Identification of an ICP27-responsive element in the coding region of a herpes simplex virus type 1 late gene.

During productive herpes simplex virus type 1 (HSV-1) infection, a subset of viral delayed-early (DE) and late (L) genes require the immediate-early (IE) protein ICP27 for their expression. However, the cis-acting regulatory sequences in DE and L genes that mediate their specific induction by ICP27 are unknown. One viral L gene that is highly dependent on ICP27 is that encoding glycoprotein C (gC). We previously demonstrated that this gene is posttranscriptionally transactivated by ICP27 in a plasmid cotransfection assay. Based on our past results, we hypothesized that the gC gene possesses a cis-acting inhibitory sequence and that ICP27 overcomes the effects of this sequence to enable efficient gC expression. To test this model, we systematically deleted sequences from the body of the gC gene and tested the resulting constructs for expression. In so doing, we identified a 258-bp "silencing element" (SE) in the 5' portion of the gC coding region. When present, the SE inhibits gC mRNA accumulation from a transiently transfected gC gene, unless ICP27 is present. Moreover, the SE can be transferred to another HSV-1 gene, where it inhibits mRNA accumulation in the absence of ICP27 and confers high-level expression in the presence of ICP27. Thus, for the first time, an ICP27-responsive sequence has been identified in a physiologically relevant ICP27 target gene. To see if the SE functions during viral infection, we engineered HSV-1 recombinants that lack the SE, either in a wild-type (WT) or ICP27-null genetic background. In an ICP27-null background, deletion of the SE led to ICP27-independent expression of the gC gene, demonstrating that the SE functions during viral infection. Surprisingly, the ICP27-independent gC expression seen with the mutant occurred even in the absence of viral DNA synthesis, indicating that the SE helps to regulate the tight DNA replication-dependent expression of gC.

Herpes simplex virus type 1 ICP27 regulates expression of a variant, secreted form of glycoprotein C by an intron retention mechanism.

We previously showed that herpes simplex virus type 1 (HSV-1) immediate-early (IE) protein ICP27 can posttranscriptionally stimulate mRNA accumulation from a transfected viral late gene encoding glycoprotein C (gC) (K. D. Perkins, J. Gregonis, S. Borge, and S. A. Rice, J. Virol. 77:9872-9884, 2003). We began this study by asking whether ICP27 homologs from other herpesviruses can also mediate this activity. Although the homologs from varicella-zoster virus (VZV) and human cytomegalovirus (HCMV) were inactive, the homolog from bovine herpesvirus 4 (BHV-4), termed HORF1/2, was a very efficient transactivator. Surprisingly, most of the mRNA produced via HORF1/2 transactivation was 225 nucleotides shorter than expected due to the removal of a previously undescribed intron from the gC transcript. We found that the gC mRNA produced in the absence of transactivation was also mostly spliced. In contrast, gC mRNA produced by ICP27 transactivation was predominantly unspliced. Based on these results, we conclude that ICP27 has two distinct effects on the transfected gC gene: it (i) stimulates mRNA accumulation and (ii) promotes the retention of an intron. Interestingly, the spliced transcript encodes a variant of gC that lacks its transmembrane domain and is secreted from transfected cells. As the gC splicing signals are conserved among several HSV-1 strains, we investigated whether the variant gC is expressed during viral infection. We report here that both the spliced transcript and its encoded protein are readily detected in Vero cells infected with three different laboratory strains of wild-type HSV-1. Moreover, the variant gC is efficiently secreted from infected cells. We have designated this alternate form of the protein as gCsec. As the extracellular domain of gC is known to bind heparan sulfate-containing proteoglycans and to inhibit the complement cascade via an interaction with complement component C3b, we speculate that gCsec could function as a secreted virulence factor.

A new technique for the surgical creation of aneurysms in an in vivo tortuous vessel model to test neurovascular devices.

The purpose of this study was to develop an aneurysm model that mimics the tortuous anatomy of the cerebrovasculature for the evaluation of endovascular devices. This model is an adaptation of the carotid siphon model of Georganos et al. The common carotid artery trunks in 10 swine were surgically elongated using an EXXCEL Soft ePTFE vascular graft and then sutured into position to form an S-curve, with each bend having a 5- to 10-mm radius. Following a 3- to 4-week healing period, aneurysms were surgically created from jugular vein grafts along or distal to the tortuous segment and immediately embolized with coils. In a subset (n = 6) of the arteries, a stent was also placed across the aneurysm neck. Animals were allowed to survive for 30 days. Clinical relevance and utility of the model were evaluated based on comparison to human angiographic images, physician feedback, and histopathological assessment. Tortuous anatomy was successfully created in all 10 animals, and aneurysms were added at various locations within or distal to the tortuous segment in a subset of 8 animals, creating 11 aneurysms in total. At 30 days, 18/20 vessels were patent and the bend radius was maintained. Endovascular access to aneurysms and placement of embolization coils and/or stents was successful in 10 of 11 attempts. Physician feedback indicated this tortuous model was more clinically relevant in terms of endovascular device delivery and deployment compared to established, nontortuous aneurysm models.

ICP27-dependent resistance of herpes simplex virus type 1 to leptomycin B is associated with enhanced nuclear localization of ICP4 and ICP0.

It was previously shown that herpes simplex virus type 1 (HSV-1) is sensitive to leptomycin B (LMB), an inhibitor of nuclear export factor CRM1, and that a single methionine to threonine change at residue 50 (M50T) of viral immediate-early (IE) protein ICP27 can confer LMB resistance. In this work, we show that deletion of residues 21-63 from ICP27 can also confer LMB resistance. We further show that neither the M50T mutation nor the presence of LMB affects the nuclear shuttling activity of ICP27, suggesting that another function of ICP27 determines LMB resistance. A possible clue to this function emerged when it was discovered that LMB treatment of HSV-1-infected cells dramatically enhances the cytoplasmic accumulation of two other IE proteins, ICP0 and ICP4. This effect is completely dependent on ICP27 and is reversed in cells infected with LMB-resistant mutants. Moreover, LMB-resistant mutations in ICP27 enhance the nuclear localization of ICP0 and ICP4 even in the absence of LMB, and this effect can be discerned in transfected cells. Thus, the same amino (N)-terminal region of ICP27 that determines sensitivity to LMB also enhances ICP27's previously documented ability to promote the cytoplasmic accumulation of ICP4 and ICP0. We speculate that ICP27's effects on ICP4 and ICP0 may contribute to HSV-1 LMB sensitivity.

Transactivation of a viral target gene by herpes simplex virus ICP27 is posttranscriptional and does not require the endogenous promoter or polyadenylation site.

ICP27 is an essential herpes simplex virus type 1 (HSV-1) immediate-early protein that stimulates viral mRNA expression from many viral delayed-early and late genes during infection. One HSV-1 late gene which is highly dependent on ICP27 during infection is that encoding the glycoprotein C (gC). Here we report that the gC gene is specifically transactivated by ICP27 in transfected Vero cells. Using various gC plasmid constructs, we show that ICP27's stimulatory effects are independent of the gC gene's endogenous promoter and polyadenylation site. This suggests that ICP27-responsive elements lie in the transcribed body of the gC gene. We also show that transactivation of the gC gene by ICP27 is independent of other viral proteins, as ICP27 alone can transactivate the gC gene when its transcription is mediated by the human cytomegalovirus immediate-early gene promoter. However, when gC gene expression is driven by its endogenous promoter, the stimulatory effect of ICP27 requires additional transactivators. To explore the level at which ICP27 transactivates the gC gene, we established stably transfected Vero cell lines that have integrated copies of the gC gene under control of the cytomegalovirus immediate-early gene promoter. These gC genes are not constitutively expressed but can be efficiently induced by HSV-1 infection. Using nuclear run-on transcription assays, we show that transcriptional induction of the stably transfected genes is ICP27 independent. In contrast, accumulation of gC mRNA is very highly dependent on ICP27. Together, these results demonstrate that ICP27 posttranscriptionally activates mRNA expression from a biologically relevant viral target gene.