Coxsackievirus B3 Engineered To Contain MicroRNA Targets for Muscle-Specific MicroRNAs Displays Attenuated Cardiotropic Virulence in Mice

Abstract

Coxsackievirus B3 (CVB3) is trophic for cardiac tissue and is a major causative agent for viral myocarditis, where local viral replication in the heart may lead to heart failure or even death. Recent studies show that inserting microRNA target sequences into the genomes of certain viruses can eradicate these viruses within local host tissues that specifically express the cognate microRNA. Here, we demonstrated both in vitro and in vivo that incorporating target sequences for miRNA-133 and -206 into the 5= untranslated region of the CVB3 genome ameliorated CVB3 virulence in skeletal muscle and myocardial cells that specifically expressed the cognate cellular microRNAs. Compared to wild-type CVB3, viral replication of the engineered CVB3 was attenuated in human TE671 (rhabdomyosarcoma) and L6 (skeletal muscle) cell lines in vitro that expressed high levels of miRNA-206. In the in vivo murine CVB3-infection model, viral replication of the engineered CVB3 was attenuated specifically in the heart that expressed high levels of both miRNAs, but not in certain tissues, which allowed the host to retain the ability to induce a strong and protective humoral immune response against CVB3. The results of this study suggest that a microRNA-targeting strategy to control CVB3 tissue tropism and pathogenesis may be useful for viral attenuation and vaccine development. IMPORTANCECoxsackievirus B3 (CVB3) is a major causative agent for viral myocarditis, and viral replication in the heart may lead to heart failure or even death. Limiting CVB3 replication within the heart may be a promising strategy to decrease CVB3 pathogenicity. miRNAs are ϳ21-nucleotide-long, tissue-specific endogenous small RNA molecules that posttranscriptionally regulate gene expression by imperfectly binding to the 3= untranslated region (UTR), the 5= UTR, or the coding region within a gene. In our study, muscle-specific miRNA targets (miRT) were incorporated into the CVB3 genome. Replication of the engineered viruses was restricted in the important heart tissue of infected mice, which reduced cardiac pathology and increased mouse survival. Meanwhile, replication ability was retained in other tissues, thus inducing a strong humoral immune response and providing long-term protection against CVB3 rechallenge. This study suggests that a microRNA-targeting strategy can potentially control CVB3 tissue tropism and pathogenesis and may be useful for viral attenuation and vaccine development. C oxsackievirus B3 (CVB3) is a clinically relevant infectious agent that causes viral myocarditis, pancreatitis, and aseptic meningitis. CVB3 is cardiotropic and efficiently replicates in the heart. We and others have shown in various animal models that CVB3 replication induces a direct cytopathic effect on infected cells, leading to tissue damage (1, 2). At present, there is no effective therapy against CVB3, and strategies to develop such a therapy are greatly desired (3, 4). Limiting CVB3 replication within the heart may be a promising strategy to decrease CVB3 pathoge...

MiR‐375‐mediated suppression of engineered coxsackievirus B3 in pancreatic cells

“…The genomic structure of picornaviruses can be divided into a 5′‐UTR, the protein coding region, and a 3′UTR, and previously it was shown that CVB3 with miR‐TS in the 5′‐UTR and in the 3′UTR are susceptible to their corresponding miRs . On the other hand, it has also been shown that certain sections within the 5′‐UTR and 3′UTR do not tolerate miR‐TS insertion, most likely because insertion of miR‐TS disturbed higher‐order RNA structures of the viral genome . In the present study, we pursued a new approach and inserted the miR‐375TS into the 5′ terminus of the polyprotein coding region of the CVB3 genome and compared it to insertion immediately downstream of the polyprotein stop codon in the 3′UTR, which is known to tolerate miR‐TS .…”

“…MiR‐mediated virus detargeting represents a powerful technique to prevent oncolytic viruses from replicating in nontargeted tissues, which makes this technique a valuable approach to increase the safety of this type of cancer therapy . Addressing prevention of undesirable CVB3 replication in the heart by insertion of muscle‐specific miR‐206TS and miR‐133TS has been carried out successfully , whereas attenuation of CVB3 in the pancreas and heart was achieved by insertion of miR‐34aTS into the CVB3 genome .…”

“…Unfortunately, and in agreement with previous studies , we found that miR‐34a was highly expressed in colorectal carcinoma cell lines, suggesting that a CVB3 equipped with miR‐34aTS would probably be detargeted not only in the pancreas and the heart, but also in the colorectal cancer cells, which would negatively impact the outcome of the cancer treatment. As miR for cardiac‐specific attenuation of CVB3 has already been defined and may be easily used in the context of oncolytic CVB3, here we focused on the development of pancreas‐attenuated CVB3. Therefore, CVB3 was equipped with miR‐TS complementary to the miR‐375, which we found is highly expressed in the pancreas but is weakly expressed in colorectal carcinoma cell lines (Fig.…”

“…Previous published studies successfully inserted miR‐TS within the 5′ and 3′UTRs of the viral genome . However, it has also been shown that certain sections within the 5′UTR and the 3′UTR do not tolerate miR‐TS insertion .…”

MicroRNA dysregulation in rhabdomyosarcoma: a new player enters the game

“…Until now, molecular mechanisms of RMS development have still not been fully elucidated . In this regard, identification of crucial biomarkers can improve our understanding of RMS tumour biology and help us discover novel targets for its therapy in clinical settings .…”