Expression of novel proteins by polyomaviruses and recent advances in the structural and functional features of agnoprotein of JC virus, BK virus, and simian virus 40

Abstract

Polyomavirus family consists of a highly diverse group of small DNA viruses. The founding family member (MPyV) was first discovered in the newborn mouse in the late 1950s, which induces solid tumors in a wide variety of tissue types that are the epithelial and mesenchymal origin. Later, other family members were also isolated from a number of mammalian, avian and fish species. Some of these viruses significantly contributed to our current understanding of the fundamentals of modern biology such as transcription, replication, splicing, RNA editing, and cell transformation. After the discovery of first two human polyomaviruses (JC virus [JCV] and BK virus [BKV]) in the early 1970s, there has been a rapid expansion in the number of human polyomaviruses in recent years due to the availability of the new technologies and brought the present number to 14. Some of the human polyomaviruses cause considerably serious human diseases, including progressive multifocal leukoencephalopathy, polyomavirus-associated nephropathy, Merkel cell carcinoma, and trichodysplasia spinulosa. Emerging evidence suggests that the expression of the polyomavirus genome is more complex than previously thought. In addition to encoding universally expressed regulatory and structural proteins (LT-Ag, Sm t-Ag, VP1, VP2, and VP3), some polyomaviruses express additional virus-specific regulatory proteins and microRNAs. This review summarizes the recent advances in polyomavirus genome expression with respect to the new viral proteins and microRNAs other than the universally expressed ones. In addition, a special emphasis is devoted to the recent structural and functional discoveries in the field of polyomavirus agnoprotein which is expressed only by JCV, BKV, and simian virus 40 genomes.

Keywords: Agnoprotein; BKV; DNA replication; Merkel cell carcinoma polyomavirus; NMR; SV40; dimer and oligomer formation; polyomavirus JCV; progressive multifocal leukoencephalopathy; transcription and viroporin.

FIGURE 1

Circular map of JC virus. (a) The genome of JC virus contains three functional regions. 1, Control region, which contains the origin of DNA replication and promoter–enhancer elements for early and late genes. 2, Early coding region, which encodes LT‐Ag, Sm t‐Ag, T’proteins (T’165, T’135, and T’136) due to cis‐alternative splicing of the early pre‐mRNA. 3, Late coding region which encodes capsid proteins (VP1, VP2, and VP3) and a regulatory protein, agnoprotein. In addition, late region also encodes two additional proteins ORF1 and ORF2 due to trans‐splicing of the late pre‐mRNA. (b) The coding regions of ORF1 and ORF2 are created by insertion of a partial coding region of VP1 in between agnoprotein and VP2 coding regions. A schematic demonstration of the mechanism of the creation of ORF1 and ORF2, open reading frames (see text for details; Saribas, DeVoto, et al., 2018). LT‐Ag: large tumor antigen; mRNA: messenger RNA; Sm t‐Ag: small tumor antigen [Color figure can be viewed at wileyonlinelibrary.com]

FIGURE 2

Predicted and validated functional motifs of JCV agnoprotein. (a) Phosphorylation of Ser7, Ser11, and Thr21 was previously demonstrated (Johannessen et al., 2008; Sariyer et al., 2006). The website, https://wolfpsort.hgc.jp/ was used to predict the functional motif or domains. (b) For prediction of mitochondrial targeting sequences, the following website was used: http://mitf.cbrc.jp/MitoFates/cgi-bin/top.cgi. JCV: JC virus, ER: Endoplasmic reticulum [Color figure can be viewed at wileyonlinelibrary.com]

FIGURE 3

The 3D structure of the full‐length JC virus agnoprotein was recently resolved by NMR (Coric et al., 2017). According to this 3D structure, agnoprotein contains a minor and a major α‐helix located at amino acid position Leu6‐Lys13 and Arg24-Phe39, respectively. The rest of the protein adopts an intrinsically unstructured conformation (Met1‐Gln5, Val14‐Lys23, and Cyt40-Thr71). 3D: three dimensional; NMR: nuclear magnetic resonance [Color figure can be viewed at wileyonlinelibrary.com]

FIGURE 4

Theoretical dimer formation by agnoprotein through the hydrophobic face I and II, both of which are located at the major α‐helix domain. (a, b) The first homodimer is formed through the interface of Leu29, Leu32 and Leu36 residues located at the hydrophobic face I and is illustrated by a radial (a) and axial (b) view. The side chains of the residues are represented in a stick conformation. The backbone of each α‐helix is represented in a ribbon representation and colored in purple. Amino acids Leu29, Leu32, and Leu36 located at the interface are colored in yellow and aromatic residues Phe31, Phe35, and Phe39 forming an aromatic cluster are colored in green. (c, d) The second homodimer structure is formed through the interface of Ile30, Leu33, and Leu37, which are located on the hydrophobic face II and is also represented in a radial (c) and axial (d) view. The backbone of the antiparallel α‐helices is illustrated in a ribbon representation and colored in purple. The hydrophobic residues Ile30, Leu33, and Leu37 forming the interface are illustrated in a stick representation and colored in orange. Phenylalanine residues, Phe31, Phe35, and Phe39, forming an aromatic cluster are colored in green. The data used to build each homodimer structure of agnoprotein are the same as the one used to build the homodimer structure for the short fragment of agnoprotein (aa 17–52; Coric et al., 2014). The structure of each theoretical dimer was calculated using simulated annealing with restrained molecular dynamics and energy minimization with the X‐PLOR program (Arkin, Sukharev, Blount, Kung, & Brunger, 1998; Fleming et al., 1998) implemented with a total of 453 distance restraints for dimer A (a) and 447 distance restraints for dimer C (c). To build each dimer, 232 intermolecular nuclear overhauser effect (NOE)‐derived distances were unambiguously defined to build dimer A with interface L29, L32, and L36 (a) and 68 for dimer C with interface I30, L33, and L37 (c) respectively [Color figure can be viewed at wileyonlinelibrary.com]

FIGURE 5

Surface fill‐in representation of the theoretical homodimeric structure of agnoprotein formed through the interface Leu29, Leu32, and Leu36. Two opposite faces with equivalent properties are shown colored according their hydrophobic properties. (a) The first hydrophobic face is constituted of the hydrophobic residues Leu29, Ile30, Leu33 and Leu37. (b) The opposite face of the dimer is also constituted of the hydrophobic residues Ala25, Ile28, Phe31, Leu32, Phe35, Leu36, and Phe39. (c) The surface fill‐in representation of the theoretical dimeric structure of agnoprotein formed through the interface of Ile30, Leu33, and Leu37. The surface is colored according to the hydrophobic properties of the amino acids. Two opposite faces with different properties can be distinguished. The first one is constituted of the hydrophilic residues Lys23, Arg24, Gln26, Arg27, Glu34, and Asp38 (c) while the opposite one is also composed of more hydrophobic residues Leu29, Ile30, Phe31, Leu32, Leu33, Phe35, Leu36, Leu37, and Phe39; and forms a relatively larger hydrophobic face (d) [Color figure can be viewed at wileyonlinelibrary.com]

FIGURE 6

Hydrophilic residues located on the major α‐helix domain play important roles in release of agnoprotein. Three-dimensional structure of the full‐length agnoprotein is illustrated in a “surface fill‐in” representation. The hydrophilic residues located on the hydrophilic face of the major α‐helix domain of agnoprotein, including Lys22, Lys23, Glu34, and Asp38; and an aromatic residue (Phe31), which is adjacent to this hydrophilic surface were determined to be critical for the release of agnoprotein from agnoprotein‐positive cells (Saribas, White, et al., 2018). Amino acid residues involved in agnoprotein release are encased in boxes and named. Note that Lys23, Lys23, Phe31, and Glu34 are all conversed between JCV, BKV, and SV40 agnoproteins. JCV Asp38 is substituted for Glu and Gln for BKV and SV40 agnoproteins, respectively, at the same position (Saribas, White, et al., 2018). BKV: BK virus; JCV: JC virus; SV40: simian virus 40 [Color figure can be viewed at wileyonlinelibrary.com]