells were freeze-thawed at −80°C to release virus particles and the viral titer was determined using the TCID
50 on RAW264.7 cells. In all cases, the expression of the viral RNA polymerase NS7 24 h after transfection of the cDNA construct was analyzed by Western blot to confirm equal transfection efficiency.
VPg-RNA synthesis assays.
The MNV RdRp and VPg coding regions were amplified using PCR from the MNV-1 infectious clone pT7:MNV 3′Rz (GenBank accession number
DQ285629.1) and cloned into the pUNO vector (InvivoGen, San Diego, CA). All VPg mutants were generated using specific forward and reverse primers and the QuikChange mutagenesis kit (Agilent Technologies). All plasmid constructs were confirmed by DNA sequencing using the BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems).
The norovirus (NoV)-5BR assay was used to determine the efficiency of RNA synthesis (
45). In this assay, HEK293T cells were transiently transfected with plasmids to express the NoV RdRp and the components needed to detect the RNAs produced by the NoV polymerase and, where appropriate, with VPg. The RNAs synthesized by the NoV RdRp activate signal transduction by the innate immune receptor RIG-I, which leads to expression of the firefly luciferase reporter that is under the control of the beta interferon (IFN-β) promoter. The cells also express the
Renilla luciferase under the control of a constitutive promoter to monitor transfection efficiency and cytotoxicity. Briefly, 1 × 10
5 HEK293T cells were seeded in a Costar 96-well plate with Dulbecco's modified Eagle medium (DMEM) and 10% fetal bovine serum for 24 h prior to cotransfection of the plasmids that can express VPg, NS7
pol, RIG-I, and the two reporter luciferases. At 36 h posttransfection, luciferase levels were determined using the Dual-Glo luciferase assay system (Promega) and a Synergy 2 microtiter plate reader (BioTek). RNA synthesis efficiency is reported as the ratio of firefly to
Renilla luciferase activities.
A VPg-RNA electrophoretic mobility shift assay (EMSA) was used to determine whether the NoV RdRp used VPg as a protein primer for RNA synthesis. HEK293T cells (1.0 × 10
6/ml in 6-well plates [BD Falcon]) were transfected with hemagglutinin (HA)-tagged WT or mutant MNV VPgs along with FLAG-tagged NS7
pol 24 h posttransfection. VPg and VPg-RNA were immunoprecipitated using anti-HA polyclonal antibody covalently linked to Dynabeads M-270 epoxy resin, resolved on a 4 to 12% NuPAGE Novex Bis-Tris gel and morpholinepropanesulfonic acid-SDS running buffer (Invitrogen), and transferred to a polyvinylidene fluoride (PVDF) membrane, which was then probed with anti-HA antibodies to detect VPg (
45).
Protein structure accession numbers.
Coordinates and NMR data have been deposited with the Protein Data Bank. The accession numbers are 2M4G (MNV VPg 11-85) and 2M4H (FCV VPg 9-79).
DISCUSSION
In this paper, we report the first high-resolution structural analysis of calicivirus VPg proteins. These structures are surprising in several ways. We find that the VPg proteins of FCV and MNV contain α-helical cores flanked by long unstructured termini, in contrast to the intrinsically disordered nature of smaller picornavirus VPg (
49) and the larger VPg proteins from RNA viruses of plants, such as potyvirus (
52,
54). Although there is some evidence (from circular dichroism measurements, protease sensitivity assays, and NMR analyses) for the presence of a compact domain within potyvirus VPg, a defined tertiary structure has not been detected; rather, the core within this protein appears to have the properties of a dynamic molten globule (
54).
These new observations for calicivirus VPg proteins emphasize the structural diversity within this broad grouping of VPg proteins, which likely also reflects their different functions. While the VPgs of picornaviruses, caliciviruses, and potyviruses all have a role in viral replication by serving as a protein primer for the initiation of RNA synthesis, their additional functions vary (
55,
72). Calicivirus VPgs are known to be critical for the initiation of viral protein synthesis (
25) by recruiting host cell initiation factors to the viral RNA (
35–37). In contrast, the short ∼22-amino-acid VPg from picornaviruses is not involved in translation initiation; instead, this function is assumed by an elaborate RNA structure, the internal ribosome entry site (IRES), within the 5′ untranslated region of the viral genome (
72). Potyvirus VPg is different again; although the potyvirus RNA genome also contains an IRES that is required for translation initiation (
55), its VPg protein makes an interaction with the eukaryotic initiation factor eIF4E that is critical for infection (
39,
41). However, it is not yet clear whether this interaction is needed to initiate translation; it may alternatively serve to suppress host-cell protein synthesis (
73). More-recent data have revealed interactions between potyvirus VPg (or its precursors) and other components of the translation initiation machinery, such as PABP, eIF4G, and eIF4A, that may be functionally important (reviewed in reference
55).
There are clear differences between the VPg structures for MNV and FCV. While the VPg core from the vesivirus FCV has a three-helix bundle, the murine norovirus VPg core contains just the first two helices of this structure (
Fig. 2 and
4). Strikingly, despite significant structural variation, the hydrophobic core residues in helix α1 and helix α2 of FCV VPg are largely conserved in the MNV structure: residues L21, F29, Y40, I42, Y45, and L46 of MNV VPg overlay closely with L19, W27, L38, V40, F43, and L44 of FCV VPg, respectively (
Fig. 4C and
D). Given that FCV and MNV are representative members of the two major clades that caliciviruses can be divided into on the basis of VPg nucleotide sequences (
74), the structures reported should serve as useful models for other calicivirus VPg proteins; in particular, helices α1 and α2 are likely to be conserved features.
Aside from the helix content, one of the most notable differences between FCV and MNV VPg occurs within the sequence of helix 2. In FCV VPg, the helix α2 sequence
40VEDFLMLRHRAAL
52 is replaced in the MNV protein by
42IDDYLADREREEELL
56 (
Fig. 3A), which contains many more charged residues. Whereas in FCV VPg, hydrophobic residues on one flank of this helix, notably L46 and A50, make apolar contacts with helix α3, the equivalent residues in MNV VPg (D48 and E52) are polar and contribute instead to electrostatic stabilization between helices α2 and α1 (
Fig. 4E).
An important common feature of calicivirus VPg proteins is the conserved acid-rich motif (E/DEYDEΩ, where Ω denotes F, Y, or W) that contains the Tyr residue nucleotidylated by the viral NS7 polymerase to form primers for RNA synthesis (
72). Our results for FCV and MNV VPg reveal that this motif is found in a conserved location, embedded within the first helix of the structure core such that the Tyr side chain points away from the core (
Fig. 4C). Remarkably, despite this exposed position, modeling studies suggest that the structure of the core would prevent the Tyr from binding close enough to the active site of NS7
pol to be nucleotidylated by the enzyme (
Fig. 8).
To probe how the observed structure relates to function, we examined the effects of VPg mutations on MNV infectivity and on the VPg-NS7
pol interaction using both a cell-based assay that reported the VPg-mediated stimulation of polymerase activity and an EMSA to monitor the production of VPg-RNA (
45,
71). Some care needs to be taken in the comparison of results from these assays because of the significant differences in their methodologies. First, it is important to appreciate that the two assays have very different dynamic ranges. The infectivity assay can detect differences in MNV plaque formation over several logs, while the two assays for VPg-NS7
pol interaction have a range of only a few fold. Second, the modulatory effects of VPg on RNA synthesis in the reporter assay rely on RIG-I detection of RNA synthesized by NS7
pol, followed by signal transduction leading to the activation of the IFN-β promoter. Thus, the readout is an indirect assessment of RNA synthesis by NS7
pol and the NS7
pol-VPg interaction.
However, even with these caveats in mind, we believe that results from these assays are relevant for understanding how VPg structure impacts the functions of the protein. It is notable that the majority of the results from the infectivity assay agreed with those from the interaction assays: mutations that impaired the functional interaction of VPg with NS7
pol generally abrogated infectivity (
Table 2). Exceptionally, within the VPg N-terminal tail, mutants K3A and L21A retained interaction with NS7
pol but were not infectious in the context of the MNV replicon. It is possible that residues K3 and L21 mediate other activities of the VPg required for MNV infectivity, such as RNA encapsidation or modulation of translation initiation. The effects of the D48R mutant in the infectivity assay and the NS7
pol-VPg interaction assays are more difficult to explain, given that this mutant apparently retained wild-type infectivity but was unable to interact with the viral polymerase. We can only speculate that the interactions within the more-elaborate RNA replication complexes that form in infected cells might suppress the observed defect in the interaction of NS7
pol and VPg detected by our assays.
By analogy with results for FMDV 3D
pol, which is structurally very similar to MNV NS7
pol (
Fig. 8A and
B) (RMSD in backbone C
α positions, 3.9 Å) and binds FMDV VPg in an extended polypeptide conformation that positions the nucleotidylated Tyr (Tyr3) within the polymerase active site (
75), we reasoned that the calicivirus VPg core would have to unfold to allow the nucleotide acceptor Tyr within helix α1 to adopt a similarly extended conformation. This hypothesis appeared to be consistent with observations that mutation of three Tyr residues in HuNV VPg (Y30A, Y41A, Y46A), all of which we would expect to destabilize the core of the protein, enhanced the nucleotidylation of the protein in
in vitro assays (
43). However, although alanine substitution of the equivalent residues in MNV VPg (F29, Y40, Y45) was confirmed to destabilize the protein core by NMR analysis (
Fig. 6 and
Table 2), these mutations generally impaired the interaction with the polymerase—although the effects for Y45A were relatively modest. The Y40F mutation, which resulted in a less-destabilized VPg core structure, showed evidence of good interaction with NS7
pol and wild-type infectivity. Together, these findings appear to suggest that the stability of the VPg core structure contributes to VPg function.
However, a counterpoint to this interpretation is the observation that the Y45F mutation, which preserves the core structure, appeared to completely abrogate interaction with the polymerase and virus infectivity. It may be that Y45 has an important function beyond stabilization of the VPg core. Alternatively, the Y45F substitution may actually enhance the stability of the VPg core, which may impair its ability to interact functionally with the polymerase, assuming that interaction requires at least partial unfolding of the tertiary structure (see below). Finally, the mutation R32D that should disrupt formation of the salt bridge between the two core α-helices within MNV VPg did not impair infectivity (
Table 2 and
Fig. 7). Collectively, our results are consistent with the notion that the two-helix core structure of MNV VPg represents a finely balanced state of the VPg structure that may undergo modest conformational changes to participate in nucleotidylation by NS7
pol and successful viral infection.
Manual docking trials with MNV VPg into the crystal structure of the MNV NS7
pol (
Fig. 8) suggest that, with minor side chain adjustments, there may be room in the substrate-binding channel to accommodate helix α1 in such a way that the Tyr26 acceptor is positioned in the active site, but only if this helix is detached from the VPg core. Alternatively, if the VPg core structure remains intact upon binding to the polymerase, the thumb and fingers domains of NS7
pol would have to move apart to accommodate VPg in a productive complex. A proper test of these ideas awaits the determination of a structure of an NS7
pol-VPg complex. Until that time, the definition of the boundaries between structured and unstructured domains of the FCV and MNV VPg proteins and the fold of the structured cores reported here provide an improved framework for the design of mutagenesis experiments to continue probing VPg function.