The Splicing History of an mRNA Affects Its Level of Translation and Sensitivity to Cleavage by the Virion Host Shutoff Endonuclease during Herpes Simplex Virus Infections

Abstract

During lytic herpes simplex virus (HSV) infections, the virion host shutoff (Vhs) (UL41) endoribonuclease degrades many cellular and viral mRNAs. In uninfected cells, spliced mRNAs emerge into the cytoplasm bound by exon junction complexes (EJCs) and are translated several times more efficiently than unspliced mRNAs that have the same sequence but lack EJCs. Notably, most cellular mRNAs are spliced, whereas most HSV mRNAs are not. To examine the effect of splicing on gene expression during HSV infection, cells were transfected with plasmids harboring an unspliced renilla luciferase (RLuc) reporter mRNA or RLuc constructs with introns near the 5' or 3' end of the gene. After splicing of intron-containing transcripts, all three RLuc mRNAs had the same primary sequence. Upon infection in the presence of actinomycin D, spliced mRNAs were much less sensitive to degradation by copies of Vhs from infecting virions than were unspliced mRNAs. During productive infections (in the absence of drugs), RLuc was expressed at substantially higher levels from spliced than from unspliced mRNAs. Interestingly, the stimulatory effect of splicing on RLuc expression was significantly greater in infected than in uninfected cells. The translational stimulatory effect of an intron during HSV-1 infections could be replicated by artificially tethering various EJC components to an unspliced RLuc transcript. Thus, the splicing history of an mRNA, and the consequent presence or absence of EJCs, affects its level of translation and sensitivity to Vhs cleavage during lytic HSV infections.

Importance: Most mammalian mRNAs are spliced. In contrast, of the more than 80 mRNAs harbored by herpes simplex virus 1 (HSV-1), only 5 are spliced. In addition, synthesis of the immediate early protein ICP27 causes partial inhibition of pre-mRNA splicing, with the resultant accumulation of both spliced and unspliced versions of some mRNAs in the cytoplasm. A common perception is that HSV-1 infection necessarily inhibits the expression of spliced mRNAs. In contrast, this study demonstrates two instances in which pre-mRNA splicing actually enhances the synthesis of proteins from mRNAs during HSV-1 infections. Specifically, splicing stabilized an mRNA against degradation by copies of the Vhs endoribonuclease from infecting virions and greatly enhanced the amount of protein synthesized from spliced mRNAs at late times after infection. The data suggest that splicing, and the resultant presence of exon junction complexes on an mRNA, may play an important role in gene expression during HSV-1 infections.

FIG 1

Spliced TPI/RLuc/TPI mRNAs are less sensitive to Vhs than are unspliced mRNAs. (A) Structures of intron-containing and intronless TPI/RLuc/TPI constructs. The RLuc open reading frame and TPI exons 6 and 7 are represented by white, gray, and black rectangles, respectively. The intron between TPI exons 6 and 7 is depicted by jagged lines. The transcriptional start site is shown by a rightward arrow, and the poly(A) site and start and stop codons are indicated by vertical lines. (B and C) HeLa cells were transfected with the no-intron, 5′ intron, or 3′ intron TPI/RLuc/TPI constructs. Forty-eight hours later, the cells were infected with 20 PFU/cell of wild-type (WT) HSV-1 or mock infected in the presence of 10 μg/ml of actinomycin D. Cultures were harvested 5 h after infection or mock infection and processed to determine the relative levels of TPI/RLuc/TPI mRNA (B) or RLuc activity (C), normalized to the levels in mock-infected cells. Error bars represent standard errors of the means.

FIG 2

Spliced TCR-β minigene mRNAs are less sensitive to Vhs degradation than are unspliced mRNAs. (A) Structures of TCR-β minigene constructs containing portions of three TCR-β exons and no intron, one intron, or two nonidentical introns. The three exons are depicted by light gray, dark gray, and black rectangles. Introns are represented by jagged lines. The transcriptional start site and polyadenylation site are indicated, as are the translational start and stop codons. (B) HeLa cells were transfected with the no-intron (lane a), one-intron (lane b), or two-intron (lane c) TCR-β constructs to yield approximately equal amounts of the spliced and unspliced mRNAs. Forty-eight hours after transfection, cells were infected with 20 PFU/cell of wild-type HSV-1 or mock infected, both in the presence of 10 μg/ml of actinomycin D. Cultures were harvested 5 h later and processed to determine the relative amounts of TCR-β mRNA, normalized to the levels in mock-infected cells. Error bars represent standard errors of the means.

FIG 3

HSV-1 stimulates the accumulation of spliced TPI/RLuc/TPI mRNAs and RLuc activity during productive infections. HeLa cells were transfected with the no-intron (A and D), 5′ intron (B and E), or 3′ intron (C and F) TPI/RLuc/RPI constructs to yield approximately equal amounts of spliced and unspliced mRNAs. Forty-eight hours later, cells were infected with 20 PFU/cell of wild-type HSV-1 or mutant strain Vhs 1 or were mock infected. Replicate cultures were harvested 3 h, 6 h, or 9 h after infection or mock infection and processed to determine the relative amounts of RLuc enzymatic activity (A to C) or TPI/RLuc/TPI mRNA (D to F). For each type of mRNA, the levels of RLuc activity and mRNA at various times in infected and mock-infected cells were normalized to the levels at 3 h in mock-infected cells. Error bars represent the standard errors of the means.

FIG 4

Vhs stimulates the accumulation and expression of spliced TPI/RLuc/TPI mRNAs in the absence of other viral gene products. HeLa cells were transfected with the no-intron or 5′ intron TPI/RLuc/TPI constructs along with either a plasmid expressing wild-type Vhs or the empty expression vector. Replicate cultures were harvested 48 h later and processed to determine the levels of TPI/RLuc/TPI mRNA (A) and RLuc activity (B), normalized to the levels in cells that received the empty expression vector. Error bars represent standard errors of the means.

FIG 5

Effect of HSV-1 infection on unspliced mRNAs tethered to components of the exon junction complex. (A) Structures of an RLuc reporter plasmid encoding an mRNA with 6 copies of the RNA-binding site for the MS2 coat protein and a control FLuc reporter lacking MS2-binding sites. Plasmids encoding fusion proteins with the MS2 RNA-binding domain fused to various EJC components are shown at the right. (B and C) HeLa cells were transfected with equal amounts of the RLuc and FLuc reporter plasmids along with either a plasmid encoding only the MS2 RNA-binding domain or a plasmid encoding a fusion protein with the MS2 RNA-binding domain fused to the indicated EJC component. The cells were infected 48 h later with 20 PFU/cell of wild-type HSV-1 or mock infected. Cell lysates were prepared 3 h (B) or 9 h (C) after infection and analyzed for RLuc and FLuc activities, and RLuc activity was normalized to that of FLuc. (D) HeLa cells were transfected with equal amounts of the RLuc and FLuc reporter plasmids along with a plasmid encoding either the MS2 RNA-binding domain or the MS2 RNA-binding domain fused to eIF4AIII. Cell lysates were prepared 3 or 9 h after infection and analyzed for the relative amounts of RLuc mRNA. Error bars represent standard errors of the means.

FIG 6

Vhs stimulates eIF4AIII-dependent expression of an intronless RLuc mRNA in the absence of other HSV-1 gene products. HeLa cells were transfected with equal amounts of the RLuc and FLuc reporter plasmids along with either a plasmid encoding the MS2 RNA-binding domain (lane a) or a plasmid encoding an MS2-eIF4AIII fusion protein (lanes b to g). Transfection mixtures also contained plasmids harboring wild-type Vhs (lane c); the Vhs T214I (lane d), D194N (lane e), or D215N (lane f) mutant allele; the immediate early protein ICP0 (lane g); or the empty expression vector (lanes a and b). Cell lysates were prepared 48 h after transfection and analyzed for RLuc and FLuc activities, and RLuc activity was normalized to that of FLuc. Error bars represent standard errors of the means.