Restriction of Flaviviruses by an Interferon-Stimulated Gene SHFL/C19orf66

Abstract

Flaviviruses (the genus Flavivirus of the Flaviviridae family) include many arthropod-borne viruses, often causing life-threatening diseases in humans, such as hemorrhaging and encephalitis. Although the flaviviruses have a significant clinical impact, it has become apparent that flavivirus replication is restricted by cellular factors induced by the interferon (IFN) response, which are called IFN-stimulated genes (ISGs). SHFL (shiftless antiviral inhibitor of ribosomal frameshifting) is a novel ISG that inhibits dengue virus (DENV), West Nile virus (WNV), Zika virus (ZIKV), and Japanese encephalitis virus (JEV) infections. Interestingly, SHFL functions as a broad-spectrum antiviral factor exhibiting suppressive activity against various types of RNA and DNA viruses. In this review, we summarize the current understanding of the molecular mechanisms by which SHFL inhibits flavivirus infection and discuss the molecular basis of the inhibitory mechanism using a predicted tertiary structure of SHFL generated by the program AlphaFold2.

Keywords: AlphaFold2; C19orf66; SHFL; flavivirus; interferon-stimulated antiviral gene.

Figure 1

Genome structure and replication cycle of flavivirus. (A) Flavivirus RNA contains a single ORF flanked by 5′ and 3′ UTRs. A polyprotein precursor expressed from the single ORF is co- and post-translationally cleaved by viral and cellular proteases, yielding three structural and seven non-structural (NS) proteins. (B) Overview of flavivirus replication. After the binding of E glycoproteins to cell-surface entry receptors, the attached virion is internalized into the cell via clathrin-dependent endocytosis. Then, acidification of the endosomal vesicles triggers a structural rearrangement of the E protein, resulting in a fusion between the viral and endosomal membranes. A single precursor protein is translated from the released viral RNA, which is, in turn, cleaved to produce functional proteins. The viral RNA amplification process takes place on the ER membrane. Subsequently, the nucleocapsids, composed of C proteins and synthesized viral RNA, are assembled with prM and E heterodimers and cellular lipid bilayers to form immature (i.e., non-infectious) particles. However, conformational changes in the glycoproteins occur during the transport through the Golgi apparatus, and eventually, mature (i.e., infectious) virions are released by exocytosis.

Figure 2

Secondary structure of SHFL. SHFL (291 amino acids) is predicted to be composed of ten α-helixes (orange) and nine β-strands (shown by navy blue). Amino acid sequence-based protein motif prediction programs also show that SHFL contains putative NLS (green, by cNLS Mapper [http://nls-mapper.iab.keio.ac.jp/cgi-bin/NLS_Mapper_form.cgi], accessed on 28 September 2022) and NES (red, by NetNES [https://services.healthtech.dtu.dk/service.php?NetNES-1.1], accessed on 28 September 2022). A characteristic E-rich domain (blue) is located next to the NES sequence. The α-helixes (orange) and β-strands (navy blue) are also indicated in the amino acid sequence of SHFL (bottom part).

Figure 3

Three-dimensional structure of SHFL predicted by AlphaFold2. (A) Stereodiagram of the overall structure. The zinc ribbon domain, E-rich domain, helix-rich domain, and flexible domain are colored green, magenta, cyan, and orange, respectively. Red and blue squares indicate areas in Figure 3B,C, respectively. (B) Enlarged view of the core region. The position of the Zn²⁺ was manually adjusted to be equidistant from the S atoms of the four cysteine residues (Cys112, Cys115, Cys132, and Cys135). Brown and red dotted lines represent electrostatic interactions and coordination bonds to Zn²⁺, respectively, with distances (Å) between the atoms. (C) Enlarged view of the putative hydrophobic interaction between two loops.