Selective inhibition of miRNA processing by a herpesvirus-encoded miRNA, 2022, Prusty et al

[Andy]

Abstract

Herpesviruses have mastered host cell modulation and immune evasion to augment productive infection, life-long latency and reactivation1,2. A long appreciated, yet undefined relationship exists between the lytic–latent switch and viral non-coding RNAs3,4.

Here we identify viral microRNA (miRNA)-mediated inhibition of host miRNA processing as a cellular mechanism that human herpesvirus 6A (HHV-6A) exploits to disrupt mitochondrial architecture, evade intrinsic host defences and drive the switch from latent to lytic virus infection. We demonstrate that virus-encoded miR-aU14 selectively inhibits the processing of multiple miR-30 family members by direct interaction with the respective primary (pri)-miRNA hairpin loops. Subsequent loss of miR-30 and activation of the miR-30–p53–DRP1 axis triggers a profound disruption of mitochondrial architecture. This impairs induction of type I interferons and is necessary for both productive infection and virus reactivation. Ectopic expression of miR-aU14 triggered virus reactivation from latency, identifying viral miR-aU14 as a readily druggable master regulator of the herpesvirus lytic–latent switch.

Our results show that miRNA-mediated inhibition of miRNA processing represents a generalized cellular mechanism that can be exploited to selectively target individual members of miRNA families. We anticipate that targeting miR-aU14 will provide new therapeutic options for preventing herpesvirus reactivations in HHV-6-associated disorders.

Paywall, https://www.nature.com/articles/s41586-022-04667-4

 

[Andy]

Link to full article, https://www.nature.com/articles/s41...RXsAWDcJLWWoWkzZUhoY0daRa75DfXbv2URmsEmUneI4=, given by lead author in this tweet

 

[InitialConditions]

ScienceDaily piece: https://www.sciencedaily.com/releases/2022/05/220504110408.htm

 

[SNT Gatchaman]

I think this is an important paper to read through, but it is in Nature Nature and not open-access. It may be that the team have now found this mechanism occurs in other herpesvirus family members, including EBV. I'll attempt some summary quotes by section along with a glossary.

Executive summary —

HHV6A produces its own microRNA (aka mIR) and by so doing interferes with the host cell's production of its own mIR-30 family members. The loss of mIR-30c leads to upregulation of p53 and therefore DRP1, which causes mitrochondrial fission and also consequently downregulates type I interferon.

 

[SNT Gatchaman]

Glossary

DRP1 - dynamin related protein 1, a GTPase
GFP - green fluorescent protein
HHV6A - human herpesvirus type 6A
JAK/STAT - janus kinase / signal transducer and activator of transcription proteins
miRNA - microRNA, aka miR (a capitalized "miR-" refers to the mature form of the miRNA, while the uncapitalized "mir-" refers to the pre-miRNA and the pri-miRNA)
mitoGFP - mitochondrial targetted green fluorescent protein
p53 - protein product of TP53 tumour suppressor gene, pro-apoptotic
pre-mIR - precursor microRNA, second step in processing (post-transcription), following asymmetric cleavage of the hairpin loop
pri-miR - primary microRNA, first step in processing (nuclear transcription), with two limbs connected by a hairpin loop
RIG-I-MAV - retinoic inducible gene I / mitochondrial antiviral signalling protein
TSA - trichostatin-A, histone deacetylase inhibitor, induces latent herpesvirus reactivation

For background on microRNAs see Overview of MicroRNA Biogenesis, Mechanisms of Actions, and Circulation (2018, Frontiers in Endocrinology)

 

[SNT Gatchaman]

Introduction

miRNAs are important regulators of gene expression that are implicated in all major cellular processes of life, ranging from embryonic development to tissue homeostasis and cancer. Accordingly, their biogenesis is tightly regulated at all levels. Shortly after the discovery of cellular miRNAs, a number of viruses, predominantly of the herpesvirus family, were identified to encode and express their own set of viral miRNAs.

Here, we identify miRNA-mediated inhibition of miRNA processing as mechanism that HHV-6A exploits to disrupt mitochondrial architecture, evade the induction of type I interferons and facilitate virus reactivation from latency.

Section - HHV-6 induces mitochondrial fission

To examine whether HHV-6A affects mitochondrial architecture, we infected primary human umbilical vein endothelial cells (HUVEC) with wild-type HHV-6A and imaged mitochondria using a constitutively expressed, mitochondrially targeted GFP (mitoGFP). Lytic HHV-6A infection resulted in extensive mitochondrial fragmentation by 24 hours post-infection.

Mitochondrial fusion–fission dynamics are governed by the activity of dynamin-related protein (DRP1). Helical oligomers of DRP1 form a ring around the outer mitochondrial membrane and fragment it. Mitochondrial fragmentation was reflected by increased DRP1 expression during both lytic HHV-6A infection and virus reactivation, as well as colocalization of DRP1 on mitochondrial surfaces in the virus-reactivated cells.

DRP1 levels are directly controlled at the transcriptional level by the p53 tumor suppressor protein. Accordingly, both lytic HHV-6A infection and virus reactivation resulted in increased p53 expression indicating that HHV-6A induces mitochondrial fragmentation via the canonical p53–DRP1 axis.

 

[SNT Gatchaman]

Section - HHV-6 inhibits miR-30 processing

Human miR-30 family members regulate mitochondrial fusion and fission by targeting p53 and its downstream target DRP1. Northern blots revealed impaired miRNA processing of various miR-30 family members (miR-30a, miR-30c, miR-30d and miR-30e) upon lytic HHV-6A infection. Loss of miR-30c was accompanied by a concomitant increase in pri-miR-30c levels indicating that HHV-6A infection affects pri-miR-30c processing.

Section - miR-aU14 inhibits miR-30 processing

Manual sequence inspection revealed a complementarity between HHV-6A miR-U14 and the hairpin loops of precursor (pre)-miR-30c, pre-miR-30a and pre-miR-30d. This viral miRNA is expressed at very high levels during both productive infection and virus reactivation. Because it is encoded antisense to the U14 open reading frame (ORF), we refer to it here as miR-aU14.

To further validate that miR-aU14 was responsible for the miR-30c processing defect, we generated HeLa cells with a doxycycline (dox)-inducible miR-aU14 expressed from a RNA polymerase III (Pol III) promoter-driven short hairpin RNA (shRNA) and a mutant version thereof (HeLa-Mut).

 

[SNT Gatchaman]

Section - miR-aU14 induces mitochondrial fission

To validate this effect in the virus context, we generated a mutant virus genome with discrete nucleotide substitutions within miR-aU14.

In contrast to HHV-6A-WT, we were unable to reconstitute the miR-aU14 mutant virus despite multiple attempts, indicating that the loss of miR-aU14 severely reduced viral fitness.

Upon virus reactivation with TSA, HHV-6A-WT but not HHV-6A-Mut impaired pri-miR-30c processing, induced DRP1 expression and triggered mitochondrial fission.

 

[SNT Gatchaman]

Section - Mechanism of the miRNA processing defect

We next tested whether the presence of the pre-miR-30c hairpin loop was sufficient to mediate its inhibitory effects on miRNA processing. Two artificial target pre-miRNAs were designed that carried the original hairpin loop sequence of pre-miR-30c but contained artificial miRNA stem duplex sequences (designated miR-A and miR-B).

Consistent with the predicted interaction of miR-aU14 with the pre-miR-30c hairpin loop, induction of miR-aU14, but not of the mutant, strongly repressed both miR-A and miR-B processing.

Section - miR-aU14 inhibits the interferon response

Mitochondria have an important physiological role in intrinsic immunity. Upon activation of toll-like or RIG-I-like receptors, mitochondria serve as antiviral signalling hubs that govern the production of type I interferons (IFNs).

Enforced mitochondrial fission dampens RIG-I–MAVS signalling and reduces the induction of type I IFNs. We thus tested whether miR-aU14-mediated mitochondrial fragmentation affects the induction of the type I IFN, IFNβ.

We next tested whether miR-aU14 also has a role in suppressing the production of IFNβ upon HHV-6A reactivation. In addition to inducing virus reactivation by TSA, we treated cells with the JAK/STAT inhibitor ruxolitinib to prevent secondary IFNβ-mediated effects on virus reactivation.

Ruxolitinib treatment enhanced TSA-induced virus reactivation, resulting in a concordantly greater loss of miR-30c.

 

[SNT Gatchaman]

Section - miR-aU14 triggers virus reactivation

Considering the observed effects of miR-aU14 on the induction of type I IFNs, we investigated whether ectopic expression of miR-aU14 could augment productive wild-type virus infection and rescue reactivation of the mutant virus.

Transfection of miR-aU14 mimic efficiently rescued reactivation of the mutant virus even in the absence of TSA. Combination of both TSA and miR-aU14 showed enhanced virus reactivation, indicating synergistic effects between the two.

We then tested whether mitochondrial fragmentation, impaired IFN response and HHV-6A reactivation were indeed mediated by the effects of miR-aU14 on miR-30. Both transfection of a miR-30c inhibitor and expression of a miR-30c sponge decreased mature miR-30c levels, induced p53 and DRP1 expression and triggered mitochondrial fragmentation.

Section - Targeting human miRNA processing

In principle, miRNA-mediated inhibition of miRNA processing should be applicable to other cellular miRNAs. This is of particular interest, as many important cellular miRNAs exist as miRNA families.

Many of the let-7 family members carry relatively large hairpin loops, which may comprise up to 30 nucleotides. Hence, we designed synthetic miRNA mimics targeting two different regions of the hairpin loop of pre-let-7d. Upon transfection into cells, both miRNA mimics efficiently reduced mature let-7d levels, consistent with impaired miRNA processing.

 

[SNT Gatchaman]

Discussion

RNA-binding protein-mediated regulation of miRNA processing thus constitutes an important regulatory network that shapes miRNA activity and function. Here we show that miRNA mimics can take over similar functions and selectively inhibit miRNA processing in a sequence-specific manner

The miR-aU14-mediated loss of miR-30c was accompanied by a marked increase of pri-miR-30c levels. This implies that the inhibition occurs at the level of pri-miRNA processing within the nucleus, consistent with previous reports that miRNAs may affect pri-miRNA processing by binding to distal sequence elements in the respective pri-miRNAs.

Notably, both lytic HHV-6A infection and virus reactivation also increased pre-miR-30d levels within the cell. This is consistent with data from our in vitro processing assay, which indicated inhibition of miR-30 processing at the pre-miRNA level.

Viral miR-aU14-mediated inhibition of miR-30 processing explained mitochondrial fragmentation during both lytic HHV-6A infection and virus reactivation via the miR-30–p53–DRP1 axis. This in turn impairs the induction of type I IFN and augments productive virus infection.

The most notable finding, however, was that transfection of miR-aU14 triggered virus reactivation from latency at least as efficiently as the commonly employed histone deacetylase inhibitor TSA.

In summary, our findings reveal a miRNA-mediated mechanism that a prevalent human herpesvirus has hijacked to interfere with intrinsic immunity, govern the lytic–latent switch and augment productive infection.