Regulation of KSHV lytic switch protein expression by a virus-encoded microRNA: an evolutionary adaptation that fine-tunes lytic reactivation

Abstract

Herpesviruses encode numerous microRNAs (miRNAs), most of whose functions are unknown. The Kaposi's sarcoma-associated herpesvirus (KSHV) encodes 17 known miRNAs as part of its latency program, suggesting that these RNAs might function to regulate the latent state. Here we show that one of these KSHV miRNAs, miRK9( *), targets a sequence in the 3' untranslated region (UTR) of the mRNA encoding the major lytic switch protein (RTA), which controls viral reactivation from latency. Ectopic expression of miRK9( *) impairs RTA synthesis, while its specific antagonism in latently infected cells enhances spontaneous lytic reactivation frequency by 2- to 3-fold. Mutation of the recognition sequence in the RTA 3'UTR abolishes RTA downregulation by miRK9( *). We propose that miRNA targeting of RTA, while not the primary regulator of the lytic switch, functions like a safety mechanism on the trigger of lytic reactivation, preventing stochastic variations in basal RTA transcription from activating inappropriate entry into the lytic cycle.

Figure 1. miR-K9* targets the RTA 3′ UTR and inhibits RTA function

A. HEK293 cells were cotransfected with a psiCHECK2 vector in which the 3′UTR of RTA was cloned downstream of the Renilla luciferase gene, and individual KSHV-encoded microRNAs (indicated along the X-axis). The Renilla/Firefly ratio was normalized to a vector that did not contain the 3′UTR and to a negative control microRNA. Bars depict the mean values of the normalized luciferase activities from triplicate independent transfections. The difference between the negative control and miRK9* samples is highly significant (p= 0.003 by t-test). B. HFF cells latently infected with rKSHV.219 were transfected with individual inhibitors to individual KSHV-encoded microRNAs (indicated along the X-axis). 48 hours later, spontaneous lytic reactivation was measured as a % of RFP-positive cells from a population of healthy, GFP-expressing (infected) cells. Mean values of the normalized % RFP-positive cells were from five independent transfections for miRK9* and negative control inhibitors and from two independent transfections for the other microRNA inhibitors. The P-value between the negative control and miRK9* samples is 0.0013 as determined by T-test.

Fig 2. Inhibition of miRK9* during lytic growth raises levels of RTA

HFF/rKSHV.219 cells were transfected with the indicated antagomir, then induced with butyrate. Extracts prepared at the indicated times post-induction were immunoblotted for RTA (and for β-actin as a loading control).

Figure 3. miR-K9* inhibits the expression of RTA protein in a transient transfection assay

A genomic RTA expression vector bearing the full 3′UTR sequences was cotransfected with the indicated individual KSHV-encoded microRNAs; 48 hrs later, lysates were prepared and tested for RTA protein by immunoblotting. As a loading control, the same blots were probed with antibodies against β-actin.

Figure 4. Mapping the target site for miR-K9* within the 3′UTR of RTA

A. Predicted base pairing between miR-K9* and its putative site within the 3′UTR of RTA is shown. Watson-Crick base pairings are shown as vertical lines. Mutations made in the 3′UTR and miR-K9* are indicated above or below the wildtype sequences, respectively. B. HEK293 cells were cotransfected with negative control 1 (C1), negative control 2 (C2), miR-K9* (K9*) or mutated miR-K9* (K9*m) microRNA together with the luciferase reporter plasmid containing the wildtype (W) or mutated (M) 3′UTR of RTA. The Renilla/Firefly ratio was normalized to a vector that did not contain the 3′UTR and to a negative control microRNA. Mean values of the normalized luciferase activities are from triplicate independent transfections. P-values taken from a t-test between C1 and K9* transfected with wildtype 3′UTR is 0.019 and between C1 and K9*m transfected with mutated 3′UTR is 0.014, reflecting statistically significant values. C. Top: Schematic physical map of the WT and 3′Δ deletion mutant of KSHV RTA region. Bottom: HEK293 cells were cotransfected with plasmids encoding WT or 3′Δ mutant transcript of RTA and with negative control or miR-K9* (K9*) microRNA. 48 hours later cell lysates were probed for RTA protein and for β-actin as a loading control.