A common strategy for host RNA degradation by divergent viruses

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.

Fig 1

Only Pol II-transcribed RNAs are degraded by each of the host shutoff factors. (A and B) HEK293T cells were transfected with 200 ng/ml Pol II-driven GFP together with either 300 ng/ml of Pol III-GFP (A) or 400 ng/ml of Pol I-GFP (B) constructs. In addition, cells were transfected with an empty vector (pCDEF3) bearing a Pol II promoter [(−)] or with increasing amounts of SOX (200 to 400 ng/ml), muSOX (200 to 300 ng/ml), BGLF5 (200 to 400 ng/ml), vhs (100 to 200 ng/ml), or nsp1 (200 to 300 ng/ml) and a corresponding amount of the empty vector to bring the total concentration of plasmid per transfection to 1 μg/ml. Reporter RNA levels were visualized by a Northern blot with probes against GFP. 18S was used as a loading control. Blots shown are representative of three independent repeats.

Fig 2

Binding of the 40S ribosomal subunit is selectively required for nsp1-triggered mRNA degradation. (A) HEK293T cells were transfected with 200 ng/ml of a control GFP reporter with an elongated 5′ UTR, 200 ng/ml of a GFP construct bearing a strong hairpin (hp-GFP; folding Gibbs free energy [ΔG] = −61 kcal/mol) approximately three nucleotides 3′ of the cap, and increasing amounts of the indicated host shutoff factors as described for Fig. 1 and/or empty vector [(−)] to a final total concentration of 1 μg/ml for each transfection. Reporter mRNA levels were visualized by a Northern blot with probes against GFP. 18S was used as a loading control. The blot shown is representative of three independent repeats. (B) HEK293T cells were transfected with increasing amounts (100, 200, and 400 ng/ml) of either a control GFP reporter or hp-GFP. The levels of GFP were assessed by a Western blot, and actin served as a loading control. (C) HEK293T cells were transfected with 200 ng/ml of a control GFP reporter or GFP constructs bearing a hairpin 3 nucleotides (hp-GFP) or 23 nucleotides (hp23-GFP) 3′ of the cap together with 800 ng/ml of empty vector alone or 600 ng/ml empty vector and 200 ng/ml of the nsp1-expressing construct. Reporter mRNA levels were visualized by a Northern blot with probes against GFP. 18S was used as a loading control.

Fig 3

Each host shutoff factor requires Xrn1 for completion of mRNA degradation. (A) Diagram of the GFP-3′SLII construct and the role of the flaviviral SLII element in blocking the cellular exonuclease Xrn1. (B) HEK293T cells were transfected with 100 ng/ml of the GFP-3′SLII construct, plasmids expressing the indicated host shutoff factors (SOX, 200 ng/ml; BGLF5, 200 ng/ml; muSOX, 100 ng/ml; vhs, 100 ng/ml; or nsp1, 100 ng/ml), and a corresponding amount of the empty vector [(−)] to bring the total concentration of plasmid per transfection to 750 ng/ml. Reporter mRNA levels were visualized using a Northern blot with probes against the 3′ UTR of the reporter or 18S (as a loading control). The arrowhead denotes a protected fragment. The blot shown is representative of three independent repeats.

Fig 4

Depletion of Xrn1 reveals different patterns of primary RNA cleavage by the host shutoff factors. (A and B) HEK293T cells were treated with 70 nM control siRNAs or Xrn1 siRNAs. They were then transfected with a DsRed2 reporter (A) or a shortened GFP reporter with nt 239 to 709 deleted to improve gel resolution (sGFP) (B) together with either empty vector [(−)] or the indicated host shutoff factor as detailed in the legend for Fig. 3B. RNA was Northern blotted with probes against the 3′ UTR of the reporters or 18S (as a loading control). Relative total reporter RNA levels were quantified using the ImageJ software. Blots shown are representative of three repeats. (C) Model of mRNA degradation by the host shutoff factors. The host shutoff factors are recruited to mRNAs either through the ribosome (nsp1) or the cap-binding complex (vhs) or through yet-unknown factors (SOX, muSOX, and BGLF5). They then mediate endonucleolytic mRNA cleavage near the cap (muSOX, nsp1) or at one or more internal positions (SOX, BGLF5, vhs), whereupon host Xrn1 completes degradation of the mRNA body.