Silencing of neurotropic flavivirus replication in the central nervous system by combining multiple microRNA target insertions in two distinct viral genome regions

Abstract

In recent years, microRNA-targeting has become an effective strategy for selective control of tissue-tropism and pathogenesis of both DNA and RNA viruses. Here, using a neurotropic flavivirus as a model, we demonstrate that simultaneous miRNA targeting of the viral genome in the open reading frame and 3′-noncoding regions for brain-expressed miRNAs had an additive effect and produced a more potent attenuation of the virus compared to separate targeting of those regions. Multiple miRNA co-targeting of these two distantly located regions completely abolished the virus neurotropism as no viral replication was detected in the developing brain of neonatal mice. Furthermore, no viral antigens were detected in neurons, and neuronal integrity in the brain of mice was well preserved. This miRNA co-targeting approach can be adapted for other viruses in order to minimize their replication in a cell- or tissue-type specific manner, but most importantly, to prevent virus escape from miRNA-mediated silencing.

MicroRNA-based control of tick-borne flavivirus neuropathogenesis: Challenges and perspectives

“…5) (p<0.02 or 0.001 for 1 and 10 PFU, correspondingly). Interestingly, the level of attenuation of TBEV/LGTV virus with miRNA target sequences in the ORF (vTL6) was comparable with attenuation level of TBEV/DEN4 virus, having a similar set of miRNA-target sequences (vE1) (Teterina et al, 2014), whereas the attenuating effect of miRNA-targeting in the 3′NCR of TBEV/LGTV (vTL1, vTL7, vTL8 and vTL10) on viral neurovirulence in suckling mice was significantly lower than that observed for miRNA-targeted TBEV/DEN4-derived viruses (for example, TBEV/DEN4 mir-9T-124aT-124aT(1,2,3) virus has the IC LD 50 of 490 PFU that indicates a 613- fold reduction of its neurovirulence compared to that of parental TBEV/DEN4 virus) (Heiss et al, 2012). The reason for such difference is not clear and might be related to the fact that TBEV/LGTV 3′NCR was found to be more restrictive for the placement of target sequences.…”

“…TBEV/LGTV virus carrying the miRNA targets in the ORF, in the region between protein E and NS1 genes, was generated as previously described (Bonaldo et al, 2007; Teterina et al, 2014). Three tandem targets (mir-124T-9T-124T) for miRNAs were placed between duplicated sequences encoding the C-terminal stem-anchor (SA) region of protein E (Fig.…”

“…Analysis of viral antigen distribution and assessment of the extent of virus-induced neuronal damage in the brain were performed as previously described (Heiss et al, 2012; Teterina et al, 2014). Three brains of suckling SW mice inoculated IC with 10 3 PFU of the virus were collected on days 13 and 20 p.i.…”

“…Using chimeric tick-borne encephalitis/dengue virus (TBEV/DEN4) as a model neurotropic flavivirus, we showed that cellular miRNAs abundantly expressed in the brain could be successfully exploited to selectively restrict replication of this virus in the CNS by inserting miRNA targets into the viral genome (Heiss et al, 2011; Heiss et al, 2012; Teterina et al, 2014). The present study focused on investigating whether this approach could effectively attenuate the more pathogenic flavivirus, a tick-borne encephalitis/Langat virus (TBEV/LGTV).…”

Engineered Mammalian RNAi Can Elicit Antiviral Protection that Negates the Requirement for the Interferon Response

“…In contrast, many RNA viruses readily escape RNAi targeting and thus do not require a mechanism to inhibit such a response. Experiments applying exogenous siRNAs as a means to treat virus infection, while generally effective, often result in the emergence of escape mutants (Barnes et al, 2008; Boden et al, 2003; Das et al, 2004; Gitlin et al, 2002; Heiss et al, 2011; Heiss et al, 2012; Teterina et al, 2014; Wilson and Richardson, 2005). While virus escape was sometimes the result of an excision event (Pham et al, 2012), in general, single mutations in a given small RNA target were sufficient to relieve silencing (van Rij and Andino, 2006).…”