Conserved Herpesvirus Kinase ORF36 Activates B2 Retrotransposons during Murine Gammaherpesvirus Infection

Abstract

Short interspersed nuclear elements (SINEs) are RNA polymerase III (RNAPIII)-transcribed, retrotransposable noncoding RNA (ncRNA) elements ubiquitously spread throughout mammalian genomes. While normally silenced in healthy somatic tissue, SINEs can be induced during infection with DNA viruses, including the model murine gammaherpesvirus 68 (MHV68). Here, we explored the mechanisms underlying MHV68 activation of SINE ncRNAs. We demonstrate that lytic MHV68 infection of B cells, macrophages, and fibroblasts leads to robust activation of the B2 family of SINEs in a cell-autonomous manner. B2 ncRNA induction requires neither host innate immune signaling factors nor involvement of the RNAPIII master regulator Maf1. However, we identified MHV68 ORF36, the conserved herpesviral kinase, as playing a key role in B2 induction during lytic infection. SINE activation is linked to ORF36 kinase activity and can also be induced by inhibition of histone deacetylases 1 and 2 (HCAC 1/2), which is one of the known ORF36 functions. Collectively, our data suggest that ORF36-mediated changes in chromatin modification contribute to B2 activation during MHV68 infection and that this activity is conserved in other herpesviral protein kinase homologs.IMPORTANCE Viral infection dramatically changes the levels of many types of RNA in a cell. In particular, certain oncogenic viruses activate expression of repetitive genes called retrotransposons, which are normally silenced due to their ability to copy and spread throughout the genome. Here, we established that infection with the gammaherpesvirus MHV68 leads to a dramatic induction of a class of noncoding retrotransposons called B2 SINEs in multiple cell types. We then explored how MHV68 activates B2 SINEs, revealing a role for the conserved herpesviral protein kinase ORF36. Both ORF36 kinase-dependent and kinase-independent functions contribute to B2 induction, perhaps through ORF36 targeting of proteins involved in controlling the accessibility of chromatin surrounding SINE loci. Understanding the features underlying induction of these elements following MHV68 infection should provide insight into core elements of SINE regulation, as well as disregulation of SINE elements associated with disease.

Keywords: B2; MHV68; RNA polymerase III; SINE; herpesvirus; noncoding RNA.

FIG 1

B2 SINE transcription is upregulated in B cells, primary macrophages, and NIH 3T3 cells upon MHV68 infection. (A) MHV68 latently infected A20-HE-RIT B cells, or parental uninfected A20 B cells, were treated with doxycycline (dox) and phorbol ester (PMA) to induce lytic reactivation. Total RNA was isolated at the indicated time points postreactivation and subjected to primer extension for B2 SINEs or 7SK for loading. (B) RT-qPCR was performed to detect the indicated RNAPIII transcript levels using RNA extracted from mock or reactivated A20-HE-RITs. (C) A20-HE-RITs were reactivated in the presence or absence of phosphonoacetic acid (PAA) to block viral DNA replication, and total RNA was subjected to primer extension as described above. (D) BMDMs or (E) NIH 3T3 were either mock infected or infected with MHV68 for the indicated time periods, whereupon total RNA was isolated and subjected to primer extension as described above.

FIG 2

Paracrine signaling does not induce B2 SINE induction. (A) NIH 3T3 cells or (B) primary BMDMs were incubated with supernatants harvested from 24-h-infected NIH 3T3 cells, either in crude form or filtered to remove whole virus, for the indicated time period. Total RNA was isolated from cells at 24 h or 48 h postincubation and subjected to primer extension for B2 SINEs or 7SK, respectively.

FIG 3

B2 SINE upregulation is dependent on RNAPIII but independent of the RNAPIII master regulator Maf1. (A) BMDMs were transfected with the indicated concentrations of either control or Brf1 siRNA pools and harvested 24 to 48 h later. Then, 30 μg of total protein lysates was resolved by SDS-PAGE and Western blotting with antibodies against Brf1 or GAPDH (as a loading control). (B) Total RNA was harvested from mock- or MHV68-infected BMDMs and NIH 3T3 fibroblasts following control or Brf1 siRNA treatment at the indicated time points. Total RNA was subjected to primer extension using primers for B2 SINEs or 7SK (as a control). (C) WT or (D) Maf1^–/– BMDMs were mock infected or infected with MHV68 for the indicated times, whereupon total RNA was harvested and subjected to primer extension as described in panel B.

FIG 4

B2 SINE induction occurs independently of innate immune signaling. (A to C) WT or the indicated innate immune factor knockout BMDMs were mock or MHV68 infected for 24 to 48 h. Total RNA was then harvested and subjected to primer extension using primers for B2 SINEs or 7SK (as a control).

FIG 5

The MHV68 kinase ORF36 induces B2 SINE transcription. (A) Schematic representing the method for testing the MHV68 ORF library. (B) NIH 3T3 cells were transfected with plasmid(s) containing the indicated ORF(s) or a GFP control for 24 h, whereupon total RNA was extracted and subjected to primer extension using primers for B2 SINEs or 7SK (as a control). (C) NIH 3T3 cells were transfected with plasmids expressing either wild-type (WT) ORF36 or a kinase-null mutant (K107Q) for 24 h, and then total RNA was isolated and subjected to primer extension as described above. (D) BMDMs were infected with WT MHV68, kinase-null (KN), or ORF36 stop (S) virus. Total RNA was isolated at 48 hpi and subjected to primer extension as described in panel B. (E) NIH 3T3 cells were infected with WT MHV68, KN, or S virus at an MOI of 5. At 24 hpi, total RNA was isolated and subjected to primer extension as described in panel B.

FIG 6

Functional conservation of B2 SINE upregulation by several MHV68 ORF36 homologs. NIH 3T3 cells were transfected with plasmids containing FLAG-tagged MHV68 ORF36 or the indicated HA-tagged ORF36 homolog from Kaposi’s sarcoma-associated herpesvirus (KSHV ORF36), Epstein-Barr virus (EBV BGLF4), varicella zoster virus (VZV ORF47), or human cytomegalovirus (HCMV UL97). These cells were then harvested for total RNA for B2 and 7SK primer extension (A) or protein lysates, which were analyzed with Western blotting with antibodies against HA and FLAG, or GAPDH as a loading control (B). The dashed line indicates where an irrelevant lane was removed from the image.

FIG 7

Inhibitors of chromatin repression cause B2 SINE upregulation. (A) NIH 3T3 cells were treated with the indicated inhibitor(s) for 24 h, whereupon total RNA was isolated and subjected to primer extension for B2 SINEs or 7SK. (B) NIH 3T3 cells were subjected to pretreatment with DMSO or the indicated inhibitors for 1 h prior to infection with MHV68 WT, KN, or S virus for 24 h, whereupon total RNA was isolated and subjected to primer extension as described in panel A.