Deep Sequencing Reveals Direct Targets of Gammaherpesvirus-Induced mRNA Decay and Suggests That Multiple Mechanisms Govern Cellular Transcript Escape

Abstract

One characteristic of lytic infection with gammaherpesviruses, including Kaposi's sarcoma-associated herpesvirus (KSHV), Epstein-Barr virus (EBV) and murine herpesvirus 68 (MHV68), is the dramatic suppression of cellular gene expression in a process known as host shutoff. The alkaline exonuclease proteins (KSHV SOX, MHV-68 muSOX and EBV BGLF5) have been shown to induce shutoff by destabilizing cellular mRNAs. Here we extend previous analyses of cellular mRNA abundance during lytic infection to characterize the effects of SOX and muSOX, in the absence of other viral genes, utilizing deep sequencing technology (RNA-seq). Consistent with previous observations during lytic infection, the majority of transcripts are downregulated in cells expressing either SOX or muSOX, with muSOX acting as a more potent shutoff factor than SOX. Moreover, most cellular messages fall into the same expression class in both SOX- and muSOX-expressing cells, indicating that both factors target similar pools of mRNAs. More abundant mRNAs are more efficiently downregulated, suggesting a concentration effect in transcript targeting. However, even among highly expressed genes there are mRNAs that escape host shutoff. Further characterization of select escapees reveals multiple mechanisms by which cellular genes can evade downregulation. While some mRNAs are directly refractory to SOX, the steady state levels of others remain unchanged, presumably as a consequence of downstream effects on mRNA biogenesis. Collectively, these studies lay the framework for dissecting the mechanisms underlying the susceptibility of mRNA to destruction during lytic gammaherpesvirus infection.

Silencing the shutoff protein of Epstein–Barr virus in productively infected B cells points to (innate) targets for immune evasion

“…2). These findings are in agreement with promiscuous shutoff by c-herpesvirus AE proteins, deduced from mRNA target analysis (Clyde & Glaunsinger, 2011) and metabolic labelling experiments (Rowe et al, 2007). Still, some gene products escape shutoff and TLR4 appears to be one of them (Fig.…”

“…AE proteins are conserved throughout the herpesvirus family, reflecting their critical DNase function in processing of newly synthesized viral genomes; their additional RNase-based shutoff function is unique to c-herpesviruses (Glaunsinger & Ganem, 2004;Rowe et al, 2007). Shutoff appears broadly active and affects expression of most cellular proteins (Clyde & Glaunsinger, 2011). As such, herpesvirus-induced shutoff provides a general strategy to dampen antiviral immune activation.…”

The “Bridge” in the Epstein-Barr Virus Alkaline Exonuclease Protein BGLF5 Contributes to Shutoff Activity during Productive Infection

“…HSV vhs not only degrades cellular mRNAs but also viral mRNAs, facilitating the transition between the immediate early, early, and late phases of infection (33,37,38,41). Some selectivity is likely, as a small subset of cellular transcripts, including interleukin-6, escapes KSHV SOX-induced degradation (6,7,19).…”

Nuclease escape elements protect messenger RNA against cleavage by multiple viral endonucleases

“…Based on our previous finding that the IL-6 SRE required binding by a complex of cellular proteins including NCL, we mined a published RNAseq dataset for transcripts that were not depleted in SOX expressing 293T cells and were known to be bound by NCL [22,[49][50][51]. A transcript that fit these criteria was growth arrest and DNA damage-inducible 45 beta (GADD45B).…”

“…Studying these 'escapees' in aggregate is complicated, however, by the fact that multiple mechanisms can promote apparent escape. These include lack of a targeting motif, indirect transcriptional effects, and active evasion of cleavage [22,[24][25][26][27][28]. This latter phenotype, termed dominant escape, is particularly notable as it involves a specific RNA element whose presence in the 3' UTR of an mRNA protects against SOX cleavage, regardless of whether the RNA contains a targeting motif.…”