HSV-1 miRNAs are post-transcriptionally edited in latently infected human ganglia
Authors: Zubković Andreja , Gomes Cristina , Parchure Adwait , Cesarec Mia , Ferenčić Antun , Rokić Filip , Jakovac Hrvoje , Whitford Abigail L. , Dochnal Sara A. , Cliffe Anna R. , Cuculić Dražen , Gallo Angela , Vugrek Oliver , Hackenberg Michael , Jurak Igor 
Published: September 2023

ABSTRACT
Viruses use miRNAs to enable efficient replication, control host defense mechanisms, and regulate latent infection. Herpes simplex virus 1 (HSV-1) expresses multiple miRNAs whose functions are largely unknown. The evolutionary conservation of many HSV-1 miRNAs in the closely related HSV-2 suggests their functional importance. miRNAs, similar to other transcripts, can undergo various post-transcriptional modifications that may affect their biogenesis, stability, and targeting. To investigate whether editing occurs in HSV-1 miRNAs, we sequenced samples from latently infected human ganglia. We show that one of the six HSV-1 miRNAs (miR-H2 to miR-H8) that define HSV-1 latency, miR-H2, exhibits adenosine-to-inosine hyperediting within the miRNA seed sequence. We observed the same specific miR-H2 hyperediting phenomenon in miRNAs isolated from the ganglia of latently infected mice and, to a lesser extent, during productive infection in cultured cells. Curiously, we found no evidence of editing of the encoded HSV-2 homolog in latently infected mice or in cultured cells. The efficient loading of the edited miRNAs onto the RNA-inducing silencing complex indicates their ability to function as miRNAs. To investigate the potential of the edited miRNA to alter mRNA targeting, we predicted the host and viral targets for the modified miRNAs. Nucleotide substitution in the seed region significantly increased the number of potential host and viral targets. Most notably, the mRNA that encodes ICP4, an essential viral protein, was predicted to be an additional target. Using transfection assays, we demonstrated that edited miRNAs have the potential to reduce ICP4 steady-state protein levels in addition to ICP0. Our study identifies a specific hyperedited HSV-1 mRNA, miR-H2, and highlights how the virus can use a single miRNA to target multiple transcripts during persistent, latent infection.

IMPORTANCE
Herpes simplex virus 1 is an important human pathogen that has been intensively studied for many decades. Nevertheless, the molecular mechanisms regulating its establishment, maintenance, and reactivation from latency are poorly understood. Here, we show that HSV-1-encoded miR-H2 is post-transcriptionally edited in latently infected human tissues. Hyperediting of viral miRNAs increases the targeting potential of these miRNAs and may play an important role in regulating latency. We show that the edited miR-H2 can target ICP4, an essential viral protein. Interestingly, we found no evidence of hyperediting of its homolog, miR-H2, which is expressed by the closely related virus HSV-2. The discovery of post-translational modifications of viral miRNA in the latency phase suggests that these processes may also be important for other non-coding viral RNA in the latency phase, including the intron LAT, which in turn may be crucial for understanding the biology of this virus.