Shiftless, a Critical Piece of the Innate Immune Response to Viral Infection

Abstract

Since its initial characterization in 2016, the interferon stimulated gene Shiftless (SHFL) has proven to be a critical piece of the innate immune response to viral infection. SHFL expression stringently restricts the replication of multiple DNA, RNA, and retroviruses with an extraordinary diversity of mechanisms that differ from one virus to the next. These inhibitory strategies include the negative regulation of viral RNA stability, translation, and even the manipulation of RNA granule formation during viral infection. Even more surprisingly, SHFL is the first human protein found to directly inhibit the activity of the -1 programmed ribosomal frameshift, a translation recoding strategy utilized across nearly all domains of life and several human viruses. Recent literature has shown that SHFL expression also significantly impacts viral pathogenesis in mouse models, highlighting its in vivo efficacy. To help reconcile the many mechanisms by which SHFL restricts viral replication, we provide here a comprehensive review of this complex ISG, its influence over viral RNA fate, and the implications of its functions on the virus-host arms race for control of the cell.

Keywords: C19ORF66; FLJ11286; IRAV; ISG; RNA granules; RNA stability; RyDEN; SVA-1; innate immune response; ribosomal frameshift; shiftless; translation.

Figure 1

Shiftless Protein Structure. Shiftless is a 291aa long protein of 33 kDa molecular weight. (A) Highlighted in Red is the PABPC-binding domain (PABPC-BD) which also encompasses the Zinc-Ribbon Domain (112–135) and the Nuclear Localization Signal (121–173). Highlighted in Blue is the -1 Programmed Ribosomal Frameshift (PRF) (169–199). Highlighted in Yellow is the C-terminal domain containing the Glutamic Acid (E)-Rich Domain (270–286) and the Nuclear Export Signal (261–269). (B) Alphafold2 predicted protein structure of Shiftless. The PABPC-BD, -1PRF Domain, and the C-terminal Domain are Red, Blue, and Yellow respectively. Also highlighted in orange is the Nuclear Localization Signal and in pink is the Nuclear Export Signal [26,27].

Figure 2

Shiftless Mechanisms of Restricting Viral Infection. Shiftless (SHFL) has been demonstrated to be a potent broad-spectrum anti-viral factor that is upregulated in response to viral infection and interferon signaling. SHFL restricts viral infection through several mechanisms summarized here: (1) SHFL directly interfaces with viral genomic RNA and viral mRNA and restricts viral gene expression at various stages between RNA stability and translation. For flaviviruses, SHFL binds to viral genomic RNA at the 3′ end and may relocalize it to Processing bodies to trigger RNA decay or directly interfere with polyprotein translation. (2) SHFL is one of the first human proteins shown to directly restrict the function of the -1 programmed ribosomal frameshift (-1PRF), a cis-RNA element that extends the coding capacity of several retro- and RNA viruses. The -1PRF signal triggers a non-canonical ribosome rotation, signaling the recruitment of SHFL and eukaryotic ribosome release factors (eRF1/eRF3), which synergistically halt and then trigger the premature release of the ribosome from frameshifting viral RNA. (3) For HCV and YFV, SHFL expression decreases the level of PI(4)P in the cell, a lipid precursor that directly contributes to the formation of the viral membranous web, a collection of reorganized lipid membranes that house viral replication compartments. SHFL was also shown to interact with and localize to Stress Granules during HCV infection. (4) Lastly, SHFL has been recently shown to bind to and trigger the degradation of select viral proteins through lysosomal or ubiquitination based pathways. Abbreviations: Shiftless (SHFL), Dengue virus (DENV), Encephalomyocarditis virus (EMCV), Yellow Fever virus (YFV), Hepatitis C virus (HCV), Kaposi’s sarcoma-associated herpesvirus (KSHV), Human Immunodeficiency virus 1 (HIV-1), Japanese Encephalitis virus (JEV), Zika virus (ZIKV), Porcine Epidemic Diarrhea virus (PEDV), Poly-A Binding Protein (PABP), La-associated RNA binding protein (LARP), eukaryotic Initiation Factor 4G (eIF4G), eukaryotic Initiation Factor 4E (eIF4E), Processing bodies (P-bodies).