A non-coding RNA of insect HzNV-1 virus establishes latent viral infection through microRNA

Abstract

Heliothis zea nudivirus-1 (HzNV-1) is an insect virus previously known as Hz-1 baculovirus. One of its major early genes, hhi1, is responsible for the establishment of productive viral infection; another gene, pag1, which expresses a non-coding RNA, is the only viral transcript detectable during viral latency. Here we showed that this non-coding RNA was further processed into at least two distinct miRNAs, which targeted and degraded hhi1 transcript. This is a result strikingly similar to a recent report that herpes simplex virus produces tightly-regulated latent specific miRNAs to silence its own key early transcripts. Nevertheless, proof for the establishment of viral latency by miRNA is still lacking. We further showed that HzNV-1 latency could be directly induced by pag1-derived miRNAs in cells infected with a pag1-deleted, latency-deficient virus. This result suggests the existence of a novel mechanism, where miRNAs can be functional for the establishment of viral latency.

Figure 1. Interactions of hhi1 and pag1 by northern analysis.

(A) The expression of pag1 transcripts in the productive infected, latent infected, and pKSpP1 transfected cells were probed. (B, C) pKShHI was either co-transfected with pKSpP1 (B), or co-infected with HzNV-1 (C), then subjected to northern analysis using hhi1 probe. Actin signal were used as a loading control.

Figure 2. Mapping of the predicted miRNAs in pag1 transcript.

Schematic drawing of the pag1 transcript to show its 5 predicted miRNAs against hhi1, and 2 miRNAs, hv-miR-246 and hv-miR-2959, proved to be expressed by virus in the infected Sf21 cells.

Figure 3. Cloning and analysis of the predicted miRNA by stem-loop PCR and northern blot.

Stem-loop PCR was performed to clone and analyze the proper expression of the predicted miRNAs in (A) HzNV-1 productively infected cells, (B) pag1-transfected cells, and (C) HzNV-1 latently infected cells. (D, E) Predicted secondary structures of hv-miR-246 5P (D), and hv-miR-2959 5p (E), precursors. (F) Small RNAs harvested from HzNV-1 productively infected cells at various time points were analysis by northern blots with probes against predicted HzNV-1 miRNAs (top panels) or let-7a miRNA as a positive control (bottom panels).

Figure 4. Down-regulation of hhi1 expression by pag1, hv-miR-246, and hv-miR-2959.

(A, C) sequences of hv-miR-2959 and hv-miR-246 were shown, and miRNAs with mutations were denoted as hv-miR-246m and hv-miR-2959m, separately. (B, D) levels of hhi1 transcript in various treatments were analyzed by northern hybridization (left panel) and RT-PCR (right panel).

Figure 5. Establishment of latent viral infection by miRNAs.

(A) Sf21 cells were transfected with or without miRNA followed by HzNV-1 infection. (B) Column representation of the results of panel (A). (C) Confirmation of hhi1 and pag1 expressions in various cells by RT-PCR.

Figure 6. Suppression of HzNV-1 viral latency by knocking down pag1 expression.

(A) RT-PCR showed that artificial siRNA can efficiently suppress pag1 expression in HzNV-1 infected cells. (B) pag1 expression is not detectable by RT-PCR in the pag1-null HzNV-1-infected cells. (C) Formation of latent colony is not observed by the infection of pag1-null HzNV-1.

Figure 7. A comprehensive model for the establishment of productive and latent HzNV-1 viral infections through hhi1 and pag1 interactions.