The Disorderly Nature of Caliciviruses

Abstract

An intrinsically disordered protein (IDP) or region (IDR) lacks or has little protein structure but still maintains function. This lack of structure creates flexibility and fluidity, allowing multiple protein conformations and potentially transient interactions with more than one partner. Caliciviruses are positive-sense ssRNA viruses, containing a relatively small genome of 7.6-8.6 kb and have a broad host range. Many viral proteins are known to contain IDRs, which benefit smaller viral genomes by expanding the functional proteome through the multifunctional nature of the IDR. The percentage of intrinsically disordered residues within the total proteome for each calicivirus type species can range between 8 and 23%, and IDRs have been experimentally identified in NS1-2, VPg and RdRP proteins. The IDRs within a protein are not well conserved across the genera, and whether this correlates to different activities or increased tolerance to mutations, driving virus adaptation to new selection pressures, is unknown. The function of norovirus NS1-2 has not yet been fully elucidated but includes involvement in host cell tropism, the promotion of viral spread and the suppression of host interferon-λ responses. These functions and the presence of host cell-like linear motifs that interact with host cell caspases and VAPA/B are all found or affected by the disordered region of norovirus NS1-2. The IDRs of calicivirus VPg are involved in viral transcription and translation, RNA binding, nucleotidylylation and cell cycle arrest, and the N-terminal IDR within the human norovirus RdRP could potentially drive liquid-liquid phase separation. This review identifies and summarises the IDRs of proteins within the Caliciviridae family and their importance during viral replication and subsequent host interactions.

Keywords: NS1-2; RdRP; VPg; calicivirus; intrinsically disordered protein; norovirus; sapovirus; vesivirus.

Figure 1

Calicivirus ORF configurations [81,88]. (A) The calicivirus genera Bavovirus, Nebovirus, Sapovirus, Salovirus, Nacovirus, Minovirus, Lagovirus and Valovirus encode a non-structural polyprotein (ORF1) that includes the VP1 capsid protein. The VP1 (capsid) protein is primarily initiated from the VP1 start codon from the subgenomic RNA. The minor structural protein VP2 is encoded by ORF2. Some sapoviruses may also contain an ORF3. (B) Norovirus, Recovirus and Vesivirus genera produce the non-structural proteins as an ORF1 polyprotein with VP1 and VP2 encoded by ORF2 and ORF3, respectively. Subgenomic RNA is produced from both genome configurations. Variations within the family include a small leader sequence encoded ahead of VP1 in vesivirus. Also, murine norovirus produces a virulence factor from an alternate reading frame within ORF2 (ORF4) [89]. Yellow shading indicates putative or identified disordered regions that have been expanded into schematic flDPnn disorder plots [90] for porcine sapovirus (PSaV), murine norovirus (MNV), feline calicivirus (FCV) and human norovirus (HuNV). Protein names are shown where function has been elucidated.

Figure 2

Percent of predicted intrinsic disorder within the type species for each genus from the Caliciviridae family. RIDAO analysis, used to efficiently analyse intrinsic disorder within whole proteomes (ridao.app) [100], calculated the percentage of disordered residues using six disorder prediction software outputs (VL-XT [101], VSL2B [102], VL3 [103], IUPred-Short, IUPred-Long [104] and PONDR-FIT [105]). For each output, the percentage of disordered residues (score > 0.5) against the total proteome residues was calculated. The percentage for each of the six prediction software algorithms was plotted as a single point for each proteome with the different symbols representing each genus. The mean percentage of intrinsic disorder residues is defined as a horizontal line. Accession numbers: bavovirus HQ010042.1, lagovirus M67473.1, minovirus KX371097, nacovirus JQ347522.1, nebovirus DQ013304.1, norovirus M87661.2, recovirus EU391643.1, salovirus KJ577139.1, sapovirus HM002617.1, valovirus FJ355928.1 and vesivirus M86379.1.

Figure 3

Key features of norovirus NS1-2. Schematic diagram of HuNV GII.4 (Sydney 2012) (A) and MNV (CW1) (B) NS1-2. Yellow shading represents the predicted disordered regions by flDPnn. Pink arrows identify the host cell caspase cleavage sites. The H-Box and NC motif (red), transmembrane domain (green), VAP-A binding site and nuclear magnetic resonance spectroscopy (NMR) characterised structured region (58–114aa) of MNV NS1 [96,114] are highlighted. NS4 binding and putative LC3 recruitment are indicated for HuNV GII.4 NS1-2. To affirm and visualise the flDPnn disorder prediction the putative HuNV GII.4 (Sydney 2012) NS1 (C) and MNV NS1 (D) are illustrated by plDDT confidence score and coloured by bfactor palette AlphaFold in ChimeraX [115]. Blue = very confident of structure prediction, yellow = low confidence (plDDT score ≤ 60), and red = very low confidence. This was performed using ColabFold v1.5.5: AlphaFold2 using MMseqs2 [116,117,118,119,120].

Figure 4

Key features of VPg. Schematic diagram of the VPg proteins from FCV, PSaV and MNV. Yellow shading represents regions of conserved disorder. Two or three tightly packed alpha-helices are identified within the core for each VPg protein (teal), and grey boxes define structured regions predicted by flDPnn, not identified within the alpha-helices. The nucleotidylylated tyrosine (Y) is indicated within the first alpha-helix, and phosphorylated sites at position T₈₀ and S₁₀₇ have been identified in FCV VPg. For MNV VPg, an N-terminal conserved basic amino acid patch has been implicated in binding of NTPs, RNA and induction of a G0/G1 cell cycle arrest. Interaction of the eIF4G HEAT-1 domain occurs at the C-terminus of MNV VPg [87], a motif conserved in all noroviruses.