ICP27 interacts with the RNA export factor Aly/REF to direct herpes simplex virus type 1 intronless mRNAs to the TAP export pathway

Herpes simplex virus type 1 (HSV-1) protein ICP27 facilitates the export of viral intronless mRNAs. ICP27 shuttles between the nucleus and cytoplasm, which has been shown to require a leucine-rich nuclear export sequence (NES). ICP27 export was reported to be sensitive to the CRM1 inhibitor leptomycin B (LMB) in HSV-1-infected cells but not in Xenopus oocytes, where ICP27 interacts with the export factor Aly/REF to access the TAP export pathway. Here, we show that ICP27 interacts with Aly/REF in HSV-1-infected mammalian cells and that Aly/REF stimulates export of viral intronless RNAs but does not cross-link to these RNAs. During infection, Aly/REF was no longer associated with splicing factor SC35 but moved into structures that colocalized with ICP27, suggesting that ICP27 recruits Aly/REF from spliceosomes to viral intronless RNAs. Further, ICP27 was found to interact in vivo with TAP but not with CRM1. In vitro export assays showed that ICP27 export was not sensitive to LMB but was blocked by a dominant-negative TAP deletion mutant lacking the nucleoporin interaction domain. These data suggest that ICP27 uses the TAP pathway to export viral RNAs. Interestingly, the leucine-rich N-terminal sequence was required for efficient export, even though ICP27 export was LMB insensitive. Thus, this region is required for efficient ICP27 export but does not function as a CRM1-dependent NES.     

Amino acid substitution mutations in the herpes simplex virus ICP27 protein define an essential gene regulation function.

ICP27 is an essential herpes simplex virus type 1 (HSV-1) alpha protein that is required for the transition from the beta to the gamma phase of infection. To identify functional regions of ICP27, we constructed 16 plasmids that contain nucleotide substitution mutations in the ICP27 gene. The mutations created XhoI restriction sites, altered one or two codons, and were spaced at semiregular intervals throughout the coding region. Three mutations completely inactivated an essential function of ICP27, as demonstrated by the inability of the transfected plasmids to complement the growth of an HSV-1 ICP27 deletion mutant. These mutations, M11, M15, and M16, mapped in the carboxyl-terminal one-third of ICP27 at residues 340 and 341, 465 and 466, and 488, respectively. In cotransfection assays, all three defective-plasmid mutants retained the transrepression function of ICP27 but were defective at transactivation. To define the lytic functions that are mediated by the transactivation activity of ICP27, we engineered HSV-1 recombinants containing the M11, M15, or M16 mutation. All three viral mutants failed to grow in Vero cells and possessed similar phenotypes. The viral mutants replicated their DNA similarly to the wild-type virus but showed several defects in viral gene expression. These were a failure to down-regulate alpha and beta genes at late times after infection and an inability to induce certain gamma-2 genes. Our results demonstrate that the transactivation function of ICP27 (as it is defined in cotransfection assays) mediates an essential gene regulation function during the HSV-1 infection. This activity is not required for ICP27-dependent enhancement of viral DNA replication. Our work supports and extends previous studies which suggest that ICP27 carries out two distinct regulatory activities during the HSV-1 infection.     

Protein arginine methyltransferase 1 regulates herpes simplex virus replication through ICP27 RGG-box methylation

Protein arginine methylation is involved in viral infection and replication through the modulation of diverse cellular processes including RNA metabolism, cytokine signaling, and subcellular localization. It has been suggested previously that the protein arginine methylation of the RGG-box of ICP27 is required for herpes simplex virus type-1 (HSV-1) viral replication and gene expression in vivo. However, a cellular mediator for this process has not yet been identified. In our current study, we show that the protein arginine methyltransferase 1 (PRMT1) is a cellular mediator of the arginine methylation of ICP27 RGG-box. We generated arginine substitution mutants in this domain and examined which arginine residues are required for methylation by PRMT1. R138, R148 and R150 were found to be the major sites of this methylation but additional arginine residues serving as minor methylation sites are still required to sustain the fully methylated form of ICP27 RGG. We also demonstrate that the nuclear foci-like structure formation, SRPK interactions, and RNA-binding activity of ICP27 are modulated by the arginine methylation of the ICP27 RGG-box. Furthermore, HSV-1 replication is inhibited by hypomethylation of this domain resulting from the use of general PRMT inhibitors or arginine mutations. Our data thus suggest that the PRMT1 plays a key role as a cellular regulator of HSV-1 replication through ICP27 RGG-box methylation.     

The conserved carboxyl-terminal half of herpes simplex virus type 1 regulatory protein ICP27 is dispensable for viral growth in the presence of compensatory mutations

ICP27 is an essential herpes simplex virus type 1 (HSV-1) immediate-early protein that regulates viral gene expression by poorly characterized mechanisms. Previous data suggest that its carboxyl (C)-terminal portion is absolutely required for productive viral infection. In this study, we isolated M16R, a second-site revertant of a viral ICP27 C-terminal mutant. M16R harbors an intragenic reversion, as demonstrated by the fact that its cloned ICP27 allele can complement the growth of an HSV-1 ICP27 deletion mutant. DNA sequencing demonstrated that the intragenic reversion is a frameshift alteration in a homopolymeric run of C residues at codons 215 to 217. This results in the predicted expression of a truncated, 289-residue molecule bearing 72 novel C-terminal residues derived from the +1 reading frame. Consistent with this, M16R expresses an ICP27-related molecule of the predicted size in the nuclei of infected cells. Transfection-based viral complementation assays confirmed that the truncated, frameshifted protein can partially substitute for ICP27 in the context of viral infection. Surprisingly, its novel C-terminal residues are required for this activity. To see if the frameshift mutation is all that is required for M16R's viability, we re-engineered the M16R ICP27 allele and inserted it into a new viral background, creating the HSV-1 mutant M16exC. An additional mutant, exCd305, was constructed which possesses the frameshift in the context of an ICP27 gene with the C terminus deleted. We found that both M16exC and exCd305 are nonviable in Vero cells, suggesting that one or more extragenic mutations are also required for the viability of M16R. Consistent with this interpretation, we isolated two viable derivatives of exCd305 which grow productively in Vero cells despite being incapable of encoding the C-terminal portion of ICP27. Studies of viral DNA synthesis in mutant-infected cells indicated that the truncated, frameshifted ICP27 protein can enhance viral DNA replication. In summary, our results demonstrate that the C-terminal portion of ICP27, conserved widely in herpesviruses and previously believed to be absolutely essential, is dispensable for HSV-1 lytic replication in the presence of compensatory genomic mutations.     

Mutational analysis of the herpes simplex virus type 1 ICP0 C3HC4 zinc ring finger reveals a requirement for ICP0 in the expression of the essential alpha27 gene.

The herpes simplex virus type 1 (HSV-1) immediate-early (IE) protein ICP0 has been implicated in the regulation of viral gene expression and the reactivation of latent HSV-1. Evidence demonstrates that ICP0 is an activator of viral gene expression yet does not distinguish between a direct or indirect role in this process. To further our understanding of the function of ICP0 in the context of the virus life cycle, site-directed mutagenesis of the consensus C3HC4 zinc finger domain was performed, and the effects of these mutations on the growth and replication of HSV-1 were assessed. We demonstrate that alteration of any of the consensus C3HC4 cysteine or histidine residues within this domain abolishes ICP0-mediated transactivation, alters the intranuclear localization of ICP0, and significantly increases its stability. These mutations result in severe defects in the growth and DNA replication of recombinant herpesviruses and in their ability to initiate lytic infections at low multiplicities of infection. These viruses, at low multiplicities of infection, synthesize wild-type levels of the IE proteins ICP0 and ICP4 at early times postinfection yet exhibit significant decreases in the synthesis of the essential IE protein ICP27. These findings reveal a role for ICP0 in the expression of ICP27 and suggest that the multiplicity-dependent growth of alpha0 mutant viruses results partially from reduced levels of ICP27.     

Host MOV10 is induced to restrict herpes simplex virus 1 lytic infection by promoting type I interferon response

Moloney leukemia virus 10 protein (MOV10) is an interferon (IFN)-inducible RNA helicase implicated in antiviral activity against RNA viruses, yet its role in herpesvirus infection has not been investigated. After corneal inoculation of mice with herpes simplex virus 1 (HSV-1), we observed strong upregulation of both MOV10 mRNA and protein in acutely infected mouse trigeminal ganglia. MOV10 suppressed HSV-1 replication in both neuronal and non-neuronal cells, and this suppression required the N-terminus, but not C-terminal helicase domain of MOV10. MOV10 repressed expression of the viral gene ICP0 in transfected cells, but suppressed HSV-1 replication independently of ICP0. MOV10 increased expression of type I IFN in HSV-1 infected cells with little effect on IFN downstream signaling. Treating the cells with IFN-α or an inhibitor of the IFN receptor eliminated MOV10 suppression of HSV-1 replication. MOV10 enhanced IFN production stimulated by cytoplasmic RNA rather than DNA. IKKε co-immunoprecipitated with MOV10 and was required for MOV10 restriction of HSV-1 replication. Mass spectrometry identified ICP27 as a viral protein interacting with MOV10. Co-immunoprecipitation results suggested that this interaction depended on the RGG box of ICP27 and both termini of MOV10. Overexpressed ICP27, but not its RGG-Box deletion mutant, rendered MOV10 unable to regulate HSV-1 replication and type I IFN production. In summary, MOV10 is induced to restrict HSV-1 lytic infection by promoting the type I IFN response through an IKKε-mediated RNA sensing pathway, and its activity is potentially antagonized by ICP27 in an RGG box dependent manner.     

Herpesvirus protein ICP27 switches PML isoform by altering mRNA splicing

Viruses use alternative splicing to produce a broad series of proteins from small genomes by utilizing the cellular splicing machinery. Since viruses use cellular RNA binding proteins for viral RNA processing, it is presumable that the splicing of cellular pre-mRNAs is affected by viral infection. Here, we showed that herpes simplex virus type 2 (HSV-2) modifies the expression of promyelocytic leukemia (PML) isoforms by altering pre-mRNA splicing. Using a newly developed virus-sensitive splicing reporter, we identified the viral protein ICP27 as an alternative splicing regulator of PML isoforms. ICP27 was found to bind preferentially to PML pre-mRNA and directly inhibit the removal of PML intron 7a in vitro. Moreover, we demonstrated that ICP27 functions as a splicing silencer at the 3′ splice site of the PML intron 7a. The switching of PML isoform from PML-II to PML-V as induced by ICP27 affected HSV-2 replication, suggesting that the viral protein modulates the splicing code of cellular pre-mRNA(s) governing virus propagation.     

A single amino acid substitution in the ICP27 protein of herpes simplex virus type 1 is responsible for its resistance to leptomycin B

Leptomycin B (LMB) is a specific inhibitor of Crm1-dependent nuclear export of proteins. The replication of herpes simplex virus (HSV) is normally highly sensitive to LMB; a resistant HSV variant, however, was isolated by serial passages of the virus. Analysis of marker transfer and viral DNA sequences revealed that a single amino acid substitution within the ICP27 gene is responsible for conferring this resistance.     

Herpes Simplex Virus Type 1 ICP27 Induces p38 Mitogen-Activated Protein Kinase Signaling and Apoptosis in HeLa Cells

The herpes simplex virus type 1 (HSV-1) protein ICP27 has been implicated in a variety of functions important for viral replication including host shutoff, viral gene expression, activation of mitogen-activated protein kinases p38 and Jun N-terminal protein kinase (JNK), and apoptosis inhibition. In the present study we sought to examine the functions of ICP27 in the absence of viral infection by creating stable HeLa cell lines that inducibly express ICP27. Here, we characterize two such cell lines and show that ICP27 expression is associated with a cellular growth defect. The observed defect is caused at least in part by the induction of apoptosis as indicated by caspase-3 activation, annexin V staining, and characteristic changes in cellular morphology. In an effort to identify the function of ICP27 responsible for inducing apoptosis, we show that ICP27 expression is sufficient to activate p38 signaling to a level that is similar to that observed during wild-type HSV-1 infection. However, ICP27 expression alone is unable to lead to a strong activation of JNK signaling. Using chemical inhibitors, we show that the ICP27-mediated activation of p38 signaling is responsible for the observed induction of apoptosis in the induced cell lines. Our findings suggest that during viral infection, ICP27 activates p38 and JNK signaling pathways via two distinct mechanisms. ICP27 directly activates p38 signaling, leading to stimulation of the host cell apoptotic pathways. In contrast, robust activation of JNK signaling by ICP27 requires one or more delayed early or late viral gene products and may be associated with the inhibition of apoptosis.     

The acidic amino-terminal region of herpes simplex virus type 1 alpha protein ICP27 is required for an essential lytic function.

The herpes simplex virus type 1 (HSV-1) alpha protein ICP27 regulates the transition between the delayed-early and late phases of the viral infection. Previous genetic analyses have suggested that the important functional domains of ICP27 map to its carboxyl-terminal half. One striking feature of the primary sequence of ICP27, however, is an extremely acidic region near its amino terminus. To determine whether this region is required for ICP27 function, we deleted the sequences in the ICP27 gene which encode it (codons 12 through 63). In transient expression assays, the deletion mutant was unable to efficiently repress the expression of a cotransfected reporter gene or to efficiently complement the growth of d27-1, an HSV-1 ICP27 null mutant. These results suggested that the acidic region of ICP27 is involved in a regulatory function required for lytic growth. To test this possibility further, we introduced the mutant allele into the HSV-1 genome by marker transfer. Two independently derived isolates of the mutant virus, designated d1-2a and d1-2b, were recovered and analyzed. Both isolates were defective for growth in Vero cells, exhibiting a 100-fold reduction in virus yield compared with the wild-type infection. Vero cells infected with the d1-2 isolates showed a three- to eightfold reduction in viral DNA replication, a moderate reduction in the expression of viral gamma genes, and a delay in the repression of beta genes. The phenotype of the d1-2 isolates differs substantially from the phenotypes of previously isolated ICP27 mutants, which show much more severe defects in viral gene expression. Our results demonstrate that the amino-terminal half of ICP27 participates in its regulatory activities in both infected and transfected cells.     

Herpes simplex virus ICP27 is required for virus-induced stabilization of the ARE-containing IEX-1 mRNA encoded by the human IER3 gene

Herpes simplex virus (HSV) stifles cellular gene expression during productive infection of permissive cells, thereby diminishing host responses to infection. Host shutoff is achieved largely through the complementary actions of two viral proteins, ICP27 and virion host shutoff (vhs), that inhibit cellular mRNA biogenesis and trigger global mRNA decay, respectively. Although most cellular mRNAs are thus depleted, some instead increase in abundance after infection; perhaps surprisingly, some of these contain AU-rich instability elements (AREs) in their 3'-untranslated regions. ARE-containing mRNAs normally undergo rapid decay; however, their stability can increase in response to signals such as cytokines and virus infection that activate the p38/MK2 mitogen-activated protein kinase (MAPK) pathway. We and others have shown that HSV infection stabilizes the ARE mRNA encoding the stress-inducible IEX-1 mRNA, and a previous report from another laboratory has suggested vhs is responsible for this effect. However, we now report that ICP27 is essential for IEX-1 mRNA stabilization whereas vhs plays little if any role. A recent report has documented that ICP27 activates the p38 MAPK pathway, and we detected a strong correlation between this activity and stabilization of IEX-1 mRNA by using a panel of HSV type 1 (HSV-1) isolates bearing an array of previously characterized ICP27 mutations. Furthermore, IEX-1 mRNA stabilization was abrogated by the p38 inhibitor SB203580. Taken together, these data indicate that the HSV-1 immediate-early protein ICP27 alters turnover of the ARE-containing message IEX-1 by activating p38. As many ARE mRNAs encode proinflammatory cytokines or other immediate-early response proteins, some of which may limit viral replication, it will be of great interest to determine if ICP27 mediates stabilization of many or all ARE-containing mRNAs.     

The herpes simplex virus type 1 regulatory protein ICP27 is required for the prevention of apoptosis in infected human cells

The herpes simplex virus type 1 (HSV-1) ICP27 protein is an immediate-early or alpha protein which is essential for the optimal expression of late genes as well as the synthesis of viral DNA in cultures of Vero cells. Our specific goal was to characterize the replication of a virus incapable of synthesizing ICP27 in cultured human cells. We found that infection with an HSV-1 ICP27 deletion virus of at least three separate strains of human cells did not produce immediate-early or late proteins at the levels observed following wild-type virus infections. Cell morphology, chromatin condensation, and genomic DNA fragmentation measurements demonstrated that the human cells died by apoptosis after infection with the ICP27 deletion virus. These features of the apoptosis were identical to those which occur during wild-type infections of human cells when total protein synthesis has been inhibited. Vero cells infected with the ICP27 deletion virus did not exhibit any of the features of apoptosis. Based on these results, we conclude that while HSV-1 infection likely induced apoptosis in all cells, viral evasion of the response differed among the cells tested in this study.     

Herpes Simplex Virus 1 ICP22 but Not US1.5 Is Required for Efficient Acute Replication in Mice and VICE Domain Formation

The herpes simplex virus 1 (HSV-1) immediate-early protein, infected cell protein 22 (ICP22), is required for efficient replication in restrictive cells, for virus-induced chaperone-enriched (VICE) domain formation, and for normal expression of a subset of viral late proteins. Additionally, ICP22 is important for optimal acute viral replication in vivo. Previous studies have shown that the U_S1 gene that encodes ICP22, produces an in-frame, N-terminally truncated form of ICP22, known as U_S1.5. To date, studies conducted to characterize the functions of ICP22 have not separated its functions from those of U_S1.5. To determine the individual roles of ICP22 and U_S1.5, we made viral mutants that express either ICP22 with an M90A mutation in the U_S1.5 initiation codon (M90A) or U_S1.5 with three stop codons introduced upstream of the U_S1.5 start codon (3×stop). Our studies showed that, in contrast to M90A, 3×stop was unable to replicate efficiently in the eyes and trigeminal ganglia of mice during acute infection, to efficiently establish a latent infection, or to induce VICE domain formation and was only mildly reduced in its replication in restrictive HEL-299 cells and murine embryonic fibroblasts (MEFs). Both mutants enhanced the expression of the late viral proteins virion host shutoff (vhs) and glycoprotein C (gC) and inhibited viral gene expression mediated by HSV-1 infected cell protein 0 (ICP0). When we tested our mutants'' sensitivity to type I interferon (beta interferon [IFN-β]) in restrictive cells, we noticed that the plating of the ICP22 null (d22) and 3×stop mutants was reduced by the addition of IFN-β. Overall, our data suggest that U_S1.5 partially complements the functions of ICP22.     

The PK Domain of the Large Subunit of Herpes Simplex Virus Type 2 Ribonucleotide Reductase (ICP10) Is Required for Immediate-Early Gene Expression and Virus Growth

The large subunit of herpes simplex virus (HSV) ribonucleotide reductase (RR), RR1, contains a unique amino-terminal domain which has serine/threonine protein kinase (PK) activity. To examine the role of the PK activity in virus replication, we studied an HSV type 2 (HSV-2) mutant with a deletion in the RR1 PK domain (ICP10ΔPK). ICP10ΔPK expressed a 95-kDa RR1 protein (p95) which was PK negative but retained the ability to complex with the small RR subunit, RR2. Its RR activity was similar to that of HSV-2. In dividing cells, onset of virus growth was delayed, with replication initiating at 10 to 15 h postinfection, depending on the multiplicity of infection. In addition to the delayed growth onset, virus replication was significantly impaired (1,000-fold lower titers) in nondividing cells, and plaque-forming ability was severely compromised. The RR1 protein expressed by a revertant virus [HSV-2(R)] was structurally and functionally similar to the wild-type protein, and the virus had wild-type growth and plaque-forming properties. The growth of the ICP10ΔPK virus and its plaque-forming potential were restored to wild-type levels in cells that constitutively express ICP10. Immediate-early (IE) genes for ICP4, ICP27, and ICP22 were not expressed in Vero cells infected with ICP10ΔPK early in infection or in the presence of cycloheximide, and the levels of ICP0 and p95 were significantly (three- to sevenfold) lower than those in HSV-2- or HSV-2(R)-infected cells. IE gene expression was similar to that of the wild-type virus in cells that constitutively express ICP10. The data indicate that ICP10 PK is required for early expression of the viral regulatory IE genes and, consequently, for timely initiation of the protein cascade and HSV-2 growth in cultured cells.