The genome-linked protein VPg of vertebrate viruses - a multifaceted protein

Abstract

Several vertebrate positive-sense RNA viruses, namely the Picornaviridae and Caliciviridae have evolved to use a protein-primed mechanism of genome replication. This results in the covalent linkage of a virus encoded protein, VPg (viral protein genome-linked), to the 5' end of viral RNA. Recent studies have highlighted the pivotal role VPg plays in the life cycle of these viruses, which in the case of the Caliciviridae, includes a role in viral protein synthesis. This article provides an overview of the current knowledge of the functions of vertebrate RNA virus VPg proteins, illustrating their diverse function and the parallels they share with plant virus VPg proteins.

Figure 1. Schematic of the genomes of representative members of the Caliciviridae and Picornaviridae

Viral RNA is illustrated as the blue line with the individual mature proteins boxed in grey. The VPg proteins linked to the 5′ end of the viral RNA are shown as red filled circles. (A) The murine norovirus genome is shown highlighting the four open reading frames note however that the majority of caliciviruses possess only three open reading frames. In addition to the viral genomic RNA (gRNA), calicivirus replication results in the production of a ~2.5Kb subgenomic RNA (sgRNA), which in the case of murine norovirus, expresses the major and minor capsid proteins, VP1 and VP2 respectively. The NS1-7 nomenclature is used to highlight the mature nonstructural proteins made from the processing of the ORF1 polyprotein. Other synonyms widely used in the literature are displayed below the viral genome in italics. Note that in addition to the mature nonstructural proteins, a variety of precursors are produced during virus replication. For simplicity however, only those containing VPg are displayed. (B) The poliovirus genome is shown as a representative member of the Picornaviridae family. The large viral polyprotein is displayed along with the various mature proteins produced as a result of polyprotein processing. Note that for simplicity only the VPg containing precursor proteins are shown. The positions of the cis-acting RNA replication elements is not shown but are illustrated in figure 4. The figure is not drawn to scale.

Figure 2. Diagrammatic model of the calicivirus VPg-containing translation initiation complex

Models based on published and unpublished data for two members of the Caliciviridae, namely feline calicivirus and murine norovirus are illustrated. Initiation factors that interact directly with VPg and play a functional role in viral protein synthesis are highlighted with a solid red surrounding line, whereas those that bind VPg directly but the function remains to be determined are highlighted with a dashed red surrounding line. Factors that do not interact directly with VPg or for which no data exists, yet are functionally important for calicivirus translation initiation are displayed with a solid black surrounding line. The interaction of poly A binding protein (PABP) with the 3′ poly A tail is inferred from published literature on other (+) RNA viruses and the presence of a poly A tail on the calicivirus genome.

Figure 3. Model of the picornavirus VPg-uridylylation complex

The positions of the three well characterized cis-acting RNA replication signals are illustrated as OriL, Oril and OriR, for Origin Left, Internal and Right respectively. For simplicity the minimal components of the uridylylation complex are shown. The VPg protein primer is synthesized by the Oril (also referred to as the 2C^CRE) templated addition of two U residues to tyrosine 3 in VPg (shown in red). In addition, the reaction requires the viral RNA polymerase (3D^Po1) and two copies of the 3C protease in its mature or precursor form (3CD). Note that experimental evidence suggest that precursor forms of the various components play an essential role in the formation of the VPg-pUpU-OH primer. A More in-depth description and analysis of this complex can be found in published literature ([6,22,50] and references therein).

Figure 4. Features of the picornavirus and calicivirus VPg proteins

Sequence alignment of the VPg proteins from representative members of the Picornaviridae (A) and Caliciviridae (B). The site of RNA linkage in the picornavirus VPg is highlighted in green. Abbreviations: PV1 poliovirus type 1, CVB3 Coxsackievirus type 3, FMDV foot and mouth disease virus. B) The site of RNA linkage in the calicivirus VPg highlighted in turquoise with position 117 of the murine norovirus (MNV) VPg protein, known to be important for nucleotidylation in vitro highlighted in yellow. The absolutely conserved signature motif of the calicivirus VPg proteins is shown (E/DEYDEΩ). Basic residues in the N-terminus of calicivirus VPg proteins are shown in red. Abbreviations: MNV murine norovirus, Norwalk human norovirus GI, FCV feline calicivirus, RHDV rabbit hemorrhagic disease virus.