No abstract

Infection with gammaherpesviruses, alphaherpesviruses, and betacoronaviruses can result in widespread mRNA degradation, in each case initiated predominantly by a single viral factor. Although not homologous, these factors exhibit significant mechanistic similarities. In cells, each targets translatable RNAs for cleavage and requires host Xrn1 to complete RNA degradation, although the mechanism of targeting and the position of the primary cleavage differ. Thus, multiple host shutoff factors have converged upon a common mRNA degradation pathway.

Several viruses encode factors that promote host mRNA degradation to silence gene expression. It is unclear, however, whether cellular mRNA turnover pathways are engaged to assist in this process. In Kaposi's sarcoma-associated herpesvirus this phenotype is enacted by the host shutoff factor SOX. Here we show that SOX-induced mRNA turnover is a two-step process, in which mRNAs are first cleaved internally by SOX itself then degraded by the cellular exonuclease Xrn1. SOX therefore bypasses the regulatory steps of deadenylation and decapping normally required for Xrn1 activation. SOX is likely recruited to translating mRNAs, as it cosediments with translation initiation complexes and depletes polysomes. Cleaved mRNA intermediates accumulate in the 40S fraction, indicating that recognition occurs at an early stage of translation. This is the first example of a viral protein commandeering cellular mRNA turnover pathways to destroy host mRNAs, and suggests that Xrn1 is poised to deplete messages undergoing translation in mammalian cells.

Lytic infection with the two human gammaherpesviruses, Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV), leads to significant depletion of the cellular transcriptome. This host shutoff phenotype is driven by the conserved herpesviral alkaline exonuclease, termed SOX in KSHV and BGLF5 in EBV, which in gammaherpesviruses has evolved the genetically separable ability to target cellular mRNA. We now show that host shutoff is also a prominent consequence of murine gammaherpesvirus 68 (MHV68) infection, which is widely used as a model system to study pathogenesis of these viruses in vivo. The effector of MHV68-induced host shutoff is its SOX homolog, here termed muSOX. There is remarkable functional conservation of muSOX host shutoff activities with those of KSHV SOX, including the recently described ability of SOX to induce mRNA hyperadenylation in the nucleus as well as cause nuclear relocalization of the poly(A) binding protein. SOX and muSOX localize to both the nucleus and cytoplasm of infected cells. Using spatially restricted variants of these proteins, we go on to demonstrate that all known host shutoff-related activities of SOX and muSOX are orchestrated exclusively from the cytoplasm. These results have important mechanistic implications for how SOX and muSOX target nascent cellular transcripts in the nucleus. Furthermore, our findings establish MHV68 as a new, genetically tractable model to study host shutoff.Efficient viral replication and immune evasion often require manipulation of cellular gene expression either in a targeted or in a global manner. Widespread inhibition of host gene expression, termed host shutoff, is a conserved feature of a diverse set of viruses, including picornaviruses, coronaviruses, orthomyxoviruses, and herpesviruses (3,13,23,32,37). Host shutoff likely confers a selective advantage to these viruses, perhaps by facilitating redirection of cellular resources toward the virus or by dampening immune stimulatory signals that could restrict viral replication. Within the herpesvirus family, both alphaherpesviruses (alpha-HVs) and gammaherpesviruses (gammaHVs) induce a prominent host shutoff phenotype (13). Interestingly, while both subfamilies potently decrease the population of cellular mRNA, the viral host shutoff factors and their mechanisms of action are distinct. Herpes simplex viruses (HSV), of the alpha subfamily, encode an RNase termed vhs that degrades cytoplasmic mRNA (6,7,26,45,53) as well as a second factor, ICP27, which inhibits splicing of cellular messages later in infection (16,17,36,42). Kaposi's sarcomaassociated herpesvirus (KSHV) and Epstein-Barr virus (EBV), two oncogenic human viruses of the gamma subfamily, target cellular mRNA via the activity of the viral alkaline exonuclease (DNase), termed SOX in KSHV and BGLF5 in EBV (12,41). In contrast to vhs, SOX and BGLF5 lack in vitro RNase activity and are presumed to instead promote host shutoff by activating cellular mRNA turnover pathways. Thus, different members of the herpesvirus family...

SUMMARY Developing strategies that promote the resolution of vascular inflammation and atherosclerosis remains a major therapeutic challenge. Here, we show that exosomes produced by naive bone marrow-derived macrophages (BMDM-exo) contain anti-inflammatory microRNA-99a/146b/378a that are further increased in exosomes produced by BMDM polarized with IL-4 (BMDM-IL-4-exo). These exosomal microRNAs suppress inflammation by targeting NF-κB and TNF-α signaling and foster M2 polarization in recipient macrophages. Repeated infusions of BMDM-IL-4-exo into Apoe −/− mice fed a Western diet reduce excessive hematopoiesis in the bone marrow and thereby the number of myeloid cells in the circulation and macrophages in aortic root lesions. This also leads to a reduction in necrotic lesion areas that collectively stabilize atheroma. Thus, BMDM-IL-4-exo may represent a useful therapeutic approach for atherosclerosis and other inflammatory disorders by targeting NF-κB and TNF-α via microRNA cargo delivery.