TRIM69 inhibits Vesicular Stomatitis Indiana Virus (VSIV)

Abstract

word count: 210 16 Text character count: 58,351 17 18 ABSTRACT 19Vesicular Stomatitis Indiana Virus (VSIV) is a model virus that is exceptionally 20 sensitive to the inhibitory action of interferons. Interferons induce an antiviral state by 21 stimulating the expression of hundreds of interferon stimulated genes (ISGs). These 22 ISGs constrain viral replication, limit tissue tropism, reduce pathogenicity and inhibit 23 viral transmission. Because VSIV is used as a backbone for multiple oncolytic and 24 vaccine strategies, understanding how ISGs restrict VSIV, not only helps in 25 understanding VSIV-pathogenesis, but helps evaluate and understand the safety and 26 efficacy of VSIV-based therapies. Thus there is a need to identify and characterize 27 the ISGs that possess anti-VSIV activity. Using arrayed ISG expression screening, 28 solely responsible for limiting VSIV replication in other organs (15). Thus, other ISGs 85 must play key roles in limiting VSIV tissue tropism. Importantly, VSIV causes 86 neurological disease in multiple species following intracranial inoculation (17, 18), 87suggesting that the ability of ISGs to prevent VSIV from initially accessing the CNS is 88 the cornerstone in limiting VSIV neuropathology across multiple species (19). 89VSIV's low pathogenicity in humans, its rapid replication, and ease of genetic 90 manipulation have made this virus the basis of multiple therapeutic strategies. For 91 example, VSIV can be modified to express antigens from heterologous viruses that 92 can be utilised as vaccine strategies (20). This approach has achieved recent notable 93 success in conferring protection from Ebola virus infection (21). Similarly, VSIV has 94 been used as the backbone of multiple oncolytic strategies (22). Just like natural 95 VSIV infection, IFNs and ISGs appear to be critical for preventing oncolytic viruses 96 from invading healthy tissues (23, 24) and could be critical determinants governing 97 whether oncolytic vesiculoviruses will be efficacious (25). Furthermore, ISGs likely 98 play a key role in limiting the replication of VSIV-based vaccines and are an 99 important safety feature of this immunization strategy. 100The key roles that ISGs play in constraining VSIV pathogenesis and limiting 101 VSIV replication (in natural infection, oncolytic therapies and vaccine strategies) 102 means there is a need to better understand how ISGs inhibit VSIV. Using arrayed 103 ISG expression screening, we identified that TRIM69, a relatively poorly 104 characterised TRIM protein, has anti-VSIV activity. Through exogenous expression 105 and CRISPR Cas9 knockout, we demonstrate that both exogenous and endogenous 106 TRIM69 have potent anti-VSIV activity. Importantly, the inhibition is highly specific 107 for VSIV and multiple other viruses were not inhibited by TRIM69. Notably, TRIM69 108 shows strong signatures of positive selection and multiple common alleles circulate in 109 human populations. Interestingly, murine orthologues of TRIM69 had no detectable 110 anti-VSIV activity whereas ...

Timing and magnitude of the type-I interferon response are determinants of disease tolerance in arbovirus infection

“…Six of the 7 genes identified in the 16 hpi screen were also identified as restrictive at 32 hpi. Given our experience with ISG screens against a variety of viruses [56][57][58][59], we ruled out from subsequent analyses ISGs with (i) known signalling properties (IRF2, IFIH1, DDX58), or (ii) able to induce expression of a reporter gene under the control of an ISRE (interferon stimulated response element) promoter in the same type of assays (IRF-1, CDADC1, CXCR4, ISG20, STARD8), or (iii) known to affect the general cell metabolism (IDO1) [61]. We also excluded SLFN12L as it was identified only in the 16 hpi timepoint and not confirmed in the 32 hpi screen.…”

“…To identify bovine ISGs which could negatively impact BTV replication, we used a medium throughput lentivirus-based overexpression screening platform. This system was previously used to identify antiviral genes acting against a range of viruses [56][57][58][59] and relies on lentivirus vectors co-expressing the ISG of interest and a fluorescent protein (RFP). HEK-293 cells were challenged with BTV expressing the green fluorescent protein (GFP), and the level of virus replication in the presence of each individual ISG was assessed via flow cytometry (Fig 2A -B).…”