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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.
Many viruses globally dampen cellular gene expression as a way to reduce activation of immune pathways and reallocate resources. This host shutoff phenotype is enacted by a variety of mechanisms, but select viruses from at least three different viral subfamilies, alphaherpesviruses, gammaherpesviruses, and betacoronaviruses, block host gene expression by promoting global mRNA degradation (2, 6, 11, 13, 19). Although they all target a similar stage of gene expression and exhibit several mechanistic similarities, the viral factors that cause mRNA degradation are unrelated. RNA turnover in alphaherpesviruses, such as herpes simplex virus 1 (HSV-1), is induced by vhs (14, 18), a nuclease of the FEN1 family (3). In contrast, gammaherpesviruses use their alkaline exonuclease homolog, a member of the PD-(D/E)XK restriction endonuclease superfamily, termed SOX in Kaposi's sarcoma-associated herpesvirus (KSHV) (6), BGLF5 in Epstein-Barr virus (EBV) (19), and muSOX in murine herpesvirus 68 (MHV68) (2). mRNA degradation induced by the betacoronavirus severe acute respiratory syndrome (SARS) coronavirus (SCoV) is carried out by nsp1, a protein with no known similarity to cellular or viral nucleases (11). Although host mRNA degradation has not directly been tested during infection with other betacoronaviruses, the nsp1 proteins of some bat betacoronaviruses can also cause mRNA degradation (21).
Interestingly, each of these proteins has been described through studies in vitro or in cells to be active in and/or localized to translation complexes (1, 5, 10, 20), suggesting that they may have evolved similar strategies to target mRNA. Moreover, each triggers mRNA degradation by a primary endonucleolytic cleavage (1, 4, 10). In the case of KSHV SOX, this enables recruitment of the cellular Xrn1 5′-to-3′ exonuclease, which then completes degradation of the cleaved intermediates (1). Although a similar involvement of host enzymes like Xrn1 has been proposed for the activity of vhs, nsp1, BGLF5, and muSOX, roles for cellular nucleases in host shutoff by these factors remain untested. Additionally, some of these factors have been characterized in vitro, but less is known about their specificity and activity in cells. Here, we sought to directly compare the activities of these distinct viral proteins in cells with regard to their selectivity for mRNA targeting, their translational requirements for cleavage, and their engagement of the host Xrn1 nuclease to complete mRNA degradation. Our results indicate that there exists a remarkable degree of conservation in the overall mechanism of mRNA degradation induced by these unrelated proteins, although there are some key differences in their initial recruitment and primary cleavage events.
We evaluated the specificity of RNA targeting by each of these viral factors in cells by first comparing their abilities to degrade green fluorescent protein (GFP) RNA reporters transcribed by RNA polymerase (Pol) I, II, and III. RNAs transcribed by Pol I and Pol III complexes lack a 7-methylated 5′ cap and 3′ poly(A) tail and are thus translated very inefficiently, if at all (7, 15). Indeed, we have not been able to detect any protein production with our Pol I and Pol III reporter constructs (data not shown). HEK293T cells were transfected with plasmids expressing KSHV SOX (pCDEF3-SOX ), MHV68 muSOX (pCDEF3-HA-muSOX ), EBV BGLF5 (pCDEF3-HA-BGLF5 ), HSV-1 vhs (pCDNA3.1-vhs ), and SCoV nsp1 (pCAGGS-nsp1 ) together with the indicated GFP reporters (described in reference 1). Total RNA was extracted approximately 24 h after transfection and examined by a Northern blot using a 32P-labeled GFP DNA probe. None of the shutoff factors could direct degradation of the Pol I- and Pol III-driven transcripts (Fig. 1A and andB),B), indicating that nontranslatable RNAs are not targets for these proteins within cells. In contrast, the cotransfected Pol II-driven GFP was readily degraded by all the viral factors. These data confirm a link between the translational competence of RNA and its ability to be targeted in cells by each host shutoff factor.
We also sought to examine whether mRNA targeting occurred before or after ribosome recruitment. In reticulocyte lysates, nsp1 has been shown to bind directly to the 40S ribosomal subunit to mediate mRNA cleavage (10), whereas ribosomes are dispensable for vhs activity (4). It is not known whether the other viral factors target mRNAs before or after ribosome engagement in cells. To test this, we cloned a strong hairpin (hp7) (12) directly adjacent to the 5′ cap (3 nucleotides [nt] downstream of the predicted transcription start site) of the pd2-eGFP-N1 reporter mRNA (hp-GFP). Placement of hairpins close to the cap has been reported to block ribosome 40S subunit binding to RNAs (12), and we found that the hairpin completely blocked translation from the hp-GFP mRNA (Fig. 2B). Surprisingly, all of the host shutoff factors except nsp1 were still able to degrade hp-GFP (Fig. 2A). This suggests that the host shutoff factors from the alpha- and gammaherpesviruses are recruited to mRNAs in a ribosome-independent manner. For vhs, this may occur via its reported interactions with the initiation factors eIF4H and eIF4A (5, 16). In contrast, nsp1 is likely recruited to RNA through its interactions with the 40S subunit (10). Although it is possible that the hairpin location masks the region of the mRNA in which cleavage occurs in nsp1-expressing cells, we disfavor this hypothesis because nsp1 has been shown to promote cleavage within secondary structures (8). We also considered the possibility that the hairpin does not inhibit nsp1-induced cleavage and instead blocks subsequent 5′-to-3′ exonucleolytic degradation of the message by Xrn1 (see below). However, RNA degradation was reinstated when the same hairpin was moved 23 nt downstream of the predicted transcription start site to block ribosome scanning but not binding (12) (hp23-GFP) (Fig. 2C), indicating that the hairpin structure itself does not impede degradation. We confirmed that GFP protein production was blocked as predicted by hp23 (data not shown).
Finally, we evaluated whether, similar to KSHV SOX, the other host shutoff factors also engage the cellular Xrn1 nuclease to complete degradation of the endonucleolytically cleaved mRNAs. We first tested this using a GFP construct containing a flavivirus-derived Xrn1 blocking element (SLII) (17) in its 3′ untranslated region (UTR) (Fig. 3A). This construct was used previously to demonstrate that Xrn1 participates in the degradation of cleaved mRNA fragments in KSHV SOX-expressing cells (1). After cotransfection of HEK293T cells with the GFP-3′SLII reporter and each host shutoff factor, total RNA was extracted and Northern blotted with a 32P-labled probe against the GFP 3′ untranslated region (UTR). Sequences following the SLII should be protected specifically in cases in which Xrn1 activity is required to complete degradation of the cleaved mRNA. As shown in Fig. 3B, a protected fragment was visible in cells expressing each of the host shutoff factors, indicating a role for Xrn1 in exonucleolytic degradation of the mRNA body after the primary endonucleolytic cleavage. To confirm this observation, we examined degradation of two different reporter mRNAs by each viral protein in cells subjected to small interfering RNA (siRNA)-mediated depletion of Xrn1. HEK293T cells were transfected twice with the indicated siRNAs, as described previously (1), and then, 48 h later, with constructs expressing each viral factor and either a shortened version of GFP (sGFP; used to achieve sufficient resolution of the degradation intermediates) or DsRed2. Under these conditions, we routinely observe 60 to 80% knockdown of the Xrn1 protein (data not shown). Degradation of sGFP and DsRed2 was visualized by a Northern blot of total RNA with 32P-labeled probes directed against the 3′ UTR of each reporter. In agreement with the GFP-3′SLII results, depletion of Xrn1 altered the mRNA degradation profile in each of the samples containing the host shutoff factors (Fig. 4A and andB).B). The degradation profile followed one of two patterns upon Xrn1 depletion: either there was stabilization of near-full-length mRNA or there was an accumulation of degradation intermediates of differing sizes, perhaps depending on the location of the primary cleavage. The first scenario was observed in cells expressing nsp1 and muSOX, and we hypothesize that this occurs as a result of the primary endonucleolytic cleavage being close to the 5′ end of the mRNA, consistent with previous reports of nsp1 activity in cell lysates (8). The second scenario was seen in cells expressing SOX, BGLF5, and vhs. It is interesting and unexpected that SOX, muSOX, and BGLF5 produced different degradation patterns given that these proteins are homologous (muSOX is 40% identical to both SOX and BGLF5). This suggests that even among highly related proteins, distinct sequences or elements may direct cleavage.
In conclusion, a comparison of these host shutoff factors shows that the overall modes of action are remarkably similar but that the specific molecular mechanisms bear significant differences (Fig. 4C). All factors selectively target RNAs that are transcribed by RNA polymerase II but likely employ different strategies to identify these RNAs. SCoV nsp1 uses the 40S ribosome subunit to access the mRNAs, whereas the other viral proteins act prior to ribosome binding. Importantly, we demonstrate for the first time that all the host shutoff factors are dependent on the host mRNA degradation machinery, requiring Xrn1 in particular to complete mRNA degradation. However, the specific cleavages that trigger mRNA degradation differ among even the closely related gammaherpesvirus homologs. Thus, these viruses have hijacked the same process of endonuclease-triggered mRNA destruction in at least three separate biochemical ways. It is possible that this strategy is advantageous because it is rapid, resembles some host pathways, and is not subject to the conventional regulation of mRNA degradation via deadenylation and decapping.
We thank Shinji Makino for the SCoV nsp1 expression construct, James Smiley for the HSV-1 vhs expression construct, and Jon Portman for assistance with cloning the hp-GFP construct. We also thank the Glaunsinger lab for critical reading of the manuscript.
This research was supported by NIH R01 CA160556 and a W.M. Keck Foundation Distinguished Young Scholar award to B.A.G.
Published ahead of print 27 June 2012